食品与发酵工业 >

2024 , Vol. 50 >Issue 7: 1 - 8

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.035668

研究报告

水相条件下合成短链脂肪酸酯霉菌角质酶的筛选与催化特性研究

  • 林良才 ,
  • 梁梦帆 ,
  • 杜荣菲 ,
  • 郑佳 ,
  • 肖冬婷 ,
  • 张煜行 ,
  • 彭志云 ,
  • 张翠英
展开
  • 1(天津科技大学 生物工程学院,天津,300457)
    2(河北衡水老白干酿酒(集团)有限公司博士后科研工作站,河北 衡水,053000)
    3(宜宾五粮液股份有限公司,中国轻工业浓香型白酒固态发酵重点实验室,四川 宜宾,644000)
第一作者:博士,副教授(林良才副教授和张翠英教授为共同通信作者,E-mail:lclin@tust.edu.cn;cyzhangcy@tust.edu.cn)

收稿日期: 2023-03-30

  修回日期: 2023-05-10

  网络出版日期: 2024-05-09

基金资助

天津市科技计划项目(22ZYJDSS00050);中国轻工业浓香型白酒固态发酵重点实验室开放基金项目资助(2021JJ011);天津科技大学研究生科研创新项目资助(KYS202131)

收起

Screening and characterization of fungal cutinases for synthesis of short chain fatty esters in aqueous phase

  • LIN Liangcai ,
  • LIANG Mengfan ,
  • DU Rongfei ,
  • ZHENG Jia ,
  • XIAO Dongting ,
  • ZHANG Yuxing ,
  • PENG Zhiyun ,
  • ZHANG Cuiying
Expand
  • 1(School of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China)
    2(Postdoctoral Research Workstation of Heng Shui Liquor Brewing (Group) Co.Ltd., Hengshui 053000, China)
    3(Sichuan Yibin Wuliangye Group Limited, Key Laboratory of Solid State Fermentation of Luzhou Flavor Baijiu in China Light Industry, Yibin 644000, China)

Received date: 2023-03-30

  Revised date: 2023-05-10

  Online published: 2024-05-09

Fold

摘要

短链脂肪酸酯是白酒重要风味物质,挖掘适用于白酒酿造体系的酯化酶对提升白酒品质具有重要的应用价值。该文在大肠杆菌BL21(DE3)中成功表达了7个霉菌角质酶,研究了其在单一酸和混酸条件下水相中催化合成风味酯的能力。研究结果表明角质酶AFLACUT-2在水相条件下具有较好的短链脂肪酸酯合成能力,其在30 ℃、pH 4的条件下酶活力最高。进一步酶学特性分析显示,当反应温度升到40 ℃时,该酶催化合成己酸乙酯的活力不变,而合成丁酸乙酯活力显著下降。角质酶AFLACUT-2催化合成己酸乙酯的最佳反应时间为18 h,比催化合成丁酸乙酯缩短了25%。此外,酸醇比例的提高有助于短链脂肪酸酯的合成,但过高的酸浓度会抑制酶活力,AFLACUT-2对已酸的耐受性优于丁酸。这些结果不仅拓宽了对白酒风味酯合成机制的认识,也为利用酯化生香技术定向调控基酒风味提供了新的策略。

关键词： 白酒; 短链脂肪酸酯; 角质酶; 水相催化体系

本文引用格式

林良才 , 梁梦帆 , 杜荣菲 , 郑佳 , 肖冬婷 , 张煜行 , 彭志云 , 张翠英 . 水相条件下合成短链脂肪酸酯霉菌角质酶的筛选与催化特性研究[J]. 食品与发酵工业, 2024 , 50(7) : 1 -8 . DOI: 10.13995/j.cnki.11-1802/ts.035668

