Cloning and Characterization of a Ginsenoside-Hydrolyzing α-L-Arabinofuranosidase, CaAraf51, From Cellulosimicrobium aquatile Lyp51.

Current Microbiology
Sha-Sha ZuoMeng-Liang Wen

Abstract

Moutai Jiuqu is a famous aromatic raw material of Maotai flavor liquor in China. It is brewed at high temperature and contains many kinds of bacteria, molds, and yeasts. There are many useful glycoside hydrolases in these microfloras, from which efficient glycoside hydrolases can be screened for biotransformation of natural saponins. In this study, an α-L-arabinofuranosidase gene (CaAraf51, 1524 bp, 507 amino acid, 55.07 kDa, and pI = 4.8) was cloned from Cellulosimicrobium aquatile Lyp51, which was isolated from the Maotai Jiuqu. The CaAraf51 was heterogeneously expressed in E. coli BL21 (DE3) and purified by N-terminal His-tag with the Ni2+-affinity column chromatography. The results show that purified CaAraf51 has a 6.8-fold purification factor and specific activity of 15 U/mg. Under optimal conditions (pH 5.0, temperature 40 °C), kinetic parameters Km of CaAraf51 for pNPαAraf and Rc were 1.1 and 0.57 mM, the Vmax were 25 and 6.25 μmol/min/mg, respectively. 90% of 0.87 mg Rc substrate can be transformed by 9.6 U purified CaAraf51 in 1 mL reaction system under suitable conditions (30 °C, pH 7.5 phosphate buffer, 1 h). In addition, we also tested the effects of metal ions and chemical agents on the activity of CaAraf51. Accord...Continue Reading

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Citations

Apr 4, 2021·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Ru ZhangZhi Yong Luo

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Datasets Mentioned

BETA
CP020857.1

Methods Mentioned

BETA
glycosylation
protein assay
PCR
protein concentration assay
column chromatography

Software Mentioned

ExPASy
ProtScale
BLAST search

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