毕赤酵母展示稻米脂肪酶催化乙酸乙酯研究
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摘要
论文利用展示在毕赤酵母表面的稻米脂肪酶作为催化剂在正己烷中催化合成乙酸乙酯,考察了稻米脂肪酶的酶活、底物摩尔比、反应温度、反应时间和3A分子筛添加量对合成乙酸乙酯反应的影响.结果表明,在15 m L正己烷体系中,n(乙酸/乙醇,摩尔比)为1.2∶1,稻米脂肪酶的酶活为15 750 U/g,发酵时间为4 h,反应温度为40℃,3A分子筛添加质量为8%是合成乙酸乙酯的最佳条件。在此条件下,稻米脂肪酶催化合成乙酸乙酯的转化率达到90.92%。
In this study,the rice lipase(RL) was displayed on the surfaceof Pichia pastoris to catalyze synthesis of ethyl acetate in n-hexane,the influence of some factors such as the enzyme activity of rice lipase,substrate concentration,reaction temperature,reaction time,the amount of 3 A molecular sieve on the synthesis of ethyl acetate was investigated. The study showed that,acetic acid/ethanol(mol/mol) ratio1.2 ∶1,reaction time of 4 h,reaction temperature of 40 ℃,the enzyme activityof rice lipase 15 750 U/g,the adding amount of 3 A molecular sieve8 % in 15 m L n-hexane was the optimum condition. Under the condition,the reaction conversion of rice lipase catalyzed synthesis of ethyl acetate reached 90.92%.
In this study,the rice lipase(RL) was displayed on the surfaceof Pichia pastoris to catalyze synthesis of ethyl acetate in n-hexane,the influence of some factors such as the enzyme activity of rice lipase,substrate concentration,reaction temperature,reaction time,the amount of 3 A molecular sieve on the synthesis of ethyl acetate was investigated. The study showed that,acetic acid/ethanol(mol/mol) ratio1.2 ∶1,reaction time of 4 h,reaction temperature of 40 ℃,the enzyme activityof rice lipase 15 750 U/g,the adding amount of 3 A molecular sieve8 % in 15 m L n-hexane was the optimum condition. Under the condition,the reaction conversion of rice lipase catalyzed synthesis of ethyl acetate reached 90.92%.
引文
[1]荣杰.催化反应精馏合成乙酸乙酯新工艺研究[D].北京:北京化工大学,2003.
[2]XU Yan,ZHANG Kechang.On synthesis of liquor flavor ester catalysed by microbial lipase[J].Wine,1998,(3):8-13.(in Chinese)
[3]GE Qingxiu,CHEN Jianping,HUANG Zhuxing,et al.On syntheses of ethyl acetate in heptane catalyzed by the alkaline lipase[J].Science and Technology of Food Industry,2006,(2):156-158.(in Chinese)
[4]ZHU Jing,XIE Ding,SHENG Min.Properties of rice lipase displayed in Pichia pastoris surface[J].Food and Machinery,2014,30(3):13-15.(in Chinese)
[5]JIN Zi,LIN Ying,HUANG Dengfeng,et al.Synthesis of flavor esters catalyzed by CALB-displaying Pichia pastoris whole-cells in non-aqueous phase[J].Biotechnology,2009,25(12):1927-1932.(in Chinese)
[6]JIANG Huifang,WANG Yaqin,LIU Chunguo.Comparison and improvement of three determination methods for lipase activity[J].Chemistry and Bioengineering,2007,24(8):72-75.(in Chinese)
[7]LUO Shaohua,XIONG Ze,SHAO Wei,et al.Determination of ethyl hexanoate in lipase catalysis reaction system by gas chromatography[J].China Brewing,2014,33(1):137-140.(in Chinese)
[8]SHEN Yongmei,XIE Xiaona,XUN Erna,et al.Research on preparation of ethyl butyrate catalyzed by lipase[J].Journal of Chendo University(Natural Science Edition),2012,31(1):4-7.(in Chinese)
[9]PAN Hong,WANG Jian,ZHENG Jianyong,et al.Lipase catalyzed synthesis of cinnamyl cinnamate in organic solvents[J].Fermentation Technology Communication,2014,43(2):10-14.(in Chinese)
[10]XU Yan,ZHANG Kechang,WANG Yafei.Flavor ester synthesis by microbial lipases in heptane phase[J].Chinese Journal of Biotechnology,1998,14(2):214-219.(in Chinese)
[11]王超,侯涛.一种乙酸乙酯脱水除杂的方法:中国,102070444[P].2011-05-25.
[12]HU Jianhua,WANG Xuliang,SHENG Li,et al.Screening of a yeast strain with high-yield of ethyl acetate and its application in the production of Niulanshan Liquor[J].Liquor-Making Science&Technology,2013,(1):69-74.(in Chinese)
[13]耿博.脂肪酶催化合成乙酸肉桂酯的应用研究[D].天津:天津大学,2012.
[14]XU Yan,WANG Yafei,ZHANG Kechang.Short-chain aliphatic ester synthesis by microbial lipases in nonaqueous phases[J].Joural of Wuxi University of Light Industry,1998,17(1):24-27.(in Chinese)
[15]SOK-JOONG K,SANG-MIN J,YONG-CHEOL P,et al.Lipase catalyzed transesterification of soybean oil using ethyl acetate,an alternative acyl acceptor[J].Biotechnology and Bioprocess Engineering,2007,12:441-445.
