A highly active phosphoglucomutase from Clostridium thermocellum: cloning, purification, characterization and enhanced thermostability

Authors

  • Y. Wang,

    1.  Biological Systems Engineering Department, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA
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  • Y.-H.P. Zhang

    1.  Biological Systems Engineering Department, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA
    2.  Institute for Critical Technology and Applied Science (ICTAS), Virginia Polytechnic Institute and State University, Blacksburg, VA, USA
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Y.-H. Percival Zhang, Biological Systems Engineering Dept., Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA. E-mail: ypzhang@vt.edu

Abstract

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). Mg2+ and Mn2+ were activators. Ser144 was identified as an essential catalytic residue through site-directed mutagenesis. The kcat and Km 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. t1/2 = 72 h) at 60°C with addition of bovine serum albumin, Triton X-100, Mg2+and Mn2+.

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).

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