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A highly active phosphoglucomutase from Clostridium thermocellum : cloning, purification, characterization and enhanced thermostability
Author(s) -
Wang Y.,
Zhang Y.H.P.
Publication year - 2010
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/j.1365-2672.2009.04396.x
Subject(s) - thermostability , clostridium thermocellum , biochemistry , phosphoglucomutase , thermophile , enzyme , biology , escherichia coli , active site , chemistry , cellulase , gene
Aims: Discovery and utilization of highly active and thermostable phosphoglucomutase (PGM) would be vital for biocatalysis mediated by multiple enzymes, for example, high‐yield production of enzymatic hydrogen. Methods and Results: The thermophilic cellulolytic bacterium Clostridium thermocellum was hypothesized to have a very active PGM because of its key role in microbial cellulose utilization. The Cl. thermocellum ORF Cthe1265 encoding a putative PGM was cloned and expressed in Escherichia coli . The purified enzyme appeared to be a monomer with an estimated molecular weight of 64·9 kDa. This enzyme was found to be a dual‐specificity enzyme – PGM/phosphomannomutase (PMM). Mg 2+ and Mn 2+ were activators. Ser144 was identified as an essential catalytic residue through site‐directed mutagenesis. The k cat and K m of PGM were 190 s −1 and 0·41 mmol l −1 on glucose‐1‐phosphate and 59 s −1 and 0·44 mmol l −1 on mannose‐1‐phosphate, respectively, at 60°C. Thermostability of PGM at a low concentration (2 nmol l −1 , 100 U l −1 ) was enhanced by 12‐fold (i.e. t 1/2 = 72 h) at 60°C with addition of bovine serum albumin, Triton X‐100, Mg 2+ and Mn 2+ . Conclusions: The ORF Cthe1265 was confirmed to encode a PGM with PMM activity. This enzyme was the most active PGM reported. Significance and Impact of the Study: This highly active PGM with enhanced thermostability would be an important building block for in vitro synthetic biology projects (complicated biotransformation mediated by multiple enzymes in one pot).