[16]SEON A C,JINHEE J,JIN H C,et al.Characterization of putative glycosylphosphatidylinositol-anchoring motifs for surface display in the methylotrophic yeast Hansenulapolymorpha[J].Biotechnol Let,2014,36:2085-2094.
[17]LUCHAI B,MONTRI J,SUJITAR J,et al.HS-SPME-GC-MS analysis of volatile aromatic compounds in alcohol related beverages made with mulberry fruits[J].Food Sci Biotechnol,2011,20(4):1021-1032.
[18]ALEXEY K,PAIVI M A,MATTHIAS R,et al.Mechanism of the chemo-bio catalyzed cascade synthesis of R-1-phenylethyl acetate over Pd/Al2O3,lipase,and Ru-catalysts[J].Res chemintermed,2010,36:193-210.
[19]KONDO A,UEDA M.Yeast cell-surface display-applications of molecular display[J].Applmicrobiolbiotechnol,2004,64:28-40.
[2]XU Yan,ZHANG Kechang.On synthesis of liquor flavor ester catalysed by microbial lipase[J].Wine,1998,(3):8-13.(in Chinese)
[3]GE Qingxiu,CHEN Jianping,HUANG Zhuxing,et al.On syntheses of ethyl acetate in heptane catalyzed by the alkaline lipase[J].Science and Technology of Food Industry,2006,(2):156-158.(in Chinese)
[4]ZHU Jing,XIE Ding,SHENG Min.Properties of rice lipase displayed in Pichia pastoris surface[J].Food and Machinery,2014,30(3):13-15.(in Chinese)
[5]JIN Zi,LIN Ying,HUANG Dengfeng,et al.Synthesis of flavor esters catalyzed by CALB-displaying Pichia pastoris whole-cells in non-aqueous phase[J].Biotechnology,2009,25(12):1927-1932.(in Chinese)
[6]JIANG Huifang,WANG Yaqin,LIU Chunguo.Comparison and improvement of three determination methods for lipase activity[J].Chemistry and Bioengineering,2007,24(8):72-75.(in Chinese)
[7]LUO Shaohua,XIONG Ze,SHAO Wei,et al.Determination of ethyl hexanoate in lipase catalysis reaction system by gas chromatography[J].China Brewing,2014,33(1):137-140.(in Chinese)
[8]SHEN Yongmei,XIE Xiaona,XUN Erna,et al.Research on preparation of ethyl butyrate catalyzed by lipase[J].Journal of Chendo University(Natural Science Edition),2012,31(1):4-7.(in Chinese)
[9]PAN Hong,WANG Jian,ZHENG Jianyong,et al.Lipase catalyzed synthesis of cinnamyl cinnamate in organic solvents[J].Fermentation Technology Communication,2014,43(2):10-14.(in Chinese)
[10]XU Yan,ZHANG Kechang,WANG Yafei.Flavor ester synthesis by microbial lipases in heptane phase[J].Chinese Journal of Biotechnology,1998,14(2):214-219.(in Chinese)
[11]王超,侯涛.一种乙酸乙酯脱水除杂的方法:中国,102070444[P].2011-05-25.
[12]HU Jianhua,WANG Xuliang,SHENG Li,et al.Screening of a yeast strain with high-yield of ethyl acetate and its application in the production of Niulanshan Liquor[J].Liquor-Making Science&Technology,2013,(1):69-74.(in Chinese)
[13]耿博.脂肪酶催化合成乙酸肉桂酯的应用研究[D].天津:天津大学,2012.
[14]XU Yan,WANG Yafei,ZHANG Kechang.Short-chain aliphatic ester synthesis by microbial lipases in nonaqueous phases[J].Joural of Wuxi University of Light Industry,1998,17(1):24-27.(in Chinese)
[15]SOK-JOONG K,SANG-MIN J,YONG-CHEOL P,et al.Lipase catalyzed transesterification of soybean oil using ethyl acetate,an alternative acyl acceptor[J].Biotechnology and Bioprocess Engineering,2007,12:441-445.
[16]SEON A C,JINHEE J,JIN H C,et al.Characterization of putative glycosylphosphatidylinositol-anchoring motifs for surface display in the methylotrophic yeast Hansenulapolymorpha[J].Biotechnol Let,2014,36:2085-2094.
[17]LUCHAI B,MONTRI J,SUJITAR J,et al.HS-SPME-GC-MS analysis of volatile aromatic compounds in alcohol related beverages made with mulberry fruits[J].Food Sci Biotechnol,2011,20(4):1021-1032.
[18]ALEXEY K,PAIVI M A,MATTHIAS R,et al.Mechanism of the chemo-bio catalyzed cascade synthesis of R-1-phenylethyl acetate over Pd/Al2O3,lipase,and Ru-catalysts[J].Res chemintermed,2010,36:193-210.
[19]KONDO A,UEDA M.Yeast cell-surface display-applications of molecular display[J].Applmicrobiolbiotechnol,2004,64:28-40.