蛋白质半理性设计提高Candida Rugosa Lipase 1的热稳定性
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摘要
褶皱假丝酵母脂肪酶(Candida rugosa lipase 1 , CRL1)广泛应用于油脂、食品、化学制药等领域来催化脂肪水解、酯合成、转酯反应、手性拆分等反应,是目前工业应用最为广泛的脂肪酶之一。但是天然酶的性质往往不足以满足实际应用的需要,需要进一步用人工手段优化来提高其实用性。对CRL1而言,其作为一种常温酶,热稳定性欠佳,最适温度为40℃,在50℃的半衰期不足一小时,并且在80℃时迅速失活。这一不足限制了其在工业高温环境下的实际应用。本研究的目的旨在提高CRL1的热稳定性。
目前在蛋白质工程中对酶分子改造的方法主要有定向进化、理性设计与半理性设计。前两种方法各有其优势和缺点。理性设计的成功率虽高,但是思路设计必须依赖于蛋白质结构-功能关系的深入了解。定向进化虽然可以避免考虑这个问题,但是需要建立高效而灵敏的筛选方法,并对容量巨大的突变库进行筛选。半理性设计的方法结合了这两种方法的优点。它在定向进化的基础上加入了理性设计的元素,将突变限制到一个或几个位点之上,可以在较小的库容量中得到好的突变体。
在本研究中,我们从本实验室前期构建的成功的重组质粒pPicZαA-CRL1出发,利用半理性设计的方法来提高CRL1的热稳定性。我们以B值作为选取突变位点的依据。B值定义为电子偏离其平衡位置的统计学平均值,是在蛋白质晶体结构中反映原子位置不确定性的参数,当这些代表着原子中电子密度的模糊度的参数被换算成蛋白质氨基酸残基的B值时,就可以代表氨基酸残基的刚性大小。由于酶的活性中心部位的柔性更高。并且,在高温环境使酶整体过渡为去折叠状态之前,酶活性中心部位通常先发生变化而使酶失去活力。我们把突变位点限制在距催化活性中心Ser209 10?以内的60个氨基酸中。
在后续的突变位点的设计之中,以B值的大小为基本原则,对排名前三位的氨基酸残基进行了分析。预测出Glu126与Leu302可能是提升CRL1稳定性的关键突变位点。下一步的工作将利用定点饱和突变的方法对这两个位点分别进行了突变,筛选得到了热稳定性上升的突变体。
? Candida rugosa lipase 1 (CRL1)is one of the most efficient catalyst in hydrolizations, stereoselective transformations and polyester synthesis in the industries of dairy foods, chemicals, pharmaceutical. . As a mesophilic lipase, CRL1 has contradiction in excellent catalysis activity and low thermostability which limit it practical application.
Enzymes are subjected to three approaches to optimize the themostability in protein engineering: directed evolution, rational design, semi-rational design. The approaches of directed evolution and rational design both have advantage and disadvantage. The rational design usually has high efficiency but demands detailed understanding the structure information, catalytic mechanism and the structure-function relationship while the directed evolution needs high-though put screening in large mutant library. The semi-rational design can avoid the disadvantage of these two approaches by adding the rational element to directed evolution in order to limit the library to specified sites.
In our study, we used the semi-rational design approach to increase the thermostability. This study was based on crystal structure analysis of CRL1 (wild type) and the atomic displacement parameters with the name of B-factor obtained from X-ray data. The B-factor was a parameter reflecting the smearing of atomic electron densities from their equilibrium positions as a result of thermal motion and positional disorder, corresponding to the parameter of flexibility. The residues with high B-factor were identified as suitable target sites along with the position and function of the target sites. The residues near the catalytic triad always change its native conformation before the entirety protein. So we focus on the residues within 10 ? from the catalytic triad Ser209.
The B-factor and rational design method were used to predict the mutant sites and Leu302 and Glu126 were chose as the key amino acids to enhance the stability of CRL1.
In the next step, the two sites will be mutated using saturation mutagenesis, respectively.
