Bacterial strain aeromonassalmonicida subsp. salmonicida, a producer of collagenase 4459, subfamily of peptidases of class m9a

Abstract

FIELD: biotechnology.SUBSTANCE: strain of Aeromonassalmonicida subsp. salmonicida 4459, deposited in the Federal Budgetary Institution of Science State Research Center of Applied Microbiology and Biotechnology under number B-25756. Produces collagenase of the M9A subfamily. Collagenase is active in a wide range of temperatures: from +2 to +30 °C.EFFECT: strain allows to improve the quality of meat products, including poultry.1 cl, 1 dwg

Description

[0001] The invention relates to genetic engineering, biotechnology, microbiology and is a new strain of Aeromonas salmonicida subsp. salmonicida 4459, producing collagenase of the M9A subfamily, which can be used in the meat industry for hydrolysis and modification of collagen-containing raw materials in order to improve the quality of meat products (soften them), expand their range, as well as in the medical, food and light industries.

[0002] From the article - New approaches to the selection of peptidases of the M9 and M4 families for the enzymatic processing of meat and collagen-containing raw materials / M. Yu. Minaev, A. A. Eremtsova / / All about meat. - 2016. - No. 2. - P. 30-33 The M9 family of metallopeptidases is known from the microorganisms Clostridium histolyticum and Vibrio alginolyticus. These collagenases are distinguished by their ability to attack native triple-helix collagens types I, II and III to a mixture of small peptides, acting on the "X-Gly" bond, under physiological conditions. However, the introduction of such enzymes into the technological process is associated with a number of difficulties. The industrial use of microbial collagenases is limited by safety considerations associated with their potential toxicity and other adverse effects described by Zhang, Y.-Z. Diversity, Structures, and Collagen-Degrading Mechanisms of Bacterial Collagenolytic Proteases / Y.-Z. Zhang, L.-Y. Ran, C.-Y. Li, X.-L. Chen / / Applied and Environmental Microbiology. - 2015. - No. 81 (18) - P. 6098-6107.DOI: 10.1128 / AEM.00883-15.

[0003] To improve the quality of low-grade meat products characterized by a high content of connective tissue, enzymes with collagenase activity are required, the optimal activity of which is achieved at low positive temperatures corresponding to the conditions of raw material salting, namely at 2±2°C.

[0004] It is known that most collagenase-producing microorganisms are mesophilic and have an optimum temperature for enzyme activity in the range of 20-40°C. For example, the microorganism Achromobacter iophagus produces collagenase that is active in this temperature range, which was described in the article "Degradation of bovine muscle collagen by collagenase produced by the microorganism Achromobacter iophagus" (Bonnet M., J. Kopp, Proceedings European Meeting of Meat Research Workers, 1984, Bristol, Great Britain).

[0005] Some microorganisms, such as members of the genus Bacillus subtilis, are capable of forming spores, which creates additional difficulties when used in industrial settings. This aspect was discussed in the article "Collagenolytic Activity of Bacillus Species Isolated from Various Environmental Sources" (Slabospitskaya A.T., Krymovskaya S.S., Reznik S.R., Microbiological Journal, 1989, No. 6, pp. 54-56).

[0006] In addition, a number of microorganisms, such as Clostridium perfringens and Entomophthora coronata, are pathogenic, which limits their use in the food industry. These microorganisms and their enzymes are described in the article "Purification and characterization of a Clostridium histolyticum 120-kilodalton collagenase and nucleotide sequence of the corresponding gene" (Matsushita O., Yoshihara K., Natayama S. J. et al., J. Bacteriol., 1994, Vol. 176, pp. 149-156).

[0007] Collagenase enzyme preparations isolated from the hepatopancreas of the Kamchatka crab also exist; however, their temperature optimum is in the range of 60-65°C, making them unsuitable for use at low positive temperatures. This enzyme was studied in the work "Development of a Technology for Molded Beef Products Using an Enzyme Preparation" (Fedonin, M.Yu., Dissertation for the Degree of Candidate of Technical Sciences, Moscow, 2000, p. 159).

[0008] Thus, known enzymes with collagenase activity have a temperature optimum of action in the range from 20 to 65°C, which limits their use for processing raw materials at low positive temperatures required for curing meat products.

[0009] Serratia proteamaculans strain 94 is known to produce collagenase active at 4°C. However, this enzyme belongs to the Helicobacter-type subfamily, exhibits general proteolytic activity, and lacks sufficient specificity for use in processing collagen-containing raw materials. Data on this enzyme are available in the MEROPS database (https: / / www.ebi.ac.uk / merops / cgi-bin / pepsum?id=U32.002).

[0010] The aim of the invention is to obtain the Aeromonas salmonicida subsp. salmonicida 4459 strain (Figure 1)—a producer of M9A subfamily collagenase—for the development of biotechnology for the transformation of meat and collagen-containing raw materials. It acts on the Pro-X-Gly bond (where X is Leu, Ala, or Pro). It exhibits high specificity for collagen types 1 and 2. It exhibits no general proteolytic activity.

[0011] The strain is morphologically represented by Gram-negative, non-motile, ovoid rods measuring 0.8-1.3 x 1.3-2.0 µm. It does not form spores. The strain is a facultative aerobe, preferring to obtain energy by using oxygen as a terminal electron acceptor. The optimal growth temperature for this bacterium is from 22 to 25°C. The maximum temperature at which it can grow is 35°C. Approximately 24 hours after the onset of growth, the colonies reach the size of a pinhead. The colonies also have a brown pigment, which appears after 48-72 hours of growth. The indole formation test, Voges-Proskauer test, citrate utilization test, coagulase test, starch hydrolysis, triglyceride hydrolysis, phospholipid hydrolysis, hydrogen sulfide formation, citrate utilization test, and phenylalanine test give negative results.The oxidase test, lysine decarboxylase test, methyl red test, gelatin hydrolysis test and catalase test are positive.

[0012] Contains gelatinase enzyme (hydrolyzes gelatin), hydrolyzes casein.

[0013] When sown on blood agar, it forms gray-white, translucent colonies (1-3 mm in diameter) of an oily consistency without hemolysis. It forms a brown, water-soluble pigment on agar containing tyrosine or phenylalanine. It causes hemolysis.

[0014] The strain is stored as a lyophilized culture or in an agar medium under vaseline oil.

[0015] The strain produces collagenase across the entire temperature range.

[0016] Collagenase in the culture fluid is determined using the ninhydrin method. Native collagen (Sigma) is used as a substrate for determining collagenase activity. The optimum pH for collagenase activity is 7.5, and the optimal temperature is 25°C. Collagenolytic activity is maintained over a temperature range of +2°C to 30°C and a pH range of 5.5 to 8.5.

[0017] Example 1. For the cultivation of seed material and the fermentation process of the Aeromonas salmonicida subsp. salmonicida 4459 strain, a liquid nutrient medium LB (10 g / L peptone, 5 g / L yeast extract, 5 g / L sodium chloride) is used.

[0018] To obtain the inoculum, use test tubes containing 20% ​​gelatin, seeded with the culture (by injection). After dissolving the gelatin, 1 cm 3 The culture fluid is subcultured into flasks containing 100 ml of LB broth. Cultivation is carried out on a shaker (180-250 rpm) for 24 hours at a temperature of 20°C.

[0019] The collagenase activity in the culture fluid towards the Pro-Leu-Gly-Met-Trp-Ser-Arg substrate should be at least 16 U / μl.

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