Matti Leisola, Jouni Jokela, Ossi Pastinen, Ossi Turunen



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6.54.2.10 INDUSTRIAL USE OF ENZYMES


Matti Leisola, Jouni Jokela, Ossi Pastinen, Ossi Turunen
Laboratory of Bioprocess Engineering, Helsinki University of Technology, Finland, and
Hans Schoemaker, DSM Research, MD Geleen, The Netherlands


Keywords:

Industrial enzymes, speciality enzymes, protein engineering, enzyme technology, enzyme production, biocatalysis, fine chemicals




Contents

1. Historical background

2. Enzyme classification

3. Enzyme production

3.1. Microbial production strains

3.2. Enzyme production by microbial fermentation

4. Protein engineering

5. Enzyme technology



6. Large scale enzyme applications

    1. Detergents

    2. Starch

    3. Drinks

    4. Textiles

    5. Animal feed

    6. Baking

    7. Pulp and paper

    8. Leather

  1. Speciality enzymes

    1. Enzymes in analytics

    2. Enzymes in personal care products

    3. Enzymes in DNA-technology

  2. Enzymes in fine chemical production

    1. Chirally pure amino acids and aspartame

    2. Rare sugars

    3. Semi synthetic penicillins

    4. Lipase based reactions

    5. Asymmetric synthesis

    6. Enzymatic oligosaccharide synthesis

9. Future trends in industrial enzymology

Glossary


Alkaline phosphatase: An enzyme that degrades ester bonds in alkaline conditions.

Amino acid amidase: An enzyme that is used in manufacturing optically pure amino acids. It hydrolyses an amide bond in natural amino acid amides.

Amylase: A group of enzymes that hydrolyse chemical bonds between glucose molecules present in starch. This group includes alpha-, beta- and glucoamylase.

Aspartame: A low calorie high intensive sweetener.

Beta-glucanase: An enzyme that degrades beta-glucan commonly found e.g. in barley.

Biocatalyst: Isolated enzyme or a whole cell (living or dead)

Bromelain: A protein-degrading enzyme from plants.

Catalase: An enzyme that degrades hydrogen peroxide to oxygen and water.

Cellulases: A group of enzymes that synergistically degrade cellulose fibers to glucose.

CLEC: Enzyme crystal that has been made insoluble by chemical cross-linking; a method to immobilise and stabilise enzymes.

Chirally pure: Many organic molecules can have two chemically identical but structurally mirror image forms. Chirally pure means that only one of the forms is present.

Dextran sucrase: An enzyme, present in some lactic acid bacteria, that forms a glucose. polymer and fructose from the disaccharide sucrose.

Dextran: Glucose containing branched polymer used e.g. in blood replacements.

DNA-polymerases: An enzyme that synthesizes DNA polymers.

Fermentor: A biological reactor for cultivation of microorganisms.

Ficin: A protein-degrading enzyme from plants.

Formate dehydrogenase: An enzyme that oxidises formate to carbon dioxide and NAD.

Glucoamylase: An enzyme that splits glucose molecules from starch.

Glucose oxidase: An enzyme that uses oxygen to oxidise glucose to gluconic acid and hydrogen peroxide.

Glycosyltransferases: Catalyse the transfer of monosaccharides from a donor to saccharide acceptors.

GRAS-status: is given to an organism that is Generally Regarded as Safe.

Hydrolases: Enzymes that break chemical bonds by adding water. They can be used to form chemical bonds in the absence of water.

Hydroxynitrile lyase: An enzyme that catalyses the addition of HCN to aldehydes and ketones.

Immunoassay: This is an analytical method in which antibodies are used to detect specific molecules.

Isomerases: Enzymes that catalyse intramolecular reactions.

Laccase: A polyphenol oxidase from fungi. This enzyme can use oxygen to oxidise different types of aromatic molecules and to form lignin type of aromatic polymers from phenolic compounds.

Lactase: his enzymes degrade milk-sugar lactose to glucose and galactose. Lactose intolerant people can consume such milk.
Ligases: Enzymes that synthesize chemical bonds.

Lipoxygenase: A lipid oxidising enzyme extracted usually from soybeans.

Lyases: Enzymes that remove chemical groups from their substrates without addition of water

Nitrile hydratases: Enzymes that catalyse addition of water to nitrales resulting in amide formation

Oxidoreductases: Enzymes that oxidise or reduce chemical compounds.

Papain: A protein degrading enzyme from animal gut.

Penicillin: An antibiotic substance extracted from molds.

Pepsin: An enzyme that degrades proteins and is isolated from animals.

Peroxidase: An oxidative heme-containing enzyme that uses hydrogen peroxide to oxidise aromatic compounds. It is responsible for lignin biosynthesis in plants and initiates lignin biodegradation by certain rot-fungi.

Phytase: A phosphatase enzyme that hydrolyses phosphoester bonds in phytic acid. Is widely used in animal feeds.

Protein engineering: Improvement of enzyme protein by genetic methods.

Rare sugar: A sugar that is rare in nature.

Rennin: An aspartic protease which coagulates milk protein. It is used in cheese manufacturing and isolated from calf stomach or produced by recombinant fungi.

Restriction enzymes: Enzymes that recognise specific 4-8 nucleotides long sequencies from DNA. They are important tools in gene technology.

Transferases:

Trypsin: An enzyme that degrades proteins and is isolated from animals.

Xylanase: A group of enzymes that degrade plant fibers made of xylose-sugars

to xylose monomers.



