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

摘要

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 30 deg C 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 (alphaG 1F) as alternate glucosyl donor in place of alpha-D-glucose 1-phosphate (alphaGlP) is exploited for the synthesis of beta-l,4-glucosides under thermodynamic control in close to 100% yield.