肠炎沙门氏菌B群重组磷酸甘露糖变位酶和GDP-α-D-甘露糖焦磷酸化酶的表达、纯化及特性研究，用于从D-甘露糖合成GDP-α-D-甘露糖

Expression, purification and characterization of recombinant phosphomannomutase and GDP-alpha-D-mannose pyrophosphorylase from Salmonella enterica, group B, for the synthesis of GDP-alpha-D-mannose from D-mannose.

作者信息

Elling L, Ritter J E, Verseck S

机构信息

Institut für Enzymtechnologie, Heinrich-Heine-Universität DAsseldorf im Forschungszentrum Jülich, Germany.

出版信息

Glycobiology. 1996 Sep;6(6):591-7. doi: 10.1093/glycob/6.6.591.

DOI:10.1093/glycob/6.6.591

PMID:8922954

Abstract

The genes rfbK and rfbM from the rfb cluster (O-antigen biosynthesis) of Salmonella enterica, group B, encoding for the enzymes phosphomannomutase (EC 5.4.2.8) and GDP-alpha-D-mannose pyrophosphorylase (EC 2.7.7.13) were overexpressed in E.coli BL21 (DE3) with specific activities of 0.1 U/mg and 0.3-0.6 U/mg, respectively. Both enzymes were partially purified to give specific activities of 0.26 U/mg and 2.75 U/mg, respectively. Kinetic characterization of the homodimeric (108 kDa) GDP-alpha-D-mannose pyrophosphorylase revealed a K(m) for GTP and mannose-1-P of 0.2 mM and 0.01 mM with substrate surplus inhibition constants (Kis) of 10.9 mM and 0.7 mM, respectively. The product GDP-alpha-D-mannose gave a competitive inhibition with respect to GTP (Ki 14.7 microM) and an uncompetitive inhibition with respect to mannose-1-P (Ki 115 microM). Both recombinant enzymes were used for repetitive batch synthesis of GDP-alpha-D-mannose staring from D-mannose and GTP. In three subsequent batches 581 mg (960 mumol) GDP-alpha-D-mannose was synthesized with 80% average yield. The overall yield after product isolation was 22.9% (329 mumol, 199 mg).

摘要

来自肠炎沙门氏菌B组rfb簇（O抗原生物合成）的rfbK和rfbM基因，编码磷酸甘露糖变位酶（EC 5.4.2.8）和GDP-α-D-甘露糖焦磷酸化酶（EC 2.7.7.13），在大肠杆菌BL21（DE3）中过表达，比活性分别为0.1 U/mg和0.3 - 0.6 U/mg。两种酶都经过部分纯化，比活性分别达到0.26 U/mg和2.75 U/mg。对同二聚体（108 kDa）的GDP-α-D-甘露糖焦磷酸化酶的动力学特性进行表征，结果显示其对GTP和甘露糖-1-P的K(m)分别为0.2 mM和0.01 mM，底物过剩抑制常数（Kis）分别为10.9 mM和0.7 mM。产物GDP-α-D-甘露糖对GTP表现出竞争性抑制（Ki 14.7 microM），对甘露糖-1-P表现出非竞争性抑制（Ki 115 microM）。两种重组酶都用于从D-甘露糖和GTP重复分批合成GDP-α-D-甘露糖。在随后的三批反应中，合成了581 mg（960 μmol）的GDP-α-D-甘露糖，平均产率为80%。产物分离后的总产率为22.9%（329 μmol，199 mg）。