目前在蛋白质工程中对酶分子改造的方法主要有定向进化、理性设计与半理性设计。前两种方法各有其优势和缺点。理性设计的成功率虽高,但是思路设计必须依赖于蛋白质结构-功能关系的深入了解。定向进化虽然可以避免考虑这个问题,但是需要建立高效而灵敏的筛选方法,并对容量巨大的突变库进行筛选。半理性设计的方法结合了这两种方法的优点。它在定向进化的基础上加入了理性设计的元素,将突变限制到一个或几个位点之上,可以在较小的库容量中得到好的突变体。
在本研究中,我们从本实验室前期构建的成功的重组质粒pPicZαA-CRL1出发,利用半理性设计的方法来提高CRL1的热稳定性。我们以B值作为选取突变位点的依据。B值定义为电子偏离其平衡位置的统计学平均值,是在蛋白质晶体结构中反映原子位置不确定性的参数,当这些代表着原子中电子密度的模糊度的参数被换算成蛋白质氨基酸残基的B值时,就可以代表氨基酸残基的刚性大小。由于酶的活性中心部位的柔性更高。并且,在高温环境使酶整体过渡为去折叠状态之前,酶活性中心部位通常先发生变化而使酶失去活力。我们把突变位点限制在距催化活性中心Ser209 10?以内的60个氨基酸中。
在后续的突变位点的设计之中,以B值的大小为基本原则,对排名前三位的氨基酸残基进行了分析。预测出Glu126与Leu302可能是提升CRL1稳定性的关键突变位点。下一步的工作将利用定点饱和突变的方法对这两个位点分别进行了突变,筛选得到了热稳定性上升的突变体。
? Candida rugosa lipase 1 (CRL1)is one of the most efficient catalyst in hydrolizations, stereoselective transformations and polyester synthesis in the industries of dairy foods, chemicals, pharmaceutical. . As a mesophilic lipase, CRL1 has contradiction in excellent catalysis activity and low thermostability which limit it practical application.
Enzymes are subjected to three approaches to optimize the themostability in protein engineering: directed evolution, rational design, semi-rational design. The approaches of directed evolution and rational design both have advantage and disadvantage. The rational design usually has high efficiency but demands detailed understanding the structure information, catalytic mechanism and the structure-function relationship while the directed evolution needs high-though put screening in large mutant library. The semi-rational design can avoid the disadvantage of these two approaches by adding the rational element to directed evolution in order to limit the library to specified sites.
In our study, we used the semi-rational design approach to increase the thermostability. This study was based on crystal structure analysis of CRL1 (wild type) and the atomic displacement parameters with the name of B-factor obtained from X-ray data. The B-factor was a parameter reflecting the smearing of atomic electron densities from their equilibrium positions as a result of thermal motion and positional disorder, corresponding to the parameter of flexibility. The residues with high B-factor were identified as suitable target sites along with the position and function of the target sites. The residues near the catalytic triad always change its native conformation before the entirety protein. So we focus on the residues within 10 ? from the catalytic triad Ser209.
The B-factor and rational design method were used to predict the mutant sites and Leu302 and Glu126 were chose as the key amino acids to enhance the stability of CRL1.
In the next step, the two sites will be mutated using saturation mutagenesis, respectively.
引文
[1] Fariha Hasan, Aamer Ali Shah, Abdul Hameed. Industrial applications of microbial lipases. Enzyme and Microbial Technology, 2006: 39(26), 235-251.
[2] Brian A. Kelch and David A. Agard. Mesophile versus Thermophile: Insights Into the Structural Mechanisms of Kinetic Stability. J. Mol. Biol, 2007, 370(4):784–795.
[3] Abbas Razvi, J. Martin Scholtz. Lessons in stability from thermophilic proteins. Protein Science, 2006, 15(7): 1569–1578.
[4] Karen M Polizzi1, Andreas S Bommarius, James M Broering. Stability of biocatalysts. Current Opinion in Chemical Biology, 2007, 11(2): 220–225.
