出芽短梗霉中α-淀粉酶基因的克隆表达及生物信息学分析
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摘要
为获知出芽短梗霉(Aureobasidium pullulans)α-淀粉酶基因及其功能,为以后的体外功能验证奠定理论基础,对α-淀粉酶基因的克隆及其结构、表达、性质与功能进行了初步分析,采用反转录PCR结合普通PCR技术成功克隆出芽短梗霉α-淀粉酶基因,并对其进行生物信息学分析。结果表明:克隆的出芽短梗霉α-淀粉酶基因cDNΑ的全长1 782bp,编码区长度1 692bp,编码的蛋白质含有563个氨基酸,分子质量约为63.32kDa,理论等电点(pI)为7.24;预测该蛋白是亲水性蛋白,无跨膜结构域,无信号肽切割位点,在47～453个氨基酸存在淀粉酶结构域。α-淀粉酶的二级结构主要由无规则卷曲组成,其中无规则卷曲占47.42%,α-螺旋占27.35%,延伸链占20.06%,β-转角占4.62%。成功克隆α-淀粉酶基因并在各个时段均有表达,在108h表达量最高。出芽短梗霉普鲁兰多糖的产量随培养时间增加而增加,而粘度随着培养时间增加而降低。
The cloning,structure,expression,character and function ofα-amylase gene from Aureobasidium pullulans are preliminarily analyzed and the bioinformatics of clonedα-amylase gene from A.pullulans by reverse transcription PCR and routine PCR technology was analyzed to learn the structure and function ofα-amylase gene fromA.pullulans and lay a theoretical foundation for its function in vitro.Result:Total length of cDNA of clonedα-amylase gene fromA.pullulans is 1 782 bp with coding region length of 1 692 bp.The encoded protein contains 563 amino acids and the molecular mass and theoretical isoelectric point are about 63.32 kDa and 7.24 separately.The forecast analysis reveals that the protein is a hydrophily protein without transmembrane domain and signal peptide cleavage sites and there exists 47～453 amino acids in the structural domain ofα-amylase.The secondary structure ofα-amylase is composed of random coil mainly and the random coil,α-helix and extended strand andβ-turn account for 47.42%,27.35%,20.06% and 4.62% respectively.The genetic expression analysis shows that the clonedα-amylase gene can express in each time period and the maximum expression is in 108 htime period.The yield and viscosity of pulullan increase and decrease with increase of culture time separately.
The cloning,structure,expression,character and function ofα-amylase gene from Aureobasidium pullulans are preliminarily analyzed and the bioinformatics of clonedα-amylase gene from A.pullulans by reverse transcription PCR and routine PCR technology was analyzed to learn the structure and function ofα-amylase gene fromA.pullulans and lay a theoretical foundation for its function in vitro.Result:Total length of cDNA of clonedα-amylase gene fromA.pullulans is 1 782 bp with coding region length of 1 692 bp.The encoded protein contains 563 amino acids and the molecular mass and theoretical isoelectric point are about 63.32 kDa and 7.24 separately.The forecast analysis reveals that the protein is a hydrophily protein without transmembrane domain and signal peptide cleavage sites and there exists 47～453 amino acids in the structural domain ofα-amylase.The secondary structure ofα-amylase is composed of random coil mainly and the random coil,α-helix and extended strand andβ-turn account for 47.42%,27.35%,20.06% and 4.62% respectively.The genetic expression analysis shows that the clonedα-amylase gene can express in each time period and the maximum expression is in 108 htime period.The yield and viscosity of pulullan increase and decrease with increase of culture time separately.
引文
[1]李尔汉,彭思露,李汉昕,等.解淀粉芽孢杆菌α-淀粉酶基因的克隆与表达[J].生物技术,2014,24(6):13-18.
[2]MATSUBARA T,BEN A Y,ANINDYAWATI T,et al.Degradation of raw starch granules by alphaamylase purified from culture of Aspergillus awamori KT-11.[J].Journal of Biochemistry&Molecular Biology,2004,37(4):422-428.
