黑曲霉内切β-1,4-半乳聚糖酶AghA的分子克隆与特征解析
|
摘要
利用内切β-1,4-半乳聚糖酶(endo-β-1,4-galactanase,EC 3. 2. 1. 89)水解土豆浆制备低聚半乳糖,有望解决产品回收率低的问题。为此,该研究通过分子克隆技术,将黑曲霉β-1,4-半乳聚糖酶基因agh A在毕赤酵母中进行克隆表达,构建获得了重组菌GS115(p PIC-aghA)。摇瓶培养条件下,重组酶Agh A的酶活为130 U/m L,高于大多数已报道的半乳聚糖酶的酶活。酶学性质的研究表明,该酶的最适反应pH值和温度分别为4. 5和45℃,且在pH 4. 0～6. 0或30～50℃具有较好稳定性。大部分金属离子和EDTA对重组酶Agh A的酶活没有显著影响; Fe3+对其活性有强烈的抑制作用;而Hg2+则可使Agh A几乎完全失活。该酶对半乳聚糖的最大反应速率Vmax和米氏常数Km分别为400 mg/(m L·min)和0. 08 mg/m L。此外,重组酶Agh A还可以水解土豆浆,产生半乳二糖、半乳三糖和少量半乳四糖等低聚半乳糖。
High yields of galactooligosaccharides may be obtained by enzymatic hydrolysis of potato pulp using endo-β-1,4-galactanase(EC 3. 2. 1. 89). In present study,the gene aghA encoding endo-β-1,4-galactanase from Aspergillus niger CICIM F0510 was thus cloned and expressed in Pichia pastoris,which generated the recombinant strain GS115(pPIC-aghA). In shaking flask experiments,the activity of recombinant enzyme AghA was 130 U/m L. The optimal pH and temperature of AghA were 4. 5 and 45 ℃,respectively,and it was stable at pH 4. 0-6. 0 or at 30-50 ℃. Most of chemical ions and EDTA had no significant effects on the activity of AghA,while it was strongly inhibited by Fe3 +and nearly completely lost in the presence of Hg2 +. The Vmaxand Kmof AghA towards galactan(potato)were determined to be 400 mg/(m L·min) and 0. 08 mg/m L,respectively. Besides,potato pulp was hydrolyzed by AghA into galactobiose,galactotrioses and a small amount of galactotetraose. The results obtained paved the way for large scale production of galactanase AghA and its applications in preparing galactooligosaccharides from potato pulp.
High yields of galactooligosaccharides may be obtained by enzymatic hydrolysis of potato pulp using endo-β-1,4-galactanase(EC 3. 2. 1. 89). In present study,the gene aghA encoding endo-β-1,4-galactanase from Aspergillus niger CICIM F0510 was thus cloned and expressed in Pichia pastoris,which generated the recombinant strain GS115(pPIC-aghA). In shaking flask experiments,the activity of recombinant enzyme AghA was 130 U/m L. The optimal pH and temperature of AghA were 4. 5 and 45 ℃,respectively,and it was stable at pH 4. 0-6. 0 or at 30-50 ℃. Most of chemical ions and EDTA had no significant effects on the activity of AghA,while it was strongly inhibited by Fe3 +and nearly completely lost in the presence of Hg2 +. The Vmaxand Kmof AghA towards galactan(potato)were determined to be 400 mg/(m L·min) and 0. 08 mg/m L,respectively. Besides,potato pulp was hydrolyzed by AghA into galactobiose,galactotrioses and a small amount of galactotetraose. The results obtained paved the way for large scale production of galactanase AghA and its applications in preparing galactooligosaccharides from potato pulp.
引文
[1]卢丽丽,李正义,肖敏.微生物酶法合成低聚半乳糖的新进展[J].微生物学报,2008,48(7):980-985.
[2]孙槐胜.基于乳酸克鲁维酵母菌的高纯度低聚半乳糖绿色生产工艺开发[D].天津:天津大学,2016.
[3] OSMAN A,TZORTZIS G,RASTALL R A,et al. High yield production of a soluble bifidobacterialβ-galactosidase(Bbg IV)in E. coli DH5αwith improved catalytic efficiency for the synthesis of prebiotic galactooligosaccharides[J]. Journal of Agricultural&Food Chemistry,2013,61(9):2 213-2 223.
[4] JOHANNSEN H,PRESCOTT S L. Practical prebiotics,probiotics and synbiotics for allergists:how useful are they?[J]. Clinical and Experimental Allergy,2009,39(12):1 801-1 814.
[5] TORPENHOLT S,DE MARIA L,OLSSON M H M,et al.Effect of mutations on the thermostability of Aspergillus aculeatus beta-1,4-galactanase[J]. Computational and Structural Biotechnology Journal,2015,13:256-264.
[6] LIMA E A D,MACHADO C B,ZANPHORLIN L M,et al. GH53 endo-beta-1,4-galactanase from a newly isolated Bacillus licheniformis CBMAI 1609 as an enzymatic cocktail supplement for biomass saccharification[J]. Applied Biochemistry&Biotechnology,2016,179(3):415-426.
