Fermentation Conditions, Purification And Characterization Of α-Transglucosidase From Aspergillus Niger

A.niger M-l which was screened in our laboratory produced a strongly a-transglucosidase. In this paper,studies on the fermentation conditions, purification and characterization of a-transglucosidase and its necessary groups was carried out in this dissertation. The main reports were as following:The fermentation conditions in shaking flasks were investigated by the method of single-factor analysis, the suitable main medium was achieved: which contained 4% A,2% B and 1% G ; the A.niger M-l was inoculated into 100mL medium in flask, shaking in 33 C at 140r/min for four days, with initial pH6.5 and 6% inocula volume ; adding 0.1 mmol/L methyl a-D-glucopyranoside had inductive effect on enzyme formation , the a-transglucosidase activity amounted to 296.05u/ml.By ammonium sulphate fractional precipitation, DEAE-cellulose 52, DEAE-sepharose CL-6B anion exchange chromatographpy, sephadex G-100 gel filtration and SAS-PAGE, a a-transglucosidase was purified from extract content of A.niger M-l, the molecular weight of the enzyme was about110kDa.Some properties of the enzyme were undertaken, the results were as follows : The steadiest pH of a-transglucosidase for methyl a-D-gluco-pyranoside was 6.0, it was stable under 60 C; the Michaelis-Menten constant (Km) was 4.32 mmol/L for methyl A-D-glucopyranoside; the a-transglucosidase was activated by Ca2+and Mg2+ ion, but was inhibited by Cu2+,Ag+ and Fe2+ ion. The partial N-terminal amino acid sequences of the purified enzyme were identified as N-SVPGTEYVV-9.The a-transglucosidase was selectively modified by pCMB,ME ,EDC, ClAc, acetyl acetone and NBS, and changes in the activities of the enzyme have been detected. The reaction of a-transglucosidase with pCMB, ME, EDC and ClAc resulted in a strong inhibition of the enzyme activities which decreased with the increase of modifier concentration. The acetyl acetone and NBS were found without inhibition effect. Studies of inactivation of a-transglucosidase by chemical modification demonstrated that Cys, Asp(Glu) and His residues were indispensable groups of the a-transglucosidase. Partial disulfide bonds were the function of this enzyme.