2^nd International Conference on Genetic and Protein Engineering November 14-16, 2016 Atlanta, Georgia, USA

Biography:

Mr. Peng Luan has worked on protein therapeutics for 20 years in the biotech/pharmaceutical industry. He holds a Master of Science degree from University of Nebraska-Lincoln and is currently a Sr. Scientific Researcher at Genentech, Inc.  His recent work has been involvedin developing high throughput purifications that enable large antibody discovery campaigns. He is a co-inventor on five issued U.S. patents and had several journal publications on therapeutic protein engineering and productions.

Abstract:

Due to the revolutionary impact of monoclonal antibodies in the field of therapeutics and diagnostics, more pharmaceutical companies are expanding their antibody discovery efforts. In early research, various types of IgGs from multiple species are generated through hybridoma, phage library and other antibody discovery technologies. In large campaigns, thousands of antibodies are often expressed in micro-scale (~ 1 mL) by high throughput (HTP) hybridoma culture or transient expression in 96 well culture blocks. Availability of purified antibodies significantly improves the reliability of “hit identification” from HTP screening assays. To improve efficiency and consistency, we have developed automated HTP workflows using two liquid handling platforms, resulting purification capacity of ~2000 antibodies in a single day with just one operator. In addition to supporting antibody discovery or engineering campaigns with thousands of purified proteins, these HTP work flows have also enabled rapid in-parallel testing of purification conditions using design of experiment (DOE) principles. For instance, purification of certain antibody formats (such as Fabs) and some murine or rat antibodies (such as Rat IgG2a) necessitates pH and/or salt adjustment with commonly used resins (Protein A or G), making the process impractical for HTP implementation. Using our HTP approaches we have identified a commercially available,engineered protein G resinthat can capture all rat IgGs under physiological conditions and have optimized elution conditions to maximize antibody recovery.

Biography:

Dr. Broersen completed her doctorate in the field of protein aggregation at Wageningen University in The Netherlands in 2005. After her postdoctoral study at the MRC-LMB in the UK, she joined the Free University of Brussels (VUB)/Flanders Institute for Biotechnology (VIB) in 2007. Here she headed a research team that studied the molecular mechanism of Alzheimer’s disease. This led to the discovery of molecular pathways of a number of risk factors that affect Alzheimer’s disease pathobiology. Subsequently, she joined the Nanobiophysics Group at the University of Twente/MIRA Institute in The Netherlands as an assistant professor investigating further the impact of protein structures on human health with her team. 

Abstract:

One of the most prominent hallmarks detected in the brains of patients suffering from Alzheimer’s disease is the deposition of amyloidogenic plaques. These plaques are largely composed of the amyloid-beta peptide. It has been demonstrated that, even though these plaques comprise an important and recurring feature for disease, that precursor forms of these plaques, called ‘oligomers’ or ‘protofibrils’ more potently affect neuronal functioning. Accumulation and subsequent assembly of amyloid-beta peptide into aggregated forms has been reported to be partly brought about by an impaired clearance of the amyloid-beta peptide. Lowering the amyloid-beta peptide burden by increasing clearance provides a promising avenue for treatment of Alzheimer’s disease.

A number of proteases have been reported to cleave amyloid-beta peptide in vivo or in vitro. Drug compounds are under development that can modulate enzymatic activity to selectively enhance amyloid-beta peptide degradation. In view of the development of proteolytic-based therapies, more insight into the amyloid-beta peptide-degrading capacity of enzymes is required. For example, the effect of amyloid-beta proteolytic enzymes on various misfolded forms of the peptide has been little documented in the literature. Also the effect of in vivo occurring combinations of enzymes has not been explored. Moreover, the properties of enzyme-induced fragments from Aβ are currently unknown. To extend our understanding of the potential therapeutic utility of amyloid-beta peptide proteolytic enzymes, we investigated enzyme-mediated cleavage of amyloid-beta peptide in vitro using biophysical and biochemical assays and we identified new cleavage sites, cooperative activity of enzymes and characterized the potential aggregation behavior of resulting fragments.

