Wu Fang-Chen, Chang Chen-Wei, Shih Ing-Lung
Department of Bioindustry Technology, Da-Yeh University, Changhua, Taiwan.
Springerplus. 2013 Dec;2(1):33. doi: 10.1186/2193-1801-2-33. Epub 2013 Jan 31.
Suitable medium for production of milk clotting enzyme (MCE) by Bacillus subtilis (natto) Takahashi in submerged liquid-state fermentation was screened, the nutrient factors affecting MCE production was optimized by response surface methodology. The MCE production by B. subtilis (natto) Takahashi was increased significantly by 428% in the optimal medium developed. The MCE was filtered and concentrated by ultrafiltration. The retentate after tandem filtration carried out with the combined membranes of MWCO 50kDa and 5 kDa showed two major bands between 25kDa and 30kDa on SDS-PAGE, and the MCA and MCA/PA improved significantly in comparison with those in the initial broth. The crude enzyme thus obtained showed MCA and MCA/PA ratio of 48,000 SU/g and 6,400, which are commensurate with those (MCA 26,667 SU/g and MCA/PA 6,667) of the commercial rennet. It had optimal pH and temperature at pH 6 and 60°C, and showed excellent pH and thermal stability.
筛选了适合纳豆芽孢杆菌在深层液态发酵中生产凝乳酶（MCE）的培养基，并通过响应面法优化了影响MCE产量的营养因素。在优化后的培养基中，纳豆芽孢杆菌产生的MCE产量显著提高了428%。通过超滤对MCE进行过滤和浓缩。使用截留分子量为50kDa和5kDa的复合膜进行串联过滤后的截留物在SDS-PAGE上显示出25kDa至30kDa之间的两条主要条带，与初始发酵液相比，凝乳活性（MCA）和凝乳活性/蛋白酶活性（MCA/PA）显著提高。由此获得的粗酶的MCA和MCA/PA比值分别为48,000 SU/g和6,400，与商业凝乳酶的比值（MCA 26,667 SU/g和MCA/PA 6,667）相当。其最适pH和温度分别为pH 6和60°C，并且具有出色的pH和热稳定性。
