基于分段氨基酸组成预测β-琼胶酶的最适温度
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摘要
为了探究氨基酸组成对β-琼胶酶催化底物时的最适温度的影响,分别统计分析了酶全长、N端、M段及C端序列中20种氨基酸出现的频次。以支持向量机回归构建了氨基酸组成与β-琼胶酶最适温度的模型,10-倍交叉验证获得最小平均绝对误差(MAE),线性核函数的M端为4.36℃,RBF核函数的M端为4.16℃,独立样本测试得到N端的MAE分别为3.12℃和4.43℃。上述结果表明,该酶N端和M端是影响其最适温度的重要因素。通过比较最适温度为60℃和30℃β-琼胶酶的高级结构,推测M端和N端中3个螺旋结构是影响该酶最适温度的重要因素。
To explore how the composition of amino acids influence the optimum temperature of β-agarases,we counted and calculated the frequences the amino acids for full-length,N terminal,M section and C-terminal sequence of β-agarases respectively.And then,we constructed support vector regression model to represent the relationships between amino acid composition and the optimum temperature.Based on the mean absolute error(MAE)of 10-fold cross-validation,the M section of linear and RBF kernel is the best,with the MAEs of 4.36℃ and 4.16℃,respectively.While the the minimum MAE of the independent test was obtained by the N terminal,with the MAEs of 3.12℃ and 4.43℃ respectively.The results showed that N terminal and M section of β-agarases might have great influence on its optimum temperature.By comparing the 3D structure of the β-agarases with the optimum temperature of 30℃ and 60℃,we speculated that there were 3 helical structure differences in N terminal and M section,which should be the key factors that affect the thermostability of the β-agarases.
To explore how the composition of amino acids influence the optimum temperature of β-agarases,we counted and calculated the frequences the amino acids for full-length,N terminal,M section and C-terminal sequence of β-agarases respectively.And then,we constructed support vector regression model to represent the relationships between amino acid composition and the optimum temperature.Based on the mean absolute error(MAE)of 10-fold cross-validation,the M section of linear and RBF kernel is the best,with the MAEs of 4.36℃ and 4.16℃,respectively.While the the minimum MAE of the independent test was obtained by the N terminal,with the MAEs of 3.12℃ and 4.43℃ respectively.The results showed that N terminal and M section of β-agarases might have great influence on its optimum temperature.By comparing the 3D structure of the β-agarases with the optimum temperature of 30℃ and 60℃,we speculated that there were 3 helical structure differences in N terminal and M section,which should be the key factors that affect the thermostability of the β-agarases.
引文
1 Oh C,Nikapitiya C,Lee Y,et al.Molecular cloning,characterization and enzymatic properties of a novelβ-agarase from a marine isolate Psudoalteromonas SP.AG52.Braz J Microbiol.2010,41(4):876-889.
2 Morrice L M,Mc Lean M W,Williamson F B,et al.beta-agarases I and II from Pseudomonas atlantica,purifications and some properties.Eur J Biochem.1983,135(3):553-558.
3 Ma C,Lu X,Shi C,et al.Molecular cloning and characterization of a novel beta-agarase,Aga B,from marine Pseudoalteromonas sp.CY24.J Biol Chem.2007,282(6):3747-54.
4 Fu X T,Kim S M.Agarase:review of major sources,categories,purification method,enzyme characteristics and applications.Mar Drugs.2010,8(1):200-218.
5 Xie W,Lin B,Zhou Z,et al.Characterization of a novelβ-agarase from an agar-degrading bacterium Catenovulum sp.X3.Appl Microbiol Biotechnol.2013,97(11):4907-15.
6 Suzuki H,Sawai Y,Kawai K,et al.Purification and characterization of an extracellularβ-agarase from Bacillus sp.MK03.J Biosci Bioeng,2003,95(4):328-34
7 Dong J,Tamaru Y,Araki T.A unique beta-agarase,Aga A,from a marine bacterium,Vibrio sp.strain PO-303.Appl Microbiol Biotechnol,2007,74(6):1248-55.
8 Yang J I,Chen L C,Shih Y Y,et al.Cloning and characterization of beta-agarase Aga YT from Flammeovirga yaeyamensis strain YT.J Biosci Bioeng,2011,112(3):225-32.
9 Henrissat B.A classfication of glycosyl hydrolases based on amino acid similarities.Eur J Biochem,1991,280(2):309-316.
10 Chi W J,Chang Y K,Hong S K.Agar degradation by microorganisms and agar-degrading enzymes.Appl Microbiol Biotechnol,2012,94(4):917-930.
