高产β-葡聚糖酶里氏木霉的诱变选育、酶学性质及体外模拟消化研究

Mutation breeding,enzymatic properties,and in vitro digestibility of Trichoderma reesei with high yield ofβ-glucanase

β-葡聚糖酶可水解大麦等谷物中的葡聚糖,降低葡聚糖在单胃动物消化道内产生的抗营养作用。为获得高产β-葡聚糖酶菌株,采用常压室温等离子体(atmospheric and room temperature plasma,ARTP)诱变里氏木霉(Trichoderma reesei),经初筛、复筛、遗传稳定性及酶学特性表征,突变株ARTP-9较出发菌株β-葡聚糖酶活力提高54.38%,为43.75 U/mL,产酶能力稳定遗传,酶的最适反应温度及pH分别为50℃和6.0,耐受温度为40~80℃,pH 2.5~6.5时酶活力稳定,Fe^(2+)、Mg^(2+)对酶活力有促进作用,Fe^(3+)、Mn^(2+)、Zn^(2+)、Ca^(2+)、Cu^(2+)对酶活力有抑制作用。以β-葡聚糖为底物,该粗酶的K m值和V max值分别为1.59 mg/mL和6.99μmol/(mg·min)。体外模拟消化实验表明,ARTP-9固态发酵酶制剂在胃期4 h黏度最低,为2.23 mPa·s;肠期21 h还原糖为0.70 mg/mL,是空白的3.2倍,差异显著(P<0.05),其大麦粉体外消化率为48.17%,较空白提升9.02%,差异极显著(P<0.01)。研究结果表明ARTP诱变技术能够显著提高里氏木霉β-葡聚糖酶活力,为其工业化生产奠定基础。

Theβ-glucanase can hydrolyze glucan in cereals such as barley and reduce the anti-nutritional effects of glucan in the digestive tract of monogastric animals.In order to obtain a strain with highβ-glucanase production,Trichoderma reesei was mutated by atmospheric and room temperature plasma(ARTP).The mutant was verified by preliminary screening,rescreening,genetic stability,and enzymatic properties.Compared with the original strain,theβ-glucanase activity of mutant ARTP-9 increased by 54.38%,reaching 43.75 U/mL,and its enzyme-producing ability was genetically stable.The optimum reaction temperature and pH of the enzyme were 50℃and 6.0,respectively.The enzyme activity was stable in the temperature of 40-80℃,and pH of 2.5-6.5.The activity of this enzyme was enhanced by Fe^(2+),Mg^(2+)but inhibited by Fe^(3+),Mn^(2+),Zn^(2+),Ca^(2+),Cu^(2+).Withβ-glucan as substrate,the K m and V max values of the crude enzyme were 1.59 mg/mL and 6.99μmol/(mg·min),respectively.The in vitro digestion viscosity treated with the mutant ARTP-9 solid fermentation enzyme preparation was the lowest,which was 2.23 mPa·s at 4 h of gastric phase,and 0.70 mg/mL of reducing sugar at 21 h of intestinal phase was 3.2 times that of blank.There was significant difference(P<0.05).Compared with the blank,the in vitro digestibility of barley flour treated by ARTP-9 solid fermentation enzyme preparation significantly increased by 9.02%,reaching 48.17%,and the difference was extremely significant(P<0.01).As shown by these results,the ARTP mutagenesis method could significantly improve theβ-glucanase activity of T.reesei,which laid the foundation for its industrial production.