Xu Chunmeng, Tang Lingjun, Liang Youxiang, Jiao Song, Yu Huimin, Luo Hui
Key Laboratory of Industrial Biocatalysis, Ministry of Education, Beijing 100084, China.
Department of Chemical Engineering, Tsinghua University, Beijing 100084, China.
Molecules. 2020 Feb 24;25(4):1002. doi: 10.3390/molecules25041002.
For large-scale bioproduction, thermal stability is a crucial property for most industrial enzymes. A new method to improve both the thermal stability and activity of enzymes is of great significance. In this work, the novel chaperones GroEL and GroES from , a nontypical actinomycete with high organic solvent tolerance, were evaluated and applied for thermal stability and activity enhancement of a model enzyme, nitrilase. Two expression strategies, namely, fusion expression and co-expression, were compared in two different hosts, and . In the host, fusion expression of nitrilase with either GroES or GroEL significantly enhanced nitrilase thermal stability (4.8-fold and 10.6-fold, respectively) but at the expense of enzyme activity (32-47% reduction). The co-expression strategy was applied in via either a plasmid-only or genome-plus-plasmid method. Through integration of the nitrilase gene into the genome at the site of nitrile hydratase (NHase) gene via CRISPR/Cas9 technology and overexpression of GroES or GroEL with a plasmid, the engineered strains TH3 dNHase::Nit (pNV18.1-P-Nit-P-GroES) and TH3 dNHase::Nit (pNV18.1-P-Nit-P-GroEL) were constructed and showed remarkably enhanced nitrilase activity and thermal stability. In particular, the GroEL and nitrilase co-expressing mutant showed the best performance, with nitrilase activity and thermal stability 1.3- and 8.4-fold greater than that of the control TH3 (pNV18.1-P-Nit), respectively. These findings are of great value for production of diverse chemicals using free bacterial cells as biocatalysts.
对于大规模生物生产而言，热稳定性是大多数工业酶的关键特性。一种提高酶的热稳定性和活性的新方法具有重要意义。在本研究中，对来自具有高有机溶剂耐受性的非典型放线菌的新型伴侣蛋白GroEL和GroES进行了评估，并将其应用于提高模型酶腈水解酶的热稳定性和活性。在两种不同的宿主中比较了两种表达策略，即融合表达和共表达。在宿主中，腈水解酶与GroES或GroEL的融合表达显著提高了腈水解酶的热稳定性（分别提高了4.8倍和10.6倍），但以酶活性为代价（降低了32 - 47%）。共表达策略通过仅质粒法或基因组加质粒法应用于宿主。通过CRISPR/Cas9技术将腈水解酶基因整合到腈水合酶（NHase）基因位点的宿主基因组中，并通过质粒过表达GroES或GroEL，构建了工程菌株TH3 dNHase::Nit（pNV18.1 - P - Nit - P - GroES）和TH3 dNHase::Nit（pNV18.1 - P - Nit - P - GroEL），它们表现出显著提高的腈水解酶活性和热稳定性。特别是，GroEL和腈水解酶共表达突变体表现出最佳性能，腈水解酶活性和热稳定性分别比对照TH3（pNV18.1 - P - Nit）高1.3倍和8.4倍。这些发现对于使用游离细菌细胞作为生物催化剂生产多种化学品具有重要价值。
