Mutation And Application Of Raw Starch-Digesting Glucoamylase Strain

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%.