by Arkady SINITSYN, Dr. Sc. (Chemistry), head of the Laboratory of Physicochemical Transformation of Polymers, Department of Chemistry, Lomonosov Moscow State University; also, head of the Laboratory of Biotechnology of Enzymes, Bach Institute of Biochemistry (Russian Academy of Sciences), Moscow, Russia

It is hard to visualize the present-day world without biotechnology and its products. Hence further advancement of this line of research and creation of an adequate industrial base are an essential condition for competiveness of Russian goods on domestic and international markets. Yet only few promising projects are in practical demand. Dr. Arkady Sinitsyn, however, is hopeful about the future, as he told our reporter Yevgeniya Sidorova in an interview.

-- You and your colleagues are in search of new effective enzymes to be used as catalysts in many industries. Publications of your associates and postgraduate students show that research laboratories are building up their innovation potential. How do you manage to expand the list of your innovative products, even though the funding situation has changed for the worse in this past decade? Your success story?

-Ours is a unique collective, to begin with. These are both specialists of the laboratories I am heading, and col-

Three-dimensional models of enzymes hydrolyzing the plant raw material.

leagues of allied laboratories at the Pushchino-based Skryabin Institute of Biochemistry and Physiology of Microorganisms, and at the Research Institute of Food Biotechnology attached to the Russian Academy of Agricultural Sciences. In formal terms we are brought together by the Collective Use Center set up in 2006 on the basis of the RAS Bach Institute of Biochemistry and equipped with facilities for microbial synthesis and enzymic research. Each group is responsible for its domain of research. For instance, our colleagues at Pushchino are involved with mutagenesis* and optimi-zation of enzymatic processes. The Biochemistry Institute is concerned with gene engineering, and our university laboratory--with enzymology. Altogether, we are exploring avenues for practical, industrial uses of enzymes.

There are two ways of changing the properties and productivity of microorganisms, and these are (1) the

* Mutagenesis, targeted changes (mutations) in a DNA nucleotide sequence.--Ed.

classic one, mutagenesis proper brought out by chemical or radiation effects, and (2) gene engineering. What concerns mutagenesis, we have joined hands with the National Research Center "Kurchatov Institute". Yet we cannot tell how radiation will affect a particular object. After irradiation we have to analyze the activity of hundreds and thousands of clones* before selecting a high-activity mutant strain. But gene engineering is a short cut.

In this case we prepare a host microorganism strain with desired characteristics imparted afterwards. Also, we get a vector system for gene cloning: a DNA molecule, the hereditary information carrier. Simultaneously we take another microorganism selected for a particular purpose (obtaining cellulases** for example) and find a gene responsible for the output of an enzyme of interest

* Clone, an artificially designed organism (cell) genetically identical with its mother original.--Ed.

** Cellulases, glucosyl-hydrolase enzymes hydrolyzing cellulose to oligosaccharides and glucose.-Auth.

to us; we clone this gene and have it expressed in the host microorganism (the host acquires a desired character thereby). Our arsenal includes methods of getting silent genes in the host to "talk". In either case we get stable producers of desired genes.

The host microorganism is so designed that it can secrete plenty of protein. Such are the strains of micellar fungi of the genus Penicillium that we have selected: they are very active, and the cellulolytic enzymes they produce digest more cellulose per unit of time than the other available analogs.

--Do they use other producers of cellulolytic enzymes abroad?

--They use strains of Hyphomycetales (Fungi imperfecti) of the genus Trichoderma and higher mold fungi of the genus Aspergillus. As far as we are concerned, working within the framework of the project of the RF Ministry of Education and Science (2005-2006)*, we have evolved micellar fungal strains of our own, our "champions", so to speak. We have patented our discovery, and keep working on Penicillia, upgrading their potential for hands-on objectives.

