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

doi:10.1007/s40242-013-3074-y

Chemical Research in Chinese Universities ›› 2013, Vol. 29 ›› Issue (5): 917-923.doi: 10.1007/s40242-013-3074-y

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

LIU Yang^1,2, LI Quan-shun¹, ZHU Hong-liang³, MENG Zhao-li^1,2, XIANG Hong-yu^1,2, XIE Qiu-hong^1,2

1. Key Laboratory for Molecular Enzymology and Engineering, Ministry of Education, Changchun 130012, P. R. China;
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

Received:2013-02-27 Revised:2013-06-13 Online:2013-10-01 Published:2013-09-17
Contact: XIANG Hong-yu E-mail:hyxiang@jlu.edu.cn
Supported by:
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).

Abstract

Abstract:

Two thermostable glucoamylases were produced from Aspergillus niger B-30 by submerged fermentation. The two glucoamylases GAM-1 and GAM-2 were purified by ammonium sulfate precipitation, diethylaminoethylcellulose fast flow(DEAE FF) and Superdex G-75 gel filtration columns. The molecular weights of GAM-1 and GAM-2 were determined as 9.72×10⁴ and 7.83×10⁴ by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), while the molecular weights of GAM-1 and GAM-2 were determined to be 8.05×10⁴ and 7.04×10⁴ 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 temperature were 4.0―4.6 and 70 ℃ for both. The two glucoamylases were maintained 100% relative activity after incubation 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 oligosaccharides 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 attractive for biotechnological application.

Key words: Glucoamylase, Thermostability, Kinetic analysis, Glycosylation

LIU Yang, LI Quan-shun, ZHU Hong-liang, MENG Zhao-li, XIANG Hong-yu, XIE Qiu-hong. Purification and Characterization of Two Thermostable Glucoamylases Produced from Aspergillus niger B-30[J]. Chemical Research in Chinese Universities, 2013, 29(5): 917-923.

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

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