偏肿革裥菌锰过氧化物酶酶学性质和染料脱色
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摘要
【目的】研究白腐菌偏肿革裥菌(Lenzites gibbosa)所产锰过氧化物酶(MnP)纯酶的序列特征、酶学性质及其对染料的脱色能力,为其应用于木质素降解、染料脱色奠定基础。【方法】将纯化后的样品经SDS-PAGE检测后获得的唯一L.gibbosa MnP条带切割,采用Nano液相色谱-电喷雾串联质谱法联用(Nano LC-ESI-MS/MS)多肽测序技术对蛋白氨基酸序列进行测定。利用该SDS-PAGE图谱,建立蛋白分子量与迁移率的标准直线,从标准直线上求出MnP纯酶样品的表观分子量;参照酶活测定方法检测最适反应温度及温度稳定性、最适反应p H值及p H稳定性;采用lineweaver-Burk双倒数作图法求解米氏动力学常数Km值。研究L.gibbosa菌种培养液和MnP纯酶液对蒽醌类的茜素红、偶氮类的刚果红、杂环类的中性红和三苯基甲烷类的结晶紫等4种不同类型染料的脱色能力。【结果】串联质谱扫描出2条MnP的保守序列肽段,与克隆到的基因Lg-mnp1编码的Lg-MnP1(Gen Bank登录号:ACO92620)序列完全吻合,表明其所代表的酶蛋白Lg-MnP1就是唯一电泳带中主要的蛋白。Lg-MnP1的表观分子量为45.39 k Da,最适反应温度为35℃,在低于40℃的条件下都具有一定的催化能力,但温度超过50℃后迅速失活;其最适反应pH值为3.5,p H超过4.5反应程度就迅速下降,在p H为2~3时最稳定;在以2,6-DMP为底物时,在21℃条件下其米氏常数Km为6.124 mmol·L~(-1)。L.gibbosa培养液对染料在1 h的脱色率分别为茜素红100%、中性红22.17%、刚果红19.09%,对结晶紫的脱色率很低,在36 h脱色率仅为0.018%。纯化后的Lg-MnP1溶液对染料的最大脱色率在2 h分别为中性红100%、刚果红95.55%、茜素红75.85%和结晶紫36.57%。【结论】SDS-PAGE得到的唯一一条电泳带中的MnP是Lg-MnP1。Lg-MnP1是一种中温性、偏酸性的酶,与2,6-DMP的亲和力较大。L.gibbosa含有菌丝的培养液对茜素红具有彻底的降解效果,但Lg-MnP1纯酶液对中性红、刚果红和结晶紫的降解效率要远高于同时含有菌丝、漆酶和MnPs的培养液。
【Objective】In order to get a clear picture of the sequence signature,characteristics,and decolorizing ability to four types of dyes of the purified manganese peroxidase (MnP) produced by white-rot basidiomycete Lenzites gibbosa strain CB-1.【Method】A single MnP protein band detected in the SDS-PAGE electrophoresis of the purified sample was cut and sequenced by Nano LC-ESI-MS/MS peptide sequencing technology.The characteristics of the purified MnP were studied,the apparent molecular weight of the purified MnP was calculated by the standard curve of protein molecular weight and mobility according to SDA-PAGE pattern,the optimal reaction temperature and thermal stability,the optimal reaction p H and p H stability were determined by enzymatic activity measuringmethod,Michaelis-constant Kmwas solved by lineweaver-Burk double bottom figure.The decolorization ability of culture fluid and purified MnP of L.gibbosa to four types s of dyes,namely,alizarin red,neutral red,congo red,and crystal violet were studied,respectively.【Result】Two conserved amino acid sequences of MnP scanned by Tandem MS perfectly match with Lg-MnP1 (Gen Bank Accession No.ACO92620) sequence,indicating that Lg-MnP1 represented by two conserved peptides is the main protein in the only electrophoresis band.The apparent molecular weight of LgMnP1 is 45.39 kDa.Its optimal reaction temperature is about 35 ℃,it has some catalytic ability and stability during 40 ℃ butrapidly becomes inactivated beyond 50 ℃.Its optimal reaction pH value is 3.5,its catakytic activity rapidly decreases when pH value is above 4.5,and it is the most stable in pH 2-3 when it is kept for more than 10 hours.Its Michaelis-constant Kmis 6.124 mmol·L~(-1) at 21 ℃ with 2,6-DMP as substrate.The decolorization percent of culture fluid is 100% to alizarin,22.17% to neutral red,and 19.09% to congo red at 1 h,respectively,but low to crystal violet and only 0.018% at 36 h,whereas that of the purified Lg-MnP1 solution at 2 h is 100% to neutral red,95.55% to congo red,75.85% to alizarin,and 36.57% to crystal violet.【Conclusion】The MnP in the only band of SDS-PAGE is Lg-MnP1.Lg-MnP1 is a mesophile and partial acidic enzyme and with higher affinity to 2,6-DMP.The culture fluid with mycylia of L.gibbosa has a thoroughly decolorizing ability to anthraquinone dye,while the purified Lg-MnP1 has a higher decolorizing efficiency to neutral red,congo red,and crystal violet than the culture fluid with mycylia,laccases and MnPs.
