新型耐热耐酸普鲁兰酶的分离纯化与酶学特性分析
|
摘要
从泡盛曲霉(Aspergillus awamori) XF发酵液中分离纯化普鲁兰酶(PulXF)并研究其酶学性质。通过硫酸铵沉淀、阴离子交换层析、凝胶过滤层析和疏水层析,纯化得到一种电泳纯的PulXF。纯化倍数8.16倍,比活力为309.28 U/mg。SDS-PAGE检测得单条带,表明PulXF为单亚基蛋白,分子量63.7 kDa。在50-80℃,pH4.5~8.0范围内,酶能保持高活性,最适反应温度60℃,最适pH5.0。在较广的pH范围内(pH3.0~8.0),28℃下,经过24 h酶活能保持80%以上活性。K_m值和V_(max)分别为0.92 mg/mL和11.90μmol/min。酶活测定及TLC分析显示PulXF对普鲁兰多糖有强水解活性,终产物麦芽三糖;对支链淀粉和可溶性淀粉有较弱活性,对直链淀粉、α-环糊精、β-环糊精和糖原无活性。表明从泡盛曲霉XF分离的PulXF属于Ⅰ型普鲁兰酶。Ca~(2+)、Zn~(2+)和Mg~(2+)能够促进酶的活性,其中Ca~(2+)激活作用最强。而PulXF在5%浓度的SDS、CTAB、Tween 80和30%的乙醇中保持高稳定性。基于PulXF在苛刻环境下的高稳定性及高活性,很有希望在淀粉加工、食品饮料等领域以及需要在高温、高酸环境下使用的生物技术工业中使用。
This study reports on the purification and characterization of the pullulanase from a newly isolated Aspergillus awamori XF( PulXF). The enzyme was purified to apparent electrophoretic homogeneity through ammonium sulphate precipitation,anion exchange chromatography,gel chromatography and hydrophobicity chromatography. The crude enzyme was purified 8.16 times with 309.28 U/mg specific activity. SDS-PAGE revealed that the enzyme is monomeric,with a molecular weight of 63.7 kDa.PulXF was highly active in the temperature range of 50 ~80 ℃,with an optimum activity at 60 ℃. It was highly active in the pH range of 4.5~8.0,with optimum pH at 5.0,and retained more than 80% of its original activity in a broad pH range of 3~8 for 24 h at 28 ℃.The enzyme displayed Kmand Vmaxvalues,for pullulan of 0.92 mg/m L and 11.90 μmol/min,respectively,demonstrate its greater affinity.PulXF hydrolyzed pullulan,producing maltotriose,and low hydrolytic activities were also detected with amylopectin and soluable starch,but almost not with amylose,α-cyclodextrine,β-cyclodextrine and glycogen.This proved that the purified PulXF from Aspergillus awamori XF was classified under pullulanase type I.The activity of PulXF could be promoted by Ca~(2+),Zn~(2+)and Mg~(2+). Among them,Ca~(2+)had the most significant effect. The enzyme had a tolerance for surfactants,which was relatively stable in the presence of 5% SDS,5% CTAB,5% Tween 80 and 30% ethanol.The excellent physical and chemical properties make PulXF suitable in the fields of starch processing,food and beverage and biotechnological industries that require high activity at high temperature and acid conditions.
This study reports on the purification and characterization of the pullulanase from a newly isolated Aspergillus awamori XF( PulXF). The enzyme was purified to apparent electrophoretic homogeneity through ammonium sulphate precipitation,anion exchange chromatography,gel chromatography and hydrophobicity chromatography. The crude enzyme was purified 8.16 times with 309.28 U/mg specific activity. SDS-PAGE revealed that the enzyme is monomeric,with a molecular weight of 63.7 kDa.PulXF was highly active in the temperature range of 50 ~80 ℃,with an optimum activity at 60 ℃. It was highly active in the pH range of 4.5~8.0,with optimum pH at 5.0,and retained more than 80% of its original activity in a broad pH range of 3~8 for 24 h at 28 ℃.The enzyme displayed Kmand Vmaxvalues,for pullulan of 0.92 mg/m L and 11.90 μmol/min,respectively,demonstrate its greater affinity.PulXF hydrolyzed pullulan,producing maltotriose,and low hydrolytic activities were also detected with amylopectin and soluable starch,but almost not with amylose,α-cyclodextrine,β-cyclodextrine and glycogen.This proved that the purified PulXF from Aspergillus awamori XF was classified under pullulanase type I.The activity of PulXF could be promoted by Ca~(2+),Zn~(2+)and Mg~(2+). Among them,Ca~(2+)had the most significant effect. The enzyme had a tolerance for surfactants,which was relatively stable in the presence of 5% SDS,5% CTAB,5% Tween 80 and 30% ethanol.The excellent physical and chemical properties make PulXF suitable in the fields of starch processing,food and beverage and biotechnological industries that require high activity at high temperature and acid conditions.
引文
[1]Roy A,Messaoud E B,Bejar S.Isolation and purification of an acidic pullulanase type II from newly isolated Bacillus sp.US149[J].2003,33(5):720-724.