Biography:

As a molecular biologist focusing on protein engineering and cancer imaging and therapy, Dr. Papo joined the BGU faculty in 2011, and his research focuses on the development of new nono- and multi-specific proteins and protein-small molecule conjugates that promise to aid in both the diagnosis and treatment of cancer. Dr. Papo’s areas of expertise include protein engineering, angiogenesis, metastasis, cancer biology, targeted cancer therapy, in vivo imaging, directed evolution, ligand-receptor interactions, molecular recognition, protein sequence-structure-function relationships, and synthetic biology. More generally, Dr. Papo and his research team are developing methods that allow us to design nono- and multi-functional proteins with optimized and targeted pharmaceutical properties, such as the protein’s distribution in the body and how long it remains, resulting in enhanced alternatives to antibodies that will benefit both cancer therapy and diagnostics. 

Abstract:

Mesotrypsin, an enzyme that contributes to progression and metastasis ofmany cancers,constitutes a compelling therapeutic target. However, with its unique capability for cleavage and inactivation of proteinaceous inhibitors, mesotrypsin presents a formidable challenge to the development of biologic inhibitors.Our study identifies a promising mesotrypsin inhibitor – a triple mutant of the human amyloid precursor protein Kunitz protease inhibitor domain (APPI) with superior affinity, specificity, and proteolytic stability – as a starting point for the development of anticancer protein therapeutics. We demonstrate that the mutant acts as a functional inhibitor of mesotrypsin-dependent prostate cancer cellular invasiveness. Additionally, the crystal structure of the mutant/mesotrypsin complex provides new insights into the structural and mechanistic basis for the mutant's improved binding and proteolytic resistance. Finally, the study establishes proof-of-principle for a novel library screening approach that is widely applicable for simultaneously evolving proteolytic stability and a desired functionality for diverse protein scaffolds.

Biography:

Prof Dr Frédéric CADET (PEACCEL, Paris, France): Vice President Research & Development of the company Peaccel (Protein Engineering ACCELerator: http://www.peaccel.com). Ph.D (specialization in: Protein Engineering, Data mining, Biosimulation). From 2004 to 2008, as an “Executive School, University & Research Commissioner”, he managed a budget of 1.3 billion Euros and was responsible for 32,000 employees. Former Chairman of the ERA Nets (European Research Area Networks) NetBIOME. He has developed pioneering research activities in bioinformatics. Author of over 70 publications and referee for 17 international scientific journals. Organizing Committee Member for « 2nd International Conference on "Genetic and Protein Engineering”, November 14-16, 2016 Atlanta, USA.

Abstract:

The knowledge of organisms and their metabolic pathways allowed to construct biological systems for the production of chemicals and pharmaceuticals such as antibiotics and biofuels. Synthetic biology expands the number of these biological systems by the assembly of artificial metabolic pathways, called synthetic pathways, not present in natural organisms. Synthetic pathways could be integrated in modified micro-organisms or in biocatalyst systems. A biocatalyst system is an in vitro assembly composed only of purified enzymes and metabolites that are useful for the production of a desired metabolic compound through a biochemical reaction network. This in vitro assembly, as compared to cellular system, has several advantages, such as the production of only desired metabolites and a great engineering flexibility. We explored an in silico approach to identify and analyse new biocatalyst systems for the production of target metabolic compounds. This approach proceeds in several steps. The first step is the enumeration of several biocatalyst systems that could synthesize a target product from a desired starting substrate. Next, a selection based on several criteria is applied to choose a biocatalyst system among the group of biocatalyst systems identified in the enumeration step. The last step is the modelling of the selected biocatalyst system to evaluate the production rate and the yield of the target product. This communication explains in more detail the modus operandi for the different steps of our in silico approach.

Biography:

Duqing has completed his PhD at the year 2015 in the major of pharmacognosy from the institute of medicinal plant development ,Chinese academy of Medical Sciences(CAMS) and Peking Union Medical College(PUMC) and got the doctor degree in 2016.Duqing begin the postdoctoral studies in the institute of genetics and developmental biology,Beijing,China from the July of 2015 to August of 2016.I am the member of American Society of Plant Biologists and Chinese Society for Cell Biology.Moreover,I am a pharmacist and the member of Chinese Pharmacist Association. We have published more than 5 papers in the BMC and Chinese famous journals as the research group. 

