Enzymology Lecture 7

Introduction Enzymes found in nature have been used since ancient times in the production of food products, such as cheese, sourdough, beer, wine and vinegar, and in the manufacture of commodities such as leather, indigo and linen. All of these processes relied on either enzymes produced by spontaneously growing microorganisms or enzymes present in added preparations such as calves’ rumen or papaya fruit. The development of fermentation processes during the later part of the last century, aimed specifically at the production of enzymes by use of selected production strains, made it possible to manufacture enzymes as purified, well- characterized preparations even on a large scale. This development allowed the introduction of enzymes into true industrial products and processes, for example, within the detergent, textile and starch industries. The use of recombinant gene technology has further improved manufacturing processes and enabled the commercialization of enzymes that could previously not be produced. Furthermore, the latest developments within modern biotechnology, introducing protein engineering and directed evolution, have further revolutionized the development of industrial enzyme.

The development of enzymes

Usage in the industry

The majority of currently used industrial enzymes are hydrolytic in action, being used for the degradation of various natural substances. Proteases remain the dominant enzyme type, because of their extensive use in the detergent and dairy industries. Various carbohydrases, primarily amylases and cellulases, used in industries such as the starch, textile, detergent and baking industries, represent the second largest group. The technical industries, dominated by the detergent, starch, textile and fuel alcohol industries, account for the major consumption of industrial enzymes. Overall, the estimated value of the worldwide use of industrial enzymes has grown from $1 billion in 1995 to $1.5 billion in The fastest growth over the past decade has been seen in the baking and animal feed industries.

Application of Enzymes in Various industries Excel sheet

Use in the detergent Industry Their use as detergent additives still represents the largest application of industrial enzymes, both in terms of volume and value. The major component is proteases, but other and very different hydrolases are introduced to provide various benefits, such as the efficient removal of specific stains. To save energy, the temperature used in household laundering and automated dishwashers has been reduced in recent years. This often results in problems with efficient cleaning and stain removal that enzyme technology can help overcome.

Recent examples of second-generation detergent enzymes include the development of novel amylases that have enhanced activity at lower temperatures and alkaline pH, while maintaining the necessary stability under detergent conditions. These enzymes were developed by the combined use of microbial screening and rational protein engineering. Proteases displaying activity at low temperatures have been isolated from nature, but have also been evolved in the laboratory. Furthermore, from a starting material of 26 subtilisin proteases Ness and coworkers utilized one round of DNA shuffling to isolate new proteases with various improved properties. The improvements included characteristics very relevant for detergent proteases (i.e. improved activity and stability at alkaline pH). The most recent introduction of a new enzyme class into a detergent has been the addition of a mannanase — the result of a joint development between Procter and Gamble and Novozymes. This enzyme helps remove various food stains containing guar gum, a commonly used stabilizer and thickening agent in food products.

Enzymes for starch conversion The enzymatic conversion of starch to high fructose corn syrup is a well-established process and provides a beautiful example of a bioprocess in which the consecutive use of several enzymes is necessary. The first step in the process is the conversion of starch to oligomaltodextrins by the action of α -amylase. The concomitant injection of steam puts extreme demands on the thermostability of the enzyme. Using traditional α -amylases, the pH has to be adjusted to an undesirable high level and calcium must be added to stabilize the enzyme. New α -amylases with optimized properties, such as enhanced thermal stability, acid tolerance, and ability to function without the addition of calcium, have recently been developed and offering obvious benefits to the industry. Engineering efforts have also been undertaken to develop improved versions of the enzymes used later in the process (i.e. glucoamylase and glucose isomerase).

Fuel alcohol production In the alcohol industry, the use of enzymes for the production of fermentable sugars from starch is also well established. Over the past decade, there has been an increasing interest in fuel alcohol as a result of increased environmental concern, higher crude oil prices. Therefore, intense efforts are currently being undertaken to develop improved enzymes that can enable the utilization of cheaper and partially utilized substrates such as lignocellulose, to make bio-ethanol more competitive with fossil fuels. The cost of enzymes needed to turn lignocellulose into a suitable fermentation feed-stock is a major issue, and current work focuses both on the development of enzymes with increased activity and stability as well as on their efficient production.

Application in textile industry

Application in the feed industry The use of enzymes as feed additives is also well established. For example, xylanases and β -glucanases have been used throughout the past decade in cereal-based feed for monogastric animals which, contrary to ruminants, are unable to fully degrade and utilize plant-based feeds containing high amounts of cellulose and hemicellulose. During recent years focus has been on the utilization of natural phosphorus bound in phytic acid in cereal-based feed for monogastrics. Better utilization of total plant phosphorus, of which 85–90% is bound in phytic acid, is only obtained by adding the enzyme phytase to the feed.

Application in the food industry Much work has been carried out on the application of transglutaminase as a texturing agent in the processing of, for example, sausages, noodles and yoghurt. At present only the transglutaminase from Streptoverticillium sp. is commercially available at a reasonable scale, and work is ongoing to increase the availability of the enzyme by recombinant production in Escherichia coli.

Within the baking industry there is an increasing focus on lipolytic enzymes. Recent findings suggest that (phospho)lipases can be used to substitute or supplement traditional emulsifiers, as the enzymes degrade polar wheat lipids to produce emulsifying lipids in situ. Studies have confirmed previous findings showing that water-binding capacity and retention in the starch and hemicellulose fractions of the bread, being the substrates of α -amylases and xylanases, respectively, to be critical for maintaining softness and elasticity. The recently determined three-dimensional structure of the widely applied amylase for antistaling (Novamyl TM ) provided further insight into the mechanism of enzyme action. This amylase is probably capable of degrading amylopectin to a degree that prevents re-crystallization after gelatinization, without completely degrading the amylopectin network which provides the bread with elasticity.

The use of laccase for clarification of juice (laccases catalyze the cross-linking of polyphenols, resulting in an easy removal of polyphenols by filtration) and for flavor enhancement in beer are recently established applications within the beverage industry.

Enzymes for organic synthesis Chemical synthesis is an area where the use of enzyme catalysis has long been seen as having great promise. At present, however, we are seeing very significant growth in this area and enzyme-based processes are now, finally, being widely introduced for the production of a diversity of different chemicals; one key example is in the production of single-enantiomer intermediates used in the manufacture of drugs and agrochemicals. This market is characterized by a very high degree of fragmentation, as very few enzymes have applicability in a broad range of different processes. Recently introduced enzyme-based processes include the use of lipases for the production of enantiopure alcohols and amides, nitrilases for the production of enantiopure carboxylic acids, and acylases for the production of new semisynthetic penicillins. As many companies are currently at an early stage in the exploitation of enzyme-based catalysis, many new developments are expected in this area over the next few years.