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Enzyme catalysis and related technologies are used in a variety of fields-say, in bread baking, in beer brewing and alcohol distilling, juice and beverage making, in broiler-type chicken raising. Enzyme catalysis is also indispensable in the textile industry, in cellulose production and in many other areas. The list is open-ended indeed. Therefore effective and low-cost enzymes are a very important part of the job. Our correspondent Yevgeniya Viktorova has interviewed Arkady Sinitsyn, a laboratory head at Lomonosov Moscow State University, and holder of the doctorate in chemistry. His laboratory is involved with physico-chemical conversions of polymers. We follow with a transcript of their conversation.

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State of the world market of technical enzymes.

A three-dimensional structure from endoglucanase III (Penicillium funiculosum).

Now your laboratory is going deep into biocatalysis, and the results you have obtained are getting top marks from experts. In fact, one of the very first publications about the work on cellulose enzymes carried out at the Chair of Chemical Enzymology at Moscow University's Department of Chemistry was published by our magazine (Science in the USSR then) back in 1981.

- The idea was born in the beginning of the 1980s-to use the renewable natural resources (like wood or annual plants) for obtaining a spectrum of products usually made from petroleum and its derivatives. The idea was backed at the government level. Accordingly, research scientists of our department under Ilya Berezin, Corresponding Member of the USSR Academy of Sciences, got down to work on cellulose bioconversions. Our research team - in 1991 it was upgraded to a laboratory status- concentrated on a search for microorganisms producing a corresponding complex of enzymes. We were to identify and pick out the most active strains.

We are going ahead with our work on enzymes breaking up polymers of native origin, such as cellulose, hemicellulose, starch and pectins. Thereby polysaccharides are converted to monosaccharides - for instance cellulose is turned into glucose which, when acted upon by microorganisms, can then be transformed into ethanol, butanol and other alcohols, and also into organic and amino acids, and other products which, in their turn, are converted via chemical or enzyme catalysis. We are dealing with a long chain of conversions, and we should act upon enzyme producers in getting them to yield enzymes or a complex of

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enzymes we need by finding optimal culture media and temperatures, by varying conditions of aeration. We make use of selection and gene engineering, too.

You have as much said that by using enzyme catalysis it is possible to get the same products as those from petroleum. How do we stand today?

- The interest in this idea slackened for a time, but it has become popular again in Europe and the United States. They have come up with tax benefits, and that has promoted a fairly wide use of bioethanol. Industrial countries are seeking to bring down their dependence on the petroleum market.*

But is it realistic to replace petroleum, the "black gold" with alternative products obtained at specialized plants? Can we tackle this job in the near future? To begin with, we should build up a raw material base, and thereto is a good potential for that. For instance, in the United States they get starch from Indian corn, but do not utilize the corncob, stem and shells, which are depositary of nonstarch polysaccharides. In California they don't know how to go about the leftover straw on rice paddies. Canada and Russia have lots of wood chips in cutting areas and elsewhere. However, the enzyme-activated hydrolysis of this waste proceeds sluggishly unless it is pretreated (there are several essentially different pre-treatment techniques): the thing is that crystalline cellulose mixed with lignin (that works like glue) is a very hard staff. As a matter of fact, the slow reaction is ecologically justified because otherwise assisted by their enzymic systems, fungi and mycellia would have "devoured" every kind of vegetation. Mechanical or chemical pre-treatments are quite energy-intensive and thus costly.

What are state-of-the-art technologies today?

- If we take ethanol, the technologies are based on the use of grain raws preheated at temperatures above the melting point of starch; the reaction mixture is supplemented with alpha amylase, an enzyme digesting the starch to oligosaccharides. Thereupon glucoamylase is added, this enzyme turns oligosaccharides to glucose and yeast. A fermentation process sets in.

Industrial enterprises are already set up to make ethanol from plant raw materials. In Western countries physicists, chemists and production engineers are trying to optimize pretreatment technologies, though so far they have not succeeded in developing breakthrough procedures. But certain headway has been made in search of new enzymes. The Iogen Company of Canada has built the first full-scale plant in its home country to produce ethanol from straw subjected to heat shock. Next, a complex of enzymes is added, an optimally balanced one for the digestion of a set of polysaccharides present in the reaction mixture. Before getting to the production of biofuel so much popular today, the company began producing enzymatic preparations for treating textiles, denim in particular.

It is now a standard procedure to obtain low-cost glucose in an enzyme-catalyzed reaction and use this glucose for getting ethanol with the aid of free or immobilized cells of yeast and bacteria. Glucose is utilized in the production of rubber, while ethanol, for the production of ethylene by means of hydrogenic catalysis. But may 1 repeat: pretreatment of the stock raw material still remains a sticking point.

We can get cellulose raws that need no pretreatment, yes. These are short-fiber wastes of the wood-pulp and


See: A. Yanenko, "Priorities of Industrial Biotechnology", Science in Russia, No. 5, 2006. - Ed.

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paper industry no good for paper. And this is also waste paper. In fact, paper is a ready-made raw material for enzymic hydrolysis (lignin eliminated). But at the present stage it is best to use waste paper for its designated purpose. One ton of dry cellulose costs about 500 USD. But it is not ruled out either that this material will be in demand for the production of biofuel, what with the ongoing rise in petroleum prices.