Abstract:

Terpeniod compound can be classified into monoterpenes((C5H8)₂),sesquiterpenes((C5H8)₃),

diterpenoids((C5H8)₄),triterpenoids((C5H8)₆) and polyterpenes((C5H8)₂,n>6) according to the numbers of Isoprene or isopentane((C5H8)n) and the derivative of having the oxygen and different saturation degree from the structure.The biosynthesis of different terpeniod compound can be produced depends on cross-talk between the MEP pathway and the mevalonate (MVA) pathway. IDI（Isopentenyl diphosphate isomerase）is the common and key enzyme related to the biosynthesis of terpeniod and gibberellin compounds.It is well known that the IDI gene families exit in the diverse species of the earth and have been carried out the research works in the plants of  Arabidopsis annua L./Artemisia apiacea、the fungus of Ganoderma lucidum etc.Why do I think the essential and important of IDI gene families regarding the biosynthesis of terpenoids compound?Because IDI gene families locate in the upstream of the pathway.The function and change of it can bring the influences or actions to the whole course of metabolism pathway and downstream products.We can not ignore the essence of research courses or dynamics on the direction of research projects.The quantities of IDI gene families are found it is not same or diverse because of the assay and characteristics involved in the species.Moreover,IDI gene families has the certain function during the course of reciprocal transformation between the gene families of IPP and DMAPP.I will give the review in the way of presentation from the aspects of exist formation,identification,cloning sequences,tissue-specific expression,protein structures and functions and applications of the different creatures so as to acquire the supports and good advice coming from the experts who are interested and has the famous experience about the research projects.

Biography:

Xiao-Qing Qiu has completed his PhD in 1991 from Emory University and postdoctoral studies from Albert Einstein College of Medicine at NYC in 1996. He is the director of Lab. of Biomembrane & Membrane Proteins and full Professor of Sichuan University. He has published more than 10 papers in reputed journals. Mainly engaged in developing a novel fusion-peptide antibiotic, pheromonicin, against drug-resistant pathogens. 56 related patents have been approved by China, US, and other countries patent offices.    

Abstract:

A new strategy in the development of antibiotics against the threat of antibiotic resistance is to simply adopt defense strategies of bacteria themselves. Bacteriocins, antimicrobial peptides produced by bacteria, are an essential entity of such strategies. Colicins are channel-forming bacteriocins produced by Escherichia coli. They are bactericidal to other E. coli strains by forming a voltage-activated channel in the cell membrane.

Here we constructed a peptide consisting of a 28-residue antibody mimetic fused to the channel-forming domain of colicin Ia and named it pheromonicin. By altering native targeting of colicins, antibody mimetic targets fused colicin to form a lethal channel in the cell membrane to lead the leak/death of targeted pathogens.

Pheromonicins have nano-molar efficacy against thousand clinical isolates of antibiotic-resistant Gram-positive and -negative pathogens while they left enteric probiotic bacteria, lactococcus, lactobacillus and Bifidobacterium unaffected at the dosages that effectively killed pathogens. In thousand rodents, poultries, ungulates and non-human primate models, pheromonicin-treated animals survived administration of MRSA, or VRE, or MDR P. aerugenosa, or MDR M.tuberculosis at doses that were lethal to controls.

With the inheritance of colicin’s native advantages, nano-molar efficacy, the ability to act in the host circulation, no toxicity and easy genetically engineered, pheromonicin application cleared fatal bacterial infections in vivo that did not respond to currently employed antibiotics (vancomycin, amoxicillin, oxacillin, carbapenem etc). Pheromonicin may be of value as a novel antibiotic against MDR pathogens with targeted bactericidal and cytokine modulating activities.

Biography:

D.L.Savithramma has completed her PhD at the age of 35 years from University of Agricultural Sciences, Bangalore, India and postdoctoral studies as a Biotechnology National Associate from Indian Institute Science, Bangalore, India. She is a Professor of Genetics and Plant Breeding at University of Agricultural Sciences, Bangalore, India, one of the premier Agricultural University in India. She has published more than 70 papers in reputed journals and has released seven varieties in Vegetable cowpea, seed cowpea, Peanut and Chrysanthemum

Abstract:

