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Biocatalysts for Amides and Carboxylic Acids Synthesis

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Biotechnological Method for a Synthesis of Amides and Carboxylic Acids, Acrylic Monomers and Oligomers

Tech Area / Field

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

Status
3 Approved without Funding

Registration date
22.03.2004

Leading Institute
Institute of Ecology and Genetics of Microorganisms, Russia, Perm reg., Perm

Supporting institutes

  • Federal State Unitary Enterprise "Perm Plant named after S.M.Kirov", Russia, Perm reg., Perm

Collaborators

  • Napier University, UK, Edinburgh\nEvonik Industries AG, Germany, Hanau-Wolfgang

Project summary

This project is aimed at the development of fundamental and applied microbiology and biotechnology, namely, the investigation of new strains of microorganisms – producers of nitrile hydratase and nitrilase enzymes as well as at selection of microorganisms, essential for syntheses of wide range of acrylic monomers.

Fundamental implications for the investigation of nitrile hydratase and nitrilase producers include the determination of physiological special features of given microorganisms, persity, specificity and genetic regulation of expression of enzymes from nitrile metabolism. Practical interest appears to be the production of new strains – producers of nitrile hydratases and nitrilases possessing properties beneficial for the industrial processes: improved growth and catalytic characteristics, novel specificity or stereo selectivity of the reactions being catalyzed by them.

Growing interest in the world within recent decades to bacteria capable to transform the nitrile is generated by their successful application at large-scale synthesis of acrylamide from acrylic acid nitrile, and also the perspective of their use in the synthesis of a number of amide compounds at industrial level that are involved in the production of various polymers and in pharmacology.

Acrylamide, acrylic acid, and their derivatives are widely used in the production of polymers and co-polymers for various purposes. Acrylic polymers find the application in many fields of economy (preparation of potable and technical water, purification of industrial wastes, machine building, highly accurate instrument making, optical instruments and devices, radio-electronics, rocket-space engineering, minerals’ output, chemical, processing, pulp and paper and textile industries, power engineering, medicine, production of building materials, and construction). World community demand in these products is enormous, incompletely provided, and annually increases by 4-5%.

Unlike the conventional processes of chemical catalysis, the biotechnological method of the organic compounds conversion is a one-staged, ecologically safe, highly specific, wasteless process. The possibility of bacteria involvement in the production of some other products, e.g. nicotinamide, nicotinic and acrylic acids also attracts the attention of the researchers. Therefore, the search for and the selection of new strains converting carboxylic acid nitriles seem to be highly urgent.

Now the investigations of enzymes from the carboxylic acid nitrile pathway are growing rapidly. Strains-destructors of nitriles are represented by microorganisms from different taxonomic groups (Pseudomonas, Alcaligenes, Agrobacterium, Bacillus, Rhodococcus, Brevibacterium, Arthrobacter, etc.). There are two major steps for nitrile biotransformation:


1) successive hydrolysis to amides, and then – to corresponding carboxylic acids provided by two enzymes – nitrile hydratase and amidase;
2) hydrolysis of nitriles directly to carboxylic acids with the use of one enzyme – nitrilase.

There are well-known microorganisms (e.g. industrial strain R. rhodochrous J1) that contain both enzymatic systems. A number of microorganisms-producers of nitrile hydratases (P. chlororophis B23, Rhodococcus sp. N744, R. rhodochrous J1, R. rhodochrous M33) are used in acrylamide synthesis at the industrial scale.

The goal of the this project:

Production and characterization of catalytic activity of strains promising for the biotechnological synthesis of amides, particularly acrylamide and nicotinamide; carboxylic acids, including acrylic and nicotinic acid and wide range of acrylic monomers and ethers of acrylic acids. The development of a technology for biocatalyst production.

The aims of the project:

1) Physiological characteristic of microorganisms-producers of nitrile hydratases and nitrilases.


2) Enzymatic examination of nitrile hydratases and nitrilases.
3) Improvement of cultural and catalytic characteristics of strains-producers of nitrile hydratases and nitrilases by selection and genetic engineering methods.
4) Investigation of the influence of various medium factors on the nitrile hydratase, amidase and nitrilase activity.
5) Enhancement of technological properties of a strain-producer of nitrile hydratase, and the production of biocatalyst suitable for large-scale acrylamide synthesis.
6) Selection of bacterial strains capable of synthesis of acrylic acids.
7) Search and selection of novel strains for synthesis of acrylic copolymers, stereo selective synthesis.
8) Development of technology of acrylic oligomers synthesis.

