International Journal of Biotechnology and Recent Advances

ISSN: 2639-4529

International Biotechnology and Research Conference

April 25-27, 2018, Rome, Italy
Accepted Abstracts
DOI: 10.18689/2639-4529.a1.004

Type III Polyketide Synthase from Indian Gooseberry: An Ideal Candidate for Metabolic Engineering to Develop Novel Nutraceuticals

E V Soniya

Rajiv Gandhi Centre for Biotechnology, India

Emblica officinalis Gaertn (Indian Gooseberry) is one of the most important medicinal plants used in various Ayurvedic formulations and traditional medicine. The plant is native to India, also grows in the tropical and subtropical regions including China, Pakistan, South East Asia and Srilanka. It has beneficial role in cancer, diabetes, liver treatment, anaemia and has application as antioxidant, immunomodulatory, antipyretic, analgesic etc. The plant is rich in secondary metabolites like flavonoids, phenols, tannins, alkaloids and vitamin C. Because of the numerous health benefits attributed to the consumption of E. officinalis as tonic and medicine, there is considerable interest in defining the pathways that control the accumulation of active components. It has been suggested that type III polyketide synthase (PKS) family of enzymes has specific role in the biosynthesis of natural products like flavonoids, alkaloids and curcuminoids. We could successfully isolate and characterise a novel type III polyketide synthase from E. officinalis. Along with this focus is also given to improve the substrate specificity of the identified type III PKS with the aid of site-directed mutagenesis. The findings of the in vitro and in planta functional characterization of the mutants based on high-throughput metabolomics is one of the interesting outcomes of the study. Heterologous expression and in vitro enzymatic assay of EoPKS1 mutants resulted in the formation of quinolone/acridone precursors which can contribute to the large scale combinatorial biosynthesis of pharmaceutically relevant compounds in vitro.

Biography:
Soniya was awarded doctorate from the University of Kerala and worked two years at Central Tuber Crops Research Institute, Sreekaryam, Thiruvananthapuram before joining Rajiv Gandhi Centre for Biotechnology. She is the Fellow of National Academy of Science, India. She is the INSA/DFG visiting Scientist in Max Planck Institute of Chemical Ecology, Jena, Germany. She organized the EMBO Conference in Plant Science in RGCB, Thiruvananthapuram. Her area of research is Characterisation of small functional non-coding RNAs and Type III polyketide synthases for exploiting its potential use in metabolic engineering. She has published morethan 20 research articles in peer reviewed International Journals and presented her research works in so many international conferences. She wrote several chapters in the Plant biology field.

Sensitivity of ITT1Δ and RPS1AΔ Yeast Strains to Arsenite Reveals their Role in Translation of URE2, a Key Gene Involved in Metal Detoxification

Houman Moteshareie1*, Bahram Samanfar1,2, Alex Mulet1 and Ashkan Golshani1

1Department of Biology, Carleton University, Canada
2Ottawa Institute of Systems Biology, Canada

Heavy metal and metalloid contaminations are among the most concerning pollutions in the world. Many parts of the world are polluted with toxic heavy metals and metalloids. Consequently, investigating the molecular mechanisms that drive the detoxification pathways in living organisms is of importance. A variety of genes have known functions in detoxification. The expression of these genes is controlled at both transcriptional and translational levels. When under heavy-metal stress, 5ʼ cap-dependent translation is often down-regulated or even completely inhibited. In bakerʼs yeast, Saccharomyces cerevisiae, resistance to a wide range of toxic metals is regulated by glutathione S-transferase products. Yeast URE2 protein has glutathione peroxidase activity and is homologous to mammalian glutathione S-transferases. In addition to 5ʼ cap-dependent translation, yeast URE2 mRNA is also translated through an internal structure, free of 5ʼ cap and eIF4E. It possesses a unique Internal Ribosome Entry Site (IRES) element between nucleotides 205 and 309 in its coding region. URE2 deletion mutants are hypersensitive to toxic metals such as arsenic, nickel and cadmium. Here, we report on the finding of two genes, ITT1, inhibitor of translation termination, and RPS1A, ribosomal protein 10 whose deletion strains exhibit similar drug sensitivity phenotype as URE2 mutant. Neither of these genes was previously linked to metal toxicity. Our gene expression analysis illustrates that these two genes affect URE2 mRNA expression at the translational level. Subsequent investigations suggest that the deletion of ITT1 or RPS1A may negatively regulate the translation of URE2 mRNA through its IRES element. Polysome profile analysis suggest a strong tie between candidate genes and protein synthesis mainly at the initiation step. Our genetic interaction analysis also showed strong communication between our two candidate genes and the genes involved in the regulation of translational and translation initiation.

Biography:
Houman Moteshareie completed his Bachelorʼs degree in Cambridge, UK in Medical Genetics. He continued his education with obtaining a Masterʼs degree from University of Wales, UK in Biotechnology and Molecular Genetics. He is now in the final year of his PhD at Carleton University, Canada in Molecular Genetics and Systems Biology. Houman is currently a lecturerat Carleton University and Algonquin College. His research focuses on identification and investigation ofnovel genes that are involved in protein biosynthesis. In particular, he is working on Internal Ribosome Entry Sites (IRES) utilizing bakerʼs yeast, Saccharomyces cerevisiae as a model organism. During his graduate education, Houman has been involved in many research and collaborated with excellent scientists, which resulted in many scientific publications.

Continuous Multilayered Composite PVA Hydrogel as Cartilage Substitute

Gemma Leone1*, Marco Consumi1, Stefania Lamponi1, Elisa Boanini2, Adriana Bigi2 and Agnese Magnani1

1University of Siena and INSTM Siena Research Unit, Italy
2University of Bologna, Italy

Cartilage is a highly organized avascular soft tissue that assembles from nano-to macro-scale to produce a complex structural network. To mimic cartilage tissue, we developed a stable multilayered composite material (MSC), characterized by a tailored gradient of mechanical properties. MSCwas obtained through a multistep procedure. A mixture of PVA and HA nanocrystals (nHA/PVA molar ratio of 0.015) was crosslinked using tri-sodium tri-metaphosphate (STMP), the crosslinking agent, added in a molar ratio 1:1 with PVA (PS11HA)(first layer). The second layer was obtained by crosslinking PVA directly on the surface of first layer without addition of the inorganic phase but with the same PVA/STMP molar ratio of the first layer (PS11). The same procedure was then applied to crosslink a third layer, which was produced with a greater PVA/STMP molar ratio (2:1). MSC can be considered a promising potential substitute for damaged cartilage tissue, since it mimics the gradient of water content and rheological properties strictly comparable with those of cartilage in terms of complex modulus (G*: 0.032±0.003 MPa; cartilage: G*: 0.03±0.003 MPa) and recovery (70% recovery after just 0.1 s). The presence of nano-hydroxyapatite in its bottom layer stimulate the adhesion to bone, whereas the uppermost soft layer represents an ideal environment for interaction with cartilage guaranteeing a lubricant action as confirmed by the good cytocompatibility shown by MSC (layer PS21) and MSC (layer PS11HA) towards chondrocytes and osteoblasts, respectively, and by the increased BALP production in samples containing nHA in comparison with samples without nHA.

Biography:
Gemma Leone is a researcher at the University of Siena. Her research activity is focused on the synthesis and the physico-chemical and rheological characterization of natural or synthetic macromolecular based bi-dimensional or three-dimensional surfaces, which can be utilized as cell scaffolds or drug vehicles. Her main research fields concern: I) Development of new polysaccharide –based hydrogels for soft tissue regeneration II) Protein adsorption studies on biomaterials III) Synthesis and physico-chemical and rheological characterization of polyvinylic hydrogels as ophthalmological devices IV) Development of model systems for saccharide based biosensors

Medical Pluralism- Practice of Alternative Medicine in Rural Communities of Nepal

Himal Luitel1,2*, Tatyana Novoyatleva1, Akylbek Sydykov1, Aleksandar Petrovic1, Argen Mamazhakypov1, Bhuminanda Devkota2, Malgorzata Wygrecka1, Hossein Ardeschir Ghofrani1, Ralph Theo Schermuly1 and Djuro Kosanovic1

1Universities of Giessen and Marburg Lung Center, Germany
2Agriculture and Forestry University, Center for Biotechnology, Nepal

Alternative medicine has been practiced and still widely accepted in Chinese as well as south Asian medical system. Use of both complementary and alternative medicine (CAM) is quite popular in rural Nepal where people are using both alternative as well as allopathic medicine. Several plants, animal and other natural resources are widely used as either therapeutic options or nutritional supplements. Owing to the huge geographical diversity of Nepal, it is rich in flora and many of them have medical importance. There is not systematic study of Nepalese flora in terms of active ingredients and their medicinal properties. Efficacy of Yarsagumba and its bioactive compound cordycepin in Pulmonary Hypertension (PH) was tested by well-designed systematic experimental setting in cell culture as well as murine model.

Entomogenous fungus Yarsagumba (Cordycepssinensis) and its biochemically active constituents such as cordycepin, which have among others the anti-oxidant, anti-inflammatory and vasodilatory effects could be the therapeutic options for this incurable disease. We investigated the effect of Yarsagumba extract and its purified bioactive compound cordycepin on human pulmonary artery smooth muscle cells (hPASMCs) and in murine model of isolated lungs to investigate their still unknown anti-proliferative and vasorelaxant properties in the context of Pulmonary Hypertension (PH). Both Yarsagumba extract and cordycepin significantly reduced proliferation of hPASMCs derived from donor and PH subjects. However, only Yarsagumba extract, and not cordycepin, showed the vasodilatory outcome, suggesting the existence of other active metabolites present in Yarsagumba which may be responsible for this effect. Further research in the field is needed to provide the detailed and mechanistic insights about the exact therapeutic potency of Yarsagumba extract and whether it can be used as an option to treat PH patients.

Biography:
Himal Luitel is an academician and researcher, currently serving as Assistant Professor in Center for Biotechnology, Agriculture and Forestry University, Chitwan, Nepal. He is veterinary graduate from Tribhuvan University, Nepal, Masters in Molecular Biology from Interuniversity Program Molecular Biology, Belgium, and PhD, Post-Doctorate from Excellence Cluster Cardio-Pulmonary System, Justus-Liebig University, Germany. He has expertise in in vivo (lab animals) and in vitro testing of pharmacologically active compounds. Currently, he is establishing new lab in Center for Biotechnology, Agriculture and Forestry University, Chitwan, Nepal where he is planning to do research in natural products in laboratory animals and in vitro settings. Currently, he is conducting research on common bacterial diseases of poultry for development of rapid molecular diagnostic tools and identifies candidate genes for vaccine development.

Study of Translational Gaps to Address the Affordable and Accessible Priority Indigenous Technologies: Emerging Aid to Indiaʼs Health Sector

Smita Sahu

Amity University, India

Medical Devices and Diagnostics cater an integral component of the health care system with a mandate of ‘access and equity’. India is battling with the issues of accessibility, affordability and availability of quality health for Public. Indian medical heritage dated back to 3000BC unveils the rich knowledge pool which has undergone a perceptible change over years, such as eradication of many Communicable diseases, changing statistics of individual awareness of quality health and import driven medical device market etc. Despite slew of initiatives the holistic slogan of ‘health for all’ remains the concern of nation.

The present scenario exhibiting demographic dividend and geographical segmentation fostering varied needs of people as per their regional conditions of climate, disease prevalence, nutrition and sanitation requires reinforcing the potential of indigenous technologies in coordination with prevailing health issues in sectoral/regional/territory of country. This paper proposes to adopt technology transfer as a strategy to establish a vibrant ecosystem for identifying and upscaling the indigenous medical technologies with diligent handholding to address the diagnostic capacity building with robust regulatory regime to mitigate the challenges of accessibility in resource poor settings, import dependency in the conducive promising initiative of government like Make- In-India, which opens new avenues to a flourishing future of indigenous medical health technologies and innovations for delivering affordable healthcare to Indiaʼs billion-plus population.

