Abstract
Nitriles derived from glucosinolates (GSLs) in rapeseed meal (RSM) can cause lesions on animal liver and kidneys. Nitrilase converts nitriles to carboxylic acids and NH3, eliminating their toxicity. Here we describe a nitrilase, BnNIT2, from Brassica napus (optimal temperature, 45 °C; pH, 7.0) that is stable at 40 °C and has a wide substrate specificity. Recombinant BnNIT2 converted the three main nitriles from GSLs (3-hydroxy-4-pentenenitrile, 3-butenenitrile, and 4-pentenenitrile), with the highest specific activity (58.6 U/mg) for 4-pentenenitrile. We used mutagenesis to improve the thermostability of BnNIT2; the resulting mutant BnNIT2-H90M had an ~ 14.5% increase in residual activity at 50 °C for 1 h. To verify the functionality of BnNIT2, GSLs were extracted from RSM and converted into nitriles at pH 5.0 in the presence of Fe2+. Then, BnNIT2 was used to degrade the nitriles from GSLs; ultimately, ~ 80% of nitriles were removed. Thus BnNIT2 is a potential enzyme for detoxification of RSM.
Key points
• Functional identification of the plant nitrilase BnNIT2.
• Identified a mutant, H90M, with improved thermostability.
• BnNIT2 was capable of degrading nitriles from transformed GSLs.
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Funding
This research was supported by the National Key Research and Development Program of China (2019YFD0900200), China Agriculture Research System of MOF and MARA (CARS-41), the State Key Laboratory of Animal Nutrition Project (2004DA125184G2101), and the National Key Research and Development Program of China (SQ2021YFC210010).
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H.L. and H.H. designed and supervised the research, analyzed the data, and revised the manuscript. H.Z. performed the experiments, analyzed the data, and wrote the manuscript. H.Z., X.Q., X.L.W., Y.W., T.T., Y.R.W., and B.Y. contributed new materials or analytical tools.
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Zhang, H., Zhang, H., Qin, X. et al. Biodegradation of nitriles derived from glucosinolates in rapeseed meal by BnNIT2: a nitrilase from Brassica napus with wide substrate specificity. Appl Microbiol Biotechnol 106, 2445–2454 (2022). https://doi.org/10.1007/s00253-022-11844-y
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DOI: https://doi.org/10.1007/s00253-022-11844-y