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食品与发酵工业  2021, Vol. 47 Issue (12): 17-22    DOI: 10.13995/j.cnki.11-1802/ts.026522
  研究报告 本期目录 | 过刊浏览 | 高级检索 |
重组枯草芽孢杆菌全细胞催化合成钙二醇的初步研究
李雨虹1,2, 耿鹏1,2, 刘建民3, 时祎1,2, 辛瑜1,2, 石贵阳1,2, 张梁1,2*
1(粮食发酵工艺与技术国家工程实验室(江南大学),江苏 无锡,214122)
2(江南大学 生物工程学院,江苏 无锡,214122)
3(山东惠仕莱生物科技有限公司,山东 济南,250101)
Whole-cell biosynthesis of 25-hydroxy vitamin D3 by recombinant Bacillus subtilis
LI Yuhong1,2, GENG Peng1,2, LIU Jianmin3, SHI Yi1,2, XIN Yu1,2, SHI Guiyang1,2, ZHANG Liang1,2*
1(National Engineering Laboratory for Cereal Fermentation Technology (Jiangnan University), Wuxi 214122, China)
2(School of Biotechnology, Jiangnan University, Wuxi 214122, China)
3(Shandong Huishilai Biotechnology Co. Ltd., Jinan 250101, China)
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摘要 钙二醇(25-hydroxy vitamin D3,25-OH VD3)是一种具有广泛生理活性及药用价值的物质。该文旨在通过分子生物学手段构建1株异源表达VD3羟化酶(VD3 hydroxylase,Vdh)的重组枯草芽孢杆菌Bacillus subtilis WB600/pMA5-vdh,使其可以利用细胞催化系统合成25-OH VD3。该重组菌株经诱导表达后,首先利用CO差光谱法评价Vdh的体外活性,并将其用作全细胞催化剂合成25-OH VD3,随后通过优化温度、时间、转速及pH等全细胞转化条件,提高其转化合成25-OH VD3的产量。结果表明,37 ℃下培养24 h后,重组枯草芽孢杆菌B.subtilis WB600/pMA5-vdh的菌体裂解物酶活力达0.56 nmol/g;并且在底物质量浓度为0.1 g/L、外加18 g/L葡萄糖和22.5 g/L的2-羟丙基-β-环糊精的条件下,控制初始pH为7、反应温度30 ℃、转速200 r/min时进行全细胞转化反应48 h,得到25-OH VD3的转化率为10.36%,以上研究结果为制备和生产食品级25-OH VD3提供了新的思路。
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李雨虹
耿鹏
刘建民
时祎
辛瑜
石贵阳
张梁
关键词:  钙二醇(25-hydroxy vitamin D3, 25-OH VD3)  枯草芽孢杆菌  VD3羟化酶  异源表达  全细胞催化    
Abstract: 25-hydroxy vitamin D3 is a substance with extensive physiological activity and high medicinal value. In this study, a recombinant strain, Bacillus subtilis WB600/pMA5, was constructed via heterologous expression of VD3 hydroxylase (Vdh) and was further applied for the cellular synthesis of 25-OH VD3.The recombinant strain was utilized as a whole-cell catalyst for synthesizing 25-OH VD3 after an evaluation for the activity of Vdh through carbon-monoxide (CO) difference spectroscopy. The conditions of whole-cell transformation, including temperature, time, rotation speed and pH were optimized thereafter to improve the yield of 25-OH VD3. Based on the results, the enzyme activity of the recombinant strain lysate reached 0.56 nmol/g when having the strain incubated at 37 ℃ for 24 h. Moreover, with the initial substrate of 0.1 g/L, the conversion rate of 25-OH VD3 was achieved at 10.36% after a reaction time of 48 h, by adding extra 18 g/L glucose and 22.5 g/L 2-hydroxypropyl-β-cyclodextrin and controlling the transformation condition at a pH of 7.0, temperature of 30 ℃ and rotation speed of 200 r/min. The above findings could provide a new idea in terms of preparing and manufacturing food-grade 25-OH VD3.
Key words:  25-hyroxy vitamin D3    Bacillus subtilis    VD3 hydroxylase    heterologous expression    whole-cell catalysis
收稿日期:  2020-12-21      修回日期:  2021-01-27           出版日期:  2021-06-25      发布日期:  2021-07-22      期的出版日期:  2021-06-25
基金资助: 国家重点研发计划(2018YFA0900300)
作者简介:  硕士研究生(张梁教授为通讯作者,E-mail:zhangl@jiangnan.edu.cn)
引用本文:    
李雨虹,耿鹏,刘建民,等. 重组枯草芽孢杆菌全细胞催化合成钙二醇的初步研究[J]. 食品与发酵工业, 2021, 47(12): 17-22.
LI Yuhong,GENG Peng,LIU Jianmin,et al. Whole-cell biosynthesis of 25-hydroxy vitamin D3 by recombinant Bacillus subtilis[J]. Food and Fermentation Industries, 2021, 47(12): 17-22.
链接本文:  
http://sf1970.cnif.cn/CN/10.13995/j.cnki.11-1802/ts.026522  或          http://sf1970.cnif.cn/CN/Y2021/V47/I12/17
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