Journal of Pure and Applied MicrobiologyVol. 8 No. May 2014 Special Edition

Purification and Characterization of b-D-glucosidase Hydrolyzing Swertiamarin into Erythrocentaurin and 5-ethylidene-8-hydroxy-3, 4, 5, 6, 7, 8-hexahydro-1H-pyrano [3, 4-C]pyridine-1-one from Aspergillus niger CICC 2169

Liu Jiantao1, Zhou Bin2, Chang Jun1* and Deng Zihao3

1Jiangxi Key Laboratory of Bioprocess, Jiangxi Science & Technology Normal University, Nanchang, Jiangxi - 330013, China. 2 School of pharmacy, Jiangxi Science & Technology Normal University, Nanchang, 330013,Nanchang city, Jiangxi - 330013, China. 3Guangdong Education Publishing House, Guangzhou - 510275, China.

Received on 12 April 2014 and accepted on 09 May 2014

 

ABSTRACT

The b-D-glucosidase involved in the biotransformation of swertiamarin to 5-ethylidene-8-hydroxy-3,4,5,6,7,8-hexahydro-1H-pyrano[3, 4-c] pyridine-1-one and erythrocentaurin was purified and characterized. The enzyme had a molecular weight of 88 kDa as determined by SDS polyacrylamide gel electrophoresis. The purified enzyme was optimally active at 55-60?C and pH 5.0. The enzyme was stable at pH 5-7 and 60?C, and specified on b-(1-4)-glucosidic linkage. The affinity constants for r-NPG and swertiamarin were 0.58 and 0.26 (mM),respectively. So the catalytic constants for r-NPG and swertiamarin were 186.8 and 79.6 (?mol min-1 mg-1), respectively. Ca2+, Cu 2+, Zn2+, and Fe3+ (0~120 mM) inhibited the b-D-glucosidase, while Mg2+ and Mn2+ (each 0~120 mM) activated this enzyme. Glucose had a high inhibitory effect on the enzyme and the inhibition constant was 0.13 and thus the concentration of glucose in the broth for producing the b-D-glucosidase should be strictly controlled no more than 8mg/l. The metabolic pathway and the role of the purified b-D-glucosidase were fully discussed.

Keywords : Erythrocentaurin, 5-ethylidene-8-hydroxy-3, 4, 5, 6, 7, 8-hexahydro- 1H-pyrano [3, 4-c] pyridine-1-one, Purification, ?-D-glucosidase, Swertiamarin