假单胞菌胞外酶降解黄曲霉毒素B1的酶学性质

Characterization of an extracellular aflatoxin B degrading enzyme from a Pseudomonas strain

【背景】黄曲霉毒素B1(Aflatoxin B1,AFB1)毒性强、污染普遍,目前尚无有效的防治办法。【目的】为了发掘高效的AFB1降解菌并探索其降解特性,对红树林污泥样品中一株AFB1降解菌株(HAI2)的酶学性质进行分析。【方法】以AFB1结构类似物为唯一碳源,筛选出一株高效的AFB1降解菌,利用16S rRNA基因测序结果鉴定菌株,并结合HPLC研究菌株对AFB1的降解特性。【结果】HAI2的16S rRNA基因序列与恶臭假单胞菌(Pseudomonas putida,NR 113651.1)的相似性高达99.85%,其主要降解AFB1的活性物质为胞外蛋白质,HAI2-AFB1降解酶的最适pH值为7.0,最适温度为37℃,Fe^(2+)、Ca^(2+)和Zn^(2+)会抑制AFB1的降解率,Cu^(2+)和Mn^(2+)可以提高AFB1的降解率。HAI2上清液对100 ng/mL的AFB1降解24 h的降解率为71.52%。此外,P.putida HAI2还可抑制被黄曲霉菌感染的玉米中AFB1的合成,AFB1含量可减少63.46%。【结论】假单胞菌P.putida HAI2具有较高的降解AFB1能力,其主要降解活性物质为胞外蛋白,在食品和饲料行业具有较大的应用前景。

[Background] Aflatoxin B1(aflatoxin B1, AFB1) is a highly toxic and widespread mycotoxin. At present, there is no effective method to control its pollution. [Objective] To discover efficient AFB1 degrading bacteria and explore its degradation characteristics, enzymatic properties of an AFB1 degrading strain(HAI2) from mangrove sludge samples were analyzed. [Methods] Using the structural analogue of AFB1 as the sole carbon source, a highly efficient AFB1 degrading strain was screened out, then 16 S rRNA gene sequencing technology was used to identify the strain species, and HPLC was used to analyze the strain’s degradation characteristics to AFB1. [Results] 16 S rRNA gene sequence of HAI2 shares 99.85% homology with Pseudomonas putida(NR 113651.1). The main component of degradation AFB1 was extracellular protein. According to the analysis of enzymatic properties, the optimum pH value of HAI2-AFB1 degrading enzyme was 7.0 and the optimum temperature was 37 ℃. Fe^(2+), Ca^(2+), and Zn^(2+) could inhibit the degradation rate of AFB1, and Cu^(2+) and Mn^(2+) could increase the degradation rate of AFB1. The degradation rate of 100 ng/mL AFB1 by HAI2 supernatant was 71.52% for 24 h. Besides, P. putida HAI2 could inhibit AFB1 synthesis in corn infected by Aspergillus flavus, and the content could be reduced by 63.46%. [Conclusion] P. putida HAI2 had a high ability to degrade AFB1, and its main degradation active substance was extracellular protein, with a large application prospect in the food and feed industry.