Analysis Of The Enzymatic Properties And Applied Research Of α-transglucosidases From Aspergillus Niger

Isomaltooligosaccharide has attracted extensive attention due to its unique physiological functions, a-transglucosidase is the key enzyme to produce isomaltooligosaccharide and different kinds of a-transglucosidases exist some difference on the enzymatic properties and transglycosidation to the substrate. This paper studyed eight recombinant Pichia pastoris strains P. pastoris A5, P. pastoris A24, P. pastoris A45, P. pastoris A58, P. pastoris B74, P. pastoris B87, P. pastoris C12, P. pastoris C35 and analysed the enzyme properties and transglycosidation products of enzymes Glu-A5, Glu-A24, Glu-A45, Glu-A58, Glu-B74, Glu-B87, Glu-C12, Glu-C35 generated by the above strains. The research contents are as follows.Firstly, the method of assaying the enzyme activity of a-transglucosidase was determined and the original GB method was optimized. The generation of glucose produced by the a-glucosidase hydrolysis of the substrate was measured by biosensor, which directly reflected the enzyme activity of a-transglucosidase and simpled the measurement method.Then the eight recombinant P. pastoris strains were accumulated cultured for 24h (BMGY) and induced culture by methanol for 120h(BMMY). The fermentation broth was centrifugated and the supernatant liquid was the crude enzyme solution. Three concentration methods ammonium sulfate precipitation, acetone precipitation, ethanol precipitation were compared and finally the ethanol precipitation was defined as the optimal method. The concentration conditions are as follows:the ratio of ethanol with crude enzyme solution was 2:1{V:V), the concentration temperature was 4℃. The enzyme activity recovery reached 88%.The results of enzymatic property analysis showed:all the optimum temperature of eight a-glucosidases was 50℃, the optimum pH was between 5 and 6, the enzyme activity almost lost when the temperature reached 90℃. The activity was stimulated by K+, Ca2+, Mg2+while restrained by Fe2+, Cu2+, Al3+, Fe3+ and no obvious enzymatic properties difference among the eight a-transglucosidases.The transglycosidation conditions of eight a-glucosidases were optimized and the optimum temperature of reaction was between 50℃ and 55℃, the range of optimum pH was 5.0-5.5, the optimum time of reaction was 25-30h and the optimum enzyme dosage was 36 U/mL-42 U/mL.The transglycosidation was conducted under the optimum condition of each a-transgluco sidase and the reaction results of hydrolyzing maltose were analysed by HPLC The results showed that after the reaction of Glu-A5, Glu-A24, Glu-A45, Glu-A58, the productivity of panose was relatively high reached,21.19%-31.41% respectively. After the reaction of Glu-C12 and Glu-C35 to maltose, the productivity of isomaltotriose was high reached 15.85%-17.73% reachedrespectively. However, Glu-B74 and Glu-B87 had a lower activity and the productivity of three oligosaccharides were all less than 11%.