普鲁兰酶N端结构域CBM68对酶学性质的影响
|
摘要
碳水化合物结合模块(Carbohydrate Binding Modules,CBMs)在普鲁兰酶结构中普遍存在,对酶的催化性质起重要作用.本文将酸性普鲁兰酶Pul B的N端结构域CBM41-X45替换成耐热普鲁兰酶Pul A的N端结构域CBM68,得到重组酶Pul-11,同时将Pul-11的催化结构域替换成Pul A的催化结构域,得到重组酶Pul-12.结果显示,重组酶Pul-11和Pul-12的最适温度均较Pul B提高了10℃,最适pH分别提高了0.5和1;60℃下热稳定性分别提高了41.4%和44.0%;同时,重组酶Pul-11和Pul-12的底物亲和力和催化效率较Pul B也均有一定程度提高.可见,来源于耐热普鲁兰酶的新型底物结构域CBM68对普鲁兰酶的最适作用温度、最适作用pH及催化效率具有重要影响.
Carbohydrate Binding Modules(CBMs) are common domains in pullulanase, which play important roles on the catalytic properties of glycosyl hydrolases. In this work, the N-terminal domain CBM41-X45 of the acid pullulanase Pul B was replaced by CBM68, which is the N-terminal domain of the thermostable pullulanase Pul A. And then the catalytic domain of the recombinant pullulanase was further replaced by the catalytic domain of Pul A. The two recombinant pullulanases were named as Pul-11 and Pul-12, respectively. The same optimum temperature of Pul-11 and Pul-12 was 65 ℃, which was10 ℃ higher than that of Pul B. And the optimum pH of Pul-11 and Pul-12 were higher than that of Pul B by 0.5 and 1 unit, respectively. The residual activities of Pul-11 and Pul-12 were 41.4 % and44.0 % higher than that of Pul B at 60 ℃ for 12 h. Moreover, Pul-11 and Pul-12 also showed higher in substrate affinity and catalytic efficiency than Pul B. Therefore, the new substrate combination domain CBM68 shows important effects on the optimum temperature, the optimum pH and catalytic efficiency of the pullulanases through this study.
Carbohydrate Binding Modules(CBMs) are common domains in pullulanase, which play important roles on the catalytic properties of glycosyl hydrolases. In this work, the N-terminal domain CBM41-X45 of the acid pullulanase Pul B was replaced by CBM68, which is the N-terminal domain of the thermostable pullulanase Pul A. And then the catalytic domain of the recombinant pullulanase was further replaced by the catalytic domain of Pul A. The two recombinant pullulanases were named as Pul-11 and Pul-12, respectively. The same optimum temperature of Pul-11 and Pul-12 was 65 ℃, which was10 ℃ higher than that of Pul B. And the optimum pH of Pul-11 and Pul-12 were higher than that of Pul B by 0.5 and 1 unit, respectively. The residual activities of Pul-11 and Pul-12 were 41.4 % and44.0 % higher than that of Pul B at 60 ℃ for 12 h. Moreover, Pul-11 and Pul-12 also showed higher in substrate affinity and catalytic efficiency than Pul B. Therefore, the new substrate combination domain CBM68 shows important effects on the optimum temperature, the optimum pH and catalytic efficiency of the pullulanases through this study.
引文
1 Manners D J.Observations on the Specificity and nomenclature of starch debranching enzymes[J].Journal of Applied Glycoscience,1997,44:83-85.
2 Wang Q Y,Xie N Z,Du Q S,et al.Active hydrogen bond network(AHBN)and applications for improvement of thermal stability and p H-sensitivity of pullulanase from Bacillus naganoensis[J].Plos One,2017,12(1):e0 169 080-e0 169 085.
3 Crouch L I,Labourel A,Walton P H,et al.The contribution of non-catalytic carbohydrate binding modules to the activity of lytic polysaccharide monooxygenases[J].Journal of Biological Chemistry,2016,291(14):7 439-7 449.
4 Duan C J,Feng Y L,Cao Q L,et al.Identification of a novel family of carbohydrate-binding modules with broad ligand specificity[J].Scientific Reports,2016,6:19 392-19 397.
5 Venditto I,Najmudin S,Luis A S,et al.Family 46 Carbohydrate-Binding Modules contribute to the enzymatic hydrolysis of xyloglucan andβ-1,3-1,4-glucans through distinct mechanisms[J].Journal of Biological Chemistry,2015,290(17):10 572.
6 Armenta S,Morenomendieta S,Sánchezcuapio Z,et al.Advances in molecular engineering of carbohydrate-binding modules[J].Proteins Structure Function&Bioinformatics,2017,85(9):1 602-1 617.
7 Liu Y,Huang L,Li W,et al.Studies on properties of the xylan-binding domain and linker sequence of xylanase Xyn G1-1 from Paenibacillus campinasensis G1-1[J].Journal of Industrial Microbiology&Biotechnology,2015,42(12):1 591-1 599.
