The key Laboratory of Biotechnology for Medicinal Plant of Jiangsu Province, School of Life Sciences, Jiangsu Normal University, Xuzhou, P. R. China.
State Key Laboratory of Agrobiotechnology and MOA Key Laboratory of Soil Microbiology, College of Biological Sciences, China Agricultural University, Beijing, China.
Bioengineered. 2020 Dec;11(1):375-385. doi: 10.1080/21655979.2020.1738127.
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 . Recombinant strains with (strain containing 4-copy ) and without ER stress (strain containing 2-copy ) were obtained. However, the effective expression of pRML in 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 . The coexpression of protein folding chaperones, including and , in different strains with or without ER stress, was investigated. 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, coexpression could not improve pRML production in the 2-copy strain because it increased protein folded stress, while 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%.
脂肪酶（RML）是一种广泛应用于实验室和工业的生物催化剂。此前，已克隆并表达了带有 70 个氨基酸前肽（pRML）的 RML。获得了带有（含 4 个拷贝）和不带有内质网应激（含 2 个拷贝）的重组菌株。然而，到目前为止，还没有报道过在不带有内质网应激的情况下，通过共表达 pRML 产生菌株中的内质网相关元件，有效表达 pRML。在本研究中，探索了在 中生产功能性 pRML 的有效方法。研究了不同菌株中内质网应激情况下共表达蛋白折叠伴侣（包括 和 ）对 pRML 生产的影响。仅过表达 仅将 4 拷贝菌株中的 pRML 产量从 705 U/mL 提高到 1430 U/mL，因为它缓解了蛋白折叠压力，将蛋白浓度从 0.56 mg/mL 提高到 0.65 mg/mL，并将酶比活从 1238 U/mg 提高到 2186 U/mg。然而， 共表达不能提高 2 拷贝菌株中的 pRML 产量，因为它增加了蛋白折叠压力，而 共表达在两种菌株中都对 pRML 表达有负面影响。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 下检测到 C8-C10 作为底物时，相对活性超过 80%，而在 45°C 和 pH 8 下检测到 C8-C12 时，相对活性超过 80%。最佳酶粉制备 pH 值为 7，pRML 的最大回收率为 73.19%。
