Bose Institute, Kolkata, India.
Biodegradation. 2011 Feb;22(1):153-61. doi: 10.1007/s10532-010-9384-6. Epub 2010 Jul 2.
After 24 h of incubation with only purified pectate lyase isolated from Bacillus pumilus DKS1 (EF467045), the weight loss of the ramie fibre was found to be 25%. To know the catalytic residue of pectate lyase the pel gene encoding a pectate lyase from the strain Bacillus pumilus DKS1 was cloned in E. coli XL1Blue and expressed in E. coli BL21 (DE3) pLysS. The pel gene was sequenced and showed 1032 bp length. After purification using CM-Sepharose the enzyme showed molecular weight of 35 kDa and maximal enzymatic activity was observed at 60°C and a pH range of 8.5-9.0. Both Ca²(+) and Mn²(+) ions were required for activity on Na-pectate salt substrates, while the enzyme was strongly inhibited by Zn²(+) and EDTA. The deduced nucleotide sequence of the DKS1 pectate lyase (EU652988) showed 90% homology to pectate lyases from Bacillus pumilus SAFR-032 (CP000813). The 3D structure as well as the catalytic residues was predicted using EasyPred software and Catalytic Site Atlas (CSA), respectively. Site directed mutagenesis confirmed that arginine is an essential catalytic residue of DKS1 pectate lyase.
在仅用从解淀粉芽孢杆菌 DKS1 中分离出的纯化果胶裂解酶孵育 24 小时后，发现苎麻纤维的失重率为 25%。为了了解果胶裂解酶的催化残基，从菌株解淀粉芽孢杆菌 DKS1 克隆编码果胶裂解酶的 pel 基因在大肠杆菌 XL1Blue 中表达，并在大肠杆菌 BL21(DE3)pLysS 中表达。 pel 基因测序并显示 1032 bp 长度。使用 CM-Sepharose 纯化后，该酶显示分子量为 35 kDa，最适酶活在 60°C 和 pH 范围为 8.5-9.0 时观察到。Ca²(+)和 Mn²(+)离子均为 Na-pectate 盐底物活性所必需，而 Zn²(+)和 EDTA 强烈抑制该酶。DKS1 果胶裂解酶（EU652988）的推导核苷酸序列与解淀粉芽孢杆菌 SAFR-032（CP000813）的果胶裂解酶显示出 90%的同源性。使用 EasyPred 软件和 Catalytic Site Atlas（CSA）分别预测了 3D 结构和催化残基。定点突变证实精氨酸是 DKS1 果胶裂解酶的必需催化残基。
