Novel Chaperones RrGroEL and RrGroES for Activity and Stability Enhancement of Nitrilase in Escherichia coli and Rhodococcus ruber

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.

A high-throughput screening method for determining the substrate scope of nitrilases

We have developed a chromogenic reagent to show nitrilase activity and demonstrate its use with 23 enzymes as cell-free extracts.

Biotransformation of iminodiacetonitrile to iminodiacetic acid by Alcaligenes faecalis cells immobilized in ACA-membrane liquid-core capsules

Biotransformation of iminodiacetonitrile (IDAN) to iminodiacetic acid (IDA) was investigated with a newly isolated Alcaligenes faecalis ZJUTBX11 strain showing nitrilase activity in the immobilized form. To reduce the mass transfer resistance and to increase the toleration ability of the microorganisms to the toxic substrate as well as to enhance their ability to be reused, encapsulation of the whole cells in alginate-chitosan-alginate (ACA) membrane liquid-core capsules was attempted in the present study. The optimal pH and temperature for nitrilase activity of encapsulated A. faecalis ZJUTBX11 cells were 7.5°C and 35°C, respectively, which is consistent with free cells. Based on the Michaelis-Menten model, kinetic parameters of the conversion reaction with IDAN as the substrate were: K m = (17.6 ± 0.3) mmol L−1 and V max = (97.6 ± 1.2) μmol min−1 g−1 of dry cell mass for encapsulated cells and (16.8 ± 0.4) mmol L−1 and (108.0 ± 2.7) μmol min−1 g−1 of dry cell mass for free cells, respectively. After being recycled ten times, the whole cells encapsulated in ACA capsules still retained 90 % of the initial nitrilase activity while only 35 % were retained by free cells. Lab scale production of IDA using encapsulated cells in a bubble column reactor and a packed bed reactor were performed respectively.

Nocardia globerula NHB-2: a versatile nitrile-degrading organism

A versatile nitrile-degrading bacterium was isolated by enrichment culture from the soil of a forest near Manali, Himachal Pradesh, India, and was identified as Nocardia globerula. This organism contains 3 enzymes with nitrile-degrading activity: nitrilase, nitrile hydratase, and amidase. Nocardia globerula NHB-2 cells grown on nutrient broth supplemented with 1% glucose and 0.1% yeast extract exhibited nitrile hydratase–amidase activity specific for saturated aliphatic nitriles or amide, while addition of acetonitrile in nutrient broth yielded cells with nitrile hydratase–amidase that in addition to saturated aliphatic nitriles–amide also hydrolyzed aromatic amide. Nocardia globerula NHB-2 cultivated on nutrient broth containing propionitrile exhibited nitrilase activity that hydrolyzed aromatic nitrile and unsaturated aliphatic nitrile. The versatility of this organism in the hydrolysis of various nitriles and amides makes it a potential bioresource for use in organic synthesis.Key words: Nocardia globerula NHB-2, nitrilase, nitrile hydratase, amidase, nitrile–amide degradation.