Characterization Of Glucoamylase From Aspergillus Niger B-30and Its Thermal Stability Mechanism

Glucoamylase is an acidic glucoside hydrolase with exo-acting enzymeactivity. Glucoamylase could catalyze the hydrolysis of α-1,4glycosidic linkagesfrom the non-reducing ends of starch and oligosaccharide to produce glucose.Glucoamylase is an industrially important biocatalyst and has the most extensiveuses in the manufacture of starch sugar, food, medicine and brewing, and it hasthe largest production in China.At present, the glucoamylase has the disadvantage of low thermal stability.Hence, the starch after liquefying at high temperature has to be cooled tocontinue the following saccharification process by glucoamylase. In addition, thelong-time saccharification also causes greater loss of glucoamylase, which hasgreatly increased the cost of production. However, the mechanism for the thermalstability of glucoamylase is not clear, and meanwhile the exploitation ofglucoamylase with high thermostability is still on the experimental level. Tosolve this problem, the glucoamylases from a mutant strain Aspergillus nigerB-30were purified, and the properties and unfold mechanism induced bytemperature of these two enzymes were also characterized. Meanwhile, thethermal stability of glucoamylases was improved by adding stabilizer, and themechanism was also eluciadted. Finally, the glucoamylase were immobilized,and the properties of the immobilized glucoamylase were analyzed.First, the glucoamylases from A. niger B-30were purified and the propertiesof these enzymes were also characterized. The two different glucoamylasesGAM-1and GAM-2were purified by ammonium sulfate precipitation, DEAE Fast Flow and Superdex G-75gel filtration columns from the fermentation brothof A. niger B-30. The molecular weight values of GAM-1and GAM-2weredetermined as97.2kDa and78.3kDa by SDS-PAGE, while the correspondingvalues of GAM-1and GAM-2were determined to be80.5kDa and70.4kDa byMALDI-TOF MS, respectively. Both the enzymes were glycosylated, with10.4%and13.4%carbohydrate content. The optimal pH and temperature of these twoenzymes were4.0-4.6and70℃. Both the two glucoamylases displayed highthermal stability and pH stability, and the GAM-2was more stable than GAM-1.Thus, GAM-1and GAM-2are suitable for starch saccharification due to theirhigh catalytic acitivty and stability.Secondly, the folding and unfolding mechanisms of glucoamylases inducedby temperature were studied. The effect of temperature on the conformation ofGAM-1and GAM-2was analyzed by the method of circular dichroism spectrum,fluorescence spectrum, ultraviolet absorption spectrum, dynamic light scatteringand Native-PAGE. The results suggested that both GAM-1and GAM-2displayedsimilar unfolding mechanism. During the process of GAM-1and GAM-2, theα-helix content decreased, and β-fold, β-turn and random coil contents increased,and hydrophobic groups exposed with the aggregation of the protein. Thestructure of GAM-2was more stable during heating, which was the probablereason for that GAM-2showed higher thermal stability.Then the effect of stabilizer on the thermal stability and the mechanism werestudied.1M sorbitol and mycose were respectively added into glucoamylasereaction mixture to stabilize the conformation to maintain the activity at hightemperature. The results suggested that the activity was not obviously increasedat low temperature. On the contrary, the glucoamylase with stabilizer remainedhigher activity at high temperature compared to the negative control. To analyze the thermal stability mechanism of the stabilizer, the thermodynamics data with1M sorbitol and mycose was calculated at75℃and80℃, respectively. Theresults indicated that the kinetics data of heat inactivation obviously reduced afterthe adding of stabilizers, and the half-life and△G increased. The glucoamylaseconformation was not influenced by the stabilizer at25℃, however, at hightemperature the stabilizer could restrain the changing of the secondary andtertiary structure and aggregating of glucoamylase to maintain the naturalconformation, which would lead to higher activity, and the mycose was moreefficient than sorbitol.Finally, the glucoamylase was immobilized on two resins Sepabeads EC-ODand Sepabeads EC-HA, and the properties of the immobilized glucoamylase wereanalyzed. The results suggested that the optimal conditions of immobilizing theGAM-1onto these two supports were uniform. The optimal concentration ofenzyme, temperature, pH and ionic strength were determined as1mg/mL,25℃,4.6and25mM, respectively. The optimal concentration of glutaraldehyde usedto cross-link Sepabeads EC-HA was2%. The two immobilized enzymeEC-OD-GA and EC-HA-GA exhibited a uniform disperse. Particle sizes of thetwo immobilized enzymes were259.9±41.10and229.2±44.04μm by laserscattering analysis, respectively. The surface morphologies of the carriers and theimmobilized enzyme were studied using SEM analysis. The results provided adirect evidence of the successful adsorption of the enzyme on the resins. Theproperties analysis of the immobilized glucoamylase suggested that the optimaltemperature of immobilized and free enzyme were both70℃, but the activity ofimmobilized enzyme was higher than free enzyme at high tempreture.Furthermore, the activity of the EC-OD-GA was higher than EC-HA-GA.Compared with free GAM-1, the optimal pH of the EC-OD-GA is more acidic, and the optimal pH of EC-HA-GA did not change. Both the thermostability andpH stability of immobilized enzyme were improved, and the EC-HA-GA wasmore stable than EC-OD-GA. After immobilization using two supports, theGAM-1showed a good ability to be recycled. After eight times repeated use, theEC-HA-GA and EC-OD-GA could maintained60%and28%relative activity,respectively. Compared with the free enzyme, the storage stability of theimmobilized enzyme was largely improved.