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侯芳芳,成春燕,杨祥开,韦星明,周兴,杨克迪.重组蔗糖异构酶一步法催化合成甘油葡萄糖苷的研究[J].广西科学院学报,2026,42(2):228-239. [点击复制]
- HOU Fangfang,CHENG Chunyan,YANG Xiangkai,WEI Xingming,ZHOU Xing,YANG Kedi.Study on the One-step Catalytic Synthesis of Glucosylglycerol by Recombinant Sucrose Isomerase[J].Journal of Guangxi Academy of Sciences,2026,42(2):228-239. [点击复制]
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| 重组蔗糖异构酶一步法催化合成甘油葡萄糖苷的研究 |
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侯芳芳1, 成春燕2,3, 杨祥开2,4, 韦星明5, 周兴2, 杨克迪1
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| (1.广西大学医学院, 天然药物化学实验室, 广西南宁 530004;2.广西科学院, 广西南宁 530007;3.广西农业职业技术大学, 广西南宁 530007;4.广西化工研究院有限公司, 广西南宁 530001;5.广西科学院生物研究所有限责任公司, 广西南宁 530007) |
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| 摘要: |
| 为开发一种高效、绿色的甘油葡萄糖苷(Glucosylglycerol,GG)酶催化合成方法，本研究通过定点饱和突变技术改造大黄欧文氏菌(Erwinia rhapontici)蔗糖异构酶(Sucrose Isomerase，SI，EC 5.4.99.11)的两个苯丙氨酸残基(F297和F321),筛选出分子内转糖苷活性降低而水解活性增加的突变体，并优化诱导表达条件;以筛选出的突变体作为催化剂进行催化反应，使用高效液相色谱(HPLC)对产物进行定量分析并进一步优化转化反应条件。结果表明，重组工程菌经过ZYM-5052/Amp自诱导培养基优化，获得的整体酶活力为异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导方式的16倍。突变体F321M的GG产率较野生型菌株提高7倍，优化后的反应条件为蔗糖浓度1.0 mol/L、丙三醇浓度2.0—2.5 mol/L、反应时间20 h，在此条件下获得的GG摩尔转化率为24.54%；当底物丙三醇浓度为5.0 mol/L时，GG产量达133.6 g/L。本研究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，实现了SI一步催化合成GG，并优化了转化反应条件。 |
| 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：10.13657/j.cnki.gxkxyxb.20260604.001 |
| 投稿时间：2025-04-03修订日期：2025-06-07 |
| 基金项目:南宁高新区大健康产业—南宁国家高新区大健康产业联合创新服务平台建设与能力提升项目(桂科AD25069031)和广西非粮生物质能源技术创新中心项目(桂科ZY23055011)资助。 |
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| Study on the One-step Catalytic Synthesis of Glucosylglycerol by Recombinant Sucrose Isomerase |
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HOU Fangfang1, CHENG Chunyan2,3, YANG Xiangkai2,4, WEI Xingming5, ZHOU Xing2, YANG Kedi1
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| (1.Laboratory of Natural Medicinal Chemistry, Medical School of Guangxi University, Nanning, Guangxi, 530004, China;2.Guangxi Academy of Sciences, Nanning, Guangxi, 530007, China;3.Guangxi Vocational University of Agriculture, Nanning, Guangxi, 530007, China;4.Guangxi Research Institute of Chemical Industry Co., Ltd., Nanning, Guangxi, 530001, China;5.Institute of Biology Co., Ltd., Guangxi Academy of Sciences, Nanning, Guangxi, 530007, China) |
| Abstract: |
| To establish an efficient and environmentally friendly enzymatic synthesis route for glucosylglycerol (GG),a site-saturation mutagenesis strategy was applied to modify two phenylalanine residues (F297 and F321) of Erwinia rhapontici Sucrose Isomerase (SI,EC 5.4.99.11).Mutants exhibiting reduced intramolecular transglycosylation activity but enhanced hydrolytic activity were selected.Following mutant screening,expression conditions were systematically optimized.The selected mutants were then employed as biocatalysts for GG synthesis,and the resulting products were quantitatively analyzed by High-Performance Liquid Chromatography (HPLC).The results showed that,using ZYM-5052/Amp auto-induction medium,recombinant expression was enhanced,yielding an overall enzymatic activity approximately 16-fold higher than that induced by isopropyl β-D-thiogalactopyranoside (IPTG).Among the mutants,F321M demonstrated a 7-fold increase in GG yield compared to the wild-type enzyme.Under optimized reaction conditions—sucrose concentration at 1.0 mol/L,glycerol concentration at 2.0—2.5 mol/L,and a reaction time of 20 h the molar conversion rate of GG reached 24.54%.Furthermore,when the initial glycerol concentration was raised to 5.0 mol/L,the GG yield increased to 133.6 g/L.This study successfully demonstrates a one-step enzymatic synthesis of GG using immobilized engineered cells,with sucrose as the glycosyl donor and glycerol as the acceptor,providing both a simplified production route and refined reaction conditions. |
| Key words: glucosylglycerol sucrose isomerase site-saturation mutagenesis enzyme catalysis matrix-entrapped cells |
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