Transcriptome Profiling in Gastric Cancer Prakash C. Sharma1*, Anil Agarwal2, Puja Sakhuja3 and Renu Verma1

1University School of Biotechnology, Guru Gobind Singh Indraprastha University, India
2Department of Gastointestinal Surgery, Govind Ballabh Pant Hospital, India
3Department of Pathology, Govind Ballabh Pant Hospital, India

Gastric cancer (GC) is the fifth most common cancer and third most common cause of cancer-related mortality in the world. The symptoms of GC occur in the late stages making early diagnosis a challenging task compromising disease management. Therefore, efforts are underwayto develop effective biomarkers for early diagnosis of the disease and to identify new targets of drug development. Recently, Next Generation Sequencing (NGS) based RNA Seq approachhave provided a cutting edge over traditional methods inunderstanding molecular pathogenesis of human diseases and disorders. We have performed comparative transcriptome profiling of tumor and normal tissues using Illumina RNA-Seq to identify differentially expressed genes (DEGs) and single nucleotide polymorphisms (SNPs) involved in the occurrence of gastric tumorigenesis. Over 390 million high quality reads covering 90% of total reads were obtained from twelve libraries, six each fortumor and normal tissue. We identified 2034 differentially expressed genes in cancerous tissue in comparison to normal tissue. The top ten upregulated and downregulated genes were CDH3, DKC1, NAT9, KRT5, CDCA5, BATF2, GNA15, ENG, KRT16, LAMP3 and LIPF, PRKAR2B, VIT, SLC28A2, FBLN2, ACACB, MAP7D2, MTURN, SASH3, CDS1, respectively. The DEGs were accommodated in various ontology categories including digestion, digestive system processes, and digestive system and digestive tract development. The level of expression of selected DEGs in tumor tissues has been validated through real-time PCR. Our findings contribute towards understanding molecular events occurring during gastric tumorigenesis and useful leads for developing future strategies management ofgastric cancer.

Biography:
Prof. Prakash Chand Sharma has teaching and research experience of over 35 years in the field of genetics, molecular biology and biotechnology. He has contributed significantly towards development and use of molecular markers, particularly microsatellite markers, for diverse applications in different crop plants. Recently, his group has exploited NGS-based transcriptome profiling to identify abiotic stress responsive elements in seabuckthorn, an important medicinal plant, and to understand molecular pathogenesis of early phase hypobaric hypoxia and gastric cancer. Dr. Sharma has published over 100 research papers, executed a number of research projects, and supervised a large number of Ph.D., M.Phil., M.Sc., B.Tech./M.Tech. students.

Performance of Microbial Desalination Cell in High Organic Load

Surajbhan Sevda

Department of Bioscience and Bioengineering, Indian Institute of Technology Guwahati, India

Microbial desalination cell presents a novel concept for wastewater treatment, salt removal and bioelectricity generation in a single system. In this study, the effect of organic load on salt removal in air cathode up-flow microbial desalination cell was investigated in batch mode. Four different organic concentrations were fed to the anodic chamber of microbial desalination cell. The initial chemical oxygen demand in the anodic chamber were 2.02, 4.05, 6.07 and 8.10g/L. A real seawater was used in the desalination chamber of UMDC. The obtained results showed that the organic load 3 (initial COD of 6.07 g/L) produced a maximum percentage of salt removal compared to other organic loads tested. The maximum power density produced (1.77 W/m2), the maximum percentage COD removal (95%) occurred with 48 % salt removal in the microbial desalination cell using the organic load of 6.10mg/L. The obtained results show that microbial desalination cell is a unique system that provides wastewater treatment and salt removal in a single system. Organic load is a very important factor for the operation of UMDC, there is an optimum value that achieve the best performance of the UMDC system.

Biography:
Dr Surajbhan Sevda is a technical officer- Department of bioscience and bioengineering at the Indian Institute of Technology Guwahati, India. He has a doctorate in Biochemical engineering and a Master of Technology in Fermentation technology from IIT Delhi and ICT Mumbai respectively. He has a engineering degree in Biotechnology engineering. His main research area bioelectrochemical system, biological wastewater treatment, Bioenergy from biomass. He has published 20 International research paper and presented his work in 22 international conferences. His other expertise includes mathematical modeling, writing and executing scientific projects. He also guided 2 master students and 11 undergraduate students for their projects.

Phenotypic and Molecular Characterization and Rapid Evaluation of Oil-Degrading Native Bacteria Isolated from Different Habitats in UAE

Ismail Saadoun1*, Dana Ajneibi1, Aisha Al Harthi1, Sara Aljunaibi1, Nouf Al Dulijan1, Ban Aljoubori1, Ayssar Nahle2 and Paul Rostron3

1Department of Applied Biology, University of Sharjah, UAE
2Department of Chemistry, University of Sharjah, UAE
3Petroleum Institute, UAE

Despite the beneficial value of crude oil and its derivatives to the countryʼs economy, it could have a huge adverse impact on the environment specifically when accidently spilled to the water and soil; thus, makes it as an environmental catastrophe. This study addresses the role of identified microorganisms as hydrocarbon degraders to be used as an eco-friendly solution in oil spill bioremediation. Successfully, 19 bacterial isolates were recovered from different habitats including crude oil wells, soil treated with diesel, oil contaminated seawater and surface hydrocarbon sediment. The isolates were assessed for degradation of different hydrocarbon compounds by agar hole-plate diffusion method. Results indicated the recovery of 9 isolates namely (2A, 1D, So1, S1A, S3, KF1, SO2, AJ1 and 2B) which were identified as positive degraders for one or more of the tested hydrocarbon compounds including diesel, pentane, hexane, heptane and tetradecane. These isolates were identified biochemically using VITEK 2 microbiology system as 2A (Pseudomonas stutzeri 97%),1D (Kocuriakristinae 92%), SO1 (Staphylococcus aureus 93%), S1A (Leuconostocmesenteroides ssp. Cremoris 90%), KF1 (Rhizobium radiobacter 99%), and S3 (Staphylococcus hominis 96%). Almost all of these isolates were able to utilize heptane as a sole carbon source for their survival with the isolates SO2, S1A and S3 being the most potent ones observed by their growth around the agar hole-plate. PCR analysis of the positive hydrocarbon degrading isolates for the presence of alkBgene showedtwo groups with different band size products; group 1 (G1) (~330 bp) and group 2 (G2) (multiple of 330 pb). This may imply that alkB gene can be found in multiple homologues as shown in G2 and each one may cover degradation of specific carbon number range in the tested hydrocarbon compound. Rapid evaluation of hydrocarbon compounds degradation by the native microbial communities was shown to be successful with considerable biodegradation role exploited by the recovered isolates.

Biography:
Prof. Saadoun, completed his Ph.D. in Microbiology from Auburn University, Alabama-USA in 1995. He is a member of the American Society for Microbiology (ASM) from 1992-2009, Jordan Society for Scientific Research (JSSR), and a Fellow of Society of Biology/UK. Prof. Saadoun has published several scientific papers through different research proposals granted as principal and associate investigator, in different abstracted, refereed and indexed Journals. Most of these papers were in the area of inhibitory/metabolites-producing Streptomyces spp. and their genetic determinants. In 2000, he has been awarded Abdel-Hameed Shuman Award for Arabian Young Researchers for Biological Sciences.
Prof. Saadoun has been appointed as chair for the Dept. of Biotechnology and Genetic Engineering at Jordan University of Science & Technology/Irbid-Jordan from 2000 to 2001 and from 2003 to 2005. Also, he has been appointed as a chairman for the Dept. of Biotechnology and Genetic Engineering at Philadelphia University/Amman-Jordan from 2001 to 2002, and for the Dept. of Applied Biology at University of Sharjah, United Arab Emirates from 2009 to 2014 where he acted during this period as the Biotechnology Program Coordinator with the University of Sheffield/UK. Currently he is the Vice Dean of College of Sciences.

Fermentative Production and Statistical Optimization of Xylitol Using Novel Isolates of Candida parapsilosis Strain BKR1 in Indigenously Designed Multiphase Reactor

Balakrishnaraja R

Department of Biotechnology, Bannari Amman Institute of Technology, India

Xylitol is a natural polyol and most widely known for its sugar substitute properties in diabetic patients. It is also used against the oral bacterial population. Most fascinating approach for commercial production of xylitol involves the suitable yeast fermentation. In this present investigation, factorials Optimization of these medium and process conditions are studied. Xylitol production by Candida parapsilosis strain BKR1 using Plackett-Burman and RSM are reported in modified minimal medium. The Plackett-Burman screening design reports the significant medium components are Xylose, yeast extract, Potassium Dihydrogen phosphate and magnesium sulphate. Further factorial optimization using face centered central composite design reveals the optimum levels of the significant medium components as Xylose – 104.69 g/l, Yeast Extract – 4.12 g/l; KH2PO4 – 2.84 g/l and MgSO4·7H2O – 2.09 g/l. Also the process parameters such as agitation, pH, temperature and inoculum level were optimized and validated as Agitation: 107 rpm, pH – 5, Temperature – 29.9°C, Inoculum level – 1 ml. Bioreactor was designed and a pilot scale study was being carried out.

Valorization of Wastes of Organic Food Industries for the Amendment of Agricultural Crops

D. Lakhal1*, B. Bahlaouan1,2, N. Boutaleb1, A. Fathi1, S. Lazar1 and S. El Antri1

1University Hassan II Casablanca, Laboratory of Biochemistry, Morocco
2Higher Institutes of the Nursing Professions and Techniques of Health of Casablanca, Morocco

Waste of agri-food industries is very varied and varies according to the seasons. They are mainly composed of organic waste generated by the processing activities of plant and animal products. This waste has a negative impact on the environment; nevertheless, his transformation, especially through the biological process of biotransformation, can generate a new economic source by creating a stable and balanced product that can be used as fertilizer because of its content of basic elements: nitrogen, potassium and phosphorus.

This work presents the results of a valorization test of certain waste of agro-food industry, three different wastes were combined: Fish waste, molasses and peels of potato. Several balanced mixtures were developed and underwent natural biotransformation for 2 weeks to produce a fertilizer of good nutritional and microbiological quality.

Quality control parameters (pH, temperature, conductivity, dry matter, phosphorus, nitrogen, total carbon ...) were monitored during the time stipulated for biotransformation.

The results show that the fertilizer generated from the mentioned waste is mature from the 8th day, it has a good hygienic quality, due to the absence of the spoilage microorganisms, rich in nitrogen, phosphorus, potassium, carbon and characterized by a stable optimal pH. Application tests on a barley crop confirmed its effect. Mathematical modeling, in the form of a ternary diagram, of the quality of the final product according to the composition of the initial mixture is established to build in practice a predictive operational model for industrial waste management.

The Mechanistic Role of Active Site Residues in Non-Stereospecific Haloacid Dehalogenase E (Dehe) using Computational Approach

Azzmer Azzar

International Islamic University, Malaysia

Dehalogenase E (DehE) is a non-stereospecific haloacid dehalogenase produced by soil bacteria, Rhizobium sp. RC1. This enzyme has been hypothesized to facilitate ‘direct attack mechanism’ whereby its activated water molecule will directly attack the α-carbon of haloacid compound, thus releasing the halogen. The similar mechanism was proved and identified for Dehalogenase I (DehI) from Pseudomonas putida PP3 which also classified under the same group with DehE. For a better understanding of the DehE catalytic mechanism, this enzyme was docked with D- and L- stereoisomer of 2-Chloropropionic acid (2CP) and the their complex structures were simulated using GROMACS 5.1.2 for 50 nano-seconds. Upon completion, the distance of water towards Asn114, Asp189 and the α-carbon of the 2CP were calculated. The same procedure was also applied to DehI as it serves as the benchmark. Analysis of DehE catalysis was revealed information on the presence of water molecule, water activation potential and direct attack mechanism. In this study, the catalytic water was found located nearby Asn114 and a hydrogen bond was formed between these molecules. Asn114 was seen to provide the water molecule that needed for the mechanism and play role as ‘waterbearerʼ. For water activation, the distance of Asp189 towards water molecule was found in the range of ~2 to ~8 Å which is favorable for the activation to occur. This aspartate residue was proposed to activate the catalytic water into hydroxide ion. While for the direct attack, the distance of water molecule towards the α-carbon of 2CP was approximately at 8 Å, which also the similar range that was observed in DehI-2CP. In conclusion, DehE is strongly suggested based on in silico analysis to facilitate ‘direct attack mechanismʼ for haloacid catalysis. This study information will provide a platform for isotope labeling experiment and protein engineering for non-stereo haloaciddehalogenase of Rhizobium sp. RC1.