【Objective】In order to get a clear picture of the sequence signature,characteristics,and decolorizing ability to four types of dyes of the purified manganese peroxidase (MnP) produced by white-rot basidiomycete Lenzites gibbosa strain CB-1.【Method】A single MnP protein band detected in the SDS-PAGE electrophoresis of the purified sample was cut and sequenced by Nano LC-ESI-MS/MS peptide sequencing technology.The characteristics of the purified MnP were studied,the apparent molecular weight of the purified MnP was calculated by the standard curve of protein molecular weight and mobility according to SDA-PAGE pattern,the optimal reaction temperature and thermal stability,the optimal reaction p H and p H stability were determined by enzymatic activity measuringmethod,Michaelis-constant Kmwas solved by lineweaver-Burk double bottom figure.The decolorization ability of culture fluid and purified MnP of L.gibbosa to four types s of dyes,namely,alizarin red,neutral red,congo red,and crystal violet were studied,respectively.【Result】Two conserved amino acid sequences of MnP scanned by Tandem MS perfectly match with Lg-MnP1 (Gen Bank Accession No.ACO92620) sequence,indicating that Lg-MnP1 represented by two conserved peptides is the main protein in the only electrophoresis band.The apparent molecular weight of LgMnP1 is 45.39 kDa.Its optimal reaction temperature is about 35 ℃,it has some catalytic ability and stability during 40 ℃ butrapidly becomes inactivated beyond 50 ℃.Its optimal reaction pH value is 3.5,its catakytic activity rapidly decreases when pH value is above 4.5,and it is the most stable in pH 2-3 when it is kept for more than 10 hours.Its Michaelis-constant Kmis 6.124 mmol·L~(-1) at 21 ℃ with 2,6-DMP as substrate.The decolorization percent of culture fluid is 100% to alizarin,22.17% to neutral red,and 19.09% to congo red at 1 h,respectively,but low to crystal violet and only 0.018% at 36 h,whereas that of the purified Lg-MnP1 solution at 2 h is 100% to neutral red,95.55% to congo red,75.85% to alizarin,and 36.57% to crystal violet.【Conclusion】The MnP in the only band of SDS-PAGE is Lg-MnP1.Lg-MnP1 is a mesophile and partial acidic enzyme and with higher affinity to 2,6-DMP.The culture fluid with mycylia of L.gibbosa has a thoroughly decolorizing ability to anthraquinone dye,while the purified Lg-MnP1 has a higher decolorizing efficiency to neutral red,congo red,and crystal violet than the culture fluid with mycylia,laccases and MnPs.
引文
慕庆峰,赵敏,钱程.2005.血红密孔菌对刚果红脱色的共培养体系优化的研究.中国造纸学报,20(2):95-100.(Mu Q F,Zhao M,Qian C.2005.Optimization of Pycnoporus sanguineus to dye degradation co-culturing system.Transactions of China Pulp and Paper,20(2):95-100.[in Chinese])
闫洪波,池玉杰,吴书景.2014.偏肿草裥苗Lg-mnp1基因克隆及表达研究.安徽农业科学,42(11):3186-3190.(Yan H B,Chi Y J,Wu S J.2014.Cloning of Lg-mnp1 gene from Lenzites gibbosa and heterologous expression.Journal of Anhui Agri Sci,42(11):3186-3190.[in Chinese])
赵丽红.2008.糙皮侧耳(Pleurotus ostteatus)降解棉浆黑液木质素的研究.大连:大连理工大学博士学位论文.(Zhao L H.2008.Study on the degradation of lignin in cotton pulp black liquor by Pleurotus ostreatus.Dalian:Ph D thesis of Dalian University of Technology.[in Chinese])
Bermek H,Yazici H,Oztürk H,et al.2004.Purification and characterization of manganese peroxidase from wood-degrading fungus Trichophyton rubrum LSK-27.Enzyme and Microbial Technology,35(1):87-92.
Cai Y,Wu H,Liao X,et al.2010.Purification and characterization of novel manganese peroxidase from Rhizoctonia sp.SYBC-M3.Biotechnology and Bioprocess Engineering,15(6):1016-1021.
