A Whole-Process Visible Strategy for the Preparation of Rhizomucor miehei Lipase with Escherichia coli Secretion Expression System and the Immobilization

作者：Mingjun Yang, Xiao‐Chuan Su, Jun Yang, Zhiwen Lu, Jie Zhou, Fei Wang, Yang Liu, Lixin Ma, Chao Zhai · 发表于：Microbial Cell Factories · 年份：2024 · DOI：10.1186/s12934-024-02432-y · 被引用次数：9 · 研究领域：Enzyme Catalysis and Immobilization、Microbial Metabolic Engineering and Bioproduction、Fungal and yeast genetics research

BACKGROUND: Rhizomucor miehei (RM) lipase is a regioselective lipase widely used in food, pharmaceutical and biofuel industries. However, the high cost and low purity of the commercial RM lipase limit its industrial applications. Therefore, it is necessary to develop cost-effective strategies for large-scale preparation of this lipase. The present study explored the high-level expression of RM lipase using superfolder green fluorescent protein (sfGFP)-mediated Escherichia coli secretion system. RESULTS: mutant was fused to the C-terminus of RM lipase to mediate its secretion expression. The yield of the fusion protein reached approximately 5.1 g/L with high-density fermentation in 5-L fermentors. Unlike conventional secretion expression methods, only a small portion of the target protein was secreted into the cell culture while majority of the fusion protein was still remained in the cytoplasm. However, in contrast to intracellular expression, the target protein in the cytoplasm could be transported efficiently to the supernatant through a simple washing step with equal volume of phosphate saline (PBS), without causing cell disruption. Hence, the approach facilitated the downstream purification step of the recombinant RM lipase. Moreover, contamination or decline of the engineered strain and degradation or deactivation of the target enzyme can be detected efficiently because they exhibited bright green fluorescence. Next, the target protein was immobilized with anion-exchange...