Keywords: Dehalogenase E, haloaciddehalogenase, Rhizobial enzyme, molecular docking, molecular dynamics simulation.

Biography:
Azzmer Azzar bin Abdul Hamid obtained his Ph.D from IIUM in Biotechnology and upon completion of his Ph.D, he was appointed as a lecturer at the Department of Biotechnology, Kuliyyah of Science, IIUM Kuantan Campus. Presently, Dr Azzmer is an Assistant Professor at the Department of Biotechnology, Kulliyyah of Science, International Islamic University Malaysia (IIUM) Kuantan Campus. His research work is towards the dehalogenation of toxic compounds by microbial enzyme. Recently, he obtained an endowment grant as a principal researcher and fundamental research scheme grant (FRGS) as a coresearcher. He has published some articles regarding protein modeling, rational design, molecular biology and enzyme kinetics at several journals. Apart from that, he served the Ministry of Education as an examiner for Matriculation programme and also as a reviewer for Journal of Molecular Modeling.

Engineering Bugs for the Discovery of New Drugs Against Neurodegenerative Diseases

Georgios Skretas1*, Ilias Matis1,2, Dafni Chrysanthi Delivoria1,2, Barbara Mavroidi3, Nikoletta Papaevgeniou1,4, Stefania Panoutsou1,5, Stamatia Bellou1, Zacharoula I. Linardaki1,6, Alexandra V. Stavropoulou5, Spiros Efthimiopoulos5, Maria Pelecanou3 and Niki Chondrogianni1

1Institute of Biology, National Hellenic Research Foundation, Greece
2School of Chemical Engineering, National Technical University of Athens, Greece
3Institute of Biosciences & Applications, National Center for Scientific Research “Demokritos”, Greece
4Faculty of Biology and Pharmacy, Friedrich Schiller University of Jena, Germany
5Faculty of Biology, National and Kapodistrian University of Athens, Greece
6Department of Biology, University of Patras, Greece

Protein misfolding is a common pathological feature for many human diseases, such as Alzheimerʼs disease (AD), Parkinsonʼs disease, type II diabetes and others. We will describe the development and application of an integrated and generalizable bacterial platform for facile discovery of macrocyclic rescuers of disease-associated protein misfolding. In this system, large combinatorial libraries of macrocycles are biosynthesized in Escherichia coli cells and simultaneously screened for their ability to rescue pathogenic protein misfolding using a genetic assay based on fluorescence-activated cell sorting. We will first describe the effectiveness of this approach through the identification of drug-like, head-to-tail cyclic peptides that modulate the aggregation of the amyloid β peptide (Aβ) of AD. By using a series of biochemical, biophysical and biological assays using isolated Αβ, primary mammalian neurons and various established AD models in the nematode Caenorhabditis elegans, we have found that the selected macrocycles potently inhibit the formation of neurotoxic Αβ aggregates. Further, to showcase the generality of our approach, we will describe the application of the same platform for the identification of misfolding rescuers of mutant Cu/Zn superoxide dismutase 1 (SOD1), a protein whose misfolding and aggregation is associated with inherited forms of amyotrophic lateral sclerosis. Overall, our approach represents a straightforward strategy for the discovery of molecules that rescue the misfolding of polypeptides known to be associated with disease effectively.

Biography:
Georgios Skretas graduated from the School of Chemical Engineering of the National technical University of Athens (Greece) in 1998 and received his PhD in Chemical Engineering from Princeton University (USA) in 2006. He then moved on to the University of Texas at Austin (USA) to carry out post-doctoral research training under the guidance of Prof. George Georgiou. Since 2009, Dr. Skretas has been the principal investigator of the Laboratory of Enzyme & Synthetic Biotechnology at the Institute of Biology, Medicinal Chemistry & Biotechnology of the National Hellenic Research Foundation (Greece), where he currently holds the rank of Research Associate Professor.

Search for Regions with Periodicity Using the Random Position Weight Matrices in Different Genomes

Eugene Korotkov

Institute of Bioengineering, Research Center of Biotechnology of the Russian Academy of Sciences, Russia
National Research Nuclear University “MEPhI”, Russia

The present study developed a mathematical method for determining tandem repeats in a DNA sequence. A multiple alignment of periods was calculated by direct optimization of the position-weight matrix (PWM) without using the pair wise alignments or searching for similarity between periods. A new mathematical algorithm for periodicity search was developed using the random PWMs. The developed algorithm was applied in analyzing the DNA sequences of the different genome. A total of more than 60x103 regions were found to possess a periodicity with the length of 2 to 50 bases. On the average, a periodicity of ~5000 nucleotides was found to be associated with each region. A significant portion of the revealed regions possess periods consisting of 10 and 11 nucleotides, multiple of 10 nucleotides and periods in the vicinity of 35 nucleotides. Only ~30% of the periods found were discovered previously. This study discussed the origin of periodicity with insertions and deletions.

Biography:
Eugene V. Korotkov is a professor at the Department of Applied Mathematics in Moscow Engineering Physics Institute and Principal Investigator in Bioinformatics Department of Bioengineering Centre, Russian Academy of Sciences. He graduated from the National Nuclear Research University (MEPI), Department of Experimental and Theoretical Physics in 1974. He completed PhD thesis in radiobiology. Then, from 1980 to 1995 he worked at the Institute of Chemical Physics, NN Semenov and since 1995, he is working with the Center ‘Bioengineeringʼ, Russian Academy of Sciences. His research interests are in developing new methods for the analysis of symbolical sequences and time series.

Manipulation of tRNA Genes to Enhance Cellular Productivity of Biologics

Robert J. White

University of York, UK

Recombinant monoclonal antibodies are a powerful class of therapeutics that are used to treat a wide range of diseases, including cancers and inflammatory disorders. However, they are expensive to produce and this restricts their availability to patients; health authorities sometimes cannot afford the costs of these therapies. Lowering production costs may increase access to potentially life-saving treatments. Through synthetic cell bioengineering, we are developing novel approaches to improve production of therapeutic proteins. One of our strategies is to manipulate tRNA expression to enhance translation of recombinant products. Our progress in this regard will be described. We aim to reduce costs and thereby increase availability to patients of therapeutics that at present are prohibitively expensive.

Biography:
Bob White studied Biochemistry at University of Oxford and Molecular Biology at the National Institute for Medical Research. He then spent 5 years at University of Cambridge before establishing his own laboratory at University of Glasgow. In 2013 he became Chair of Biochemistry at University of York. His research focuses on gene expression and has been published in Nature, Science and Cell (>6400 citations, h-index 44). He has received several national and international awards and is a Fellow of The Royal Society of Edinburgh, The Academy of Medical Sciences and The European Academy of Cancer Sciences.

Nanomaterials for Enzymatic Immobilization

Rebecca Pogni

Department of Biotecnology, Università di Siena, Italy

In recent years nanoparticles and nanoscale materials have generated a great deal of interest from scientists and engineers of nearly all disciplines. This interest has been motivated to a large extent by reports that a number of physical properties including optical and magnetic properties, specific heats, melting points, and surface reactivity are size-dependent. These size-dependent properties are widely believed to be a result of the high ratio of surface to bulk atoms as well as the bridging state they represent between atomic and bulk materials. The interest in biocatalysts for biotechnological applications continues to grow. The fragile nature, high cost, and high loadings required for commercial production limits the use of free enzymes. Enzyme immobilization is utilized to surmount the stability, recovery, and recyclability disadvantages of using enzymes in solution, making them industrially and commercially viable. Nanomaterials are particularly suitable for enzymatic immobilization.

Among nanomaterials, nanofibers represent one of the most attractive nano-device for the production of high added value products. When the diameters of polymer fiber materials are shrunk from micrometers to sub-microns or nanometers there appear several amazing characteristics such as very large surface area to volume ratio flexibility in surface functionalities, and superior mechanical performance (stiffness and tensile strength) compared with any other known form of the material. Membranes of electrospun nylon and polyurethane nanofibers mesoporous silicas or magnetic nanoparticles can be synthesized, functionalized and used for enzymatic immobilization. Different examples will be shown.

Biography:
Rebecca Pogni is Associate Professor of Physical Chemistry. She is a participant in European and national projects and coordinator of the EU project BISCOL “Bioprocessing for sustainable production of coloured textiles”. She is an author of more than 80 papers on international journals on structure-activity relationship studies on enzymatic systems and protein radicals, enzymatic immobilization and synthesis of novel compounds and LCA studies. She is a representative for the University of Siena in the Italian cluster SPRING (Sustainable Processes and Resources for Innovation and National Growth). She is a member of the National Consortium on the Development of Systems with Great Interface (CSGI) and member of the Life Cycle Assessment (LCA) network.

Purification of α-Amylase from Thermophilic Bacillus licheniformis and its Application in the Production of Ethanol

Reena Gupta* and Kalpana Hiteshi

Department of Biotechnology, Himachal Pradesh University, India

An α-amylase from thermophile Bacillus licheniformis was purified by using DEAE- Cellulose column chromatography followed by Sephadex G-75 column chromatography. Native and SDS-PAGE analyses showed a single band for the purified enzyme, with an apparent molecular weight of 33 kDa. This showed that the enzyme was purified to homogeneity, was a monomer and had no subunits. The α-amylase immobilized on WhatmanTM filter paper showed maximum activity at 55°C of incubation temperature, 10 min of incubation time and pH 8.5 of 0.1M Tris-HCl buffer. The immobilized enzyme gave maximum activity with 0.175% (w/v) of substrate (starch) concentration and showed Km and Vmax value of 0.74 mM and 6.39 µmol/g/min respectively. Immobilized enzyme retained almost 50.25% of its original activity after 9th cycle of reusability. During storage, immobilized enzyme retained relative activity of 90.87% and 56.28% upto 10 and 40 days respectively. The immobilized enzyme produced maximum amount of reducing sugar from maize stalk juice extract when incubated at 55°C of temperature at pH 8.5 and for 20 min of incubation. It was observed that there was a 50-fold increase in production of ethanol from extract of maize stalk treated with immobilized α-amylase (27.33%) as compared to the untreated sample (18.92%).

Biography:
Reena Gupta is presently working as Professor in the Department of Biotechnology, Himachal Pradesh University, Shimla. Her area of specialization is Biochemistry, Immunology and Enzyme Technology. She had completed B.Sc. (Hons.) and M.Sc. (Hons.) in Biochemistry from Panjab University, Chandigarh in the year 1985 and 1987 respectively. She did her Ph.D. in 1992 from Panjab Unversity, Chandigarh and Post Doctorate from May 1993 to Oct. 1995 from IMTECH (Institute of Microbial Technology), Chandigarh. She has 22 years of teaching and 29 years of research experience. She completed four Research Projects funded by UGC, New Delhi and one Project funded by DEST, Shimla. Her total number of publications in peer reviewed Journals of international repute are 87 (National and International). She is a Member of five National Professional Bodies.

Efficient Retting of Natural Fibre Using Microbial Enzyme

Wan Zuhainis Saad*, Paridah Md. Tahir and Rosfarizan Mohamad

Universiti Putra Malaysia, Malaysia

The global natural fibre composites market is forecast to grow 8.2% from 2015 to 2020. The major driver for the growth of this market is; The rise in demand for lightweight and environmentally sustainable composite materials in various applications, such as automotive, building & construction, and others. In this market, natural fibre (hemp, flax, jute, kenaf, and others) are the major raw materials used for producing natural fibre composites. Kenaf also known as Hibiscus cannabinus is an annual fibre plant closely related to cotton. The kenaf bast fibre found its application in many industries such as textile industry, automotive industries, structural and building materials and made into biocomposite consumer products. The process of separating the bast from the core by degrading the pectin rich middle lamella is known as retting. The traditional method of water retting requires a longer retting time and caused pollution while dew retting produced fibres with poor quality. Hence, there is a need to seek for an environmentalfriendly approach to produce high-quality kenaf bastfibres. In this study, pectinolytic fungi were isolated from various sources and screened for their pectinase activity. A potential pectinase producing strain was chosen and identified as Aspergillus fumigatus R6 by amplification of the Internal Transcribed Spacer region. It was found the retting process using the enzyme has reduced the retting time from 3 weeks to 3 days. Kenaf bast fibres quality can improve by further optimisation of the enzyme formulation. Aspergillus fumigatus R6 pectinase enzyme shows potential to be used in kenaf bast bio retting process to produce strong and high quality kenaf long bast fibres.

