1st Edition
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.
Introduction
Enzymes
Etymology and historical aspects of Industrial enzymes
Industrial enzymes: development of bio-industrial sector
Classification of Enzymes
Industrially important enzymes
Kinetic model of lipolysis, structure of lipase, stability and cofactors
Classification of bacterial lipolytic enzymes
Application of Lipases
Detergent industry
Food processing, flavor development and improving quality
Bakery industry
Fat and oleochemical industry
Textile industry
Cosmetic industry
Pulp and paper industry
Oil biodegradation
Production of biodegradable polymers
Diagnostic tool
Degreasing of leather
Waste/effluent/sewage treatment
Production of biodiesel
Tea processing
Biosensors
Environmental Management
Major obstacles and future prospect of microbial lipases
Metagenomics and Unculturable Bacteria
Microbial communities
Soil as a microbial habitat
Unculturable microorganisms
Culturable microorganisms
Microbial species for beneficial products and processes
Metagenomics
Soil metagenomics for desirable genes
Soil metagenomics: tool for novel compounds
Sequence based metagenomics
Function driven metagenomics
Metagenomic library construction and its analysis
Approaches to metagenomics
Accessing Metagenomics
Methods
Expression vector/host system
Functional screening of metagenomic libraries
Metagenomics for Lipase
Collection of environmental samples
Extraction, purification and quantification of soil metagenomic DNA
Isolation and purification of mgDNA from gram+ and gram- bacteria
Polymerase chain reaction
Restriction digestion by Eco RI, Hind III and Bam HI
Metagenomic DNA library construction
Sequence based metagenomic approach
Construction of 16S rDNA gene libraries
Preparation of E. coli competent cells and transformation
Analysis of transformation efficiency using non-recombinant plasmid
Screening of recombinants by a-complementation
Purification of PCR products
Transformant confirmation by colony PCR using universal primers
Isolation and restriction of recombinant/non-recombinant plasmid DNAs
Analysis of 16S rDNA gene
Phylogenetic analysis
Genbank submission and accession numbers
Phylogeny
Functional Approach for Metagenomic Library Construction
Restriction digestion of mgDNA
Transformation of E. coli BL21 competent cells
Functional screening of metagenomic libraries for the lipase gene
Isolation and restriction of recombinant plasmid
Sequencing of cloned DNA
Sequence analysis and phylogenetic tree construction
Analysis of the cloned lipase gene
Phylogeny of recombinant protein
Amino acid sequence of recombinant protein
Multiple sequence alignment
Storage of recombinant plasmid
Overexpression of Recombinant Protein
Purification of expressed protein
Determination of active lipase using zymographic study
Determination of specific activity, % yield and protein fold purification
Homology model and validation for protein structure prediction
Biochemical Characterization of Purified Lipase
Dose dependent enzyme activity
Substrate specificity and effect of substrate concentration
Enzyme Kinetics
Effect temperature and pH
Effect of surfactants
Effect of other factors
Genomic Study of Culture Dependent Bacteria
Isolation and screening for lipase producing culturable bacteria
Pure culture of lipase producing bacterial isolates
Inoculum preparation
Zone of hydrolysis
Growth kinetics and lipase production
Thermal stability
Evaluation of crude enzyme for detergent formulation
Stability of culture supernatant under storage
Taxonomic identification of lipase producing bacteria
Biochemical characterization
Extracellular enzyme activity
Spectrophotometric growth determination
Genomic Study of Culturable Bacteria
Genomic DNA extraction of lipase producing culturable bacteria
Molecular genetic assessment of lipase producing bacteria
Phylogenetic analysis
Optimization of culture condition for lipase production
Effect of carbon and nitrogen sources for the growth of culturable bacterial
Time course with lipase production
Effect of other factors
Microbial Assay of Culture Supernatant Containing Crude Lipase
Determination of antibacterial activity
Determination of antifungal activity
Critical Observations
Biography
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.
