摘要
自上世纪90年代以来,定向进化技术作为一种体外优化蛋白质性质的方法,取得了令人瞩目的成就。定向进化技术将基因突变和高通量筛选相结合,在实验室中模拟自然进化中的关键步骤:突变、重组和筛选,加快了分子进化过程,能在短期内获得具有优良性质的突变酶;同时,对突变体性质变化的结构基础和分子机理进行分析,加深了对蛋白质结构-功能关系的理解,因此定向进化技术也成为人类获取蛋白质化学知识的重要途径之一。本文选定两种具有重要研究及应用价值的酶:嗜热酰基肽水解酶及糖基转移酶作为研究对象,运用定向进化对其性质进行优化,并通过对突变体的酶学性质表征、计算机同源建模、分子动力学模拟等技术对突变体性质变化的分子机制进行了深入的探讨。
(一)通过对来自嗜热古菌Aeropyrum pernix K1的超嗜热酰基肽水解酶apAPH进行结构分析发现,其活性中心附近的Arg526在POP家族中高度保守,但在与其相关的脂肪酶家族成员中的对应位点均为疏水残基。为了探讨该位点对于酶催化及底物选择性的重要作用,我们运用半理性设计策略构建了526位点的饱和突变库,通过筛选获得了底物选择性发生重大变化的突变体,并且通过酶动力学及计算生物学手段阐明了526位点对于肽酶/酯酶活力转换的重要作用,揭示了蛋白酶及脂肪酶家族的进化相关性;进一步的结构分析显示,Arg526参与到了apAPH催化活性中心附近的离子网络中,与推进器结构域上的Glu88形成了一对保守的、跨结构域的盐桥。为了阐明这个盐桥对于酶催化的影响及其在酶家族中保守的原因,我们针对Glu88和Arg526位点设计了一系列盐桥突变体,通过定点突变、酶动力学和计算生物学等手段,证明了Glu88-Arg526这对跨结构域盐桥对于酶的催化功能及保持酶在高温下的正确折叠具有重要作用。
(二)我们以来自于Campylobacter jejuni的β-1,3-糖基转移酶CgtB为模型,建立了针对糖基转移酶的超高通量筛选方法(>108克隆/天),是目前唯一可以对糖基转移酶活力进行高效筛选的方法,为这个重要的酶家族的定向进化提供了强有力的工具。应用此种筛选方法,我们成功地提高了CgtB的催化活力。最优突变体的相对活力比野生型提高了28倍。酶动力学分析表明,进化突变体的Km值均较野生型明显下降,即突变体通过提高酶与底物的结合力,促进了酶催化的进行。
Enzymes are very useful tools for the synthesis of complex chiral molecules and hold a tremendous promise for the green and economical chemistry. However, while the number of biocatalysts has undergone an explosive growth over the past several years, there are disproportionately few examples of commercial scale applications of biocatalysts in industrial manufacturing and process development. This is mainly due to the fact that enzymes isolated directly from nature are evolved for a purpose other than being the catalysts of industrial processes, hence are usually not optimal for a certain application. Directed evolution, as a powerful tool for protein engineering, has made great achievement in enhancing various properties of enzymes since it has emerged in 1990s. By conjunction of consecutive rounds of diversification of target gene and selection for the desired catalytic activity, directed evolution mimics the key steps of natural evolution: mutagenesis, recombination and natural selection. It great accelerates the speed of molecular evolution and has the ability to obtain a protein with desired properties in months or even weeks, which usually takes millions of year in nature. More importantly, directed evolution is also a powerful tool to broaden our knowledge of protein chemistry. By analyzing the structure and property change of the mutants, directed evolution could provide valuable information of the underlying mechanism for enzyme catalysis, substrate specificity and stability.
In this work, two important enzymes, acylaminoacyl peptidase from Aeropyrum pernix K1 andβ-1,3-galactosyltransferase CgtB from Campylobacter jejuni, were subjected to directed evolution. These two enzymes are extremely valuable for both research and industry applications. Different strategies of directed evolution were employed accroding to their different features. Several mutants with altered catalytic activity were obtained and fully characterized. Further kinetics and molecular modeling studies allow us have a deeper insight into the mechanism of mutation effect and the possible structural basis for the activity change.
ApAPH from thermophilic archaeon Aeropyrum pernix K1 belongs to the prolyl oligopeptidase family (POP family), which represents a relative new class of serine protease. ApAPH catalyzes the removal of an N-acylated amino acid from the blocked peptides with different acyl groups at the N-terminus, and it also hydrolyzes p-nitrophenyl alkanoate esters of various alkyl chain lengths. The recombinant protein (apAPH) shows an optimal temperature at 90oC and is extremely stable. Its half life at 90oC is 18.6 hours. Recently, we have determined the crystal structure of apAPH and surprisingly found that its catalytic domain is very simliar to that of the hormone-sensitive lipase family. In addition, there is the canonical catalytic motif of lipase family (GXSXG) in the active site of apAPH, suggesting that apAPH has the potential to be evolved to an esterase.
It has been shown that highly conserved residue that forms crucial structural elements of a catalytic center may be used to account for the evolutionary history of enzymes. Arg526, which is adjacent to the catalytic Asp524, is completely conserved among all APHs. In fact, there is an obviously Arg bias at this position in the whole POP family. The special location and the highly conservation suggests that the 526 residue may play a crucial role in substrate recognition and/or transition state stabilization. Here, we have chosen Arg526 for saturation mutagenesis and made a semi-rational design library of apAPH. Screening of the mutation library by high-throughput screening method resulted in mutants with up-to-6-fold increased esterase activities and decreased peptidase activities, converting apAPH from an enzyme with dual functions to an esterase with high specificity. Kinetic characterization of mutants and wild type showed that the activity changes were achieved by an increased kcat for the esterase activity and an increased Km for peptidase activity. Molecular dynamics simulation suggested that Arg526 plays crucial role in stabilizing the enzyme-substrate complex for the peptidase activity. On the other hand, bulky hydrophobic side chains at 526 position is more favorable for esterase activity since the hydrophobic side chain results in a more stable enzyme-substrate complex. This study provides the first direct evidence that the POP family and lipase family are evolutional related; and Arg526 may be an evolutional marker.
Structure-based alignment indicated that the spatial position of the Glu88-Arg526 salt bridge is highly conserved among the POP family, implys that this interaction plays important role in this family. However, the effects of this inter-domain interactions on catalytic efficiency, substrate specificity, and stability are not clear yet. In order to study the functional and structural roles of this inter-domain salt bridge, a series of mutants on these two residues were created and characterized. The results had shown that E88A mutation on theβ-propeller domain almost abolished the catalysis for both of the ester and peptide substrate, indicating that Glu88 mutation is not only helps Arg526 to bind the substrate, but more importantly, it also plays crucial role in stabilizing the active site conformation. In contrast, E88A/R526V exhibited similar catalytic capability to R526V, suggesting that the Glu88 regulates the activity via an inter-domain salt bridge with the Arg526. Analysis of the pH dependence of the variants’reaction kinetics showed that the mutations make great influence on the electrostatic environment of the active site. In addition, chloride ions strongly modulated the activity of E88A mutant and the electrostatic environment of the active site. These phenomena imply that the activity change caused by E88A is closely related to the electrostatic environment near the active site. To further understand the experimental results, molecular dynamics simulation studies were undertaken with selected apAPH mutants. The results showed that the E88A mutant breaks the inter-domain salt bridge and results in the rotation of the guanidine side chain of Arg526, thus interfering with the charge-relay system of the catalytic triad. However, when sodium chloride was added to the system, the chloride anion restricts the guanidine group of Arg526 by electrostatic attraction and neutralizes the positive charge, therefore rescue the charge-relay system from disorder.
