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Purification and characteristics of a new milk-clotting enzyme from Bacillus licheniformis BL312

Author:
Zhang, Yao, Xia, Yongjun, Ding, Zhongyang, H. Lai, Phoency F., Wang, Guangqiang, Xiong, Zhiqiang, Liu, Xiaofeng, Ai, Lianzhong
Source:
Lebensmittel-Wissenschaft + [i.e. und] Technologie 2019 v.113 pp. 108276
ISSN:
0023-6438
Subject:
Bacillus licheniformis, alphaS-casein, ammonium sulfate, beta-casein, calcium chloride, calves, cheesemaking, chymopapain, fractionation, gels, hydrolysates, hydrolysis, kappa-casein, mass spectrometry, milk clotting, pH, polyacrylamide gel electrophoresis, rennet, reversed-phase high performance liquid chromatography, sodium dodecyl sulfate, temperature
Abstract:
A new milk-clotting enzyme (MCE) was produced from Bacillus licheniformis BL312. The characteristics of BL312 MCE were compared with those of Calf rennet, R. miehei MCE, and Chymopapain. BL312 MCE showed a remarkable activity of 5291 SU/mg after purification by ammonium sulfate fractionation and DEAE-Sepharose Fast Flow, which yielded 5.09-fold purification with a recovery of 39.24%. The purified BL312 MCE gave a single protein band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel, corresponding to 27 kDa. The αs-casein (αs-CN) and β-casein (β-CN) hydrolysates generated by BL312 MCE were different from those by Calf rennet, R. miehei MCE, and Chymopapain through SDS-PAGE and reversed-phase HPLC (RP-HPLC) analyses. The main cleavage sites in κ-casein (κ-CN) were the Met106-Ala107 and Asn123-Thr124 bonds for BL312 MCE, as determined by mass spectrometry analysis. BL312 MCE hydrolyzed αs-CN to a greater level than β-CN. It was stable at a wide pH range of 5.5–11.0 and temperature below 45 °C. The milk-clotting activity (MCA) reached the maximum when the substrate contained 50 mM CaCl2 at pH 5.5. The optimal temperature of MCE was 55 °C. The successful application in Monascus-fermented cheese production confirmed the usefulness of BL312 MCE in food and dairy processing.
Agid:
6473979