Effect Of N-Deglycosylation On Thermostability And Spatial Structure Of Tannase

Glycosylation,one of the major naturally occurring modifications of the covalent structure of proteins,has been found to effect the structure and function of protein.To research the effects of glycosylation on the catalytic characteristic and structure of enzyme,tannase from Aspergillus niger was purified and glycosylation level was analysised.The catalytic characteristic and kinetics of the tannase was investigated,and we also studied the effect of glycosylation on the thermal stability and conformation stability tannase.Main results are as follows:(1)Ammonium sulphate precipitation,DEAE-Sepharose Fast Flowcolumn chromatography and Sephadex G-150 column chromatograph were sequentially carried out to obtain an electrophoretically pure tannase(tannin acyl hydrolase E.C.3.1.1.20)of Aspergillus niger,with 20.2-fold and a yield of 35.95%.The molecular mas of tannase was deter mined to be 81.9 kDa,consisted of approximately 45.0 and 36.9 kDa sub-units.(2)The optimum temperature and pH of catalysis reaction active of purified tannase were 50 ? and 5.0,respectively.The enzyme was found stable in a range of temperature(20-60 ?)and pH(4.0-6.5),pH and temperature have influence on the secondary structure of tannin enzyme.Under 4 ?,the storage stability of crude tannase is better than purified tannase,probably because of some material in the crude enzyme protected enzyme activity.The influence of additives to the activity of tannase were studied.The enzyme was activated by some metal ions(Mg2+,Mn2+,Ca2+,Ba2+,Zn2+),inhibited by other metal ions(Sn2+,Hg2+Al3+,Fe2+,Fe3+,Cu2+)and organic reagent(methanol,ethanol,acetone,formaldehyde,sherwood oil),and induced by Na+,K+,Li+,tween 20,tween 60,tween 80,triton-100,EDTA and SDS.The michaelis constant(Km)of the tannase for propyl gallate(PG)was 9.03 mmol/L with Vmax 0.10 mmol/min.E.of these reactions and temperature dependence(at 30-70?)of ?Gd,?Hd,?Sd for the enzyme and substrate were deter mined.As the temperature increased,the stability of tannase got poor.(3)After deglycosylated of tannase at the optimization condition(PNGase F dosage 0.5%(v/v),37? for 12 h),the molecular weight of two subunits of tannase were 45.0 kDa and 29.1 kDa.The activity of deglycosylated tannase lossed 34.4%at 30 ?,and the deglycosylated tannase displlayed a significant decrease in its thermostability than the glycosylated counterpart.The results of circular dichroism(CD)spectra,fluorescence chromatogram and atomic force microscopy(AFM)showed the secondary structure,tertiary structure and microstructure changed at different degree after deglycosylated by PNGase-F,indicated that it maybe occur molecular rearrangement in tannase decreased the enzyme activity after deglycosylated by PNGase-F.