In addition, we have found that this inter-domain salt bridge also plays important roles in stabilized the protein. Compared with the wild type apAPH, the half life of E88A, R526V and E88A/R526V mutants at 90℃have decreased 2-, 3- and 5-fold, respectively. And the DSC results showed that the melting temperatures of the mutants decreased 5-10℃. In addition, the Cm value of these mutants also decreased 0.4-0.6M. The observed alterations in the unfolding process and reductions in chemical stability of the mutants indicate that the evolutionary pressures promoting the conservation of the Glu88-Arg526 are due to the requirements for structural stability and efficient folding of apAPH. Therefore, this inter-domain salt bridge plays two major roles in the function of the enzyme: (1) neutralizing the positive charge of Arg526, thereby increasing the enzymatic activity and anchoring the substrate, and (2) forming the Glu88-Arg526 salt bridge, thereby stabilizing the protein. Our results provide a rare example of the regulation of catalytic activity and thermodynamic stability by a single residue on the non-catalytic domain. These new insights should be useful for the rational design of multi-domain enzymes.
Glycosyltransferases (GT) are ubiquitous enzymes that catalyze the transfer reaction of a monosaccharide unit from an activated sugar phosphate (known as the "glycosyl donor") to an acceptor molecule. GTs are important biological catalysts in cellular systems which generating complex cell surface glycans including monosaccharide glycoside, oligosaccharide, or polysaccharide. GTs are now playing a key role for in vitro synthesis of oligosaccharides and has a number of important applications in pharmacal industry. However, assaying for GT activity is extremely challenging as no obvious change in fluorescence or absorbance is associated with glycosidic bond formation. In practical, the GT activity is usually determined by indirect ways, which make it difficult to be applied in a high-throughput manner. So far, the high-throughput screening method for GT activity is very rare, and screened library size is usually no more than 1000. To our knowledge, there is no ultra high-throughput screening method (>106) for GT has been not reported yet. In this thesis, we usedβ-1,3-galactosyltransferase CgtB from Campylobacter jejuni as a model enzyme to develop the first ultra high-throughput screening system for GT. CgtB transfers the galactose residue from UDP-Gal to GalNAc to form aβ-1,3 linkage. CgtB is the only enzyme that could be used in both of the synthesis of two important glycans: gangliosides GM1 and T-antigen (core 1 O-glycan). However, the native activity of CgtB is very low compared with other glycosyltransferases, which limit its utility in synthetic chemistry. In this work, we have successfully increased the activity of CgtB by directed evolution using this screening method.
Two fluorescent substrates GalNAc-Bodipy and GalNAc-Coumarin were synthesized by chemical ways. Both of these substrates could transport both into and out of the cell freely by the lactose permease located on the cell membrane. When there is CgtB activity inside the cell, the enzyme transfers the galactose from UDP-Gal to the fluorescent acceptors to form a disaccharide. While the monosaccharide substrate could be removed from the cell, the fluorescent disaccharide is not recognized by lactose permease and thus trapped inside the cell. The trapped fluorescence can be detected by flow cytometry with ultra high-throughput (up to 107 per hour). By screening a random library of CgtB with 2~5 mutations per gene, 11 mutants with enhanced activity were isolate from 2×107 colonies. Sequencing results showed that 3 hot spots (K151E, L227G, E234D) occurred more than once in the mutants, suggesting that they play important roles in the increases of the activities. The mutant E14 has 5 mutation site(sN26K, C40S, K50R, K151E, L227G)and shows a kcat/Km value 8.73-fold higher than the wild type CgtB. And then, one round of DNA shuffling was performed to remove the negative mutation and optimize the combination of positive mutation sites. Screening of the shuffling library resulted in the best mutant S42, which has a kcat/Km value 16.12-fold higher than the wild type. Kinetic analysis showed that the activity increase was mainly due to the decrease of Km, which has decreased 22.34- and 82.02-fold in E14 and S42 compared to that of the wild type CgtB, respectively.
We further constructed the single mutants of the“hot spots”(K151E, L227G, E234D) to understand their effects on the activity change. All of these mutants showed increased activities, but the kinetic results suggested that they have different effect on the kinetic parameters. K151E increase the kcat value twice, but L227G, E234D strongly decreased the Km value. The activities of the double mutation K151E/E234D were higher than that of both of the single mutant K151E and E234D but lower than the S42 mutant which contains all of the three mutation sites (K151E, L227G and E234D), suggesting that the effects of this mutation are additive.
In this work, we have developed a novel screening method for the detection of the activity of glycosyltransferases. To our knowledge, this is the only method to date to detect the activity of this important enzyme at an ultra high-through rate. This approach can also be applied for screening the substrate specificity, stability and other properties of glycosyltransferases after simple modification, providing a powerful tool for the directed evolution of the enzymes of this important family.
引文
[1] Arnold FH. Directed evolution: creating biocatalysts for the future. Chemical Engineering Science. 1996, 51: 5091-5102.
[2] Turner NJ. Directed evolution of enzymes for applied biocatalysis. Trends Biotechnol. 2003, 21: 474-478.
[3] Chica RA, Doucet N, Pelletier JN. Semi-rational approaches to engineering enzyme activity: combining the benefits of directed evolution and rational design. Curr Opin Biotechnol. 2005, 16: 378-384
[4] Chaparro-Riggers JF, Polizzi KM, Bommarius AS. Better library design: data-driven protein engineering. Biotechnol J. 2007, 2:180-191.
[5]朱国萍.用蛋白质工程方法设计和改造工业用酶.安徽师大学报. 1998, 22: 402-405.
[6]吴梧桐.蛋白质工程技术与新型生物催化剂设计.药物生物技术. 2004, 11:1-6.
[7]吴华昌,邓静,吴晓莉,韩莎.蛋白质工程在改造工业用酶中的应用.四川理工学院学报. 2004, 2: 11-14.
[8] Masui A, Fujiwara N, Imanaka T, Stabilization and rational design of serine protease Aprm under highly alkaline and hightemperature conditions. Appl Environ Microbiol, 1994, 60: 3579-3584.
[9] Zhu GP, Xu C, Teng MK, Tao LM. Increasing the thermostability of D-xylose isomerase by introduction of a proline into the turn of a random coil. Protein Eng. 1999, 12: 635-638.
[10] Lehmann M, Kostrewa D, Wyss M, Brugger R, D'Arcy A, Pasamontes L, van Loon AP. From DNA sequence to improved functionality: using protein sequence comparisons to rapidly design a thermostable consensus phytase. Protein Eng. 2000, 13: 49-57.
[11] Declerck N, Machius M, Huber R, Caillardin C. Probing structural determinanAts specifying high thermostability in Bacillus licheniformisα-amylase. J. Mol. Biol. 2000, 301: 1041-1057.
[12] Ueda T, Masumoto K, Ishibashi R, Imoto T. Remarkable thermal stability of doubly intramolecularly cross-linked hen lysozyme. Protein Eng. 2000, 13: 193-196.
