米曲霉具有较高的蛋白酶活性,是酱油、豆瓣酱等豆类发酵食品制曲中应用最广泛的发酵剂。然而,目前对米曲霉关键蛋白酶的研究仍然缺乏。在前期研究基础上,该研究旨在将Aspergillus oryzae BL18菌株在制曲过程中表达量最高的关键蛋白酶GME238_g基因在毕赤酵母GS115进行异源表达以探究其酶学性质,并评价其在酱油发酵中的应用效果。结果表明,GME238_g的最适温度和最适pH值分别为45 ℃和pH 8,其在pH 5~6环境中具有一定耐受性;该酶能在180 g/L盐度下处理6 d仍有活性,说明该酶具有一定耐盐性;与此同时,该酶对球蛋白和清蛋白的降解率分别为35.2%和28.2%。最后,将GME238_g应用于酱油发酵初始阶段,结果发现与对照组相比,添加GME238_g的酱油中游离氨基酸含量提高了21%,丰富了挥发性风味物质在数量和浓度,且具有更高的风味感官评分。
Aspergillus oryzae is the most widely used starter in the production of bean-based fermented foods (such as soy sauce and broad bean paste) due to its strong ability to produce proteolytic enzymes.However, the researches on the key proteolytic enzymes from A.oryzae are still lacking.Based on our previous research, this study aimed to heterologously express the key proteolytic enzyme GME238_g from A.oryzae BL18 in Komagataella phaffii GS115, to characterize its enzymatic properties and to evaluate its application in soy sauce fermentation.The results showed the optimal temperature and optimal pH of GME238_g were 45 ℃ and pH 8, which also displayed preferable stability in pH 5-6.This enzyme also showed good stability towards high salinity since GME238_g was still active after treatment at 180 g/L salinity for 6 days.Moreover, the hydrolysis degree of GME238_g towards globulin and albumin reached 35.2% and 28.2%.Finally, this enzyme was applied in the initial stage of soy sauce fermentation.Compared with control group, the concentration of free amino acids in soy sauce with the addition of GME238_g was increased by 21% while the volatile flavour substances were enriched in both quantity and concentration.The addition of GME238_g also enhanced the sensory evaluation scores of soy sauce.
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