杂合β-甘露聚糖酶AuMan5A~(loop)的H321对其酶学性质的影响
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摘要
目的:将loop置换杂合β-甘露聚糖酶AuMan5A~(loop)的H321突变回野生型酶AuMan5A对应的Gly,以分析杂合酶的酶学性质与H321的相关性。方法:采用大引物PCR技术将AuMan5A~(loop)基因(Auman5Aloop)编码H321的密码子CAC突变为Gly的GGT,构建突变酶基因Auman5A~(loop/H321G);借助表达质粒pPICZαA将该突变酶基因在Pichia pastoris GS115中实施表达,并分析重组表达产物AuMan5A~(loop) H321G的酶学性质。结果:AuMan5A~(loop/H321G)置换前后的最适温度T_(opt)均为75℃,高于AuMan5A的65℃;AuMan5A~(loop/H321G)在70℃的半衰期t_(1/2)~(70)为300 min,介于AuMan5A(10 min)和AuMan5A~(loop)(480 min)之间;AuMan5A~(loop/H321G)比活性分别为AuMan5A和AuMan5A~(loop)的12.8和1.43倍;催化效率k_(cat)/K_m为后两者的14.1和1.12倍。结论:通过H321G置换及对3种酶的温度特性、比活性和催化效率的测定及比较,证实了H321对AuMan5A~(loop)的酶学性质有一定的影响。
Objective: AuMan5A~(loop),a hybrid β-mannanase,was constructed by substituting the loop structure of wild-type AuMan5A with the corresponding one of Aspergillus fumigatus β-mannanase. To analyze the correlation between the enzymatic properties and amino acid H321 of hybrid β-mannanase,its H321 was mutated into the corresponding Gly of AuMan5A. Methods: Using the mega primer PCR method,the mutant enzyme gene,Auman5A~(loop/H321G) G,was constructed by mutating a H321-encoding codon CAC of Auman5 Aloopinto a Glyencoding GGT. Then,Auman5A~(loop/H321G) Gwas expressed in Pichia pastoris GS115 mediated by expression plasmid pPICZαA,and the enzymatic properties of expressed recombinant enzyme,AuMan5A~(loop) / H321 G,were analyzed.Results: Before and after the H321 G substitution, the temperature optimum(T_(opt)) of AuMan5A~(loop) or AuMan5A~(loop/H321G) was 75℃, higher than that(65℃) of AuMan5A. The half-life at 70℃( t_(1/2)~(70)) of AuMan5A~(loop/H321G) was 300 min,between AuMan5A(10 min) and AuMan5A~(loop)(480 min). Besides,its specific activity was 12. 8 and 1. 43 fold those of AuMan5A and AuMan5A~(loop),and its catalytic efficiency(k_(cat)/K_m) was 14. 1 and 1. 12 fold those of the latter two,respectively. Conclusion: After H321G substitution as well as determination and comparison of temperature characteristics,specific activity and catalytic efficiency of three enzymes,the certain effect of H321 on the enzymatic properties of AuMan5A~(loop) was confirmed.
Objective: AuMan5A~(loop),a hybrid β-mannanase,was constructed by substituting the loop structure of wild-type AuMan5A with the corresponding one of Aspergillus fumigatus β-mannanase. To analyze the correlation between the enzymatic properties and amino acid H321 of hybrid β-mannanase,its H321 was mutated into the corresponding Gly of AuMan5A. Methods: Using the mega primer PCR method,the mutant enzyme gene,Auman5A~(loop/H321G) G,was constructed by mutating a H321-encoding codon CAC of Auman5 Aloopinto a Glyencoding GGT. Then,Auman5A~(loop/H321G) Gwas expressed in Pichia pastoris GS115 mediated by expression plasmid pPICZαA,and the enzymatic properties of expressed recombinant enzyme,AuMan5A~(loop) / H321 G,were analyzed.Results: Before and after the H321 G substitution, the temperature optimum(T_(opt)) of AuMan5A~(loop) or AuMan5A~(loop/H321G) was 75℃, higher than that(65℃) of AuMan5A. The half-life at 70℃( t_(1/2)~(70)) of AuMan5A~(loop/H321G) was 300 min,between AuMan5A(10 min) and AuMan5A~(loop)(480 min). Besides,its specific activity was 12. 8 and 1. 43 fold those of AuMan5A and AuMan5A~(loop),and its catalytic efficiency(k_(cat)/K_m) was 14. 1 and 1. 12 fold those of the latter two,respectively. Conclusion: After H321G substitution as well as determination and comparison of temperature characteristics,specific activity and catalytic efficiency of three enzymes,the certain effect of H321 on the enzymatic properties of AuMan5A~(loop) was confirmed.