[5] W.F. Li, X.X. Zhou, P. Lu. Structural features of thermozymes. Biotechnology Advances, 2005, 23(4): 271–281.
[6] Benjamin Folch, Yves Dehouck, Marianne Rooman. Thermo- and Mesostabilizing Protein Interactions Identified byTemperature-Dependent Statistical Potentials. Biophysical Journal, 2010, 98(4): 667–677.
[7] Liang Zhu Li, Tian Hong Xie, Mao Sheng Sun. Enhancing the thermostability of Escherichia coli l-asparaginase II by substitution with pro in predicted hydrogen-bonded turn structures. Enzyme and Microbial Technology, 2007, 41(4): 523–527.
[8] Sameh Ben Mabrouk, Nushin Aghajari, Mamdouh Ben Ali. Enhancement of the thermostability of the maltogenic amylase MAUS149 by Gly312Ala and Lys436 Arg substitutions. Bioresource Technology, 2011, 102(2): 1740–1746.
[9] Toshihisa Ohshima, Norimichi Nomura, Hideaki Tsuge. Structure of a Hyperthermophilic Archaeal Homing Endonuclease, I-Tsp061I: Contribution of Cross-domain Polar Networks to Thermostability.J.Mol.Biol.2007, 365(2): 362-378.
[10] Mi Young Jeong, Sanguk Kim, Cheol-Won Yun. Engineering a de novo internal disulfide bridge to improve the thermal stability of xylanase from Bacillus stearothermophilus. Journal of Biotechnology, 2007, 127(2): 300–309.
[11] Narayana Annaluru , Seiya Watanabe, Seung Pil Pack. Thermostabilization of Pichia stipitisxylitol dehydrogenase by mutation of structural zinc-binding loop. Journal of Biotechnology, 2007, 129(4): 717–722.
[12] Fulvio Baldasseroni, Stefano Pascarella. Subunit interfaces of oligomeric hyperthermophilic enzymes display enhanced compactness. International Journal of Biological Macro- molecules, 2009,44(4): 353–360.
[13] Abolfazl Barzegar1, Ali A. Moosavi-Movahedi1, Jens Z. Pedersen, Mehran Miroliae. Comparative thermostability of mesophilic and thermophilic alcohol dehydrogenases: Stability-determining roles of proline residues and loop conformations. Enzyme and Microbial Technology, 2009, 45(2):73-79.
[14] Pablo Dominguez de Maria , Aristotelis Xenakis , Haralambos Stamatis. Lipase factor (LF) as a characterization parameter to explain the catalytic activity of crude lipases from Candida rugosa, free or immobilized in microemulsion-based organogels. Enzyme and Microbial Technology, 2004, 34: 277–283.
[15] Yang Yong , Yong Xiao Bai , Chun Gu Xia. Characterization of Candida rugosa lipase immobilized onto magnetic microspheres with hydrophilicity. Process Biochemistry, 2008, 43(11): 1179–1185.
[16] Palackal N, Brennan Y, Callen WN, Dupree P, Frey G, Goubet F, Hazlewood GP, Healey S, Kang YE, Kretz KA. An evolutionary route to xylanase process fitness. Protein. Sci. 2004, 13(2): 494-503.
[17] Vincent G.H. Eijsink, Sigrid Ga seidnes , Bertus van den Burg. Directed volution of enzyme stability. Biomolecular Engineering, 2005, 22: 21–30.
[18] Dawn Elizabeth Stephens, Karl Rumbold, Suren Singh. Directed evolution of the thermostable xylanase from Thermomyces lanuginosus. Journal of Biotechnology, 2007, 127: 348–354.
[19] Wen-Chen Suen, Ningyan Zhang, Aleksey Zaks. Improved activity and thermostability of Candida antarctica lipase B by DNA familyshuffling. Protein Engineering, Design & Selection , 2004,17: 133-140.
[20] Wei Ning Niu, Zhao Peng Li, Tian Wei Tan. Improved thermostability and the optimum temperature of Rhizopus arrhizus lipase by directed evolution. Journal of Molecular Catalysis B: Enzymatic, 2006, 43: 33–39.
