蔗糖磷酸化酶的分离纯化及其催化合成α-熊果苷的研究

Purification of Sucrose Phosphorylase and Enzymatic Synthesis of α-arbutin

考察了肠膜明串珠菌(Leuconostoc mesenteroides)G123厌氧发酵产蔗糖磷酸化酶下游的分离纯化工艺。收集的菌体经超声破碎得到粗酶液,通过硫酸铵沉淀、透析、阴离子交换层析分离后获得了电泳纯的蔗糖磷酸化酶,酶活回收率为31.7%,酶的分子量约为55.7 kD,纯化后的蔗糖磷酸化酶比活为115.3 U/mg。该酶在中性及偏酸性(pH5.5-8.0)情况下,酶稳定性较好,较报道的肠膜明串珠菌(Leuconostoc mesenteroides)B-1149的pH稳定范围宽。同时该酶在37℃保存2 h,酶活几乎没有下降。利用获得的纯酶以氢醌和蔗糖为底物催化合成α-熊果苷,在23 U/mL的酶反应体系中,60%蔗糖、5%氢醌、pH7.5,37℃,反应12 h,氢醌转化率达到16.3%,α-熊果苷的产量为20g/L。

The isolation and purification of sucrose phosphorylase from Leuconostoc mesenteroides G123 anaerobic fermentation was in- vestigated. The cell recovered from the broth was disrupted by sonication to get crude enzyme solution. The sucrose phosphorylase was purified by precipitation with ammonium sulfate and ion exchange chromatography,leading to 15-fold purification with 31.7% recovery rate. The product showed eletrophoretic homogeneity,as identified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis（SDS-PAGE） ,and the specific activity of the purified sucrose phosphorylase reached 115. 3 U/rag. SDS-PAGE analysis indicated that the relative molecular mass of the protein was about 55. 7 kD. The enzyme was stable in the pH range of 5. 5 - 8.0. The pH stability of the sucrose phosphorylase G123 was larger than the enzyme of Leuconostoc mesenteroides B-1149 reported by Jin-Ha Lee. After kept in the condition temperature 37℃ ,sucrose phos- phorylase G123 activity was hardly declined. In the reaction of synthesis α-arbutin by purified sucrose phosphorylase ,the sucrose was servered as glucosyl donor and the hydroquinone as acceptor. The optimal condition of synthesis of α-arbutin by sucrose phosphorylase G123 was deter- mined 50% sucrose ,5% hydroquinone, pHT. 5.37℃ ,23 U/mL sucrose phosphorylase, and the reaction time was 12 hours, under which the transfer ratio of hydroquinone reached 16. 3% ,and the yield of α-arbutin was about 20 g/L. Key words： Sucrose phosphorylase α-arbutin Transglueosylation Transghicosidase