Candida rugosa脂肪酶同工酶的选择固定化

采用DEAE－Sepharose CL-6B离子交换层析，将Candida rugosa脂肪酶（CRL）分成了含同工酶CRL A和CRL B的3个组份，CRL A和CRL B在低水－有机溶剂双液相体系中催化（R，S）－普生甲酯的不对称水解反应，具有不同的对映体选择性。SDS聚丙烯酰胺凝胶电泳分析发现，CRL A和CRL B的分子量几乎相同（约为62000－64000Da)。等电聚焦显示CRL A在等电点pI5.6处有单一条带，CRL B在等电点pI4.2附近有2条带。圆二色谱分析未发现明显结构差异，但在极低的离子强度下它们在疏水载体YWG－C6H5上的吸附速度明显不同。50％异丙醇处理发现，CRL B可解离为CRL A和小分子酸性化合物。据此认为CRL A和CRL B在结构组成上存在着轻微差别，CRL A的活性位点处疏水腔的开放程度较CRL B大，CRL B上非共价结合有一些小分子酸性化合物。据此，分别将其选择性地固定在了疏水性不同的载体YWG－C6H5和YWG－NH2上。本文通过一个简单易行的选择吸附步骤、同时达到了同工酶的分离纯化及固定化目的，提供了一种分离结构上相近的同工酶的便利方法。

SELECTIVE IMMOBILIZATION OF Candida rugosa LIPASE ISOENZYME

By anion-exchange chromatography on DEAE-Sepharose CL-6B column, commercial Candida rugosa lipase (CRL) was fractionated into three fractions containing two isoenzyme (CRL A and CRL B). they have different enantioselectivity for asymmetric hydrolysis of (R, S)-Naproxen methyl ester in the low aqueous-organic solvent biphase system. As analyzed on polyacrylamide gel electrophoresis in denaturing conditions (SDS-PAGE), CRL A and CRL B both have the same molecular weight range of 62000-64000Da. On isoelectric focusing, CRL A showed a single band corresponding to an isoelectric point (pI) of 5.6 and CRL B was resolved in two bands having pI values around 4.2. No obvious difference had been find between CRL A and CRL B by circular dichroism spectroscopy, however, the two iso-enzyme have obvious different adsorption rates on hydrophobic support YWG-C6H5 at very low ionic strength. CRL B can be converted irreversibly into CRL A and some low molecular weight acidic compound by organic solvent (50% 2-propanol) treatment. By these analysis, slight structural difference between two iso-enzymes had been find, the open extent of hydrophobic pocket around active site of CRLA is bigger than that of CRLB, and the CRLB is associated noncovalently with low molecular weight acidic components. Due to the slight structural difference, two iso-enzymes have been adsorbed selectively on different hydrophobic supports (YWG-C6H5 and YWG-NH2). Via a simple and easily performed selective adsorption, we are able to combine the purification with the immobilization. This method is suitable for separation of slightly structure different iso-enzymes.