Molecular Cloning, Expression and Characterization of Pectin Methylesterase (CtPME) from Clostridium thermocellum
Molecular Cloning, Expression and Characterization of Pectin Methylesterase (CtPME) from Clostridium thermocellum
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热纤梭菌果胶甲酯酶 (CtPME) 的分子克隆、表达和表征
DOI：
10.1007/s12033-017-9997-7
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发表时间：
2017
影响因子：
2.6
通讯作者：
A. Goyal
中科院分区：
文献类型：
--
作者：
Vikky Rajulapati;A. Goyal
许多植物病原微生物，如细菌和真菌，在植物入侵过程中会产生果胶甲酯酶（PME）。植物和昆虫也会产生PME来降解植物细胞壁。在本研究中，克隆、表达和纯化了来自热纤梭菌的耐热果胶甲酯酶（CtPME），该酶属于家族8碳水化合物酯酶（CE8）。 CtPME 的氨基酸序列与菊欧文氏菌果胶甲酯酶相似，一致性为 38%。将编码CtPME的基因克隆到pET28a(+)载体中并使用大肠杆菌BL21(DE3)细胞表达。重组 CtPME 表达为可溶性蛋白，并在 SDS-PAGE 凝胶上显示出分子量约为 35.2 kDa 的单条带。该酶的分子量为 35.5 kDa，也通过 MALDI-TOF MS 分析得到证实。值得注意的是，与 LB 培养基 (1.5 mg/mL) 相比，自诱导培养基中的 CtPME 蛋白浓度最高 (11.4 mg/mL)。 CtPME 对柑橘果胶表现出最大活性 (18.1 U/mg)，甲基酯化率 >85%。 CtPME 活性的最佳 pH 值和温度分别为 8.5 和 50 °C。该酶在 pH 值 8.0–9.0 范围内稳定，在 45 至 70 °C 之间热稳定。 5 mM Ca2+ 或 Mg2+ 离子使 CtPME 活性增加 40%。 CtPME 的蛋白质熔解曲线在 80 °C 处出现峰值。在 5 mM Ca2+ 离子存在下，峰移至 85 °C，添加 5 mM EDTA 将熔化峰移回到 80 °C。 CtPME 可潜在用于食品和纺织工业应用。
Many phytopathogenic micro-organisms such as bacteria and fungi produce pectin methylesterases (PME) during plant invasion. Plants and insects also produce PME to degrade plant cell wall. In the present study, a thermostable pectin methylesterase (CtPME) from Clostridium thermocellum belonging to family 8 carbohydrate esterase (CE8) was cloned, expressed and purified. The amino acid sequence of CtPME exhibited similarity with pectin methylesterase from Erwinia chrysanthemi with 38% identity. The gene encoding CtPME was cloned into pET28a(+) vector and expressed using Escherichia coli BL21(DE3) cells. The recombinant CtPME expressed as a soluble protein and exhibited a single band of molecular mass approximately 35.2 kDa on SDS-PAGE gels. The molecular mass, 35.5 kDa of the enzyme, was also confirmed by MALDI-TOF MS analysis. Notably, highest protein concentration (11.4 mg/mL) of CtPME was achieved in auto-induction medium, as compared with LB medium (1.5 mg/mL). CtPME showed maximum activity (18.1 U/mg) against citrus pectin with >85% methyl esterification. The optimum pH and temperature for activity of CtPME were 8.5 and 50 °C, respectively. The enzyme was stable in pH range 8.0–9.0 and thermostable between 45 and 70 °C. CtPME activity was increased by 40% by 5 mM Ca2+ or Mg2+ ions. Protein melting curve of CtPME gave a peak at 80 °C. The peak was shifted to 85 °C in the presence of 5 mM Ca2+ ions, and the addition of 5 mM EDTA shifted back the melting peak to 80 °C. CtPME can be potentially used in food and textile industry applications.
影响因子：
3.8
作者：
Ridley, BL;O'Neill, MA;Mohnen, DA
通讯作者：
Mohnen, DA
影响因子：
1.6
作者：
Studier, FW
通讯作者：
Studier, FW