为开发新型高产β-葡萄糖苷酶的微生物菌种资源,本实验从腐木中分离获得1株产β-葡萄糖苷酶的青霉菌株L1;经等离子-硫酸二乙酯复合诱变后,利用七叶苷平板法初筛,摇瓶发酵复筛,最终获得1株可稳定遗传的突变菌株D-6,经单因素试验、Plackett-Burman试验、最陡爬坡试验和响应面试验确定了其发酵产酶最佳条件。结果表明,最佳产酶条件是:KH2PO4 6 g/L、MgSO4·7H2O 1 g/L、CaCl2 0.5 g/L、FeSO4 0.1 g/L,初始pH 5.2,接种量5%(孢子浓度108个/mL),碳源添加量(X1) 玉米秸秆45.74 g/L、氮源添加量(X2) (NH4)2SO4 7.23 g/L、装液量(X5) 63 mL/250 mL,发酵温度28 ℃,摇床转速160 r/min,发酵时间132 h,D-6菌株的β-葡萄糖苷酶活力为142.92 U/mL,较出发菌株L1提高了274.4%。研究结果为产β-葡萄糖苷酶菌株发酵条件优化提供技术参考,同时为该类菌株的开发和应用提供有效的菌种资源。
A Penicillium strain named L1 capable of producing β-glucosidase was isolated from rotten wood with the aim to study and develop microbial origin β-glucosidase resources. The strain was treated by atmospheric and room temperature plasma (ARTP) and diethyl sulfate (DES) mutation, followed by esculin agar plate screening and enzyme activity re-screening, a β-glucosidase high-yield mutant D-6 with genetic stability was obtained. By one-factor-at-a-time method, Plackett-Burman design, steepest ascent path and response surface methodology, the optimum conditions for β-glucosidase production in fermentation were determined. The optimal fermentation conditions were KH2PO4 6 g/L, MgSO4·7H2O 1 g/L, CaCl2 0.5 g/L and FeSO4 0.1 g/L; pH value of 5.2, inoculum 5% (spore concentration 108/mL), corn straw(X1) 45.74 g/L, (NH4)2SO4 7.23 g/L, packing volume(X5) 63 mL/250 mL, temperature 28 ℃, shaking speed is 160 r/min, incubation 132 h. Under the above conditions, the β-glucosidase activity of D-6 was 142.92 U/mL, with an increase of 274.4% compared with the original strain L1. The results obtained in the study were meaningful for technical reference and microbial resources of β-glucosidase producing strain.
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