Novel Chaperones GroEL and GroES for Activity and Stability Enhancement of Nitrilase in and .

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.