Collagenolytic proteases from Bacillus subtilis B13 and B. siamensis S6 and their specificity toward collagen with low hydrolysis of myofibrils
Introduction
Tenderness is the most important quality trait among sensorial characteristics of meat for consumer acceptability. There are two primary muscle proteins impacting meat tenderness, the myofibrils (or the actomyosin effect) or the connective tissue contribution (or the background effect). After exsanguination and rigor development, a meat becomes to post-mortem aging phase in which the calpain system is the primary enzyme responsible for structure disruption of myofibrillar proteins through z-disk degradation and then meat tenderization (Taylor, Geesink, Thompson, Koohmaraie, & Goll, 1995). However, the collagen that is the main component of connective tissue is not degraded by calpain (Purslow, 2005). In case of any meat cuts with high collagen content, the toughness is still present. Due to its typical structure, collagen is resistant to most common proteases but can be degraded by collagenolytic proteases from a few types of mammalian matrix metalloproteases (MMPs), mammalian cysteine proteases, and some bacterial proteases (Zhang, Ran, Li, & Chen, 2015).
Among various sources of bacterial collagenolytic proteases, proteases derived from Clostridium histolyticum, Vibrio alginolyticus and Porphyromonas gingivalis (Duarte, Correia, & Esteves, 2016; Petrova, Shishkov, & Vlahov, 2006) play an important role in controlling biotechnological processes. However, these bacteria are pathogens that potentially produce toxins, leading to the limit application of collagenolytic proteases in food or meat (Duarte et al., 2016). Among bacterial non-pathogenic strains, Bacillus is the most investigated species producing extracellular proteases. Previous studies have been remarkable progressed in identifying new bacterial collagenolytic proteases, including B. subtilis FS-2 (Nagano & To., 2000), B. pumilus Col-J (Wu, Li, Li, Chen, & Shuliang, 2010), B. licheniformis F11.4 (Baehaki, Sukarno, Syah, Setyahadi, & Suhartono, 2014), and B. polyfermenticus SCD (Choi et al., 2013). However, bacterial proteases from those studies were screened for general application with different hydrolysis profiles toward protein substrates. In the case of meat, to prevent over-tenderization, the ideal of meat tenderizer would be proteases specific toward only connective tissue with little degradation of myofibrillar protein. This concept must be proved before applying to meat application, but there is limited research concerning this evidence. Furthermore, bacteria isolated from different sources may contribute to a novel discovered collagen-degrading enzyme. Therefore, this research aimed to screen, identify, and characterize the collagenolytic proteases produced by selected strains with high hydrolytic properties toward collagen but low myofibrillar protein cleavage.
Section snippets
Materials
Collagen from bovine Achilles tendon (C9879), elastin from bovine neck ligament (E1625), collagenase from Clostridium histolyticum (C9891), papain from papaya latex (P3375), bromelain from pineapples (1016651), and phenylmethylsulfonyl fluoride (PMSF) were purchased from Sigma-Aldrich. Protease inhibitor set (11206893001) was purchased from Roche. Perfect Protein™ Markers (69,079–3) was purchased from Millipore. Microbiological media were purchased from Merck. GF-1 bacterial DNA extraction kit
Isolation and screening of bacterial collagenolytic proteases
Among 400 strains that isolated from soil, fish docks, fermented fishery products, and beef from a butcher shop, bacterial strains with collagenolytic protease producing ability above 0.5 U/mL were observed in 20 strains (Fig. 1). Moreover, out of 20 stains, only six isolated displayed higher collagenolytic activity than 4 U/mL in which the strain B13 showed the highest activity, followed by S6, B4, B12, S13, and KP11, respectively. These strains were selected for further bacterial
Conclusion
Collagenolytic proteases producing bacteria, B. subtilis B13 and a novel protease from B. siamensis S6, were isolated from beef butchery and soil, respectively. These fractionated proteases had the optimum pH at pH 7.5. Protease from S6 had the optimum temperature at 60 °C, while B13 showed a lower optimum temperature (50 °C). The protease's inhibitors patterns revealed that these proteases mainly belonged to the serine proteinases with some metalloproteases. Unlike other nonspecific proteases
CRediT authorship contribution statement
Supaluk Sorapukdee: Conceptualization, Methodology, Investigation, Writing - review & editing. Punnanee Sumpavapol: Resources, Methodology. Soottawat Benjakul: Resources. Pussadee Tangwatcharin: Methodology, Investigation.
Acknowledgments
This work is supported by the Thailand Research Fund (TRF) through Research Grant for New Scholar (grant number of MRG6180240). The authors would like to sincerely thanks to King Mongkut's Institute of Technology Ladkrabang, Thailand (grant number of 2562-02-04-002) for the additional financial support.
References (33)
- et al.
Characterisation of kiwifruit and asparagus enzyme extracts, and their activities toward meat proteins
Food Chemistry
(2013) - et al.
Discovery of marine Bacillus species by 16S rRNA and rpoB comparisons and their usefulness for species identification
Journal of Microbiological Methods
(2009) - et al.
Thermostable collagenolytic activity of a novel thermophilic isolate, Bacillus sp. strain NTAP-1
Journal of Bioscience and Bioengineering
(2000) Intramuscular connective tissue and its role in meat quality
Meat Science
(2005)- et al.
Application of exogenous enzymes to beef muscle of high and low-connective tissue
Meat Science
(2010) - et al.
Production and properties of two collagenases from bacteria and their application for collagen extraction
New Biotechnology
(2011) - et al.
Hydrolysis of insoluble collagen by deseasin MCP-01 from deep-sea Pseudoalteromonas sp. SM9913: Collagenolytic characters, collagen-binding ability of C-terminal polycystic kidney disease domain, and implication for its novel role in deep-sea sedimentary particulate organic nitrogen degradation
Journal of Biological Chemistry
(2008) - et al.
Production and characterization of collagenolytic protease from Bacillus licheniformis F11.4 originated from Indonesia
Asian Journal of Chemistry
(2014) - et al.
Handbook of proteolytic enzymes
(2004) - et al.
Foodborne pathogens, hazards, risk analysis and control
(2009)
Benson's microbiological applications: Laboratory manual in general microbiology, concise version
Adding enzymes to improve beef tenderness. Beef fact. product enhancement
Purification and characterization of heat-tolerant protease produced by Bacillus polyfermenticus SCD
Journal of Microbiology and Biotechnology
Purification and characterization of proteases from Bacillus amyloliquefaciens isolated from traditional soybean fermentation starter
Journal of Agricultural and Food Chemistry
An overview of Bacillus proteases: From production to application
Critical Reviews in Biotechnology
Bacterial collagenase-A review
Critical Reviews in Microbiology
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