Plant root is one of the major organs for water and nutrient uptake from soil. Root development and amount of water absorption from the soil are closely related. Under drought condition the success of crop plants often depend on growth of roots. In the present study gravimetric experiment was conducted to evaluate the yield potential of released and pre released groundnut genotypes.Root growth parameters such as root length, shoot length, shoot weight, leaf length, root volume and dry root weight were recorded in five genotypes viz., KCG-2, TMV-2, GKVK-5, GKVK-3 and GKVK-13 under controlled and water stress conditions in pot experiment at University of Agricultural Sciences, Bangalore, India. Stress was imposed for 20 days during flowering stage by balancing the amount of water applied to make it 50% stress. Analysis of variance revealed highly significant differences for the traits observed indicating the existence of variability in the material selected. In controlled condition all the root traits exhibited higher mean values over stress condition in the genotypes studied except in GKVK-5 for root dry weight. In stress treatment GKVK-5, GKVK-3 and GKVK-13 genotypes exhibited higher mean values for root volume, root dry weightand root length. Genotypes GKVK-5, TMV-2 and KCG-2 recorded higher mean for shoot weight and leaf length. Among all genotypes GKVK-5 was identified as high yielding with 2.8 to 3.0 t/ ha, with low to moderate Δ¹³C (17.53) and high δ¹⁸O (31.24) and low specific leaf area(107.76cm²/g)and was found to have higher Water Use Efficiency or to be drought tolerant genotype indicating effective partitioning of the accumulated biomass towards root and shoot, while diverting relatively lesser towards shoot biomass. Hence, breeding for root development proved to be effective in increasing crop Water Use Efficiency and drought tolerance.

Biography:

D.L.Savithramma has completed her PhD at the age of 35 years from University of Agricultural Sciences, Bangalore, India and postdoctoral studies as a Biotechnology National Associate from Indian Institute Science, Bangalore, India. She is a Professor of Genetics and Plant Breeding at University of Agricultural Sciences, Bangalore, India, one of the premier Agricultural University in India. She has published more than 70 papers in reputed journals and has released seven varieties in Vegetable cowpea, seed cowpea, Peanut and Chrysanthemum

Abstract:

 Simple sequence repeat (SSR) markers are a major molecular tool for genetic and genomic research that have been extensively developed and used in major crops. However, few are available in mungbean (Vigna radiata(L.) Wilczek), an economically important protein rich(22-24%) edible food legume of South and Southeast Asia. Mung bean is a self-pollinating diploid grain legume with the genome size of 560 Mb. Therefore, the application of molecular markers can play key role in direct improvement of mung bean. Development and use of molecular markers in the species are limited. The major objective of this study was development for a set of simple sequence repeat (SSR) markers based on construction of SSR-enriched genomic DNA libraries, sequencing, and validation of designed primers. Mungbean genotypes Chinamung and BL 849 were used to isolate DNA for constructing SSR-enriched genomic libraries. More than 200 positive clones were purified and they were sequenced using vector specific primer.Quality of all DNA sequences was analyzed. After trimming off the vector sequences, all the sequences were placed in a fasta file for further analysis with the CAP3 program to categorize the sequences into contiguous sequences (contigs) and singletons(non repeating). The two categories of sequences were run separately with the SSR Locator program for SSR detection and primer designing. The parameters for the primer design were given as  amplicon size in 140–350 bp, primer length of 18–22 bases with 20 as the optimum, annealing temperature of 55–61°C with an optimum of 59°C, GC clamp 0, G/C content 45–50 %, start and end point automatic scan and end stability at 250. A total of 40 primer pairs were designed from the microsatellite-containing sequences. Newly synthesized 40 mung bean SSR primers were used for genetic diversity studies with 24 genotypes and dendrogram was constructed. Out of 40 designed primer pairs 26 primer pairs were found to amplify bands of the expected sizes while eleven primers failed to amplify and three primers produced monomorphic bands. The overall size of the amplified product varied from 110 to 400 bp. The coefficient of genetic dissimilarity ranged from 0.159 to 0.536. Pairwise estimates of dissimilarity ranged from 0.14 to 0.90 and the average dissimilarity among all 24 genotypes was 0.365. Two genotypes LM 192 and IC325738 were the closest related genotypes with the lowest dissimilarity index of 0.159. The highest dissimilarity (0.536) was observed between genotypes WBM and SML 348. The SSR markers developed in the study are highly valuable for molecular and traditional breeding research.

