枯草芽孢杆菌和戊糖片球菌乙酰乳酸合成酶的异源表达及其酶学性质比较

摘要/Abstract

摘要： 乙偶姻是一种具有重要应用价值的生物基平台化合物，其在不同微生物菌种中的合成效率存在显著差异，其中乙酰乳酸合成酶（ALS）是调控乙偶姻生物合成的关键限速酶。本研究以实验室筛选获得的高产乙偶姻枯草芽孢杆菌JN17（Bacillus subtilis JN17）和戊糖片球菌JXQ20（Pediococcus pentosaceus JXQ20）为研究对象，对其ALS基因（BsALS和PpALS）进行了克隆和异源表达，并对其酶学性质与结构特征进行了系统表征。结果显示，枯草芽孢杆菌JN17的乙偶姻产量为18.1581 g/L，显著高于戊糖片球菌JXQ20（0.0442 g/L）。BsALS和PpALS的比活力分别为184.87 U/mg和2.57 U/mg；其最适温度分别为50 ℃和30 ℃，最适pH值分别为7.0和6.0，且BsALS在热稳定性和pH值稳定性方面表现更优。Cs+对BsALS的抑制作用最强，Cu2+对PpALS的促进作用最显著，而十二烷基硫酸钠（sodium dodecyl sulfate，SDS）使两者完全失去活性。在乙醇体积分数20%条件下，BsALS保持76.03%的相对活性，而PpALS在乙醇体积分数15%条件下完全失去活性；在氯化钠质量分数5%条件下，BsALS的相对活性为52.42%，而PpALS仅为21.04%。BsALS和PpALS的米氏常数（Km）分别为77.54 mmol/L和255.98 mmol/L，最大反应速率（Vmax）分别为595.14 U/mg和19.04 U/mg。结构分析表明，两种ALS在与焦磷酸硫胺素（thiamine diphosphate，ThDP）和Mg2?结合的关键氨基酸残基及氢键数量上存在差异，可能是导致其催化性能和稳定性显著不同的分子基础。本研究从酶学和结构层面揭示了不同微生物乙偶姻合成能力差异的内在机制，为乙酰乳酸合成酶的理性改造及其在食品发酵和工业生物合成中的应用提供了理论依据。

关键词: 乙酰乳酸合成酶, 枯草芽孢杆菌, 戊糖片球菌, 异源表达, 酶学性质

Abstract: Acetoin is a bio-based platform compound with significant application value, and its synthesis efficiency varies greatly among different microbial strains, with acetolactate synthase (ALS) being the key rate-limiting enzyme regulating acetoin biosynthesis. This study focuses on the high-yield acetoin-producing strains Bacillus subtilis JN17 and Pediococcus pentosaceus JXQ20, obtained through laboratory screening. The ALS genes (BsALS and PpALS) were cloned and heterologously expressed, and their enzymatic properties and structural characteristics were systematically characterized. The results showed that B. subtilis JN17 produced an acetoin yield of 18.1581 g/L, significantly higher than the 0.0442 g/L yield of P. pentosaceus JXQ20. The specific activities of BsALS and PpALS were 184.87 U/mg and 2.57 U/mg, respectively; their optimal temperatures were 50 ℃ and 30 ℃, and optimal pH values were 7.0 and 6.0, with BsALS demonstrating superior thermal and pH stability. Cs+ showed the strongest inhibition on BsALS, while Cu2+ significantly enhanced PpALS activity, and sodium dodecyl sulfate (SDS) completely inactivated both enzymes. BsALS retained 76.03% relative activity at 20% ethanol volume fraction, while PpALS lost all activity at 15% ethanol volume fraction. At 5% NaCl mass fraction, BsALS showed 52.42% relative activity, whereas PpALS had only 21.04%. The Michaelis constants (Km) for BsALS and PpALS were 77.54 mmol/L and 255.98 mmol/L, respectively, with maximum reaction rates (Vmax) of 595.14 U/mg and 19.04 U/mg. Structural analysis indicated differences in key amino acid residues and hydrogen bond numbers for thiamine diphosphate (ThDP) and Mg2+ binding between the two ALS, possibly explaining the significant differences in their catalytic performance and stability. This research reveals the intrinsic mechanisms of differences in acetoin synthesis capabilities among different microorganisms from enzymatic and structural perspectives, offering a theoretical foundation for the rational modification of acetolactate synthase and its application in food fermentation and industrial biosynthesis.

Key words: acetolactate synthase, Bacillus subtilis, Pediococcus pentosaceus, heterologous expression, enzymatic properties

中图分类号:

Q814.9

刘裕峰李圆鑫袁思棋王浩黄楠刘君. 枯草芽孢杆菌和戊糖片球菌乙酰乳酸合成酶的异源表达及其酶学性质比较[J]. 食品科学.

