摘要
采用“界面亲和层析” ,从商品Candidarugosa脂肪酶 (CRL)中分离到三个同工酶 (CRL 1、CRL 2和CRL 3) ,它们在水 有机溶剂双液相体系中催化 (R ,S) 萘普生甲酯的不对称水解反应 ,具有不同的立体选择性 .分析表明 :CRL 1和CRL 2上不同程度地非共价结合有小分子的酸性化合物 ,阻碍了其活性位点处疏水腔的完全开放 ;CRL 3上不含有该小分子酸性化合物 ,活性位点处疏水腔可处于完全开放构象 .据此分别将CRL同工酶选择性地固定在不同的载体 (GDX10 1和YWG NH2 )上 .通过简单易行的选择吸附步骤 ,可同时达到同工酶的分离及固定化目的 。
By “interfacial affinity chromatography” on GDX101 column, commercial Candida rugosa lipase (CRL) is fractionated into four fractions containing three isoenzymes (CRL 1, CRL 2 and CRL 3). They have different enantioselectivity for the asymmetric hydrolysis of (R, S) Naproxen methyl ester in the aqueous organic solvent biphase system. As analyzed on SDS polyacrylamide gel electrophoresis, isoelectric focusing and organic solvent treatment, slight structural difference among these isoenzymes has been found. CRL 1 and CRL 2 are associated noncovalently with low molecular mass acidic components in different degree, CRL 3 is disassociated with the low molecular mass acidic components. Because of the perhaps hamper of the low molecular mass acidic components, the open extent of hydrophobic pocket around active site on CRL 3 is bigger than on CRL 1 and CRL 2. According to the slight structural difference, isoenzymes with different enantioselectivity for hydrolysis of Naproxen ester have been immobilized selectively on different hydrophobic supports (GDX101 and YWG NH 2). Via a sample and easily performed selectivity adsorption step, the purification can be combined with the immobilization. This new method seems to be very suitable for an easily separation of such isoenzymes with slightly different structure.
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2000年第5期513-516,共4页
Progress In Biochemistry and Biophysics
基金
中国科学院重点项目!(KY95 S1 2 6 2 ) .
关键词
CRL同工酶
分离
界面亲和层析
选择性固定化
CRL isoenzymes, separation,interfacial affinity chromatography, selectivity immobilization