Biography:

Abstract:

Biography:

D.L.Savithramma has completed her PhD at the age of 35 years from University of Agricultural Sciences, Bangalore, India and postdoctoral studies as a Biotechnology National Associate from Indian Institute Science, Bangalore, India. She is a Professor of Genetics and Plant Breeding at University of Agricultural Sciences, Bangalore, India,  one of the premier Agricultural University in India. She has published more than 70 papers in reputed journals and has released seven varieties in Vegetable cowpea, seed cowpea, Peanut and Chrysanthemum

Abstract:

Pulse crops have been shown to be an excellent source of dietary protein important for the human diet and play a key role in crop rotation due to their ability to fix nitrogen. Since all recommended greengram varieties are susceptible to the disease, plant breeders are interested in developing resistant varieties through both conventional and marker-assisted breeding. The genetic nature of a trait, phenotypic data and genotypic data from molecular markers was studied by detecting associations between markers and traits and determined the number and nature of a gene/quantitative trait locus (QTL) controlling a trait. Mung bean germplasm screening was done for powdery mildew disease resistance and yield related traits and genotypes were selected. Hybridization was done between powdery mildew resistant BL 849 (male parent) and Chinamung (female parent) which issusceptible but high yielding. The F₁ generation was developed and forwarded to F₂. Individuals of F₂generation of this cross were screened for powdery mildew disease. The F₂ individuals were also classified into three marker classes. The mean disease score of the 94 F₂ individuals belonging to each of the marker classes were computed. The significance of differences among the three marker classes for disease score and productivity traits were tested using F test through ANOVA approach. The relationship between molecular markers and phenotypic scores were analysed by single marker analysis to identify SSR markers that had significant association with powdery mildew disease reaction severity scoresin greengram.For the markers DMBSSR 130 and VM 27, chi square (χ2) was non significant suggesting that the distribution of the genotypic data fit Mendel’s segregation ratio of 1:2:1. Single marker analysis for SSR marker DMBSSR 130 and VM 27 showed very high association among the marker classes and the powdery mildew resistant type in F₂ mapping population.  In the single marker analysis, the markers GBSSRMB 14 and DMBSSR 34 were found to be highly linkedwithtraitsviz., number of pods per plant, pod yield per plant and seed yield per plant in F₂ segregating population. This indicates that the same gene is controlling the expression of these characters. Moreover, phenotypically these characters have more association with each other. Hence these markers may be useful for marker assisted breeding programme. GBSSRMB 14 was also linked with number of pods per plant and number of clusters per plant. MBSSR 42 was associated with seed yield, pod yield and number of clusters per plant. The marker GBSSR MB 87 was found to be associated with number of branches per plant.  Molecular markers linked with QTL/major genes for traits of interest are being routinely developed in several crops. However, non-availability of mapping populations and substantial time needed to develop such populations are sometimes major limitations in the identification of molecular markers for specific traits. Another limitation is the absence of tight linkage between marker and traits observed in these studies. Also, it is difficult to eliminate false positives with available methods. Therefore, markers identified during the present study need to be subjected to validation and/or functional analysis of respective traits.

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Cowpea (Vigna unguiculata) with chromosome number 2n=22 is a multipurpose grain legume crop grown throughout sub-tropical areas of the world. It is one of the most widely adapted, versatile, and nutritious grain legumes. It’s a good source of protein, carbohydrate, fat, vitamins and phosphorous. Cowpea is a cheapest source of vegetarian protein, development of high yielding cowpea varieties with high zinc, iron and protein will help fighting against malnutrition. Introduction of biofortified agricultural products and crop varieties with superior nutritional value are a prime low-cost crop improvement strategy for increasing the food sustainability. Though several options are available for augmenting mineral deficiencies of the soil, development of nutrient efficient genotypes seems be eco-friendly and long sustaining option to fight malnutrition. Keeping these aspects in mind, present investigation was carried at Department of Genetics and Plant Breeding, University of Agricultural Sciences, Bengaluru. Two hundred cowpea genotypes obtained from different sources were field screened for their phenotypic performance and also screened for zinc, iron, protein. Iron and Zinc contents in seeds of elite cowpea genotypes were analyzed using Atomic absorption spectrophotometry (AAS). The seed protein content was estimated using Kjeldahl method described in AOAC (1984).

The analysis of variance (ANOVA) indicated significant difference for all the characters under consideration justifying the selection of genotypes for the study. Genetic variability estimates, Phenotypic co-efficient of variation (PCV) and Genotypic co-efficient of variation (GCV) were high for plant height, clusters per plant, pods per plant, pod yield per plant and seed yield per plant indicating the predominance of additive gene action and these traits offer scope for direct selection.

