重组大肠杆菌产蔗糖磷酸化酶的酶学性质及其催化合成α-熊果苷

万月佳 等/重组大肠杆菌产蔗糖磷酸化酶的酶学性质及其催化合成α-熊果苷

Chinese Journal of Biotechnology December 25, 2012, 28(12): 1450−1459 /cjbcn

©2012 Chin J Biotech, All rights reserved

Received : July 4, 2012; Accepted : September 11, 2012

Supported by : National Natural Science Foundation of China (No. 21076105), National Basic Research Program of China (973 Program) (No. 2009CB724701), the Priority Academic Program Development of Jiangsu Higher Education Institutions.

Corresponding author : Jiangfeng Ma. Tel: +86-25-83172078; Fax: +86-25-84172062; E-mail: bioengine@

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。

生物工程学报

重组大肠杆菌产蔗糖磷酸化酶的酶学性质及其催化合成α-熊果苷

万月佳，马江锋，徐蓉，贺爱永，姜岷，陈可泉，姜引

南京工业大学生物与制药工程学院 材料与化学工程国家重点实验室，江苏 南京 211816

万月佳, 马江锋, 徐蓉, 等. 重组大肠杆菌产蔗糖磷酸化酶的酶学性质及其催化合成α-熊果苷. 生物工程学报, 2012, 28(12): 1450−1459.

Wan YJ, Ma JF, Xu R, et al. Properties of recombinant sucrose phosphorylase from Escherichia coli and enzymatic synthesis of α-arbutin. Chin J Biotech, 2012, 28(12): 1450−1459.

摘 要: 利用重组大肠杆菌Escherichia coli Rosetta(DE3)/pET-SPase 发酵生产蔗糖磷酸化酶 (EC 2.4.1.7，

Sucrose phosphorylase ，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59 kDa 。该酶在不高于37 ℃，pH 6.0~6.7的条件下比较稳定，最适催化温度与最适催化pH 分别为37 ℃，pH 6.7，该酶对蔗糖的米氏常数 (K m ) 为7.3 mmol/L ，最大反应速率 (V max ) 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α-熊果苷。其最佳反应条件为：20％蔗糖，200 U/mL 的酶液，1.6％氢醌，pH 6.0~6.5，25 ℃，反应21 h 。α-熊果苷的摩尔产率为78.3％，α-熊果苷的产量为31 g/L 。

关键词: 重组蔗糖磷酸化酶，纯化，酶学性质，α-熊果苷

万月佳 等/重组大肠杆菌产蔗糖磷酸化酶的酶学性质及其催化合成α-熊果苷

cjb@

Properties of sucrose phosphorylase from recombinant Escherichia coli and enzymatic synthesis of α-arbutin

Yuejia Wan, Jiangfeng Ma, Rong Xu, Aiyong He, Min Jiang, Kequan Chen, and Yin Jiang

State Key Laboratory of Materials-Oriented Chemical Engineering , College of Biotechnology and Pharmaceutical engineering , Nanjing University of Technology , Nanjing 211816, Jiangsu , China

Abstract: 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 af finity 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 °C, and pH stability between pH 6.0 and 6.7. The optimum temperature and pH were 37 °C and 6.7, respectively. The K m of SPase for sucrose was 7.3 mmol/L, and V max was 0.2 µmol/(min·mg). Besides, α-arbutin was synthesized from sucrose and hydroquinone by transglucosylation with recombinant SPase. The optimal conditions for synthesis of α-arbutin were 200 U/mL of recombinant SPase, 20% of sucrose, and 1.6% hydroquinone at pH 6−6.5 and 25 °C for 21 h. Under these conditions, α-arbutin was obtained with a 78.3% molar yield with respect to hydroquinone, and the concentration of α-arbutin was about 31 g/L.

Keywords : recombinant sucrose phosphorylase, purification, properties, α-arbutin

蔗糖磷酸化酶 (EC2.4.1.7，Sucrose phosphorylase ，SPase) 属于糖基水解酶13家族，是一种催化转移葡萄糖苷键的酶，能够催化蔗糖和无机磷酸盐合成1-磷酸-葡萄糖[1-2]。该酶主要以蔗糖、1-磷酸-葡萄糖为供体，多类物质如多羟基的糖和糖醇、酚羟基、羧基等为受体，催化合成各种糖苷[3]。据报道，蔗糖磷酸化酶主要存在于肠膜明串珠菌Leuconostoc mesenteroides [4]、变异链球菌Stococcus mutans [5]、嗜糖假单胞菌Pseudomonas saccharophila [6]、长双歧杆菌Bifidobacterium longum [7]、青春双歧杆菌Bifidobacterium adolescentis [8]等微生物中，通过生物发酵获得，但由于产量和生产效率都比较低，因此通过基因工程手段构建重组菌株，过量表达蔗糖磷酸化酶对于进行工业化大量生产十

分必要。

利用蔗糖磷酸化酶催化合成α-熊果苷是该酶的重要应用之一。α-熊果苷是一种新型的皮肤增白剂，能够通过抑制酪氨酸酶的活性，从而减少黑色素的生成，而对表皮细胞的正常生长以及酪氨酸酶的表达没有影响，并且其美白效果是β-熊果苷10倍以上，是21世纪最有竞争力的美白添加剂之一[9-11]。研究表明，α-熊果苷只能通过不同的微生物的酶进行糖转移反应，让一分子葡萄糖和一分子的氢醌结合形成[12]。

本文利用重组菌株E . coli Rosetta(DE3)/ pET-SPase [13]发酵生产SPase ，

并对其进行分离纯化以及酶学性质的研究，此外还利用该酶以氢醌和蔗糖为底物催化合成α-熊果苷，其反应式如图1所示。