Collagenolytic proteases from Bacillus subtilis B13 and B. siamensis S6 and their specificity toward collagen with low hydrolysis of myofibrils

Highlights

• Proteases from Bacillus subtilis B13 and B. siamensis S6 effectively degraded collagen.

• A novel collagenolytic protease from B. siamensis was firstly reported.

• Two collagenolytic proteases specifically hydrolyzed collagen and elastin than myofibrillar protein.

• Proteases secreted by non-pathogenic strains could be potential meat tenderizer.

Abstract

To obtain proteases with high collagenolytic activity, 400 bacteria isolates were screened. The secreted proteases from Bacillus subtilis subsp. subtilis B13 (isolated from beef butchery) and B. siamemsis S6 (isolated from soil) which showed the two highest activity to hydrolyze collagen from bovine Achilles tendon were partially characterized. After enzyme fractionation, both enzymes had the optimum pH for the collagenolytic activity at pH 7.5. However, the optimum temperature of enzyme produced by S6 (60 °C) was higher than B13 (50 °C). The inhibitor study revealed that these proteases were mainly a member of serine proteases and some metalloproteases. Both enzymes were able to degrade collagen with multiple cleavage sites as observed by electrophoretic patterns. In contrast to papain and bromelain, these collagenolytic proteases showed strong hydrolysis toward collagen and elastin as well as beef intramuscular collagen with low beef myofibrillar protein degradation. Moreover, the lower digestion of myofibrillar protein and casein by these enzymes as compared with collagenase from Clostridium histolyticum were observed. These proteases could be applied as a meat tenderizer when collagen or connective tissue protein was responsible for the meat toughness.

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.