Biotechnology of Microbial Enzymes

Production, Biocatalysis and Industrial Applications

Goutam Brahmachari, Goutam Brahmachari, Arnold L Demain, Jose L Adrio

• Publisher: Academic Press
• Year: 2016
• eBook ISBN: 9780128037461
• Topic: Biological Sciences
• Subtopic: Microbiology
• Index: Biological Sciences

Biotechnology of Microbial Enzymes: Production, Biocatalysis and Industrial Applications provides a complete survey of the latest innovations on microbial enzymes, highlighting biotechnological advances in their production and purification along with information on successful applications as biocatalysts in several chemical and industrial processes under mild and green conditions. Applications of microbial enzymes in food, feed, and pharmaceutical industries are given particular emphasis. The application of recombinant DNA technology within industrial fermentation and the production of enzymes over the last 20 years have produced a host of useful chemical and biochemical substances. The power of these technologies results in novel transformations, better enzymes, a wide variety of applications, and the unprecedented development of biocatalysts through the ongoing integration of molecular biology methodology, all of which is covered insightfully and in-depth within the book. - Features research on microbial enzymes from basic science through application in multiple industry sectors for a comprehensive approach - Includes information on metabolic pathway engineering, metagenomic screening, microbial genomes, extremophiles, rational design, directed evolution, and more - Provides a holistic approach to the research of microbial enzymes

Chapter 1

Useful Microbial Enzymes—An Introduction

Sergio Sanchez¹ and Arnold L. Demain², ¹Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México (UNAM), Mexico City, Mexico, ²Research Institute for Scientists Emeriti (RISE), Drew University, Madison, NJ, United States

Abstract

Enzymes are important due to their many useful properties. Their development, to a great extent, has been possible due to the availability of microbial sources. Microorganisms are of much attention because they can be produced economically and are amenable to genetic improvement. Microbial enzymes have replaced many plant and animal enzymes. They have found application in many industries including foods, beverages, pharmaceuticals, detergents, textiles, leather, chemicals, biofuels, animal feed, personal care, pulp and paper, diagnostics, and therapy. New molecular methods, including genomics and metagenomics, are being employed for the discovery of new enzymes from microbes. The development of recombinant DNA technology has had a major effect on production levels of enzymes and represent a way to overproduce industrially important microbial, plant, and animal enzymes. It has been estimated that between 50–60% of the world enzyme market is supplied by recombinant enzymes. In addition, directed evolution techniques have allowed design of enzyme specificities and better performance.

Keywords

Microbial enzymes; improvement; discovery; industry

1.1 The Enzymes: A Class of Useful Biochemicals

According to the International Union of Biochemistry (IUB), and based on their nature of reaction, enzymes are divided into six classes: oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases. The use of enzymes in industrial processes has been of crucial importance since they can eliminate the use of high temperatures, extreme pH values, organic solvents, and at the same time, offer high substrate specificity, low toxicity, product purity, reduced environmental impact, and ease of termination of activity. Microorganisms constitute the major source of enzymes as they produce high concentrations of extracellular enzymes. Screening for the best enzymes is simple, allowing the examination of thousands of cultures in a short period of time. Microorganisms used for enzyme production include around 50 GRAS bacteria and fungi. Bacteria are mainly represented by Bacillus subtilis, Bacillus licheniformis, and various Streptomyces species. Fungi are usually represented by Aspergillus, Mucor, and Rhizopus. Microorganisms can be cultured in large quantities in a relatively short period by established methods of fermentation. Microbial enzyme production is economical on a large scale due to inexpensive culture media and short fermentation cycles.

There are more than 3000 different enzymes known but only 5% are commercially used (Binod et al., 2013). Over 500 commercial products are made using enzymes (Johannes and Zhao, 2006). In regard to the total enzyme market, its global figures depend on the consulted source. In one case, the market reached $5.1 billion in 2009 and is predicted to rise 6.45 per annum to grasp $6.9 billion in 2017 (The Freedonia Group, Inc., 2014). In a second report, it was estimated to be $3.3 billion in 2010 and to reach $4.4 billion by 2015 (BBC Research, 2011; Binod et al., 2013). The major technical enzymes are used in bulk form for manufacture of detergents, textiles, leather, pulp, paper, biofuels, and the market for these enzymes reached $1.2 billion in revenues in 2011 and is still on the rise. Other applications include household care, foods, animal feed, fine chemicals, and pharmaceuticals. Enzymes have unique properties such as rapid action, high specificity, biodegradability, high yields, ability to act under mild conditions, and reduction in generation of waste materials. These properties offer flexibility with respect to operating conditions in the reactor.

Sales of feed enzymes are expected to reach $730 million in 2015. They are used to increase nutrient digestibility, and to degrade unacceptable components of feed. Included, mainly for poultry and swine, are proteases, phytases, glucanases, alpha-galactosidases, alpha-amylases, and polygalacturonases. Recent emphasis has been on development of heat-stable enzymes, economical and rapid assays that are more reliable, improvement of activity, and discovery of new nonstarch polysaccharide-degrading enzymes.