11 Hou Y P,Chen X L,Zeng R Y,et al.Expression and characterization of a thermostable and p H-stableβ-agarase encoded by a new gene from Flammeovirga pacifica WPAGA1.Process Biochem,2015,doi:10.1016.
13 Cui F,Dong S J,Shi X,et al.Overexpression and characterization of a novel thermostableβ-Agarase YM01-3 from marine bacterium Catenovulum agarivorans YM01(T).Mar Drugs,2014,12(5):2731-47.
14 Dong J,Tamaru Y,Araki T.Molecular cloning,expression,and characterization of a beta-agarase gene,aga D,from a marine bacterium,Vibrio sp.strain PO-303.Biosci Biotechnol Biochem,2007,71(1):38-46.
15 Dong J,Hashikawa S,Konishi T,et al.Cloning of the novel gene encoding beta-agarase C from a marine bacterium,Vibrio sp.strain PO-303,and characterization of the gene product.Appl Environ Microbiol,2006,72(9):6399-401.
16 Fu X T,Lin H,Pan C H,et al.Gene cloning,expression,and characterization of a beta-agarase,Aga B34,from Agarivorans albus YKW-34.J Microbiol Biotechnol,2009,19(3):257-64.
17 Lu X,Chu Y,Wu Q,et al.Cloning,expression and characterization of a new agarase-encoding gene from marine Pseudoalteromonas sp.Biotechnol Lett,2009,31(10):1565-70.
18 Ohta Y,Hatada Y,Nogi Y,et al.Enzymatic properties and nucleotide and amino acid sequences of a thermostable beta-agarase from a novel species of deep-sea Microbulbifer.Appl Microbiol Biotechnol,2004,64(4):505-14.
19 Takagi E,Hatada Y,Akita M,et al.Crystal structure of the catalytic domain of a GH16 beta-agarase from a deep-sea bacterium,Microbulbifer thermotolerans JAMB-A94.Biosci Biotechnol Biochem,2015,79(4):625-32.
20 Ohta Y,Hatada Y,Nogi Y,et al.Cloning,expression and characterization of a glycoside hydrolase family 86 beta-agarase from a deep-sea Microbulbifer-like isolate.Appl Microbiol Biotechnol.2004,66(3):266-75.
21 Ohta Y,Hatada Y,Ito S,et al.High-level expression of a neoagarobiose-producingβ-agarase gene from Agarivorans sp.JAMB-A11 in Bacillus subtilis and enzymic properties of the recombinant enzyme.Biotechnol Appl Biochem,2005,41(2):
183 -91.22 Sugano Y,Matsumoto T,Noma M.Sequence analysis of the aga B gene encoding a new beta-agarase from Vibrio sp.strain JT0107.Biochim Biophys Acta,1994,1218(1):105-8.
23 Ariga O,Inoue T,Kubo H,et al.Cloning of agarase gene from non-marine agarolytic bacterium Cellvibrio sp.J Microbiol Biotechnol,2012,22(9):1237-44.
24 Shi Y L,Lu X Z,Yu W G.A newβ-agarase from marine bacterium Janthinobacterium sp.SY12.World J Microbiol Biotechnol,2008,24(11):2659-64.
25 Liang S S,Chen Y P,Chen Y H,et al.Characterization and overexpression of a novel beta-agarase from Thalassomonas agarivorans.J Appl Microbiol,2014,116(3):563-72.
26 Lee D G,Park G T,Kim N Y,et al.Cloning,expression,and characterization of a glycoside hydrolase family 50 beta-agarase from a marine Agarivorans isolate.Biotechnol Lett,2006,28(23):1925-32.
27 Young B S,Yan L,Chi W J,et al.Heterologous expression of a newly screenedβ-agarase from Alteromonas sp.GNUM1 in Escherichia coli and its application for agarose degradation.Process Biochem,2014,49(3):430-436.
28 Chi W J,Lee C R,Park J S,et al.Biochemical characterization of a novel iron-dependent GH16β-agarase,Aga H92,from an agarolytic bacterium Pseudoalteromonas sp.H9.FEMS Microbiol Lett,2015,362(7),doi:10.1093.
29 Biasini M,Bienert S,Waterhouse A,et al.SWISS-MODEL:modelling protein tertiary and quaternary structure using evolutionary information.Nucleic Acids Res,2014,42(1):252-258.
30 Shatsky M,Nussinov R,Wolfson H J.A method for simultaneous alignment of multiple protein structures.Proteins,2004,56(1):143-56.