But we have to show our paces and work fast, what with the cutoff dates and all that. But we shall cope, for we have a large database on different strains of microorganisms and on the characteristics of their enzymes. We have collected this database over many

* See: A. Sinitsyn, "Versatile Enzymes", Science in Russia, No. 4, 2007.--Ed.

years, and we are expanding it. Each time as we find or obtain a new producer microorganism, we get busy with basic research: we isolate from the culture medium all enzymes it secretes and see in what amounts it does that. We study their characteristics, their specificity and mechanism of action, their structure. We look into their possible uses.

--What about the practical applications?

--We have as many as 50 products in our bag. But only a few have found practical application for a variety of reasons. It may be the low activity of strains which, therefore, cannot compete with analogs on the world market. In most cases, however, we have no partners among biochemical enterprises at home. There is but one enzyme-producing plant, the Sibbiofarm at Berdsk in the Novosibirsk region.

As soon as we get a microorganism up to desired standards, we have to cultivate it as enzyme producer. As I have said, our fellow workers from the Skryabin Institute of Biochemistry and Physiology of Microorganisms and the Institute of Food Biotechnology are optimizing this process under laboratory conditions. They should make the producer strain active and get it to put out dozens of grams of enzyme per liter of the culture liquid. Optimal cultivation conditions are likewise important, for instance, low-cost components of the culture medium containing carbon, nitrogen and phosphorus.

Once a sufficient amount of the microorganic biomass is obtained and the enzyme synthesis is completed, we

take the culture medium containing extracellular enzymes out of the fermenter, isolate it from other components, purify it, say, by ultrafiltration and select stabilizing agents to avoid attacks of the ambient microflora during storage. If the cultivated microorganism shows high productivity, we can get to its commercial production in the liquid, dry or granulated form.

--Can you use the productive capacity of the enzyme farm in full on domestic material? Is this a real proposition?

--The Sibbiofarm enterprise is still producing several enzymic preparations hailing from the Soviet times (cellulase, pectinase, protease, amylase). We could offer a much wider mix of cheap enzymes consumed in large amounts--say, for biofuel production. Yet to market such products we should offer not only good quality, we should learn how to sell. The Russian market is glutted with foreign analogs.

--Does the Biotech-2030 program for revival of the biotechnological industry provide for ways of coping with the downturn?

--The Biotech-2030*--Bioindustry and Bioresources program--is consolidating biotechnologists working in Russia. The co-chairman of this program is Mikhail Kirpichnikov, member of the Russian Academy of Sciences and head of the Bioengineering Chair of the Department of Biology at Moscow State University, and Pyotr Kanygin, Director General of the RT-Biotechprom Association (part of the Rostechnologies State

* See: A. Yanenko, "Prospects of National Bioindustry", Science in Russia, No. 5, 2011.--Ed.

Corporation). Among the progenitors of this program is Vladimir Popov, head of the Bach Institute of Biochemistry and RAS Corresponding Member. The authors of this action program have chartered the basic development trends of the branch. The state and business should provide the required wherewithal.

The RT-Biotechprom Association was set up in 2009. It was to materialize the idea of biofuel production from renewable hydrocarbons (wood, annual plants), the idea born at the government level early in the 1980s.

A brief retrospective look will be in place here. Outbursts of interest in substituting renewable raw materials for petroleum always concur with petroleum price fluctuations. I could name three price hikes like that--in the early 1970s, in the 1980s and today. I began my research work in the 1980s at the Chair of Chemical Enzymology at the Department of Chemistry of Lomonosov Moscow State University under Dr. Anatoly Klesov, now a Harvard University professor in the United States. Our collective took up this new line of research not only because of its practical side, but because of the overriding interest in the scientific side of the matter. As a rule, enzymologists make use of a soluble low-molecular substrate. But we had to deal with polymer and insoluble substrates like cellulose, hemicellulose and other polysaccharides. Since processes implicated in their adsorption play a big part in the behavior of enzymes, we had to get down to brass tacks and see how it happens. It came out that one sole enzyme is unable to digest polysaccharides--a concert-

A plot describing cellulose degradation by penicillia and trichoderma enzymes.

ed action of an enzymic complex is needed. We had to deal with another problem--lignin hydrolysis, and increasing the reactivity of the native cellulose-containing raw material through its pretreatment. The "kitchen" of this scientific inquiry was thrilling indeed.