[3]VUJICIZAGAR A,DIJKSTRA B W.Monoclinic crystal form of Aspergillus niger alpha-amylase in complex with maltose at 1.8angstroms resolution.[J].Acta Crystallographica,2006,62(8):716-721.
[4]库米拉·马吉提,王伟,张晓燕,等.响应面法优化枯草芽孢杆菌发酵产低温淀粉酶的工艺条件[J].新疆农业大学学报,2012,35(6):478-483.
[5]CHAKRABORTY K,BHATTACHARYYA B K,SEN S K.Purification and characterization of a thermostable alpha-amylase from Bacillus stearothermophilus[J].Folia Microbiologica,2000,45(3):207-210.
[6]李松,王正祥.真菌α-淀粉酶的研究进展[J].生物技术通报,2011(3):66-71.
[7]DR K,FT B,CC B.loning,characterization,and expression of two alpha-amylase genes fromAspergillus niger var.awamori[J].Curr Genet,1990,17(3):203-212.
[8]IEFUJI H,CHINO M,KATO M,et al.Rawstarch-digesting and thermostable alpha-amylase from the yeast Cryptococcus sp.S-2:purification,characterization,cloning and sequencing[J].Biochemical Journal,1996,318(3):989-996.
[9]李金霞,蔡恒,路福平,等.地衣芽孢杆菌耐高温α-淀粉酶基因在大肠杆菌中的克隆、表达及其产物的分泌[J].食品与发酵工业,2004,30(3):70-73.
[10]惠秀娟,朱晓东.解淀粉芽孢杆菌α-淀粉酶基因的分子克隆及其在大肠杆菌中表达的初步研究[J].辽宁大学学报(自然科学版),1993(2):69-73.
[11]尹清强,韩彪,郑秋红,等.枯草杆菌的淀粉酶基因在大肠杆菌中的表达[J].畜牧与兽医,2005(6):7-9.
[12]郭建强,李运敏,岳丽丽,等.超耐热酸性α-淀粉酶基因的克隆及其在酵母细胞中的表达[J].生物工程学报,2006,22(2):237-242.
[13]LEATHERS T D.Substrate regulation from Aureobasidiumand specificity of amylases strain NRRL Y-12,974[J].FEBS Lett,1993,10:217-222.
[14]CATLEY B J.Pullulan,a relationship between molecular weight and fine structure[J].FEBS Lett,1970,10(3):190-193.
[15]LINARDI V R,MACHADO K M.Production of amylases by yeasts[J].Canadian Journal of Microbiology,1990,36(11):751.
[16]POLLOCK T J,THORNE L,ARMENTROUT RW.Isolation of New Aureobasidium Strains That Produce High-Molecular-Weight Pullulan with Reduced Pigmentation[J].Applied and Environmental Microbiology,1992,58(3):877-883.
[17]MANITCHOTPISIT P,SKORY C D,LEATHERST D,et al.α-Amylase activity during pullulan production andα-amylase gene analyses of Aureobasidium pullulans[J].Journal of Industrial Microbiology&Biotechnology,2011,38(9):1211-1218.
[18]PRASONGSUK S,BERHOW M A,DUNLAP C A,et al.Pullulan production by tropical isolates of Aureobasidium pullulans[J].Journal of Industrial Microbiology&Biotechnology,2006,34(1):55-61.
[19]WANG D H,NI T F,JU X M,et al.Sodium chloride improves pullulan production by Aureobasidium pullulans but reduces the molecular weight of pullulan[J].Appl Microbiol Biotechnol,2018,102(20):8921-8930.
[20]王晓红,池振明.产淀粉酶海洋酵母菌的筛选及鉴定[J].中国海洋大学学报,2007,37(S2):95-100.
[21]LI H,CHI Z,WANG X,et al.Amylase production by the marine yeast Aureobasidium pullulans N13d[J].Journal of Ocean University of China,2007,6(1):60-65.
[22]LI H,CHI Z,WANG X,et al.Purification and characterization of extracellular amylase from the marine yeast Aureobasidium pullulans N13dand its raw potato starch digestion[J].Enzyme and Microbial Technology,2007,40(5):1006-1012.
[23]李海峰.海洋酵母普鲁兰类酵母一种新型生淀粉酶生产和特性研究[D].青岛:中国海洋大学,2007.
