Characterization of a recombinant mannobiose 2-epimerase from Spirochaeta thermophila that is suggested to be a cellobiose 2-epimerase

Abstract

A purified recombinant enzyme from Spirochaeta thermophila, that is suggested to be a cellobiose 2-epimerase, was a 47 kDa monomer with a specific activity of 29.2 U min(-1) for mannobiose. The epimerization activity of the recombinant enzyme for mannobiose was maximal at pH 7.0 and 60 °C with a half-life of 124 h. The enzyme exhibited a higher epimerization activity for mannose or the mannose moiety at the reducing end of β- and α-1,4-glycosyl-mannose than for glucose or the glucose moiety of β- and α-1,4-glycosyl-glucose. The enzyme was identified as a mannobiose 2-epimerase by evaluating its substrate specificity with not only glucose-containing sugars but also mannose-containing sugars. The activities of the reported cellobiose 2-epimerases from Caldicellulosiruptor saccharolyticus, Dictyoglomus turgidum and Ruminococcus marinus for mannobiose were higher than those for cellobiose, strongly suggesting that these enzymes are not cellobiose 2-epimerases but are mannobiose 2-epimerases.

Identification and distribution of cellobiose 2-epimerase genes by a PCR-based metagenomic approach

“…Functional CEs have been identified from R. albus (RaCE) ), E. cellulosolvens (EcCE) , B. fragilis (BfCE) (Senoura et al 2009), C. saccharolyticus (CsCE) (Park et al 2011), R. marinus (RmCE) (Ojima et al 2011), D. turgidum (DtCE) (Kim et al 2012), and S. thermophila (StCE) (Park et al 2013). In this study, the environmental ce genes md1 and md2 were functionally expressed in E. coli cells.…”

“…Among them, functionally expressed mD1 and mD2 had low K m values and high catalytic efficiencies (k cat /K m ) for mannobiose compared with cellobiose ( Table 2), suggesting that they function physiologically as an MBE rather than a CE. Park et al (2013) also suggested that StCE from S. thermophila is an MBE. Therefore, it is possible that the proposed mannan catabolic pathway is widely distributed in bacteria, as we have The values in parentheses indicate percentages, taken each activity toward mannobiose as 100 % suggested previously (Senoura et al 2011).…”

“…We also characterized CEs in ruminal Eubacterium cellulosolvens (EcCE) ) and enteric Bacteroides fragilis (BfCE) (Senoura et al 2009). After our studies, CEs were found in several bacteria, including those from Caldicellulosiruptor saccharolyticus (CsCE) (Park et al 2011), Rhodothermus marinus (RmCE) (Ojima et al 2011), Dictyoglomus turgidum (DtCE) (Kim et al 2012), and Spirochaeta thermophila (StCE) (Park et al 2013). These reported CEs show very low amino acid sequence homology to N-acetyl-D-glucosamine 2-epimerases (AGEs; EC 5.1.3.8) from porcine kidney (Maru et al 1996) and an Anabaena sp.…”

Functional reassignment of Cellvibrio vulgaris EpiA to cellobiose 2-epimerase and an evaluation of the biochemical functions of the 4-O-β-d-mannosyl-d-glucose phosphorylase-like protein, UnkA

“…However, the amino acid sequence of EpiA shows a high sequence identity (32-42%) to those of several other reported CEs. [9][10][11][12][13][14][15][16] UnkA, whose function currently remains unknown, has been classified into glycoside hydrolase family 130 based on its amino acid sequence. 17) Glycoside hydrolase family 130 contains MGP, β-1,4-mannooligosaccharide phosphorylase, and β-1,4-D-mannosyl-N-acetyl-D-glucosamine phosphorylase (EC 2.4.1.320).…”

“…EpiA had high epimerization activity to β-(1→4)-linked disaccharides, and β-(1→4)-mannobiose is the best substrate of β-(1→4)-linked disaccharides as other reported CEs. 4,13,16,20) EpiA was consequently designated as CvCE.…”