Biography:
Wan Zuhainis Saad is an associate professor in Microbiology. Research activities include drug discoveries from thermophilic fungi and lactic acid bacteria, microbial enzymes technology in biopolymers and pulp and papers. She awarded three patents on isolation and preservation techniques of thermophilic fungi and enzyme for bioretting of kenaf. An Educational Technology enthusiast. Practices E-learning and active learning approach in enhancing studentsʼ engagement for effective and meaningful learning. Shortlisted for the QS Wharton Reimagine Education Award 2015 and 2016. QS Wharton Reimagine Education Awards Judge 2017. Awarded Vice-Chancellor Fellowship Award (Young Educator) 2012 and Vice-Chancellor Fellowship Award (Excellent Educator) 2016.

Regulation of Virulence in P. Aeruginosa by Mechanical Cues

Siryaporn

University of Pennsylvania, USA

We explore how bacteria detect and respond to mechanical forces. During the course of an infection, bacteria encounter a variety of mechanical forces such as adhesive forces during contact with the host cells that they infect and shear stresses in fluidic environments. We have developed a biophysical approach using microfluidics and fluorescence lifetime imaging microscopy to explore how bacteria interpret mechanical cues to detect the presence of host cells and to guide the expansion of large bacterial populations within host organisms. In particular, we found that the pathogen Pseudomonas aeruginosa detects the presence of hosts using a mechano-sensitive mechanism, akin to a bacterial sense of touch. This response activates virulence and consequently P. aeruginosa, unlike other pathogens, relies on mechanical input rather than exclusively on chemical signals for infection. This model provides a long-sought explanation for understanding how P. aeruginosa can infect a broad range of hosts including humans, animals and plants. The ubiquity and diversity of mechanical forces in all aspects of a bacteriumʼs life have far-reaching consequences that we are just beginning to comprehend.

Biography:
Dr. Siryaporn received his Ph.D. in Physics and Astronomy at the University of Pennsylvania with Prof. Mark Goulian in 2008 studying cross-talk between signaling networks in bacteria. As a postdoc in the Department of Molecular Biology at Princeton University, he investigated physical mechanisms that regulate the ability of bacteria to infect. He received the NIH Kirschstein NRSA postdoctoral fellowship and the NIH K Career Transition Award for his work. Dr. Siryaporn is currently an assistant professor in the Physics and Astronomy and Molecular Biology and Biochemistry Departments at the University of California, where his lab studies bacterial pathogenesis at the intersection of biophysics and molecular biology.

Breast Cancer Gene Expression Profile Analysis in terms of Genes Specific to Non-breast tissues

Hatice Büşra Konuk1* and Alper Yılmaz2

1Gebze Technical University, Turkey
2Yildiz Technical University, Turkey

Investigation of cancer gene expression profile in context of tissue-specific genes has not been studied in detail, although it provides important signatures for cancer. In this study, we aim to understand genetic alteration of tumors in context of tissue-specific genes.

Specific genes for various healthy tissues were determined using extended tau which is a robust and rigorous method generated in our previous study. Tissue-specific genes for all tissues were joined with breast cancer expression data downloaded from TCGA. Utilizing a statistical approach, we identified genes which are specific to a tissue other than breast but having high expression in cancerous breast, not in normal breast. After applying all criteria, we pinpointed 34 genes specifically expressed in breast cancer, although they are specific to ovary, placenta or testis. This unsuspected phenomenon was also observed in different cancers.

Some of cancer-testis genes which are only specific to testis but expressed in various tumors shown in literature such as MAGE, TEX and PAGE family members have been confirmed by our study and we observed additional cancer-testis genes such as CT83, SPANX family members. Some placenta-specific genes CRISP2, DKKL1 and CST9L had been identified as cancer-testis genes before. Several placenta and testis-specific genes are highly expressed in only breast cancer, while some of them are expressed in breast, lung and liver cancer.

As conclusion, detected genes have potential to be adopted as early diagnostic markers and immunogenic therapeutics. Germline and placental genes should be studied in detail to reveal cancer cells characteristic features.

Biography:
Hatice Büşra Konuk has completed her undergraduate and graduate studies in Yildiz Technical University, Bioengineering Department, Turkey in 2015 and 2018, respectively. Her master thesis was about calculation of tissue-specific genes and investigation of intra-tumoralheterogeneity to understand cancer cell molecular mechanisms to reveal biomarkers for diagnosis and targeted therapy in solid tumors using computational approaches. She is now a PhD student and Research Assistant in Gebze Technical University Bioengineering Department/Bioinformatics and System Biology Program. Her keywords are data analysis, human genome, cancer genetics and computational approaches.

New Tool for Enzymatic Analysis in Raw Materials

Julia Schückel* and Stjepan K. Kracun

GlycoSpot, Denmark

Industries, such as pharma, feed, and food, are increasingly looking for enzymes to solve production and product issues. Correct and adequate information on enzyme activity would help producers avoid prolonged production times, reduce waste, improve end-products, prolong shelf-life and save costs for overtreatment.

Most of the production processes turning raw materials into products are under the influence of a wide variety of enzyme combinations. Existing tools for screening of enzymatic activity, are cumbersome, time-consuming and often requires expert staff. Each assay needs to be constructed from the bottom up forcing the user to aliquot substrates with high precision into a large number of reaction vessels manually. Furthermore, all existing assays measure the activity of one enzyme only at a time.

We have developed a ready-to-use screening technology, where it is possible to detect important enzymatic activity in raw material. We will show different examples of endogenous enzymatic activity in the grain, flour and malt. This data helps to minimize uncertainties when using technical aids and eventually, desired properties of the final product are ensured.

Biography:
Dr. Julia Schückel holds a M.Sc. degree in chemistry from the Technical University of Dresden, Germany and a PhD degree in biochemistry from the University of York, United Kingdom. She developed together with her colleague Stjepan K. Kracun the screening technology for testing enzyme activities in raw material.

CRISPR/CAS9 Targeting MicroRNA-24 in Chinese Hamster Ovary Cells Increases Growth and Boosts Productivity

Kevin Kellner1*, Nga T Lao1, Paula Meleady1 and Niall Barron2

1National Institute for Cellular Biotechnology, Ireland
2National Institute for Bioprocessing Research and Training, Ireland

Chinese Hamster Ovary (CHO) cells are the prominent cell line used in biopharmaceutical production. Although optimisation efforts have led to a vast increase in productivity, CHO cells yield less than other expression systems like yeast or bacteria. To improve yields and find beneficial bioprocess phenotypes, genetic engineering plays an essential role in recent research. The miR-23 cluster with its genomic paralogues (miR-23a and miR-23b) was first identified as differentially expressed during temperature shift, suggesting its role in proliferation and productivity. The common approach to deplete miRNAs is the use of a sponge decoy which, requires the introduction of reporter genes. As an alternative this work aims to knockdown miRNA expression using the recently developed CRISPR/Cas9 system which does not require a reporter transcript. This system consists of two main components: the single guide RNA (sgRNA) and an endonuclease (Cas9) which induces double strand breaks (DSBs). These DSBs can result in insertion or deletion (indels) of base pairs which can disrupt miRNA function and processing.

A CHO-K1 cell line stably expressing an IgG was used for knockdown experiments. SgRNAs were designed to target the seed region of miR-24-3p and stable mixed populations were generated.

It was shownthat miRNA expression for miR-24-3p as well as miR-24-5p was significantly reduced in mixed populations. Furthermore, an increase in pre-miR-24 was exhibited suggesting impaired processing by Drosha or Dicer. A knockdown up to 95% was achieved for miR-24 as well as the passenger miRNA (Figure 1A, B and C). The other members of the cluster which are located proximal to miR-24 were not affected by the knockdown (Figure 1D). Depletion of miR-24 showed increased proliferation in batch culture as well as boosted productivity (Figure 1E and F). However, faster growth led to increased nutrient demand of miR-24 depleted cultures and a reduction in culture time was exhibited. Quantitative label-free LC/MS was used to identify 81 more abundant protein targets. Pathway analysis revealed proteins potentially involved in enhanced ribosomal RNA biogenesis, recycling and assembly of ribosomal subunits. Furthermore, proteins involved in catalysing the loading of cognate aminoacyl-tRNAs and release of deacetylated tRNAs were higher expressed. These targets were highlighted as potential cell line engineering targets to improve productivity of CHO cells.

Out of all 81 upregulated proteins, 50 were predicted targets of either miR-24-3p or miR-24-5p. In this work, we have shown that CRISPR/Cas9 can be successfully applied as a tool to knockdown miRNA expression in CHO cells. The data was generated using mixed pools and it remains to be established if both alleles can be successfully targeted e.g. using next-generation sequencing ofindividual clones.

Microbial Decolorization of Reactive Azo Dyes by Bacillus Spp. Under Anaerobic Condition

Maulin P Shah

Industrial Waste Water Research Laboratory, Enviro Technology Limited, India

A bacterial strain Bacillus spp. with remarkable ability to decolourize the reactive azo dyes such as reactive red, reactive yellow, reactive brilliant red and reactive brilliant blue was isolated from the textile effluent contaminated site. The effluent sample was collected from local text textile industry, Ankleshwar, Gujarat, India Static conditions with 10 g/l glucose, pH 9, 37°C, 20% inoculum concentration, 50 mg/l of dye concentration, 3 g/l of NH4NO3, were considered to be the optimum decolourizing conditions. Bacillus spp. grew well in these optimum conditions, resulting in 82% decolourization extent 7 days of incubation. Phenotypic characterization and phylogenetic analysis of the 16S rRNA sequence indicated that the bacterial strain belonged to the genus Bacillus. UV (Ultra Violet) analysis of bacterial isolate suggested that it exhibited decolourizing activity, rather than inactive surface adsorption. This decolourization extent and facile conditions show the potential for this bacterial strain to be used in the biological treatment of textile effluent or dyes.

Biography:
Maulin P Shahʼs laboratory is trying to assess the impact of industrial pollution on microbial diversity of waste water following cultivation dependant and cultivation independent analysis. His major work involves isolation, screening, identification and Genetic Engineering of high impact of Microbes for the degradation of hazardous materials. Dr. Maulin P Shah has more than 150 research publication in highly reputed national & international journals. He is Editorin-Chief of Research Journal of Microbiology, Journal of Biotechnology & Bio materials.

Molecular Insights into Membrane Trafficking by the SNX27-retromer Complex

Mintu Chandra*, Thomas Clairfeuille, Nathan J Pavlos, Victor Anggono, Rohan D Teasdale and Brett Collins

The University of Queensland, Institute for Molecular Bioscience, Australia

Compartmentalisation is a defining feature of all eukaryotic cells, and we have evolved highly sophisticated protein machineries to control the flow of transmembrane moleculesand membrane lipids between different organelles. Disruption of these processes are linkedto numerous diseases including neurodegenerative disorders, pathogen invasion andcancer. We are determining how these trafficking machineries are assembled and regulatedat the molecular level through a combination of structural biology, biophysical, and cellbiology approaches. In my talk, I will describe our most recent work on critical protein sortingmachineries – the retromer complex and the sorting nexins (SNXs) - regulating endosomalmembrane recycling and cellular homeostasis. We have defined sorting signals requiredfor endosomal recycling by the SNX27-retromer complex, how this is regulated by post translation alphosphorylation, and the structural basis for SNX27-retromer-cargoassembly.

Health Disparities in Females with Rheumatic Heart Disease in Central Afghanistan

Ruhina Najem

Walden University, USA

In developing countries, and sporadically in developed countries, Rheumatic Heart Disease (RHD) persists in being the main health condition, and yet the medical management of RHD throughout the years has not changed. The acquisition of RHD as well as its pathogenesis remains elusive. What is known is that Anonsuppurative indication of group A streptococcal (GAS) pharyngitis causes rheumatic fever (RF) which develops into RHD when untreated. Secondary prophylaxis proves ineffective in preventing death from this disease. Because an anti-streptococcal vaccine does not yet exist, ancillary prophylaxis is not possible (Vijayalakshmi, 2011). The attempt to invent such a vaccine using the M protein has been unsuccessful over the last 40 years (Vijayalakshmi, 2011). Although developing countries have reduced the incidence of RHD, the disease still remains one of the leading causes of mortality and morbidity in developing countries worldwide. This study offers strategies to eliminate health disparities in women with RHD in Central Afghanistan, where the life expectancy for women is 44 years old. By increasing the number of trained healthcare professionals, as there are presently 695 female doctors for 4,595,000 women, and by increasing the health quality of life in a region where 85% of women are illiterate, the incidence of RHD in women in Central Afghanistan can be significantly reduced (Arnold, Teijlingen, Ryan, & Holloway, 2014; “Trust In Education.” 2011; Bezruchka, 2012).