[24]LI H,CHI Z,DUAN X,et al.Glucoamylase production by the marine yeast Aureobasidium pullulans N13dand hydrolysis of potato starch granules by the enzyme[J].Journal of ocean university of China,2007,42(3):462-465.
[25]MULAY Y R,DEOPURKAR R L.Purification,Characterization of Amylase from Indigenously Isolated Aureobasidium pullulans Cau 19and Its Bioconjugates with Gold Nanoparticles[J].Applied Biochemistry and Biotechnology,2018,184(2):644-658.
[26]LI B,ZHANG N,PENG Q,et al.Production of pigment-free pullulan by swollen cell in Aureobasidium pullulans NG which cell differentiation was affected by pH and nutrition[J].Applied Microbiology and Biotechnology,2009,84(2):293-300.
[27]孔维甲,徐琼,靳建忠,等.环境pH诱导出芽短梗霉细胞多形化[J].微生物学杂志,2011,31(2):7-12.
[28]赵乐.出芽短梗霉(UVMU3-1)漆酶性物质性质分析及其降解氯酚的研究[D].沈阳:沈阳农业大学,2017.
[29]KUMAR S,GADAGKAR S R.Efficiency of the Neighbor-Joining Method in Reconstructing Deep and Shallow Evolutionary Relationships in Large Phylogenies[J].Journal of Molecular Evolution,2000,51(6):544-553.
[30]陈丽静,颜楠楠,尹元元,等.岷江百合LiGPAT基因的克隆及生物信息学分析[J].分子植物育种,2016,14(11):2984-2993.
[31]NIELSEN H,ENGELBRECHT J,BRUNAK S,et al.Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites[J].Protein Eng,1997,10(1):1-6.
[32]苏顺宗,吴锋锴,刘丹,等.一个玉米Pht1家族磷转运蛋白基因克隆和功能分析[J].核农学报,2013(7):885-894.
[33]SONNHAMMER E L,von HEIJNE G,KROGH A.A hidden Markov model for predicting transmembrane helices in protein sequences[J].Proc Int Conf Intell Syst Mol Biol,1998,6:175-182.
[34]安超,马赛箭,薛文娇,等.无机氮源对出芽短梗霉发酵普鲁兰多糖分子量的影响[J].现代食品科技,2017(5):213-219.
[35]李春季.掷孢酵母类胡萝卜素合成关键酶基因的克隆表达与功能分析[D].沈阳:沈阳农业大学,2016.
[36]LIU G,WANG D,WANG L,et al.Mig1is involved in mycelial formation and expression of the genes encoding extracellular enzymes in Saccharomycopsis fibuligera A11[J].Fungal Genetics and Biology,2011,48(9):904-913.
[37]戴凌燕,唐呈瑞,殷奎德,等.甜高粱SUT1基因克隆表达及生物信息学分析[J].核农学报,2015,12(29):2276-2286.
[38]滕利荣,洪水声,孟庆繁,等.普鲁兰多糖的粘度性质研究[J].食品科学,2003(10):32-35.
[39]惠秀娟.解淀粉芽孢杆菌α-淀粉酶基因的分子克隆及其在大肠杆菌中表达的初步研究[J].辽宁大学学报(自然科学版),1993,20(2):69-72.
[40]尹清强,韩彪,郑秋红,等.枯草杆菌的淀粉酶基因在大肠杆菌中的表达[J].畜牧与兽医,2005,37(6):7-9.
[41]蔡恒,陈忠军,路福平,等.地衣芽孢杆菌耐高温α-淀粉酶基因在大肠杆菌中的克隆及表达[J].食品与发酵工业,2004,30(12):15-18.
[42]LIU N,CHI Z,LIU G,et al.α-Amylase,glucoamylase and isopullulanase determine molecular weight of pullulan produced by Aureobasidium melanogenum P16[J].International Journal of Biological Macromolecules,2018,117:727-734.
[2]MATSUBARA T,BEN A Y,ANINDYAWATI T,et al.Degradation of raw starch granules by alphaamylase purified from culture of Aspergillus awamori KT-11.[J].Journal of Biochemistry&Molecular Biology,2004,37(4):422-428.
