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Proteolytic Enzymes for Biotechnology

#3038


New Generation of Highly Purified Proteolytic Enzymes for Biotechnological Purposes

Tech Area / Field

  • BIO-IND/Industrial Biotechnology/Biotechnology
  • BIO-CHM/Biochemistry/Biotechnology
  • BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology

Status
3 Approved without Funding

Registration date
31.03.2004

Leading Institute
Institute of Genetics and Selection of Industrial Microorganisms, Russia, Moscow

Project summary

Objectives of the project:

- To develop modern technology of highly purified carboxypeptidase A (CpA), carboxypeptidase B (CpB) and trypsin isolation from mammalians pancreas.


- To search for the relative enzymes from other available natural sources to substitute mammalian proteinases.
- To develop the approaches to obtaining of recombinant bacterial enzymes, new generation of proteolytic preparations for biotechnology.

Proteolytic enzymes of animal origin (CpA, CpB and trypsin) are widely applied in food-processing industry for manufacturing beer and clarification fault; in medicine, as components of anticarcinogenic preparations; in bioindustry, for obtaining biologically active peptides from precursors; and in biochemical laboratories, for establishing protein structure. The modern technology of obtaining gene-engineered insulin including a partial proteolysis of the proinsulin precursor is one of the most important current application of these enzymes.

The commercial preparations containing the indicated enzymes are basically manufactured from cattle pancreas. However, thus obtained purified enzymes are very expensive. In this connection improving of the isolation procedure of the cattle enzymes by the use of specific sorbents or more efficient methods of fractionation still attracts the attention of investigators.

The recently reported proteolytic enzyme complexes from fish and arthropods seem to be promising alternative to the mammalian enzymes, because they can be isolated from inexpensive see sources.

At the same time design of recombinant bacterial strain producers of enzymes with preset characteristics is currently regarded as the most promising way of manufacturing of proteolytic preparations. Apart from economic reasons, the use of bacterial producers of animal enzymes can facilitate epidemiological survey taking into account a recent state of the distribution of dangerous infectious diseases. For example, bacterial carboxypeptidase T (CpT) from Thermoactinimyces vulgaris can be successfully substituted for cattle CpB if a wide specificity of the first is narrowed to increase the portion of B-type activity.

We now succeeded in isolation of CpB from cattle pancreas using a modern biochemical method (our know-how). The obtained enzyme preparation can be successfully used in processing of proinsulin, because it is free of trypsin, chymotrypsin, and elastase admixtures, and contains a very low amount of CpA. The presence of the above admixtures drastically reduces the quality of the commercial enzyme preparations.

We have developed a preliminary approach to CpA and trypsin isolation from pancreas of large horned livestock.

We have isolated carboxypeptidase from Kamchatka crab and studied the properties of this enzyme to show its mixed type specificity. A proteolytic complex of Pylorin preparation from fish pancreas was also analyzed to detect the enzyme with CpB specificity.

CpT from Thermoactinomyces vulgaris has also been found out and characterized in our laboratory. A thorough characterization of this exopeptidase by means of enzyme kinetics was carried out, specific peptide substrates for determining the activity of the enzyme were developed, and a spatial structure of the enzyme was established in co-operation with The Institute for Crystallography (Moscow). A possibility in principal to use this enzyme for proinsulin processing was shown.

To attain the planned thresholds of the project, the following experimental tasks must be dissolved:


- To develop the technology of CpA, CpB and trypsin isolation and purification from cattle pancreas.
- To synthesize new affinity sorbents efficient for isolation of the target enzyme with a high yield.
- To carry out testing of the enzyme preparation for large-scaled obtaining of gene-engineered insulin.
- To investigate a proteolytic complex of sea animals as potential sources of CpA, CpB and trypsin.
- To develop approaches to modify the CpT specificity and create the bacterial analogue of mammalian CpB.
- To develop the appropriate specifications and technical documents.

The proposed project belongs to the area of fundamental and applied biochemistry and molecular biology.

Results of theoretical importance:


- Study of proteolytic complexes of sea animals pancreas.
- Development and improvement of the methods of CpA, CpB and trypsin purification from cattle pancreas.
- Study of molecular basis of dual substrate specificity of CpT. Design of bacterial enzyme with specificity similar pancreatic CpB.

Results of applied importance:


- Development of the technology of homogeneous enzyme (CpA, CpB and trypsin) isolation from cattle pancreas.
- Manufacturing of CpB and trypsin preparations for their use in the gene-engineered insulin production.
- Obtaining of enzyme preparations from sea animals.
- Increase in the efficiency and profitability of CpB and trypsin manufacturing.
- Decrease in the cost price of gene-engineered insulin preparations.

Results of commercial importance:


- Prospects of the development of a new generation bacterial enzyme which similar in activity to CpB.
- Increase in the efficiency and profitability of manufacturing of CpB and trypsin preparations.
- Decrease in the cost price of gene-engineered insulin preparations.


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