Biography:
Ruhina Najem is a foreign medical graduate from Afghanistan, He came to USA as a refugee hoping for a better future. His long life dream to attain my Doctoral degree is coming to light at Walden University. He is currently an experienced Administrator & Faculty at Peralta Colleges, Merritt College with a demonstrated history of working in the higher education industry, Strong education professional skilled in Event Planning, Customer Service, Instructional Design, Public Speaking, and Curriculum Development. He is a coauthor while working at Stanford University on Mycophenolic acid concentrations in longterm heart transplant patients: relationship with calcineurin antagonists and acute rejection.

Enhanced Production, Purification and Thermodynamic Characterization of Endotoxin Free Antileukemic Recombinant L-Asparaginase of Escherichia Coli K-12

Santosh Kumar Jha* and Hare Ram Singh

Department of Bioengineering, Birla Institute of Technology, Mesra, India

L-Asparaginase (E.C. 3.5.1.1) is a well accepted chemotherapeutic agent against the acute lymphoblastic leukemia and lymphosarcoma. The recombinant L-asparaginase enzyme was produced by the over-expression of ansB gene of E. coli K-12 in E. coli BL21. Different carbon sources, nitrogen sources, minerals and additives having yield enhancing effect, were screened by Plackett Burman Design (PBD). Their optimum level was identified by the using the orthogonal array method of Taguchi design of experiment. The screening of media components by PBD proposed the glucose as main carbon source, tryptone and yeast extract as organic nitrogen source, NH4Cl as inorganic nitrogen source, NaCl and K2HPO4 as mineral source have significant effect on the production of enzyme. Glycerol was identified as the most influential effect on the recombinant L-asparaginase production among the all additives. The addition of small amount (0.6% v/v) of it, significantly increased the enzyme yield. After the complete optimization of the selected process parameters 121.8% enhanced production of L-asparaginase was observed at shake flask level. There was further 14.8% enhancement in the enzyme production after the scale up the process in 5L bioreactor. The volumetric yield of 3.58 X 105 U/L of L-asparaginase with the specific activity of 6.97 X 103 U/mg in fermentation broth was reported. The enzyme was purified by affinity chromatography followed by three-phase partitioning. The endotoxin level was estimated by LAL-assay. The purified recombinant enzyme was further used to study the thermodynamic parameters. The enzyme showed highest stability at 28°C than at higher temperatures with a half-life of 46 hrs which is quite significant. Its deactivation energy was found to be 60.64 kJ/mol. The value of thermodynamic parameters including ΔH, ΔS and ΔG were found to be -49.23kJ/mol, 0.09kJ/mol.K and 73.12-74.78 kJ/mol respectively implying that there are no significant processes of aggregation and the enzymatic reaction was exothermic and spontaneous in nature. The antileukemic potential of the enzyme was accessed by using human leukaemia cell line HL-60 through viability study, cell and morphological studies.

Biography:
Dr. Santosh Kumar Jha is an academician cum researcher and presently serving as an Assistant Professor in the Birla Institute of Technology, Mesra, India. He has expertise in the process development and designing of bioprocess for the production of biotherapeutics. Presently he is working on the bioprocess development of various biotherapeutics, development of nanobiocomposite based nanobiotics and tissues scaffolds for various biomedical applications.

Biosorption and Removal of Cr(VI) from Aqueous Solutions by Nigelle Sativa Seeds Biomass

Abdelhamid Addala1*, NoureddineBelattar1 and Maria Elektorowicz2

1Applied Biochemistry Laboratory, Department of Bioche mistry, Ferhat Abbas University, Algeria
2Department of Building, Civil and Environmental Engineering, Concordia University, Canada

Every year millions tons of biomasses issues from agriculture activities and food factories are discarded as a waste. These bulk vegetal biomasses constitute a potential and promoting material in heavy metal pollutants remediation. This first study investigated the efficiency removal of Cr(VI) from solution effluents using Nigella sativa seeds waste biomass. In this context, the study aims to assess biosorbing properties by batch procedure of waste biomass toward Cr (VI) by varyingcontact time, initial Cr (VI) concentration, initial pH and biosorbent dose. The biosorption data was analyzed using Langmuir and Freundlich isotherms. The optimal conditions such as equilibrium contact time for the removal of Pb(II), amount of adsorbentdose and pH were 90 min, 0.025g and pH >2. Under these conditions, the corresponding response for maximum biosorption yield was 0.150mg/g. The Langmuir model and pseudo-second order kinetic fitted well to the adsorption experimental data. Based on the results, the Nigella sativa seeds waste biomass appears as a promising biosorbent for remediation of lead pollutant from wastewaters.

Green Synthesis of Silver Nanoparticles from Salacia chinensis and Analysis of its Antioxidant and Antimicrobial Potential

Abhijith P*, Bhavya G, Ashika P and Geetha N

Eco Biotech Lab, DOS in Biotechnology, University of Mysore, India

In the current era of research Green synthesized nanoparticles are playing a vital role in medicine and other field. The current research was concentrated on green synthesis of Ag-NPSʼ by exploiting Salacia chinensis leaf extract. The leaf extract was synthesized by boiling water method, followed by incubation at room temperature in light conditions (usually synthesis of AgNPs are carried out in dark) in AgNO3 solution. Synthesized particles were characterized using XRD, SEM-EDX, FTIR and UV-Vis spectroscopy. This reveled that synthesized particle had a size of 56 to 151nm, face centered cubic - crystalline structure, with polymorphic shape; rod, spherical, round and polygonal. The antioxidant potential of the synthesized nanoparticles was compared against crude extract using DPPH assay, it was found that G-AgNPs has potent scavenging activity than crude. Antibacterial potential of the particles tested against E. coli, Vibrio sp, Salmonella sp, Candida albicans and Candida tropicalis by micro-titer plate method, showed that the G-AgNPs had moderate growth inhibition on E. coli but Vibrio sp. and Salmonella sp. showed more resistance but potent antifungal activity against C. albicans and C. tropicalis, which are found to be resistant to clinical antifungal agents like Fluconazole. These findings could lead to a better understanding of the antifungal properties of AgNPs synthesized through green synthesis mode using Salacia chinensis, also this could pave way to developing better drugs for resistant pathogenic yeasts and bacterial strains.

Bioprospecting of Yeast Strains for Efficient Conversion of Lignocellulose to Bioethanol through Simultaneous Saccharification and Fermentation

Ajay Kumar Pandey* and Naseem A. Gaur

Yeast Biofuel Group, DBT-ICGEB Center for Advanced Bioenergy Research, India

Lignocellulosic hydrolysate is a mixer of C6/C5 sugars and inhibitors (Furans, weak acids and phenolics) generated during the pretreatment. Therefore, robust yeast isolates with characteristics of C6/C5 fermentation and pretreatment inhibitor tolerance are pre-requisite forlignocellulosic ethanol production. Moreover, use of thermotolerant yeast isolates will reduce cooling cost, contaminations during fermentation, and also required for developing SSF and SSCF processes. Therefore, we evaluated the growth and fermentation performances of yeast isolates isolated from diverge natural habitats. The growth and fermentation performances were evaluated at 30°C and 40°C along with tolerance towards pretreatment inhibitors (Furfural, HMF, Acetic acid and Ethanol). K. marxianus NGY8 and O. thermophila NGY11 were able to grow onwide range of C6/C5 sugars including arabinose and cellobiose. S. cerevisiae NGY10 isolate, isolated from sugarcane bagasse distillery waste, produced maximum ethanol yield of 47.59 ± 2.34 g/l and 46.8 ± 3.11 g/l with the efficiency of 94.11% and 93.73% at 30°C and 40°C respectively, in 24 hours with glucose as a carbon source. This isolate produced ethanol yield of 8.17 ± 0.14g/l with fermentation efficiency of 93.23% at 40°C, when rice straw enzymatic hydrolyzate was used as carbon source and displayed furfural (1.5 g/l), HMF (3.0 g/l), acetic acid (0.2% v/v) and ethanol (10.0% v/v) tolerant phenotypes. Together, NGY10 isolate could be potential yeast isolate for lignocellulosic ethanol production, C5 metabolic engineering and developing strategies for SSF and SSCF processes.

Applications of Antibacterial Substances Produced by LAB from Fermented Metata Ayib

Alazar Essayas

Sharda University, India

Recently, there have been increases in antibiotic resistant strains of human pathogens, threats effectiveness of currently applicable drugs and significantly causing treatment failure of microbial infections. These days more attention is given to searching new antimicrobial drugs to combat the war against pathogenic microbes. Traditional fermented cottage of Metata Ayib that contains lactic acid bacteria (LAB) may have antimicrobial activity against various human pathogens to preserve Ayib for a long time. However, there was no scientific report on the antimicrobial activity of lactic acid bacteria isolated from Metata Ayib. Therefore, the objective of this study was to evaluate lactic acid bacteria from Metata Ayib and study antibacterial activity against clinical and standard human pathogens. The study was laboratory based experiment conducted from October to June 2014. Antibiotic production by the LAB was performed by inoculating the LAB isolates into 6.0 ml MRS medium and incubating at 30 °C. Cell free supernatants (CFS) were collected by centrifugation (10,000 rpm for 15 min at 4 °C) of the six day fermented broth cultures. The pH of the CFS was adjusted to 6.5 with 4 N NaOH to eliminate the effect of organic acids. The susceptibility of produced antibiotic against test organisms was done by growing on Muller Hinton agar in triplicate using well diffusion method and the inhibition zone was recorded. Preceddingly, MIC and MBC were determined using standard methods. The antibiotic substance was shown antimicrobial activity towards standard and drug resistant bacteral strains with the inhibition zone ranges upto 25.33±3.21 mm. The result of MIC against tested ornaisnms was shown a considerable antimicrobial activity of the antibiotic substance withn the range values 6.25% to 12.5%. In addition, the result of this study was shown that LAB obtained from Metata Ayib exhibits antimicrobial activity against standard and pathogenic pathogenic test bacterial species ranged from 12.5-25% of MBC value. This might be due to the production of organic acids, but also other compounds, such as ethanol, hydrogen peroxide, diacetyl, reuterin and bacteriocins. The results of this investigation can also provide baseline for information for future studies about the application of antibacterial substances produced by LAB from fermented Metata ayib.

Biography:
Alazar Essayas, a young PhD student in Sharda University, India. He was a lecturer in Wollo University, Dessie, Ethiopia before joing the PhD class. He got my MSc degree in biotechnology and bachelor degree in applied biology from governmental university, University of Gondar. Now he is doing PhD in Industrial biotechnology from one of Indian prestigious university, Sharda University. MSc thesis was on, antibacterial activity of lactic acid bacteria from Metata ayib (traditional fermented cheese) which triggers him to continue his study on industrial biotechnology.

Control of Sigatoka Disease using a Ribonuclease T2 from G.lucidum: The Way to Improve Colombian Banana Production

Mosquera A1*, Arias M1, Palacios A1, Areiza D1, Zapata P2, Salazar V3, Boix S3, Sanchez J4 and Atehortúa L1

1Grupo de Biotecnologia, Antioquia University, Colombia
2Grupo de investigación en ciencias farmacéuticas ICIF, CES University, Colombia
3Human RNasesinvolved in host defense, AutonomousUniversity of Barcelona, Spain
4Centro de Investigaciones del Banano, Colombia

On banana crops, Mycosphaerella. fijiensis is the most important pathogens causing the black sigatoka disease. At list nearly of 50% of the banana yield it is lost, due to a reduction of the photosynthetic area of the leaves, poor quality of bunchand decrease of planted area per farm, causing the greatest amount of losses on commercial plantations. Previously, we have patent to a novel process for potentiating the production of substances with antifungal activity obtained from Ganodermalucidum. However, the reology of the submerged culture fermentation of filamentous fungi makes harder this process. Therefore, recombinant technology becomes a biotechnological tool for the rapid, efficient and economic production of specific enzymes from G. lucidum. A 60kDa RNase, with a tag of eGFP was produced in E. coli the gen was isolated from submerged culture of G.lucidum. The purification process involves affinity chromatography on IMAC cartridges. It suppressed fragmented mycelium upgrowth of M. fijiensiswith an IC50 of 7.6 ppm compared to Mancozeb with an IC50 of 1.8 ppm. However, it does notinhibite the elongation of germ tube but it is true that has antifungal activity, it does not have the capacity to cross bacterial and yeastʼsmembranes.

