赭曲霉毒素A降解菌株的筛选及脱毒酰胺水解酶的基因克隆和表达

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

摘要:

【背景】赭曲霉毒素A (ochratoxin A, OTA)是一种可以致癌的真菌毒素，其污染严重影响食品安全，危害人类健康。生物降解法去除OTA污染是近些年的研究热点，发掘高效的OTA降解脱毒酶资源具有重要的意义。【目的】筛选高效的OTA降解菌株并从中克隆降解基因，为生物脱毒方法的开发提供基因和酶资源。【方法】利用OTA为唯一碳源的筛选培养基从土壤中筛选纯化OTA降解菌株，通过16S rRNA基因序列分析确定其分类地位，利用高效液相色谱(high performance liquid chromatography, HPLC)分析其降解产物。通过同源序列比对的方法克隆降解基因并与载体pET-29a(+)相连，然后在大肠杆菌BL21(DE3)中表达。利用Ni²⁺亲和层析对表达产物进行纯化，研究其对OTA的降解活性和酶学特征。【结果】筛选到一株高效的OTA降解菌株，在12 h内能够完全降解1 μg/mL的OTA；初步鉴定该菌株属于Niastella，编号为JX-6；菌株JX-6通过酰胺键断裂途径降解OTA生成无毒的OTα；从菌株JX-6中鉴定了一个OTA酰胺水解酶，命名为NcOTase；NcOTase与已报道的OTA酰胺水解酶序列相似性较低，仅为31%–53%；纯化的NcOTase具有OTA水解活性，比酶活为60.3 U/mg，活性显著高于大部分已报道的OTA降解酶。【结论】NcOTase是一个高效的OTA降解脱毒酶，在去除食品和饲料中OTA污染方面具有很好的应用前景。

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.