生淀粉糖化酶产生菌的选育与应用
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摘要
根霉2是本实验室保存的一株产生淀粉糖化酶活力较高的菌株,本实验将根霉2产的生淀粉糖化酶应用于生料发酵,研究了37℃木薯生料发酵生成酒精的工艺。通过单因素试验和响应面优化,得到其最优发酵条件为：pH 4.6、发酵时间68.9 h、加酶量173.00 U/g、料液比1：3.5、酵母接种量108/mL、尿素0.2%(w/v),在此条件下发酵,酒精度为9.95%(v/v),与预测值9.90%(v/v)相差不大。另外摇床发酵、添加糖化酶和添加α-淀粉酶对发酵没有明显的提高效果。初步研究了木薯浓醪发酵酒精,采用料液比为1：2.5,发酵时间60 h,酒精度达到最大12.89%(v/v)。
以根霉2为出发菌株,筛选得到突变株UL16,其生淀粉糖化酶活力比出发菌株平均提高28.29%。同时研究了添加2-D-脱氧葡萄糖以筛选抗性突变株,得到突变株K7,酶活平均提高了25.44%。UL16和K7经传代试验,产酶稳定。
对生淀粉糖化酶进行了硫酸铵初步分离纯化,并研究了生淀粉糖化酶的部分酶学性质。结果表明：最适反应温度为40℃；最适反应pH范围为4.0～4.8；50℃内较稳定,超过此温度酶活急剧下降；pH4.8～9.0范围内较稳定,酶活力都能保持80%以上；金属离子Hg2+、Ni2+、Ag+和Fe3+对酶活有抑制作用,K+、Fe2+和Na+对酶活基本没有影响,Li+、Co2+、Zn2+、Cu2+、Mg2+、Pb2+、Ca2+和Mn2+对酶活有激活作用,其中Mn2+对酶活提高幅度最大,达到88.42%。
Rhizopus sp.2 stored in our laborator is a strain that can produce raw starch-digesting glucoamylase at high level. In this experiment, raw starch-digesting glucoamylase produced by Rhizopus sp.2 was applied in ethanol fermentation, and the conditions of ethanol fermentation under 37℃using raw cassava srarch with was studied. By single factor experiment and response surface analysis optimization, the optimum fermentation conditions were:pH 4.6, fermentation time 68.9 h, enzyme 173.00 U/g, material/liquid 1:3.5, yeast inoculum size 108/mL, urea 0.2%(w/v). Fermenting under these conditions, the predictive value of alcohol concentration was 9.90%(v/v), and the actual alcohol concentration was 9.95%(v/v). Besides, shaker fermentation、adding glucoamylase and adding a-amylase had no significant improvement on the alcohol fermentation. High-gravity fermentation of cassava ethanol fermentation was preliminary studied，the material water ratio was 1:3.5，when fermentation time was 60 h, the maximum alcohol concentration reached 12.89%(v/v)
Using Rhizopus sp.2 as the starting strain, mutant strain UL16 was obtained, and its raw starch-digesting glucoamylase activity increased by 28.29% average than that of the starting strain. At the same time, resistant mutant strain was screened by adding 2-D-deoxyglucose, mutant strain K7 was obtained, its enzyme activity rised in an average of 25.44%. The enzyme production of UL16 and K7 were very steady through passage experiments.
Raw starch-digesting glucoamylase was initially purified by ammonium sulfate, and enzymology properties of raw starch-digesting glucoamylase was partly studied. The optimum temperature was 40℃. The optimum pH was in the range of 4.0～4.8. Enzyme was more stable under 50℃, and its activity declined sharply when over this temperature. The enzyme performed stably in range of pH 4.8 to 9.0 and the residual enzyme activity maintained more than 80%. Hg2+、Ni2+、Ag+ and Fe3+ showed inhibition to the enzyme activity, K+、Fe2+ and Na+ had no effect on the enzyme, Li+、Co2+、Zn2+、Cu2+、Mg2+、Pb2+、Ca2+ and Mn2+ could activate the enzyme activity, Mn2+ had great activation on the enzyme activity especially, which increased by 88.42%.
以根霉2为出发菌株,筛选得到突变株UL16,其生淀粉糖化酶活力比出发菌株平均提高28.29%。同时研究了添加2-D-脱氧葡萄糖以筛选抗性突变株,得到突变株K7,酶活平均提高了25.44%。UL16和K7经传代试验,产酶稳定。
对生淀粉糖化酶进行了硫酸铵初步分离纯化,并研究了生淀粉糖化酶的部分酶学性质。结果表明：最适反应温度为40℃；最适反应pH范围为4.0～4.8；50℃内较稳定,超过此温度酶活急剧下降；pH4.8～9.0范围内较稳定,酶活力都能保持80%以上；金属离子Hg2+、Ni2+、Ag+和Fe3+对酶活有抑制作用,K+、Fe2+和Na+对酶活基本没有影响,Li+、Co2+、Zn2+、Cu2+、Mg2+、Pb2+、Ca2+和Mn2+对酶活有激活作用,其中Mn2+对酶活提高幅度最大,达到88.42%。
Rhizopus sp.2 stored in our laborator is a strain that can produce raw starch-digesting glucoamylase at high level. In this experiment, raw starch-digesting glucoamylase produced by Rhizopus sp.2 was applied in ethanol fermentation, and the conditions of ethanol fermentation under 37℃using raw cassava srarch with was studied. By single factor experiment and response surface analysis optimization, the optimum fermentation conditions were:pH 4.6, fermentation time 68.9 h, enzyme 173.00 U/g, material/liquid 1:3.5, yeast inoculum size 108/mL, urea 0.2%(w/v). Fermenting under these conditions, the predictive value of alcohol concentration was 9.90%(v/v), and the actual alcohol concentration was 9.95%(v/v). Besides, shaker fermentation、adding glucoamylase and adding a-amylase had no significant improvement on the alcohol fermentation. High-gravity fermentation of cassava ethanol fermentation was preliminary studied，the material water ratio was 1:3.5，when fermentation time was 60 h, the maximum alcohol concentration reached 12.89%(v/v)
Using Rhizopus sp.2 as the starting strain, mutant strain UL16 was obtained, and its raw starch-digesting glucoamylase activity increased by 28.29% average than that of the starting strain. At the same time, resistant mutant strain was screened by adding 2-D-deoxyglucose, mutant strain K7 was obtained, its enzyme activity rised in an average of 25.44%. The enzyme production of UL16 and K7 were very steady through passage experiments.
Raw starch-digesting glucoamylase was initially purified by ammonium sulfate, and enzymology properties of raw starch-digesting glucoamylase was partly studied. The optimum temperature was 40℃. The optimum pH was in the range of 4.0～4.8. Enzyme was more stable under 50℃, and its activity declined sharply when over this temperature. The enzyme performed stably in range of pH 4.8 to 9.0 and the residual enzyme activity maintained more than 80%. Hg2+、Ni2+、Ag+ and Fe3+ showed inhibition to the enzyme activity, K+、Fe2+ and Na+ had no effect on the enzyme, Li+、Co2+、Zn2+、Cu2+、Mg2+、Pb2+、Ca2+ and Mn2+ could activate the enzyme activity, Mn2+ had great activation on the enzyme activity especially, which increased by 88.42%.
引文
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