胶原蛋白酶产生菌的筛选及酶的分离纯化

Screening of collagenase-producing strain and purification of Bacillus cereus collagenase

摘要:

从堆积骨骼的土样中筛选出高产胶原蛋白酶的MBL13菌株，经鉴定为蜡样芽孢杆菌Bacillus cereus。对其所产的胶原蛋白酶BCC进行分离纯化，并进行酶学性质的研究。从菌株的发酵液中纯化出分子量约为38.0 kDa BCC酶。酶反应的最适温度为40℃，最适pH为8.0。在50℃以下稳定，60℃保温1 h酶活仅保留10％；在pH 7.0~8.5活性较稳定；金属离子Ca2+、Zn2+、Mg2+对酶有激活作用，金属离子Cu2+对酶有显著的抑制作用。EDTA和EGTA能抑制该酶，表明BCC酶为一种金属蛋白酶。酶的底物特异性表明该酶为骨胶原蛋白酶，且对Ⅰ型骨胶原蛋白水解能力极显著高于Ⅱ型胶原蛋白和Ⅲ型胶原蛋白。将纯化的BCC酶应用于骨胶原蛋白的水解可以得到不同链长的多肽，其水解能力高于标准品胶原酶Ⅰ型。本研究为工业酶提供了新的菌株和新型胶原蛋白酶，为胶原蛋白酶的开发提供了重要的理论依据。

Abstract:

We isolated the strain MBL13 with high collagenase productivity from the soil of piled up animal bones. It was identified as Bacillus cereus. We purified and characterized Bacillus cereus collagenase (BCC). The molecular weight of BCC was 38.0 kDa and the optimum temperature and pH for the enzyme activity were 40?C and 8.0 respectively. The enzyme was stable when the temperature was below 50?C, but only retained 10% activity when kept at 60?C for 1 h. The enzyme activity was stable between pH 7.0–8.5. Some metal ions such as Ca2+, Zn2+, Mg2+ enhanced the enzyme activity, and Cu2+ brought the obvious inhibition. In addition, EDTA and EGTA could inhibit the enzyme activity. We suggested that the purified enzyme was a member of the metalloproteases. Based on the experiment of substrate specificity, we found that the purified enzyme was bone collagenolytic protease, and had a much stronger capacity of hydrolysis for type I collagen than that for type II collagen and type III collagen. By BCC hydrolyzing bone collagen, we obtained polypeptides with different chain lengths. The comparative test indicated that the hydrolysis capacity of BBC was higher than that of standard type I collagenase. The results introduced a new strain and a novel collagenolytic protease for industrial enzyme.