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食品与发酵工业  2020, Vol. 46 Issue (13): 1-9    DOI: 10.13995/j.cnki.11-1802/ts.023866
  研究报告 本期目录 | 过刊浏览 | 高级检索 |
蔗糖磷酸化酶的半理性设计及生产α-熊果苷的条件优化
沈洋1, 吕雪芹1, 林璐1,2, 李江华2, 堵国成1, 刘龙1*
1(未来食品科技中心,江南大学,江苏 无锡,214122)
2(糖化学实验室,江南大学,江苏 无锡,214122)
Semi-rational design of sucrose phosphorylase and optimization of conditionsfor α-arbutin production
SHEN Yang1, LYU Xueqin1, LIN Lu1,2, LI Jianghua2, DU Guocheng1, LIU Long1*
1(Science Center for Future Foods, Jiangnan University, Wuxi 214122, China)
2(Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China)
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摘要 α-熊果苷是一种高价值糖苷,在化妆品和制药行业具有广泛的应用前景。酶法是生产α-熊果苷的主要手段,但由于缺乏高催化效率的酶而限制了α-熊果苷的生产。为了提高来自Streptococcus mutans UA159的蔗糖磷酸化酶(SmSP)的生物转化效率,进而提高α-熊果苷的产量,筛选了4种在Bacillus subtilis WB600中表达SmSP的载体,其中重组质粒pP43NMK-P43-gtfA在全细胞转化20 h后获得了最高的α-熊果苷产量(40.2 g/L)。其次,对SmSP催化活性中心周围的loop A进行了定点饱和突变,以提高酶和受体之间的亲和力,得到了突变体SmSPI336L,其α-熊果苷产量和底物对苯二酚(hydroquinone,HQ)摩尔转化率分别为71.7 g/L和72.4%,产量相比对照提高了78.4%。最后,对重组菌株B. subtilis WB600/pP43NMK-P43-SmSPI336L全细胞转化合成α-熊果苷反应条件进行优化,α-熊果苷的产量达到了110.3 g/L,HQ摩尔转化率为88.7%,产量相比对照提高了2.74倍。高催化效率重组菌株的构建,以及对突变体动力学的分析,对生物转化合成α-熊果苷具有重要的研究意义和应用价值。
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沈洋
吕雪芹
林璐
李江华
堵国成
刘龙
关键词:  蔗糖磷酸化酶  α-熊果苷  定点饱和突变  全细胞催化    
Abstract: α-arbutin is a high-value glycoside and has a wide range of applications in the cosmetics and pharmaceutical industries. The enzymatic method is the main way for the production of α-arbutin, but the production of α-arbutin is limited due to the lack of enzymes with high catalytic efficiency. In order to improve the biotransformation efficiency of sucrose phosphorylase (SmSP) from Streptococcus mutans UA159 and further improve the production of α-arbutin, four vectors expressing SmSP in Bacillus subtilis WB600 were screened, among which the recombinant plasmid pP43NMK-P43-gtfA achieved the highest α-arbutin yield (40.2 g/L) after 20 h of whole cell transformation. Secondly, site-directed saturation mutagenesis was performed on the loop A around the catalytic active center of SmSP to improve the affinity between the enzyme and the receptor, and a mutant SmSPI336L was obtained. Its α-arbutin production and the molar conversion of substrate hydroquinone (hydroquinone,HQ) were 71.7 g/L and 72.4%, respectively. The production increased by 78.4% compared to the control. Finally, the reaction conditions for α-arbutin synthesis were optimized by the recombinant strain B. subtilis WB600/pP43NMK-P43-SmSPI336L.The final α-arbutin productionreached110.3 g/L,and the molar conversion rate of HQ was 88.7%. The production was increased by 2.74 times compared to the control. The obtained high-catalytic efficiency recombinant strains and the analysis of mutant kinetics were of important research significance and application value for the biotransformation of α-arbutin.
Key words:  sucrose phosphorylase    α-arbutin    site-specific saturation mutation    biotransformation
收稿日期:  2020-03-05                出版日期:  2020-07-15      发布日期:  2020-08-04      期的出版日期:  2020-07-15
基金资助: 国家自然科学基金面上项目(31870069);国家自然科学基金面上项目(21676119)
作者简介:  硕士研究生(刘龙教授为通讯作者,E-mail:longliu@jiangnan.edu.cn)
引用本文:    
沈洋,吕雪芹,林璐,等. 蔗糖磷酸化酶的半理性设计及生产α-熊果苷的条件优化[J]. 食品与发酵工业, 2020, 46(13): 1-9.
SHEN Yang,LYU Xueqin,LIN Lu,et al. Semi-rational design of sucrose phosphorylase and optimization of conditionsfor α-arbutin production[J]. Food and Fermentation Industries, 2020, 46(13): 1-9.
链接本文:  
http://sf1970.cnif.cn/CN/10.13995/j.cnki.11-1802/ts.023866  或          http://sf1970.cnif.cn/CN/Y2020/V46/I13/1
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