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Home > Archive>Volume 22, Issue 6, 2022 >83-94. DOI:10.16429/j.1009-7848.2022.06.009
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Heterologous Expression and Enzymatic Analysis of Alcohol Dehydrogenase I in β-Phenylethanol Biosynthesis Pathway from Huangjiu Yeast
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  • Yang Qilin 1,4,5

    Yang Qilin

    School of Food Science and Technology， National Engineering Research Center of Cereal Fermentation and Food Biomanufacturing， Jiangnan University， Wuxi 214122， Jiangsu;Jiangsu Provincial Engineering Research Center for Bioactive Product Processing， Jiangnan University，Wuxi 214122， Jiangsu;Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province， Wuxi 214122， Jiangsu
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  • Liu Shuangping 1,2,3,4,5

    Liu Shuangping

    School of Food Science and Technology， National Engineering Research Center of Cereal Fermentation and Food Biomanufacturing， Jiangnan University， Wuxi 214122， Jiangsu;Jiangnan University Shaoxing Industrial Technology Research Institute， Shaoxing 312000， Zhejiang;National Engineering Research Center of Huangjiu， Zhejiang Guyuelongshan Shaoxing Wine Co.， Ltd.，Shaoxing 312000， Zhejiang;Jiangsu Provincial Engineering Research Center for Bioactive Product Processing， Jiangnan University，Wuxi 214122， Jiangsu;Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province， Wuxi 214122， Jiangsu
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  • Zhao Yuzong 1,4,5

    Zhao Yuzong

    School of Food Science and Technology， National Engineering Research Center of Cereal Fermentation and Food Biomanufacturing， Jiangnan University， Wuxi 214122， Jiangsu;Jiangsu Provincial Engineering Research Center for Bioactive Product Processing， Jiangnan University，Wuxi 214122， Jiangsu;Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province， Wuxi 214122， Jiangsu
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  • Sun Hailong 1,4,5

    Sun Hailong

    School of Food Science and Technology， National Engineering Research Center of Cereal Fermentation and Food Biomanufacturing， Jiangnan University， Wuxi 214122， Jiangsu;Jiangsu Provincial Engineering Research Center for Bioactive Product Processing， Jiangnan University，Wuxi 214122， Jiangsu;Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province， Wuxi 214122， Jiangsu
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  • Mao Jian 1,2,3,4,5

    Mao Jian

    School of Food Science and Technology， National Engineering Research Center of Cereal Fermentation and Food Biomanufacturing， Jiangnan University， Wuxi 214122， Jiangsu;Jiangnan University Shaoxing Industrial Technology Research Institute， Shaoxing 312000， Zhejiang;National Engineering Research Center of Huangjiu， Zhejiang Guyuelongshan Shaoxing Wine Co.， Ltd.，Shaoxing 312000， Zhejiang;Jiangsu Provincial Engineering Research Center for Bioactive Product Processing， Jiangnan University，Wuxi 214122， Jiangsu;Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province， Wuxi 214122， Jiangsu
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Affiliation:

(1.School of Food Science and Technology， National Engineering Research Center of Cereal Fermentation and Food Biomanufacturing， Jiangnan University， Wuxi 214122， Jiangsu;2.Jiangnan University (Shaoxing) Industrial Technology Research Institute， Shaoxing 312000， Zhejiang;3.National Engineering Research Center of Huangjiu， Zhejiang Guyuelongshan Shaoxing Wine Co.， Ltd.，Shaoxing 312000， Zhejiang;4.Jiangsu Provincial Engineering Research Center for Bioactive Product Processing， Jiangnan University，Wuxi 214122， Jiangsu;5.Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province， Wuxi 214122， Jiangsu)

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    Abstract:

    Alcohol dehydrogenase is a key enzyme in the final synthesis of β-phenylethanol in Huangjiu yeast， which comes from the Ehrlich pathway. To explore the differences and enzymatic analysis of Adh1pHJ， the ADH1 of Saccharomyces cerevisiae HJ and Saccharomyces cerevisiae S288C were used as templates respectively to amplify the target gene by PCR. The expression plasmid pET-28a(+)-ADH1 were constructed， and transformed into Escherichia coli BL21(DE3) cells to obtain high expression induced by IPTG. The crude enzymes were obtained after ultrasonic fragmentation， and then purified by affinity chromatography with AKTA pure. Finally the enzymatic analysis were explored. Using phenylacetaldehyde as substrate， it was found that the enzyme activity(231.51 U/g) of Adh1pHJ was 13.79% higher than that of Adh1pS288C (203.48 U/g)， and the Km value of Adh1pHJ (0.524 μmol/L) was less than that of Adh1pS288C (0.759 μmol/L)， which indicated that the affinity between Adh1pHJ and substrate was higher. From the kcat/Km value， it is found that the catalytic efficiency of Adh1pHJ[0.406 L/(μmol·min)] is higher. The tolerance of Adh1pHJ to β-phenylethanol and ethanol was higher than that of Adh1pS288C. This study provides a method and theoretical basis for the analysis of the structure and properties of Adh1p， and also provides a reference for the industrial production and development of β- phenylethanol.

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History
  • Received:June 11,2021
  • Online: July 19,2022
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