Does the idea of the byconversion of raw material find a response here in Russia?

- This technology is practicable with us in the production of various monomers and amino acids, but not of motor fuel, though. Many experts agree that the stumbling block is in the high excise-duty on ethanol alcohol that doubles its price: if sold at cost price, ethanol would be much in demand among car drivers.

Why not produce bioethanoi for export?

- A few countries, Kazakhstan among them, want to export this product. Kazakhstan has built a plant producing as much as 30,000 hecaliters of bioethanoi from grain. By long-standing tradition ethanol alcohol is produced in our country from grain. We have but 100 distilleries, true, but their number could be increased- we have grain well enough, and its output could be boosted further. Some wineries-distilleries, too-run shops for the production of enzymes needed for starch hydrolysis. Yet no more than 25 percent of enterprises consume this produce because of the influx of imported enzymatic preparations. Russian scientists are developing new technologies and selecting more productive strains of microorganisms.

Recently we had a lucky break indeed: we got an exceptionally active strain producing a set of cellulolytic enzymes - its efficiency is the world's highest today. Just 1 mg of its protein processes a top amount of cellulose per time unit. Our next objective was to elucidate the cause of the singular characteristics of such microorganisms and enhance it via genetic manipulations.

Would you spell it out?

- The Russian Ministry of Education and Science launched a biennial project that came to a close at the end of 2006 and brought an array of new technical enzymes above the world standard.

What do you mean by "technical enzymes"?

- These are effective low-cost enzymes much in use in many fields of the economy. Hundreds and thousands of tons of them are produced for the food, alcoholic beverage, and meat and dairy industries, for bakehouses, breweries and for the production of juices and nonalcoholic beverages as well as for the textile and wood-pulp and paper industries. Proteases help clean soiled wool fabrics, and cellulases give a "stone-washed" look to denim.

Such technologies are based on high-performance strains (as a rule, these are microscopic fungi or else bacteria producing a set of extracellular enzymes- cellulases, hemicellulases, proteases, pectinases, and so forth). A genetically modified substance usually produces one particular enzyme. That's the point. Our project did not pursue this objective-we were selecting strains in the canonical mode, without gene engineering. This is an important consideration for food industry technologies.

We were cooperating with the RAS Institute of Biochemistry and Physiology of Microorganisms named after G. Skryabin (based in the town of Pushchino south of Moscow) and with the Research Institute of Food Biotechnology set up under the auspices of the Russian Academy of Agricultural Sciences (RAAS) and involved with high-efficiency mutant strains of microorganisms. Taking part in the project was also the A.N. Bach Institute of Biochemistry-it provided a base for our Joint Pool.

This is a joint research pool, isn't it?

- Yes. Thanks to the concerted efforts of the participants in the project we have established facilities for microbial synthesis and enzymological studies.

The project launched by the Ministry of Education and Science has given you technologies for obtaining enzymic complexes. What are their application domains?

- We have come up with six groups of enzymes. The first one includes heat resistant alpha amylases designed for starch fluidification at the initial stage of ethanol production from grain. The second group combines glucoamylases, and catalysts for glucose derived from starch and maltodextrins. The third and fourth groups comprise cellulases and associated enzymes for feed additives at poultry farms. The point is that in our northern land the rye and wheat fed to the

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Minireactors for biopolishing and depigmentation of cotton fabric at laboratory conditions. A - denim (elimination of indigo), B - cotton fabric (biopolishing).

Biopolishing of cotton fabric: A - prior to cellulase treatment, B - after such treatment.

fowl contain a highly viscous nonstarch polysaccaride, xylan, which is but poorly assimilated if unaided by enzymes, xylanases in particular. These hydrolyze xylan. Associated enzymes do the same thing to other nonstarch polysaccarides of grain. Today as good as all feed is put out with enzymatic additives.

The fifth group inclines alkaline bacterial proteases added to detergents. The sixth offers fungal acid proteases meant for the food industry, namely for getting readily assimilated protease hydrolyzates from yeast or from animal protein. This product is good for athletes, for one.

As you see, we are working out different technologies. Those related to hydrolysis of nonstarch polysaccharides have been adopted in industries producing food additives, foodstuffs, beer and fabrics, too.

One of the enzymatic complexes obtained by your research group is of interest for the production of bioethanol. How?

- Celulases originally designated as feed additives for animal husbandry have proved to be quite valuable in quite another sphere. This is a good example of universality proper to enzymes. We are pushing ahead in studying the characteristics of the unique microscopic fungus producing this complex. The high performance of its commercial strain has to be sustained all along: it seeks to relapse into its original, native state, while we want to prevent that. Our Western partners, the United States above all-out to boost the output of biofuel - are showing a great interest in our work. Here in Russia we are using cellulases only in the textile, wood-pulp and paper and food industries. So far.

Which means our biotechnologists are integrated in world science?

- I must say that Russian experts enjoy much respect in the world, there are but few research collectives like us. We are filling orders for our partners abroad, and we are learning from them, too, on a give-and-take basis. As to the bioconversion of plant raw material in this country, this problem is drawing interest from agricultural companies involved with fodder and mineral fertilizer output, and from wood-pulp and paper mills seeking to utilize the piling up waste. This means an interest in scientific research, too. What we need is support at the government level, a government decision to increase biofuel output. I hope catalytic enzymes we are getting shall be in demand at home.


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