-- Your experience holds today: petroleum prices still on the rise...

--That's true. Several years ago the United States started building plants for processing renewable plant raw materials, it invested big money, what with the growing oil prices. At about this time Dr. Yevgeny Davidov, one of the leading researchers of the GosNIIProtein Synthesis center (years ago he organized work in building plants processing paraffins into protein) talked the country's leadership into using raw materials at idle biochemical enterprises (hydrolysis mills for example) for making new useful products. It was a bargain proposition. He told government official about bioconversion. And so in 2007 the Rostechnologies state corporation approached us with a proposal to conceptualize enzyme-producing complexes for obtaining biofuel and other products from wood.

We turned to and got busy with a comprehensive project off to a good start in 2011. The RT-Biotechprom Company is in charge of this project, and it is financed in part by the state, and in part by RT-Biotechprom, that is not from the budget. The aim is to build a pilot enterprise on the basis of the former butanol-producing hydrolytic plant at Tulun in the Irkutsk region. The new plant, East Siberian Integrated Works, will be producing alcohols and sugars by biotechnological methods.

First we asked our partners to provide a substrate. Now, the biofactory concept (current in Europe, it amounts to replacing hydrocarbons with carbohydrates) has one essential drawback, the low reactivity. Otherwise the microflora would have devoured all woodlands. So, as I have said, pretreatment of the stock material is needed--it should be ground and treated with acid or alkali so as destroy the lattice. These are old methods, but they should be brought to a technology. Fortunately Rostechnologies and GosNII Protein Synthesis have agreed to take care of that.

Then, using enzymatic hydrolysis, we shall get the primary "building bricks", that is glucose, from plant polysaccharides; from glucose, by fermentation, we get ethanol, or ethyl alcohol, in a reaction catalyzed by yeast; or else we get butanol (used for this purpose are bacteria of the genus Clostridium).

-- Which means that butanol, or butyl alcohol, can be used as a biofuel?

--Sure. It is better than ethanol in physicochemical characteristics: it does not mix with water and gives off

more energy in burning. Yet in technical terms its production is more expensive compared with ethyl alcohol, and thus state subsidies are needed. The point is that the butanol-producing Clostridia get poisoned by butanol once its concentration is up, and the process comes to a stop. Yet this process was optimized in the end and made continuous. Four enterprises producing butyl alcohol from corncobs were commissioned still in the Soviet Union. Their products were used in the chemical industry, among other things, as a raw material for polymer synthesis. The Tulun enterprise will work on fine acid-hydrolyzed sawdust. The quality of wood doesn't matter--even wood contaminated by phytopathogenes will be good.

-- Will you keep working on microorganisms in your database only? Or will you pick out new strains producing cellulolytic enzymes?

--We are upgrading the strains that we have patented and studying enzyme complexes they produce. We are coming to understand why enzymes of these microorganisms are more effective than those produced by trichoderms and used by such international companies as Jennencor and Danisco (both incorporated within the American Dupont chemical concern). Here we are able to compete with their experts. By the way, there are Moscow University graduates and our lab fellows among them. But all of us shall not stop at what has been achieved.

--Any other projects in your portfolio of innovations?

-We are involved in a number of small projects. We in Russia are having at least three industries ready to make use of biotechnology achievements, and these are the wood-pulp and paper, and food industries as well as one producing feed for cattle farms. Enzymes are likewise used in paper bleaching and in cellulose pretreatment. The cultivation technological standards are quite stringent: high temperature, alkaline pH of solutions, and primer enzymes vigorous enough to keep their original characteristics. Their adaptation is no simple job. Needless to say, biotechnological approaches will work significant changes in production, but first they should become part of the process. Here we are cooperating with one of the leaders of the wood-pulp and paper industry, the St. Petersburg-based "Ilim" company.