8 Xu J,Ren F,Huang C H,et al.Functional and structural studies of pullulanase from Anoxybacillus sp.LM18-11[J].Proteins:Structure,Function,and Bioinformatics,2014,82(9):1 685-1 693.
9 Duan X,Wu J.Enhancing the secretion efficiency and thermostability of a bacillus deramificans pullulanase mutant(D437H/D503Y)by N-Terminal domain truncation[J].Appl Environ Microbiol,2015,81(6):1 926-1 931.
10 Jane?ek,MajzlováK,Svensson B,et al.The starch-binding domain family CBM41-an in silico analysis of evolutionary relationships[J].Proteins-structure Function&Bioinformatics,2017,85(8):1 480-1 492.
11 Khan M I,Sajjad M,Sadaf S,et al.The nature of the carbohydrate binding module determines the catalytic efficiency of xylanase Z of Clostridium thermocellum[J].Journal of Biotechnology,2013,168(4):403-408.
12 Liu S,Ding S.Replacement of carbohydrate binding modules improves acetyl xylan esterase activity and its synergistic hydrolysis of different substrates with xylanase[J].Bmc Biotechnology,2016,16(1):73-76.
13 Mandels M,Hontz L,Nystrom J,et al.Enzymatic hydrolysis of waste cellulose[J].Biotechnology&Bioengineering,2010,105(1):1-25.
14 陈阿娜,刘秀霞,戴晓峰,等.N端截短对嗜酸普鲁兰芽孢杆菌普鲁兰酶酶学特性及功能的影响[J].生物工程学报,2016,32(3):355-364.
2 Wang Q Y,Xie N Z,Du Q S,et al.Active hydrogen bond network(AHBN)and applications for improvement of thermal stability and p H-sensitivity of pullulanase from Bacillus naganoensis[J].Plos One,2017,12(1):e0 169 080-e0 169 085.
3 Crouch L I,Labourel A,Walton P H,et al.The contribution of non-catalytic carbohydrate binding modules to the activity of lytic polysaccharide monooxygenases[J].Journal of Biological Chemistry,2016,291(14):7 439-7 449.
4 Duan C J,Feng Y L,Cao Q L,et al.Identification of a novel family of carbohydrate-binding modules with broad ligand specificity[J].Scientific Reports,2016,6:19 392-19 397.
5 Venditto I,Najmudin S,Luis A S,et al.Family 46 Carbohydrate-Binding Modules contribute to the enzymatic hydrolysis of xyloglucan andβ-1,3-1,4-glucans through distinct mechanisms[J].Journal of Biological Chemistry,2015,290(17):10 572.
6 Armenta S,Morenomendieta S,Sánchezcuapio Z,et al.Advances in molecular engineering of carbohydrate-binding modules[J].Proteins Structure Function&Bioinformatics,2017,85(9):1 602-1 617.
7 Liu Y,Huang L,Li W,et al.Studies on properties of the xylan-binding domain and linker sequence of xylanase Xyn G1-1 from Paenibacillus campinasensis G1-1[J].Journal of Industrial Microbiology&Biotechnology,2015,42(12):1 591-1 599.
8 Xu J,Ren F,Huang C H,et al.Functional and structural studies of pullulanase from Anoxybacillus sp.LM18-11[J].Proteins:Structure,Function,and Bioinformatics,2014,82(9):1 685-1 693.
9 Duan X,Wu J.Enhancing the secretion efficiency and thermostability of a bacillus deramificans pullulanase mutant(D437H/D503Y)by N-Terminal domain truncation[J].Appl Environ Microbiol,2015,81(6):1 926-1 931.
10 Jane?ek,MajzlováK,Svensson B,et al.The starch-binding domain family CBM41-an in silico analysis of evolutionary relationships[J].Proteins-structure Function&Bioinformatics,2017,85(8):1 480-1 492.
11 Khan M I,Sajjad M,Sadaf S,et al.The nature of the carbohydrate binding module determines the catalytic efficiency of xylanase Z of Clostridium thermocellum[J].Journal of Biotechnology,2013,168(4):403-408.
12 Liu S,Ding S.Replacement of carbohydrate binding modules improves acetyl xylan esterase activity and its synergistic hydrolysis of different substrates with xylanase[J].Bmc Biotechnology,2016,16(1):73-76.
13 Mandels M,Hontz L,Nystrom J,et al.Enzymatic hydrolysis of waste cellulose[J].Biotechnology&Bioengineering,2010,105(1):1-25.
14 陈阿娜,刘秀霞,戴晓峰,等.N端截短对嗜酸普鲁兰芽孢杆菌普鲁兰酶酶学特性及功能的影响[J].生物工程学报,2016,32(3):355-364.