33 Li C,Heatwole J,Soelaiman S,et al.Crystal structure of a thermophilic alcohol dehydrogenase substrate complex suggests determinants of substrate specificity and thermostability.Proteins.1999,37(4):619-27.
34 Yamamoto T,Mukai K,Yamashita H,et al.Enhancement of thermostability of kojibiose phosphorylase from Thermoanaerobacter brockii ATCC35047 by random mutagenesis.J Biosci Bioeng,2005,100(2):212-5.
35 Takano K,Yamagata Y,Yutani K.Role of non-glycine residues in left-handed helical conformation for the conformational stability of human lysozyme.Proteins,2001,44(3):233-43.
12 林源清,王粲,张光亚.β-甘露聚糖酶理化性质与其最适p H的相关性[J].计算机与应用化学,2013,30(11):111-115.
31 卢柏松,王国力,黄培堂.嗜热和嗜常温微生物的蛋白质氨基酸组成比较[J].微生物学报,1998,38(1):20-25.
32 吴穗洁,吴文林,刘斌.嗜热酶耐热机制及提高酶热稳定性的研究进展[J].亚热带农业研究,2006,2(4):293-297.
36 陈路清,张秀艳,唐彦捷,何国庆.嗜热酶的稳定机制和稳定策略的研究进展[J].科技通报,2008,24(6):792-798.
2 Morrice L M,Mc Lean M W,Williamson F B,et al.beta-agarases I and II from Pseudomonas atlantica,purifications and some properties.Eur J Biochem.1983,135(3):553-558.
3 Ma C,Lu X,Shi C,et al.Molecular cloning and characterization of a novel beta-agarase,Aga B,from marine Pseudoalteromonas sp.CY24.J Biol Chem.2007,282(6):3747-54.
4 Fu X T,Kim S M.Agarase:review of major sources,categories,purification method,enzyme characteristics and applications.Mar Drugs.2010,8(1):200-218.
5 Xie W,Lin B,Zhou Z,et al.Characterization of a novelβ-agarase from an agar-degrading bacterium Catenovulum sp.X3.Appl Microbiol Biotechnol.2013,97(11):4907-15.
6 Suzuki H,Sawai Y,Kawai K,et al.Purification and characterization of an extracellularβ-agarase from Bacillus sp.MK03.J Biosci Bioeng,2003,95(4):328-34
7 Dong J,Tamaru Y,Araki T.A unique beta-agarase,Aga A,from a marine bacterium,Vibrio sp.strain PO-303.Appl Microbiol Biotechnol,2007,74(6):1248-55.
8 Yang J I,Chen L C,Shih Y Y,et al.Cloning and characterization of beta-agarase Aga YT from Flammeovirga yaeyamensis strain YT.J Biosci Bioeng,2011,112(3):225-32.
9 Henrissat B.A classfication of glycosyl hydrolases based on amino acid similarities.Eur J Biochem,1991,280(2):309-316.
10 Chi W J,Chang Y K,Hong S K.Agar degradation by microorganisms and agar-degrading enzymes.Appl Microbiol Biotechnol,2012,94(4):917-930.
11 Hou Y P,Chen X L,Zeng R Y,et al.Expression and characterization of a thermostable and p H-stableβ-agarase encoded by a new gene from Flammeovirga pacifica WPAGA1.Process Biochem,2015,doi:10.1016.
13 Cui F,Dong S J,Shi X,et al.Overexpression and characterization of a novel thermostableβ-Agarase YM01-3 from marine bacterium Catenovulum agarivorans YM01(T).Mar Drugs,2014,12(5):2731-47.
14 Dong J,Tamaru Y,Araki T.Molecular cloning,expression,and characterization of a beta-agarase gene,aga D,from a marine bacterium,Vibrio sp.strain PO-303.Biosci Biotechnol Biochem,2007,71(1):38-46.
15 Dong J,Hashikawa S,Konishi T,et al.Cloning of the novel gene encoding beta-agarase C from a marine bacterium,Vibrio sp.strain PO-303,and characterization of the gene product.Appl Environ Microbiol,2006,72(9):6399-401.
16 Fu X T,Lin H,Pan C H,et al.Gene cloning,expression,and characterization of a beta-agarase,Aga B34,from Agarivorans albus YKW-34.J Microbiol Biotechnol,2009,19(3):257-64.