Siqi YUAN. Heterologous Expression and Comparative Enzymatic Properties of Acetolactate Synthase from Bacillus subtilis and Pediococcus pentosaceus[J]. FOOD SCIENCE.

参考文献

[1] CUI Z, WANG Z, ZHENG M, et al. Advances in biological production of acetoin: a comprehensive overview[J]. Critical Reviews in Biotechnology, 2022, 42(8): 1135-1156. DOI: 10.1080/07388551.2021.1995319.
[2] LUO Y, LAI H, LIN S, et al. Screening and characterization of acetoin-producing lactic acid bacteria and their effect on the flavor quality of soy sauce[J]. LWT, 2025, 231: 118323. DOI: 10.1016/j.lwt.2025.118323.
[3] BAO W, SHEN W, PENG Q, et al. Metabolic engineering of Zymomonas mobilis for acetoin production by carbon redistribution and cofactor balance[J]. Fermentation, 2023, 9(2): 113. DOI: 10.3390/fermentation9020113.
[4] LIU H, CHEN X, LU J, et al. Evaluation of the differences between low-salt solid-state fermented soy sauce and high-salt diluted-state fermented soy sauce in china: from taste-active compounds and aroma-active compounds to sensory characteristics[J]. Journal of the Science of Food and Agriculture, 2024, 104(1): 340-351. DOI:10.1002/jsfa.12924.
[5] SHENG L, MADIKA A, LAU M S H, et al. Metabolic engineering for the production of acetoin and 2,3-butanediol at elevated temperature in Parageobacillus thermoglucosidasius ncimb 11955[J]. Frontiers in Bioengineering and Biotechnology, 2023, 11: 1191079. DOI: 10.3389/fbioe.2023.1191079.
[6] SHI L, LIN Y, SONG J, et al. Engineered Bacillus subtilis for the production of tetramethylpyrazine,(R,R)-2,3-butanediol and acetoin[J]. Fermentation, 2023, 9(5): 488. DOI: 10.3390/fermentation9050488.
[7] JI X, HUANG H, OUYANG P. Microbial 2,3-butanediol production: a state-of-the-art review[J]. Biotechnology Advances, 2011, 29(3): 351-364. DOI: 10.1016/j.biotechadv.2011.01.007.
[8] ZHU C, SHEN T, LIU D, et al. Production of liquid hydrocarbon fuels with acetoin and platform molecules derived from lignocellulose[J]. Green Chemistry, 2016, 18(7): 2165-2174. DOI: 10.1039/C5GC02414E.
[9] XIAO Z, LU J R. Generation of acetoin and its derivatives in foods[J]. Journal of Agricultural and Food Chemistry, 2014, 62(28): 6487-6497. DOI: 10.1021/jf5013902.
[10] MAINA S, PRABHU A A, VIVEK N, et al. Prospects on bio-based 2,3-butanediol and acetoin production: recent progress and advances[J]. Biotechnology Advances, 2022, 54: 107783. DOI: 10.1016/j.biotechadv.2021.107783.
[11] KOCHIUS S, PAETZOLD M, SCHOLZ A, et al. Enantioselective enzymatic synthesis of the α-hydroxy ketone (R)-acetoin from meso-2,3-butanediol[J]. Journal of Molecular Catalysis B: Enzymatic, 2014, 103: 61-66. DOI: 10.1016/j.molcatb.2013.08.016.
[12] LI S, GAO X, XU N, et al. Enhancement of acetoin production in Candida glabrata by in silico-aided metabolic engineering[J]. Microbial Cell Factories, 2014, 13(1): 55. DOI: 10.1186/1475-2859-13-55.
[13] XIAO Z, LU J R. Strategies for enhancing fermentative production of acetoin: a review[J]. Biotechnology Advances, 2014, 32(2): 492-503. DOI: 10.1016/j.biotechadv.2014.01.002.
[14] BAE S, KIM S, PARK H J, et al. High-yield production of (R)-acetoin in Saccharomyces cerevisiae by deleting genes for NAD(P)H-dependent ketone reductases producing meso-2,3-butanediol and 2,3-dimethylglycerate[J]. Metabolic Engineering, 2021, 66: 68-78. DOI: 10.1016/j.ymben.2021.04.001.
[15] LIANG Y, LONG Z, ZHANG Y, et al. The chemical mechanisms of the enzymes in the branched-chain amino acids biosynthetic pathway and their applications[J]. Biochimie, 2021, 184: 72-87. DOI: 10.1016/j.biochi.2021.02.008.