[13] Lee LC, Chen YT, Yen CC, Chiang TC, Tang SJ, Lee GC, Shaw JF. Altering the Substrate Specificity of Candida rugosa LIP4 by Engineering the Substrate-Binding Sites. J Agric Food Chem. 2007, 55: 5103-5108.
[14] Rotticci D, Rotticci-Mulder JC, Denman S, Norin T, Hult K. Improved enanAtioselectivity of a lipase by rational protein engineering. Chembiochem. 2001, 2: 766-770.
[15] Gunnar E H?st,Bengt-Harald Jonsson. Converting human carbonic anhydrase II into a benzoate ester hydrolase through rational redesign. Biochimica Biophysica Acta. 2008, 1784: 811–815.
[16] Kapoli P, Axarli IA, Platis D, Fragoulaki M, Paine M, Hemingway J, Vontas J, Labrou NE. Engineering sensitive glutathione transferase for the detection of xenobiotics. Biosens Bioelectron. 2008, 24: 498-503.
[17] Broun P, Shanklin J, Whittle E, Somerville C. Catalytic plasticity of fatty acid modification enzymes underlying chemical diversity of plant lipids. Science. 1998, 282: 1315-1317.
[18] Xiang H, Luo L, Taylor KL, Dunaway-Mariano D. Interchange of catalytic activity within the 2-enoyl-coenzyme A hydratase/isomerase superfamily based on a common active site template. Biochemistry. 1999, 38: 7638-7652.
[19] Kamondi S, Szilágyi A, Barna L, Závodszky P. Engineering the thermostability of a TIM-barrel enzyme by rational family shuffling. Biochem Biophys Res Commun. 2008, 374: 725-730.
[20] Boersma YL, Pijning T, Bosma MS, van der Sloot AM, Godinho LF, Dr?ge MJ, Winter RT, van Pouderoyen G, Dijkstra BW, Quax WJ. Loop Grafting of Bacillus subtilis Lipase A:Inversion of EnanAtioselectivity. Chem Biol. 2008, 15: 782-789.
[21] You L, Arnold FH. Directed evolution of subtilisin E in Bacillus subtilis to enhance total activity in aqueous dimethylformamide. Protein Eng. 1996, 9: 77-83.
[22] Caldwell RC, Joyee GF. Randomization of genes by PCR mutagenesis. PCR Methods Appl. 1992, 2: 28-33.
[23] Stemmer WPC. DNA shuffling by random fragmentation and reassembly in vitro recombination for molecular evolution. Proc Natl Acad Sci. 1994, 91: 747-751.
[24] Kikuchi M, Ohnishi K, Harayama S An effective family shuffling method using single stranded DNA. Gene. 2000, 243: 133-137.
[25] Shao Z., Zhao H., Giver L. Random priming in vitro recombination : an effective tool for directed evolution. Nucl Acids Res. 1998, 26: 681-685.
[26] Sieber V., Martinez CA., Arnold FH. Libraries of hybrid proteins fromdistantly related sequence. Nat Biotechnol. 2001, 19: 456-460.
[27] Zhao H., Giver L., Shao Z. Molecular evolution by staggered extension process (StEP) in vitro recombination. Nat Biotechnol. 1998, 16: 258-261.
[28] Coco WM. RACHITT: Gene family shuffling by Random Chimeragenesis on Transient Templates. Methods Mol Biol. 2003, 231:111-127.
[29] Horswill AR, Naumann TA, Benkovic SJ. Using incremental truncation to create libraries of hybrid enzymes. Methods Enzymol. 2004; 388: 50-60.
[30] Moore GL., Maranas CD, Gutshall KR. Modeling and optimization of DNA recombination. Computers and Chemical Engineering. 2000, 24:693-699.
[31] Voigt CA, Mayo SL, Arnold FH, Wang ZG. Computational method to reduce the search space for directed protein evolution. Proc Natl Acad Sci USA. 2001, 3: 778-783.
[32] FernanAdez Gacio A.,Uguen M., Fastrez J. Phage display as a tool for the directed evolution of enzymes. Trends Biotechnol. 2003, 21: 408-414.
[33] Raberts BL., Markland W., Ley AC. Directed evolution of a protein : selection of potent neutrophil elastase inhibitors displayed on M13 fusion phage. Proc Natl Acad Sci USA. 1992, 2: 429-433.
[34] Schaffitzel C., Hanes J., Jermutus L. Ribosome display: an in vitro method for selection and evolution of antibodies from libraries Journal of Immunological Methods.1999, 231: 119-135.
[35] Warren MS., Marolewski AE., Beukovic SJ. A rapid screen of active site mutants in glycinamide ribonucleotide. Biochem. 1996, 35: 8855-8886.
[36] Becker S, Schmoldt HU, Adams TM, Wilhelm S, Kolmar H. Ultra-high-throughput screening based on cell-surface display and fluorescence-activated cell sorting for the identification of novel biocatalysts. Curr Opin Biotechnol. 2004, 15: 323-9
[37] Farinas ET. Fluorescence activated cell sorting for enzymatic activity. Comb Chem High Throughput Screen. 2006, 9: 321-8.
[38] Eklund BI, Edalat M, Stenberg G, Mannervik B. Screening for recombinanAt glutathione transferases active with monochlorobimane. Anal Biochem. 2002, 309: 102-8.
[39] Griswold KE, Kawarasaki Y, Ghoneim N, Benkovic SJ, Iverson BL, Georgiou G. Evolution of highly active enzymes by homology-independent recombination. Proc Natl Acad Sci U S A. 2005, 102: 10082-10087.
[40] Griswold KE, Aiyappan NS, Iverson BL, Georgiou G. The evolution of catalytic efficiency andsubstrate promiscuity in human theta class 1-1 glutathione transferase. J Mol Biol. 2006, 364:400-410.
[41] Olsen MJ, Stephens D, Griffiths D, Daugherty P, Georgiou G, Iverson BL. Function-based isolation of novel enzymes from a large library. Nat Biotechnol. 2000, 18:1071-1074.
[42] Varadarajan N, Gam J, Olsen MJ, Georgiou G, Iverson BL. Engineering of protease variants exhibiting high catalytic activity and exquisite substrate selectivity. Proc Natl Acad Sci U S A. 2005, 102: 6855-6860.
[43] Varadarajan N, Rodriguez S, Hwang BY, Georgiou G, Iverson BL. Highly active and selective endopeptidases with programmed substrate specificities. Nat Chem Biol. 2008, 4: 290-294
[44] Varadarajan N, Georgiou G, Iverson BL.An engineered protease that cleaves specifically after sulfated tyrosine. Angew Chem Int Ed Engl. 2008, 47: 7861-7863.
[45] Dalby PA. Optimising enzyme function by directed evolution. Curr Opin Struct Biol. 2003, 13: 500-505.
[46] Park S, Morley KL., Horsman GP, Holmquist M, Hult K, Kazlauskas RJ Focusing mutations into the P. fluorescens esterase binding site increases enanAtioselectivity more effectively than distant mutations. Chem Biol. 2005, 12: 45-54.
[47] Strausberg SL, Ruan B, Fisher KE, Alexander PA, Bryan PN. Directed coevolution of stability and catalytic activity in calcium-free subtilisin. Biochemistry 2005, 44: 3272-3279.
[48] Morley KL, Kazlauskas RJ. Improving enzyme properties:when are closer mutations better Trends Biotechnol 2005, 23: 231-237.