Fundamental aspects of the project are related to the study of biochemical, physiological, and catalytic properties of a number of strains possessing high nitrile hydratase or nitrilase activity.

The requirement in experimental evaluation of these microorganisms is also determined by the necessity in using of introducing the ecological full-value biotechnologies for production of acrylic monomers and oligomers on defense enterprises of special technical chemistry (FSUE “Perm plant after S.M. Kirov”, Perm, chemical enterprise "Russia", Kamensk-Shachtinsk, as well as chemical enterprises "Beraton", Beresniky, "Orgsynthes", Dzerzhinsk and others.).

The participants of the project have experience in experimental investigations in this field of the science. Since 1999 the laboratory of chemical mutagenesis at IEGM UB RAS has been carrying out the research work on the production and characterization of the catalytic activity of the strains that are promising for the biotechnological amide synthesis, acrylamide in particular, and carboxylic acids, acrylic acids in particular, and also the development of novel biocatalysts based on biomass from microbial degraders of nitriles. As a result of screening of natural populations, chemical mutagenesis, and selection a number of bacterial clones possessing high nitrile hydratase or nitrilase activity were produced.

The group of strains that hydrolyze nitriles by nitrile hydratase pathway that were identified as Pseudomonas, Bacillus, Rhodococcus and Brevibacterium genera was selected. The selection resulted in the isolation of Bacillus mycoides B5, Rhodococcus sp. gt1, and Rhodococcus sp. E84 that produced nitrile hydratases. Clones of these strains exhibited nitrile hydratase activity to acrylonitrile of over 200 units was obtained. Model synthesis with the use of experimental samples of bacterial biomass provided the acrylamide solution with concentration of 48%.

Cultures were tested for the ability to grow on various aliphatic nitriles and aromatic nitriles, and amides. The kinetics of the utilization with these strains of a number of nitriles and amides (20 compounds) at 0,1% concentration was analyzed. Growth characteristics of selected strains and the dependence of nitrile hydratase activity from the growth phase on a salt medium containing 1% glucose and 1% acetonitrile were also examined.

It was determined that genes of alpha and beta subunits of nitrile hydratase in all strains tested possessed chromosomal localization and occurred in one locus. DNA loci contained genes of nitrile hydratase subunits were isolated. Their sizes fell within the sizes of nucleotide sequences of similar genes from other bacteria.

Rhodococcus sp. P1, P-3, P5-3220 and Alcaligenes sp. C-328 strains capable to nitrile conversion to carboxylic acids, acrylic acid in particular, was obtained. Also Alcaligenes sp. strains possessed thermostable nitrilase activity was selected. Nitrilase of these strains has shown also high technological stability. Specific activity of the cultures comprised 16 mol/mg/min that comparable with activity of best analogues.

The proposed project is intended to provide:

- detailed investigation of chemical factors’ (structure and concentration of the carbon and nitrogen source, concentration of phosphates and microelements, acidity, aeration, etc.) and temperature effects on the growth, culture yield, enzymatic activity of bacteria - producers of nitrile hydratases and nitrilase;

- investigation of the influence of concentration of the components in reaction medium, substrate, and reaction product, and also the potential enzyme stabilizers and inhibitors, temperature, and pH on the activity and stability of purified preparations of nitrile hydratase and nitrilase;

- optimization of medium composition and cultivation conditions for the growth of bacteria biomass with high nitrile hydratase activity;

- the use of selection and genetic engineering methods for production of stable in the process of cultivation and highly active clones of the strains;

- development of optimal conditions for active biomass flocculation and isolation of biocatalyst paste;

- development of methods of immobilizations of bacteria on porous carbon, polymeric and other carriers.

The framework of the project is also intended to design a laboratory technology to provide a biocatalyst, and to test this process at pilot-industrial scale to produce biomass at FSUE “Perm plant after S.M. Kirov”.

The implementation of fundamental and applied investigations that are proposed in the project suggests the creation of the experimental basis for carrying out of the research work that supposes purchasing of modern scientific-research equipment.

Part of the total investigation on the project will be fulfilled by “military” scientists from the scientific-research centre FSUE “Perm plant after S.M. Kirov”, that were involved in the development of rocket technologies (rocket materials, propellants, composites), and the technology for biotechnological acrylamide synthesis at industrial scale.

Close collaboration with a number of foreign teams that expressed the interest to the implementation of this work will be provided within accomplishing of the given project.


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