Cellobiose phosphorylase from Cellulomonas uda:: gene cloning and expression in Escherichia coli, and application of the recombinant enzyme in a 'glycosynthase-type' reaction

We have cloned and sequenced the gene encoding cellobiose phosphorylase from Cellulomonas uda and report high yield production in Escherichia coli of a functional recombinant enzyme containing an N-terminal metal affinity fusion peptide. Use of heterologous gene expression increases the space-time yield of active phosphorylase by three orders of magnitude, compared to production of the enzyme with the natural organism. The full-length phosphorylase is a 91.3 kDa protein that consists of 821 amino acids and whose primary structure shares significant residue identity with different members of glycosyltransferase family 36. Purified enzyme was obtained in 39% overall yield by using copper-chelate and hydroxyapatite chromatographies. A comparative steady-state kinetic analysis for enzymatic reactions in the directions of phosphorolysis and synthesis of cellobiose at 30degreesC and pH 6.6 demonstrates that the catalytic properties of the natural enzyme are retained completely in the recombinant cellobiose phosphorylase. The ability of the phosphorylase to utilize alpha-D-glucose 1-fluoride (alphaG1F) as alternate glucosyl donor in place of alpha-D-glucose 1-phosphate (alphaG1P) is exploited for the synthesis of beta-1,4-glucosides under thermodynamic control in close to 100% yield. (C) 2004 Elsevier B.V. All rights reserved.