Xylitol: A tooth-friendly sugar alcohol used in chewing gums.
Summary

Enzymes have been used since the dawn of mankind in cheese manufacturing and indirectly via yeasts and bacteria in food manufacturing. Isolated enzymes were first used in detergents in the year 1914, their protein nature proven in 1926 and their large-scale microbial production started in 1960s. Industrial enzyme business is steadily growing due to improved production technologies, engineered enzyme properties and new application fields. The major part of enzymes is produced by with GRAS-status microorganisms in large biological reactors called fermentors. Usually the production organism and often also the individual enzyme have been genetically engineered for maximal productivity and optimised enzyme properties. Large volume industrial enzymes are usually not purified but sold as concentrated liquids or granulated dry products. Enzymes used in special applications like diagnostics or DNA-technology need to be highly purified. Isolated enzymes have found several applications in fine chemical industry. Enzymes are used in production of chirally pure amino acids and rare sugars. They are also used in production of fructose and penicillin derivatives as well as several other chemicals. Enzymes should be considered as a part of a rapidly growing biocatalyst industry also involving genetically optimised living cells as chemical production factories.



1. Historical background
Most of the reactions in living organisms are catalysed by protein molecules called enzymes. Enzymes can rightly be called the catalytic machinery of living systems. Man has indirectly used enzymes almost since the beginning of human history. Enzymes are responsible for the biocatalytic fermentation of sugar to ethanol by yeasts, a reaction that forms the bases of beer and wine manufacturing. Enzymes oxidise ethanol to acetic acid. This reaction has been used in vinegar production for thousands of years. Similar microbial enzyme reactions of acid forming bacteria and yeasts are responsible for aroma forming activities in bread making and in preserving activities in sauerkraut preparation.
The fermentative activity of microorganisms was discovered only in 18th century and finally proved by the French scientist Louis Pasteur. The term “enzyme” comes from Latin words, which literally mean “in yeast”. This name was given since enzymes where closely associated with yeast activity. The study of enzymes is a fairly recent activity. Scientists who found out that an alcohol precipitate of malt extract contained a thermo labile substance, which converted starch into sugar, made the first clear recognition of enzymes in 1833. They called the substance diastase. We know now that it was an enzyme nowadays called amylase. Sumner finally proved the protein nature of enzymes in 1926 when he was able to crystallize urease enzyme from jack bean.
Probably the first application of cell free enzymes was the use of rennin isolated from calf or lamb stomach in cheese making. Rennin is an aspartic protease (see Mechanisms of Enzyme Action) which coagulates milk protein and has been used for hundreds of years by cheese makers. Röhm in Germany prepared the first commercial enzyme preparation in 1914. This trypsin enzyme isolated from animals degraded proteins and was used as a detergent. It proved to be so powerful compared to traditional washing powders that the original small package size made the German housewives suspicious so that the product had to be reformulated and sold in larger packages. The real breakthrough of enzymes occurred with the introduction of microbial proteases into washing powders. The first commercial bacterial Bacillus protease was marketed in 1959 and became big business when Novozymes in Denmark started to manufacture it and major detergent manufactures started to use it around 1965.
In food industry - in addition to cheese manufacturing - enzymes were used already in 1930 in fruit juice manufacturing. These enzymes clarify the juice. They are called pectinases, which contain numerous different enzyme activities. The major usage of microbial enzymes in food industry started in 1960s in starch industry. The traditional acid hydrolysis of starch was completely replaced by alpha-amylases and glucoamylases, which could convert starch with over 95%, yield to glucose. Starch industry became the second largest user of enzymes after detergent industry.
Presently the industrial enzyme companies sell enzymes for a wide variety of applications. The estimated value of world enzyme market is presently about US $ 1.3 billion and it has been forecasted to grow to almost US $ 2 billion by 2005. Detergents (37%), textiles (12%), starch (11%), baking (8%) and animal feed (6%) are the main industries, which use about 75% of industrially, produced enzymes. Enzymes are also indirectly used in biocatalytic processes involving living or dead and permeabilised microorganisms. This review concentrates on the use of isolated enzyme preparations in large scale and speciality applications and chemical manufacturing. The use of microorganisms as biocatalysts in chemical production is, however, an interesting and growing field. The techniques of genetic, protein and pathway engineering are making chemical production by living cells an interesting green alternative to replace traditional chemical processes.
2. Enzyme classification
Presently more than 2000 different enzyme activities have been isolated and characterized [see Enzymology; Concept and Scope of Enzyme Action]. The sequence information of a growing number of organisms opens the possibility to characterise all the enzymes of an organism on a genomic level. The smallest known organism, Mycoplasma genitalium, contains 470 genes of which 145 are related to gene replication and transcription. Baker’s yeast has 7000 genes coding for about 3000 enzymes. Thousands of different variants of the natural enzymes are known. The number of reported 3-dimensional enzyme structures is rapidly increasing. In the year 2000 the structure of about 1300 different proteins were known. The enzymes are classified into six major categories based on the nature of the chemical reaction they catalyse:


    1. Oxidoreductases catalyse oxidation or reduction of their substrates

    2. Transferases catalyse group transfer

    3. Hydrolases catalyse bond breakage with the addition of water

    4. Lyases remove groups from their substrates

    5. Isomerases catalyse intramolecular rearrangements

    6. Ligases catalyse the joining of two molecules at the expense of chemical energy

Only a limited number of all the known enzymes are commercially available and even smaller amount is used in large quantities. More than 75% of industrial enzymes are hydrolases. Protein-degrading enzymes constitute about 40% of all enzyme sales. Proteinases have found new applications but their use in detergents is the major market. More than fifty commercial industrial enzymes are available and their number increases steadily.






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