Novel Chaperones RrGroEL and RrGroES for Activity and Stability Enhancement of Nitrilase in Escherichia coli and Rhodococcus ruber
Novel Chaperones RrGroEL and RrGroES for Activity and Stability Enhancement of Nitrilase in Escherichia coli and Rhodococcus ruber
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DOI：
10.3390/molecules25041002
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发表时间：
2020-02
期刊：
影响因子：
4.6
通讯作者：
Chunmeng Xu;Lingjun Tang;Youxiang Liang;Song Jiao;Huimin Yu;Hui Luo
中科院分区：
文献类型：
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作者：
Chunmeng Xu;Lingjun Tang;Youxiang Liang;Song Jiao;Huimin Yu;Hui Luo
对于大规模的生物生产，热稳定性是大多数工业酶的关键特性。因此，寻找一种既能提高酶的热稳定性又能提高酶活力的新方法具有重要意义。本研究以一株具有高有机溶剂耐受性的非典型放线菌红球菌（Rhodococcus ruber）为出发菌株，对其分子伴侣RrGroEL和RrGroES进行了研究，并将其应用于腈水解酶的热稳定性和活性增强研究。比较了融合表达和共表达两种表达策略在两种不同宿主大肠杆菌中的表达效果。coli和R.鲁伯。在急诊在大肠杆菌宿主中，腈水解酶与RrGroES或RrGroEL的融合表达显著增强了腈水解酶的热稳定性（分别为4.8倍和10.6倍），但以酶活性为代价（降低32-47%）。将共表达策略应用于R. ruber通过质粒或基因组加质粒的方法。通过将腈水解酶基因整合到R.通过CRISPR/Cas9技术在Ruber基因组的腈水合酶（NHase）基因位点处插入RrGroES或RrGroEL，用质粒过表达RrGroES或RrGroEL，工程菌株R.构建了红TH 3 dNHase：：RrNit（pNV18.1-Pami-RrNit-Pami-RrGroES）和TH 3 dNHase：：RrNit（pNV18.1-Pami-RrNit-Pami-RrGroEL），显示出显着增强的腈水解酶活性和热稳定性。特别地，RrGroEL和腈水解酶共表达突变体显示出最好的性能，腈水解酶活性和热稳定性分别是对照TH 3（pNV18.1-Pami-RrNit）的1.3倍和8.4倍。这些发现对于利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。
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 RrGroEL and RrGroES from Rhodococcus ruber, 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, E. coli and R. ruber. In the E. coli host, fusion expression of nitrilase with either RrGroES or RrGroEL 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 R. ruber via either a plasmid-only or genome-plus-plasmid method. Through integration of the nitrilase gene into the R. ruber genome at the site of nitrile hydratase (NHase) gene via CRISPR/Cas9 technology and overexpression of RrGroES or RrGroEL with a plasmid, the engineered strains R. ruber TH3 dNHase::RrNit (pNV18.1-Pami-RrNit-Pami-RrGroES) and TH3 dNHase::RrNit (pNV18.1-Pami-RrNit-Pami-RrGroEL) were constructed and showed remarkably enhanced nitrilase activity and thermal stability. In particular, the RrGroEL 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-Pami-RrNit), respectively. These findings are of great value for production of diverse chemicals using free bacterial cells as biocatalysts.