Maqbool Qurrat A, Johri Sarojini, Verma Lata, Riyaz-ul-Hassan S, Verma Vijeshwar, Koul Surrinder, Taneja Subhash C, Parshad Rajinder, Qazi Ghulam N
Biotechnology Division, Regional Research Laboratory, Canal Road, Jammu Tawi-180001, India.
Biotechnol Appl Biochem. 2002 Dec;36(3):227-34. doi: 10.1042/ba20020045.
Screening of the micro-organisms from an in-house microbial culture repository, identified a bacterial strain bearing membrane-bound, inducible ester hydrolase activity. The strain designated as RRL-BB1 has been identified as Bacillus subtilis by 16 S rRNA typing. Its application in the kinetic resolution of several racemates, including drug intermediates, showed moderate to high enantioselectivity. The enzyme, designated as BBL, exhibited high enantioselectivity (ee approximately 99%) with acyl derivatives of unsubstituted and substituted 1-(phenyl)ethanols and 1-(6-methoxy-2-naphthyl)ethanols. With acyl derivatives of 2-(6-methoxy-2-naphthyl)propan-1-ol, moderate enantioselectivity was observed. The enzyme also showed moderate enantioselectivity with alkyl esters of carboxylic acids i.e. 2-bromopropanoic acid and 2-hydroxy-4-phenylbutanoic acid. The enzyme was purified to >90% purity from cell-free extract of RRL-BB1 with 26% overall yield. The purified enzyme exhibited hydrolase activity without any noticeable decrease in the rate of hydrolysis or the enantioselectivity profile. A specific activity of 450 units/mg protein resulted after at least a 200-fold purification of the crude cell-free extract. The key purification step was the irreversible adsorption of the salt-precipitated crude enzyme on hydrophobic resin, in the presence of a low salt concentration, and desorption of the enzyme with a linear gradient of 1% sodium cholate. The purified enzyme was a 45 kD monomer as shown by SDS/PAGE. The N-terminal amino acid sequence of the purified enzyme was determined as Thr-Lys-Leu-Thr-Val-Gln-Thr-Arg-Asp-Gly-Ala-Leu-Arg-Gly-Thr. The N-terminal sequence did not bear any homology with other known bacterial lipases. BBL is maximally active at 37 degrees C, pH 8.0 and fairly stable up to 40 degrees C, pH 6-10. The enzyme is insensitive to EDTA but inhibited by serine protease inhibitor PMSF. Its activity (72%) was retained in the presence of the anionic detergent SDS at a concentration of 0.2% (w/v).
从内部微生物培养库中筛选微生物时，鉴定出一种具有膜结合、可诱导酯水解酶活性的细菌菌株。命名为RRL - BB1的菌株通过16S rRNA分型被鉴定为枯草芽孢杆菌。它在包括药物中间体在内的几种外消旋体的动力学拆分中的应用显示出中等至高的对映选择性。命名为BBL的酶对未取代和取代的1 -（苯基）乙醇以及1 -（6 - 甲氧基 - 2 - 萘基）乙醇的酰基衍生物表现出高对映选择性（ee约为99%）。对于2 -（6 - 甲氧基 - 2 - 萘基）丙 - 1 - 醇的酰基衍生物，观察到中等对映选择性。该酶对羧酸的烷基酯即2 - 溴丙酸和2 - 羟基 - 4 - 苯基丁酸也表现出中等对映选择性。该酶从RRL - BB1的无细胞提取物中纯化至纯度>90%，总产率为26%。纯化后的酶表现出水解酶活性，水解速率或对映选择性谱没有明显降低。粗无细胞提取物经过至少200倍纯化后，比活性达到450单位/毫克蛋白质。关键的纯化步骤是在低盐浓度下，盐沉淀的粗酶不可逆地吸附在疏水树脂上，然后用1%胆酸钠的线性梯度洗脱酶。如SDS/PAGE所示，纯化后的酶是一种45 kD的单体。纯化酶的N端氨基酸序列确定为苏氨酸 - 赖氨酸 - 亮氨酸 - 苏氨酸 - 缬氨酸 - 谷氨酰胺 - 苏氨酸 - 精氨酸 - 天冬氨酸 - 甘氨酸 - 丙氨酸 - 亮氨酸 - 精氨酸 - 甘氨酸 - 苏氨酸。N端序列与其他已知细菌脂肪酶没有任何同源性。BBL在37℃、pH值8.0时活性最高，在40℃、pH值6 - 10范围内相当稳定。该酶对EDTA不敏感，但被丝氨酸蛋白酶抑制剂PMSF抑制。在0.2%（w/v）的阴离子洗涤剂SDS存在下，其活性保留了72%。
