Purification and properties of milk‐clotting enzyme from Bacillus subtilis K‐26

Abstract

A milk-clotting enzyme from Bucillirs sirhrilis K-26 was purified by gel filtration and ion-exchange chromatography resulting in a 24-fold increase in specific activity with an 80% yield. Polyacrylamide gel electrophoresis and ultracentrifugal analysis revealed that the purified enzyme was homogeneous and had a molecular weight of 27,000 and a K , of 2.77 mgiml for K-casein. The enzyme was most stable at pH 7.5 and showed increasing clotting activity with decrease in milk pH up to 5.0. The maximum milk-clotting activity was obtained at 60°C. but the enzyme was inactivated by heating for 30 min at 60°C. The enzyme was irreversibly inhibited by EDTA and unaffected by DFP. Heavy-metal ions (Hg2+, Pb2+) inactivated the enzyme.

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

“…faecalis enzyme was about 34,000-36,000, which is similar to those of MCE from Irepex lacteus (34,000-36,000) (Kawai, 1971;Kobayashi et al, 1983), E. parasitica (37,500) (Hagemeyer et al, 1968), M. miehei (34,000-39,000) (Sternberg, 1971), and M. pusillus (29,000-30,600) (Arima, 1968), but different from that from B. subtilis K-26 (27,000) (Rao & Mathur, 1979). The isoelectric point of the Ec.…”

“…In addition, the pH stability of the Ec. faecalis enzyme (pH 5.5-10) was also different from those of the MCE from M. miehei (pH 4-8) (Sternberg, 1971), M. pusillus (pH 4-8) (Iwasaki et al, 1967a), I. lacteus (pH 3-6) (Kawai, 1971;Kobayashi, 1983), and B. subtilis K-26 (most stable at pH 7.5) (Rao & Mathur, 1979). As in the case of MCE from B. subtilis, the Ec.…”

“…These have been purified, and their enzymatic properties investigated in detail. However, reports on bacterial MCE are limited to those from Bacillus (Melachouris & Tuckey, 1968;Srinivasan et al, 1964;Rao & Mathur, 1979), and there is yet no report on the production of MCE by LAB.…”

Production of milk-clotting enzyme by Bacillus subtilis B1 from wheat bran

“…The MCE from B. subtilis B1 was proved as an acid protease with optimum pH 5.5, although, commercial proteases from the genus Bacillus were mainly neutral and alkaline (Dutt et al, 2008). Its optimum pH was consistent with that of M. miehei, E. faecalis TUA2495L and B. subtilis K-26 (Rao and Mathur, 1979;D'souza and Pereira, 1982;Sato et al, 2004). Gels made in low pH appeared to have a denser and more interconnected structure (Esteves et al, 2003).…”

“…Most of the microorganisms mentioned were screened from soil, natto, milk or glutinous rice wine samples (Rao and Mathur, 1979;D'souza and Pereira, 1982;Sato et al, 2004;Dutt et al, 2008). Microorganisms from different sources allow a variety of cheeses with different rheological and organoleptic properties (Silva and Malcata, 2005;Ageitos et al, 2006).…”