[16] LIANG Y, NIU Z, WU Z, et al. Catalytic insights of acetolactate synthases from different bacteria[J]. Archives of Biochemistry and Biophysics, 2025, 764: 110248. DOI: 10.1016/j.abb.2024.110248.
[17] ZHAO T, LI Y, YUAN S, et al. Structure-based design of acetolactate synthase from Bacillus licheniformis improved protein stability under acidic conditions[J]. Frontiers in Microbiology, 2020, 11: 582909. DOI: 10.3389/fmicb.2020.582909.
[18] FAN X, WU H, JIA Z, et al. Metabolic engineering of Bacillus subtilis for the co-production of uridine and acetoin[J]. Applied Microbiology and Biotechnology, 2018, 102(20): 8753-8762. DOI: 10.1007/s00253-018-9316-7.
[19] PETROV K, PETROVA P. Current advances in microbial production of acetoin and 2,3-butanediol by Bacillus spp.[J]. Fermentation, 2021, 7(4): 307. DOI: 10.3390/fermentation7040307.
[20] ZHANG X, YANG T, LIN Q, et al. Isolation and identification of an acetoin high production bacterium that can reverse transform 2,3-butanediol to acetoin at the decline phase of fermentation[J]. World Journal of Microbiology and Biotechnology, 2011, 27(12): 2785-2790. DOI: 10.1007/s11274-011-0754-y.
[21] ZHANG Y, LI S, LIU L, et al. Acetoin production enhanced by manipulating carbon flux in a newly isolated Bacillus amyloliquefaciens[J]. Bioresource Technology, 2013, 130: 256-260. DOI: 10.1016/j.biortech.2012.10.036.
[22] ZHAO J, MENG Z, MA X, et al. Characterization and regulation of the acetolactate synthase genes involved in acetoin biosynthesis in Acetobacter pasteurianus[J]. Foods, 2021, 10(5): 1013. DOI: 10.3390/foods10051013.
[23] 潘婉舒, 林晓芳, 杨小平, 等. 乳酸胁迫对Pichia kudriavzevii固态发酵特性的影响[J]. 食品工业科技, 2024, 45(4): 116-122. DOI: 10.13386/j.issn1002-0306.2023050193.
[24] LIU X, YANG W, GU H, et al. Optimization of fermentation conditions for 2,3,5-trimethylpyrazine produced by Bacillus amyloliquefaciens from Daqu[J]. Fermentation, 2024, 10(2): 112. DOI: 10.3390/fermentation10020112.
[25] LIU Y, LIU L, YANG S, et al. Molecular characterization and functional analysis of a pathogenesis-related β-1,3-glucanase gene in spruce (Picea asperata)[J]. European Journal of Plant Pathology, 2022, 164(2): 177-192. DOI: 10.1007/s10658-022-02547-1.
[26] YANG J, FAN D, ZHAO F, et al. Characterization of d-allulose-3-epimerase from Ruminiclostridium papyrosolvens and immobilization within metal-organic frameworks[J]. Frontiers in Bioengineering and Biotechnology, 2022, 10: 869536. DOI: 10.3389/fbioe.2022.869536.
[27] 林源, 秦伟帅, 王佳琳, 等. 枯草芽孢杆菌利用玉米秸秆水解液生产乙偶姻[J]. 食品与发酵工业, 2022, 48(19): 225-229. DOI: 10.13995/j.cnki.11-1802/ts.030430.
[28] 杨铭, 郝晓娜, 罗天淇, 等. 功能基因分析辅助筛选产双乙酰和乙偶姻乳酸菌[J]. 食品科学, 2020, 41(10): 117-123. DOI: 10.7506/spkx1002-6630-20190418-231.
[29] LEE S, JUNG I, BAIG I A, et al. Mutational analysis of critical residues of FAD-independent catabolic acetolactate synthase from Enterococcus faecalis v583[J]. International Journal of Biological Macromolecules, 2015, 72: 104-109. DOI: 10.1016/j.ijbiomac.2014.08.002.
[30] GUO X W, ZHANG Y H, CAO C H, et al. Enhanced production of 2,3‐butanediol by overexpressing acetolactate synthase and acetoin reductase in Klebsiella pneumoniae[J]. Biotechnology and Applied Biochemistry, 2014, 61(6): 707-715. DOI: 10.1002/bab.1217.
[31] SOMMER B, von MOELLER H, HAACK M, et al. Detailed structure-function correlations of Bacillus subtilis acetolactate synthase[J]. ChemBioChem, 2015, 16(1): 110-118. DOI: 10.1002/cbic.201402541.
