Rhizomucor miehei lipase (RML) is a biocatalyst that widely used in laboratory and industrial. Previously, RML with a 70-amino acid propeptide (pRML) was cloned and expressed in P. pastoris. Recombinant strains with (strain containing 4-copy prml) and without ER stress (strain containing 2-copy prml) were obtained. However, the effective expression of pRML in P. pastoris by coexpressing ER-related elements in pRML-produced strain with or without ER stress has not been reported to date. In this study, an efficient way to produce functional pRML was explored in P. pastoris. The coexpression of protein folding chaperones, including PDI and ERO1, in different strains with or without ER stress, was investigated. PDI overexpression only increased pRML production in 4-copy strain from 705 U/mL to 1430 U/mL because it alleviated the protein folded stress, increased the protein concentration from 0.56 mg/mL to 0.65 mg/mL, and improved enzyme-specific activity from 1238 U/mg to 2186 U/mg. However, PDI coexpression could not improve pRML production in the 2-copy strain because it increased protein folded stress, while ERO1 coexpression in the two strains all had a negative effect on pRML expression. We also investigated the effect of the propeptide on the substrate specificity and the condition for pRML enzyme powder preparation. Results showed that the relative activity exceeded 80% when the substrates C8-C10 were detected at 35°C and pH 6, and C8-C12 at 45°C and pH 8. The optimal enzyme powder preparation pH was 7, and the maximum recovery rate for pRML was 73.19%.

中文翻译：

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通过在巴斯德毕赤酵母中共表达蛋白质折叠分子伴侣，改善了重组根瘤菌米黑脂酶的生产，从而触发了内质网应激。

Rhizomucor miehei脂肪酶（RML）是一种生物催化剂，广泛用于实验室和工业领域。以前，具有70个氨基酸的前肽（pRML）的RML被克隆并在巴斯德毕赤酵母中表达。获得具有（含有4个拷贝的prml的菌株）和没有ER应力（含有2个拷贝的prml的菌株）的重组菌株。然而，迄今尚未报道过通过在pRML产生的菌株中共表达ER相关元件在有或没有ER应激的情况下在巴斯德毕赤酵母中有效表达pRML。在这项研究中，在巴斯德毕赤酵母中探索了一种生产功能性pRML的有效方法。研究了蛋白质折叠分子伴侣（包括PDI和ERO1）在有或没有内质网应激的不同菌株中的共表达。PDI过度表达只会将4拷贝菌株的pRML产量从705 U / mL增加到1430 U / mL，因为它减轻了蛋白质折叠的压力，将蛋白质浓度从0.56 mg / mL增加到0.65 mg / mL，并将酶比活性从1238 U / mg提高到2186 U / mg。但是，PDI共表达不能提高2拷贝菌株中pRML的产生，因为它增加了蛋白质折叠的压力，而ERO1共表达在两个菌株中都对pRML表达产生了负面影响。我们还研究了前肽对底物特异性和pRML酶粉制备条件的影响。结果表明，在35°C和pH 6下检测底物C8-C10，在45°C和pH 8下检测底物C8-C12时，相对活性超过80％。最佳酶粉制剂pH为7，最大回收率pRML率为73.19％。但是，PDI共表达不能增加2拷贝菌株中pRML的产生，因为它增加了蛋白质折叠的压力，而ERO1共表达在两个菌株中都对pRML表达产生了负面影响。我们还研究了前肽对底物特异性和pRML酶粉制备条件的影响。结果表明，当在35°C和pH 6下检测底物C8-C10，在45°C和pH 8下检测底物C8-C12时，相对活性超过80％。最佳酶粉制剂pH为7，最大回收率pRML率为73.19％。但是，PDI共表达不能提高2拷贝菌株中pRML的产生，因为它增加了蛋白质折叠的压力，而ERO1共表达在两个菌株中都对pRML表达产生了负面影响。我们还研究了前肽对底物特异性和pRML酶粉制备条件的影响。结果表明，当在35°C和pH 6下检测底物C8-C10，在45°C和pH 8下检测底物C8-C12时，相对活性超过80％。最佳酶粉制剂pH为7，最大回收率pRML率为73.19％。我们还研究了前肽对底物特异性和pRML酶粉制备条件的影响。结果表明，在35°C和pH 6下检测底物C8-C10，在45°C和pH 8下检测底物C8-C12时，相对活性超过80％。最佳酶粉制剂pH为7，最大回收率pRML率为73.19％。我们还研究了前肽对底物特异性和pRML酶粉制备条件的影响。结果表明，当在35°C和pH 6下检测底物C8-C10，在45°C和pH 8下检测底物C8-C12时，相对活性超过80％。最佳酶粉制剂pH为7，最大回收率pRML率为73.19％。