发酵乳杆菌多铜氧化酶的异源表达及酶学性质
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摘要
生物胺是一种存在于发酵食品中的含氮小分子有机化合物,过量摄入可能引起过敏或其他不良反应。利用酶法降解是减少发酵食品中生物胺含量从而保障食品安全的有效方法之一。文中成功克隆了来源于发酵乳杆菌的多铜氧化酶基因,在大肠杆菌中表达的酶活水平为484 U/L。通过镍柱亲和层析方法获得了此多铜氧化酶的纯酶。该多铜氧化酶的最适反应温度为50℃,最适反应pH为3.5,其K_m为1.3 mmol/L,V_(max)为7.67×10~(-2) mmol/(L·min)。对酶的应用特性研究表明,来源于发酵乳杆菌的多铜氧化酶对18%(W/F)NaCl有一定的耐受性,并可降解包括色胺、苯乙胺、腐胺、尸胺、组胺、酪胺和亚精胺在内的7种生物胺。其中它对组胺和酪胺的降解能力最高,分别为51.6%和40.9%。此外,该酶对酱油中的生物胺也有普遍降解作用,使用较低酶量(500 U/L)时,对酱油中总胺的降解率达到10.6%。多铜氧化酶具备降解发酵食品中生物胺的潜力,为进一步实现这类食品酶的实际应用奠定基础。
Biogenic amines(BAs)are low molecular weight organic compounds that present in fermented foods.Large amount of ingested biogenic amines can cause allergy or significant symptoms.Reduction of BAs by enzymatic reaction in fermented foods is one of the most efficient methods for removal of biohazard compounds and assurance food safety.In this study,the multicopper oxidase(MCO)gene in the genome of Lactobacillus fermentum was successfully cloned in Escherichia coli BL21 and expressed at 484 U/L.The recombinant MCO was purified by the immobilized metal affinity chromatography method.The optimal reaction temperature and pH for this enzyme was detected to be 50 ℃ and 3.5.The K_m and V_(max) values of the recombinant MCO was determined to be 1.30 mmol/L and 7.67×10~(-2) mmol/(L·min).Moreover,this MCO dramatically degrades histamine and tyramine by 51.6% and 40.9%,and can degrade other BAs including tryptamine,phenylethylamine,putrescine,cadaverine and spermidine,and was found to be tolerant to 18%(W/V)NaCl.The recombinant MCO is also capable of degrading BAs in soy sauce.The degradation rate of total BAs in soy sauce reaches 10.6% though a relatively low level of enzyme(500 U/L)is used.Multicopper oxidase has the potential to degrade biogenic amines in fermented foods,which lays a foundation for the further application of this kind of food enzymes.
Biogenic amines(BAs)are low molecular weight organic compounds that present in fermented foods.Large amount of ingested biogenic amines can cause allergy or significant symptoms.Reduction of BAs by enzymatic reaction in fermented foods is one of the most efficient methods for removal of biohazard compounds and assurance food safety.In this study,the multicopper oxidase(MCO)gene in the genome of Lactobacillus fermentum was successfully cloned in Escherichia coli BL21 and expressed at 484 U/L.The recombinant MCO was purified by the immobilized metal affinity chromatography method.The optimal reaction temperature and pH for this enzyme was detected to be 50 ℃ and 3.5.The K_m and V_(max) values of the recombinant MCO was determined to be 1.30 mmol/L and 7.67×10~(-2) mmol/(L·min).Moreover,this MCO dramatically degrades histamine and tyramine by 51.6% and 40.9%,and can degrade other BAs including tryptamine,phenylethylamine,putrescine,cadaverine and spermidine,and was found to be tolerant to 18%(W/V)NaCl.The recombinant MCO is also capable of degrading BAs in soy sauce.The degradation rate of total BAs in soy sauce reaches 10.6% though a relatively low level of enzyme(500 U/L)is used.Multicopper oxidase has the potential to degrade biogenic amines in fermented foods,which lays a foundation for the further application of this kind of food enzymes.
引文
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[14]Siddiqui JA,Shoeb SM,Takayama S,et al.Purification and characterization of histamine dehydrogenase from Nocardioides simplex IFO 12069.FEMS Microbiol Lett,2000,189(2):183-187.
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[22]Li ZJ.Methods for determination of genetic amines in food and amine-producing bacteria and its application[D].Qingdao:Ocean University of China,2007(in Chinese).李志军.食品中生物胺及其产生菌株检测方法研究[D].青岛:中国海洋大学,2007.
[23]Del Rio B,Redruello B,Linares DM,et al.The dietary biogenic amines tyramine and histamine show synergistic toxicity towards intestinal cells in culture.Food Chem,2017,218:249-255.
