| 摘要: |
| 为了开发一种高效、绿色的甘油葡萄糖苷(Glucosylglycerol, GG)酶催化合成方法，通过定点饱和突变技术改造大黄欧文氏菌蔗糖异构酶(Sucrose isomerase, EC 5.4.99.11 SI)的F297和F321两个苯丙氨酸残基，筛选出分子内转糖苷活性降低而水解活性增加的突变体，并优化表达条件实现高效分泌表达，最终以筛选优化后的工程菌固定化细胞为酶催化材料、蔗糖为糖基供体、丙三醇为糖基受体，实现蔗糖异构酶一步催化合成GG。通过高效液相色谱(HPLC)对产物进行定量分析并优化反应条件。重组工程菌经过ZYM-5052自诱导培养基优化，实现了整体酶活为IPTG诱导的16倍。突变体F321M的GG产率较野生菌株提高7倍，通过优化反应条件（蔗糖浓度1 mol/L、丙三醇浓度2-2.5 mol/L、反应时间20 h），最终实现GG的摩尔转化率为24.54%，当底物甘油浓度为5 mol/L时，GG产量达133.5 g/L。 |
| 关键词: 甘油葡萄糖苷,蔗糖异构酶,点饱和突变，酶催化，固定化细胞 |
| DOI： |
| 投稿时间：2025-04-03修订日期：2025-06-07 |
| 基金项目: |
|
| Enzymatic Synthesis of Glucosylglycerol Catalyzed by Recombinant Sucrose Isomerase |
|
hou fangfang1, zhouxing2
|
| (1.Guangxi University;2.Guangxi Academy of Sciences) |
| Abstract: |
| To develop an efficient and environmentally friendly enzymatic method for the synthesis of glucosylglycerol (GG), site-saturation mutagenesis was employed to modify two phenylalanine residues (F297 and F321) in Erwinia rhapontici sucrose isomerase (EC 5.4.99.11). Mutants with reduced intramolecular transglycosylation activity and enhanced hydrolytic activity were screened and further optimized for high-level secretory expression. Using immobilized cells of the engineered strain as the biocatalyst, with sucrose as the glycosyl donor and glycerol as the glycosyl acceptor, a one-step enzymatic synthesis of GG was achieved. High-performance liquid chromatography ( HPLC ) was used for quantitative analysis of the products, and reaction conditions were systematically optimized. Through optimization of culture conditions in ZYM-5052 auto-induction medium, the total enzymatic activity was increased by 16-fold compared to IPTG induction. The F321M mutant exhibited a 7-fold improvement in GG yield compared to the wild-type strain. Under optimized conditions (1 mol/L sucrose, 2-2.5 mol/L glycerol, 20 h reaction time), the maximum molar conversion rate of GG reached 24.54% with a maximum yield of 133.5 g/L. |
| Key words: glucosylglycerol, sucrose isomerase, site-saturation mutagenesis, enzymatic reaction, matrix-entrapped cells |