Biography:
Angela Rocio started this process in the Institute for the study of Inborn Errors of Metabolism in 2009 working as a Bacteriologist on the clinical diagnosis of these diseases. Since 2010 by degree he has been inclined for the study of the production of recombinant proteins, starting this process in the purification of recombinant GALNS protein produced in E.coli; analyzed its stability and its uptake capability for HEK293 cells. Then, he will be tied to work of developed of purification model for Hexosaminidase A produced in P.pastoris. On the other hand, in 2014 as Ph.D research, he started the study of Ribonucleases from mushroom expressed in E. coli and P. pastoris systems at Antioquia University.

Green Synthesis of Silver Nanoparticles (AgNPs) Using a Local Bio-derived Product-BSH

Ghada Youssef

Alexandria University, Egypt

This research article represents a strong approach of green synthesis of silver nanoparticles (AgNPs) using a local bio-derived product-honey called black seed honey (BSH). Antioxidant activity of BSH had been determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay and found to be 91.34% scavenging activity. Antibacterial activity of BSH and AgNPs was investigated against six pathogenic strains; Escherichia coli, Proteus mirabilis, Klebsilla pneumoniae, Pseudomonas aeruginosa (Gramnegative), Staphylococcus aureus, Streptococcus mutans (Gram-positive). BSH can reduce silver ions into silver nanoparticles after 20 min with stirring the reaction mixture. Nanoparticles of different sizes could be obtained and the solution turned to black by time. Characterization of the AgNPs was done by UV-Visible Spectroscopy, Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM) and scanning electron microscope (SEM) imaging. The colloid obtained at a pH of 9 was found to be spherical in shape distributed with some agglomeration. An intense surface plasmon resonance band at 400 nm in the UV–visible spectrum clearly revealed the formation of AgNPs after 72h. Transmission electron microscopy (TEM) showed spherical shaped of AgNPs, and the size of nanoparticles was in range of 25 ~70 nm. The results suggest that the synthesized AgNPs act as an effective antibacterial agent more than natural black seed honey, and can potentially be used in human contacting areas.

Appraisal of Microcarrier Suspension in a Single-use Shaken Bioreactor with Conical Bottom

G. Rodriguez1*, A. Ducci2 and M. Micheletti1

1University College London, Department of Biochemical Engineering, UK
2University College London, Department of Mechanical Engineering, UK

The pharmaceutical industry is at the forefront of the production of drug products using mammalian cell-based cultures, some of which rely on anchorage dependant cells, with single-use technology bioreactors gaining prominence. Whilst often adapted to suspensioncultures, hybridoma and other cells give increased product yields when culturedattached to a substrate, such as microcarriers, resulting in smaller fermentation runs at higher densities. Microcarriers are also used in tissue engineering andcell for therapy culture. Stem cells are adherent-dependantcells and traditional 2-dimensional static cultures rely on disposable multilayervessels, which have become the common route for stem cell expansion. It has been a recent industry trend to use shaken bioreactors at large scale in the upstream process, performing mammalian cell culture in single-use bags which eliminate the need for cleaning in place, offer flexibility and lower production down-times. Whilst currently still to be fully developed, large scale production of stem cells would require the use of 3-dimensional dynamic culture methods by employing microcarrier technology, asdemonstrated by Frauenschuh et al. (2007). Microcarriers consist of sphericalbeads with a size of 100-300 µm, and can be made of a wide variety of materials, amongst others: plastics, glass, acrylamide, silica, silicone rubber, cellulosedextran and collagen. Medium to large scale single-use devices would greatly benefit the consistency of stem cell culture whilst maintaining potency.

Whilst most studies have focused on investigating optimal compositions of microcarrier materials, their concentration and cell culture media, little researchhas been undertaken on the engineering aspects of microcarrier use. The aim of the work is to characterize the suspension dynamics of microcarriers in a cylindrical orbitally shaken bioreactor (OSR) with conical bottoms of different heights. This study builds upon previous works of the research group (1-4) for a flat bottom reactor, where increases in Froude number were found to determine a mean flow transition which was found to be instrumental in determining the just-suspended speed.

The dynamicsof solid suspension is studied using commercially available Cytodex-3,stained with trypan for improved visual contrast and image acquisition. The experimental procedure allows to estimate not only the speed required for the solids to lift from the vessel bottom, but also the conditions at which a homogenous distribution of microcarreirs is obtained. Preliminary results indicate that the presence of the conical bottom improves solid suspensionby requiring lower agitation rates for the microcarriers to lift fromthe bottom completely. The critical Froude, which determines the flow type controlling the bioreactor, can be used to scale the suspension of microcarriers in OSRs.

Enzyme Stabilization by Immobilization for an Industrial Uses

Hadjer Zaak1*, Naima Zaak1, Mohamed Sassi1 and Roberto Fernandez-Lafuente2

1Faculty of Science of Nature and Life, Ibn Khaldun University, Algeria
2Department of Biocatalysis, Institute of Catalysis and Petrochemistry-CSIC, Spain

Background and Aim: Lipases are among the industrial enzymes most used because of their high activity and stability as well as the wide range of substrates they can catalyze. Their use requires immobilization to allow their reuse and easy separation of the reaction mixture. This technique may also improve other enzymatic properties such as stability or activity and selectivity, etc. Stability is a key factor that can limit the use of all enzymes. In the present study, phospholipase Lecitase Ultra (LU) and Thermomyceslanuginosus lipase (TLL) were immobilized in octyl-agarose (OC) beads. The aim was to find a strategy to improve the stability of immobilized enzymes and Limiting their desorption in the reaction medium.

Methods: The immobilization of the lipase used in this study is based on the interfacial activation of the lipase on the hydrophobic support surface. Phospholipase Lecitase Ultra (LU) and Thermomyceslanuginosus lipase (TLL) were immobilized on Octylagarose using two enzyme charges. Subsequently, all preparations were treated with glutaraldehyde (Glu), polyethyleneimine (PEI) or sequentially with Glu and PEI. These treatments can make it possible to stabilize the physically immobilized lipases by avoiding enzymatic desorption by intermolecular crosslinking.

Results: During these treatments, LU retained about 90% of the activity, and TLL over 80%, and the PEI bound to these biocatalysts was maintained fairly. Thus, this new strategy has probed to be useful for co-immobilizing two enzymes, thereby allowing the recovery and reuse of the most stable enzyme in other hydrolysis or synthetic processes without any other activation.

Conclusion: The treatment with PEI was more effective in increasing enzymatic stability, while glutaraldehyde had a significant stabilizing effect. This enzymatic stabilization was more significant when using heavily loaded preparations where intermolecular crosslinking was easier to achieve.

Enzymatic immobilization; Polyethyleneimine; Glutaraldehyde; Enzymatic desorption; Thermal stability

Enhanced Production of L-glutaminase from Bacillus licheniformis by Taguchi DOE

Hare Ram Singh* and Santosh Kumar Jha

Department of Bioengineering, Birla Institute of Technology, India

L-glutaminase (L-glutamine amidohydrolase, EC 3.5.1.2) is an important enzyme due to its property such as, it enhances the flavour of food and can also act as an anti-leukemic agent and as a biosensor. L-glutaminase is produced by micro-organisms like bacteria, fungi, yeast and including humans and animals. L-glutaminase hydrolyses glutamine to glutamic acid and ammonia. The objective of the present investigation is to qualitative and quantitative screening of potential L-glutaminase producers. The primary screening of L-glutaminase is performed by rapid plate assay method on the basis of pH dependent analysis. Bacillus licheniformis is observed as the maximum producer of L-glutaminase, which is then used for the further investigation. The secondary screening is performed under optimized conditions. Taguchi orthogonal method of optimization of six factors viz. Carbon source, Nitrogen source, Salts, Incubation period, pH and Temperature was used for the maximum production. The maximum enzyme production of L-glutaminase was observed at dextrose (2.5g/L), L-glutamine (0.9g/L), MgSO4.7H2O (0.4 g/L), NaCl (0.35 g/L), KH2PO4 (2.5 g/L), CaCl2.2H2O (0.9 g/L), Na2HPO4.2H2O (5 g/L), pH 7.0 at 37°C. An optimized enzyme production ensures high profitability and authentic significance in terms of its usage.

Biography:
Dr. Hare Ram Singh having a long experience in the field of bioprocess engineering. He is actively engaged in the bioprocess development for the industrially important biomolecules using the microbial system. He has expertise in the process optimization, downstream processing and mathematical modelling of the bioprocess. By profession he is an academician cum researcher and presently serving as an Assistant Professor in the Birla Institute of Technology, Mesra, India.

Effect of Nitrogen and BAP in the Tissue Culture of Lupinuspubescensbenth, an Emblematic Quito Plant

Ivonne Vaca1*, Thaly Benavides2, Adriana Córdova2, Felipe Andrade3, Alicia Morales3 and Tatiana Jaramillo3

1BIOARN Research Group, Salesian Polytechnic University, Ecuador
2Universidad PolitécnicaSalesiana, Engineering Degree in Biotechnology of Natural Resources, Ecuador
3Botanical garden of Quito, Ecuador

LupinuspubescensBenth, also known by the name of Ashpachocho, is a native ornamental plant of Ecuador, due to the beauty of its flowers, it was declared as one of the species of patrimonial and emblematic flora of the Metropolitan District of Quito. The present work was developed in Quito - Ecuador. The current study was performed to evaluate three total nitrogen concentrations, coming from the Murashige and Skooog (MS) salts, components of culture media, during the tissue culture adaptation phase and, to determine the effect of the presence of BAP (Benzil amino purine) in the media, for the period of the multiplication. The seeds exposed to 4.88mM of the total nitrogen concentration presented the highest germination percentage among all the treatments (100%). Adding BAP (1ppm) to the medium, during multiplication phase achieved a higher average multiplication index (3.22). The results show that the reduction of total nitrogen in the culture medium was a determining factor in the increase of germination, besides that the presence of BAP in the culture medium favors the production of shoots per plant.

Biography:
Ivonne Vaca Suquillo did specialization in Agricultural Engineering and Masters in Agricultural Biotechnology. With 10 years of experience in the development of plant tissue culture, in different species, among these Andean fruits, tropical fruits, ornamental plants, plants of conservation interest, among others. With 9 years of experience in university teaching, in chairs such as Botany, Plant Biology and Plant Biotechnology. She is a Director and collaborator of research projects in different areas of the agricultural sector, biological control and molecular biology

Study of the In Vitro Germination of Immature Embryos of Orange Trees (Citrus sinensis)

Karim Mahmoudi1,2*, Najat Handaji1, Mohammed Ibriz2, Najat Arsalane1, Tarik Aderdour1,2, Kawtar Label1,2, and Hamid Benyahya1

1Laboratoire dʼamélioration et conservation des ressources phytogénétiques, Institut National de la Recherche Agronomique Kenitra, Morraco
2Laboratoire de Génétique et Biométrie, Université Ibn Tofail, Morraco

Introduction and objectives: Oranges constitute the major part of the production of citrus fruit, which is the most important fruit group in international trade. The creation of new triploid hybrids via the rescue of immature embryos allows diversification of the varietal profile of orange trees. The objective of this study is to optimize the in vitro germination of immature embryos according to the chemical composition of four in vitro culture media in two varieties of orange trees (Pineapple and Parson Brown).

Methods: At the maturity stage, the fruit was harvested and the extracted seeds were classified according to their size. Only small or flat seeds were cultured in a base medium of Murashig and Tuker (MT) under sterile conditions. The different growth regulator concentrations were tested to obtain the best medium for seedling development: M1 (MT + 1 mg / l GA3), M2 (MT + 1mg / l Kenitin + 0.5 mg / l BAP + 0.1 mg / l ANA), M3 (MT + 25 mg / l adenine sulphate), M4 (MT + 0.5 mg / l Kenitin + 0.5 mg / l BAP + 1 mg / l GA3).

