Lipase: An Industrial Enzyme Through Metagenomics, 1st Edition (Hardback) book cover

Lipase

An Industrial Enzyme Through Metagenomics, 1st Edition

By B.K. Konwar, Kalpana Sagar

Apple Academic Press

214 pages | 14 Color Illus. | 63 B/W Illus.

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Hardback: 9781771886185
pub: 2018-05-24
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Description

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.

Table of Contents

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 α-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

About the Authors

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.

Subject Categories

BISAC Subject Codes/Headings:
MED008000
MEDICAL / Biochemistry
MED071000
MEDICAL / Pharmacology
SCI049000
SCIENCE / Life Sciences / Biology / Molecular Biology