[7] BJOERNVAD M E,CLAUSEN I G,SCH LEIN M,et al.Bacterial galactanases and use thereof:US 2003/0022347A1[P]. 2001-02-06.
[8] LE NOURS J,DE MARIA L,WELNER D,et al. Investigating the binding of beta-1,4-galactan to Bacillus licheniformis beta-1,4-galactanase by crystallography and computational modeling[J]. Proteins-Structure Function and Bioinformatics,2009,75(4):977-989.
[9] ZAVALETA V,EYZAGUIRRE J. Penicillium purpurogenum produces a highly stable endo-beta-(1,4)-galactanase[J]. Applied Biochemistry&Biotechnology,2016,180(7):1 313-1 327.
[10]董自星,李伟国,佟新新,等.黑曲霉内切β-1,3(4)-葡聚糖酶的基因克隆与酶学特性分析[J].食品与发酵工业,2016,42(11):58-64.
[11]诸葛健,王正祥.工业微生物实验技术手册[M].北京:中国轻工业出版社,1994:413-450.
[12]奥斯伯F,金斯顿R,塞德曼J,等.精编分子生物学实验指南[M].北京:科学出版社,2008:42-50.
[13] MICHALAK M,THOMASSEN L V,ROYTIO H,et al.Expression and characterization of an endo-1,4-beta-galactanase from Emericella nidulans in Pichia pastoris for enzymatic design of potentially prebiotic oligosaccharides from potato galactans[J]. Enzyme and Microbial Technology,2012,50(2):121-129.
[14] SAKAMOTO T,ISHIMARU M. Peculiarities and applications of galactanolytic enzymes that act on type I and II arabinogalactans[J]. Applied Microbiology and Biotechnology,2013,97(12):5 201-5 213.
[15] NAKANO H,TAKENISHI S,WATANABE Y. Purification and properties of two galactanases from Penicillium citrinum[J]. Journal of the Agricultural Chemical Society of Japan,2014,49(12):3 445-3 454.
[16] SAKAMOTO T,NISHIMURA Y,MAKINO Y,et al. Biochemical characterization of a GH53 endo-beta-1,4-galactanase and a GH35 exo-beta-1,4-galactanase from Penicillium chrysogenum[J]. Applied Microbiology and Biotechnology,2013,97(7):2 895-2 906.
[17] YANG H,ICHINOSE H,YOSHIDA M,et al. Characterization of a thermostable endo-beta-1,4-D-galactanase from the hyperthermophile Thermotoga maritima[J]. Bioscience Biotechnology and Biochemistry,2006,70(2):538-541.
[18] CHRISTGAU S,SANDAL T,KOFOD L V,et al. Expression cloning,purification and characterization of a beta-1,4-galactanase from Aspergillus aculeatus[J]. Current Genetics,1995,27(2):135-141.
[19] LARSEN D M,NYFFENEGGER C,SWINIARSKA M M,et al. Thermostability enhancement of an endo-1,4-beta-galactanase from Talaromyces stipitatus by site-directed mutagenesis[J]. Applied Microbiology and Biotechnology,2015,99(10):4 245-4 253.
[20] LE NOURS J,RYTTERSGAARD C,LO LEGGIO L,et al. Structure of two fungal beta-1,4-galactanases:Searching for the basis for temperature and p H optimum[J].Protein Science,2003,12(6):1 195-1 204.
[21] THOMASSEN L V,MEYER A S. Statistically designed optimisation of enzyme catalysed starch removal from potato pulp[J]. Enzyme and Microbial Technology,2010,46(3/4):297-303.
[22] BONNIN E,LAHAYE M,VIGOUROUX J,et al. Preliminary characterization of a new exo-beta-(1,4)-galactanase with transferase activity[J]. International Journal of Biological Macromolecules,1995,17(6):345-351.
[23] ZHANG J H,PAKARINEN A,VIIKARI L. Synergy between cellulases and pectinases in the hydrolysis of hemp[J]. Bioresource Technology,2013,129(2):302-307.
[24] RYTTERSGAARD C,LE NOURS J,LO LEGGIO L,et al. The structure of endo-beta-1,4-galactanase from Bacillus licheniformis in complex with two oligosaccharide products[J]. Journal of Molecular Biology,2004,341(1):107-117.
[2]孙槐胜.基于乳酸克鲁维酵母菌的高纯度低聚半乳糖绿色生产工艺开发[D].天津:天津大学,2016.
[3] OSMAN A,TZORTZIS G,RASTALL R A,et al. High yield production of a soluble bifidobacterialβ-galactosidase(Bbg IV)in E. coli DH5αwith improved catalytic efficiency for the synthesis of prebiotic galactooligosaccharides[J]. Journal of Agricultural&Food Chemistry,2013,61(9):2 213-2 223.