[24]Koschorreck K,Richter SM,Ene AB,et al.Cloning and characterization of a new laccase from Bacillus licheniformis catalyzing dimerization of phenolic acids.Appl Microbiol Biotechnol,2008,79(2):217-224.
[25]Yang S,Long Y,Yan H,et al.Gene cloning,identification,and characterization of the multicopper oxidase CumA from Pseudomonas sp.593.Appl Microbiol Biotechnol,2017,64(3):345-355.
[26]Li Y,Yin J,Qu G,et al.Gene cloning,protein purification,and enzymatic properties of multicopper oxidase,from Klebsiella sp.601.Can J Microbiol,2008,54(9):725-733.
[27]Brander S,Mikkelsen JD,Kepp KP.Characterization of an alkali-and halide-resistant laccase expressed in E.coli:CotA from Bacillus clausii.PLoS ONE,2014,9(6):e99402.
[28]Kunamneni A,Plou FJ,Ballesteros A,et al.Laccases and their applications:a patent review.Recent Pat Biotechnol,2008,2(1):10-24.
[29]Ruijssenaars HJ,Hartmans S.A cloned Bacillus halodurans multicopper oxidase exhibiting alkaline laccase activity.Appl Microbiol Biotechnol,2004,65(2):177-182.
[30]Yang L,Zuo WF,Li YD,et al.Cloning of multicopper oxidase gene from Ochrobactrum sp.531 and characterization of its alkaline laccase activity towards phenolic substrates.Adv Biol Chem,2012,2(3):248-255.
[31]Callejón S,Sendra R,Ferrer S,et al.Recombinant laccase from Pediococcus acidilactici CECT 5930 with ability to degrade tyramine.PLoS ONE,2017,12(10):e0186019.
[32]Guarcello R,De Angelis M,Settanni L,et al.Selection of amine-oxidizing dairy lactic acid bacteria and identification of the enzyme and gene involved in the decrease of biogenic amines.Appl Environ Microbiol,2016,82(23):6870-6880.
[33]Yu JZ,Xu F,Xu Y.Analysis on the change rule of biogenic amines during the process of high-salt diluted soy sauce.Food Fermn Ind,2016,42(10):44-49(in Chinese).于金芝,徐峰,徐莹.高盐稀态酱油生产过程中的生物胺变化规律.食品与发酵工业,2016,42(10):44-49.
[2]Feng TT,Fang F,Yang J,et al.Formation and removal of biogenic amines in food bioprocessing.Food Sci,2013,34(19):360-366(in Chinese).冯婷婷,方芳,杨娟,等.食品生物制造过程中生物胺的形成与消除.食品科学,2013,34(19):360-366.
[3]Shah P,Swiatlo E.A multifaceted role for polyamines in bacterial pathogens.Mol Microbiol,2008,68(1):4-16.
[4]Tofalo R,Perpetuini G,Schirone M,et al.Biogenic amines:toxicology and health effect.Enc Food Heal,2016:424-429,doi:10.1016/B978-0-12-384947-2.00071-4.
[5]Kim MK,Mah JH,Hwang HJ.Biogenic amine formation and bacterial contribution in fish,squid and shellfish.Food Chem,2009,116(1):87-95.
[6]Zou Y.Study on formation mechanism and control of biogenic amines during the process of high-salt diluted soy sauce[D].Guangzhou:South China University of Technology,2012(in Chinese).邹阳.高盐稀态酱油生产过程中生物胺的形成机制及发酵调控研究[D].广州:华南理工大学,2012.
[7]Koral S,Tufan B,??avni?ar A,et al.Investigation of the contents of biogenic amines and some food safety parameters of various commercially salted fish products.Food Contr,2013,32(2):597-606.
[8]Yang LK,Fu XJ,Hu YB,et al.Screening of strains with degradation activity for biogenic amines in fish sauce.Food Sci,2012,33(11):158-162(in Chinese).杨利昆,付湘晋,胡叶碧,等.鱼露中生物胺降解菌的筛选及其特性.食品科学,2012,33(11):158-162.
[9]Callejon S,Sendra R,Ferrer S,et al.Recombinant laccase from Pediococcus acidilactici CECT 5930 with ability to degrade tyramine.PLoS ONE,2017,12(10):e0186019.
[10]Callejón S,Sendra R,Ferrer S,et al.Identification of a novel enzymatic activity from lactic acid bacteria able to degrade biogenic amines in wine.Appl Microbiol Biotechnol,2014,98(1):185-198.
[11]Fadda S,Vignolo G,Oliver G.Tyramine degradation and tyramine/histamine production by lactic acid bacteria and Kocuria strains.Biotechnol Lett,2001,23(24):2015-2019.