[49] Chaparro-Riggers JF, Polizzi KM, Bommarius AS. Better library design: data-driven protein engineering. Biotechnol J. 2007, 2: 180-191.
[50] Santoro SW, Schultz PG. Directed evolution of the site specificity of Cre recombinase. Proc Natl Acad Sci USA 2002, 99: 4185-4190.
[51] Rui L, Cao L, Chen W, Reardon KF, Wood TK. Active site engineering of the epoxide hydrolase from Agrobacterium radiobacter AD1 to enhance aerobic mineralization of cis-1,2-dichloroethylene in cells expressing an evolved toluene ortho-monooxygenase. J Biol Chem. 2004, 279: 46810-46817.
[52] Colin DY, Deprez-Beauclair P, Allouche M, Brasseur R, Kerfelec B. Exploring the active site cavity of human pancreatic lipase. Biochem Biophys Res Commun. 2008, 370: 394-398.
[53] Reetz MT, Carballeira JD, Vogel A. Iterative Saturation Mutagenesis on the Basis of B Factors as a Strategy for Increasing Protein Thermostability. Angew. Chem. Int. Ed. 2006, 45: 7745–7751.
[54] Reetz MT. Controlling the enanAtioselectivity of enzymes by directed evolution: practical and theoretical ramifications. Proc Natl Acad Sci USA 2004, 101: 5716-5722.
[55] Geddie ML., Matsumura I. Rapid evolution ofβ-glucuronidase specificity by saturation mutagenesis of an active site loop. J Biol Chem 2004, 279: 26462-26468.
[56] Hayes RJ., Bentzien J., Ary ML., Hwang MY., Jacinto JM., Vielmetter J., Kundu A., Dahiyat BI. Combining computational and experimental screening for rapid optimization of protein properties. Proc Natl Acad Sci USA 2002, 99: 15926-15931.
[57] MEROPS: the peptidase database. Nucleic Acids Res. 2006 Jan 1;34(Database issue):D270-2.
[58] Structure–Function Properties of Prolyl Oligopeptidase Family Enzymes Cell Biochem Biophys. 2006;44(3):349-65.
[59] Tsunasawa S, Narita K, Ogata K. Purification and properties of acylamino acid-releasing enzyme from rat liver. J Biochem (Tokyo). 1975 Jan 1;77(1?):89-102
[60] Jones WM, Manning LR, Manning JM. Enzymic cleavage of the blocked amino terminal residues ofpeptides. Biochem. Biophys. Res. Commun. 1986. 139: 244-250.
[61] Jones W M, Scaloni A, Bossa F, Popowicz A M, Schneewind O, Manning JM. Genetic relationship between acylpeptide hydrolase and acylase, two hydrolytic enzymes with similar binding but different catalytic specificities. Proc. Natl. Acad. Sci. USA 1991, 88: 2194-2198.
[62] Farries TC, Harris A, Auffret AD, Aitken A. Removal of N-acetyl groups from blocked peptides with acylpeptide hydrolase. Stabilization of the enzyme and its application to protein sequencing. Eur. J. Biochem. 1991, 196: 679-685.
[63] Gade W, Brown JL. Purification and partial characterization of a-N-acylpeptide hydrolase from bovine liver. J. Biol. Chem. 1978, 253: 5012-5018.
[64] Naylor SL, Marshall A, Hensel C, Martinez PF, Holley B, Sakaguchi AY. The DNF15S2 locus at 3p21 is transcribed in normal lung and small cell lung cancer. Genomics. 1989, 4: 355-361
[65] Radhakrishna G, Wold F. Purification and characterization of a-N-acylpeptide hydrolase from rabbit muscle. J. Biol. Chem. 1989, 264: 11076–11081.
[66] Sharma KK, Ortwerth BJ. Bovine lens acylpeptide hydrolase. Purification and characterization of a tetrameric enzyme resistant to urea denaturation and proteolytic inactivation. Eur. J. Biochem. 1993, 216: 631–637.
[67] Raphel V, Giardina T, Guevel L, Perrier J, Dupuis L, Gou XJ. Cloning, sequencing and further characterization of acylpeptide hydrolase from porcine intestinal mucosa. Biochim. Biophys. Acta 1999, 1432: 371–381.
[68] Mitta M, Asada K, Uchimura Y, Kimizuka F, Kato I, Sakiyama F. The primary structure of porcine liver acylamino acid-releasing enzyme deduced from cDNA sequences. J. Biochem. 1989, 106: 548-551.
[69] Kobayashi K, Lin L, Yeadon J, Klickstein L, Smith J. Cloning and sequence analysis of a rat liver cDNA encoding acyl-peptide hydrolase. J. Biol. Chem. 1989, 264: 8892-8899.
[70] Scaloni A, Jones WM, Pospischil M, Schneewind O, Popowicz A, Bossa F, Graziano SL, Manning JM. Deficiency of acylpeptide hydrolase in small-cell lung carcinoma cell lines. J. Lab. Clin. Med. 1992, 120: 546–552.
[71] Erlandsson R, Boldog F, Persson B, Zabarovsky ER, Allikmets RL, Sümegi J, Klein G, J?rnvall H. The gene from the short arm of chromosome 3, at D3F15S2, frequently deleted in renal cell carcinoma, encodes acylpeptide hydrolase. Oncogene. 1991, 6: 1293-1295
[72] Richards PG, Johnson MK, Ray DE. Identification of acylpeptide hydrolase as a sensitive site for reaction with organophosphorus compounds and a potential target for cognitive enhancing drugs. Mol. Pharmacol. 2000, 58: 577-583.
[73] Quistad GB, Klintenberg R, Casida JE. Blood acylpeptide hydrolase activity is a sensitive marker for exposure to some organophosphate toxicants. Toxicol Sci. 2005, 86: 291-299.
[74] Prolyl peptidases: a serine protease subfamily with high potential for drug discovery. Curr Opin Chem Biol. 2003 Aug;7(4):496-504
[75] The prolyl oligopeptidase family. Cell Mol Life Sci. 2002 Feb;59(2):349-62. Review.
[76] Wilk S. Prolyl endopeptidase. Life Sci. 1983, 33:2149- 2157.
[77] Mentlein R. Proline residues in the maturation and degradation of peptide-hormones and neuropeptides. FEBS Lett. 1988, 234:251-2566.
[78] Cunningham D.F. andóConnor B. Proline specific peptidases. Biochim. Biophys. Acta 1997, 1343: 160-186.
[79] Maes M., Goossens F., ScharpéS., Meltzer H. Y., ?. Hondt P. and Cosyns P. Lower serum prolylendopeptidase enzyme activity in major depression: further evidence that pep-tidases play a role in the pathophysiology of depression. Biol.Psychiatry. 1994, 35: 545-552.
[80] Maes M., Goossens F., Scharpe S., Calabrese J., Desnyder R. and Meltzer H. Y. Alteration in plasma prolyl oligopeptidase activity in depression, mania, and schizophrenia: effects of antidepressants, mood stabilizers and antipsychotic drugs. Psychiatry Res. 1995, 58: 217-225.
[81] Maes M., Monteleone P., Bencivenga R., Goossens F., Maj M., van Wes D .Lower serum activity of prolyl oligopeptidase in anorexia and bulimia nervosa. Psychoneuroendocrinology 2001, 26:17-26.
[82] Wilk S. and Orlowski M. Inhibition of rabbit brain prolyl endopeptidase byN-benzyloxycarbonyl-prolyl-prolinal, a transition state aldehyde inhibitor. J. Neurochem. 1983, 41:69-75.
