葡萄汁有孢汉逊酵母β-葡萄糖苷酶的提取与纯化
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摘要
研究葡萄汁有孢汉逊酵母β-葡萄糖苷酶的提取纯化步骤,为其增香酿造特性的研究提供技术支持。首先将优选酵母细胞在振幅为80的超声波下破碎20 min,进行β-葡萄糖苷酶的酶活定位,然后对优选菌株的培养条件和糖苷酶的分离纯化条件进行优化。结果表明,供试酵母菌株的β-葡萄糖苷酶为胞外酶,其最佳培养及提取纯化工艺:酵母在28℃发酵培养基中振荡培养72 h,4℃10 000 r/min离心10 min,收集上清液,然后经80%硫酸铵盐析、DEAE-Sepharose-FF阴离子交换层析与Sephacryl S-200凝胶过滤层析3步纯化,得到β-葡萄糖苷酶液。纯化后β-葡萄糖苷酶回收率为6.91%,比酶活由0.081 U/mg提高至1.998 U/mg,为纯化前的24.56倍,纯化效果明显。
To provide the theoretical and technical guidance for its application in the winemaking of aroma enhancement,the extraction and purification of β-glucosidase from one selected Hanseniaspora uvarum strain were studied.In the experiment,the isolate cells were broken under ultrasonic(80 amplitude) for 20 min to locate the β-glucosidase firstly.Then,the yeast cultivation conditions,the extraction and purification steps of β-glucosidase were explored and optimized.Results showed that the β-glucosidase from Hanseniaspora uvarum is extracellular enzyme,its best cultivation and extraction conditions for the crude enzyme were cultivated in fermentation medium in 28 ℃ for 72 h,and 10 000 r/min centrifuged in 4 ℃ for 10 min,collecting supernatant.Then the crude β-glucosidase was purified by 80% ammonium sulfate precipitation,DEAE-Sepharose-FF anion exchange chromatography and Sephacryl S-200 gel chromatography.After purification,the recovery of the β-glucosidase was 6.91% and the enzyme activity increased from 0.081 U/mg to 1.998 U/mg(24.56 times improved),purification effect is obvious.
To provide the theoretical and technical guidance for its application in the winemaking of aroma enhancement,the extraction and purification of β-glucosidase from one selected Hanseniaspora uvarum strain were studied.In the experiment,the isolate cells were broken under ultrasonic(80 amplitude) for 20 min to locate the β-glucosidase firstly.Then,the yeast cultivation conditions,the extraction and purification steps of β-glucosidase were explored and optimized.Results showed that the β-glucosidase from Hanseniaspora uvarum is extracellular enzyme,its best cultivation and extraction conditions for the crude enzyme were cultivated in fermentation medium in 28 ℃ for 72 h,and 10 000 r/min centrifuged in 4 ℃ for 10 min,collecting supernatant.Then the crude β-glucosidase was purified by 80% ammonium sulfate precipitation,DEAE-Sepharose-FF anion exchange chromatography and Sephacryl S-200 gel chromatography.After purification,the recovery of the β-glucosidase was 6.91% and the enzyme activity increased from 0.081 U/mg to 1.998 U/mg(24.56 times improved),purification effect is obvious.
引文
[1]BAFFI M A,TOBAL T,HENRIQUE J,et al.Anovelβ-glucosidase from Sporidiobolus pararoseus:characterization and application in winemaking[J].Journal of Food Science,2011,76(7):C997-C1002.
[2]GONZLEZ P,FARIA L,CARRAU F,et al.Anovel extracellularβ-glucosidase from Issatchenkia terricola:Isolation,immobilization and application for aroma enhancement of white Muscat wine[J].Process Biochemistry,2011,46(1):385-389.
[3]MAICAS S,MATEO J J.Hydrolysis of terpenyl glycosides in grape juice and other fruit juices:a review[J].Applied Microbiology and Biotechnology,2005,67(3):322-335.
[4]CAPALDO A,WALKER M E,FORD C M,et al Beta-glucoside metabolism in Oenococcus oeni:Cloning and characterisation of the phospho-betaglucosidase bgID[J].Food Chemistry,2011,125(2):476-482.
[5]MICHLMAYR H,MANN C S,SILVA N M,et al Isolation and basic characterization of aβ-glucosidase from a strain of Lactobacillus brevis isolated from a malolactic starter culture[J].Journal of Applied Microbiology,2010,108(2):550-559.
[6]LI Y H,FAN M T,RAN J J,et al.Influence of abiotic stress onβ-D-glucosidase activity of two typical Oenococcus oeni strains in China[J].African Journal of Microbiology Research,2012,6(37):6685-6692.
[7]VILLENA M A,IRANZO J F,OTERO R,et al Optimization of a rapid method for studying the cellular location ofβ-glycosidase activity in wine yeasts[J].Journal of Applied Microbiology,2005,99(6):558-564.