[12]Dapkevicius MLNE,Nout MJR,Rombouts FM,et al.Biogenic amine formation and degradation by potential fish silage starter microorganisms.Int J Food Microbiol,2000,57(1/2):107-114.
[13]Lee JI,Kim YW.Characterization of amine oxidases from Arthrobacter aurescens and application for determination of biogenic amines.World J Microbiol Biotechnol,2013,29(4):673-682.
[14]Siddiqui JA,Shoeb SM,Takayama S,et al.Purification and characterization of histamine dehydrogenase from Nocardioides simplex IFO 12069.FEMS Microbiol Lett,2000,189(2):183-187.
[15]Sattler J,Hesterberg R,Lorenz W,et al.Inhibition of human and canine diamine oxidase by drugs used in an intensive care unit:relevance for clinical side effects.Agents Actions,1985,16(3/4):91-94.
[16]Hoegger PJ,Kilaru S,James TY,et al.Phylogenetic comparison and classification of laccase and related multicopper oxidase protein sequences.FEBS J,2006,273(10):2308-2326.
[17]Askwith C,Eide D,van Ho A,et al.The FET3 gene of S.cerevisiae encodes a multicopper oxidase required for ferrous iron uptake.Cell,1994,76(2):403-410.
[18]Madhavi V,Lele SS.Laccase:properties and applications.Bioresources,2009,4(4):1694-1717.
[19]Solomon EI,Sundaram UM,Machonkin TE.Multicopper oxidases and oxygenases.Chem Rev,1996,96(7):2563-2606.
[20]Sheng Y,Yan L,Hong Y,et al.Gene cloning,identification,and characterization of the multicopper oxidase CumA from Pseudomonas sp.593.Biotechnol Appl Biochem,2017,64(3):345-355.
[21]Callejón S,Sendra R,Ferrer S,et al.Cloning and characterization of a new laccase from Lactobacillus plantarum J16 CECT 8944 catalyzing biogenic amines degradation.Appl Microbiol Biotechnol,2016,100(7):3113-3124.
[22]Li ZJ.Methods for determination of genetic amines in food and amine-producing bacteria and its application[D].Qingdao:Ocean University of China,2007(in Chinese).李志军.食品中生物胺及其产生菌株检测方法研究[D].青岛:中国海洋大学,2007.
[23]Del Rio B,Redruello B,Linares DM,et al.The dietary biogenic amines tyramine and histamine show synergistic toxicity towards intestinal cells in culture.Food Chem,2017,218:249-255.
[24]Koschorreck K,Richter SM,Ene AB,et al.Cloning and characterization of a new laccase from Bacillus licheniformis catalyzing dimerization of phenolic acids.Appl Microbiol Biotechnol,2008,79(2):217-224.
[25]Yang S,Long Y,Yan H,et al.Gene cloning,identification,and characterization of the multicopper oxidase CumA from Pseudomonas sp.593.Appl Microbiol Biotechnol,2017,64(3):345-355.
[26]Li Y,Yin J,Qu G,et al.Gene cloning,protein purification,and enzymatic properties of multicopper oxidase,from Klebsiella sp.601.Can J Microbiol,2008,54(9):725-733.
[27]Brander S,Mikkelsen JD,Kepp KP.Characterization of an alkali-and halide-resistant laccase expressed in E.coli:CotA from Bacillus clausii.PLoS ONE,2014,9(6):e99402.
[28]Kunamneni A,Plou FJ,Ballesteros A,et al.Laccases and their applications:a patent review.Recent Pat Biotechnol,2008,2(1):10-24.
[29]Ruijssenaars HJ,Hartmans S.A cloned Bacillus halodurans multicopper oxidase exhibiting alkaline laccase activity.Appl Microbiol Biotechnol,2004,65(2):177-182.
[30]Yang L,Zuo WF,Li YD,et al.Cloning of multicopper oxidase gene from Ochrobactrum sp.531 and characterization of its alkaline laccase activity towards phenolic substrates.Adv Biol Chem,2012,2(3):248-255.
[31]Callejón S,Sendra R,Ferrer S,et al.Recombinant laccase from Pediococcus acidilactici CECT 5930 with ability to degrade tyramine.PLoS ONE,2017,12(10):e0186019.
[32]Guarcello R,De Angelis M,Settanni L,et al.Selection of amine-oxidizing dairy lactic acid bacteria and identification of the enzyme and gene involved in the decrease of biogenic amines.Appl Environ Microbiol,2016,82(23):6870-6880.
[33]Yu JZ,Xu F,Xu Y.Analysis on the change rule of biogenic amines during the process of high-salt diluted soy sauce.Food Fermn Ind,2016,42(10):44-49(in Chinese).于金芝,徐峰,徐莹.高盐稀态酱油生产过程中的生物胺变化规律.食品与发酵工业,2016,42(10):44-49.