柚苷酶生产菌TC-01发酵培养条件的优化及其酶学性质的初步研究

Optimization of Culture Conditions and Characteristics of Naringinase from Aspergillus. niger TC-01

以黑曲霉TC-01菌株作为试验材料对其液态发酵条件及初步纯化后的柚苷酶的酶学性质进行研究。采用单因素试验优化碳、氮源对产酶的影响,利用Plackett-Burman设计筛选影响产酶的主要因素,最后通过响应面分析法对液体发酵培养条件进行优化,优化后柚苷酶酶活与初始相比提高了2.4倍,达到1 216.7 U/mL。对TC-01产柚苷酶中α-鼠李糖苷酶和β-D葡萄糖苷酶的酶学性质分别进行了研究,2个酶反应的最适反应温度均为60℃,在50℃以下保温1 h,2个酶的活力基本不受损失;2个酶最适pH值为4.0,α-鼠李糖苷酶在pH值4.0~8.0范围内稳定性较好,β-D葡萄糖苷酶在pH值5.0~7.0范围内稳定性较好;α-鼠李糖苷酶米氏方程Y=0.001 3X-4.0×10-6,Km=5.60 mmol/L,β-D葡萄糖苷酶米氏方程Y=0.001 8X-3.0×10-6,Km=1.03 mmol/L。

In this article,to Aspergillus.niger TC-01 which is screened as experimental materials,liquid experimental optimization using single factor effect of carbon and nitrogen source on production,filter design via Plackett-Burman main factors affecting enzyme production,and finally by method of response surface analysis on liquid fermentation conditions optimization,optimization naringinase enzyme activity compared with the initial enzyme increases 2.4 fold,up to 1 216.7 U/mL.The experiment studies characteristics of α-rhamnoside and β-D-glucosidase in naringinase from Aspergillus.niger TC-01 of naringinase-producing respectively,two enzymatic reactions of optimum temperature is 60 ℃,at below 50 ℃ insulation 1 h,two activities is not lost.Two enzyme optimum pH is 4.0,the pH range of α-rhamnoside active stability is 4.0~8.0,the pH range of β-D-glucosidase active stability is 5.0~7.0.Michaelis-menten equation of α-rhamnoside is Y=0.001 3X-4.0×10-6,Km=5.60 mmol/L;Michaelis-menten equation of β-D-glucosidase is Y=0.001 8X-3.0×10-6,Km=1.03 mmol/L.