Rajulapati Vikky, Goyal Arun
Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, 781039, India.
Mol Biotechnol. 2017 May;59(4-5):128-140. doi: 10.1007/s12033-017-9997-7.
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 Ca or Mg 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 Ca 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.
许多植物病原微生物，如细菌和真菌，在侵染植物的过程中会产生果胶甲基酯酶（PME）。植物和昆虫也会产生PME来降解植物细胞壁。在本研究中，从属于8族碳水化合物酯酶（CE8）的热纤梭菌中克隆、表达并纯化了一种耐热果胶甲基酯酶（CtPME）。CtPME的氨基酸序列与菊欧文氏菌的果胶甲基酯酶具有相似性，同一性为38%。将编码CtPME的基因克隆到pET28a(+)载体中，并利用大肠杆菌BL21(DE3)细胞进行表达。重组CtPME表达为可溶性蛋白，在SDS-PAGE凝胶上呈现出一条分子量约为35.2 kDa的条带。该酶的分子量为�5.5 kDa，也通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）分析得到了证实。值得注意的是，与LB培养基（1.5 mg/mL）相比，在自诱导培养基中获得了最高的CtPME蛋白浓度（11.4 mg/mL）。CtPME对甲基酯化率>85%的柑橘果胶表现出最大活性（18.1 U/mg）。CtPME活性的最佳pH值和温度分别为8.5和50℃。该酶在pH值8.0 - 9.0范围内稳定，在45至70℃之间耐热。5 mM的Ca或Mg离子使CtPME活性提高了40%。CtPME的蛋白质解链曲线在80℃出现一个峰值。在存在5 mM Ca离子的情况下，该峰值移至85℃，而添加5 mM乙二胺四乙酸（EDTA）使解链峰又移回到80℃。CtPME在食品和纺织工业应用中具有潜在用途。
