Lipase An Industrial Enzyme Through Metagenomics

Microbial lipases are industrially important and have gained attention due to their stability, selectivity, and broad substrate specificity. Lipases are used as medicine, and they also aid in indigestion, heartburn, allergy to gluten in wheat products (celiac disease), Crohn’s disease, and cystic fibrosis. This volume considers the industrial demand for new sources of lipases with different catalytic characteristics that stimulate the isolation, growth, and development of new microbial strains. The volume narrates the challenging metagenomic approach with the isolation of the lipase gene, its cloning into Escherichia coli, culture of the recombinant bacteria, and extraction and assessment of the lipase enzyme. Lipase-producing bacteria are available in different habitats, such as industrial wastes, vegetable oil processing factories, dairy plants, and soils contaminated with oil and oil seeds, among others. This volume is the effort of the authors to document the scientific findings carried out over the last eight years in the area of un-culturable soil microorganisms. The book presents the physic-chemical features of lipases and their specific applications in different commercial industries. The in-depth study looks at metagenomics for lipases from all angles and provides a truly informative resource. It describes the biochemical characterization of lipase enzymes with the high activity in the presence of 1% tributyrin. A wide review has been presented in the book on lipase enzymes purified from a large collection of microbes present in soil, seawater, waste-dumping sites, animal systems (including human beings), and the atmosphere. Stability of enzymes over changing environments of the industry is indeed a big issue, and the book deals at length with the changing temperatures and pH and metal ion concentrations.

IntroductionEnzymesEtymology and historical aspects of Industrial enzymesIndustrial enzymes: development of bio-industrial sectorClassification of EnzymesIndustrially important enzymesKinetic model of lipolysis, structure of lipase, stability and cofactorsClassification of bacterial lipolytic enzymesApplication of LipasesDetergent industryFood processing, flavor development and improving qualityBakery industryFat and oleochemical industryTextile industryCosmetic industryPulp and paper industryOil biodegradationProduction of biodegradable polymersDiagnostic toolDegreasing of leatherWaste/effluent/sewage treatmentProduction of biodieselTea processingBiosensorsEnvironmental ManagementMajor obstacles and future prospect of microbial lipasesMetagenomics and Unculturable BacteriaMicrobial communitiesSoil as a microbial habitatUnculturable microorganismsCulturable microorganismsMicrobial species for beneficial products and processesMetagenomicsSoil metagenomics for desirable genesSoil metagenomics: tool for novel compoundsSequence based metagenomicsFunction driven metagenomicsMetagenomic library construction and its analysisApproaches to metagenomicsAccessing Metagenomics MethodsExpression vector/host systemFunctional screening of metagenomic librariesMetagenomics for LipaseCollection of environmental samplesExtraction, purification and quantification of soil metagenomic DNAIsolation and purification of mgDNA from gram+ and gram- bacteriaPolymerase chain reactionRestriction digestion by Eco RI, Hind III and Bam HIMetagenomic DNA library constructionSequence based metagenomic approachConstruction of 16S rDNA gene librariesPreparation of E. coli competent cells and transformationAnalysis of transformation efficiency using non-recombinant plasmidScreening of recombinants by a-complementationPurification of PCR productsTransformant confirmation by colony PCR using universal primersIsolation and restriction of recombinant/non-recombinant plasmid DNAsAnalysis of 16S rDNA genePhylogenetic analysisGenbank submission and accession numbersPhylogenyFunctional Approach for Metagenomic Library ConstructionRestriction digestion of mgDNATransformation of E. coli BL21 competent cellsFunctional screening of metagenomic libraries for the lipase geneIsolation and restriction of recombinant plasmidSequencing of cloned DNASequence analysis and phylogenetic tree constructionAnalysis of the cloned lipase genePhylogeny of recombinant proteinAmino acid sequence of recombinant proteinMultiple sequence alignmentStorage of recombinant plasmidOverexpression of Recombinant ProteinPurification of expressed proteinDetermination of active lipase using zymographic studyDetermination of specific activity, % yield and protein fold purificationHomology model and validation for protein structure predictionBiochemical Characterization of Purified LipaseDose dependent enzyme activitySubstrate specificity and effect of substrate concentrationEnzyme KineticsEffect temperature and pHEffect of surfactantsEffect of other factorsGenomic Study of Culture Dependent BacteriaIsolation and screening for lipase producing culturable bacteriaPure culture of lipase producing bacterial isolatesInoculum preparationZone of hydrolysisGrowth kinetics and lipase productionThermal stabilityEvaluation of crude enzyme for detergent formulationStability of culture supernatant under storageTaxonomic identification of lipase producing bacteriaBiochemical characterizationExtracellular enzyme activitySpectrophotometric growth determinationGenomic Study of Culturable BacteriaGenomic DNA extraction of lipase producing culturable bacteriaMolecular genetic assessment of lipase producing bacteriaPhylogenetic analysisOptimization of culture condition for lipase productionEffect of carbon and nitrogen sources for the growth of culturable bacterialTime course with lipase productionEffect of other factorsMicrobial Assay of Culture Supernatant Containing Crude LipaseDetermination of antibacterial activityDetermination of antifungal activityCritical Observations

B. K. Konwar, PhD, is currently Professor in the Department of Molecular Biology and Biotechechnology at Tezpur University (Central), Assam, India. He has worked as a lecturer, assistant professor, and associate professor at the university. Former appointments include Senior Scientist, Tocklai Experimental Station, Tea Research Association; Professor and Department Head, Molecular Biology and Biotechnology at Tezpur University (Central), India; Head, Centre for Petroleum Biotechnology; Dean, School of Science and Technology, Tezpur University; and Vice- Chancellor, Nagaland University (Central), Nagaland, India. Dr. Konwar has carried out 12 research projects of national importance as the principal investigator. He and his research group so far have deposited 11 gene (DNA) sequences in gene banks and have published 142 research papers in reputed international/national journals, along with many papers in seminar and conference proceedings. Kalpana Sagar, PhD, is currently a Research Associate at Delhi University. She has earned her PhD in molecular biology and biotechnology from Tezpur University under the supervision of Prof. Konwar.