白酒杂醇油酯化酶的分子构建
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摘要
为降低白酒中杂醇油含量,构建高活性杂醇油酯化酶,酯化杂醇油为较高沸点的酯从而降低酒体中杂醇油含量。该研究利用逆转录-聚合酶链式反应(RT-PCR)扩增酯化酶基因,酶切、以其为模板,进行易错聚合酶链式反应,连接、转化大肠杆菌(Escherichia coli)DH5α,筛选酶活显著提升的酯化酶。结果表明,酯化酶氨基酸中146位缬氨酸突变为天冬氨酸,198位苏氨酸突变为异亮氨酸,376位天冬氨酸突变为谷氨酸。初始酯化酶和构建酯化酶合成乙酸正丙酯、乙酸异丁酯、乙酸异戊酯酶活分别为10.83 U/L、24.72 U/L,2.58 U/L、6.24 U/L,3.83 U/L、8.78 U/L,构建酯化酶酶活分别是初始酯化酶酶活的2.28倍、2.42倍、2.29倍。构建杂醇油酯化酶在白酒生产中具有极大的应用潜力。
In order to decrease fusel oil of Baijiu, the esterifying enzyme was reconstructed to efficiently transform fusel oil into the esters with higher boiling points. The gene of the esterifying enzyme was amplified by reverse translation-polymerase chain reaction(RT-PCR). The mutated sequences were cut and linked with the plasmid cut by restricted enzymes and it was used as template and error-prone PCR was performed. The constructed vector was transformed into Escherichia coli DH5α, and the esterifying enzyme with significantly enhanced enzyme activity was selected. The results showed that the 146 site valine, 198 site theronine and 376 site aspartic acid amino acids of esterifying enzyme were mutated into aspartic acid, isoleucine and glutamic acid. The enzyme activity of n-propyl acetate, isobutyl acetate, isoamyl acetate of the original esterifying enzyme and the reconstructed esterifying enzyme was 10.83 U/L and 24.72 U/L, 2.58 U/L and 6.24 U/L, 3.83 U/L and 8.78 U/L, respectively. The esterifying enzyme activity of the reconstructed esterifying enzyme was 2.28, 2.42, 2.29 times that of the original esterifying enzyme. The esterase designed by molecular reconstruction was promising for application in the modern production of Baijiu brewing.
In order to decrease fusel oil of Baijiu, the esterifying enzyme was reconstructed to efficiently transform fusel oil into the esters with higher boiling points. The gene of the esterifying enzyme was amplified by reverse translation-polymerase chain reaction(RT-PCR). The mutated sequences were cut and linked with the plasmid cut by restricted enzymes and it was used as template and error-prone PCR was performed. The constructed vector was transformed into Escherichia coli DH5α, and the esterifying enzyme with significantly enhanced enzyme activity was selected. The results showed that the 146 site valine, 198 site theronine and 376 site aspartic acid amino acids of esterifying enzyme were mutated into aspartic acid, isoleucine and glutamic acid. The enzyme activity of n-propyl acetate, isobutyl acetate, isoamyl acetate of the original esterifying enzyme and the reconstructed esterifying enzyme was 10.83 U/L and 24.72 U/L, 2.58 U/L and 6.24 U/L, 3.83 U/L and 8.78 U/L, respectively. The esterifying enzyme activity of the reconstructed esterifying enzyme was 2.28, 2.42, 2.29 times that of the original esterifying enzyme. The esterase designed by molecular reconstruction was promising for application in the modern production of Baijiu brewing.
引文
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[21]宋海超,史学群.一种适用于RT-PCR的拮抗酵母菌总RNA提取方法[J].中国农学通报,2012,28(27):199-203.
[22]龙玮.顶空气相色谱法测定水中卤代烃类化合物[J].工业用水与水,2018,49(4):78-81.
[23]刘蓉,张永江,邓茂,等.顶空气相色谱法测定环境水样中14种挥发性卤代烃[J].西南师范大学学报(自然科学版),2018,43(3):133-138.
[24]QI Y K,LI F L,CHEN Q,et al.Protein termini relocation plus random mutation:A new strategy for finding key sites in esterase evolution[J].Mol Cataly,2018,460:94-99.
[25]HANG Y,RAN S,WANG X Y,et al.Mutational analysis and stability characterization of a novel esterase of lipolytic enzyme family Ⅵ from Shewanella sp.[J].Int J Biol Macromol,2016,93:655-664.
