Lipase

An Industrial Enzyme Through Metagenomics

B.K. Konwar, Kalpana Sagar

• Publisher: Apple Academic Press
• Year: 2018
• ISBN:
  9781351658959
• Edition: 1
• Topic: Medicine
• Subtopic: Biochemistry in Medicine

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.

CONTENTS

Preface

About the Authors

1. Introduction

1.1 Enzymes

1.2 Etymology and Historical Aspects of Industrial Enzymes

1.3 Industrial Enzymes: Development of Bioindustrial Sector

1.4 Classification of Enzymes

1.5 Lipase

1.6 Classification of Bacterial Lipolytic Enzymes

References

2. Application of Lipases

2.1 Detergent Industry

2.2 Food Processing, Flavour Development and Improving Quality

2.3 Bakery Industry

2.4 Fat and Oleochemical Industry

2.5 Textile Industry

2.6 Cosmetic Industry

2.7 Pulp and Paper Industry

2.8 Oil Biodegradation

2.9 Production of Biodegradable Polymers

2.10 Diagnostic Tool

2.11 Degreasing of Leather

2.12 Waste/Effluent/Sewage Treatment

2.13 Production of Biodiesel

2.14 Tea Processing

2.15 Biosensors

2.16 Environmental Management

2.17 Major Obstacles And Future Prospect of Microbial Lipases

References

3. Metagenomics and Unculturable Bacteria

3.1 Microbial Communities

3.2 Soil as a Microbial Habitat

3.3 Unculturable Microorganisms

3.4 Culturable Microorganisms

3.5 Microbial Species for Beneficial Products and Processes

3.6 Metagenomics

3.7 Soil Metagenomics for Desirable Genes

3.8 Soil Metagenomics: Tool for Novel Compounds

3.9 Sequence-Based Metagenomics

3.10 Function-Driven Metagenomics

3.11 Metagenomic Library Construction and its Analysis

3.12 Approaches to Metagenomics

References

4. Accessing Metagenomics

4.1 Methods

4.2 Expression Vector/Host System

4.3 Functional Screening of Metagenomic Libraries

References

5. Metagenomics for Lipase

5.1 Collection of Environmental Samples

5.2 Extraction, Purification and Quantification of Soil Metagenomic DNA

5.3 Isolation And Purification Of mgDNA from Gram⁺ and Gram^- Bacteria

5.4 Polymerase Chain Reaction

5.5 Restriction Digestion by EcoRI, HindIII and BamHI

5.6 Metagenomic DNA Library Construction

5.7 Sequence-Based Metagenomic Approach

5.8 Construction of 16S rDNA Gene Libraries

5.9 Preparation of E. coli Competent Cells and Transformation

5.10 Analysis of Transformation Efficiency Using Non-Recombinant Plasmid

5.11 Screening of Recombinants by α Complementation

5.12 Purification of PCR Products

5.13 Transformant Confirmation by Colony PCR Using Universal Primers

5.14 Isolation and Restriction of Recombinant/ Nonrecombinant Plasmid DNAs

5.15 Analysis of 16S rDNA Gene

5.16 Phylogenetic Analysis

5.17 Genbank Submission and Accession Numbers

5.18 Phylogeny

References

6. Functional Approach for Metagenomic Library Construction

6.1 Restriction Digestion Of MGDNA

6.2 Transformation of E. coli BL21 Competent Cells

6.3 Functional Screening of Metagenomic Libraries for the Lipase Gene

6.4 Isolation and Restriction Digestion of Recombinant Plasmid

6.5 Sequencing of Cloned DNA

6.6 Sequence Analysis and Phylogenetic Tree Construction

6.7 Analysis of the Cloned Lipase Gene

6.8 Phylogeny of Recombinant Protein

6.9 Amino Acid Sequence of Recombinant Protein

6.10 Multiple Sequence Alignment

6.11 Storage of Recombinant Plasmid

References

7. Overexpression of Recombinant Protein

7.1 Purification of Expressed Protein

7.2 Determination of Active Lipase using Zymographic Study

7.3 Determination of Specific Activity, % Yield and Protein Fold Purification

7.4 Homology Model and Validation for Protein Structure Prediction

References

8. Biochemical Characterization of Purified Lipase

8.1 Dose-Dependent Enzyme Activity

8.2 Substrate Specificity and Effect of Substrate Concentration

8.3 Enzyme Kinetics

8.4 Effect of Temperature and Ph

8.5 Effect of Surfactants

8.6 Effect of Other Factors

References

9. Genomic Study of Culture Dependent Bacteria

9.1 Isolation and Screening for Lipase Producing Culturable Bacteria

9.2 Pure Culture of Lipase-Producing Bacterial Isolates

9.3 Inoculum Preparation

9.4 Zone of Hydrolysis

9.5 Growth Kinetics and Lipase Production

9.6 Thermal Stability

9.7 Evaluation of Crude Enzyme in Detergent Formulation

9.8 Stability of Culture Supernatant Under Storage

9.9 Taxonomic Identification Of Lipase-Producing Bacteria

9.10 Extracellular Enzyme Activity

9.11 Spectrophotometric Growth Determination

References

10. Genomic Study of Culturable Bacteria

10.1 Genomic DNA Extraction of Lipase-Producing Culturable Bacteria

10.2 Agarose Gel Electrophoresis

10.3 Quantification and Purity Determination of the Isolated DNA

10.4 Molecular Genetic Assessment of Lipase-Producing Bacteria

10.5 Phylogenetic Analysis

10.6 Optimization of Culture Condition for Lipase Production

10.7 Effect of Carbon and Nitrogen Sources for the Growth of Culturable Bacteria

10.8 Time Course with Lipase Production

10.9 Effect of Other Factors

References

11. Microbial Assay of Culture Supernatant Containing Crude Lipase

11.1 Determination of Antibacterial Activity

11.2 Determination of Antifungal Activity

References

12 Critical Observations

Index

PREFACE

Metagenomics, a molecular method based on direct isolation and analysis of nucleic acids, proteins and lipids from environmental samples, reveals structural and functional information about microbial communities. Microbial lipases are industrially important and have gained attention due to their stability, selectivity and broad substrate specificity. Bacteria, yeast and fungi are potential sources of lipases. The industrial demand for new sources of lipases, with different catalytic characteristics, stimulates the isolation and selection of new strains. Lipase-producing bacteria have been found in different habitats such as industrial wastes, vegetable oil processing factories, dairy plants and soil contaminated with oil and oil seeds among others. Among the various kinds of bacteria, Bacillus group exhibited interesting properties that make them potential candidates for biotechnological applications. Lipases are classified into eight families, I to VIII, based on their amino acid sequences. It is a widely accepted fact that more than 99% of microorganisms present in soil samples are not readily culturable and therefore not accessible for biotechnology or basic research. The genomes of these uncultured species encode a largely untapped reservoir of novel enzymes and metabolic capabilities. As such more than 99% of microorganisms have unique and potentially very useful abilities such as waste degradation, enzyme production and synthesis of compounds that could find use as drugs or antibiotics.

The present investigation was undertaken to explore the lipase- producing b...

Table of contents

• Cover
• Halftitle Page
• Title Page
• Copyright
• Table of Contents