17 Lu X,Chu Y,Wu Q,et al.Cloning,expression and characterization of a new agarase-encoding gene from marine Pseudoalteromonas sp.Biotechnol Lett,2009,31(10):1565-70.
18 Ohta Y,Hatada Y,Nogi Y,et al.Enzymatic properties and nucleotide and amino acid sequences of a thermostable beta-agarase from a novel species of deep-sea Microbulbifer.Appl Microbiol Biotechnol,2004,64(4):505-14.
19 Takagi E,Hatada Y,Akita M,et al.Crystal structure of the catalytic domain of a GH16 beta-agarase from a deep-sea bacterium,Microbulbifer thermotolerans JAMB-A94.Biosci Biotechnol Biochem,2015,79(4):625-32.
20 Ohta Y,Hatada Y,Nogi Y,et al.Cloning,expression and characterization of a glycoside hydrolase family 86 beta-agarase from a deep-sea Microbulbifer-like isolate.Appl Microbiol Biotechnol.2004,66(3):266-75.
21 Ohta Y,Hatada Y,Ito S,et al.High-level expression of a neoagarobiose-producingβ-agarase gene from Agarivorans sp.JAMB-A11 in Bacillus subtilis and enzymic properties of the recombinant enzyme.Biotechnol Appl Biochem,2005,41(2):
183 -91.22 Sugano Y,Matsumoto T,Noma M.Sequence analysis of the aga B gene encoding a new beta-agarase from Vibrio sp.strain JT0107.Biochim Biophys Acta,1994,1218(1):105-8.
23 Ariga O,Inoue T,Kubo H,et al.Cloning of agarase gene from non-marine agarolytic bacterium Cellvibrio sp.J Microbiol Biotechnol,2012,22(9):1237-44.
24 Shi Y L,Lu X Z,Yu W G.A newβ-agarase from marine bacterium Janthinobacterium sp.SY12.World J Microbiol Biotechnol,2008,24(11):2659-64.
25 Liang S S,Chen Y P,Chen Y H,et al.Characterization and overexpression of a novel beta-agarase from Thalassomonas agarivorans.J Appl Microbiol,2014,116(3):563-72.
26 Lee D G,Park G T,Kim N Y,et al.Cloning,expression,and characterization of a glycoside hydrolase family 50 beta-agarase from a marine Agarivorans isolate.Biotechnol Lett,2006,28(23):1925-32.
27 Young B S,Yan L,Chi W J,et al.Heterologous expression of a newly screenedβ-agarase from Alteromonas sp.GNUM1 in Escherichia coli and its application for agarose degradation.Process Biochem,2014,49(3):430-436.
28 Chi W J,Lee C R,Park J S,et al.Biochemical characterization of a novel iron-dependent GH16β-agarase,Aga H92,from an agarolytic bacterium Pseudoalteromonas sp.H9.FEMS Microbiol Lett,2015,362(7),doi:10.1093.
29 Biasini M,Bienert S,Waterhouse A,et al.SWISS-MODEL:modelling protein tertiary and quaternary structure using evolutionary information.Nucleic Acids Res,2014,42(1):252-258.
30 Shatsky M,Nussinov R,Wolfson H J.A method for simultaneous alignment of multiple protein structures.Proteins,2004,56(1):143-56.
33 Li C,Heatwole J,Soelaiman S,et al.Crystal structure of a thermophilic alcohol dehydrogenase substrate complex suggests determinants of substrate specificity and thermostability.Proteins.1999,37(4):619-27.
34 Yamamoto T,Mukai K,Yamashita H,et al.Enhancement of thermostability of kojibiose phosphorylase from Thermoanaerobacter brockii ATCC35047 by random mutagenesis.J Biosci Bioeng,2005,100(2):212-5.
35 Takano K,Yamagata Y,Yutani K.Role of non-glycine residues in left-handed helical conformation for the conformational stability of human lysozyme.Proteins,2001,44(3):233-43.
12 林源清,王粲,张光亚.β-甘露聚糖酶理化性质与其最适p H的相关性[J].计算机与应用化学,2013,30(11):111-115.
31 卢柏松,王国力,黄培堂.嗜热和嗜常温微生物的蛋白质氨基酸组成比较[J].微生物学报,1998,38(1):20-25.
32 吴穗洁,吴文林,刘斌.嗜热酶耐热机制及提高酶热稳定性的研究进展[J].亚热带农业研究,2006,2(4):293-297.
36 陈路清,张秀艳,唐彦捷,何国庆.嗜热酶的稳定机制和稳定策略的研究进展[J].科技通报,2008,24(6):792-798.
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