著者

Daichi Ito Emiri Nakano Shuichi Karita Midori Umekawa Khanok Ratanakhanockchai Chakrit Tachaapaikoon

出版者

The Japanese Society of Applied Glycoscience

雑誌

Journal of Applied Glycoscience (ISSN:13447882)

巻号頁・発行日

pp.jag.JAG-2022_0001, (Released:2022-04-20)

被引用文献数

4

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Paenibacillus xylaniclasticus strain TW1, a gram-positive facultative anaerobic bacterium, was isolated as a xylanolytic microorganism from the wastes of a pineapple processing factory. A gene encoding one of its xylanolytic enzymes, a β-xylosidase, was cloned and sequenced. Sequence analysis revealed that this β-xylosidase, named PxXyl43A, was composed of a glycoside hydrolase (GH) family 43 subfamily 12 catalytic module and an unknown function module (UM). The full-length PxXyl43A (PxXyl43A) was heterologously expressed in Escherichia coli and purified. Recombinant PxXyl43A exhibited hydrolysis activity against both p-nitrophenyl-β-D xylopyranoside (pNPX) and p-nitrophenyl-α-L-arabinofuranoside at specific activities of 250 mU/mg and 310 mU/mg, respectively. The optimal reaction pH and temperature for pNPX hydrolysis were 7.1 and 54 °C, respectively. At pH 7.0 and 54 °C, the Km and kcat for pNPX were 1.2 mM and 2.8 ± 0.15 s-1, respectively. It was also discovered that the recombinant unknown function module of PxXyl43A (PxXyl43A-UM) could bind to insoluble xylans like birchwood xylan and oat spelt xylan, whereas it did not bind to cellulosic substrates such as ball-milled cellulose, carboxymethyl cellulose or lichenan. The PxXyl43A-UM’s binding constant value Ka for oat spelt xylan was 2.0 × 10-5 M-1. These results suggest that PxXyl43A possesses a novel carbohydrate-binding module, named as CBMx, specific for xylan-containing polysaccharides.

著者

Midori Umekawa Kaito Hamada Naoto Isono Shuichi Karita

出版者

The Japanese Society of Applied Glycoscience

雑誌

Journal of Applied Glycoscience (ISSN:13447882)

巻号頁・発行日

vol.67, no.4, pp.103-109, 2020-11-20 (Released:2020-11-20)

参考文献数

20

被引用文献数

6

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Hexokinases catalyze glucose phosphorylation at the first step in glycolysis in eukaryotes. In the budding yeast Saccharomyces cerevisiae, three enzymes for glucose phosphorylation have long been known: Hxk1, Hxk2, and Glk1. In this study, we focus on Emi2, a previously uncharacterized hexokinase-like protein of S. cerevisiae. Our data show that the recombinant Emi2 protein (rEmi2), expressed in Escherichia coli, possesses glucose-phosphorylating activity in the presence of ATP and Mg2+. It was also found that rEmi2 phosphorylates not only glucose but also fructose, mannose and glucosamine in vitro. In addition, we examined changes in the level of endogenous Emi2 protein in S. cerevisiae in the presence or absence of glucose and a non-fermentable carbon source. We found that the expression of Emi2 protein is tightly suppressed during proliferation in high glucose, while it is strongly upregulated in response to glucose limitation and the presence of a non-fermentable carbon source. Our data suggest that the expression of the endogenous Emi2 protein in S. cerevisiae is regulated under the control of Hxk2 in response to glucose availability in the environment.