高效降解牛奶过敏原蛋白酶菌种筛选及其重组植物过氧化氢酶的功能验证

Study on Milk-Allergen-Degradation Protease Screening and Its Recombinant Vegetative Catalase Function Verification

摘要:

αs1酪蛋白是奶制品中主要的过敏原，为了高效地去除过敏原，从3 000株左右野生菌中筛选获得一株脱敏效果好的枯草芽孢杆菌S7，并分别采用高效液相色谱技术（HPLC）和液相色谱串联二级质谱（LC-MS/MS）分析了目标菌中关键酶制备的脱敏奶制品的风味与生成的生物活性多肽。通过与另一株枯草芽孢杆菌S21对比后发现，S7具有更高的水解α_s1酪蛋白的酶活，为分析这种差异，通过肽指纹图谱鉴定，发现S7可分泌表达更多的植物过氧化氢酶，对该酶进行重组表达后，回补至S21发酵液上清液中，发现S21水解α_s1酪蛋白的酶活提升了约1.5倍，达到与S7相似的水平。结果表明，重组植物过氧化氢酶可辅助蛋白酶进行水解，从而提高了蛋白酶水解过敏原的酶活。枯草芽孢杆菌S7中的关键酶制备的脱敏奶制品具有较好的食品工业应用价值。

Abstract:

To efficiently remove αs1-casein,the primary allergen in dairy products, a strain of Bacillus subtilis S7 with decent desensitization ability was screened from 3 000 strains of wild bacteria. High performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry(LC-MS/MS) were adopted to analyze the flavor of desensitization dairy products made by the key enzyme in Bacillus subtilis S7 and the bioactive peptides produced. By comparison with another candidate Bacillus subtilis S21, it was found that S7 had a higher activity to α_s1-casein hydrolysis. To analyze this difference, all secreted enzymes were identified through peptide fingerprint. It was found that S7 could secrete and express more vegetative catalase. After the enzyme was recombinantly expressed and supplemented into the supernatant of Bacillus subtilis S21 fermentation broth, the activity of S21 to α_s1-casein hydrolysis was increased by about 1.5 times to a level similar to that of S7. The enzyme activity to allergen was improved by vegetative catalase, which helped proteases to hydrolyze. The results show that desensitized milk products prepared by vegetative catalase and subtilisin E in Bacillus subtilis S7 may have promising application in the food industry.