[83] Stone S. R., Rennex D., Wikstrom P., Shaw E. and Hofsteenge J. Inactivation of prolyl endopeptidase by a peptidylchloromethane. Kinetics of inactivation and identification of sites of modification. Biochem. J. 1991, 276:837-840.
[84] Polgár L. Structural relationship between lipases and peptidases of the prolyl oligopeptidase family. FEBS Lett.1992, 311:281-284.
[85] Kahyaoglu A., Haghjoo M., Fusheng G., Jordan F., Kettner C Felf?ldi F. Low-barrier hydrogen bond is absent in the catalytic triads in the ground state but is present in a transition state complex in prolyl oligopeptidase family of serine proteases. J. Biol. Chem. 1997, 272:25547-25554.
[86] Ollis D. I., Cheah E., Cygler M., Dijkstra B., Frolow F., Franken S. M. Theα/βhydrolase fold. Protein Eng. 1992, 5:197-211.
[87] Polgár L. Structure and function of serine proteases. New Comp. Biochem. 1987, 16: 159-200.
[88] Polgár L.Mechanisms of Protease Action, CRC Press 1989, 6: 87-122.
[89] Schwartz NB (1982) In: Varma RS (ed) Glycosaminoglycans and Proteoglycans in Physiological and Pathological Processes of Body Systems. Karger, Basel, New York,p 41-54
[90]孙之荣,王钰,胡胜民,郭青,贺福初.丝氨酸蛋白酶超家族分子结构进化研究.生物物理学报, 1999, 15: 530-535.
[91] Perona J. J. and Craik C. S. Structural basis of substrate specificity in the serine proteases. Protein Sci. 1995, 4:337-360.
[92] Fül?p V., Szeltner Z. and Polgár L. Catalysis of serine oligopeptidases is controlled by a gating filter mechanism. EMBO Reports 2000, 1:277-281.
[93] Ishikawa K, Ishida H, Koyama Y, Kawarabayasi Y, Kawahara J, Matsui E. Acylamino acid-releasing enzyme from the thermophylic archaeon Pyrococcus horikoshii. J. Biol. Chem. 1998, 273: 17726–17731.
[94] R Gao, Y Feng, K. Ishikawa, H. Ishida, S. Ando, Y. Kosugi, S. Cao, Cloning, purification and properties of a hyperthermophilic esterase from archaeon Aeropyrum pernix K1. Journal of Molecular Catalysis B: Enzymatic. 2003, 24-25: 1-8.
[95] Bartlam M, Wang G, Yang H, Gao R, Zhao X, Xie G, Cao S, Feng Y, Rao Z. Crystal structure of an acylpeptide hydrolase/esterase from Aeropyrum pernix K1. Structure. 2004 Aug;12(8):1481-8.
[96] Sheldon RA. Chirotechnology: Industrial Synthesis of Optically Active Compounds, Marcel Dekker, New York. NY, 1993.
[97] Faber K, Franssen MC. Prospects for the increased application of biocatalysts in organic transformations, Trends Biotechnol. 1993, 11: 461-470.
[98] Ohmer N, Orrenius C, Mattson A, Norin T, Hult K. Kinetic resolutions of amine and thiol analogues of secondary alcohols catalyzed by the Candida antarctica lipase B. Enzyme Microb. Technol. 1996,9: 328-331.
[99] Sage CI, Jenner AJ, Kurmeier AH, Detlef P, Escher C, Poetsch E. Alkylbyphenyloxyacetic acid esters and their use in smectic liquid crystal materials, United States Patent, 1990, 4911863.
[100] Oda J, Amano M, Toda H, Koyagi T, Optical resolution of racemic alcohols, United States Patent, 1991, 4996158.
[101] Saccomano N, Enzymatic resolution of endo-bicyclo[2.2.1]heptan-2-ol and derived pharmaceutical agents, United States Patent, 1993, 5270206.
[102] Peter. Seufer-Wasserthal, Mayrhofer H, Wirth I, Pochlauer P, Enzymatic resolution of asymmetric alcohols by means of vinyl esters of polybasic carboxylic acids, United States Patent, 1996, 5534436.
[103] T. Mine, H. Mineta, T. Yui, M. Johno, Process for the production of an optically active alcohol and a novel optically active alcohol, United States Patent, 2000, 6103517.
[104] N. Yoshida, H. Morita, K. Oyama, H.-H. Lee, Chem. Express. 1989, 4: 721-725.
[105]林文逸,洪伟泽,黄振球,旋光性材料/诱导层列型复合系液晶的变色效应,计划编号:NSC 90-2216-E-155-004
[106] Piao G, Akagi K, Shirakawa H, Chiroptical titanium complexes as catalytically active chiral dopants available for asymmetric acetylene polymerization, Synthetic Metals. 1999, 101: 92-93.
[107] Gaoa X, Cao S, Zhang K. Production, properties and application to nonaqueous enzymatic catalysis of lipase from a newly isolated Pseudomonas strain, Enzyme Microb Tech. 2000, 27: 74-82.
[108]殷敬华,莫志深,编现代高分子物理(上册).科学出版社, 2001: 393-395.
[109] MacDonald RT, Pulapura S, Svirkin Y, Gross RA, Kaplan DA, Akkara J, et al. Enzyme catalyzedε-caprolactone ring-opening polymerization. Macromolecules 1995, 28: 73-78.
[110] Dong H, Cao SG, Li ZQ, Han SP, You DL, Shen JC. Study on the enzymatic polymerization mechanism of lactone and the strategy for improving the degree of polymerization. J Polym Sci Part A: Polym Chem 1999, 37: 1265-1275.
[111] Loeker FC, Duxbury CJ, Kumar R, Gao W, Gross RA, Howdle SM. Enzyme-catalyzed ring-opening polymerization ofε-caprolactone in supercritical carbon dioxide. Macromolecules 2004, 37: 2450-2453.
[112] Cordova A, Iversen T, Hult K, Martinelle M. Lipase catalyzed formation of macrocycles by ring-opening polymerization ofε-caprolactone. Polymer 1998, 39: 6519-6524.
[113] Kennedy S. The role of proteomics in toxicology: Identification of biomarkers of toxicity by protein expression analysis. Biomarkers. 2002, 7: 269–290.
[114] Scaros O, Fisler R.. Biomarker technology roundup: From discovery to clinical applications, a broad set of tools is required totranslate from the lab to the clinic. Biotechniques, 2005, 38(Suppl.): 30–32.
[115]王秋岩,杨广宇,刘艳莉,王艳平,冯雁。嗜热酯酶APE1547催化活性的定向进化研究,微生物学报,2005,46:259-262
[116] Schwartz NB (1982) In: Varma RS (ed) Glycosaminoglycans and Proteoglycans in Physiological and Pathological Processes of Body Systems. Karger, Basel, New York,p 41–54
[117] Geetha-Habib M, Campbell SC, Schwartz NB. Subcellular localization of the synthesis and glycosylation of chondroitin sulfate proteoglycan core protein. J Biol Chem. 1984, 259:7300-7310.
[118] Frederiksen S, Malling H, Klenow H. Isolation of 3’-Deoxyadenosine (Cordycepin) from the liquid medium of Cordyceps militaris (L. Ex. Fr.). link. Biochim Biophys Acta. 1965, 95: 189-193.
