Kim Do Young, Kim Hyun Chul, Kim Sun Young, Rhee Young Ha
Department of Microbiology, School of Bioscience and Biotechnology, Chungnam National University, Daejeon 305-764, Republic of Korea.
J Microbiol. 2005 Jun;43(3):285-94.
A bacterial strain M4-7 capable of degrading various polyesters, such as poly(epsilon-caprolactone), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(3-hydroxyoctanoate), and poly(3-hydroxy-5-phenylvalerate), was isolated from a marine environment and identified as Pseudomonas alcaligenes. The relative molecular mass of a purified extracellular medium-chain-length poly(3-hydroxyalkanoate) (MCL-PHA) depolymerase (PhaZ(PalM4-7)) from P. alcaligenes M4-7 was 28.0 kDa, as determined by SDS-PAGE. The PhaZ(PalM4-7) was most active in 50 mM glycine-NaOH buffer (pH 9.0) at 35 degrees C. It was insensitive to dithiothreitol, sodium azide, and iodoacetamide, but susceptible to p-hydroxymercuribenzoic acid, N-bromosuccinimide, acetic anhydride, EDTA, diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, Tween 80, and Triton X-100. In this study, the genes encoding MCL-PHA depolymerase were cloned, sequenced, and characterized from a soil bacterium, P. alcaligenes LB19 (Kim et al., 2002, Biomacromolecules 3, 291-296) as well as P. alcaligenes M4-7. The structural gene (phaZ(PalLB19)) of MCL-PHA depolymerase of P. alcaligenes LB19 consisted of an 837 bp open reading frame (ORF) encoding a protein of 278 amino acids with a deduced M((r)) of 30,188 Da. However, the MCL-PHA depolymerase gene (phaZ(PalM4-7)) of P. alcaligenes M4-7 was composed of an 834 bp ORF encoding a protein of 277 amino acids with a deduced Mr of 30,323 Da. Amino acid sequence analyses showed that, in the two different polypeptides, a substrate-binding domain and a catalytic domain are located in the N-terminus and in the C-terminus, respectively. The PhaZ(PalLB19) and the PhaZ(PalM4-7) commonly share the lipase box, GISSG, in their catalytic domains, and utilize 111Asn and 110Ser residues, respectively, as oxyanions that play an important role in transition-state stabilization of hydrolytic reactions.
从海洋环境中分离出一株能够降解多种聚酯的细菌菌株M4 - 7，这些聚酯包括聚（ε-己内酯）、聚（3-羟基丁酸酯- co - 3-羟基戊酸酯）、聚（3-羟基辛酸酯）和聚（3-羟基-5-苯基戊酸酯），并鉴定为产碱假单胞菌。通过SDS - PAGE测定，产碱假单胞菌M4 - 7纯化的细胞外中链长度聚（3-羟基链烷酸酯）（MCL - PHA）解聚酶（PhaZ(PalM4 - 7)）的相对分子质量为28.0 kDa。PhaZ(PalM4 - 7)在35℃的50 mM甘氨酸 - NaOH缓冲液（pH 9.0）中活性最高。它对二硫苏糖醇、叠氮化钠和碘乙酰胺不敏感，但对对羟基汞苯甲酸、N - 溴代琥珀酰亚胺、乙酸酐、EDTA、二异丙基氟磷酸、苯甲基磺酰氟、吐温80和曲拉通X - 100敏感。在本研究中，从土壤细菌产碱假单胞菌LB19（Kim等人，2002年，《生物大分子》3，291 - 296）以及产碱假单胞菌M4 - 7中克隆、测序并表征了编码MCL - PHA解聚酶的基因。产碱假单胞菌LB19的MCL - PHA解聚酶的结构基因（phaZ(PalLB19)）由一个837 bp的开放阅读框（ORF）组成，编码一个278个氨基酸的蛋白质，推导的分子量为30,188 Da。然而，产碱假单胞菌M4 - 7的MCL - PHA解聚酶基因（phaZ(PalM4 - 7)）由一个834 bp的ORF组成，编码一个包含277个氨基酸的蛋白质，推导的Mr为30,323 Da。氨基酸序列分析表明，在这两种不同的多肽中，底物结合结构域和催化结构域分别位于N端和C端。PhaZ(PalLB19)和PhaZ(PalM4 - 7)在其催化结构域中共同拥有脂肪酶框GISSG，并分别利用111Asn和110Ser残基作为氧阴离子，它们在水解反应的过渡态稳定中起重要作用。
