| Raw-starch-digesting amylase is capable of cooking without raw starch granule gelatinization can exhibit strong hydrolytic activity of enzymes. Due to raw-starch-digesting amylase without cooking turn raw starch can be directly translated into glucose and fermentable sugar for microbial growth and metabolism, its than traditional high temperature cooking saccharification save 25%-30% of energy consumption. It is because of raw-starch-digesting amylase has potential industrial application value and some special purpose, it has been the subject of many researchers concern at home and abroad.This topic from raw-starch-digesting amylase Monascus M2 for starting produce bacteria strains, through the optimization of fermentation conditions and medium, the strain of further improve the capacity of producing enzymes and the crude enzyme liquid produced by the separation and purification, and some properties of purified enzyme liquid was studied. The main research results are as follows:(1). Solid state fermentation of Monascus M2 raw-starch-digesting amylase is optimized. The solid state fermentation conditions optimization results show that:the temperature of 32℃, pH value 5.0, inorganic salt content is 14mL, incubation time 8d, under the condition of the monascus M2 can be obtained the maximum amylase enzyme activity is 445.37 U/g. Medium optimization of the single factor results show that the optimal carbon sources is lactose, nitrogen source is (NH4)2SO4, optimal inorganic salt is K2HPO4, further through the Box-Bohnken center of experimental design and response surface analysis, finally to determine the appropriate culture conditions for:lactose:1.80g,(NH4)2SO4:1.40g,K2HPO4: 0.2g. Under these optimal conditions,the predicted and experimental production of the activity of raw-starch-digesting amylase was high up to 680.29 U/g and 662.21 U/g,respectively.(2) The raw-starch-digesting amylase from solid-state fermentation culture of Monascus M2 was purified by with water extraction,ammonium sulfate precipitation,DEAE-Sepharose fast flow and Sephacryl S-200 gel filtrateion chromatography. A purified raw-starch-digesting amylase,showing a single band on Tricine-SDS-PAGE, was obtained with specific enzyme activity of 396.61U/mg, the purification multiple was 6.86 and the recovery rate is 12.70%. The molecular weight of the raw-starch-digesting amylase was determined as 67.81kD on Tricine-SDS-PAGE.(3) The purified Monascus raw-starch-digesting amylase were characterization,experimen-tal results show that:the optimum temperature is 50℃,60℃ for 2h can still maintain the enzyme activity of nearly 70%. the optimum pll is 5.0. in the pH3.0-7.0 environment for 30min can still maintain the enzyme activity of more than 60%. Its kinetics coefficients Km and Vmax with raw corn flour as substrate were 14.96mg·ml-1and 10.79mg·mL-1 min-1,respectively. Its activity was activated by Mn2+,Ca2+and Ba2+, while Na+, K+,Mg2+, Zn2+ have no effect of it and strongly activated by Fe3+. Its activity was activated by L-cysteine, while SDS, DMSO, DTT and β-mercaptoethanol have no effect of it, EDTA and EGTA can inhibit the enzyme activity is weak and strongly activated by PMSF,KMnO4, Chloramine-T and Triton X-100.In summary, this study through the solid state fermentation of raw-starch-digesting amylase, improved Monascus M2, having raw-starch-digesting amylase, produced by the pH stability under the condition of experiment not only has the greatly strengthened, but also can adapt to a wide pH range. At the same time most of metal ions and chemicals to the influence of the enzyme is little, so it is of high theoretical and practical value. |
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