Enzymes in Human and Animal Nutrition

Principles and Perspectives

Carlos Simões Nunes, Vikas Kumar

• Publisher: Academic Press
• Year: 2018
• Print ISBN: 9780128054192
• eBook ISBN: 9780128094266
• Topic: Technology & Engineering
• Subtopic: Zoology
• Index: Technology & Engineering

Enzymes in Human and Animal Nutrition is a detailed reference on enzymes covering detailed information on all relevant aspects fundamental for final use of enzymes in human and animal nutrition. Topics explored include selection, engineering and expression of microbial enzymes, effects of probiotics on enzymes in the digestive tract, potential new sources of enzymes, valorization of plant biomass by food and feed enzymes. Economics and intellectual property issues are also examined.- Examines the role of enzymes in nutrition and in the production of food and animal feed so that food industry and academic researchers can understand applications of enzymes in the health of humans and animals- Begins with a thorough overview of selection, engineering and expression of microbial enzymes- Examines extremophile organisms as a potential new source of enzymes- Includes discussion of analytics, economics and intellectual property to increase applicability of the rest of the book outside of the lab

Chapter 1

Selection, engineering, and expression of microbial enzymes

Patrícia Poeta^1,2, Albino A. Dias¹, Gilberto Igrejas^1,2, Vanessa Silva¹, Rui Bezerra¹ and Carlos Simões Nunes³, ¹University of Trás-os-Montes and Alto Douro, Vila Real, Portugal, ²Nova University of Lisbon, Caparica, Portugal, ³CSN Consulting, Versailles, France

Abstract

Native and recombinant enzymes are tools in major sectors of the biopharmaceutical, agricultural, and chemical industries, acting in very diverse areas of medicine, diagnostics, food, feed, detergents, textiles, leather, paper, pulp, and plastics. Nowadays, microorganisms are the main source of commercial enzymes and are continuously optimized by selection, recombination, expression, and production of hydrolases for practical use. Biotechnology (genetic engineering) has the more accurate available methods for the optimization of enzyme production, starting from the transfer of the encoding genetic sequence from a plant, animal, or microorganism to the expressing microbe, yeast, or fungi.

The safety of the source organism is the fundamental element to be taken into account in the evaluation of a target as an enzymatic source. Success with DNA polymerase has allowed an extensive increase in biotechnological utilization of enzymes from archaea (extremozymes). The diversity of proteins from extremophiles allows the expansion of the ranges of optimal enzyme general hydrolytic performance as well as enables enzymatic biocatalysis under unfavorable conditions.

Priorities in the screening and development of improved and/or new enzymes are detergents, drugs, bioremediation, waste treatment, and valorization of nonedible plant biomass. The use of enzymes in “white biotechnology” is expected to grow to over 8 million metric tons in 2018. The initial process of enzyme discovery and optimization is still a limiting factor in the adoption of new biobased industrial processes. There is therefore an increasing opportunity to promote newly discovered archaeal enzymes for sustainable manufacturing.

Techniques to achieve such goals are metagenomic and metatranscriptomics-based approaches, high-throughput screening, genetic engineering, and ideal expression organisms.

Keywords

Enzymes; extremozymes; genomic; screening; recombinant; expression; bacteria; yeast; fungi; archaea; metagenomics

1.1 Introduction

Enzymes are biological molecules that catalyze the essential chemical reactions that maintain life. Enzymes are synthesized by all living organisms. Native and recombinant enzymes are tools in major sectors of the biopharmaceutical, agricultural, and chemical industries, acting in very diverse areas of medicine, diagnostics, food, feed, detergents, textiles, leather, paper, pulp, and plastics. Enzymes extracted from edible plants and animal tissues as well as those produced by microorganisms (bacteria, archaea, yeasts, and fungi) have been used in food production for centuries. For example, rennet, which is a mixture of natural enzymes obtained from calf stomach, has been used for a long time in the production of cheese, and enzymes produced by yeasts are utilized for fermentation of grape juice in wine production and of barley in that of beer.

Isolation of enzymes from living cells started in the 20th century, driving their large-scale utilization in the food industry. Nowadays, microorganisms are the main source of commercial practical enzymes. Enzyme sealers are continuously optimizing selection, recombination, expression and production of enzymes for industrial use (Fig. 1.1).

