Solid State Fermentation And Purification Of Acid Stable α-amylase From Aspergillus Niger PZ301

Acid stable α-amylase could catalyze the hydrolysis of the a-1,4glycosidic linkagesof starch at random, and oligosaccharides, maltoses, dextrines were obtained at low pH.Compared with α-amylase, application of acid stable α-amylase in starch industry andbrewage industry had more value to development, due to its stability in acid conditions.According the morphological identification and microscopic observation, a strain offungi PZ301producing acid stable α-amylases was identified as Aspergillus niger.Aacid stable α-amylase producing strain PZ301was used as parent strain and mutatedby microwave and NTG. The results showed that the spores of PZ301were treated with250μg/mL NTG for90min and microwave radiation (2450HZ) for30s, the death ratereached86.6%, and the positive mutation frequency get maximum. Under the aboveconditions, one mutant with high acid stable α-amylase-producing capacity was bredthrough primary screening with transparent circle method, compared with original enzymeactivity, the enzyme activity was1.985IU/mL,23%higher than acid stable α-amylaseproduced by parent strain.Solid state fermentation conditions for PZ301producing acid stable α-amylase werestudied. The results showed that optimal medium was composed of bran7g, complexcarbon source (glucose+starch (1/4))0.28g;(NH4)2SO40.14g, and the ratio of material towater for1:2;1mL spore suspension (107spores per mL) was inoculated into medium andPZ301was grown at35℃for72hours, the enzyme activity reached40IU/g, and it wasincreased by55%.The quantitative relationship between nucleic acid and biomass was established byA260absorbance assay method. Based on the assay method, the biomass curve of PZ301insolid state fermentation was determined. Production kinetics was studied through studyingthe relation between the curve of strain PZ301and the time of acid stable α-amylaseproduction. The result indicated that the type of acid stable α-amylase synthesizing wassynchronous synthesizing.Solid-state fermentation medium was extracted by5times volume of0.05mol/L citricacid-disodium phosphate buffer (pH4.2). After being filtrated and centrifuged, Crudeenzyme was obtained. The extracts were precipitated by50-80%degree of saturationammonium sulfate, decolorized by3%carbon powder and treated by ultra filtration(100kDa). Then the enzyme was separated on Sephadex DEAE A-50chromatography atpH5.4and HPLC chromatography. PAGE and SDS-PAGE were used to identify the purity and the molecular weight of the acid stable α-amylase. The results showed that a singleband appeared on PAGE gel which suggested that relatively pure acid stable α-amylase hasbeen obtained, and the molecular weight of fucoidanase was58.74kDa.Crude enzyme properties were also studied. The results showed that the optimumtemperature and pH were50℃and4.2, respectively;50%inactivation of the acid stableα-amylase activity occurred at65℃for90min, around pH2.2~8.0the enzyme have theprime stability.