Klomklang Wilailak, Tani Akio, Kimbara Kazuhide, Mamoto Rie, Ueda Takashi, Shimao Masayuki, Kawai Fusako
Research Institute for Bioresources, Okayama University, Kurashiki, Okayama 710-0046, Japan.
Faculty of Nutrition, Kobegakuin University, Kobe, Hyogo 651-2180, Japan.
Microbiology (Reading). 2005 Apr;151(Pt 4):1255-1262. doi: 10.1099/mic.0.27655-0.
Oxidized polyvinyl alcohol hydrolase (OPH) and polyvinyl alcohol dehydrogenase were found to be constitutively present in the periplasm of Sphingomonas sp. strain 113P3 (formerly Pseudomonas sp. 113P3). The OPH was purified to homogeneity with a yield of 40 % and a 5.9-fold increase in specific activity. The enzyme was a homodimer consisting of 35 kDa subunits. Its activity was inhibited by PMSF, Hg(2+) and Zn(2+). The enzyme hydrolysed oxidized polyvinyl alcohol (oxidized PVA) and p-nitrophenyl acetate (PNPA), but did not hydrolyse any of the mono- or diketones tested. K(m) and V(max) values for oxidized PVA and PNPA were 0.2 and 0.3 mM, and 0.1 and 3.4 micromol min(-1) mg(-1), respectively. The gene for OPH was cloned and sequenced. Sequencing analysis revealed that the open reading frame consisted of 1095 bp, corresponding to a protein of 364 amino acids residues, encoding a signal peptide and a mature protein of 34 and 330 amino acids residues, respectively. The presence of a serine-hydrolase motif (a lipase box; Gly-X-Ser-X-Gly) strongly suggested that the enzyme belongs to the serine-hydrolase family. The protein exhibited homology with OPH of the Pseudomonas sp. strain VM15C (63 % identity) and the polyhydroxybutyrate depolymerases from Mesorhizobium loti, Rhizobium sp. and Sinorhizobium meliloti (29-32 % identity). The oph gene was expressed in Escherichia coli under the control of the lac promoter. The recombinant protein had the same molecular mass and N-terminal amino acid sequence as the purified OPH from strain 113P3.
在鞘氨醇单胞菌属菌株113P3（以前称为假单胞菌属113P3）的周质中发现氧化聚乙烯醇水解酶（OPH）和聚乙烯醇脱氢酶是组成型存在的。OPH被纯化至同质，产率为40%，比活性增加了5.9倍。该酶是由35 kDa亚基组成的同型二聚体。其活性受到苯甲基磺酰氟（PMSF）、汞离子（Hg(2+)）和锌离子（Zn(2+)）的抑制。该酶能水解氧化聚乙烯醇（氧化PVA）和对硝基苯乙酸酯（PNPA），但不能水解所测试的任何单酮或二酮。氧化PVA和PNPA的米氏常数（K(m)）和最大反应速度（V(max)）值分别为0.2和0.3 mM，以及0.1和3.4 μmol min(-1) mg(-1)。OPH基因被克隆并测序。序列分析表明，开放阅读框由1095 bp组成，对应于一个364个氨基酸残基的蛋白质，分别编码一个信号肽和一个由34个和330个氨基酸残基组成的成熟蛋白。丝氨酸水解酶基序（脂肪酶框；甘氨酸- X -丝氨酸- X -甘氨酸）的存在强烈表明该酶属于丝氨酸水解酶家族。该蛋白质与假单胞菌属菌株VM15C的OPH具有同源性（63%的同一性），与百脉根中生根瘤菌、根瘤菌属和苜蓿中华根瘤菌的聚羟基丁酸酯解聚酶具有同源性（29 - 32%的同一性）。oph基因在乳糖启动子的控制下在大肠杆菌中表达。重组蛋白具有与从菌株113P3纯化的OPH相同的分子量和N端氨基酸序列。
