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Biocatalytic Synthesis of Fine Chemicals

#A-692


Extremozymes: Biocatalytic Synthesis of Fine Chemicals

Tech Area / Field

  • BIO-IND/Industrial Biotechnology/Biotechnology
  • BIO-CHM/Biochemistry/Biotechnology
  • BIO-MIB/Microbiology/Biotechnology

Status
3 Approved without Funding

Registration date
12.03.2001

Leading Institute
Institute of Microbiology, Armenia, Abovian

Collaborators

  • CNRS / Centre de Recherches sur les Macromolecules Vegetales, France, Nantes\nConsejo Superior de Investigaciones Cientificas / Instituto de Catalisis y Petroleoquimica, Spain, Madrid\nRuhr Universität Bochum / Fakultät für Biologie, Germany, Bochum\nIstituto di Ricerca su Membrane e Modellistica di Reattori Chimici, Italy, Arcavacata di Rende

Project summary

The Project concerns the isolation and characterization of extremophilic and hyperextremophilic microbial cultures-producers of specific enzymes and investigation their ability to produce various fine chemicals.

Enzymes catalyze a rich variety of metabolic transformations, and do so with very high catalytic rates under mild conditions with high reaction selectivity and stereospecificity. They are important in the armory of tools for clean product synthesis.

Owing to their unique properties some of enzymes are widely used in various industrial areas for many years.

However, recently a number of causes have restricted to date more extensive technological use of enzymes. The most important ones are:

– the difficulties of enzyme separation from the initial reagents and reaction products after process termination which permits the usage of the enzymes for a single time only;


– instability of the enzymes on storage, and also during various interactions;
– the difficulties of enzyme purification in sufficient active form, and as a result, high cost of the homogeneous enzymes with high activity.

In addition, different agents, like temperature and chemicals, promote enzyme inactivation. Inactivation by chemicals can often be avoided rather easily by keeping them out of the reaction medium. Temperature, however, produces opposed effects on enzyme activity and stability and is therefore a key variable in any biocatalytic process. Biocatalyst stability, i.e. the capacity to retain activity through time, is undoubtedly the limiting factor in most bioprocesses, biocatalyst stabilization being then a central issue of biotechnology. In fact, biocatalyst operational stability will determine to a large extent the viability of the process, be it new or faced to compete with an already existing technology.

Recently the paths of overcoming these difficulties were ascertained. They are connected with the so-called immobilized enzymes and cells of microorganisms, which have some advantages comparing with biocatalysts in a free state. Following factors are of significance: decreasing of the expenses on separation and purification of enzymes and reaction products, higher stability, the possibility of the creation of continuous automated processes, prolonged function, high efficiency and the possibility to establish of wasteless technologies. Other strategies are at hand to increase operational stability: the use of stabilizing additives, chemical modification of enzyme structure, derivatization, crystallization, cross-linking and medium engineering. However, as stability requirements vary dramatically depending on the end-use and storage conditions, it doesn't seem to be possible to insist on one particular treatment or formulation even for single enzyme species.

Besides, the majority of enzymes used to date, have been obtained from mesophilic organisms and, despite their many advantages, the application of these enzymes is restricted because of their limited stability to extremes of temperature, pH, ionic strength, in the presence of organic solvents, etc. The fact leads to the additional difficulties.

On the other hand, the extremophilic microorganisms, which are able to live at extremes of temperature and pH, at high pressure, or at very high salt concentrations, may be so useful as the sources for enzymes needed. Enzymes and proteins that function under extreme conditions mediate metabolic processes and specific biological functions of these microorganisms. The enzymes that have been isolated from these exotic microorganisms show unique features, are extremely thermostable and usually resistant against chemical denaturants such as detergents, chaortopic agents, organic solvetns and extreme of pH.

