Purification and Characterization of an Extracellular Proteinase Having Milk-Clotting Activity from Enterococcus faecalis TUA2495L

Abstract

Lactic acid bacteria (157 stock cultures) were screened for their ability to produce extracellular proteinase with milk-clotting activity. A strain identified as Enterococcus faecalis TUA2495L showed the highest ratio of milk-clotting activity (MCA) to proteinase activity (PA). The molecular weight of the purified enzyme from the strain was estimated to be 34-36 kDa by gel filtration and by SDS-PAGE. Its isoelectric point was about 5.4, and the K m value on casein (Hammarsten) was 0.61% (w/v). The optimum temperature was 70˚C for MCA and 50˚C for PA. The MCA increased with a decrease in the pH from 7.8 to 5.8 but the PA was optimum at pH 8.0-9.0. Both MCA and PA were stable within the pH range of 5.

Purification and partial characterization of milk-clotting enzyme extracted from glutinous rice wine mash liquor

“…3). This result is in accordance with the findings of other researchers who also reported enzymes with almost similar molecular weight, like milk-clotting enzymes of Enterococcus faecalis TUA2495L (34.0-36.0 kDa) [33], E. paraitica (37.5 kDa) [34] and M. miehei (34.0-39.0 kDa) [24]. Furthermore, calf rennet which is considered to be the best milk-clotting enzyme for cheese production, also has a similar molecular weight of 35.65 kDa [35].…”

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

“…While T. clypeatus enzyme was especially active on j-casein initially as depicted in Fig. 3c block c, its degradation patterns were very similar to those of R. miehei milk clotting enzyme and Enterococcus faecalis TUA2495L protease (Sato, Tokuda, Koizumi, & Nakanishi, 2004). Thus, the cleavage sites of j-casein by E. faecalis enzyme, R. miehei enzyme and AcP s are almost the same.…”

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

“…2, lane 1). MCE from different sources has been purified using a range of chromatographic techniques [7,12,18,27], but only limited numbers can be used as calf chymosin substitutes, in which even few can be used in yak cheese industry. The results suggested that MCE YS-1 has a great potential to be used in yak cheese industry.…”

“…The proteolytic behavior of MCE isolated from Rhizomucor pusillus and B. subtilis YB-3 was examined as described [18]. A solution (200 ll, 0.4%) of a-casein, b-casein, and j-casein (Sigma, St. Louis, USA) in 0.5 mM phosphate buffer (pH 6.0) was mixed with 10 ll of enzyme solution (300 SU/ml).…”