Enzymes for food and beverage manufacture are a major part of the industrial enzyme market, reaching sales of almost $1.2 billion in 2011. Lipases constitute a major portion of the usage, targeting fats and oils. In order to maximize flavor and fragrance, control of lipase concentration, pH, temperature, and emulsion content is necessary. Lipases are potentially useful as emulsifiers for foods, pharmaceuticals, and cosmetics. Aspergillus oryzae is used as a cloning host to produce fungal lipases, such as those from Rhizomucor miehi, Thermomyces lanuginosus, and Fusarium oxysporum.

Important detergent additives include proteases, lipases, oxidases, amylases, peroxidases, and cellulases which catalyze the breakdown of chemical bonds upon the addition of water. The useful ones are active at thermophilic temperatures (c. 60^οC) and alkalophilic pH (9–11), and in the presence of components of washing powders.

Over 60% of the worldwide enzyme market is devoted to proteases. These enzymes are involved in the manufacture of foods, pharmaceuticals, leather, detergents, silk, and agrochemicals. Their use in laundry detergents constitute 25% of global enzyme sales. They include (1) the B. licheniformis alkalase Biotex, (2) the first recombinant detergent lipase called Lipolase, made by cloning the lipase from Humicola lanuginose into A. oryzae, (3) the Pseudomonas mendocina lipase (Lumafast), and (4) the Pseudomonas alcaligenes lipase (Lipomax).

Natural enzymes are often unsuitable for use as industrial biocatalysis and need modifications for industrial use. The production strains are usually modified by genetic manipulation to gain improved properties, including high production levels. With the introduction of recombinant DNA technology, it has been possible to clone genes encoding enzymes from microbes and expressing them at levels tens and hundreds times higher than those produced by unmodified microorganisms. Because of this, the enzyme industry rapidly accepted the technology and moved enzyme production from strains not suited for industry into industrial strains (Galante and Formantici, 2003). Genomics, metagenomics, proteomics, and recombinant DNA technology are employed to facilitate the discovery of new enzymes from microbes in nature, and to create or evolve improved enzymes. A number of new and useful enzymes have been obtained by metagenomics (Ferrer et al., 2007).

Directed evolution of proteins includes DNA shuffling, whole genome shuffling, heteroduplex, random chimeragenesis of transient templates, assembly of designed oligonucleotides, mutagenic and unidirectional reassembly, exon shuffling, Y-ligation-based block shuffling, nonhomologous recombination, and the combination of rational design with directed evolution (Yuan et al., 2005; Siehl et al., 2005; Bershstein and Tewfic, 2008; Reetz, 2009). Directed evolution has yielded increased activity, stability, solubility, and specificity of enzymes. For example, it increased the activity of glyphosate-N-acetyltransferase 10,000-fold and, at the same time, its thermostability by 5-fold.

1.2 Microbial Enzymes for Industry

According to their applications, microbial enzymes have been applied to make numerous biotechnology products and in processes commonly encountered in the production of laundry, food and beverages, paper and textile industries, clothing, etc. The use of enzymes as detergent additives represents a major application of industrial enzymes. The detergent market for enzymes has grown strongly in the last 25 years. In the year 2003, it was around $0.79 billion, with proteases as the major detergent enzyme product. The detergent industry uses more than 25% of the total enzyme production.

Proteases, lipases, amylases, oxidases, peroxidases, and cellulases are added to the detergents where they catalyze the breakdown of chemical bonds on the addition of water. For this purpose, they must be active under thermophilic (60^oC) and alkalophilic (pH 9–11) conditions, as well as in the presence of various components of washing powders (Stoner et al., 2005). The market share of detergent proteases is estimated to be at 72% of the global detergent enzyme market (Maurer, 2015). The first detergent containing a bacterial protease was introduced in 1956, and in 1960, Novo Industry A/S introduced alcalase produced by B. licheniformis (“Biotex”). Cellulase from Bacillus sp. KSM-635 has been used in detergents because of its alkaline pH optimum and insensitivity to components in laundry detergents (Ozaki et al., 1990). Later, Novozymes launched a detergent using a cellulase complex isolated from Humicolla insolence (Celluzyme). Certain microorganisms called extremophiles grow under extreme conditions such as 100^oC, 4^oC, 250 atm., pH 10, or 5% NaCl. Their enzymes, which act under such extreme conditions, are known as extremozymes. One such enzyme, called Cellulase 103, was isolated from an alkaliphile and commercialized because of its ability to break down microscopic fuzz of cellulose fibers which trapped dirt on the surface of cotton textiles. It has been used for over 10 years in detergents to return the “newness” of cotton clothes, even after many washings. As early as the mid-1990s, virtually all laundry detergents contained genetically-engineered enzymes (Stoner et al., 2005). Over 31% of the enzymes used in detergents are recombinant products (McAuliffe et al., 2007).