So, extremophiles are a source of enzymes (extremozymes) with extreme stability, and the application of these enzymes as biocatalysts is attractive because they are stable and active under conditions that were previously regarded as incompatible with biological materials. Furthermore, it is clear that some extremophiles have novel metabolic pathways and so might serve as a source of enzymes with novel activities and applications. They can hence be used not only as a model for designing and constructing proteins with new properties that are of interest for industrial applications but also as remarkable tools for development of unique reactions that are possible to carry out only in extreme conditions and in presence of large amount of organic solvents. The combinations of stabilizing methods with extremozymes may have the great potential for biocatalysis not only in aqueous but also organic media.

The use of organic solvents as reaction media for biocatalytic reactions has proven to be an extremely useful approach to expanding the range and efficiency of practical applications of biocatalysis. The advantages of using organic solvents include, for example, increased solubility of hydrophobic substrates and favorable shifts of reaction equilibrium. The method also so useful for investigations of enzymes condensing activities for the synthesis of peptides and oligosaccharides from monomers because the enzymes catalyze the velocities of the forward and the reverse reactions by the same factor. Condensation is the reverse reaction of hydrolysis. Condensation and transfer reactions are similar as the formed products by degrees of polymerization are higher than the starting substrate. The enzyme hydrolyzing, for example, a-glucosidic linkage must catalyze the formation (the condensation) of the same linkage in the same ratio. Thus, the condensation is favored by the higher concentration of substrates and by the lower water concentration. Therefore, because salt has the effect of reducing water activity, enzymes from the halophiles and alkalophiles are thought to be important biocatalysts in aqueous/organic and nonaqueous media. Studies on peptide synthesis in organic media with mesophilic enzymes have shown that low temperatures favor high yields, so the application of cold-adapted enzymes from psychrophiles in this field may have many advantages.

In this context, the main objective of the Project will be the overproduction of extremozymes, their immobilization and use in various fine chemicals synthesis. For this purpose the various enzymes, such as glycosyl hydrolases, transferases, proteases, lipases, lyases, amidases, etc. produced by extremophiles (thermophiles, halophiles, psychrophiles, alkalophiles, actinomycetes) and whole cells with specific enzymatic activities will be tested.

Institute of Microbiology of the National Academy of Sciences of Armenia (INMIA) for many years is actively engaged in the study of biosynthetic activity of many groups of microorganisms, particularly their extremophilic forms. Well-defined and characterized vast Culture Collection is maintained.

As a result of long-term researches the species specificity of enzymatic activity in different groups of microorganisms has been revealed.

The study of enzymatic activity of sporeforming bacteria permitted to establish its strict correlation with cell differentiation. In this respect the most attractive are Bacillus thuringiensis, Bacillus sphaericus and Bacillus popilliae species characterized by formation of specific crystalline polypeptides with insecticide action. The crucial point is that the biosynthesis of such polypeptides with high enzymatic condensation reaction strictly correlates with sporulation function, i.e. with the development of waterless environment within the cell. A great number of bacterial strains of species mentioned have been isolated and studied in detail. Biochemical and genetic patterns for production of such peptides have been defined.

Biosynthesis of theanine (g-glutamylethylamide) from mixture of glutamine and ethylamine with immobilized cells of Pseudomonas nitroreducens as source of theanine synthesis enzyme has been carried out.

At present the Collection of sporeforming bacterial cultures producing crystalline peptides comprises more than 2,000 strains and represents a remarkable resource for study of peptide condensation activity. The Database serves the collection with use of original approaches for its creation. The Culture Collection of carbohydrate processing enzymes extremophilic producers is available.

Proteases have been used in enzymatic peptide synthesis to catalyze the formation of new amide or ester bonds, with two main purposes: elongation of peptides for large-scale synthesis of biologically active compounds of industrial interest and specific modification of nature or fermentation-derived peptides and proteins.