Figure 1.1 Screening steps for the discovery of new proteins (enzymes).

Biotechnology (genetic engineering) has the more accurate available methods for the optimization of enzyme production, starting from the transfer of the encoding genetic sequence from a plant, animal or microorganism to the expressing microbe, yeast, or fungi. It should be underlined that, in such conditions, the produced enzyme is not genetically modified but only produced by a genetically modified organism.

The global industrial enzymes market is very competitive with Novozymes being the largest player in the industry, followed by DSM and DuPont, among others. The companies mainly compete on the basis of product quality, performance, use of intellectual property rights, and the ability to innovate, among other factors. North America and Europe are the largest consumers of industrial enzymes although the Asia Pacific region is undergoing a rapid increase in enzyme demand in China, Japan, and India, reflecting the size and strength of these countries’ economies [1].

1.2 Principal Applications of Microbial Industrial Enzymes

The development of performant and economically affordable new enzymes is the subject of permanent research efforts. It is obvious that progress in screening, genetic engineering, and expression of recombinant enzymes is fundamental for the production of new enzyme products.

Enzyme applications in medicine are growing rapidly, being already extensively used in therapeutics in health issues associated with enzymatic deficiency and digestive disorders, as well as in diagnostic procedures such as ELISA and diabetes [2]. At present, most prominent medical uses of microbial enzymes are removal of dead skin, and burns by proteolytic enzymes, and clot busting by fibrinolytic enzymes. Several new enzymes for medicinal applications are expected to be developed in the coming years based on the processes in the screening and expression techniques.

In the food industry, enzymes are used not only to improve food production but also to improve food components, such as aroma, flavor, texture, color, nutritive value, and appearance. For instance, baking enzymes are used for providing enhancement of organoleptic characteristics of more or less all types of bakery products. The baking enzymes industry is expected to reach $700 million by 2019 growing at a rate of more than 8% from 2013 to 2019. Enzymes (proteases, lipases, esterases, lactase, aminopeptidase, lysozyme, lactoperoxidase, transglutaminase, catalase, etc.) in dairy industries are parts of the tools required for the quality of the outcome products. This is also the case for alcoholic and nonalcoholic beverages. The deep understanding of the use of microbial enzymes in food industries has allowed the development of better markets of more safe products of higher quality.

The use of enzymes in animal diets exploded in the 1990s. Feed enzymes are gaining importance as they can increase the digestibility of nutrients, improve feed utilization by animals, and reduce the footprint of intensive husbandry [3]. The global market for feed enzymes is expected to reach almost $1.3 billion by 2020. Feed enzymes essentially used for monogastric animals (swine and poultry) are phytases, proteases, α-galactosidases, glucanases, xylanases, α-amylases, and polygalacturonases [4].

In vitro enzyme-catalyzed synthesis of polymers is an environmentally safe process that has several advantages over conventional chemical methods [5]. Biopolymers are environmentally friendly materials as these are synthesized from renewable carbon sources via biological processes, degrade biologically after use, and return to the natural environment as renewable resources, such as CO₂ and biomass [6]. Biopolymers, such as polyesters, polycarbonates, and polyphosphates are used in various biomedical applications, e.g., orthopedic devices, tissue engineering, adhesion barriers, control drug delivery, etc. [7].

With increasing awareness of sustainability issues, uses of microbial enzymes in the paper and pulp industry have grown steadily to reduce the adverse effects on ecosystems. Enzymes are also used to enhance deinking, and bleaching in the paper and pulp industry, and waste treatment by increasing biological oxygen demand and chemical oxygen demand [8].

The leather industry discharges and waste cause severe health hazards and environmental problems [9]. The biodegradable enzymes are efficient alternatives to improve the quality of leather and help to reduce waste [4].

The textile industry is responsible for enormous production of waste from production facilities, bleaching chemicals, and dye, and is one of the largest contributors to environmental pollution [10]. In such industrial activities, enzymes are used to allow the development of environmentally friendly technologies in fiber processing and strategies to improve the final product quality [11].

The applications of enzymes in cosmetics has been continuously increased. Enzymes are used as free radical scavengers in sunscreen cream, toothpaste, mouthwashes, hair waving and dyeing, etc. [12].

