Purification and characterization of Bacillus subtilis milk-clotting enzyme from Tibet Plateau and its potential use in yak dairy industry

Abstract

A milk-clotting enzyme named YS-1 was purified from a Bacillus subtilis (B. subtilis) YB-3, which we have isolated from Tibetan Plateau, Gansu, China. The enzyme YS-1 was identified as a metalloproteinase. SDS-PAGE and MALDI-TOF-MS analysis of the purified enzyme gave a molecular weight of 42 kDa. YS-1 was stable over a wide range of temperature from 20 to 60°C. Purified YS-1 was also active over a wide range of pH from 5.0 to 9.0. It can be activated by Ca 2? and Al 3? but inhibited by Zn 2? , Fe 2? and Cu 2? . The milk-clotting enzyme YS-1 exhibited high specificity to substrate b-casein and yak milk casein and led to a 75% more rapid coagulation of yak milk than cow milk, due to high b-casein content in yak milk. Together, our findings confirmed that the enzyme YS-1 has a potential to be used in yak cheese industry.

High Milk-Clotting Activity Expressed by the Newly Isolated Paenibacillus spp. Strain BD3526

“…The enzyme activity was completely inhibited by 5 mM of ethylenediaminetetraacetic acid (EDTA), indicating that it was a metalloprotease. This result suggested the protease was different from other reported bacterial coagulants, such as those from B. amyloliquefaciens D4 [35], B. amyloliquefaciens JNU002 [17], B. subtilis YB-3 [27] and B. licheniformis USC13 [29], in both molecular weight and type, as listed in Table 1.…”

“…The protease with MCA was accumulated in the late-exponential phase of BD3526 (12–18 h). The cultivation time required to reach the peak MCA by BD3526 was longer than those required by Enterococcus faecalis TUA2495L [26], B. subtilis YB-3 [27] and B. subtilis natto [28], but much shorter than those required by B. amyloliquefaciens JNU002 [17], B. subtilis [22], B. licheniformis USC13 [29], and fungal milk-clotting protease producers [24,30,31,32,33]. The rapid production of the enzyme by BD3526 seems to be a promising advantage for industrial purposes.…”

Purification and characterisation of κ-casein specific milk-clotting metalloprotease from Termitomyces clypeatus MTCC 5091

“…Milk clotting acid protease (religiosin) was reported from the latex of Ficus religiosa belonging to the serine protease group (Kumari, Sharma, & Jagannadham, 2010). Milk-clotting enzyme (YS-1) with high specificity to the substrate b-casein was purified and identified as a metalloproteinase from Bacillus subtilis (Li et al, 2012) and from Bacillus amyloliquefaciens D4 (He et al, 2011). Purification and characterisation of a milkclotting serine protease from Bacillus licheniformis strain USC13 was also reported (Ageitos, Vallejo, Sestelo, Poza, & Villa, 2007).…”

Optimization of the production and characterization of milk clotting enzymes by Bacillus subtilis natto

“…However, attention has been focused on the production of milk-clotting enzymes (MCEs) from microbial sources for use as rennin substitutes (Ayhan et al 2001 ; Cavalcanti et al 2004 ; Hashem 1999 ; Silveira et al 2005 ). Although there are many microorganisms that produce MCEs (Ding et al 2011 ; He et al 2011 ; Li et al 2012 ; Vishwanatha et al 2010 ), only the MCEs produced by strains of Rhizomucor miehei , Rhizomucor pusillus var. Lindt , Aspergillus oryzae and Enthothia parasitica are widely used (Birkkjaer & Jonk 1985 ; Crawford 1985 ; Thakur et al 1990a ).…”