Cheng X,Jia R,Li P,et al.2007.Purification of a new manganese peroxidase of the white-rot fungus Schizophyllum sp.F17,and decolorization of azo dyes by the enzyme.Enzyme and Microbial Technology,41(3):258-264.
Lu L,Zhao M,Zhang B B,et al.2007.Purification and characterization of laccase from Pycnoporus sanguineus and decolorization of an anthraquinone dye by the enzyme.Appl Microbiol Biotechnol,74(6):1232-1239.
PériéF H,Sheng D,Gold M H.1996.Purification and characterization of two manganese peroxidase isozymes from thewhite-rot basidiomycete Dichomitus squalens.Biochimica et Biophysica Acta,1297(2):139-148.
Rajakumar S,Gaskell J,Cullen D,et al.1996.lip-like genes in Phanerochaete sordida and Ceriporiopsis subvermispora,white rot fungi with no detectable lignin peroxidase activity.Applied and environmental microbiology,62(7):2660-2663.
Schneega I,Hofrichter M,Scheibner K,et al.1997.Purification of main manganese peroxidase isoenzyme Mn P2 from the white-rot fungus Nematoloma frowardii b19.Appl Microbiol Biotechnol,48(5):602-605.
Si J,Peng F,Cui B.2013.Purification,biochemical characterization and dye decolorization capacity of analkali-resistant and metaltolerant laccase from Trametes pubescens.Bioresource Technology,128:49-57.
Steffen K T,Hofrichter M,Hatakka A.2002.Purification and characterization of manganese peroxidase from thelitter-decomposing basidiomycetes Agrocybe praecox and Stropharia coronilla.Enzyme and Microbial Technology,30(4):550-555.
闫洪波,池玉杰,吴书景.2014.偏肿草裥苗Lg-mnp1基因克隆及表达研究.安徽农业科学,42(11):3186-3190.(Yan H B,Chi Y J,Wu S J.2014.Cloning of Lg-mnp1 gene from Lenzites gibbosa and heterologous expression.Journal of Anhui Agri Sci,42(11):3186-3190.[in Chinese])
赵丽红.2008.糙皮侧耳(Pleurotus ostteatus)降解棉浆黑液木质素的研究.大连:大连理工大学博士学位论文.(Zhao L H.2008.Study on the degradation of lignin in cotton pulp black liquor by Pleurotus ostreatus.Dalian:Ph D thesis of Dalian University of Technology.[in Chinese])
Bermek H,Yazici H,Oztürk H,et al.2004.Purification and characterization of manganese peroxidase from wood-degrading fungus Trichophyton rubrum LSK-27.Enzyme and Microbial Technology,35(1):87-92.
Cai Y,Wu H,Liao X,et al.2010.Purification and characterization of novel manganese peroxidase from Rhizoctonia sp.SYBC-M3.Biotechnology and Bioprocess Engineering,15(6):1016-1021.
Cheng X,Jia R,Li P,et al.2007.Purification of a new manganese peroxidase of the white-rot fungus Schizophyllum sp.F17,and decolorization of azo dyes by the enzyme.Enzyme and Microbial Technology,41(3):258-264.
Lu L,Zhao M,Zhang B B,et al.2007.Purification and characterization of laccase from Pycnoporus sanguineus and decolorization of an anthraquinone dye by the enzyme.Appl Microbiol Biotechnol,74(6):1232-1239.
PériéF H,Sheng D,Gold M H.1996.Purification and characterization of two manganese peroxidase isozymes from thewhite-rot basidiomycete Dichomitus squalens.Biochimica et Biophysica Acta,1297(2):139-148.
Rajakumar S,Gaskell J,Cullen D,et al.1996.lip-like genes in Phanerochaete sordida and Ceriporiopsis subvermispora,white rot fungi with no detectable lignin peroxidase activity.Applied and environmental microbiology,62(7):2660-2663.
Schneega I,Hofrichter M,Scheibner K,et al.1997.Purification of main manganese peroxidase isoenzyme Mn P2 from the white-rot fungus Nematoloma frowardii b19.Appl Microbiol Biotechnol,48(5):602-605.
Si J,Peng F,Cui B.2013.Purification,biochemical characterization and dye decolorization capacity of analkali-resistant and metaltolerant laccase from Trametes pubescens.Bioresource Technology,128:49-57.
Steffen K T,Hofrichter M,Hatakka A.2002.Purification and characterization of manganese peroxidase from thelitter-decomposing basidiomycetes Agrocybe praecox and Stropharia coronilla.Enzyme and Microbial Technology,30(4):550-555.