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Highly Alkaline Protease

#G-1769


Selection of Highly Alkaline Protease Producer Actinomycetes Strain, Isolation and Characterization of the Enzyme, Cloning and Expression of its Gene

Tech Area / Field

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

Status
3 Approved without Funding

Registration date
14.08.2009

Leading Institute
Durmishidze Institute of Biochemistry and Biotechnology, Georgia, Tbilisi

Collaborators

  • Lawrence Berkeley National Laboratory / Center for Environmental Biotechnology, USA, CA, Berkeley\nPolish Academy of Science / Institute of Biochemistry and Biophysics, Poland, Warsaw

Project summary

Selection of new extremophilic microorganisms, including alkaliphilic and haloalkaliphilic actinomycetes, revelation of their potential for biosynthesis of alkaline proteases, characterization of produced enzyme, and cloning its encoding gene is significant task of industrial biotechnology.

Alkaline proteases due to their resistance (stability) to high temperature and alkaline pH conditions, inorganic and organic detergents are considered as appropriate enzymes for industrial application (Horikoshi, 1999 Alkaliphiles: Some Applications of their Products for Biotechnology. Microbiology and Molecular Biology Review 63:735-750; Gupta et al, 2002 Bacterial Alkaline proteases: molecular approaches and industrial applications. Appl Microbiol Biotechnology 59:15-32).

Alkaliphilic and haloalkaliphilic commercial alkaline proteases are generally obtained from the genus Bacillus (Ban et al, 2003 Annals of Microbiology 53:95-103; Patel, et al 2006 World Journal of Microbiology and Biotechnology 22:375-382 Joshi et al, 2008 Biotechnology and Bioprocess Engineering13:552-559; Jaouadi et al, 2008 Biochimie 20).

Actinomycetes are extremely interesting as active producers of many primary and secondary metabolites (Krasilnikov N.A. Actinomycetes Antagonists and Antibiotic Substances. Publ. Akad. Nauk USSR, 1950). Only a few proteases of actinomycetes have been purified and characterized at the biochemical and molecular levels (Patke and Dey, 1998 Letters in Applied Microbiology 26:171-174; Thomas et al, 2007 Braz J Microbiol. 38:1-9; Jignasha T. et al, 2009 J Ind Microbiol Biotechnol 36:211–218), though their biotechnological potential is almost unknown.

At Durmishidze institute of Biochemistry and Biotechnology collection of actinomycetes isolated from different soil-climatic zones of Southern slopes of Caucasus have been created for more than 20 years. The collection contains above 1200 mesophilic, alkaliphilic and helophytic strains, out of which over 200 are alkaliphilic. Among collection cultures there are producers of proteolytic enzymes. Some of alkaliphilic actinomycetes are excreting alkaline proteases while their deep cultivation, stable at pH 10-11(Pataraya D., Gurielidze M., Berishvili T., Cholokava N., Zaalishvili G., Nutsubidze N. Halophylic Actinomycetes from Saline Soils of Eastern Georgia and their Biological Properties. Proc.Georgian Acad. Sci., 6, 2008).

The study and application of available perse and unique collection of actinomycetes, for the production of alkaline protease, which is in great demand in industry is urgent and represents novelty.

The goal of the project is selection of high alkaliphilic actinomycetes strain, producer of highly active extracellular alkaline protease, isolation, purification and characterization of the enzyme from selected strain, cloning of encoding gene and its expression.

Modern microbiological, biochemical, and enzymological methods and molecular biology tools will be used to implement the project tasks:

  1. Selection of alkaliphilic actinomycetes strains active producers of alkaline proteases.
  2. .Morphological-cultural (colony shape, size, surface color, consistence, pigment formation ability) and biochemical-physiological characterization (relation to oxygen, temperature and pH-optimum, assimilation of different sources of C- and N-, enzymes activity) of the most active and alkaline resistant strain, determination antimicrobial properties and identification of the strain.
  3. Phylogenetic characterization of the most active and stable alkaline protease producer strain.
  4. Isolation and purification of an alkaline protease from selected strain.
  5. Study of the most important industrial properties of alkaline protease: existence of different molecular forms, pH and temperature optima of action, effect of various metal ions and of inhibitors (PMS, EDTA), hydrolysis of natural protein substrates (casein, egg, albumin, BSA, gelatin), conditions for the revelation of maximum activity (Vmax), resistance to organic solvents and detergents (nonorganic, organic), compatibility with local laundry detergents, washing performance of alkaline protease in the presence of detergent.
  6. N terminal sequence determination (20 amino acids).
  7. Cloning and sequencing of an alkaline protease gene.
  8. Investigation of the expression of gene encoding alkaline protease.