The major application of proteases in the dairy industry is for the manufacturing of cheese. Four recombinant proteases have been approved by FDA for cheese production. Calf rennin had been preferred in cheese-making due to its high specificity, but microbial proteases produced by GRAS microorganisms, such as Mucor miehei, Mucor pusilis, B. subtilis, and Endothia parasitica, are gradually replacing it. The primary function of these enzymes in cheese-making is to hydrolyze the specific peptide bond (Phe105-Met106) that generates para-k-casein and macropeptides. Nearly 40,000 U/g bran of milk-clotting activity are produced by A. oryzae at 120 h by solid state fermentation (Vishwanatha et al., 2009). For many years, proteases have also been used for production of low allergenic milk proteins used as ingredients in baby milk formulas (Gupta et al., 2002).

Proteases can also be used for synthesis of peptides in organic solvents. Thermolysin is used in this way to make aspartame (Oyama et al., 1981). Aspartame sales reached $1.5 billion in 2003 (Baez-Viveros et al., 2004). In 2004, the production of aspartame amounted to 14,000 metric tons. The global sugar substitute market is the fastest growing sector of the sweetener market.

Fungal alpha-amylase, glucoamylase, and bacterial glucose isomerase are used to produce “high fructose corn syrup” from starch in a business amounting to $1 billion per year. Fructose syrups are also made from glucose by “glucose isomerase” (actually xylose isomerase) at a level of 15 million tons per year. The food industry also uses invertase from Kluyveromyces fragilis, Saccharomyces cerevisiae, and Saccharomyces carlsbergensis for manufacture of candy and jam. Beta-galactosidase (lactase), produced by Kluyveromyces lactis, K. fragilis, or Candida pseudotropicalis, is used to hydrolyze lactose in milk or whey and alpha-galactosidase from S. carlsbergensis is employed in the crystallization of beet sugar.

Microbial lipases catalyze the hydrolysis of triaclyglycerol to glycerol and fatty acids. They are commonly used in the production of a variety of products ranging from fruit juices, baked foods, pharmaceuticals, and vegetable fermentations to dairy enrichment. Fats, oils, and related compounds are the main targets of lipases in food technology. Accurate control of lipase concentration, pH, temperature, and emulsion content is required to maximize the production of flavor and fragrance. The lipase mediation of carbohydrate esters of fatty acids offers a potential market for use as emulsifiers in foods, pharmaceuticals, and cosmetics. Another application of increasing importance is the use of lipases in removing pitch (hydrophobic components of wood, mainly triglycerides and waxes). A lipase from Candida rugosa is used by Nippon Paper Industries to remove up to 90% of these compounds (Jaeger and Reetz, 1998). The use of enzymes as alternatives to chemicals in leather processing has proved successful in improving leather quality and in reducing environmental pollution. Alkaline lipases from Bacillus strains, which grow under highly alkaline conditions in combination with other alkaline or neutral proteases, are currently being used in this ...

Table of contents

• Cover image
• Title page
• Table of Contents
• Copyright
• Dedication
• List of Contributors
• Preface
• Chapter 1. Useful Microbial Enzymes—An Introduction
• Chapter 2. Production, Purification, and Application of Microbial Enzymes
• Chapter 3. Solid State Fermentation for Production of Microbial Cellulases
• Chapter 4. Hyperthermophilic Subtilisin-Like Proteases From Thermococcus kodakarensis
• Chapter 5. Enzymes from Basidiomycetes—Peculiar and Efficient Tools for Biotechnology
• Chapter 6. Microbial Production and Molecular Engineering of Industrial Enzymes: Challenges and Strategies
• Chapter 7. Metagenomics and the Search for Industrial Enzymes
• Chapter 8. The Pocket Manual of Directed Evolution: Tips and Tricks
• Chapter 9. Insights into the Structure and Molecular Mechanisms of β-Lactam Synthesizing Enzymes in Fungi
• Chapter 10. The Cellulosome: A Supramolecular Assembly of Microbial Biomass-Degrading Enzymes
• Chapter 11. Microbial Enzymes of Use in Industry
• Chapter 12. Significance of Microbial Glucokinases
• Chapter 13. Lipase-Catalyzed Organic Transformations: A Recent Update
• Chapter 14. Enzymatic Biocatalysis in Chemical Transformations: A Promising and Emerging Field in Green Chemistry Practice
• Chapter 15. Microbial Enzymes for Glycoside Synthesis: Development of Sucrose Phosphorylase as a Test Case
• Chapter 16. Industrial Applications of Multistep Enzyme Reactions
• Chapter 17. Biocatalysis for Industrial Production of Active Pharmaceutical Ingredients (APIs)
• Chapter 18. Agro-Industrial Residues and Microbial Enzymes: An Overview on the Eco-Friendly Bioconversion into High Value-Added Products
• Chapter 19. Microbial Enzymes for the Food Industry
• Chapter 20. Productive Chain of Biofuels and Industrial Biocatalysis: Two Important Opportunities for Brazilian Sustainable Development
• Index