Biochemical properties of GH94 cellodextrin phosphorylase THA_1941 from a thermophilic eubacterium Thermosipho africanus TCF52B with cellobiose phosphorylase activity

Abstract

A hypothetic gene (THA_1941) encoding a putative cellobiose phosphorylase (CBP) from Thermosipho africanus TCF52B has very low amino acid identities (less than 12%) to all known GH94 enzymes. This gene was cloned and over-expressed in Escherichia coli BL21(DE3). The recombinant protein was hypothesized to be a CBP enzyme and it showed an optimum temperature of 75 °C and an optimum pH of 7.5. Beyond its CBP activity, this enzyme can use cellobiose and long-chain cellodextrins with a degree of polymerization of greater than two as a glucose acceptor, releasing phosphate from glucose 1-phosphate. The catalytic efficiencies (k cat/K m) indicated that cellotetraose and cellopentaose were the best substrates for the phosphorolytic and reverse synthetic reactions, respectively. These results suggested that this enzyme was the first enzyme having both cellodextrin and cellobiose phosphorylases activities. Because it preferred cellobiose and cellodextrins to glucose in the synthetic direction, it was categorized as a cellodextrin phosphorylase (CDP). Due to its unique ability of the reverse synthetic reaction, this enzyme could be a potential catalyst for the synthesis of various oligosaccharides. The speculative function of this CDP in the carbohydrate metabolism of T. africanus TCF52B was also discussed.

Identification of Euglena gracilis β-1,3-glucan phosphorylase and establishment of a new glycoside hydrolase (GH) family GH149

“…Therefore, the chain length specificity of β-1,3-glucan phosphorylases might be more flexible than previously observed. This has also been observed in a recently characterized thermophilic cellodextrin phosphorylase (β-1,4-glucan phosphorylase), which uses Glc and longer cello-oligosaccharide as acceptors ( 42 ) and is distinct from GH94 cellodextrin phosphorylases, which have no detectable activity on Glc as an acceptor ( 19 , 43 ).…”

Product solubility control in cellooligosaccharide production by coupled cellobiose and cellodextrin phosphorylase

“…This makes it difficult to establish the combined application of Cu CbP and Cs CdP under optimum conditions for both enzymes. In contrast to Cs CdP (Tran et al, ) and other (hyper)thermostable CdPs (from C. thermocellum ; Arai, Tanaka, & Kawaguchi, ; Thermosipho africanus ; Wu et al, ; and Ruminococcus albus ; Sawano, Saburi, Hamura, Matsui, & Mori, ), Cc CdP offers a temperature profile of activity well compatible with Cu CbP activity at an optimal temperature of 45°C (Figure a). Reaction thermodynamic analysis with eQuilibrator (Flamholz, Noor, Bar‐Even, & Milo, ) shows that cellobiose synthesis from αGlc1‐ P and glucose is largely independent of pH in the range of 6.0 to 9.0.…”

Unraveling the subtleties of β-(1→3)-glucan phosphorylase specificity in the GH94, GH149, and GH161 glycoside hydrolase families

“…During bioinformatics analyses of GH161 sequences in the NCBI database, we came across sequence from the Gram-negative thermophilic bacterium Thermosipho africanus TCF52B (TaCDP, GenBank TM number ACJ76363.1) that had recently been reported as a cellodextrin (β-(1→4)-glucan) phosphorylase, operating on Glc and cello-oligosaccharides (37). This was at odds with our expectations and warranted further investigation, given that TaCDP phosphorolytic activity on β-(1→3)-gluco-oligosaccharide substrates had not been assessed and the glucan products for β-(1→4)-glucan extension had not been subjected to linkage analysis.…”