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Bernd Nidetzky Richard Griessler Alexandra Schwarz Barbara Splechtna 《Journal of Molecular Catalysis .B, Enzymatic》2004,29(1-6):241-248
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 °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 -
-glucose 1-fluoride (G1F) as alternate glucosyl donor in place of -
-glucose 1-phosphate (G1P) is exploited for the synthesis of β-1,4-glucosides under thermodynamic control in close to 100% yield. 相似文献
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