Results: For both Orange varieties Pineapple and Parson Brown, the germination rate is maximum in M3 medium respectively at percentages of 100% and 90%. varieties between 6 and 7 days. With respect to growth rate (mm / week), both varieties knew a variation in the four media. Similarly, the maximum acclimation rate in the M1 medium is 80% and 90% respectively for the Pineapple and Parson Brown varieties.

Conclusion: In general, the smaller the embryos, the more sensitive they are to the composition of the culture medium. It is therefore essential to optimize the components of the medium to promote their growth and their in vitro developments. Therefore, the medium M1 (MT + 1 mg / l GA3) remains the best to promote good germination in short time and a better acclimatizationrate.

Synergistic Effect of Cholesterol-Vitamin E, Solubilized in Cyclodextrins, on Frozen-Thawed Bovine Semen

Allaeddine Khellouf* and Mokrane Iguer-Ouadaa

Associated Laboratory in Marine Ecosystems and Aquaculture, Faculty of Nature and Life Sciences, Abderrahmane-Mira-University, Algeria

Cryopreservation of sperm has been developed considerably in the recent years. However, during the different phases of the cryopreservation process spermatozoa are affected by various stresses. The aim of this study was to minimize this damage, particularly at the membrane level by supplementing the freezing extender medium with bioactive molecules, vitamin E and cholesterol. The two molecules are supposed to act in a complementary manner, cholesterol to reinforce the cell membrane and to fight against the cold shock, and vitamin E to fight against the oxidative stress. As the two molecules are lipophilic, they were both preloaded in cyclodextrins to enhance their solubility. The two molecules were used alone or in association. Sperm mobility (using CASA), sperm viability (using HOST) and levels of lipid peroxidation (using TBARs) were used to analyze sperm quality. The post-thawed results showed a significant protection of all sperm parameters when vitamin E and cholesterol were used simultaneously with 57.69±0.82 µm/s, 39.87±6.3 %, 0.046±0.12 nmol/108, for VCL, viability, and oxidative stress status, respectively. Associating cholesterol and vitamin E, both preloaded in cyclodextrins, seems to offer a real opportunity to improve bovine semen quality after freezing-thawing process.

Identification of Bread Wheat Seed-specific bZIP Transcription Factors Binding Sites by Genome-wide In Vitro Binding Analysis

Koushik Shah*, Prateek Jain, Nishtha Sharma, Raminder Kaur and Vikas Rishi

National Agri - Food Biotechnology Institute, Department of Biotechnology, India

Basic leucine zipper (bZIPs) are dimeric sequence-specific transcription factors (TFs) that are unique to eukaryotes and play an important role in various biological processes including seed development and maturation, plant immunity and defense, and biotic and abiotic stress. In humans and other vertebrates bZIP TFs DNA binding sites gene promoters are well characterized but not much is known in plants. Problem is more pronounced in economically important crop like wheat. Recently an ordered draft sequence of hexaploid wheat (Triticum aestivum) genome and transcriptome analysis has opened new opportunities to study the roles of TFs proteins in gene regulation in wheat. Particularly, we are interested in analyzing the structure and functions of bZIP TFs of wheat. In last few years there has been a spurt in breakthrough technologies that identify TF-binding sites in whole genome (e.g. PBM, ChIP-seq, Bind-n-seq/DAP-seq). The new protocols are helping in identify hitherto unknown binding sites of TFs both in vivo and in vitro. We have cloned seed- specific bZIPs and adapted high-throughput bind-n-seq methodology to identify their genome-wide binding sites. Bind-n-Seq is a simple and robust method in which bZIPs are incubated with bar coded random oligonucleotides libraries (70 mer) with random binding sequences. Pure bZIP bound oligonucleotides are isolated and are sequenced using illumina platform. Based on abundance binding sites are scored. One of significant advantage of bind-n-seq over other method is that large numbers of binding sites are captured for each bZIP that is used in constructing transcription factor binding landscape.

Genomics Approach for Excavation of NAS Genes from Nutri Rich Minor Millet Crops: Transforming Perspective from Orphan Plants to Future Food Crops

Mahima Dubey*, Arun Patil and Girish Chandel

Department of Plant Molecular Biology and Biotechnology, IGKV, India

Minor millets are highly nutritious and climate resilient cereal crops. These features make them ideal candidates to excavate the physiology of the underlying mechanism. In an attempt to understand the basis of mineral nutrition in minor millets, a set of five Barnyard millet genotypes were analyzed for grain Fe and Zn content under contrasting Fe-Zn supply to identify genotypes proficient in tolerating mineral deficiency. This resulted in identification of Melghat- 1 genotype to be nutritionally superior with better ability to withstand deficiency. Expression analysis of several Nicotianamine synthase (NAS) genes showed that HvNAS1 and OsNAS2 genes were prominent in positively mediating mineral deficiency response in Barnyard millet. Further, strategic efforts were employed for fast track identification of more effective orthologous NAS genes from Barnyard millet. This resulted in identification of two genes namely EfNAS1 (orthologous to HvNAS1 of barley) and EfNAS2 (orthologous to OsNAS2 gene of rice). Sequencing and thorough characterization of these sequences revealed presence of intact NAS domain and signature tyrosine and di- leucine motifs in their predicted proteins and thus established their candidature as functional NAS genes in Barnyard millet. Moreover, EfNAS1 showed structural superiority over previously known NAS genes and is anticipated to have role in more efficient metal transport. Findings of the study provide insight into Fe-Zn deficiency response and mineral nutrition in millets. This provides millets with a physiological edge over micronutrient deficient staple cereals such as rice in withstanding Fe-Zn deficiency and subsequently accumulating higher levels of Fe and Zn in millet grains.

Design of Plastic Materials Based on Non-Stick Polypropylene for Improve Food Packaging Hygiene

Maria Mekouar1*, Nadia Boutaleb1, Bouchaib Bahlaouan1,2, Hanane El omari1 and Said El Antri1

1Laboratoire Biochimie, Faculté des Sciences et Techniques de Mohammedia, Morocco
2Institut Supérieur des Professions Infirmières et des Techniques de Santé de Casablanca, Morocco

Before being consumed, food comes into contact with many materials namely plastic materials during the various processes of production, processing, storage, preparation and serving of food. This contacting is concerned with the phenomena of physical and chemical interaction, the bonding and also the transfer of material. This can lead to adverse effects both in terms of quality, hygiene and public health.

The aim of this work is oriented towards the design of anti-adhesion polypropylene materials by modifying in the standard formula of the basic material. In this work, three different formulations incorporating known natural components of their antibacterial effect were studied. The material was developed by extrusion process. The anti-biofilm effect of the material was confirmed by adhesion tests and observations by MEB scanning electron microscopy.

The new material was analyzed by infrared in order to highlight the structural modifications of the groups exposed on the surface of pipelines. The contact angle allows the detection of any modifications of the hydrophobicity and the acid-base character which will make it possible to explain certain variabilities observed after adhesion test.

The mechanical properties of the new material were also evaluated and discussed and compared to the properties of the material developed according to the standard formula.

Antifungal Protein Production from Submerged Cultures of the Medicinal Mushroom Ganoderma Lucidum (Curtis) P. Karst

Monica Andrea Arias Londoño1*, Angela Rocío Mosquera Arevalo1, Natalia Pabón Mora1, Lucía AtehortuaGarcés1, Paola Andrea Zapata Ocampo2 and Jaiver Danilo Sánchez3

1Laboratorio de Biotecnología, Universidad de Antioquia, Colombia
2Facultad de Ciencias y Biotecnología, Universidad CES, Colombia
3Centro de Investigaciones del Banano, Conjunto Residencial Los Almendros Carepa, Colombia

The Banana is a very important edible crop in the world, however it is attacked by different pathogens, one of the most prominent is the fungus Mycosphaerellafijiensis, causal agent of the Black Sigatoka disease. The environmental and economic issues related to the pesticides used for its control have encouraged the search for cleaner alternative biomolecules. Active proteins can act as potential antifungal agents because of their ability to degrade different compounds, which constitute essential parts of a living cell. Mushrooms represent an abundant source of active proteins, particularly the mushroom Ganoderma lucidum. This is a white rot basidiomycete fungus that secretes diverse hydrolytic and oxidative enzymes. In this study, protein extracts from the mushroom G.lucidum exhibited DNase, RNase, protease, glucanase and chitinase enzymatic activities. The extracts were fractionated evaluated for inhibition capability against M. fijiensis. Mass spectrometry identification has shown the presence of different enzymes with potential antifungal capability. Besides different protein concentrations were evaluated over early stage infected banana plants, obtaining interesting results in comparison with the commercial fungicide control.

Development of Process to Produce Biofuel from Lignocellulosic Biomass Using Natural Isolate

Nidhi Adlakha

JNU, India

We report here the production of pure (R,R)- 2,3-butanediol (2,3-BDO) isomer by the non-pathogenic Paenibacilluspolymyxa ICGEB2008 using lignocellulosichydrolysate as substrate. Experimental design based on Plackett-Burman resulted in identication of Mn and K as most crucial salt elements along with the yeast extract for 2,3-BDO production. Further experiments using Box- Behnken design indicated that both KCl and yeast extract together had major impact on 2,3-BDO production. Optimized medium resulted in 2,3-BDO production with 2.3- fold higher maximum volumetric productivity (2.01 g/L/h) and similar yield (0.33 g/g sugar) as compared to rich yeast extract-peptone-dextrose medium in the bioreactor studies. Considering that the balance substrate was channeled towards ethanol, carbon recovery was close to theoretical yield between the two solvents, i.e., 2,3-BDO and ethanol. Biomass hydrolysate and corn-steep liquor was used further to produce 2,3-BDO without impacting its yield. In addition, 2,3-BDO was also produced via simultaneous saccharication and fermentation, signifying robustness of the strain.

Biography:
Nidhi Adlakha is working as Post Doctoral Fellow (Inspire Faculty) in JNU, New Delhi, India. She is working extensively towards understanding cellulose hydrolysis mechanism and developing system for efficient biofuel production. She obtained her PhD degree from International Centre for Genetic Engineering and Biotechnology, New Delhi, India. Her thesis work was on “Development of process to produce biofuel from lignocellulosic biomass using natural isolate” in which she had characterized and isolated potential cellulolytic enzyme systems from Paenibacillus polymyxa, isolated from the gut wood feeding insects. Till date, she have 10 papers published in reputed international journals such as Applied and Environmental Microbiology, Biotechnology for Biofuels and Scientific Reports. Alongwith this, she have 3 Indian patents.

Development and Characterization of Transgenic Pigeon Pea Plants Carrying Osruvb Gene against Salinity Stress Tolerance

Rakshita Singh* and Pushpa Kharb

Department of Molecular Biology, CCSHAU, India

Stress which arises due to environmental parameters such as salinity, drought, high temperature and cold disrupt the normal metabolism of plants. Almost all abiotic stress conditions generate osmotic stress in the plants. Salinity being a very vital problem for the survival of crops leads to major losses in crop productivity. Overexpression of DNA helicases like PDH 45, PDH 47 leads to abiotic stress tolerance (salinity tolerance in tobacco). However, the role of overexpression of RuvB, which is also a DNA helicase, in abiotic stress tolerance in plants has not been reported so far. Therefore, we have developed transgenic pigeonpea plants overexpressing OsRuvB gene, working under the control of CaMV35S promoter to analyse the effect of this gene on plants under saline conditions. The transgene integration in putative T0 plants has been confirmed through PCR analysis and transformation efficiency of 35-40% has been observed. The transgene integration has also been confirmed in T1 plants through PCR and these plants have been exposed to salinity stress. The physio-biochemical parameters such as relative water content, chlorophyll content, membrane stability test, proline content etc. have been studied to assess the tolerance level of the transgenic plants. The PCR positive transgenic plants are being analysed through southern hybridization and real time PCR to determine the transgene copy number.

Biography:
Rakshita Singh has been pursuing PhD from CCSHAU, Hisar, India and working on a very important problem of salinity stress in pigeonpea. Her major advisor is Dr. Pushpa Kharb and she has learnt a lot in her guidance. She is a person with scientific temperament. She love doing science and itʼs her ultimate passion and devotion. It not only excites her but also helps her being logical in her routine life.

