Isolation, Purification And Enzymatic Properties Of Aspergillus Oryzae α-amylase

Bean sauce was a traditional fermented soy product in China,which was very popular with unique flavor and aroma during a few thousand years.With the continuous development of science technology and the qulity of common lives,the system of new technology sauce continued to improve.Compared with the traditional production process,using pure enzyme to produce bean sauce not only improved the product quality,but also greatly shortened the fermentation period,which had beening become the trend of bean sauce manufacturing.This paper described the purification and characterization of a-amylase from Aspergillus oryzae HDF-10 isolated from the traditional soy sauce,which could lay foundation on the enzyme industry production.The aqueous two-phase system of PEG/(NH4)2SO4 was estabillised for α-amylase preliminary extraction and purification.The single factor,which influenced on partition coefficient and recovery rate of a-amylase in aqueous two-phase systems,was also examined.At the same time,optimum combination of those factors studied above was confirmed by orthogonal test,which was used for the extraction on a system of 30%PEG1500/9%(NH4)2SO4 with 13%NaCl at pH 6.5.The conclusion showed that it was obtained with a maximum partition coefficient of 3.738 and enzyme activity recovery rate of 85.315%.The enzyme displayed a molecular weight of 55 kDa,which was purified by PEG 1500/(NH4)2SO4 system and gel filtration chromatography-Sephadex G-75,on SDS-PAGE electrophoresis verification.The purification was 25.64 times and the recovery rate was 47.2%ultimately.Patial amino acid sequences which were compared through NCBI Protein BLAST demonstrated 93%similarity with other known α-amylases from Aspergillus spp.At the same time,enzyme characterizations were also different indicating that the presented enzyme maybe was new.The enzyme was stable over a range of pH from 5.0 to 6.0 with an optimum at 6.0;the optimum temperature for activity was found to be at 60℃ and the enzyme remained active to almost 85%to 50-60℃.The enzyme activity was strongly inhibited by Mn2+ and Cu2+,whereas the activity was strongly increased by K+ and Ca2+.Soluble starch was gradually hydrolyzed to be glucose,maltose and some oligosaccharides by thin-layer chromatography analysis,which showed that the enzyme was an incision enzyme.This paper showed that the aqueous two-phase system of PEG/(NH4)2SO4 for a-amylase preliminary extraction and purification not only simplified the purification steps to effectively reduce the loss of the enzyme,which maintained the high activity,but decreased toxics during the operation of purification.It was an efficient and convenient method of purifying a-amylase,which could be widely applied in soybean paste and other food productions.