Abstract

Short-chain fatty acid esters are important flavor substances of Baijiu.It is of great application value to explore esterifying enzymes that suitable for Baijiu fermentation system to improve the quality of Baijiu.In this paper, seven fungal cutinases were successfully expressed in Escherichia coli BL21 (DE3), and their ability to catalyze the synthesis of flavor esters in aqueous phase under single and mixed acid conditions was investigated.The results showed that the cutinase AFLACUT-2 had a strong ability to synthesize short-chain fatty acid esters under aqueous conditions, with the highest enzyme activity at 30 ℃ and pH 4.Further analysis of enzymatic properties showed that when the reaction temperature rose to 40 ℃, the catalytic activity of the enzyme for the synthesis of ethyl caproate remained unchanged, while the catalytic activity for the synthesis of ethyl butyrate significantly decreased.The optimal reaction time for the synthesis of ethyl caproate catalyzed by cutinase AFLACUT-2 was 18 h, which was 25% shorter than that for the catalytic synthesis of ethyl butyrate.In addition, an increase in the ratio of acid to alcohol was beneficial to esterification, but excessive acid could inhibit enzyme activity.Furthermore, AFLACUT-2 has a better tolerance to caproic acid than butyric acid.These results not only broaden the understanding of the mechanism of ester synthesis during Baijiu fermentation,but also provide a new strategy for the directional regulation of base liquor flavor by using fungal cutinases.

Key words： Baijiu; short chain fatty acid esters; cutinase; aqueous catalytic system