The highly efficient producers of aspartase, asparate-b-decarboxylase, fumarase, cyclomaltodextrin glucanotransferase (CGTase), b-galactosidase, a-glucosidase, b-fructofuranosidase, inulinase, invertase, amylases, glucoamylase and debranching enzymes from various groups of microorganisms have been isolated and comparatively studied for biocatalysis. The ability of such enzymes to form different oligosaccharides by transfer and condensing reactions at the definite conditions has been revealed and studied at the INMIA.

The new methods of enzyme and cells immobilization with the use of original strains of microorganisms including their extremophilic forms have been applied in the continuous processes of the preparation of some aminoacids, organic acids, peptides and oligosaccharides.

The results of these works were summarized in about 250 scientific publications, patents and three monographies.

The Institute was involved in R&D for military purposes in the Former SU and weapon scientists are engaged within the Project. Actually the Project represents the conversion of military R&D to civil needs.

On different stages of the Project some other institutions will be involved.

The Project represents an integrated complex of microbiological, biochemical, biotechnological, chemical and analytical study with application of modern technologies and comprises R&D devoted to use of extremozymes for production of new and high-value products for food and medical application.

Microbiological researches aimed to obtain and to characterize efficient extremophilic producers of certain enzymes, to develop their growth conditions and biosynthetic pathways for enzymes overproduction.

The main goals of biochemical investigations have to be the study of physico-chemical and biochemical properties of enzymes produced by extremophilic and hyperextremophilic microorganisms. The special experiments will be done on ascertaining, study and application of transferring and condensing activities of extremozymes in aqueous, low-water and organic media. In this step the methods of combinatorial biocatalysis will be applied. The attraction of combinatorial biocatalysis is the expeditious discovery of new drugs using solid stage techniques, which may be automated. In itself this technology may be classified as a clean industrial process as it minimizes the use of reagents by miniaturizing the drug-discovery process. The use of biocatalysts offers the opportunity to impart the synthetic benefits of enzymes to the combinatorial process, although it must be realized that it differs from combinatorial biosynthesis, which involves the de novo formation of compounds using whole cells. In essence, combinatorial biocatalysis provides a method for the rapid transformation of unnatural substrates in a high throughput system that offers both catalytic novelty and containment. This strategy can be applied to a wide range of transformations.

Biotechnological R&D includes the development of process-engineering for the production of fine chemicals such as, pharmaceuticals, radionuclide chelating agents, food additives, biodegradable polymers, etc. of high commercial value as well as the oligosaccharides with prebiotic activity. Some of samples will be obtained in pilot-scale conditions. The new immobilized systems for development of biocatalysis in extreme conditions and non-conventional media will be carried out.

The chemical methods will be used to prepare and to carry out a comparative study of different chemical derivatives of oligosaccharides, as well as for synthesis and characterization of new insoluble derivatives of cross-linked inulin and polymeric cyclodextrins, including modification with cyclofructins and peptides.

INMIA is now actively engaged in the study of synthesis of various physiologically active substances, cyclic- and linear sugars by biotransformation and have almost all facilities and pilot-scale installations to carry out R&D mentioned, including process-engineering.

The results expected of the Project have the scientific, practical and economical significances, as the development of biocatalysis, biochemistry of extremophilic and hyperextremophilic microorganisms as well as methods for realization of enzymatic transformations in organic and low-water media. The Project realization permits to carry out the efficient methods for the synthesis of some high-value fine chemicals.

The realization of the Project provides former weapons scientists of INMIA with opportunities to redirect their activity to peaceful needs, in particularly for manufacturing of high-value fine chemicals by biocatalysis with the potential wide spheres of application.

The Project is open for international community: join investigations and production of fine chemicals by biocatalysis using extremozymes, pilot-scale experiments and applications.

In the framework of Project Proposal the role of foreign collaborators is as follows:

– provide each other the new specific extremophilic producers for their investigation and references;


– shared use of test samples and certain equipment for characterization of compounds obtained;
– information exchange in the course of project implementation;
– specialist exchange;
– participation in joint seminars.


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