[8]BAFFI M A,TOBAL T,LAGO J H,et al.Wine aroma improvement using aβ-lucosidase preparation from Aureobasidium pullulans[J].Applied Biochemistry&Biotechnology,2013,69(2):493-501.
[9]PAULA GONZLEZ-POMBO,LAURA FARINA,FRANCISCO CARRAU,et al.A novel extracellularβ-glucosidase from Issatchenkia terricola:Isolation,immobilization and application for aroma enhancement of white Muscat wine[J].Process Biochemistry,2011,46(1):385-389.
[10]陈今朝,王剑锋,王慧超,等.热稳定酸性β-葡萄糖苷酶的分离纯化及其酶学性质[J].食品科学,2012,33(11):205-209.
[11]祝霞,盛文军,杨学山,等.红佳酿酵母β-葡萄糖苷酶的分离纯化及酶学性质[J].食品科学,2014,35(13):147-150.
[12]MOREIRA N,MENDES F,GUEDES DE P P,et al.Heavy sulphur compounds,higher alcohols and esters production profile of Hanseniaspora uvarum and Hanseniaspora guilliermondii grown as pure and mixed cultures in grape must[J].International Journal of Food Microbiology,2008,124(3):231-238.
[13]YOUNG A H,DONG H P.Role of non-Saccharomyces yeasts in Korean wines produced from Campbell Early grapes:Potential use of Hanseniaspora uvarum as a starter culture[J].Food Microbiology,2013,34(1):207-214.
[14]VIANA F,GIL J V,GENOV魪S S,et al.Rational selection of non-Saccharomyces wine yeasts for mixed starters based on ester formation and enological traits[J].Food Microbiology,2008,25(6):778-785.
[15]MATURANO Y P,RODR魱GUEZ L A,TORO ME,et al.Multi-enzyme production by pure and mixed cultures of Saccharomyces and non-Saccharomyces yeasts during wine fermentation[J].International Journal of Food Microbiology,2012,155(1/2):43-50.
[16]彭传涛.基于野生非酿酒酵母及其糖苷酶调控葡萄酒香气质量的研究[D].杨凌:西北农林科技大学,2014.
[17]MATEO J J,PERIS L,IBANEZ C,et al.Characterization of glycolytic activities from non-Saccharomyces yeasts isolated from Bobal musts[J].Journal of Industrial Microbiology&Biotechnology,2011,38(2):347-354.
[18]陶永胜,牟含,李国,等.野生胶红酵母糖苷酶水解媚丽新酒中香气糖苷研究[J].农业机械学报,2014,45(12):249-254.
[19]HU K,ZHU X L,MU H,et al.A novel extracellular glycosidase activity from Rhodotorula mucilaginosa:Its application potential in wine aroma enhancement.Letters in Applied Microbiology,2015,DOI:10.1111/lam.12527.
[20]ROSA J C,MASUI D C,LEONE F A,et al.Purification and biochemical characterization of a mycelial glucose-and xylose-stimulatedβ-glucosidase from the thermophilic fungus Humicola insolens[J].Process Biochemistry,2010,45(2):272-278.
[21]PHIMCHANOK N,DIETMAR H.Purification and characterisation of an intracellular enzyme with betaglucosidase and beta-galactosidase activity from the thermophilic fungus Talaromyces thermophilus CBS236.58[J].Journal of Biotechnology,2006,123(3):304-313.
[22]DONG M,FAN M,ZHANG Z,et al.Purification and characterization ofβ-glucosidase from Oenococcus oeni 31MBR[J].European Food Research&Technology,2014,239(6):995-1001.
[23]GUO Y,YAN Q,YANG Y,et al.Expression and characterization of a novelβ-glucosidase,with transglycosylation and exo-β-1,3-glucanase activities,from Rhizomucor miehei[J].Food Chemistry,2015,175(2):431-438.
[24]GAO L,GAO F,ZHANG D,et al.Purification and characterization of a newβ-glucosidase from Penicillium piceum and its application in enzymatic degradation of delignified cornstover[J].Bioresour Technol,2013,147(2):658-661.
[2]GONZLEZ P,FARIA L,CARRAU F,et al.Anovel extracellularβ-glucosidase from Issatchenkia terricola:Isolation,immobilization and application for aroma enhancement of white Muscat wine[J].Process Biochemistry,2011,46(1):385-389.
[3]MAICAS S,MATEO J J.Hydrolysis of terpenyl glycosides in grape juice and other fruit juices:a review[J].Applied Microbiology and Biotechnology,2005,67(3):322-335.
[4]CAPALDO A,WALKER M E,FORD C M,et al Beta-glucoside metabolism in Oenococcus oeni:Cloning and characterisation of the phospho-betaglucosidase bgID[J].Food Chemistry,2011,125(2):476-482.