[3]VUJICIZAGAR A,DIJKSTRA B W.Monoclinic crystal form of Aspergillus niger alpha-amylase in complex with maltose at 1.8angstroms resolution.[J].Acta Crystallographica,2006,62(8):716-721.
[4]库米拉·马吉提,王伟,张晓燕,等.响应面法优化枯草芽孢杆菌发酵产低温淀粉酶的工艺条件[J].新疆农业大学学报,2012,35(6):478-483.
[5]CHAKRABORTY K,BHATTACHARYYA B K,SEN S K.Purification and characterization of a thermostable alpha-amylase from Bacillus stearothermophilus[J].Folia Microbiologica,2000,45(3):207-210.
[6]李松,王正祥.真菌α-淀粉酶的研究进展[J].生物技术通报,2011(3):66-71.
[7]DR K,FT B,CC B.loning,characterization,and expression of two alpha-amylase genes fromAspergillus niger var.awamori[J].Curr Genet,1990,17(3):203-212.
[8]IEFUJI H,CHINO M,KATO M,et al.Rawstarch-digesting and thermostable alpha-amylase from the yeast Cryptococcus sp.S-2:purification,characterization,cloning and sequencing[J].Biochemical Journal,1996,318(3):989-996.
[9]李金霞,蔡恒,路福平,等.地衣芽孢杆菌耐高温α-淀粉酶基因在大肠杆菌中的克隆、表达及其产物的分泌[J].食品与发酵工业,2004,30(3):70-73.
[10]惠秀娟,朱晓东.解淀粉芽孢杆菌α-淀粉酶基因的分子克隆及其在大肠杆菌中表达的初步研究[J].辽宁大学学报(自然科学版),1993(2):69-73.
[11]尹清强,韩彪,郑秋红,等.枯草杆菌的淀粉酶基因在大肠杆菌中的表达[J].畜牧与兽医,2005(6):7-9.
[12]郭建强,李运敏,岳丽丽,等.超耐热酸性α-淀粉酶基因的克隆及其在酵母细胞中的表达[J].生物工程学报,2006,22(2):237-242.
[13]LEATHERS T D.Substrate regulation from Aureobasidiumand specificity of amylases strain NRRL Y-12,974[J].FEBS Lett,1993,10:217-222.
[14]CATLEY B J.Pullulan,a relationship between molecular weight and fine structure[J].FEBS Lett,1970,10(3):190-193.
[15]LINARDI V R,MACHADO K M.Production of amylases by yeasts[J].Canadian Journal of Microbiology,1990,36(11):751.
[16]POLLOCK T J,THORNE L,ARMENTROUT RW.Isolation of New Aureobasidium Strains That Produce High-Molecular-Weight Pullulan with Reduced Pigmentation[J].Applied and Environmental Microbiology,1992,58(3):877-883.
[17]MANITCHOTPISIT P,SKORY C D,LEATHERST D,et al.α-Amylase activity during pullulan production andα-amylase gene analyses of Aureobasidium pullulans[J].Journal of Industrial Microbiology&Biotechnology,2011,38(9):1211-1218.
[18]PRASONGSUK S,BERHOW M A,DUNLAP C A,et al.Pullulan production by tropical isolates of Aureobasidium pullulans[J].Journal of Industrial Microbiology&Biotechnology,2006,34(1):55-61.
[19]WANG D H,NI T F,JU X M,et al.Sodium chloride improves pullulan production by Aureobasidium pullulans but reduces the molecular weight of pullulan[J].Appl Microbiol Biotechnol,2018,102(20):8921-8930.
[20]王晓红,池振明.产淀粉酶海洋酵母菌的筛选及鉴定[J].中国海洋大学学报,2007,37(S2):95-100.
[21]LI H,CHI Z,WANG X,et al.Amylase production by the marine yeast Aureobasidium pullulans N13d[J].Journal of Ocean University of China,2007,6(1):60-65.
[22]LI H,CHI Z,WANG X,et al.Purification and characterization of extracellular amylase from the marine yeast Aureobasidium pullulans N13dand its raw potato starch digestion[J].Enzyme and Microbial Technology,2007,40(5):1006-1012.
