Purification and Characterization of Two Thermostable Glucoamylases Produced from Aspergillus niger B-30  

作　　者：LIU Yang LI Quan-shun ZHU Hong-liang MENG Zhao-li XIANG Hong-yu XIE Qiu-hong 

机构地区：[1]Key Laboratory for Molecular Enzymology and Engineering, Ministry of Education [2]National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun 130012, P. R. China [3]Zibo Guoao Biotechnology Co., Ltd., Zibo 255400, P. R. China

出　　处：《Chemical Research in Chinese Universities》2013年第5期917-923,共7页高等学校化学研究（英文版）

基　　金：Supported by the National Natural Science Foundation of China（No.81072564） and the Project of the Jilin Province Science and Technology Institute of China（No.20090945）.

摘　　要：Two thermostable glucoamylases were produced from Aspergillus niger B-30 by submerged fermentation. The two glueoamylases GAM-1 and GAM-2 were purified by ammonium sulfate precipitation, diethylaminoethyl- cellulose fast flow（DEAE FF） and Superdex G-75 gel filtration columns. The molecular weights of GAM-1 and GAM-2 were determined as 9.72x 104 and 7.83x104 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）, while the molecular weights of GAM-1 and GAM-2 were determined to be 8.05x 104 and 7.04x 104 by matrix assisted laser desorption ionizationtime-of-flight（MALDI-TOF） mass spectrometry, respectively. Both the enzymes were glycosylated, with 10.4% and 11.4% carbohydrate content, respectively. The optimal pH and tempera- ture were 4.0--4.6 and 70 ℃ for both. The two glucoamylases were maintained 100% relative activity after incuba- tion at 60 ℃ for 120 min. After the hydrolysis of starch for 120 min, glucose was the only product, confirming that the two enzymes were of high efficiency towards starch. The GAM-2 exhibited higher catalytic activity towards oli- gosaccharides such as maltose than GAM-1, and the kinetic analysis shows that the affinity of GAM-2 to starch was lower than that of GAM-1. The high thermostability and effectiveness make the two glucoamylases potentially attrac- tive for biotechnological application.Two thermostable glucoamylases were produced from Aspergillus niger B-30 by submerged fermentation. The two glueoamylases GAM-1 and GAM-2 were purified by ammonium sulfate precipitation, diethylaminoethyl- cellulose fast flow（DEAE FF） and Superdex G-75 gel filtration columns. The molecular weights of GAM-1 and GAM-2 were determined as 9.72x 104 and 7.83x104 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）, while the molecular weights of GAM-1 and GAM-2 were determined to be 8.05x 104 and 7.04x 104 by matrix assisted laser desorption ionizationtime-of-flight（MALDI-TOF） mass spectrometry, respectively. Both the enzymes were glycosylated, with 10.4% and 11.4% carbohydrate content, respectively. The optimal pH and tempera- ture were 4.0--4.6 and 70 ℃ for both. The two glucoamylases were maintained 100% relative activity after incuba- tion at 60 ℃ for 120 min. After the hydrolysis of starch for 120 min, glucose was the only product, confirming that the two enzymes were of high efficiency towards starch. The GAM-2 exhibited higher catalytic activity towards oli- gosaccharides such as maltose than GAM-1, and the kinetic analysis shows that the affinity of GAM-2 to starch was lower than that of GAM-1. The high thermostability and effectiveness make the two glucoamylases potentially attrac- tive for biotechnological application.

关 键 词：GLUCOAMYLASE Thermostability Kinetic analysis GLYCOSYLATION 

分 类 号：Q949.327.1[生物学—植物学] TQ223.122[化学工程—有机化工]