[119] Osborn MJ, D’Ari L. Enzymatic incorporation of N-acetylglucosamine into cell wall lipopolysaccharide in a mutant strain of Salmonella typhimurium. Biochem Biophys Res Commun. 1964, 16: 568-575.
[120] Cooper D, Manley RS. Cellulose synthesis by Acetobacter xylinum. I. Low molecular weight compounds present in the region of synthesis. Biochim Biophys Acta. 1975, 381: 78-96.
[121] McGuire EJ, Jourdian GW, Carlson DM, Roseman S. Incorporation of D-galactose into glycoproteins. J Biol Chem. 1965, 240: 4112-4115.
[122] Moczar E, Moczar M. A micro-method for the determination of hydroxylysine and its glycosylated derivatives. J Chromatogr. 1970, 51: 277-281.
[123] Apweiler R, HermjakobH, Sharon N. On the frequency of protein glycosylation, as deduced fromanalysis of the SWISS-PROTdatabase. Biochim Biophys Acta. 1999, 1473: 4-8.
[124] Campbell JA, Davies GJ, Bulone V, Henrissat B. A classification of nucleotidediphospho-sugar glycosyltransferases based on amino acid sequence similarities. Biochem J. 1997, 326 ( Pt 3): 929-939.
[125] Coutinho PM, Deleury E, Davies GJ, Henrissat B. An evolving hierarchical family classification for glycosyltransferases. J Mol Biol. 2003, 328: 307-317.
[126] Rosen ML, Edman M, Sjostrom M, Wieslander A. Recognition of fold and sugar linkage for glycosyltransferases by multivariate sequence analysis. J Biol Chem, 2004, 79(37):38683–38692
[127] Charnock SJ, Davies GJ. Structure of the nucleotide-diphospho-sugar transferase, SpsA from Bacillus subtilis, in native and nucleotide-complexed forms. Biochemistry, 1999, 38: 6380-6385
[128] Dodson E, Harding MM, Hodgkin DC, Rossmann MG. The crystal structureof insulin. 3. Evidence for a 2-fold axis in rhombohedral zinc insulin. J Mol Biol, 1966, 16: 227–241
[129] Bourne Y, Henrissat B. Glycoside hydrolases and glycosyltransferases: families and functional modules. Curr Opin Struct Biol, 2001, 11: 593-600
[130] Mulichak AM, Losey HC, Lu W, Wawrzak Z, Walsh CT, Garavito RM. Structure of the TDP-epi-vancosaminyltransferase GtfA from the chloroeremomycin biosynthetic pathway. Proc Natl Acad Sci USA. 2003, 100: 9238-9243
[131] Lairson LL, Withers SG. Mechanistic analogies amongst carbohydrate modifying enzymes. Chem Commun, 2004: 2243-2248.
[132] Breton C, ?najdrováL, Jeanneau C, Ko?a J, Imberty A. Structures and mechanisms of glycosyltransferases. Glycobiology, 2006, 16: 29R-37R.
[133] Thibodeaux CJ, Melan?on CE, Liu HW. Unusual sugar biosynthesis and natural product glycodiversification. Nature, 2007, 446: 1008-1016.
[134] Chelsky D, Parsons SM. Stereochemical course of the adenosine triphosphate phosphoribosyltransferase reaction in histidine biosynthesis. J Biol Chem, 1975, 250: 5669-5673
[135] Seibel J, Beine R, Moraru R, Behringer C, Buchholz K. A new pathway for the synthesis of oligosaccharides by the use of non-Leloir glycosyltransferases. Biocatal Biotransform 2006, 24:157–65.
[136] Skirrow MB. Campylobacter enteritis: a "new" disease. Br Med J. 1977, 2: 9-11.
[137] Ketley JM.Pathogenesis of enteric infection by Campylobacter. Microbiology. 1997, 143(Pt 1): 5-21.
[138] Bilbao A, Mauro KJ. The Guillain-Barrésyndrome.N Engl J Med. 1992, 327: 501.
[139] Gilbert M, Karwaski MF, Bernatchez S, Young NM, Taboada E, Michniewicz J, Cunningham AM,Wakarchuk WW. The genetic bases for the variation in the lipo-oligosaccharide of the mucosal pathogen, Campylobacter jejuni: Biosynthesis of sialylated ganglioside mimics in the core oligosaccharide. J Biol Chem. 2002, 277: 327–337.
[140] Yuki N, Okada M. Ganglioside mimicry as a cause of Guillain-Barr′e syndrome.Curr Opin Neurol. 2005, 18: 557-561.
[141] Godschalk PC, Kuijf ML, Li J, St Michael F, Ang CW, Jacobs BC,Karwaski MF, Brochu D, Moterassed A, Endtz HP, van Belkum A, Gilbert M. Structural characterization of Campylobacter jejuni lipooligosaccharide outer cores associated with Guillain-Barre and Miller Fisher syndromes. Infect Immun. 2007, 75: 1245-1254
[142] Moran AP, Prendergast MM, Appelmelk BJ. Molecular mimicry of host structures by bacterial lipopolysaccharides and its contribution to disease. FEMS Immunol Med Microbiol. 1996, 16: 105-115.
[143] Gilbert M, Brisson JR, Karwaski MF, Michniewicz J, Cunningham AM, Wu Y, Young NM, Wakarchuk WW. Biosynthesis of ganglioside mimics in Campylobacter jejuni OH4384. Identification of the glycosyltransferase genes, enzymatic synthesis of model compounds, and characterization of nanAomole amounts by 600-mhz (1) h and (13) c NMR analysis. J Biol Chem. 2000, 275: 3896-3906.
[144] Coutinho PM, Deleury E, Davies GJ, Henrissat B. An evolving hierarchical family classification for glycosyltransferases. J Mol Biol. 2003, 328: 307–317.
[145] Charnock SJ, Davies GJ. Structure of the nucleotide-diphospho-sugar transferase, SpsA from Bacillus subtilis, in native and nucleotide-complexed forms. Biochemistry. 1999, 38: 6380–6385.
[146] Tarbouriech N, Charnock SJ, Davies GJ. Three-dimensional structures of the Mn and Mg dTDP complexes of the family GT-2 glycosyltransferase SpsA: a comparison with related NDP-sugar glycosyltransferases. J Mol Biol. 2001, 314: 655-661.
[147] Goletz S, Cao Y, Danielczyk A, Ravn P, Schoeber U, Karsten U. Thomsen-Friedenreich antigen: the "hidden" tumor antigen.Adv Exp Med Biol. 2003, 535: 147-162.
[148] Wells L, Gao Y, Mahoney JA, Vosseller K, Chen C, Rosen A, Hart GW. Dynamic O-glycosylation of nuclear and cytosolic proteins: further characterization of the nucleocytoplasmic beta-N-acetylglucosaminidase, O-GlcNAcase. J Biol Chem. 2002, 277:1755-1761.
[149] McEver RP, Cummings RD. Perspectives series: cell adhesion in vascular biology. Role of PSGL-1 binding to selectins in leukocyte recruitment. J Clin Invest. 1997 , 100: 485-491.
[150] Brocke C, Kunz H. Synthesis of tumor-associated glycopeptide antigens. Bioorg Med Chem. 2002, 10: 3085-3112.
[151] Ju T, Cummings RD. A unique molecular chaperone Cosmc required for activity of the mammalian coreβ1, 3-galactosyltransferase. Proc Natl Acad Sci USA. 2002, 99: 16613-16618.