Detergents, which are the prime industrial application of enzymes today amounting to about 30% of the total sales of enzymes, are expected to grow fast and have contributed significantly to the growth and development of industrial enzymes. Enzymes in detergent products are used to remove several compounds such as protein, starch, oil, and fat-based stains. Detergents are used in miscellaneous applications such as dishwashing, laundering, domestic, industrial, and institutional cleaning [13].

In the past, waste was traditionally disposed of by digging a hole and filling it with the waste material, but this mode of waste disposal is impossible to sustain. Classically, waste was and is still treated by high-temperature incineration and/or chemical decomposition (base-catalyzed dechlorination and UV oxidation). These methods are complex, uneconomical, and lack public acceptance. The associated deficiencies in these methods have focused efforts toward harnessing modern-day bioremediation processes as suitable alternatives. The accumulation of biological micropollutants and toxic chemicals can lead to environmental hazards which can be minimized through bioremediation.

Bioremediation is performed by a microorganism or an enzyme-mediated transformation or degradation of contaminants into nonhazardous or less-hazardous substances. Bioremediation can be effective only where environmental conditions permit microbial growth and activity. Its application often involves the manipulation of environmental parameters to allow microbial growth and degradation to proceed at a faster rate, knowing that the process of bioremediation is very slow. Most bioremediation systems operate under aerobic conditions, but anaerobic environments may also permit microbial degradation of recalcitrant molecules. Both bacteria and fungi rely on the participation of different intracellular and extracellular enzymes, respectively, for the remediation of recalcitrant organopollutants. The use of culture-independent molecular techniques has certainly facilitated the understanding of bacterial community dynamics, and assembly, and has helped to provide understanding of the specifics of bioremediation, which has resulted in safer and more reliable techniques.

Laccases (p-diphenol: dioxygen oxidoreductases) constitute a family of multicopper oxidases produced by certain plants, fungi, insects, and bacteria, that catalyze the oxidation of a wide range of reduced phenolics. Laccases are known to occur in multiple isoenzyme forms, each of which is encoded by a separate gene [14] and, in some cases, the genes have been expressed differently depending upon the nature of the inducer [15]. Many microorganisms produce intra- and extracellular laccases capable of catalyzing the oxidation of ortho- and paradiphenols, aminophenols, polyphenols, polyamines, lignins, and aryl diamines, as well as some inorganic ions [16–18]. These enzymes are involved in the depolymerization of lignin, which results in a variety of small phenols. In addition, these compounds are utilized as nutrients for microorganisms or repolymerized to humic materials by laccase. Among the biological agents, laccases represent an interesting group of ubiquitous, oxidoreductase enzymes that show promise of offering great potential for biotechnological and bioremediation applications [19]. The substrate specificity and affinity of laccases can vary with changes in pH. Laccases can be inhibited by various reagents such as halides (excluding iodide), azide, cyanide, and hydroxide. Different laccases appear to have differing tolerances toward inhibition by halides, indicating differential halide accessibility. Laccase production is sensitive to the nitrogen concentration in the culture media. Additionally, laccase activity in fungal cultures can be amplified by the addition of diverse aromatic compounds such as gallic acid, ferulic acid, xylidine, guaiacol, syringaldazine, and veratryl alcohol, which have been extensively used to encourage laccase production.

The use of enzymes for waste management is extensive and a number of enzymes are involved in the degradation of toxic pollutants. Industrial effluents as well as domestic waste contain many chemical commodities, which are hazardous or toxic to the living being and ecosystem. Microbial enzyme(s), alone or in combinations, are used for the treatment of industrial effluents containing phenols, aromatic amines, nitriles, etc., and are becoming important tools for the degradation of several toxic pollutants such as aromatic polycyclic hydrocarbures, heavy metals, pesticides, etc.

Enzyme-based processes for production of fine chemicals are rapidly gaining practical significance owing to more economical high-purity products in an ecoenvironmentally acceptable manner...

Table of contents

• Cover image
• Title page
• Table of Contents
• Copyright
• List of Contributors
• Preface
• Acknowledgments
• Introduction
• Chapter 1. Selection, engineering, and expression of microbial enzymes
• Chapter 2. Intellectual property on selection, expression, and production of enzymes
• Part I: Phytases
• Part II: Depolymerizating Enzymes
• Part III: NSP Enzymes
• Part IV: Proteases
• Part V: Other Enzymes
• Part VI: Enzymes and New or Alternative Food- and Feedstuffs
• Index