牛凝乳酶的结晶及其二硫键的化学修饰  

Purification, Crystallization and Chemical Modification of Disulfide Bond of Calf Chymosin

作　　者：黄葵 陈世芝[2] 杨开宇[1] 

机构地区：[1]中国科学院微生物研究所 [2]中国科学院生物物理研究所

出　　处：《生物工程学报》1991年第1期77-83,共7页Chinese Journal of Biotechnology

摘　　要：为了配合凝乳酶的蛋白质工程,围绕酶的纯化,结晶和二硫键的化学修饰进行了研究。实验结果表明采用FPLC Mono Q柱代替DEAE-纤维素柱层析可以提高同工酶A,B及其降解组分C的分离效果并缩短纯化时间。以10mg/ml的纯凝乳酶B为出发材料,在4℃1.4—2.0mol/L氯化钠条件下进行培养可以获得外形近似为正立方体晶体。化学修饰的结果证明,凝乳酶的三对二硫键处于酶分子不同的空间部位。0.72mmol/L DTT在pH6.3条件下能还原处于分子表面的一对二硫键,经溴化氰降解分肽测定为Cys^(250)-Cys^(283),还原后引起活力下降25%。在二硫键之间引入汞原子其活力与还原酶,羧甲基化酶的活力大体相同,说明Cys^(250)-Cys^(283)在维持凝乳酶具有活力的空间构象中起着一定的作用。In order to carry out protein engineering of chymosin, purificationcrystallization and chemical modification of calf chymosin wereundertaken. The resoiution of iso-enzymes A,B and the degraded pro-duct, fraction C was improved and the period of purification was shor-tened by using fast protein liquid chromatograph. Crystal of approxima-tely cubic shape was obtained from 10mg/ml purified chymosin B at 4℃after successive dialysis against 1.4, 1.8 and 2.0mol/L NaCl in 0.1mol/Lsodium phosphate buffer. One disulfide bond located on the surface ofchymosin molecule was reduced by 0.72mmol/L DTT at pH6.3. Thereduced disulfide bond was identified as Cys^(250)-Cys^(285) by using themethod of cynogen bromide degradation and peptide separation. The reduction of one disulfide bond resulted in the loss of about25% activity.Carboxymethylated and mercurated derivatives exhibite thesimilar activity to that of the reduced enzyme suggesting that Cys^(250)-Cys^(283) plays some role in keeping the conformation with full activity.

关 键 词：牛凝乳酶 二硫键 化学修饰 

分 类 号：Q78[生物学—分子生物学]