微生物凝乳酶具有生产周期短和易于发酵生产的优点,是传统小牛皱胃酶的经济高效替代选择之一。为了筛选新的凝乳酶产生菌,该研究以从传统发酵腐乳分离的霉菌为出发菌株,筛选具有高凝乳活性和低蛋白水解活性的霉菌菌株。采用内源转录区间隔区结合线粒体小亚基核糖体DNA扩增测序的方法对菌株进行了分子鉴定。通过硫酸铵分级盐析沉淀、DEAE-Sepharose Fast Flow阴离子交换柱、Sephadex G100凝胶过滤层析法对酶进行了纯化,并研究了凝乳酶酶学特性。筛选出菌株CQ3具有良好的凝乳活性和低蛋白水解活性,该菌株鉴定为变色栓菌(Trametes versicolor)。CQ3凝乳酶纯化后经SDS-PAGE分析呈现出分子质量为61 kDa的唯一条带。该酶作用的最适温度为45 ℃,最适pH值为6.5,Ca2+对酶活力有显著的促进作用。该酶在pH值为4.5~7.0和温度低于50 ℃条件下有良好的稳定性。蛋白酶抑制剂试验表明,该酶为一种天冬氨酸蛋白酶。该研究结果报道了一种新的产凝乳酶菌种资源,为丰富凝乳酶产生菌株资源,进一步评价栓菌凝乳酶在干酪加工适用性的研究提供了理论基础。
Microbial milk-clotting enzymes present an economically viable alternative to traditional calf rennet, attributed to their expedited production cycles and facile fermentation processes. The objective of this study was to screen for novel microbial strains that produce milk-clotting enzymes with both high activity and minimal proteolytic effects. Molds isolated from traditional sufu served as the starting material for this selection process. Strains demonstrating high milk-clotting activity and low proteolytic activity were identified through ITS and mt SSU rDNA amplification sequencing for molecular characterization. Enzyme purification was conducted using a sequence of ammonium sulfate fractionation, DEAE-Sepharose Fast Flow anion exchange chromatography, and Sephadex G100 gel filtration chromatography. Subsequent analysis of the enzymatic properties of the purified milk-clotting enzyme was performed. Strain CQ3 emerged as a prominent candidate, showing substantial milk-clotting activity with reduced proteolytic effects, and was identified as Trametes versicolor. SDS-PAGE analysis of the purified enzyme from strain CQ3 revealed a single band corresponding to a molecular weight of 61 kDa. The enzyme exhibited optimal activity at 45 ℃ and a pH of 6.5, with Ca2+ markedly enhancing its activity. Stability tests demonstrated consistent enzyme performance within a pH range of 4.5-7.0 and at temperatures below 50 ℃. Protease inhibitor assays classified the enzyme as an aspartic protease. These results document a novel source of fungal strains for milk-clotting enzyme production, laying a theoretical groundwork for further assessment of Trametes versicolor enzymes in cheese manufacturing applications.
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