One of the demands of our clients is that we should modify cellulose by enzymic treatment so that its

Science in Russia, No.3, 2012

physicochemical properties (length of fiber, strength, degree of polymerization) comply with definite standards. Say, nanocellulose is trendy now.

--Nanocellulose: what is that?

--Microcrystal cellulose is known to everybody-it is used as food additive--added to ice cream, for example. Actually there are no crystals in it but tiny pieces of fiber, about ten microns in size. Nanocellulose is in the form of unidimensional little needles, with their tip 10 to 20 nm wide and their length, 1,000 nm and more. In fact we are dealing with a colloidal chemistry object. This material is quite good: a sheet of paper made from it will be firm and transparent. If added to ordinary paper, its quality will improve greatly.

This innovative product can also be used for obtaining biodegradable materials. If nanocellulose is added to a chemical polymer (polyethylene for instance) that is not degraded by the microflora under natural conditions, its particles get easily into the polymer structure and speed up the degradation process. Polylactides--substances derived from lactic acid--are now in use for the same purpose. This process is based on the above technological sequence: renewable plant material is degraded to sugars from which lactic acid is synthesized. Yet polylactides are quite expensive, and are ordered for special purposes only.

As to nanocellulose, we in this country first take cellulose and mince it to definite dimensions; then we follow with acid hydrolysic, which can be substituted by enzymic hydrolysis as well. It is important to select an adequate enzymes carefully. This technology has already been mastered abroad. Our home producers, seeing the advantages of the innovative technology, have turned to us with a proposal on cooperation. We are going ahead with research in this direction.

It was clear to us from the very start: we were not dealing with a group of aggressive cellulases (saccharolytics) capable of "eating" a hole in a cotton fabric and turning its cellulose into glucose, the initial "building brick"; but we were dealing with enzymes contracting the dimensions of cellulose particles and fibers without utter destruction of the woof. We call such enzymes "topolytics"--in the sense that they work on the surface of a fabric. We first learned about them fifteen years ago, as we began cooperating with the Americans within the framework of a project of identifying enzymes removing indigo when you wash your jeans. A primitive goal, it might seem; but we discovered lots of new things in what concerns the basic characteristics of enzymes. There are cellulases and cellulases, and they are used in different areas. Knowing what kind of cellulase is needed for the purpose, we pick out an adequate producer enzyme. As I have said, our rule is this: isolate all secreting enzymes from the culture medium individually, one by one, study them and get each into the database.

-- Yes, because you can get the material from your old database to solve a wide range of problems, isn't it so?

-First, we should understand the molecular and genetic nature of a phenomenon of interest to us.

To begin with, we must show great skill in simulating processes under laboratory conditions, that is on a small scale compared with those taking place in commercial production. We cannot produce an enzyme in amounts needed for denim washing in your washing machine; we cannot do it on a regular basis. We take a tiny piece of the fabric, sort out enzymes and get the most effective one. Then we go on with our search: how to make our "champion" more stable and more active? How to get a high-activity microorganism, its producer?

While fifteen years ago we were still guessing about the causes of the higher or lower activity of various enzymes, now we know the structure of genes and proteins; and building a three-dimensional model we can see why a particular enzyme performs better than its analog produced by another microorganism.

-- Undergraduates of the Moscow University Chemical Enzymological Chair are involved in this work, aren't they?

--Certainly. There are clear heads among them. Fortunately of late biotechnology, biochemistry, enzymology and gene engineering are getting ever more attractive to the younger set. What we are trying to do is to teach them to work carefully and thoughtfully. Even the tiniest shortfalls in an experiment may kill the whole thing and lead a research scientist astray.

To conclude. Even in the grim times of the crisis we did not feel a shortage of personnel. Our collective has been drawing fresh blood, the talented young people, university graduates for the most part. Many of those who have decided to devote their life to science will stay on with us.