                Highheritability estimates coupled with high genetic advance as per cent of meanwas observed for days to first flowering, days to fifty per cent flowering, plant height, primary branches per plant, clusters per plant, pods per plant, pod length, pod yield per plant, 100-seed weight and seed yield per plant suggesting that these characters are under the control of additive genes and phenotypic selection for these characters may be effective.Twenty-two lines were found to be rich zinc, iron and protein. Considering yield also, eighteen genotypes were found to be most promising. Themicronutrient range was found to be 15.8-48.6ppm, 49.6-150.2ppm and 22.03ppm to 26.21 %for zinc, iron and protein respectively. Genotype, PGCP-6 scored high zinc content of 48.6ppm, high iron of 137.2 ppm and 23.85% protein. Genotype KBC-6 was found to be rich in zinc 41.8ppm, iron 150.2ppm and 23.11 % protein. Local check KM5 scored zinc of 15.8ppm, iron 83.6ppm and protein 24.5%. The genotypes which showed high zinc were also associated with stay green colour even after maturity thus serving as phenotypic markers. Biochemical components associated with this trait needs to be identified which serve as marker to tag genotypes as zinc efficient. Molecular markers associated with stay green phenotype with high zinc needs to be identified. The two genotypes viz., PGCP-6 and KBC-6 were found to be most promising in terms of yield also thus could be used for further confirmation of their results and then used in crop improvement programmes.

Biography:

Prof. Dr. Ikram-ul-Haq (SI) has completed his Postdoc from Cornell University, New York, USA, and Ph.D. in Industrial Microbiology from University of the Punjab, Lahore, Pakistan. He has been the Dean, Faculty of Science and Technology and founding Director of Institute of Industrial Biotechnology, Government College University (GCU), Lahore. He has successfully completed 16Projects sponsored by Pakistan Science Foundation, Pakistan Atomic Energy Commission, HEC, GCU & Ministry of Science and Technology while 4 Projects are in process sponsored by MoST, PSF and Pakistan Academy of Sciences. He has published more than 309 papers in journals of international repute, with 1730 citations and has been serving as an editorial board member of reputed Journals 

Abstract:

The growing demands of bioenergy has led to the emphasis on novel cellulases to improve efficiency of biodegradation process of plant biomass. Therefore, a thermostable cellulolytic gene (CenC) with 3,675 bp was cloned from Clostridium thermocellum and over-expressed in Escherichia coli strain BL21 CodonPlus. It was attested that CenC belongs to glycoside hydrolase family 9 (GH9) with four binding domains, a processive endoglucanase. CenC was purified to homogeneity, producing a single band on SDS-PAGE corresponding to 137.11 kDa, by purification steps of heat treatment combined with ion-exchange chromatography. Purified enzyme displayed optimal activity at pH 6.0 and 70°C. CenC had a half-life of 24 min at 74°C, was stable upto 2 h at 60°C and over a pH range of 5.5-7.5. Enzyme showed high affinity towards various substrates and processively released cellobiose from cellulosic substrates confirmed by using HPLC technique. It efficiently hydrolyzed carboxymethyl cellulose (30 U/mg), β-glucan Barley (94 U/mg); also showed activity towards p-nitrophenyl-β-D-cellobioside (18 U/mg), birchwood xylan (19 U/mg), beechwood xylan (17.5 U/mg), avicel (9 U/mg), whatman filter paper (11 U/mg) and laminarin (3.3 U/mg). CenC exhibited K_m, V_max, K_cat, V_max K_m^-1 and K_cat K_m^-1 of 7.14 mM, 52.4 µmol mg^-1min^-1, 632.85 s^-1, 7.34 min^-1 and 88.63, respectively used CMC as substrate.Recombinant CenC saccharified pretreated wheat straw and bagasse to 5.12% and 7.31%, respectively at pH 7.0 and 45°C after 2 h incubation. Its thermostability, high catalytic efficiency and independence of inhibitors make CenC enzyme an appropriate candidate for industrial applications and cost-effective saccharification process

