基因工程菌Escherichia coli BL21/pET-pel重组果胶酶的纯化及酶学性质

采用超声波破碎法、镍离子亲合层析柱法，纯化重组菌Escherichia coli BL21/pET-pel表达的胞内重组果胶酶，并用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate-polyacrylamide pelelectrophoresis，SDSPAGE）检测，研究纯化的重组果胶酶酶学性质。结果表明：重组果胶酶纯化倍数为1.71，回收率为88.5%，分子质量约为44 kD。最适pH值为9.0～9.5，在pH 7.0～10.0之间催化性质较稳定；最适作用温度范围为45～55 ℃，在60 ℃下保温30 min还剩余40%的酶活力；在离子浓度为1 mmol/L时，Ca2+和Co2+对该酶有较强的促进作用，而Fe2+则对其有较强的抑制作用。

Purification and Characterization of Recombinant Pectinase from Genetically Engineering Strain Escherichia coli BL21/pET-pel

The recombinant pectinase expressed in Escherichia coli BL21/pET-pel was purified by ultrasonic cell disruption
and nickel ion affinity chromatography, and detected using SDS-PAGE. Its enzymatic characteristics were analyzed. The
results showed that purification fold of the recombinant pectinase was 1.71, and recovery was 88.5%. Molecular weight
of the recombinant pectinase was approximately 44 kD. Its optimal temperature and pH were 45–55 ℃ and 9.0–9.5. It
was stable at pH 7.0–10.0. When the recombinant pectinase was incubated for 30 min at 60 ℃, the relative activity was
approximately 40%. Different metal ions revealed different effects on the recombinant pectinase, which was activated by
Ca2+ and Co2+ but inhibited by Fe2+.