[32] URBAN A, ANSMANT I, MOTORIN Y. Optimisation of expression and purification of the recombinant Yol066 (Rib2) protein from Saccharomyces cerevisiae[J]. Journal of Chromatography B, 2003, 786(1-2): 187-195. DOI: 10.1016/S1570-0232(02)00742-0.
[33] ZENG Y, ZHANG K, LU T, et al. Structural and mechanistic basis for nitrile synthetase by an argininosuccinate synthetase-like enzyme[J]. ACS Catalysis, 2025, 15(18): 16254-16267. DOI: 10.1021/acscatal.5c04243.
[34] HUO Y, ZHAN Y, WANG Q, et al. Acetolactate synthase (AlsS) in Bacillus licheniformis WX-02: enzymatic properties and efficient functions for acetoin/butanediol and l-valine biosynthesis[J]. Bioprocess and Biosystems Engineering, 2018, 41(1): 87-96. DOI: 10.1007/s00449-017-1847-2.
[35] ARCUS V L, van der KAMP M W, PUDNEY C R, et al. Enzyme evolution and the temperature dependence of enzyme catalysis[J]. Current Opinion in Structural Biology, 2020, 65: 96-101. DOI: 10.1016/j.sbi.2020.06.001.
[36] 张显, 赵晓静, 李欣, 等. 利用代谢工程构建高产乙偶姻枯草芽孢杆菌[J]. 食品与发酵工业, 2015, 41(12): 18-25. DOI: 10.13995/j.cnki.11-1802/ts.201512004.
[37] CAO Y, ZHANG H, DU H, et al. Microorganisms and metabolic characteristics of temperature-dependent fermentation during sauce-flavor baijiu production[J]. Food Bioscience, 2025, 63: 105787. DOI: 10.1016/j.fbio.2024.105787.
[38] HU X, DU H, REN C, et al. Illuminating anaerobic microbial community and cooccurrence patterns across a quality gradient in chinese liquor fermentation pit muds[J]. Applied and Environmental Microbiology, 2016, 82(8): 2506-2515. DOI: 10.1128/AEM.03409-15.
[39] KISRIEVA Y S, SEREBRENNIKOV V M, ZAGUSTINA N A, et al. Isolation and purification of acetolactate synthase and acetolactate decarboxylase from the culture of Lactococcus lactis[J]. Applied Biochemistry and Microbiology, 2000, 36: 109-114. DOI: 10.1007/BF02737903.
[40] ZHANG H, ZHANG H, DU H, et al. Unraveling the multiple interactions between phages, microbes and flavor in the fermentation of strong-flavor baijiu[J]. Bioresources and Bioprocessing, 2025, 12(1): 14. DOI: 10.1186/s40643-025-00852-1.
[41] HE Y, SHI W, HUANG T, et al. Overproduction of organic acids affects the microbial community succession and flavor metabolism during baijiu fermentation[J]. LWT, 2024, 213: 117093. DOI: 10.1016/j.lwt.2024.117093.
[42] 符长春, 杨瑞金, 仝艳军. Bifidobacterium bifidum ATCC 29521 β-半乳糖苷酶的生物信息学分析、异源表达及其酶学性质[J]. 食品科学, 2025, 46(06): 98-107. DOI: 10.7506/spkx1002-6630-20240912-098.
[43] 陈伟, 谷新晰, 张莉娟, 等. 茶树单宁酶的原核表达及酶学性质表征[J]. 食品科学, 2022, 43(8): 74-80. DOI: 10.7506/spkx1002-6630-20210308-095.
[44] ATSUMI S, LI Z, LIAO J C. Acetolactate synthase from Bacillus subtilis serves as a 2-ketoisovalerate decarboxylase for isobutanol biosynthesis in Escherichia coli[J]. Applied and Environmental Microbiology, 2009, 75(19): 6306-6311. DOI: 10.1128/AEM.01160-09.
[45] LIU Y, ZHANG X, YUAN S, et al. Molecular identification and functional characterization of a chitinase gene in Picea asperata[J]. Discover Plants, 2025, 2(1): 145. DOI: 10.1007/s44372-025-00231-2.
[46] 李新锋, 郭彤彤, 李彬春. 鼠李糖乳杆菌LGG芳香酯酶的酶学表征与结构分析[J]. 食品科学, 2023, 44(08): 162-169. DOI: 10.7506/spkx1002-6630-20220426-332.
[47] YANG H, LIU L, SHIN H, et al. Structure-based engineering of histidine residues in the catalytic domain of α-amylase from Bacillus subtilis for improved protein stability and catalytic efficiency under acidic conditions[J]. Journal of Biotechnology, 2013, 164(1): 59-66. DOI: 10.1016/j.jbiotec.2012.12.007.
[48] 刘庆涛, 朱司宝, 王天文, 等. 降解氨基甲酸乙酯的酯酶挖掘及酶学性质表征[J]. 食品科学, 2025, 46(02): 99-107. DOI: 10.7506/spkx1002-6630-20240603-011.