[21] H. Minagawa, Y. Yoshida, H. Kaneko. Improving the thermal stability of lactate oxidase bydirected evolution. Cellular and Molecular Life Sciences, 2006, 64: 77-81.
[22] Kotzia GA, Labrou NE. Engineering thermal stability of L-asparaginase by in vitro directed evolution. FEBS J. 2009, 276: 1750-61.
[23] Richard J. Fox and Gjalt W. Huisman. Enzyme optimization: moving from blind evolution to statistical exploration of sequence–function space. Trends in Biotechnology, 2003, 26: No.3.
[24] Manfred T. Reetz, Daniel Kahakeaw, and Renate Lohmer. Addressing the Numbers Problem in Directed Evolution. ChemBioChem 2008, 9: 1797– 1804.
[25] Recombinatoric exploration of novel folded structures:A heteropolymer-based model of protein evolutionary landscapes. PNAS, 2002, 99:809-814.
[26] M.A.Dwyer,L.L.Looger,H.W.Hellinga, Science, 2004, 304:1967.
[27] Journal of Molecular Biology,366(3): 945-953.
[28] M.A.Dwyer,L.L.Looger,H.W.Hellinga, Science 304,1967,(2004).
[29] Fre′de′ric Carrie`re, Kenneth Thirstrup, Chrislaine Withers-Martinez, Lars Thim,and Robert Verger.Pancreatic Lipase Structure-Function Relationships by Domain Exchange. Biochemistry 1997, 36: 239-248.
[30] Hee-Sung Park,Sung-Hun Nam,Jin Kak Lee,Chang No Yoon,Bengt Mannervik,Stephen J.Benkovic,4 Hak-Sung Kim.Design and Evolution of New Catalytic Activity with an Existing Protein Scaffold.Science.2006, 27 : 535-538.
[31] Miroslaw Cygler , Joseph D. Schrag. Structure and conformational￡exibility of Candida rugosa lipase. Biochimica et Biophysica Acta, 1999, 1441: 205-214.
[32] Frédéric Beisson, AliTiss, Claude Rivière, Robert Verger. Methods for lipase detection and assay: a critical review. Eur. J. Lipid Sci. Technol. 2000, 133–153.
[33] Jean Louis ARPIGNY and Karl-Erich JAEGER. Bacterial lipolytic enzymes:classification and properties. Biochem.J, 1999, 343: 177-183.
[34] Broadmeadow A,Clare C,De Boer AS.An overview of the safety evaluation of the Rhizomucor miehei lipase enzyme.Food Addit Contam, 1994,11:105-19.
[35] Mirella Di Lorenzo. Improvement of lipase stability in the presence of commercial triglycerides. European Journal of Lipid Science and Technology, 2003, 105: 608–613.
[36] Pablo Dom?′nguez de Mar?′a, Jose M. Sa′nchez-Montero, Andre′s R. Alca′ntara. Understanding Candida rugosa lipases: An overview. Biotechnology Advances, 2006, 24: 180–196.
[37] Miroslaw Cygler , Joseph D. Schrag. Structure and conformational fexibility of Candida rugosa lipase. Biochimica et Biophysica Acta, 1999, 1441: 205-214.
[38] Karl-Erich Jaeger , Michael Puls , Thorsten Eggert. The lid is a structural and functional determinant of lipase activity and selectivity. Journal of Molecular Catalysis B: Enzymatic , 2006, 39: 166–170.
[39] Wei Ning Niu, Zhao Peng Li, Tian Wei Tan. Improved thermostability and the optimum temperature of Rhizopus arrhizus lipase by directed evolution. Journal of Molecular Catalysis B: Enzymatic 43 (2006) 33–39.
[40] Mar?′a L. Ru′a, Carles Casas, Carles Sola`. Characterization of the lipase and esterase multiple forms in an enzyme preparation from a Candida rugosa pilot-plant scale fed-batch fermentation. Enzyme and Microbial Technology, 1999, 45: 214–223.