参考文献

[1] 肖冬光, 赵树欣, 陈叶福, 等.白酒生产技术[M].2版.北京:化学工业出版社, 2011.
XIAO D G, ZHAO S X, CHEN Y F.Baijiu Production Technology[M].2nd.Beijing:Chemical Industry Press, 2011.
[2] WANG L L, FAN S S, YAN Y, et al.Characterization of potent odorants causing a pickle-like off-odor in Moutai-aroma type Baijiu by comparative aroma extract dilution analysis, quantitative measurements, aroma addition, and omission studies[J].Journal of Agricultural and Food Chemistry, 2020, 68(6):1666-1677.
[3] 陶新功, 孙国昌, 朱瑞康, 等.从四大酯类含量分析糟烧白酒[J].中国酿造, 2013, 32(9):120-122.
TAO X G, SUN G C, ZHU R K, et al.Characterization of arak liquor with the contents of four kinds of esters analysis[J].China Brewing, 2013, 32(9):120-122.
[4] 庄名扬. 中国白酒香味物质形成机理及酿酒工艺的调控[J].四川食品与发酵, 2007, 43(2):1-6.
ZHUANG M Y.The production mechanism of the fragrance in the Chinese spirit & the operation of the spirit brewing[J].Sichuan Food and Fermentation, 2007, 43(2):1-6.
[5] 王晓程, 徐友强, 李秀婷, 等.中国传统发酵食品来源典型小分子脂肪酸酯合成微生物及酶的研究进展[J].中国食品学报, 2021, 21(4):345-355.
WANG X C, XU Y Q, LI X T, et al.Research progress in microorganisms and enzymes for synthesis of typical small molecule fatty acid esters from traditional Chinese fermented foods[J].Journal of Chinese Institute of Food Science and Technology, 2021, 21(4):345-355.
[6] ZHU L, WANG X L, SONG X B, et al.Evolution of the key odorants and aroma profiles in traditional Laowuzeng Baijiu during its one-year ageing[J].Food Chemistry, 2020, 310:125898.
[7] KRUIS A J, LEVISSON M, MARS A E, et al.Ethyl acetate production by the elusive alcohol acetyltransferase from yeast[J].Metabolic Engineering, 2017, 41:92-101.
[8] 李锋, 陈叶福, 郭建, 等.过表达EHT1基因对酿酒酵母己酸乙酯生产能力的影响[J].现代食品科技, 2014, 30(3):93-98;176.
LI F, CHEN Y F, GUO J, et al.Effects of overexpressed EHT1 on ester production of Saccharomyces cerevisiae[J].Modern Food Science and Technology, 2014, 30(3):93-98;176.
[9] VERSTREPEN K J, VAN LAERE S D M, VANDERHAEGEN B M P, et al.Expression levels of the yeast alcohol acetyltransferase genes ATF1, Lg-ATF1, and ATF2 control the formation of a broad range of volatile esters[J].Applied and Environmental Microbiology, 2003, 69(9):5228-5237.
[10] 曾婷婷, 张志刚.白酒酿造中酯化酶的研究现状[J].酿酒, 2010, 37(6):12-14.
ZENG T T, ZHANG Z G.Present research situation of the esterifying enzyme in the production of liquor[J].Liquor Making, 2010, 37(6):12-14.
[11] 刘正, 秦培军, 卢延想, 等.酱香型白酒酿造过程中微生物多样性及代谢过程研究进展[J].中国酿造, 2022, 41(6):6-11.
LIU Z, QIN P J, LU Y X, et al.Research progress of microbial diversity and metabolic process during the fermentation of sauce-flavor Baijiu[J].China Brewing, 2022, 41(6):6-11.
[12] XU Y Q, LIU X, ZHAO J R, et al.An efficient phthalate ester-degrading Bacillus subtilis:Degradation kinetics, metabolic pathway, and catalytic mechanism of the key enzyme[J].Environmental Pollution, 2021, 273:116461.
[13] KRUIS A J, BOHNENKAMP A C, PATINIOS C, et al.Microbial production of short and medium chain esters:Enzymes, pathways, and applications[J].Biotechnology Advances, 2019, 37(7):107407.
[14] 范光森, 吴秋华, 刘朋肖, 等.脂肪酶在白酒酯类化合物合成中的作用研究进展[J].中国食品学报, 2021, 21(1):351-360.
FAN G S, WU Q H, LIU P X, et al.Research progress on the role of lipase in the synthesis of esters in Baijiu[J].Journal of Chinese Institute of Food Science and Technology, 2021, 21(1):351-360.
[15] 杨磊, 穆敏敏, 文成兵, 等.浓香型白酒提质增香技术研究进展[J].酿酒, 2022, 49(1):17-21.
YANG L, MU M M, WEN C B, et al.Research progress on improving quality and flavor of Luzhou-flavor Baijiu[J].Liquor Making, 2022, 49(1):17-21.
[16] XU Y Q, WANG X C, LIU X, et al.Discovery and development of a novel short-chain fatty acid ester synthetic biocatalyst under aqueous phase from Monascus purpureus isolated from Baijiu[J].Food Chemistry, 2021, 338:128025.
[17] ZHAO J R, XU Y Q, LU H Y, et al.Molecular mechanism of LIP05 derived from Monascus purpureus YJX-8 for synthesizing fatty acid ethyl esters under aqueous phase[J].Frontiers in Microbiology, 2023, 13:1107104.
[18] CONG S Z, TIAN K M, ZHANG X, et al.Synthesis of flavor esters by a novel lipase from Aspergillus niger in a soybean-solvent system[J].3 Biotech, 2019, 9(6):244.
[19] SU L Q, HONG R Y, GUO X J, et al.Short-chain aliphatic ester synthesis using Thermobifida fusca cutinase[J].Food Chemistry, 2016, 206:131-136.
[20] XU H B, YAN Q J, DUAN X J, et al.Characterization of an acidic cold-adapted cutinase from Thielavia terrestris and its application in flavor ester synthesis[J].Food Chemistry, 2015, 188:439-445.
[21] DUAN X J, LIU Y, YOU X, et al.High-level expression and characterization of a novel cutinase from Malbranchea cinnamomea suitable for butyl butyrate production[J].Biotechnology for Biofuels, 2017, 10:223.
[22] XU Y Q, HUANG H Q, LU H Y, et al.Characterization of an Aspergillus niger for efficient fatty acid ethyl ester synthesis in aqueous phase and the molecular mechanism[J].Frontiers in Microbiology, 2022, 12:820380.
[23] 王雅琪, 凡林林, 李文瑶, 等.黑曲霉脂肪酶Lip A的异源表达与酶学性质分析[J].食品与发酵工业, 2023, 49(5):25-31.
WANG Y Q, FAN L L, LI W Y, et al.Heterologous expression and enzymatic characterization of Aspergillus niger lipase Lip A[J].Food and Fermentation Industries, 2023, 49(5):25-31.
[24] 刘小敏, 谢翔云, 林良才, 等.黑曲霉脂肪酶tabI酶学性质及乳酸乙酯合成能力的研究[J].食品与发酵工业, 2022, 48(10):9-15.
LIU X M, XIE X Y, LIN L C, et al.Enzymatic properties of Aspergillus niger lipase tabI and its synthesis ability of ethyl lactate[J].Food and Fermentation Industries, 2022, 48(10):9-15.
Options
文章导航

/

〈 〉

技术支持：北京玛格泰克科技发展有限公司