摘要
胶原蛋白酶是特异性水解天然胶原蛋白或明胶的酶类。为制备新型的骨胶原蛋白酶并探讨其降解胶原蛋白的能力,通过采用硫酸铵分级沉淀、DEAE-Sepharose Fast Flow离子交换层析、Sephadex G-100凝胶层析等分离纯化步骤,从Bacillus cereus MBL13-U菌株的粗酶液中分离纯化出一种特异性降解牛骨胶原蛋白的胶原蛋白酶(bone-specific collagenase,BSC),对该酶的分子质量、底物特异性和降解能力进行了分析,并构建出其降解牛骨胶原蛋白的动力学模型。结果表明:分离纯化的BSC比活力为5.57×103U/mg,纯化倍数达到42.85倍,分子质量约为52.0 k Da。经特异性分析该酶为骨胶原蛋白酶,且有显著的水解I型胶原蛋白的能力。其水解能力优于其他常用的蛋白酶。构建出该酶降解牛骨胶原蛋白的动力学模型为:降解速率R=(583.813 6E_0+1.296 7S_0)exp(-0.119 0DH),水解度DH=8.403 4ln(1+69.473 8(E_0/S_0)t+0.154 3)t,酶的失活常数K4为64.115 7/h。
Collagenase is a kind of enzymes that specifically hydrolyze natural collagen or gelatin. In order to prepare a new type of collagen protease and explore its ability to degrade collagen,a novel bone specific collagenase（ BSC） was obtained from Bacillus cereus crude enzyme liquid through the purification steps of ammonium sulfate precipitation,DEAE-Sepharose Fast Flow ion exchange chromatography and Sephadex G-100 gel chromatography. Furthermore,its molecular weight of the purified enzyme,substrate specificity and degradation ability were analyzed.What＇s more,the kinetic model of hydrolyzing bovine bone collagen was constructed. The results showed that the specific activity of BSC was 5. 57 × 103 U/mg,the purification times was up to 42. 85 times,and its molecular weight was about 52. 0 k Da. The specificity analysis showed that the substrate was a bone collagen enzyme,and its ability to hydrolyze type I collagen was significant. The comparative test indicated that the hydrolysis capacity of BSC was higher than that of other proteases. The dynamic model of the enzymatic degradation of collagen was as follows： the degradation rate was R =（ 583. 813 6 E_0＋ 1. 296 7 S_0） exp（-0. 119 0 DH）,degree of hydrolysis was DH = 8. 403 4 ln（1 ＋ 69. 473 8 （E_0/S_0） t ＋ 0. 154 3）t,Enzyme inactivation constant was K_4= 64. 115 7 h^（-1）. The results provided a novel collagenolytic protease for the development of animal bone collagen.
出处
《食品与发酵工业》
CAS
CSCD
北大核心
2017年第12期13-19,共7页
Food and Fermentation Industries
基金
国家自然科学基金(31401622)
公益性行业(农业)科研专项(201303084)
河南省重点攻关项目(152102110080)
河南省教育厅自然科学研究项目(13A550255)
河南省重大专项(161100110900)
关键词
胶原蛋白
纯化
骨胶原蛋白酶
动力学
降解
collagen
purification
bone specific collagenase （BSC）
kinetics
degradation