[152] Blixt O, Razi N. Chemoenzymatic synthesis of glycan libraries. Methods Enzymol. 2006, 415: 137-153.
[153] Hoetzl S, Sprong H, van Meer G. The way we view cellular (glyco)sphingolipids. J. Neurochem. 2007, 103: 3-13.
[154] Mishra S, Joshi PG. Lipid raft heterogeneity: an enigma. J. Neurochem. 2007, 103, 135-142.
[155] Abad-Rodriguez J, Robotti A. Regulation of axonal development by plasma membrane gangliosides. J. Neurochem. 2007, 103: 47-55.
[156] Ariga T, McDonald MP, Yu RK. Role of ganglioside metabolism in the pathogenesis of Alzheimer's disease-a review. J. Lipid Res. 2008, 49: 1157-1175.
[157] Wu D, Fujio M, Wong CH. Glycolipids as immunostimulating agents. Bioorg. Med. Chem. 2007, 16: 1073-1083.
[158] De Rosa M, Park HJ, Mylvaganum M, Binnington B, Lund N, Branch DR, Lingwood CA. The medium is the message: glycosphingolipids and their soluble analogues. Biochim. Biophys. Acta. 2008, 1780: 347-352.
[159] Morales-Serna JA, Boutureira O, Díaz Y, Matheu MI, Castillón S. Recent advances in the glycosylation of sphingosines and ceramides. Carbohydr. Res. 2007, 342: 1595-1612.
[160] Vankar YD, Schmidt RR. Chemistry of glycosphingolipids-carbohydrate molecules of biological significance. Chem. Soc. Rev. 2000, 29: 201-216.
[161] Rai AN, Basu A. Sphingolipid synthesis via olefin cross metathesis: preparation of a differentially protected building block and application to the synthesis of D-erythro-ceramide. Org Lett. 2004, 6: 2861-2863.
[162] Bernatchez S, Gilbert M, Blanchard MC, Karwaski MF, Li J, Defrees S, Wakarchuk WW. Variants of the beta 1, 3-galactosyltransferase CgtB from the bacterium Campylobacter jejuni have distinct acceptor specificities. Glycobiology. 2007, 17:1333-1343.
[163] Ola Blixt, Nahid Razi, Enzymatic Glycosylation by Transferases. In: Fraser-Reid, B., Tatsuta, K., editors. Glycoscience. 2nd. Edition. 1361-1387
[164] Shaikh FA, Withers SG. Teaching old enzymes new tricks: engineering and evolution of glycosidases and glycosyl transferases for improved glycoside synthesis. Biochem Cell Biol. 2008, 86:169-177.
[165] Hancock SM, Vaughan MD, Withers SG. Engineering of glycosidases and glycosyltransferases.Curr Opin Chem Biol. 2006, 10: 509-519.
[166] Leemhuis H, Rozeboom HJ, Dijkstra BW, Dijkhuizen L. Proteins. Improved thermostability of bacillus circulans cyclodextrin glycosyltransferase by the introduction of a salt bridge. 2004, 54:128-134.
[167] van der Veen BA, Uitdehaag JC, Penninga D, van Alebeek GJ, Smith LM, Dijkstra BW, Dijkhuizen L. Rational design of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 to increase alpha-cyclodextrin production.J Mol Biol. 2000, 296: 1027-1038.
[168] Nakahara T, Hindsgaul O, Palcic MM, Nishimura S. Computational design and experimental evaluation of glycosyltransferase mutants: engineering of a blood type B galactosyltransferase with enhanced glucosyltransferase activity.Protein Eng Des Sel. 2006, 19: 571-578.
[169] Persson M, Palcic MM. A high-throughput pH indicator assay for screening glycosyltransferase saturation mutagenesis libraries. Anal Biochem. 2008, 378: 1-7.
[170] Tumbale P, Jamaluddin H, Thiyagarajan N, Acharya KR, Brew K. Screening a limited structure-based library identifies UDP-GalNAc-specific mutants of alpha-1,3-galactosyltransferase. Glycobiology. 2008, 18:1036-1043
[171] Krauth C, Fedoryshyn M, Schleberger C, Luzhetskyy A, Bechthold A. Engineering a function into a glycosyltransferase. Chem Biol. 2009, 16: 28-35.
[172] Williams GJ, Zhang C, Thorson JS. Expanding the promiscuity of a natural-product glycosyltransferase by directed evolution. Nat Chem Biol. 2007, 3: 657-662.
[173] Williams GJ, Thorson JS.A high-throughput fluorescence-based glycosyltransferase screen and its application in directed evolution. Nat Protoc. 2008, 3: 357-362.
[174] Williams GJ, Goff RD, Zhang C, Thorson JS. Optimizing glycosyltransferase specificity via "hot spot" saturation mutagenesis presents a catalyst for novobiocin glycorandomization. Chem Biol. 2008, 15: 393-401.
[175] Smith GP. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science, 1985, 228: 1315-1317.
[176] Skerra A. Engineered protein scaffolds for molecular recognition. J. Mol. Recognit. 2000, 13: 167-187.
[177] Nemoto N, Miyamoto-Sato E, Husimi Y, Yanagawa H. In vitro virus: bonding of mRNA bearingpuromycin at the 3′-terminal end to the C-terminal end of its encoded protein on the ribosome in vitro. FEBS Lett. 1997, 414: 405-408
[178] Roberts RW, Szostak JW. RNA-peptide fusions for the in vitro selection of peptides and proteins. Proc. Natl. Acad. Sci. USA. 1997, 94: 12297-12302.
[179] Hanes J, Plückthun A. In vitro selection and evolution of functional proteins by using ribosome display. Proc. Natl. Acad. Sci. USA. 1997, 94: 4937-4942.
[180] Becker S, Schmoldt HU, Adams TM, Wilhelm S, Kolmar H. Ultra-high-throughput screening based on cell-surface display and fluorescence-activated cell sorting for the identification of novel biocatalysts. Curr Opin Biotechnol. 2004, 15: 323-329
[181] Farinas ET. Fluorescence activated cell sorting for enzymatic activity. Comb Chem High Throughput Screen. 2006, 9: 321-328.
[182] Aharoni A, Thieme K, Chiu CP, Buchini S, Lairson LL, Chen H, Strynadka NC, Wakarchuk WW, Withers SG. High-throughput screening methodology for the directed evolution of glycosyltransferases. Nat Methods. 2006, 3: 609-614.
1. Chica RA, Doucet N, Pelletier JN. Semi-rational approaches to engineering enzyme activity: combining the benefits of directed evolution and rational design. Curr Opin Biotechnol. 2005, 16:378-384
2. Patrick WM, Firth AE. Strategies and computational tools for improving randomized protein libraries. Biomol Eng. 2005, 22:105-112.
3. Chaparro-Riggers JF, Polizzi KM, Bommarius AS. Better library design: data-driven protein engineering. Biotechnol J. 2007, 2:180-191.
4. Shivange AV, Marienhagen J, Mundhada H, Schenk A, Schwaneberg U. Advances in generating functional diversity for directed protein evolution. Curr Opin Chem Biol. 2009 Mar 2.
5. R Gao, Y Feng, K. Ishikawa, H. Ishida, S. Ando, Y. Kosugi, S. Cao, Cloning, purification and properties of a hyperthermophilic esterase from archaeon Aeropyrum pernix K1. Journal of Molecular Catalysis B: Enzymatic. 2003, 24-25: 1-8.