2,3-Diaryl Indenone and 2-Chloro-3-Amino Indenone Derivatives as Selective Inhibitor of DNA Repair Enzyme AlkB and AlkBH3

Richa Nigam* and Anindya Roy

Department of Biotechnology, Indian Institute of Technology Hyderabad, India

Human AlkB homologue-3 (AlkBH3) is a DNA repair enzyme that demethylases N1-methyladenine and N3-methylcytosine base lesions. AlkBH3 is an ortholog of bacterial AlkB and also known as prostate cancer antigen-1 (PCA-1) and known to express abundantly in several types of cancers, including prostate cancer. Because of its immense biological and clinical significance, extensive efforts are being directed in developing selective inhibitors for AlkBH3. Here we report synthesis, screening and evaluation of panel of arylated indenone and 2-chloro-3-amino indenone derivatives as new class of specific inhibitors of AlkB family of DNA repair enzymes. An efficient synthesis of 2,3-diaryl indenones from 2,3-dibromo indenones was achieved via Suzuki-Miyaura cross-coupling. Further, synthesis of 2-chloro-3-amino indenone derivatives was achieved from 2,3-dichloro indenones via addition elimination method. Using a robust quantitative assay, we have obtained few inhibitors that showed a unique competitive inhibition mechanism against DNA substrate and a mixed inhibition against 2OG co-substrate. These AlkBH3 inhibitor rendered human cells hypersensitive to exposure to DNA/RNA damaging alkylating agent. This discovery is the first report of an indenone derivative as inhibitors targeting bacterial and human DNA alkylation repair and provides a framework from which second-generation indenone derivatives may be developed.

Biography:
Richa Nigam is a PhD student at Dept of Biotechnology, Indian Institute of Technology Hyderabad, India. She is working under guidance of Dr. Anindya Roy. Her area of research is screening and biochemical characterization of small molecules for inhibition of AlkB family of dioxygenases.

The Generation of g-secretase Inhibitor-loaded PLGA-Fe3O4- Magnetic Nanoparticles

Bashmail R*, McKenna N, OʼShea C, Hakimjavadi R, Molony C, Kozlowska D and Cahill PA

Vascular Biology & Therapeutics Group, School of Biotechnology, Dublin City University, Ireland

Cardiovascular disease is the number one killer in Ireland and the wider EU. A hallmark of the disease is the obstruction to blood flow due to the build-up of vascular smooth muscle (SMCs)-like cells within the vessel wall. Treatment options include percutaneous transluminal coronary angioplasty (PTCA) and the insertion of a stent – a metal mesh tube – into the obstructed vessel to keep it open. However, the vessel can become re-occluded due to the accumulation of SMC-like cells within stented area. The introduction of the 1st generation drug-eluting stents (DES) has resulted in a paradigm shift for the treatment of in-stent restenosis with significant improvement in therapeutic outcomes. However, while polymer-coated DES have significantly reduced the incident of in-stent restenosis, current DESs lack the fundamental capacity for (i) adjustment of the drug dose and release kinetics and the (ii) ability to replenish the stent with a new drug on depletion. This limitation can be overcome by a strategy combining magnetic targeting via a uniform field-induced magnetization effect and a biocompatible magnetic nanoparticle (MNP) formulation designed for efficient entrapment and delivery of specific drugs that target the resident vascular stem cell source of the SMC.

Magnetic nanoparticles (MNPʼs) containing magnetite (Fe3O4) were fabricated, polymer coated with poly(DL-lactide- co-glycolide) polyvinyl alcohol [PLGA-PVA] and loaded with a-secretase inhibitor (GSI) of Notch signalling, DAPT using an oil in water emulsification technique. The free GSIʼs and GSI-loaded MNPʼs were assessed for drug release, the efficacy at controlling mesenchymal stem cell (MSC) growth (proliferation and apoptosis) and inhibiting myogenic differentiation under magnetic and non-magnetic conditions. The DAPT-loaded MNPs had an average hydrodynamic diameter of 351 d.nm Up to 40% of drug was released from MNPs within 48 h rising to 65% after 1 week under magnetic conditions. The Notch ligand, Jagged1 increased Hey1 mRNA levels and promoted myogenic differentiation of MSCs in vitro by increasing SMC differentiation markers, myosin heavy chain 11 (Myh11) and calponin1 (Cnn1) expression, respectively. This effect was significantly attenuated following treatment of cells with MNPʼs loaded with DAPT when compared to unloaded MNPʼs. Notch GSI loaded magnetic nanoparticles are functional at targeting vascular stem cells in vitro.

Biography:
-Roa Bashmail, Bsc. MS. Final year PhD student in the field of Biotechnology, previous qualifications in Master degree in the field of Anatomy and histology, Bachelor degree in Biochemistry.
-Excellent in culturing cells and in techniques like PCR,RNA isolation and Immunocytochemistry also good at the Flow Cytometer and High Performance Liquid Chromatography techniques.
-Excellent in socializing and good leader as well.
-Have won the award of best poster in Dublin City University in the Research day 2015 with title (The Generation of g-secretase inhibitor-loaded PLGA-Fe3O4- Magnetic Nanoparticles).

Utilization of Agro-Industrial Waste for Production of Protease Equipped with Industrially Desired Attributes

Satbir Singh

University of Jammu, India

Microbial proteases, due to their huge application potential, have attracted research attention and account for more than 60% of the world wide enzyme sale. However, large scale industrial application of proteases is hindered due to their poor performance under relatively hostile industrial conditions (extremes of temperature, pH), and high production cost of enzymes. The cost-effective production of a thermostable and wide range pH stable protease from a newly isolated Bacillus subtilis K-1 strain (BSK-1) was done using agro-industrial residues. Process optimization for protease production was conducted by one-variable-at-a-time and statistical approaches. The most significant variables for protease production were identified as incubation time, soybean meal, mustard cake and wheat bran. Optimization of these variables by central composite design of response surface methodology resulted in a substantial enhancement in protease yield. Purification of BSK-1 protease was done by chromatography and the purity was confirmed by zymogram and SDS-PAGE analysis. The BSK-1 protease was stable at wide pH and temperature range with optimum of pH 9.0 and 50°C. Kinetic study of protease suggested high affinity for substrate and sequence analysis of encoding gene showed that K-1 protease is a serine alkaline protease. The enzyme was stable in presence of detergents and metal ions but strongly inhibited by PMSF. It is envisaged that the isolate BSK-1 could be a potential source of alkaline and thermostable protease for applications in industries like detergent, photographic and leather industry. Moreover, exploitation of agricultural wastes as substrates may pave the way for cost-effective production of industrially-suitable protease.

Biography:
Satbir Singh has completed Ph.D. Biotechnology (2010-15) from Fermentation Biotechnology Lab, School of Biotechnology, University of Jammu J&K, India. His thesis title is “Process optimization for production of thermo-alkalistable bacterial protease and its characterization”. He has qualified Jawaharlal Nehru University combined entrance examination for Biotechnology (Masterʼs programme) and availed research fellowship from Council of Scientific and Industrial Research during his doctoral research. He has published 15 articles including research papers, reviews and book chapters in reputed journals.

Production and Characterisation of Extracellular Chitinase from a Novel Isolate Chitinophaga sp. S167

Sonia Shrama1*, Shiv Kumar1 and Ramandeep Kaur2

1Guru Nanak Dev University Amritsar, India
2Panjab University, India

Chitinases cleave β-1,4 N acetyl glucosamine linkages and thus have the ability to degrade chitin in cell wall of fungi and exoskeleton of insects. They are potential antifungal, insecticidal and nematacidal agents. Chitinases have been isolated from bacteria, fungi, plants, insects, crustaceans, animals and humans. Based on the site of action on the substrate, they are classified as exochitinases or endochitinases. An isolate S167 (GeneBank Acc. No. KP017541) showing 98.62%, 98.47 and 97.78% 16S rDNA similarity to Chitinophaga ginsengisoli, C. filiformis and C. pinensis respectively, on EzTaxon was isolated from soil rich in organic decaying matter. Chitinophaga sp. S167 shows chitinolytic activity and produces extracellular chitinase. The chitinase from Chitonophaga sp. S167 inhibited Cladosporium sp., Alternaria sp. and Fusarium sp. The enzyme was found to be optimally active at 40°C and pH 6. The chitinase was maximally induced at 72 hours by 1.5% swollen chitin when incubated in the medium of pH 8 at 35°C. The enzyme was purified using ion exchange and hydrophobic interaction chromatography. The purity of the enzyme was checked by SDS-PAGE which showed a band with an apparent molecular weight of 50 kDa and its activity was confirmed by zymography. Further studies are being carried out to characterize the enzyme and determine its substrate specificity.

Biography:
Sonia Sharma is pursuing Ph.D. in the field of Microbial Biotechnology from Guru Nanak Dev University Amritsar, India. She is interested in exploring potential chitinases from soil microbial diversity that can be employed for industrial, biomedical and environmental purposes. To enhance her skills, she has attended various short term courses and national and international conferences. Recently her work on chitinases was appreciated and got the best poster award at Him Science Congress, an international conference held in India. Currently, she is using structure biology tools to study novel chitinases from Chitinophaga spp. with an emphasis on bio-extraction of chitin from chitinous biowaste.

Engineering of the Chinese Hamster Ovary (Cho) Cell Lines to Investigate Cellular Bottlenecks during Recombinant Protein Expression

Suraksha Muneerappa

Alliance College of Engineering and Design, Alliance University, India

Cultured mammalian cells such as Chinese hamster ovary(CHO)have become the host of choice to produce and secrete biotherapeutic recombinant proteins as they encompass the potential to perform human-like PTMs and correctly fold and assemble complex proteins. However, currently one of the limitations that are observed in the production of immunoglobulins (Igs) is the low degree of N-glycans terminating in sialic acid. In order to overcome this impediment, we focussed on carrying out the below reaction so that the amount of sialic acid can be improved and so as to enhance the complete assembly of N-glycans.

R-Galactose(R-Gal)+Sialic acid(CMP-Neu5Ac)-(Sialyltransferase;STGal) → R-Gal-SA.

The study also focussed on investigating the various bottlenecks which obstruct the sialic acid pathway, such as under expression of α(2,6)STGal, decreased CMP-SA( Cyclic Monophosphate-sialic acid) and improper glycan attachments and faulty galactosylation in N,O-glycans. To investigate these features, we were successful in cloning the genes (B4GalT1 and C1GalT1 chaperone) and transfecting constructs for these into the CHO cells hence our results demonstrate that the cells expressing the genes of interest in the PCR, however did not show expression in the blots. Hence, engineering these systems enabled us to study the various bottlenecks and provide a suitable solution.

Analysis of the Expression of Three Genes Involved in the Biosynthesis of Anthocyanin Using RT-qPCR in Rubus Niveus

Viviana Chiluisa-Utreras

Direction of Biotechnology Career of Natural Resources, Salesiana University, Mexico

Biotechnological applications such as (RT-qPCR) Quantitative Polymerase Chain Reaction,are laboratory techniques that have revolutionized the world of Molecular Biology, providing results in less time and a high degree of reliability, allowing the understanding of genes, functioning and expression, as in the case of the genes of the anthocyanins, which in recent times have increased their interest due to its antioxidant activity which generates therapeutic effects such as reduction of coronary heart disease, improvement of visual acuity, anti-cancer, anti-aging and antitumor effects.

The Polymerase Chain Reaction with reverse transcription (RT-qPCR) allowed to analyze the level of expression of β-Actin, RuANS and RuMYB10, genes involved in the biosynthesis of anthocyanin in Rubus niveus, collected in three different zones of Rumiñahui-Ecuador. According to values obtained with statistical analyses such as Shapiro Wilks, Anova and Tukey test, it was noted that significant differences in the expression of three genes didnʼt exist and there were generated the following results: gene β-Actin, which has greater concentration (174, 65ngmL), followed by the RuANS gene (167, 43ngmL) and finally the RuMYB10 gene (163, 55ngmL), reaching the conclusion that the studied species presents a level of similar expression among the three analyzed genes.

This research can be considered as a starting point of projects for improving the genus Rubus(blackberry), in order to increase their nutritional value moreover grow them as a source for the production of nutraceuticals, drugs and therefore developing productivity and economic value that this crop representsfor the country.

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