摘要
利用重组大肠杆菌EscherichiacoliRosetta（DE3）／pET-SPase发酵生产蔗糖磷酸化酶（EC2．4．1．7，Sucrosephosphorylase，SPase）。收集的菌体经高压破碎后离心得到粗酶液，通过镍NTA亲和层析、超滤除盐后得到电泳纯的SPase，纯化后的SPase的比酶活是原来的2．1倍，酶活回收率达到82．7％。经SDS．PAGE电泳测定，重组SPase的分子量约为59kDa。该酶在不高于37℃，pH6．0～6．7的条件下比较稳定，最适催化温度与最适催化pH分别为37℃，pH6．7，该酶对蔗糖的米氏常数（Km）为7．3mmol／L，最大反应速率（Vmax）为0．2／μmol／（min·mg）。此外文中还以蔗糖和氢醌为底物，利用重组SPase催化合成a-熊果苷。其最佳反应条件为：20％蔗糖，200U／mL的酶液，1．6％氢醌，pH6．0～6．5，25℃，反应21h。a-熊果苷的摩尔产率为78．3％，a-熊果苷的产量为31g／L。
Sucrose phosphorylase （EC 2.4.1.7, Sucrose phosphorylase, SPase） can be produced by recombinant strain Escherichia coli Rosetta（DE3）/Pet-SPase. Crude enzyme was obtained from the cells by the high pressure disruption and centrifugation. Sucrose phosphorylase was purified by Ni-NTA affinity column chromatography and desalted by ultrafiltration. The specific enzyme activity was 1.1-fold higher than that of the crude enzyme, and recovery rate was 82.7%. The purified recombinant SPase had a band of 59 kDa on SDS-PAGE. Thermostability of the enzyme was shown at temperatures up to 37 ℃, and pH stability between pH 6.0 and 6.7. The optimum temperature and pH were 37 ℃ and 6.7, respectively. The Km of SPase for sucrose was 7.3 mmol/L, and Vmax was 0.2 μmol/（min.mg）. Besides, a-arbutin was synthesized from sucrose and hydroquinone by transglucosylation with recombinant SPase. The optimal conditions for synthesis of a-arbutin were 200 U/mL of recombinant SPase, 20% of sucrose, and 1.6% hydroquinone at pH 6-6.5 and 25 ℃ for 21 h. Under these conditions, a-arbutin was obtained with a 78.3% molar yield with respect to hydroquinone, and the concentration of a-arbutin was about 31 g/L.
出处
《生物工程学报》
CAS
CSCD
北大核心
2012年第12期1450-1459,共10页
Chinese Journal of Biotechnology
基金
国家自然科学基金(No.21076105)
国家重点基础研究发展计划(973计划)(No.2009CB724701)
江苏高校优势学科建设工程项目资助~~
关键词
重组蔗糖磷酸化酶
纯化
酶学性质
α-熊果苷
recombinant sucrose phosphorylase, purification, properties, a-arbutin