[5]MICHLMAYR H,MANN C S,SILVA N M,et al Isolation and basic characterization of aβ-glucosidase from a strain of Lactobacillus brevis isolated from a malolactic starter culture[J].Journal of Applied Microbiology,2010,108(2):550-559.
[6]LI Y H,FAN M T,RAN J J,et al.Influence of abiotic stress onβ-D-glucosidase activity of two typical Oenococcus oeni strains in China[J].African Journal of Microbiology Research,2012,6(37):6685-6692.
[7]VILLENA M A,IRANZO J F,OTERO R,et al Optimization of a rapid method for studying the cellular location ofβ-glycosidase activity in wine yeasts[J].Journal of Applied Microbiology,2005,99(6):558-564.
[8]BAFFI M A,TOBAL T,LAGO J H,et al.Wine aroma improvement using aβ-lucosidase preparation from Aureobasidium pullulans[J].Applied Biochemistry&Biotechnology,2013,69(2):493-501.
[9]PAULA GONZLEZ-POMBO,LAURA FARINA,FRANCISCO CARRAU,et al.A novel extracellularβ-glucosidase from Issatchenkia terricola:Isolation,immobilization and application for aroma enhancement of white Muscat wine[J].Process Biochemistry,2011,46(1):385-389.
[10]陈今朝,王剑锋,王慧超,等.热稳定酸性β-葡萄糖苷酶的分离纯化及其酶学性质[J].食品科学,2012,33(11):205-209.
[11]祝霞,盛文军,杨学山,等.红佳酿酵母β-葡萄糖苷酶的分离纯化及酶学性质[J].食品科学,2014,35(13):147-150.
[12]MOREIRA N,MENDES F,GUEDES DE P P,et al.Heavy sulphur compounds,higher alcohols and esters production profile of Hanseniaspora uvarum and Hanseniaspora guilliermondii grown as pure and mixed cultures in grape must[J].International Journal of Food Microbiology,2008,124(3):231-238.
[13]YOUNG A H,DONG H P.Role of non-Saccharomyces yeasts in Korean wines produced from Campbell Early grapes:Potential use of Hanseniaspora uvarum as a starter culture[J].Food Microbiology,2013,34(1):207-214.
[14]VIANA F,GIL J V,GENOV魪S S,et al.Rational selection of non-Saccharomyces wine yeasts for mixed starters based on ester formation and enological traits[J].Food Microbiology,2008,25(6):778-785.
[15]MATURANO Y P,RODR魱GUEZ L A,TORO ME,et al.Multi-enzyme production by pure and mixed cultures of Saccharomyces and non-Saccharomyces yeasts during wine fermentation[J].International Journal of Food Microbiology,2012,155(1/2):43-50.
[16]彭传涛.基于野生非酿酒酵母及其糖苷酶调控葡萄酒香气质量的研究[D].杨凌:西北农林科技大学,2014.
[17]MATEO J J,PERIS L,IBANEZ C,et al.Characterization of glycolytic activities from non-Saccharomyces yeasts isolated from Bobal musts[J].Journal of Industrial Microbiology&Biotechnology,2011,38(2):347-354.
[18]陶永胜,牟含,李国,等.野生胶红酵母糖苷酶水解媚丽新酒中香气糖苷研究[J].农业机械学报,2014,45(12):249-254.
[19]HU K,ZHU X L,MU H,et al.A novel extracellular glycosidase activity from Rhodotorula mucilaginosa:Its application potential in wine aroma enhancement.Letters in Applied Microbiology,2015,DOI:10.1111/lam.12527.
[20]ROSA J C,MASUI D C,LEONE F A,et al.Purification and biochemical characterization of a mycelial glucose-and xylose-stimulatedβ-glucosidase from the thermophilic fungus Humicola insolens[J].Process Biochemistry,2010,45(2):272-278.
[21]PHIMCHANOK N,DIETMAR H.Purification and characterisation of an intracellular enzyme with betaglucosidase and beta-galactosidase activity from the thermophilic fungus Talaromyces thermophilus CBS236.58[J].Journal of Biotechnology,2006,123(3):304-313.
[22]DONG M,FAN M,ZHANG Z,et al.Purification and characterization ofβ-glucosidase from Oenococcus oeni 31MBR[J].European Food Research&Technology,2014,239(6):995-1001.
[23]GUO Y,YAN Q,YANG Y,et al.Expression and characterization of a novelβ-glucosidase,with transglycosylation and exo-β-1,3-glucanase activities,from Rhizomucor miehei[J].Food Chemistry,2015,175(2):431-438.
[24]GAO L,GAO F,ZHANG D,et al.Purification and characterization of a newβ-glucosidase from Penicillium piceum and its application in enzymatic degradation of delignified cornstover[J].Bioresour Technol,2013,147(2):658-661.
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