引文
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[10]Xie Z H,Shi X J.Fast and almost 100%efficiency site-directed mutagenesis by the mega primer PCR method.Prog Biochem Biophys,2009,36(11):1490-1494.
[11]Li J F,Zhao S G,Tang C D,et a1.Cloning and functional expression of an acidophilicβ-mannanase gene(Anmart5A)from Aspergillus niger LW-1 in Pichia pastoris.J Agric Food Chem,2012,60(3):765-773.
[12]Bradford M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding.Anal Biochem,1976,72:248-254.
[13]Fei B J,Xu H,Cao Y,et al.A multi-factors rational design strategy for enhancing the thermostability of Escherichia coli App A phytase.J Ind Microbiol Biotechnol,2013,40(5):457-464.
[14]Missimer J H,Steinmetz M O,Baron R,et al.Configurational entropy elucidates the role of salt-bridge networks in protein thermostability.Protein Sci,2007,16:1349-1359.
[15]Ragone R.Hydrogen-bonding classes in proteins and their contribution to the unfolding reaction.Protein Sci,2001,10:2075-2082.
[16]Gray T M,Matthews B W.Structural analysis of the temperaturesensitive mutant of bacteriophage T4 lysozyme,glycine 156aspartic acid.J Biol Chem,1987,262(35):16858-16864.
[2]Zhang Y,Ju J,Peng H,et al.Biochemical and structural characterization of the intracellular mannanase Aa Man A of Alicyclobacillus acidocaldarius reveals a novel glycoside hydrolase family belonging to clan GH-A.J Biol Chem,2008,283(46):31551-31558.
[3]Wang C H,Luo H Y,Niu C F,et al.Biochemical characterization of a thermophilicβ-mannanase from Talaromyces leycettanus JCM12802 with high specific activity.Appl Microbiol Biotechnol,2015,99:1217-1228.
[4]Couturier M,Feliu J,Bozonnet S,et al.Molecular engineering of fungal GH5 and GH26 beta-(1,4)-mannanases toward improvement of enzyme activity.PLo S One,2013,8(11):e79800.
[5]Chen K,Liu S,Ma J,et al.Deletion combined with saturation mutagenesis of N-terminal residues in transglutaminase from Streptomyces hygroscopicus results in enhanced activity and thermostability.Process Biochem,2012,47(12):2329-2334.
[6]Duan X,Chen J,Wu J.Improving the thermostability and catalytic efficiency of Bacillus deramificans pullulanase by sitedirected mutagenesis.Appl Environ Microbiol,2013,79(13):4072-4077.
[7]Voutilainen S P,Murray P G,Tuohy M G,et al.Expression of Talaromyces emersonii cellobiohydrolase Cel7A in Saccharomyces cerevisiae and rational mutagenesis to improve its thermostability and activity.Protein Eng Des Sel,2010,23(2):69-79.
[8]Dong Y H,Li J F,Hu D,et al.Replacing a piece of loopstructure in the substrate-binding groove of Aspergillus usamii beta-mannanase,Au Man5A,to improve its enzymatic properties by rational design.Appl Microbiol Biotechnol,2016,100(9):3989-3998.
[9]李剑芳,董运海,胡蝶,等.β-甘露聚糖酶Au Man5A/Af酶学性质的改善与其Asp320的相关性分析.微生物学报,2016,56(2):301-308.Li J F,Dong Y H,Hu D,et al.Correlation between superior enzymatic properties ofβ-mannanase Au Man5A/Af and its residue Asp320.Acta Microbiologica Sinica,2016,56(2):301-308.
[10]Xie Z H,Shi X J.Fast and almost 100%efficiency site-directed mutagenesis by the mega primer PCR method.Prog Biochem Biophys,2009,36(11):1490-1494.
[11]Li J F,Zhao S G,Tang C D,et a1.Cloning and functional expression of an acidophilicβ-mannanase gene(Anmart5A)from Aspergillus niger LW-1 in Pichia pastoris.J Agric Food Chem,2012,60(3):765-773.
[12]Bradford M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding.Anal Biochem,1976,72:248-254.
[13]Fei B J,Xu H,Cao Y,et al.A multi-factors rational design strategy for enhancing the thermostability of Escherichia coli App A phytase.J Ind Microbiol Biotechnol,2013,40(5):457-464.
[14]Missimer J H,Steinmetz M O,Baron R,et al.Configurational entropy elucidates the role of salt-bridge networks in protein thermostability.Protein Sci,2007,16:1349-1359.
[15]Ragone R.Hydrogen-bonding classes in proteins and their contribution to the unfolding reaction.Protein Sci,2001,10:2075-2082.
[16]Gray T M,Matthews B W.Structural analysis of the temperaturesensitive mutant of bacteriophage T4 lysozyme,glycine 156aspartic acid.J Biol Chem,1987,262(35):16858-16864.