Konwar / Sagar Lipase

An Industrial Enzyme Through Metagenomics
1. Auflage 2018
ISBN: 978-1-351-65895-9
Verlag: Taylor & Francis
Format: EPUB
Kopierschutz: 0 - No protection

E-Book, Englisch, 230 Seiten

Microbial lipases are industrially important and have gained their attention due to their stability, selectivity, and broad substrate specificity. Lipases are used as a medicine and also aid in indigestion, heartburn, allergy to gluten in wheat products (celiac disease), Crohn’s disease, and cystic fibrosis. This new volume, Lipase: An Industrial Enzyme Through Metagenomics, considers the industrial demand for new sources of lipases with different catalytic characteristics that stimulate the growth and development isolation of new 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 have been found 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 physicochemical 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. The book also highlights the maximum activity of the enzyme at temperature 37°C and pH 7.5 in the presence of the divalent cations Ca2+, Mn2+, Zn2+ and Fe2+. CTAB, gum arabic, NaCl and organic solvents like ethanol, 1-propanol, acetone, acetonitrile, glycerol and DMSO.

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. The book also highlights the antifungal and antibacterial activity of the lipase enzyme.

Autoren/Hrsg.

Konwar, B.K.

Sagar, Kalpana

Fachgebiete

• Medizin | Veterinärmedizin Medizin | Public Health | Pharmazie | Zahnmedizin Medizinische Fachgebiete Pharmakologie, Toxikologie
• Medizin | Veterinärmedizin Medizin | Public Health | Pharmazie | Zahnmedizin Vorklinische Medizin: Grundlagenfächer Biochemie (med.)
• Naturwissenschaften Biowissenschaften Molekularbiologie

Inhaltsverzeichnis

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

Über Autor(innen)

B. K. Konwar, PhD, is currently Professor in the Department of Molecular Biology and Biotechechnology at Tezpur University (Central), Assam, India. He obtained his MSc agricultural degree in plant breeding and genetics from Assam Agricultural University, Jorhat, Assam, India. He has worked as a lecturer, assistant professor, and associate professor at the university. His PhD in plant biotechnology is from the Imperial College of Science, Technology and Medicine, University of London, United Kingdom. He was formerly affiliated with the Tocklai Experimental Station, Tea Research Association, Jorhat, Assam, India, as a Senior Scientist (Botany and Biotechnology). Other appointments include Professor and Department Head, Molecular Biology and Biotechnology at Tezpur University (Central), Napaam, Tezpur, Assam, India; Head of the Centre for Petroleum Biotechnology, DBT (DST, Govt of India; Dean, School of Science and Technology, Tezpur University; and Vice-Chancellor, Nagaland University (Central), HQ: Lumami (campuses: Meriema, Medziphema, Dimapur), Nagaland, India.

Dr. Konwar has carried out 12 research projects of national importance as the principal investigator and has supervised over 40 MSc students who carried out research projects at Assam Agricultural University and Tezpur University. He and his research group so far deposited 11 gene (DNA) sequences in national/international gene banks and have published 139 research papers in reputed and impact factor bearing (1-6) international/national journals, along with many papers in seminars and onference proceedings in India and abroad. He published more than 130 popular science, environment, biotechnology, history, national integration, higher education, research needs, and other articles in various Assamese magazines and newspapers, as well as more than 30 scientific articles in English magazines in addition to several books, booklets, and book chapters.

Kalpana Sagar, PhD, is currently a Research Associate at Delhi University. She earned her PhD in molecular biology and biotechnology from Tezpur University under the supervision of Prof. B. K. Konwar.