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With a paradigm shift in industry, moving from natural fuels to alternative renewable resource utilization, the need of efficient thermostable cellulases are expected to increase in future. β-glucosidase, an essential member of cellulases that plays a critical role in cellulosic biomass degradation and in many biological processes. Therefore, a novel β-glucosidase gene encodes a protein (BglA) of 446 amino acid, belonging to glycoside hydrolase family 1 (GH1), was cloned from a hyperthermophilic bacterium Thermotoga naphthophila RKU-10^T and over-expressed in Escherichia coli BL21 CodonPlus. An extracellular BglA with a molecular weight of 51.50 kDa, was purified to homogeneityby ion-exchange and hydrophobic interaction chromatography after heat treatment. Purified enzyme displayed optimal activity at pH 7.0 and 95°C. It was quite stable over a broad range of pH (6.0-9.0) and temperature (60-90°C), fairly stable up to 8 h at 80°C. Enzyme activity was stimulated by glucose concentration up to 600 mM and exhibited high glucose tolerance with a K_i value of 1200 mM.BglA showed great affinity towardsp-nitrophenyl substrates and cellobiose. The K_m, V_max and K_catvalues, against pNPG as substrate, were 1.5 mM, 297 mmol mg^-1min^-1 and 1527778 s^-1, respectively.Thermodynamic parameters for pNPG hydrolysis by BglA like ∆H*, ∆G* and ∆S* were calculated at 95°C as 25.7 kJ mol^-1, 47.24 kJ mol^-1 and -58.6 J mol^-1 K^-1, respectively. It displayed a half-life (t_1/2) of 5.21 min at 97°C with denaturation parameters of enzyme including ΔH*_D, ΔG*_D and ΔS*_D were 662.04 kJ mol^-1, 110.10 kJ mol^-1and 1.491 kJ mol^-1 K^-1, respectively. This is the first ever report on a highly glucose and thermotolerant β-glucosidase fromThermotoga naphthophilawith high catalytic efficiency and low product inhibition, also exhibited independence of detergents and metal cations. All these significant features make BglA an appropriate candidate for biotechnological and industrial applications

Biography:

Kapil Vashisht is a Senior Research Fellow at National Institute of Malaria Research, New Delhi, India and registered for Ph.D programe at Goa University, Goa, India. He is working under the supervision of Dr. Kailash C. Pandey, Scientist ‘E’, currently at National Institute for Research in Environmental Health, Bhopal, India. Recently he has submitted this interesting research in Biochemistry (Revised) as a first author and this study has also been filed for PCT at Indian Council of Medical Research, India (PCT/IN2015/000451). He is currently working to prove this concept in SCS of P.falciparum (another human parasite

Abstract:

SCS was discovered in 1958, there are extensive studies and crystal structures available, but mechanism of nucleotide specificity is still not clear. Hallmark concepts like “Lock and key” and “Induced fit” hypothesis were present for enzyme specificity. Here, we have proposed a novel strategy, employed by SCS in Blastocystis for discriminating between cognate and non-cognate ligands (ATP>P). Charged, solvent exposed residues at the entrance to substrate binding site (Gatekeeper residues), produce electrostatic dipole interactions with approaching substrates, and control their access by a novel mechanism called “Electrostatic Gatekeeper Effect”. In this proof-of-concept study we have demonstrated that nucleotide specificity of wild ATP-specific (K_m=145±47µM) SCS with positive gatekeepers(KK), can be engineered by altering electrostatic properties of its gatekeeper residues. Our enzyme kinetics results showed dual specificity [ATP(K_m=230±34µM) & GTP(K_m=143±17µM)] for gatekeeper mutant(ED) with negative gatekeepers(ED), which favored GTP access to binding site. However, nucleotide binding site mutant(LF) showed no GTP-specificity despite previously reported GTP-supporting residues(LF), and remained ATP-specific (K_m=265±50µM), because positive gatekeepers(KK) still precluded GTP access to binding site. Interestingly, combining gatekeeper mutant with nucleotide binding site mutant(ED+LF), resulted in exclusive GTP-specificity (K_m=82±12µM) and no detectable ATP-activity. This striking result was entirely due to; negatively charged gatekeeper residues(ED) favored GTP access, while nucleotide binding site residues(LF) altered ATP binding. These results were further supported by molecular modelling and simulation studies. Hence it is imperative to explore this strategy in different range of crucial enzymes (synthetases, kinases, transferases) to engineer substrate specificity for various industrial applications and substrate based drug design.

Biography:

I  have  completed bachelor at the age of  28 years from Sulaimanyah  university college of science biology department . I work as medical laboratory technologist  at the big hospital named shar hospital. I have puplished two paper .  I am young  to the research area  i will have plan to search and publish more papers .the reason to attend in the conferece to get more experience for tmy future works. 
 

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