芳香酯是食品、饮料和护理品中重要的风味成分。通过分子克隆、表达与酶学特征解析,从黑曲霉F0215中鉴定出一种新的、具有催化合成芳香酯活性的脂肪酶CutA。该酶在30℃和pH 7.0时呈现最高活性,以对硝基苯酚乙酸酯为底物的Km和Vmax值分别为19.5 mmoL/L和63.3 mmol/(mL·h)。在30℃、有机溶剂介导条件下,CutA催化丁酸和丁醇、乳酸和乙醇、辛酸和乙醇生成丁酸丁酯、乳酸乙酯或辛酸乙酯,转化率分别为17.2%、4.7%和1.8%。脂肪酶CutA可用于发酵食品的风味生成与改善。
Flavor esters are important flavor components in food, beverage and personal care products. A novel lipase (CutA) with flavor ester synthesis activity was identified from Aspergillus niger by molecular cloning, heterologous expression and biochemical characterization. CutA was revealed that the condition of the maximal activity was 30℃ and pH 7.0. With synthetic substrate pNPA, its Km and Vmax were 19.50 mmol/L and 63.30 mmol/(mL·h), respectively. CutA was able to catalyze the synthesis of butyl butyrate, ethyl caprylate and ethyl lactate by esterification of corresponding acids and alcohols with conversion efficiencies of 17.2%, 4.7% and 1.8 % respectively under 30 ℃ in the organic solvent system. The results indicated a possible application of CutA for flavor improvement/modification of certain fermented foods.
[1] LIAQUAT M, APENTEN R K O R. Synthesis of low molecular weight flavor esters using plant seedling lipases in organic media[J]. Journal of Food Science, 2000, 65(2): 295-299.
[2] BRAULT G, SHARECK F, HURTUBISE Y, et al. Short-chain flavor ester synthesis in organic media by an Escherichia coli whole-cell biocatalyst expressing a newly characterized heterologous lipase[J]. PLoS One, 2014, 9(3): 1-9.
[3] LONGO M A, SANROMÁN M A. Production of food aroma compounds: Microbial and enzymatic methodologies[J]. Food Technology and Biotechnology, 2006, 44(3): 335-353.
[4] DEBARROS D P C, AZEVEDO A M, CABRAL J M S, et al. Optimization of flavor esters synthesis by Fusarium solanipisi cutinase[J].Journal of Food Biochemistry, 2012, 36(3): 275-284.
[5] DEBARROS D P C, FERNANDES P, CABRAL J M S, et al. Synthetic application and activity of cutinase in an aqueous, mini-emulsion model system: Hexyl octanoate synthesis[J]. Catalysis Today, 2011, 173(1): 95-102.
[6] PATEL V, GAJERA H, GUPTA A, et al. Synthesis of ethyl caprylate in organic media using Candida rugosa lipase immobilized on exfoliated graphene oxide: Process parameters and reusability studies[J]. Biochemical Engineering Journal, 2015, 95: 62-70.
[7] KOUTINAS M, YIANGOU C, OSÓRIO N M, et al. Application of commercial and non-commercial immobilized lipases for biocatalytic production of ethyl lactate in organic solvents[J]. Bioresource Technology, 2018, 247: 496-503.
[8] DUAN Xiaojie, LIU Yu, YOU Xin, 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(1): 223.
[9] 吴华昌,由耀辉,卢中明,等. 脂肪酶应用于白酒催陈的初探[J]. 中国酿造, 2011, 30(3): 75-77.
[10] 封莉,邓绍林,黄明,等. 脂肪酶对中式香肠脂肪降解、氧化和风味的影响[J]. 食品科学, 2015, 36(1): 51-58.
[11] 张硕,王玉田,孟鑫. 内源性脂肪酶对奶酪风味的作用及风味成分分析[J]. 食品工业科技, 2013, 34(6): 100-103.
[12] GANDHI N N, PATIL N S, SAWANT S B, et al. Lipase-catalyzed esterification [J]. Catalysis Reviews, 2000, 42(4): 439-480.
[13] 诸葛健,王正祥. 工业微生物实验技术手册[M]. 北京:中国轻工业出版社, 1994: 413-450.
[14] GREEN M R, SAMBROOK J. Molecular Cloning: A Laboratory Manual: Three-Volume Set[M]. 4th ed. New York: Cold Spring Harbor Laboratory Press, 2012.
[15] XU Haibo, YAN Qiaojuan, DUAN Xiaojie, 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.
[16] SINGH A K, MUKHOPADHYAY M. Immobilization of lipase on carboxylic acid-modified silica nanoparticles for olive oil glycerolysis[J]. Bioprocess and Biosystems Engineering, 2018, 41(1): 115-127.
[17] GB/T 10345—2007白酒分析方法[S]. 北京:中国标准出版社, 2007.
[18] 李志斌,李净. 浓香型白酒中的重要物质-辛酸乙酯含量及其贡献分析[J]. 酿酒, 2013, 40(3): 33-35;36.
[19] 谢玉球,钟雨,谢旭,等. 乳酸菌在固态法白酒生产中的地位与作用[J]. 酿酒科技, 2008, 173(11): 83-86.
[20] WU Rina, YU Meiling, LIU Xiaoyu, et al. Changes in flavour and microbial diversity during natural fermentation of suan-cai, a traditional food made in northeast China[J]. International Journal of Food Microbiology, 2015, 211: 23-31.
[21] 段明松,胡晓龙,曾田,等. 不同空间位置的浓香型原酒中香气成分的差异性分析[J]. 酿酒科技, 2019, 295(1): 126-130;135.
[22] 郑晓吉,刘飞,任全路,等. 基于SPME-GC-MS法比较新疆哈萨克族不同居住区奶酪风味差异[J]. 食品科学, 2018, 39(8): 83-89.
[23] 戴照琪,赵见营,罗辑,等. 复合精油涂层对干腌火腿挥发性风味品质的影响研究[J]. 食品工业科技, 2018, 39(3): 243-249;258.
