Isolation of ochratoxin A-degrading bacteria strain, molecular cloning and expression of detoxification amidohydrolase

Abstract:

[Background] Ochratoxin A (OTA) is one of the most potent carcinogens, threatening food safety and human health. In recent years, biodegradation of OTA has attracted the interest of scholars, but the currently available OTA-detoxifying enzymes are limited. Therefore, it is of great significance to explore efficient OTA-degrading and -detoxifying enzyme resources. [Objective] To screen efficient OTA-degrading strain, clone the degradation-related gene, and thus to provide gene and enzyme resources for eliminating OTA. [Methods] The selective medium with OTA as the sole carbon source was used to screen OTA-degrading strain from the sludge. The strain was identified based on the 16S rRNA gene sequence analysis. The degradation products of OTA by the strain were determined by high performance liquid chromatography (HPLC). The degradation-related gene was obtained by homologous sequence alignment, cloned into the expression vector pET-29a(+), and then expressed in Escherichia coli BL21(DE3). The expressed product was purified by Ni²⁺-NTA affinity chromatography, and its OTA degradation activity and enzymatic characteristics were determined. [Results] A strain with high OTA degradation efficiency was screened out, which could completely degrade 1 μg/mL OTA within 12 h. The strain was identified to belong to Niastella and named JX-6. OTA was transformed by JX-6 through the hydrolysis of the amide bond to generate non-toxic OTα. An OTA amidohydrolase was identified in JX-6 and named NcOTase. The sequence similarity between NcOTase and the reported OTA amidohydrolase was low (31%-53%). The purified NcOTase showed OTA hydrolysis activity with specific enzyme activity of 60.3 U/mg, which was significantly higher than that most of the characterized OTA-degrading enzymes. [Conclusion] NcOTase is a highly efficient OTA-detoxifying enzyme, which has good application prospects in the removal of OTA in food and feed.

Key words:mycotoxins;ochratoxin A;biodegradation;amidohydrolase;enzymatic activity