In future purification of recombinant protein and comparison of its physical and chemical characteristics with that of natural protease will be conducted.

The expected results of the present project are:

  • An alkaliphilic actinomycetes strains, producers of alkaline protease will be selected
  • Selected alkaliphilic actinomycetes strain will be characterized phylogenetically.
  • Alkaline protease isolation and purification methods (biotechnology) will be elaborated and its industrial chemical and physical characteristics will be established
  • Application fields of the enzyme (alkaline protease) in industry will be selected on the basis of above mentioned enzyme properties.
  • Cloning of an alkaline protease encoding gene will be implemented.
  • Nucleotide sequence of the gene will be determined.
  • The optimal conditions for gene expression will be selected.

All above mentioned will enable us to clone the gene in different microorganisms in large scale via technologies of recombinant DNA, in order to acquire and enhance alkaline protease biosynthetic feature.

Cloning and expression of enzyme encoding genes, including alkaline protease encoding gene is the novel DNA –recombinant technology research direction in Georgia.

Gene cloning and expression of an alkaline protease from selected actinomycetes strain represents an essential step in the engineering of most efficient producer microorganisms.

Project supports basic research with practical significance for peaceful purposes.

Implementation of the project would result in technology for production of high alkaline stable protease, would reinforce the transition to market-based economies and contribute to development of country industry. Project would have international significance as well.

High qualification Georgian researchers guarantee successful realization of the project. Note some selected publications of project participants:

  1. Kvesitadze,G. Bezborodov A.M et al. Introduction in Biotechnology. Monograph. Ed. Nauka, Moscow, 2002, 248 p. (in Russian).
  2. Kvesitadze G. Khatisashvili G; Sadunishvili T; Ramsden J et al. Biochemical Mechanisms of Detoxification in Higher Plants. Basis of Phytoremediation. Monograph. Springer Verlag, 2006, 241 p.
  3. Sadunishvili T, Torok L, Kutateldze N. et al. Collection of extremophilic microorganisms from the area of Caucasus source for stable enzymes. Plant & Microbial Enzymes: isolation, characterization, biotechnology applications, 2007, pp. 74-79.
  4. Pataraia D. Formation of Cr(V) and Cr)III) in Arthrobacter oxydans exposed to high concentrations of Cr(VI). In: Modern Multidisciplinary Applied Microbiology (Ed. By Mendez-Vilas, A.), Wiley-VCH, Weinhem, 2006.
  5. Pataraia D. et al. Isolation o f Alkaliphilic Actinomycetes from Various Types of Soils of Georgia and their Biological Characterization. Annals of Agrarian Science, 2009.
  6. Brodzik R; Gaganidze D. et al. Transgenic plants as a potential source of an oral vaccine against Helicobacter pylori, Food Biotechnology, S. Bielecki, J. Tramper and J. Polak (Editor).Elsevier Science B..V. 2000, p. 35.
  7. Sirko A; Blaszczyk A; F. Liszewska F; Gaganidze D. Genetic engineering of oxidative stress resistance in plants. In: Kluver Academic Publishers, Dordecht, the Nederlands, 2003, pp. 245-263.
  8. Wawzynska A.; Gaganidze D. et al. Overexpression of genes involved in phytochelatin biosynthesis in E.Coli: effects of growth, cadmium accumulation and thiol level..Acta Biochimica Polonica 2005, vol. 52, N 1, p.109-116.
  9. Betsiashvili M., Sadunishvili T., Gigolashvili G., et al. Valuable food protein preparation from soybean. Advances in Food Sciences, 2002, V.24, N1.

Researchers, who previously dealt with the development of the weapon of mass decomposition, participate in the Project, and their knowledge and skills will be directed to peaceful activities.

Special attention will be paid to cooperation with western collaborators. This will include exchange of information, consultation in modern methodologies, discussion of project results, publishing and patenting of project results.

Following to preliminarily agreements with project collaborators the roles were distributed in the following way:

Professor Agnieszka Sirko (EU) will provide help and consultation in the following techniques and procedures: gene cloning and expression in other systems (bacteria, yeast), protein purification and characterization (Mass Spectrometry), monitoring gene expression (Northern).


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The International Science and Technology Center (ISTC) is an intergovernmental organization connecting scientists from Kazakhstan, Armenia, Tajikistan, Kyrgyzstan, and Georgia with their peers and research organizations in the EU, Japan, Republic of Korea, Norway and the United States.

 

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