[41] M.L. Rua. Purification and characterization of Lip2 and Lip3 isoenzymes from a Candida rugosa pilot-plant scale fed-batch fermentation. Journal of Biotechnology, 2000, 84: 163–174.
[42] Benjamin, S. and Pandey, A. Candida rugosa and its lipases—a retrospect. J. Sci. Ind. Res. 1998, 57: 1–9.
[43] Deng Shuang Huang. Prediction of protein B-factors using multi-class bounded SVM. Protein and peptide letters, 2007,14: 185-190.
[44] A. Keith Dunker. Protein flexibility and intrinsic disorder. Protein Science, 2003, 13: 71-80.
[45] Zheng Yuan, Timothy L. Bailey, Rohan D. Teasdale. Prediction of Protein B-factor Profiles. PROTEINS: Structure, Function, and Bioinformatics, 2005, 58:905–912.
[46] Manfred T. Reetz, Jos D. Carballeira, Andreas Vogel. Iterative Saturation Mutagenesis on the Basis of B Factors as a Strategy for Increasing Protein Thermostability. Angew. Chem. Int. Ed, 2006, 45: 7745–775.
[47] Hyun Sook Kim, Quang Anh Tuan Le, Yong Hwan Kim. Development of thermostable lipase B from Candida antarctica (CalB) through in silico design employing B-factor and RosettaDesign. Enzyme and Microbial Technology, 2010, 47: 1–5.
[48] Francesco Secundo, Giacomo Carrea ,Thorsten Eggert. The lid is a structural and functional determinant of lipase activity and selectivity. Journal of Molecular Catalysis B: Enzymatic 39 (2006) 166–170.
[2] Brian A. Kelch and David A. Agard. Mesophile versus Thermophile: Insights Into the Structural Mechanisms of Kinetic Stability. J. Mol. Biol, 2007, 370(4):784–795.
[3] Abbas Razvi, J. Martin Scholtz. Lessons in stability from thermophilic proteins. Protein Science, 2006, 15(7): 1569–1578.
[4] Karen M Polizzi1, Andreas S Bommarius, James M Broering. Stability of biocatalysts. Current Opinion in Chemical Biology, 2007, 11(2): 220–225.
[5] W.F. Li, X.X. Zhou, P. Lu. Structural features of thermozymes. Biotechnology Advances, 2005, 23(4): 271–281.
[6] Benjamin Folch, Yves Dehouck, Marianne Rooman. Thermo- and Mesostabilizing Protein Interactions Identified byTemperature-Dependent Statistical Potentials. Biophysical Journal, 2010, 98(4): 667–677.
[7] Liang Zhu Li, Tian Hong Xie, Mao Sheng Sun. Enhancing the thermostability of Escherichia coli l-asparaginase II by substitution with pro in predicted hydrogen-bonded turn structures. Enzyme and Microbial Technology, 2007, 41(4): 523–527.
[8] Sameh Ben Mabrouk, Nushin Aghajari, Mamdouh Ben Ali. Enhancement of the thermostability of the maltogenic amylase MAUS149 by Gly312Ala and Lys436 Arg substitutions. Bioresource Technology, 2011, 102(2): 1740–1746.
[9] Toshihisa Ohshima, Norimichi Nomura, Hideaki Tsuge. Structure of a Hyperthermophilic Archaeal Homing Endonuclease, I-Tsp061I: Contribution of Cross-domain Polar Networks to Thermostability.J.Mol.Biol.2007, 365(2): 362-378.
[10] Mi Young Jeong, Sanguk Kim, Cheol-Won Yun. Engineering a de novo internal disulfide bridge to improve the thermal stability of xylanase from Bacillus stearothermophilus. Journal of Biotechnology, 2007, 127(2): 300–309.
[11] Narayana Annaluru , Seiya Watanabe, Seung Pil Pack. Thermostabilization of Pichia stipitisxylitol dehydrogenase by mutation of structural zinc-binding loop. Journal of Biotechnology, 2007, 129(4): 717–722.
