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过程工程学报 ›› 2022, Vol. 22 ›› Issue (1): 97-107.DOI: 10.12034/j.issn.1009-606X.221036

• 研究论文 • 上一篇    下一篇

以葡萄糖为原料结合生物发酵与化学合成制备酮泛解酸

姚尧1,2， 卢希阳1， 舒琳1,2， 王卿惠1， 孙少琦1， 郝健1,2*   

  1. 1. 中国科学院上海高等研究院，上海 201210 2. 中国科学院大学，北京 100049
  • 收稿日期:2021-02-01 修回日期:2021-03-19 出版日期:2022-01-28 发布日期:2022-01-28
  • 通讯作者: 郝健 haoj@sari.ac.cn
  • 作者简介:姚尧(1996-)，女，陕西省咸阳市人，硕士研究生，生物工程专业，E-mail: 13260189106@163.com；郝健，通讯联系人，E-mail: haoj@sari.ac.cn.
  • 基金资助:
    上海市自然科学基金;国家重大研发计划;国家重大研发计划

Ketopantoate production from glucose by combining biological and chemical steps

Yao YAO1,2,  Xiyang LU1,  Lin SHU1,2,  Qinghui WANG1,  Shaoqi SUN1,  Jian HAO1,2*   

  1. 1. Shanghai Advanced Research Institute, Chinese Academy of Sciences, Shanghai 201210, China 2. University of Chinese Academy of Sciences, Beijing 100049, China
  • Received:2021-02-01 Revised:2021-03-19 Online:2022-01-28 Published:2022-01-28

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摘要： D-泛酸(维生素B5)是一种重要的维生素，在饲料、化妆品和制药行业均有广阔的市场。酮泛解酸是泛酸生物合成途径的中间体，易内酯化为酮基泛解酸内酯，后者可立体选择性地转化为D-泛解酸，并进一步用于D-泛酸的生产。本工作提供了一种采用α-酮基异戊酸与甲醛经羟醛反应合成酮泛解酸的新方法。其中，反应物α-酮基异戊酸可由葡萄糖原料经发酵法生产。结果表明，以葡萄糖为碳源，经克雷伯氏肺炎杆菌发酵能高水平生产D-泛酸生物合成途径的关键前体—α-酮基异戊酸。采用商品α-酮基异戊酸开发一种新型的酮泛解酸合成方法；分析合成反应机理，确定总反应级数为1.87；经条件优化确定酮泛解酸合成反应的最佳pH=13，最佳温度为45℃，此条件下酮泛解酸转化率达83.5%。调节酮泛解酸溶液pH至强酸性，有助于其内酯化合成酮基泛解酸内酯。之后采用前述优化的方法，由流加发酵制备的25.2 g/L α-酮基异戊酸合成酮泛解酸19.9 g/L。最后通过异丁醇萃取、活性炭脱色、浓缩结晶方法进行产物提纯，调节酮基泛解酸内酯溶液pH在7~10范围内可使其开环转化回酮泛解酸。最终得到酮泛解酸及其内酯的纯品。本研究建立了以葡萄糖为原料通过α-酮基异戊酸中间体生产酮泛解酸的方法。该方法结合生物发酵和化学合成过程，采用廉价原料，具有绿色高效的特点，有望用于工业化生产泛酸。

关键词: 酮基泛解酸, 2-酮基异戊酸, 羟醛缩合, 克雷伯氏肺炎杆菌

Abstract: D-pantothenic acid (vitamin B5) is an essential vitamin to animals and has large markets in the feed, cosmetics, and pharmaceutical industries. The biochemical role of D-pantothenic acid in all organisms is to form the core of the structure of coenzyme A. Biosynthesis of coenzyme A from pantothenate occurs in all organisms, while the synthesis of D-pantothenic acid is absent from animals. Thus D-pantothenic acid is an essential nutrient to animals. Ketopantoate is an intermediate of pantothenate biosynthesis pathway. Ketopantoate can be stereoselectivity converted to D-pantoic acid and further used for D-pantothenic acid production. However, the economic production of ketopantoate is a bottleneck of D-pantothenic acid production from ketopantoate. Hence, this study provided a novel method for synthesis of ketopantoate by aldol reaction of α-ketoisovalerate and formaldehyde, and α-ketoisovalerate was produced from fermentation with glucose as the raw material. 25.2 g/L α-ketoisovalerate was produced by an engineering Klebsiella pneumoniae strain with glucose as the main carbon source. 19.9 g/L Ketopantoate was synthesized from formaldehyde and α-ketoisovalerate by an aldol reaction at basic conditions. The reaction parameters of reaction were optimized and a conversion ratio of 83.5% was obtained at reaction conditions of pH 13 and 45℃. The ketopantoate in the solution was converted to ketopantoyl lactone at acidic conditions of pH<3. Ketopantoyl lactone was extracted to isobutanol with an extraction rate of 50.9%. The organic phase was decolourized, and ketopantoyl lactone crystal was obtained after concentration. Ketopantoyl lactone was converted back to ketopantoate in an aqueous solution in the pH range of 7~10, and ketopantoate crystal was obtained after concentration. Ketopantoate production from glucose via α-ketoisovalerate as an intermediate was set up, which suggested a novel and competitive technical route to produce ketopantoate. The whole processes were combinated biological fermentation and chemical reactions and had a high conversion ratio. This method adopted renewable and cheap original materials rather than highly toxic raw materials. The optimal temperature of the reaction was 45℃, which was in mild conditions. Overall, a novel and promising method for ketopantoate and ketopantoyl lactone production was provided.

Key words: Ketopantoate, 2-Ketoisovalerate, Aldol condensation, Klebsiella pneumoniae