[4] JOHANNSEN H,PRESCOTT S L. Practical prebiotics,probiotics and synbiotics for allergists:how useful are they?[J]. Clinical and Experimental Allergy,2009,39(12):1 801-1 814.
[5] TORPENHOLT S,DE MARIA L,OLSSON M H M,et al.Effect of mutations on the thermostability of Aspergillus aculeatus beta-1,4-galactanase[J]. Computational and Structural Biotechnology Journal,2015,13:256-264.
[6] LIMA E A D,MACHADO C B,ZANPHORLIN L M,et al. GH53 endo-beta-1,4-galactanase from a newly isolated Bacillus licheniformis CBMAI 1609 as an enzymatic cocktail supplement for biomass saccharification[J]. Applied Biochemistry&Biotechnology,2016,179(3):415-426.
[7] BJOERNVAD M E,CLAUSEN I G,SCH LEIN M,et al.Bacterial galactanases and use thereof:US 2003/0022347A1[P]. 2001-02-06.
[8] LE NOURS J,DE MARIA L,WELNER D,et al. Investigating the binding of beta-1,4-galactan to Bacillus licheniformis beta-1,4-galactanase by crystallography and computational modeling[J]. Proteins-Structure Function and Bioinformatics,2009,75(4):977-989.
[9] ZAVALETA V,EYZAGUIRRE J. Penicillium purpurogenum produces a highly stable endo-beta-(1,4)-galactanase[J]. Applied Biochemistry&Biotechnology,2016,180(7):1 313-1 327.
[10]董自星,李伟国,佟新新,等.黑曲霉内切β-1,3(4)-葡聚糖酶的基因克隆与酶学特性分析[J].食品与发酵工业,2016,42(11):58-64.
[11]诸葛健,王正祥.工业微生物实验技术手册[M].北京:中国轻工业出版社,1994:413-450.
[12]奥斯伯F,金斯顿R,塞德曼J,等.精编分子生物学实验指南[M].北京:科学出版社,2008:42-50.
[13] MICHALAK M,THOMASSEN L V,ROYTIO H,et al.Expression and characterization of an endo-1,4-beta-galactanase from Emericella nidulans in Pichia pastoris for enzymatic design of potentially prebiotic oligosaccharides from potato galactans[J]. Enzyme and Microbial Technology,2012,50(2):121-129.
[14] SAKAMOTO T,ISHIMARU M. Peculiarities and applications of galactanolytic enzymes that act on type I and II arabinogalactans[J]. Applied Microbiology and Biotechnology,2013,97(12):5 201-5 213.
[15] NAKANO H,TAKENISHI S,WATANABE Y. Purification and properties of two galactanases from Penicillium citrinum[J]. Journal of the Agricultural Chemical Society of Japan,2014,49(12):3 445-3 454.
[16] SAKAMOTO T,NISHIMURA Y,MAKINO Y,et al. Biochemical characterization of a GH53 endo-beta-1,4-galactanase and a GH35 exo-beta-1,4-galactanase from Penicillium chrysogenum[J]. Applied Microbiology and Biotechnology,2013,97(7):2 895-2 906.
[17] YANG H,ICHINOSE H,YOSHIDA M,et al. Characterization of a thermostable endo-beta-1,4-D-galactanase from the hyperthermophile Thermotoga maritima[J]. Bioscience Biotechnology and Biochemistry,2006,70(2):538-541.
[18] CHRISTGAU S,SANDAL T,KOFOD L V,et al. Expression cloning,purification and characterization of a beta-1,4-galactanase from Aspergillus aculeatus[J]. Current Genetics,1995,27(2):135-141.
[19] LARSEN D M,NYFFENEGGER C,SWINIARSKA M M,et al. Thermostability enhancement of an endo-1,4-beta-galactanase from Talaromyces stipitatus by site-directed mutagenesis[J]. Applied Microbiology and Biotechnology,2015,99(10):4 245-4 253.
[20] LE NOURS J,RYTTERSGAARD C,LO LEGGIO L,et al. Structure of two fungal beta-1,4-galactanases:Searching for the basis for temperature and p H optimum[J].Protein Science,2003,12(6):1 195-1 204.
[21] THOMASSEN L V,MEYER A S. Statistically designed optimisation of enzyme catalysed starch removal from potato pulp[J]. Enzyme and Microbial Technology,2010,46(3/4):297-303.
[22] BONNIN E,LAHAYE M,VIGOUROUX J,et al. Preliminary characterization of a new exo-beta-(1,4)-galactanase with transferase activity[J]. International Journal of Biological Macromolecules,1995,17(6):345-351.
[23] ZHANG J H,PAKARINEN A,VIIKARI L. Synergy between cellulases and pectinases in the hydrolysis of hemp[J]. Bioresource Technology,2013,129(2):302-307.
[24] RYTTERSGAARD C,LE NOURS J,LO LEGGIO L,et al. The structure of endo-beta-1,4-galactanase from Bacillus licheniformis in complex with two oligosaccharide products[J]. Journal of Molecular Biology,2004,341(1):107-117.