[12] Fulvio Baldasseroni, Stefano Pascarella. Subunit interfaces of oligomeric hyperthermophilic enzymes display enhanced compactness. International Journal of Biological Macro- molecules, 2009,44(4): 353–360.
[13] Abolfazl Barzegar1, Ali A. Moosavi-Movahedi1, Jens Z. Pedersen, Mehran Miroliae. Comparative thermostability of mesophilic and thermophilic alcohol dehydrogenases: Stability-determining roles of proline residues and loop conformations. Enzyme and Microbial Technology, 2009, 45(2):73-79.
[14] Pablo Dominguez de Maria , Aristotelis Xenakis , Haralambos Stamatis. Lipase factor (LF) as a characterization parameter to explain the catalytic activity of crude lipases from Candida rugosa, free or immobilized in microemulsion-based organogels. Enzyme and Microbial Technology, 2004, 34: 277–283.
[15] Yang Yong , Yong Xiao Bai , Chun Gu Xia. Characterization of Candida rugosa lipase immobilized onto magnetic microspheres with hydrophilicity. Process Biochemistry, 2008, 43(11): 1179–1185.
[16] Palackal N, Brennan Y, Callen WN, Dupree P, Frey G, Goubet F, Hazlewood GP, Healey S, Kang YE, Kretz KA. An evolutionary route to xylanase process fitness. Protein. Sci. 2004, 13(2): 494-503.
[17] Vincent G.H. Eijsink, Sigrid Ga seidnes , Bertus van den Burg. Directed volution of enzyme stability. Biomolecular Engineering, 2005, 22: 21–30.
[18] Dawn Elizabeth Stephens, Karl Rumbold, Suren Singh. Directed evolution of the thermostable xylanase from Thermomyces lanuginosus. Journal of Biotechnology, 2007, 127: 348–354.
[19] Wen-Chen Suen, Ningyan Zhang, Aleksey Zaks. Improved activity and thermostability of Candida antarctica lipase B by DNA familyshuffling. Protein Engineering, Design & Selection , 2004,17: 133-140.
[20] Wei Ning Niu, Zhao Peng Li, Tian Wei Tan. Improved thermostability and the optimum temperature of Rhizopus arrhizus lipase by directed evolution. Journal of Molecular Catalysis B: Enzymatic, 2006, 43: 33–39.
[21] H. Minagawa, Y. Yoshida, H. Kaneko. Improving the thermal stability of lactate oxidase bydirected evolution. Cellular and Molecular Life Sciences, 2006, 64: 77-81.
[22] Kotzia GA, Labrou NE. Engineering thermal stability of L-asparaginase by in vitro directed evolution. FEBS J. 2009, 276: 1750-61.
[23] Richard J. Fox and Gjalt W. Huisman. Enzyme optimization: moving from blind evolution to statistical exploration of sequence–function space. Trends in Biotechnology, 2003, 26: No.3.
[24] Manfred T. Reetz, Daniel Kahakeaw, and Renate Lohmer. Addressing the Numbers Problem in Directed Evolution. ChemBioChem 2008, 9: 1797– 1804.
[25] Recombinatoric exploration of novel folded structures:A heteropolymer-based model of protein evolutionary landscapes. PNAS, 2002, 99:809-814.
[26] M.A.Dwyer,L.L.Looger,H.W.Hellinga, Science, 2004, 304:1967.
[27] Journal of Molecular Biology,366(3): 945-953.
[28] M.A.Dwyer,L.L.Looger,H.W.Hellinga, Science 304,1967,(2004).
[29] Fre′de′ric Carrie`re, Kenneth Thirstrup, Chrislaine Withers-Martinez, Lars Thim,and Robert Verger.Pancreatic Lipase Structure-Function Relationships by Domain Exchange. Biochemistry 1997, 36: 239-248.