[2]Roy I,Gupta M N.Purification of a bacterial pullulanase on a fluidized bed of calcium alginate beads[J].J Chromatogr A,2002,950(1-2):131-137.
[3]Singh R S,Saini G K,Kennedy J F.Covalent immobilization and thermodynamic characterization of pullulanase for the hydrolysis of pullulan in batch system[J].Carbohydrate Polymers,2010,81(2):252-259.
[4]Singh R S,Saini G K,Kennedy J F.Maltotriose syrup preparation from pullulan using pullulanase[J].Carbohydrate Polymers,2010,80(2):401-407.
[5]Li X F J W Y.Preparation of low digestible and viscoelastic tigernut(Cyperus esculentus)starch by Bacillus acidopullulyticus pullulanase[J].International Journal of Biological Macromolecules,2017(102):651-657.
[6]肖亚朋,沈微,李婷霖,等.嗜热脂肪土芽孢杆菌普鲁兰酶基因的异源表达及重组酶性质[J].食品与发酵工业,2017(5):30-36.
[7]高涛,王芳,别小妹,等.产耐热普鲁兰酶菌株的分离、筛选与鉴定[J].食品科学,2017(8):117-121.
[8]Kunamneni A,Singh S.Improved high thermal stability of pullulanase from a newly isolated thermophilic Bacillus sp.AN-7[J].Enzyme and Microbial Technology,2006,39(7):1399-1404.
[9]Li S F,Xu J Y,Bao Y J,et al.Structure and sequence analysis-based engineering of pullulanase from Anoxybacillus sp.LM18-11for improved thermostability[J].J Biotechnol,2015,210:8-14.
[10]Kang J,Park K M,Choi K H,et al.Molecular cloning and biochemical characterization of a heat-stable type I pullulanase from Thermotoga neapolitana[J].Enzyme Microb Technol,2011,48(3):260-266.
[11]Liu J,Liu Y,Yan Q,et al.Gene cloning,functional expression and characterisation of a novel type I pullulanase from Paenibacillus barengoltzii and its application in resistant starch production[J].2016,121:22-30.
[12]Wu H,Yu X,Chen L,et al.Cloning,overexpression and characterization of a thermostable pullulanase from Thermus thermophilus HB27[J].Protein Expression and Purification,2014,95:22-27.
[13]Lu Z,Hu X,Shen P,et al.A pH-stable,detergent and chelator resistant type I pullulanase from Bacillus pseudofirmus703 with high catalytic efficiency[J].Int J Biol Macromol,2018,109:1302-1310.
[14]文留坤,缪福俊,王云松,等.一株塔宾曲霉的分离鉴定及其在地沟油鉴别中的应用[J].食品工业科技,2018(01):122-126.
[15]Zou C,Duan X,Wu J.Enhanced extracellular production of recombinant Bacillus deramificans pullulanase in Escherichia coli through induction mode optimization and a glycine feeding strategy[J].Bioresource Technology,2014,172:174-179.
[16]朱梦,孙海彦,彭明.普鲁兰酶产生菌的筛选鉴定与发酵条件的研究[J].食品研究与开发,2011(7):136-140.
[17]Bradford M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J].Analytical Biochemistry,1976,72(1):248-254.
[18]Laemmli U K.Cleavage of structural proteins during the assembly of the head of bacteriophage T4[J].Nature,1970,227(5259):680-685.
[19]刘程惠,胡文忠,王艳颖,等.鲜切生菜致腐霉菌的分离鉴定[J].食品工业科技,2016,37(3):135-138.
[20]Elleuche S,Qoura F M,Lorenz U,et al.Cloning,expression and characterization of the recombinant cold-active type-Ipullulanase from Shewanella arctica[J].Journal of Molecular Catalysis B:Enzymatic,2015,116:70-77.
[21]严伟,聂尧,徐岩.长野芽胞杆菌(Bacillus naganoensis)普鲁兰酶在大肠杆菌中的活性表达与分泌调控[J].微生物学报,2013(2):145-153.
[22]Kim C H,Nashiru O,Ko J H.Purification and biochemical characterization of pullulanase type I from Thermus caldophilus GK-24[J].FEMS Microbiol Lett,1996,138(2-3):147-152.
[23]Lee Y,Seo S,Yoon S,et al.Identification of a novel alkaline amylopullulanase from a gut metagenome of Hermetia illucens[J].International journal of biological macromolecules,2016,82:514-521.
[24]Wei W,Ma J,Guo S,et al.A type I pullulanase of Bacillus cereus Nws-bc5 screening from stinky tofu brine:Functional expression in Escherichia coli and Bacillus subtilis and enzyme characterization[J].Process Biochemistry,2014,49(11):1893-1902.
[25]魏涛,迟雷,余轩,等.嗜热细菌莱廷格热袍菌TMO普鲁兰酶TMP在大肠杆菌中的原核表达与酶学性质[J].中国食品学报,2016(8):16-22.
[26]康怀彬,肖天天,李净净,等.嗜冷普鲁兰酶产生菌NX-1的筛选及产酶条件优化[J].食品科学,2015(1):118-123.