[23]李海峰.海洋酵母普鲁兰类酵母一种新型生淀粉酶生产和特性研究[D].青岛:中国海洋大学,2007.
[24]LI H,CHI Z,DUAN X,et al.Glucoamylase production by the marine yeast Aureobasidium pullulans N13dand hydrolysis of potato starch granules by the enzyme[J].Journal of ocean university of China,2007,42(3):462-465.
[25]MULAY Y R,DEOPURKAR R L.Purification,Characterization of Amylase from Indigenously Isolated Aureobasidium pullulans Cau 19and Its Bioconjugates with Gold Nanoparticles[J].Applied Biochemistry and Biotechnology,2018,184(2):644-658.
[26]LI B,ZHANG N,PENG Q,et al.Production of pigment-free pullulan by swollen cell in Aureobasidium pullulans NG which cell differentiation was affected by pH and nutrition[J].Applied Microbiology and Biotechnology,2009,84(2):293-300.
[27]孔维甲,徐琼,靳建忠,等.环境pH诱导出芽短梗霉细胞多形化[J].微生物学杂志,2011,31(2):7-12.
[28]赵乐.出芽短梗霉(UVMU3-1)漆酶性物质性质分析及其降解氯酚的研究[D].沈阳:沈阳农业大学,2017.
[29]KUMAR S,GADAGKAR S R.Efficiency of the Neighbor-Joining Method in Reconstructing Deep and Shallow Evolutionary Relationships in Large Phylogenies[J].Journal of Molecular Evolution,2000,51(6):544-553.
[30]陈丽静,颜楠楠,尹元元,等.岷江百合LiGPAT基因的克隆及生物信息学分析[J].分子植物育种,2016,14(11):2984-2993.
[31]NIELSEN H,ENGELBRECHT J,BRUNAK S,et al.Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites[J].Protein Eng,1997,10(1):1-6.
[32]苏顺宗,吴锋锴,刘丹,等.一个玉米Pht1家族磷转运蛋白基因克隆和功能分析[J].核农学报,2013(7):885-894.
[33]SONNHAMMER E L,von HEIJNE G,KROGH A.A hidden Markov model for predicting transmembrane helices in protein sequences[J].Proc Int Conf Intell Syst Mol Biol,1998,6:175-182.
[34]安超,马赛箭,薛文娇,等.无机氮源对出芽短梗霉发酵普鲁兰多糖分子量的影响[J].现代食品科技,2017(5):213-219.
[35]李春季.掷孢酵母类胡萝卜素合成关键酶基因的克隆表达与功能分析[D].沈阳:沈阳农业大学,2016.
[36]LIU G,WANG D,WANG L,et al.Mig1is involved in mycelial formation and expression of the genes encoding extracellular enzymes in Saccharomycopsis fibuligera A11[J].Fungal Genetics and Biology,2011,48(9):904-913.
[37]戴凌燕,唐呈瑞,殷奎德,等.甜高粱SUT1基因克隆表达及生物信息学分析[J].核农学报,2015,12(29):2276-2286.
[38]滕利荣,洪水声,孟庆繁,等.普鲁兰多糖的粘度性质研究[J].食品科学,2003(10):32-35.
[39]惠秀娟.解淀粉芽孢杆菌α-淀粉酶基因的分子克隆及其在大肠杆菌中表达的初步研究[J].辽宁大学学报(自然科学版),1993,20(2):69-72.
[40]尹清强,韩彪,郑秋红,等.枯草杆菌的淀粉酶基因在大肠杆菌中的表达[J].畜牧与兽医,2005,37(6):7-9.
[41]蔡恒,陈忠军,路福平,等.地衣芽孢杆菌耐高温α-淀粉酶基因在大肠杆菌中的克隆及表达[J].食品与发酵工业,2004,30(12):15-18.
[42]LIU N,CHI Z,LIU G,et al.α-Amylase,glucoamylase and isopullulanase determine molecular weight of pullulan produced by Aureobasidium melanogenum P16[J].International Journal of Biological Macromolecules,2018,117:727-734.
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