To the content
4 . 2019

Enzymatic activity of recombinant metallopeptidase for further using in meat industry

Abstract

Enzymatic modification of meat with a high content of connective tissue is an effective mean, allowing to improve its properties and expand its use. Microbial enzymes have been extensively investigated as meat tenderizers. Compliance with safety requirements in terms of forecasting the development of various risks is essential for the use of these enzymes in food industry. The method of producing recombinant protease as a potential candidate for applications on meat tenderization was described in the article.

The aim of this study was the production of recombinant Pichia pastoris with M9 peptidase gene from Aeromonas salmonicida.

Material and methods. Objects: peptidase gene M9 (GenBank: CP000644.1 ASA3723) Aeromonas salmonicida (strain of laboratory collection, isolated from the surface of raw meat), the vector plasmid pPic9K, competent E. coli DH5a cells, competent Pichia pasto -ris GS115 cells, culture fluid (QOL) from recombinant Pichia pastoris clones, beef shank samples. To obtain a recombinant strain, genetic engineering methods, the PCR method, and the bacteriological method were used. Polyacrylamide gel electrophoresis was used to separate and analyze the components of the supernatant. Enzyme activity was evaluated by HPLC-MS/MS using synthesized peptides. The impact of the supernatant from recombinant clones on the connective tissue of raw meat was assessed by histological method.

Results and discussion. A metalloprotease M9 gene was cloned from the Aeromonas salmonicida (2748 bp) and expressed in Pichia pastoris. The molecular mass of the recombinant protein was estimated to be 120 kDa by SDS-PAGE. Histological analyses of the control and enzyme treated beef samples showed degradation intramuscular connective tissue, suggesting its effectiveness on meat tenderization.

Conclusion. The recombinant strain Pichia pastoris, which produces the recombinant M9 peptide of Aeromonas salmonicida, has a specific enzymatic activity against collagen, the main component of the connective tissue of meat. The obtained recombinant peptidase M9 can be used as an enzyme softener of raw meat with a high content of connective tissue.

Keywords:recombinant protease, M9 family, Aeromonas salmonicida, Pichia pastoris, bio modification, meat tenderization

For citation: Makhova A.A., Minaev MYu., Kulikovsky A.V., Vostrikova N.L. Enzymatic activity of recombinant metallopeptidase for further using in meat industry. Voprosy pitaniia [Problems of Nutrition]. 2019; 88 (4): 95-104. doi: 10.24411/0042-8833-2019-10047 (in Russian)

References

1. Chanalia P., Gandhi D., Attri P., Dhanda S. Extraction, purification and characterization of low molecular weight Proline imino-peptidase from probiotic L. plantarum for meat tenderization. Int J Biol Macromol. 2018; 109: 651-63.

2. Zhao G.Y., Zhou M.Y., Zhao H.L., Chen X.L., Xie B.B., Zhang X.Y., et al. Tenderization effect of cold-adapted collagenolytic protease MCP-01 on beef meat at low temperature and its mechanism. Food Chem. 2012; 134 (4): 1738-44.

3. Titov E.I., Litvinova E.V., Kidjaev S.N., Pchelkina V.A. About the microstructure of collagen-containing raw materials modified with alkaline proteinases. Myasnaya industriya [Meat Industry]. 2017; (8): 36-8. (in Russian)

4. Bagryantseva O.V., Shatrov G.N. On the need for changes in the legislation of the Eurasian Economic Union in the field of rationing enzyme preparations. In: Sbornik nauchnyh trudov GNU VNI-IPB "Perspektivnye fermentnye preparaty i biotehnologicheskie processy v tehnologijah produktov pitanija i kormov" [Proceedings of the GNU VNIIPB "Advanced enzyme preparations and biotechnological processes in food and feed technologies"]. Moscow, 2014: 101-8. (in Russian)

5. Eckhard U., Huesgen P.F., Brandstetter H., Overall C.M. Pro-teomic protease specificity profiling of clostridial collagenases reveals their intrinsic nature as dedicated degraders of collagen. J Proteomics. 2014; 100: 102-14.

6. Eckhard U., Schonauer E., Brandstetter H. Structural basis for activity regulation and substrate preference of clostridial collage-nases G, H, and T. J Biol Chem. 2013; 288: 20 184-94.

7. Nezafat, Navid & Negahdaripour, Monica & Gholami, Ahmad & Younes, Ghasemi. Computational analysis of collagenase from different Vibrio, Clostridium and Bacillus strains to find new enzyme sources. Trends Pharm Sci. 2015; 1 (4): 213-22.

8. Zhang Y.Z., Ran L.Y., Li C.Y., Chen X.L. Diversity, structures, and collagen-degrading mechanisms of bacterial collagenolytic proteases. Appl Environ Microbiol. 2015; 81 (18): 6098-107.

9. Miyoshi S., Nitanda Y., Fujii K., Kawahara K., Li T., Maehara Y., et al. Differential gene expression and extracellular secretion of the collagenolytic enzymes by the pathogen Vibrio parahaemolyticus. FEMS Microbiol Lett. 2008; 283 (2): 176-81.

10. Brazzelli M., Cruickshank M., Tassie E., McNamee P., Robertson C., Elders A., et al. Collagenase clostridium histolyticum for the treatment of Dupuytren’s contracture: systematic review and economic evaluation. Health Technol Assess. 2015; 19 (90): 201-2.

11. Arykbaeva A.B. Yeast Saccharomyces cerevisiae in genetic engineering. In: Sbornik "Al’manah nauchnyh rabot molodyh uchenyh Universiteta ITMO" [Collection "Almanac of scientific works of young scientists of ITMO University"]. Saint Petersburg, 2016: 88-91. (in Russian)

12. Rabert C., Weinacker D., Pessoa A., Farias Jr. Recombinants proteins for industrial uses: utilization of Pichia pastoris expression system. Braz J Microbiol. 2013; 44 (2): 351-6.

13. Queiroz Brito Cunha C.C., Gama A.R., Cintra L.C., Bataus L.A.M., Ulhoa C.J. Improvement of bread making quality by supplementation with a recombinant xylanase produced by Pichia pastoris. PLoS One. 2018; 13 (2): 1-14.

14. Macauley-Patrick S., Fazenda M.L., McNeil B., Harvey L.M. Heterologous protein production using the Pichia pastoris expression system. Yeast. 2005; 22: 249-70.

15. Silva V.C., Peres, M.F.S., Gattas E.A.L. Application of methylo-trophic yeast Pichia pastoris in the field of food industry - a review. J Food Agric Environ. 2009; 2: 268-73.

16. URL: https://www.ncbi.nlm.nih.gov

17. URL: https://eu.idtdna.com/calc/analyzer

18. Sun Q., Chen F., Geng F., Luo Y., Gong S., Jiang Z. A novel aspartic protease from Rhizomucor miehei expressed in Pichia pastoris and its application on meat tenderization and preparation of turtle peptides. Food Chem. 2017; 245: 570-7. URL: https:// doi.org/10.1016/j.foodchem.2017.10.113

All articles in our journal are distributed under the Creative Commons Attribution 4.0 International License (CC BY 4.0 license)

SCImago Journal & Country Rank
Scopus CiteScore
CHIEF EDITOR
CHIEF EDITOR
Viktor A. Tutelyan
Full Member of the Russian Academy of Sciences, Doctor of Medical Sciences, Professor, Scientific Director of the Federal Research Centre of Nutrition, Biotechnology and Food Safety (Moscow, Russia)

Journals of «GEOTAR-Media»