[2]Roy I,Gupta M N.Purification of a bacterial pullulanase on a fluidized bed of calcium alginate beads[J].J Chromatogr A,2002,950(1-2):131-137.
[3]Singh R S,Saini G K,Kennedy J F.Covalent immobilization and thermodynamic characterization of pullulanase for the hydrolysis of pullulan in batch system[J].Carbohydrate Polymers,2010,81(2):252-259.
[4]Singh R S,Saini G K,Kennedy J F.Maltotriose syrup preparation from pullulan using pullulanase[J].Carbohydrate Polymers,2010,80(2):401-407.
[5]Li X F J W Y.Preparation of low digestible and viscoelastic tigernut(Cyperus esculentus)starch by Bacillus acidopullulyticus pullulanase[J].International Journal of Biological Macromolecules,2017(102):651-657.
[6]肖亚朋,沈微,李婷霖,等.嗜热脂肪土芽孢杆菌普鲁兰酶基因的异源表达及重组酶性质[J].食品与发酵工业,2017(5):30-36.
[7]高涛,王芳,别小妹,等.产耐热普鲁兰酶菌株的分离、筛选与鉴定[J].食品科学,2017(8):117-121.
[8]Kunamneni A,Singh S.Improved high thermal stability of pullulanase from a newly isolated thermophilic Bacillus sp.AN-7[J].Enzyme and Microbial Technology,2006,39(7):1399-1404.
[9]Li S F,Xu J Y,Bao Y J,et al.Structure and sequence analysis-based engineering of pullulanase from Anoxybacillus sp.LM18-11for improved thermostability[J].J Biotechnol,2015,210:8-14.
[10]Kang J,Park K M,Choi K H,et al.Molecular cloning and biochemical characterization of a heat-stable type I pullulanase from Thermotoga neapolitana[J].Enzyme Microb Technol,2011,48(3):260-266.
[11]Liu J,Liu Y,Yan Q,et al.Gene cloning,functional expression and characterisation of a novel type I pullulanase from Paenibacillus barengoltzii and its application in resistant starch production[J].2016,121:22-30.
[12]Wu H,Yu X,Chen L,et al.Cloning,overexpression and characterization of a thermostable pullulanase from Thermus thermophilus HB27[J].Protein Expression and Purification,2014,95:22-27.
[13]Lu Z,Hu X,Shen P,et al.A pH-stable,detergent and chelator resistant type I pullulanase from Bacillus pseudofirmus703 with high catalytic efficiency[J].Int J Biol Macromol,2018,109:1302-1310.
[14]文留坤,缪福俊,王云松,等.一株塔宾曲霉的分离鉴定及其在地沟油鉴别中的应用[J].食品工业科技,2018(01):122-126.
[15]Zou C,Duan X,Wu J.Enhanced extracellular production of recombinant Bacillus deramificans pullulanase in Escherichia coli through induction mode optimization and a glycine feeding strategy[J].Bioresource Technology,2014,172:174-179.
[16]朱梦,孙海彦,彭明.普鲁兰酶产生菌的筛选鉴定与发酵条件的研究[J].食品研究与开发,2011(7):136-140.
[17]Bradford M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J].Analytical Biochemistry,1976,72(1):248-254.
[18]Laemmli U K.Cleavage of structural proteins during the assembly of the head of bacteriophage T4[J].Nature,1970,227(5259):680-685.
[19]刘程惠,胡文忠,王艳颖,等.鲜切生菜致腐霉菌的分离鉴定[J].食品工业科技,2016,37(3):135-138.
[20]Elleuche S,Qoura F M,Lorenz U,et al.Cloning,expression and characterization of the recombinant cold-active type-Ipullulanase from Shewanella arctica[J].Journal of Molecular Catalysis B:Enzymatic,2015,116:70-77.
[21]严伟,聂尧,徐岩.长野芽胞杆菌(Bacillus naganoensis)普鲁兰酶在大肠杆菌中的活性表达与分泌调控[J].微生物学报,2013(2):145-153.
[22]Kim C H,Nashiru O,Ko J H.Purification and biochemical characterization of pullulanase type I from Thermus caldophilus GK-24[J].FEMS Microbiol Lett,1996,138(2-3):147-152.
[23]Lee Y,Seo S,Yoon S,et al.Identification of a novel alkaline amylopullulanase from a gut metagenome of Hermetia illucens[J].International journal of biological macromolecules,2016,82:514-521.
[24]Wei W,Ma J,Guo S,et al.A type I pullulanase of Bacillus cereus Nws-bc5 screening from stinky tofu brine:Functional expression in Escherichia coli and Bacillus subtilis and enzyme characterization[J].Process Biochemistry,2014,49(11):1893-1902.
[25]魏涛,迟雷,余轩,等.嗜热细菌莱廷格热袍菌TMO普鲁兰酶TMP在大肠杆菌中的原核表达与酶学性质[J].中国食品学报,2016(8):16-22.
[26]康怀彬,肖天天,李净净,等.嗜冷普鲁兰酶产生菌NX-1的筛选及产酶条件优化[J].食品科学,2015(1):118-123.