Matti Leisola, Jouni Jokela, Ossi Pastinen, Ossi Turunen


Future trends in industrial enzymology



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  1. Future trends in industrial enzymology

Industrial enzyme market grows steadily. The reason for this lies in improved production efficiency resulting in cheaper enzymes, in new application fields and in new enzymes from screening programmes or in engineered properties of traditional enzymes. New applications are to be expected in the field of textiles, new animal diets like ruminant and fish feed. It can be expected that breakthroughs in pulp and paper will materialise. The use of cellulases to convert waste cellulose into sugars and further to ethanol by fermentative organisms has been a major study topic for years. Increasing environmental pressures and energy prices will make this application a real possibility one day.


Tailoring enzymes for specific applications will be a future trend with continuously improving tools and understanding of structure-function relationships and increased search for enzymes from exotic environments. This means that there will be a specifically tailored xylanase for baking, another for feed and a third one for pulp bleaching.
New technical tools to use enzymes as crystalline catalysts, ability to recycle cofactors, and engineering enzymes to function in various solvents with multiple activities are important technological developments, which will steadily create new applications.
Enzymes should, however, not be considered alone but rather as a part of a biocatalyst technology. Whole cell catalysts, increased ability to engineer metabolic pathways and a combination of specific biocatalytic reactions with organic chemistry form a basis to develop new technologies for chemical production.


Bibliography

Bhat M (2000) Cellulases and related enzymes in biotechnology. Biotechnology Advances 18, 355-383. [This review paper discusses present and possible future trends; xylanases were first suggested for pulp bleaching by VTT-Biotechnology in Finland, Röhm Enzyme is the leader in the field see http://www.roehmenzyme.com/]



Biochimica et Biophysica Acta (2000) 1543, 203-252 [Contains several reviews of engineered enzymes of industrial importance]

Chotani G., Dodge T., Hsu A., Kumar M., LaDuca R., Trimbur D., Weyler W. and Sandford K. (2000) The commercial production of chemicals using pathway engineering. Biochimica et Biophysica Acta 1543, 434-455. [This review article discusses the latest trends in using engineered organisms in chemical production]

Doran P.M. (1999) Bioprocess Engineering Principles. 439 pp. Academic Press. [A textbook, which describes engineering aspects of bioprocesses]

Flickinger M.C. and Drew S.W. (eds., 1999) Encyclopedia of Bioprocess Technology: fermentation, biocatalysis, and bioseparation (5 volumes), John Wiley & Sons [Good resource book on all aspects of modern bioprocess technologies]

Godfrey T. and West S. (eds, 1996) Industrial Enzymology, Macmillan [This is a basic textbook on industrial enzymes, authored by industrial experts; www.novozymes.com is a webpage of world´s largest enzyme company, second largest is Genencor Int. Inc., www.genencor.com];

Palcic M.M. (1999) Biocatalytic synthesis of oligosaccharides. Current Opinion in Biotechnology 10, 616-624 [A good review with several references about the topic]

Pastinen O, Visuri K, Schoemaker H and Leisola M (1999) Novel reactions of xylose isomerase from Streptomyces rubiginosus. Enzyme and Microbial Technology 25, 695-700. [Glucose isomerase is a traditional name; xylose isomerase would be a more correct name although it has recently been shown to catalyse many monosaccharide isomerizations]

Schmid A., Dordick J.S., Hauer B., Kiener A., Wubbolts M. and Witholt B. (2001) Industrial biocatalysis today and tomorrow. Nature 409, 258-268 [A good review article on developments of enzymatic and whole cell biocatalytic applications and trends in chemical industry; http://www.isrs.kagawa-u.ac.jp/ is a page of a recently formed International Society of Rare Sugars]



Visuri, K. (1987) Stable glucose isomerase concentrate and a process for the preparation thereof.

US Patent 4,699,882 [This patent describes the first large scale crystallization process of an intracellular industrial enzyme] and Visuri, K. (1995) Preparation of cross-linked glucose isomerase crystals. US Pat. 5437993. [This patent describes the first preparation of a cross-linked enzyme crystal catalyst]





Figure 1. A typical enzyme production scheme. Large volume industrial enzymes are usually not purified. Their recovery is often finalised by an ultrafiltration step. Speciality enzymes need more purification.



Figure 2. Three-dimensional structure of a Trichoderma xylanase II. This enzyme is used in baking to improve bread quality, in animal feed to improve digestibility of feed, in cellulose pulp bleaching to reduce the use of chlorine chemicals and in fruit juice manufacturing to facilitate juice extraction and clarification. The two active centre glutamates and the one alpha helix are shown in a green colour.


Figure 3. Cross-linked glucose isomerase crystals. The average crystal size of these crystals is 86 m. They can be used in chiral separations and as an immobilized enzyme in a backed-bed or fluidised bed column. Cross-linking makes the enzyme insoluble but it retains its activity as water containing porous material.


Scheme 1. Formation of a racemic amino acid amide (1) synthetically by Strecker reaction and enzymatic resolution of the racemic amide mixture by amidase to form L-amino acid (L-2) and D-amide (D-1) which can be hydrolysed to D-amino acid (D-2).


Scheme 2. L-aspartic acid (3) is formed from fumaric acid by aspartase which catalyses the addition of ammonia to fumaric acid. A protective group is added to form Z-aspartic acic (Z-3) which is combined using a racemic mixture of D/L-phenylalanine methyl ester (rac-4) by thermolysin to give Z-aspartame (Z-5). By removing the protective group by catalytic hydrogenation, aspartame (5) is obtained.


Table 1. Change in enzyme characteristics by protein engineering































Enzyme




Industry




Need
















xylanase




feed




temperature stability













acid activity







pulp and paper




temperature and alkali stability higher pH-optimum
















glucoamylase

starch




higher pH-optimum










glucose isomerase

fructose




substrate specificity

acid stability



thermo stability




























proteinase




detergent




thermostability













alkali stability













oxidative stability



Table 2. Tools in protein engineering


















Target







Method



















protein structure




crystallization













x-ray crystallography










NMR



















modelling and simulation




computational methods
















gene




plasmids

expression systems















targeted mutagenesis

PCR











DNA shuffling










random mutagenesis


Table 3. Large scale enzyme applications




Industry

Effect




Detergent













proteinase




protein degradation







lipase

cellulase






fat removal

color brightening







Textile













cellulase




microfibril removal







laccase




color brightening





Animal feed













xylanase




fiber solubility




phytase




release of phosphate





Starch













amylases




glucose formation

glucose isomerase

fructose formation





Pulp and paper










xylanase




biobleaching





Fruit juice











pectinase

cellulase,

xylanase


juice clarification, juice extraction







Baking













xylanase




dough conditioning




alpha-amylase

glucose oxidase



loaf volume; shelf-life

dough quality







Dairy
















rennin




protein coagulation







Brewing


lactase

glucanase

papain





lactose hydrolysis

filter aid



haze control




Table 4. Major enzymatically measured analytes



Analyte

Enzyme




alcohol

alcohol dehydrogenase

ammonia

L-glutamate dehydrogenase

carbon dioxide

phosphoenolpyruvate carboxylase

cholesterol

cholesterol oxidase

glucose

glucose oxidase

oxalate

oxalate oxidase

urea

urease

uric acid

uricase




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