[2]GIUDICI P,ROMANO P,ZAMBONELLI C.A biometric study of higher alcohol production in Saccharomyces cerevisiae[J].Can J Microbiol,1990,36(3):61-64.
[3]张跃廷,刘琼.浅淡杂醇油[J].酿酒,2002,29(5):18-20.
[4]张兴亚,高梦莎,蒋予箭.糖化发酵剂对黄酒中高级醇含量的影响[J].中国酿造,2012,31(1):130-133.
[5]KOSOWSKI G,MIKULSKI D,MACKO D,et al.Influence of various yeast strains and selected starchy raw materials on production of higher alcohols during the alcoholic fermentation process[J].Eur Food Res Technol,2015,240(1):233-242.
[6]DACK R E,BLACK G W,KOUTSIDIS G,et al.The effect of Maillard reaction products and yeast strain on the synthesis of key higher alcohols and esters in beer fermentations[J].Food Chem,2017,232(1):595-601.
[7]孔小勇,冷云伟,孙然,等.影响黄酒中高级醇含量的工艺因素探讨[J].中国酿造,2011,30(10):163-165.
[8]LIU D F,ZHANG H T,XIONG W L,et al.Effect of temperature on Chinese rice wine brewing with high concentration presteamed whole sticky rice[J].Biomed Res Int,2015,2014(1):426-429.
[9]龚耀平,周建弟,钱斌,等.不同酿造工艺对黄酒理化指标及高级醇含量影响的研究[J].中国酿造,2013,32(2):37-40.
[10]张亮,韩颖,孟宪梅.杂醇油提纯分离技术研究应用进展[J].吉林工商学院学报,2014(5):96-99.
[11]邓振坤,李万民,田洁.杂醇油分离器在酒精生产中的综合效应[J].四川食品与发酵,2006(6):36-38.
[12]贾政浩,朱红平.乙醇生产中杂醇油的分离[J].河南科技,2012(4):59.
[13]黄科林,樊晓丹,黄焕生,等.连续催化酯化-精馏联合过程合成乙酸正丙酯的研究[J].安徽化工,2002(2):16-18.
[14]周峰,陈长旭,许春建.乙酸异戊酯+异戊醇和乙酸异戊酯+正己醇体系汽液平衡[J].化工学报,2017(2):560-566.
[15]MU譙OZ R,MONT魷N J B,BURGUET M C,et al.Vapor-liquid equilibria in the temary systen isobutyl acloho+isobuty acetate+butyl propionate and the binary systerm isobutyl alcohol+butyl propionate,isobutyl acetate+butyl propionate at 101.3 kPa[J].Fluid PhASE Equilibria,2005,238(1):65-71.
[16]王成涛,丁晓英,张佳婵,等.非水相介质杂醇油酶法转化天然酯类香料的研究[J].食品与机械,2011(6):19-22,27.
[17]陈帅,郑佳,刘琨毅,等.红曲酯化酶促反应及其代谢产物特征[J].食品与发酵工业,2012,38(2):47-51.
[18]方春玉,周健,李智.红曲霉发酵液酯化酿酒尾水制备酯化液的研究[J].食品工业科技,2018,39(13):46-50.
[19]MORATA A,VEJARANO R,RIDOLFI G,et al.Reduction of 4-ethylphenol production in red wines using HCDC+yeasts and cinnamyl esterases[J].Enzyme Microbial Technol,2013,52(2):99-104.
[20]MORATA A,VEJARANO R,RIDOLFI G,et al.Reduction of 4-ethylphenol production in red wines using HCDC+yeasts and cinnamyl esterases[J].Enzyme Microbial Technol,2013,52(2):99-104.
[21]宋海超,史学群.一种适用于RT-PCR的拮抗酵母菌总RNA提取方法[J].中国农学通报,2012,28(27):199-203.
[22]龙玮.顶空气相色谱法测定水中卤代烃类化合物[J].工业用水与水,2018,49(4):78-81.
[23]刘蓉,张永江,邓茂,等.顶空气相色谱法测定环境水样中14种挥发性卤代烃[J].西南师范大学学报(自然科学版),2018,43(3):133-138.
[24]QI Y K,LI F L,CHEN Q,et al.Protein termini relocation plus random mutation:A new strategy for finding key sites in esterase evolution[J].Mol Cataly,2018,460:94-99.
[25]HANG Y,RAN S,WANG X Y,et al.Mutational analysis and stability characterization of a novel esterase of lipolytic enzyme family Ⅵ from Shewanella sp.[J].Int J Biol Macromol,2016,93:655-664.
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