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 (Km) and maximal velocity (Vmax) of the purified recombinant pectinase were 6.9 mg/mL and 67.57 μmol/mg/min, respectively.

中文翻译：

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密码子优化和启动子选择对毕赤酵母重组内聚半乳糖醛酸酶产生的影响

果胶酶是食品工业不同领域中应用最广泛的酶之一，用于不同目的，例如果汁的澄清、植物油的提取和农业基质的糖化。本研究的目的是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。本研究根据毕赤酵母的密码子使用优化了黑曲霉中编码果胶酶的基因。在研究范围内，密码子优化和天然（非密码子优化）果胶酶基因被转移到毕赤酵母 X33 菌株，并在甲醇诱导型 AOX1 和乙醇诱导型 ADH2 启动子的调控下表达。根据研究结果，表现出最高生产水平的启动子和密码子组合被确定为 ADH2 和密码子优化的果胶酶，产生 42.33 U/mL 的酶活性。最好的生产者克隆在 2 L 摇瓶中的 400 mL 培养基中培养，酶通过 His-tag 方法纯化。纯化酶的最佳工作条件为50℃，pH 5.0，60℃孵育1h后，酶活保持在60%的水平。纯化的重组果胶酶的米歇利斯-门腾常数 (Km) 和最大速度 (Vmax) 分别为 6.9 mg/mL 和 67.57 μmol/mg/min。纯化酶的最佳工作条件为50℃，pH 5.0，60℃孵育1h后，酶活保持在60%的水平。纯化的重组果胶酶的米歇利斯-门腾常数 (Km) 和最大速度 (Vmax) 分别为 6.9 mg/mL 和 67.57 μmol/mg/min。纯化酶的最佳工作条件为50℃，pH 5.0，60℃孵育1h后，酶活保持在60%的水平。纯化的重组果胶酶的米歇利斯-门腾常数 (Km) 和最大速度 (Vmax) 分别为 6.9 mg/mL 和 67.57 μmol/mg/min。