Abstract
A gene (bgl) encoding a β-glucosidase in thermophilic actinomycete Thermobifida fusca NTU 22 was cloned into a Yarrowia lipolytica expression system. Heterologous expression resulted in extracellular β-glucosidase production with activity as high as 630 U/mL in a Hinton flask culture filtrate. This recombinant β-glucosidase was purified 9.2-fold from crude culture filtrate by DEAE-Sepharose FF column chromatography as measured by its increase in specific activity. The overall yield of the purified enzyme was 47.5%. The molecular weight of the purified β-glucosidase estimated by SDS-PAGE was 45 kDa, which agreed with the predicted molecular weight based on the nucleotide sequence. About 15% enzyme activity loss was observed after the enzyme was heat-treated at 50 °C for 180 min. It was also found that the activity of the enzyme was inhibited by Hg2+, Cu2+, Ba2+, Ag+, p-chloromercuribenzene, and iodoacetate. The β-glucosidase from T. fusca had the most activity for daidzein-7-glucoside and genistein-7-glucoside among the tested flavonoid glycosides, but there was moderate or little activity for luteolin-7-glucoside, cyanidine-3-glucoside, and quercetin-3-glucoside. These properties are important for the soybean isoflavone applications of this β-glucosidase.
Highlights
Introduction βD-glucoside glucohydrolase (EC 3.2.1.21), generally called β-glucosidase, catalyzes the hydrolysis of β-glucosidic linkages of glucose or oligosaccharides conjugates [1]
The activity of the 2+ increased the β-glucosidase activity (67.4%) of the Mn
This study showed that the β-glucosidase from the Y. lipolytica transformant had a high substrate selectivity β-glucosidase from the Y. lipolytica transformant had a high substrate selectivity towards towards the 7-glucosides of genistein and the 7-glucosides of daidzein
Summary
Introduction βD-glucoside glucohydrolase (EC 3.2.1.21), generally called β-glucosidase, catalyzes the hydrolysis of β-glucosidic linkages of glucose or oligosaccharides conjugates [1]. Β-Glucosidases play pivotal roles in various biotechnological processes such as the reduction of cellobiose accumulation by the action of exo-glucanases to minimize the end-product inhibition [2] and the modification of cyanogenesis, glycolipids, and secondary metabolites [1]. They are involved in the bioconversion of lignocellulose to glucose, producing hydrogen with other exo- or endo-hydrolases and using glucose as an ingredient for fermentation [3,4]. Soybean ingredients are rich sources of flavonoids and phenolic compounds with biological activity [8].
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