[30] Hee-Sung Park,Sung-Hun Nam,Jin Kak Lee,Chang No Yoon,Bengt Mannervik,Stephen J.Benkovic,4 Hak-Sung Kim.Design and Evolution of New Catalytic Activity with an Existing Protein Scaffold.Science.2006, 27 : 535-538.
[31] Miroslaw Cygler , Joseph D. Schrag. Structure and conformational￡exibility of Candida rugosa lipase. Biochimica et Biophysica Acta, 1999, 1441: 205-214.
[32] Frédéric Beisson, AliTiss, Claude Rivière, Robert Verger. Methods for lipase detection and assay: a critical review. Eur. J. Lipid Sci. Technol. 2000, 133–153.
[33] Jean Louis ARPIGNY and Karl-Erich JAEGER. Bacterial lipolytic enzymes:classification and properties. Biochem.J, 1999, 343: 177-183.
[34] Broadmeadow A,Clare C,De Boer AS.An overview of the safety evaluation of the Rhizomucor miehei lipase enzyme.Food Addit Contam, 1994,11:105-19.
[35] Mirella Di Lorenzo. Improvement of lipase stability in the presence of commercial triglycerides. European Journal of Lipid Science and Technology, 2003, 105: 608–613.
[36] Pablo Dom?′nguez de Mar?′a, Jose M. Sa′nchez-Montero, Andre′s R. Alca′ntara. Understanding Candida rugosa lipases: An overview. Biotechnology Advances, 2006, 24: 180–196.
[37] Miroslaw Cygler , Joseph D. Schrag. Structure and conformational fexibility of Candida rugosa lipase. Biochimica et Biophysica Acta, 1999, 1441: 205-214.
[38] Karl-Erich Jaeger , Michael Puls , Thorsten Eggert. The lid is a structural and functional determinant of lipase activity and selectivity. Journal of Molecular Catalysis B: Enzymatic , 2006, 39: 166–170.
[39] Wei Ning Niu, Zhao Peng Li, Tian Wei Tan. Improved thermostability and the optimum temperature of Rhizopus arrhizus lipase by directed evolution. Journal of Molecular Catalysis B: Enzymatic 43 (2006) 33–39.
[40] Mar?′a L. Ru′a, Carles Casas, Carles Sola`. Characterization of the lipase and esterase multiple forms in an enzyme preparation from a Candida rugosa pilot-plant scale fed-batch fermentation. Enzyme and Microbial Technology, 1999, 45: 214–223.
[41] M.L. Rua. Purification and characterization of Lip2 and Lip3 isoenzymes from a Candida rugosa pilot-plant scale fed-batch fermentation. Journal of Biotechnology, 2000, 84: 163–174.
[42] Benjamin, S. and Pandey, A. Candida rugosa and its lipases—a retrospect. J. Sci. Ind. Res. 1998, 57: 1–9.
[43] Deng Shuang Huang. Prediction of protein B-factors using multi-class bounded SVM. Protein and peptide letters, 2007,14: 185-190.
[44] A. Keith Dunker. Protein flexibility and intrinsic disorder. Protein Science, 2003, 13: 71-80.
[45] Zheng Yuan, Timothy L. Bailey, Rohan D. Teasdale. Prediction of Protein B-factor Profiles. PROTEINS: Structure, Function, and Bioinformatics, 2005, 58:905–912.
[46] Manfred T. Reetz, Jos D. Carballeira, Andreas Vogel. Iterative Saturation Mutagenesis on the Basis of B Factors as a Strategy for Increasing Protein Thermostability. Angew. Chem. Int. Ed, 2006, 45: 7745–775.
[47] Hyun Sook Kim, Quang Anh Tuan Le, Yong Hwan Kim. Development of thermostable lipase B from Candida antarctica (CalB) through in silico design employing B-factor and RosettaDesign. Enzyme and Microbial Technology, 2010, 47: 1–5.
[48] Francesco Secundo, Giacomo Carrea ,Thorsten Eggert. The lid is a structural and functional determinant of lipase activity and selectivity. Journal of Molecular Catalysis B: Enzymatic 39 (2006) 166–170.