Department of Food Engineering, Erzincan Binali Yıldırım University, Erzincan 24100, Turkey.
Department of Food Engineering, Erzincan Binali Yıldırım University, Erzincan 24100, Turkey.
Enzyme Microb Technol. 2020 Sep;139:109589. doi: 10.1016/j.enzmictec.2020.109589. Epub 2020 May 12.
Pectinase is one of the most widely used enzymes in different fields of food industry for different purposes, such as clarification of fruit juice, extraction of vegetable oil and saccharification of agricultural substrates. The aim of this study is to produce recombinant pectinase and evaluate the effect of codon optimization and promoter selection on production. In this study, the gene encoding pectinase in Aspergillus niger was optimized according to the codon usage of Pichia pastoris. Within the scope of the study, codon-optimized and native (non-codon-optimized) pectinase genes were transferred to P. pastoris X33 strain and expressed under the regulation of methanol-inducible AOX1 and ethanol-inducible ADH2 promoter. As a result of the study, the promoter and codon combination which exhibited the highest production level was determined as the ADH2 and codon-optimized pectinase yielding an enzyme activity of 42.33 U/mL. The best producer clone was cultured in 400 mL media in 2 L shake-flask and the enzyme was purified by His-tag method. The optimum working conditions of the purified enzyme was 50 °C and pH 5.0, and after incubation at 60 °C for 1 -h, enzyme activity was maintained at 60% level. The Michelis-Menten constant (K) and maximal velocity (V) of the purified recombinant pectinase were 6.9 mg/mL and 67.57 μmol/mg/min, respectively.
果胶酶是食品工业中应用最广泛的酶之一，用于澄清果汁、提取植物油和糖化农业底物等不同目的。本研究旨在生产重组果胶酶，并评估密码子优化和启动子选择对生产的影响。在本研究中，根据毕赤酵母的密码子使用情况对黑曲霉中编码果胶酶的基因进行了优化。在研究范围内，将密码子优化和天然（非密码子优化）的果胶酶基因转移到毕赤酵母 X33 菌株中，并在甲醇诱导的 AOX1 和乙醇诱导的 ADH2 启动子的调控下表达。研究结果表明，表现出最高生产水平的启动子和密码子组合是 ADH2 和密码子优化的果胶酶，酶活达到 42.33 U/mL。最佳生产菌在 2L 摇瓶中的 400ml 培养基中培养，并通过 His 标签法对酶进行纯化。纯化酶的最佳工作条件为 50°C 和 pH5.0，在 60°C 孵育 1 小时后，酶活保持在 60%的水平。纯化重组果胶酶的米氏常数（K）和最大速度（V）分别为 6.9mg/mL 和 67.57μmol/mg/min。