6. Bartlam M, Wang G, Yang H, Gao R, Zhao X, Xie G, Cao S, Feng Y, Rao Z. Crystal structure of an acylpeptide hydrolase/esterase from Aeropyrum pernix K1.
7. Shindyalov IN, Bourne PE. Protein structure alignment by incremental combinatorial extension (CE)of the optimal path. Protein Eng. 1998, 11:739-747.
8. Zheng L, Baumann U, Reymond JL. An efficient one-step site-directed and site-saturation mutagenesis protocol. Nucleic. Acids Res. 2004, 32, e115.
9. Laskowski RA, MacArthur MW, Moss DS, Thornton JM. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Cryst. 1993, 26: 283-291.
10. Sander C, Schneider R. Database of homology-derived protein structures and the structural meaning of sequence alignment. Proteins. 1991, 9:56-68.
11. Doolittle RF. Similar amino acid sequences: chance or common ancestry. Science. 1981, 214: 149-159.
12. Russell RB., Barton GJ. Structural features can be unconserved in proteins with similar folds. An analysis of side-chain to side-chain contacts secondary structure and accessibility. J Mol Biol. 1994, 244: 332-350.
13. Grishin NV. Estimation of evolutionary distances from protein spatial structures. J Mol Evol. 1997, 45: 359-369.
14. Michael L. Sierk and William R. Pearson Sensitivity and selectivity in protein structure comparison. Protein Sci. 2004, 13: 773-785.
15. Chothia, C., and Lesk, AM. The relation between the divergence of sequence and structure in proteins. EMBO J. 1986, 5: 823-826.
16. Gao, R., Feng, Y., Ishikawa, K., Ishida, H., Ando, S., and Ksougi, Y. Cloning, purification and properties of a hyperthermophilic esterase from archason Aeropyrum pernix K1 J. Mol. Catal. B Enzym. 2003, 24-25, 1-8.
17. Nardini, M., Lang, D. A., Liebeton, K., Jaeger, K. E., and Dijkstra, B. W. Crystal structure of Pseudomonas aeruginosa lipase in the open conformation: the prototype for family I of bacterial lipases. J. Biol. Chem. 2000, 275: 31219–31225.
18. Rawlings, N. D., Polgár, L., and Barrett, A. J. A new family of serine-type peptidases related to prolyl oligopeptidase. Biochem. J. 1991, 279: 907–908.
19. arrett, A. J., and Rawlings, N. D. Oligopeptidases, and the emergence of the prolyl oligopeptidase family. Biol. Chem. Hoppe Seyler. 1992, 373: 353–360.
20. Polgár, L. The prolyl oligopeptidase family. Cell. Mol. Life Sci. 2002, 59: 349-362.
21. Szeltner, Z., Rea, D., Renner, V., Juliano, L., Fül?p, V., and Polgár, L. Electrostatic Environment at the Active Site of Prolyl Oligopeptidase Is Highly Influential during Substrate Binding. J. Biol. Chem. 2003, 278: 48786-48793.
[1] Fül?p V, Szeltner Z, Renner V, and Polgár L. Structures of prolyl oligopeptidase substrate/inhibitor complexes. Use of inhibitor binding for titration of the catalytic histidine residue. J. Biol. Chem. 2001, 276: 1262-1262.
[2] Szeltner Z, Rea D, Renner V, Juliano L, Fül?p V, Polgár L. Electrostatic Environment at the Active Site of Prolyl Oligopeptidase Is Highly Influential during Substrate Binding. J. Biol. Chem. 2003, 278: 48786-48793.
[3] Shan L, Mathews II, Khosla C. Structural and mechanistic analysis of two prolyl endopeptidases: Role of interdomain dynamics in catalysis and specificity Proc. Natl. Acad. Sci. 2005, 102: 3599-3604.
[4] Abbott CA, McCaughan GW, Gorrell MD.Two highly conserved glutamic acid residues in the predicted beta propeller domain of dipeptidyl peptidase IV are required for its enzyme activity. FEBS Lett. 1999, 458: 278-284
[5] Ven?l?inen JI, Juvonen RO, M?nnist? PT. Evolutionary relationships of the prolyl oligopeptidase family enzymes. Eur J Biochem. 2004 Jul;271(13):2705-15.
[6] Szeltner Z, Rea D, Juhász T, Renner V, Mucsi Z, Orosz G, Fül?p V, Polgár L. Substrate-dependent competency of the catalytic triad of prolyl oligopeptidase. J Biol Chem. 2002 Nov 22;277(47):44597-605. Epub 2002 Sep 11
[7] Harris MN, Madura JD, Ming LJ, Harwood VJ. Kinetic and mechanistic studies of prolyl oligopeptidase from the hyperthermophile Pyrococcus furiosus. J Biol Chem. 2001, 276:19310-19317.
[8] Polgár L. A potential processing enzyme in prokaryotes: oligopeptidase B, a new type of serine peptidase. Proteins. 1997, 28: 375-379.
[9] Polgár L. Oligopeptidase B: a new type of serine peptidase with a unique substrate-dependent temperature sensitivity. Biochemistry. 1999, 38: 15548-15555.
[10] Polgár L. pH-dependent mechanism in the catalysis of prolyl endopeptidase from pig muscle. Eur J Biochem. 1991, 197: 441-447
[11] Polgár L. Effects of ionic strength on the catalysis and stability of prolyl oligopeptidase. Biochem J. 1995, 312( Pt 1): 267-271.
[12] Wang Q, Yang G, Liu Y, Feng Y. Discrimination of Esterase and Peptidase Activities of Acylaminoacyl Peptidase from Hyperthermophilic Aeropyrum pernix K1 by a Single Mutation. J Biol Chem, 2006, 281: 18618-18625.
[13] Vieille C, Zeikus GJ. Hyperthermophilic enzymes: sources, uses and molecular mechanisms for thermostability, Microbiology and molecular biology reviews, 2001, 65: 1-43.
[14] Elcock AH. The stability of salt bridges at high temperatures: implications for hyperthermophilic proteins. J. Mol. Biol., 1998, 284: 489-502.
[15] Zhou HX, Toward the physical basis of thermophilic proteins: linking of enriched polar interactions and reduced heat capacity of unfolding, Biophysical Journal, 2002, 83: 3126-3133.
1. Bernatchez S, Gilbert M, Blanchard MC, Karwaski MF, Li J, Defrees S, Wakarchuk WW. Variants of the beta 1, 3-galactosyltransferase CgtB from the bacterium Campylobacter jejuni have distinct acceptor specificities. Glycobiology. 2007, 17:1333-1343.
2. Blixt O, Razi N. Chemoenzymatic synthesis of glycan libraries. Methods Enzymol. 2006, 415: 137-153.
3. Bernatchez S, Gilbert M, Blanchard MC, Karwaski MF, Li J, Defrees S, Wakarchuk WW. Variants of the beta 1, 3-galactosyltransferase CgtB from the bacterium Campylobacter jejuni have distinct acceptor specificities. Glycobiology. 2007, 17:1333-1343.
4. Ola Blixt, Nahid Razi, Enzymatic Glycosylation by Transferases. In: Fraser-Reid, B., Tatsuta, K., editors. Glycoscience. 2nd. Edition. 1361-1387
5. Shapiro HM. Practical Flow. Cytometry. 2003. Wiley, Hoboken
6. Georgiou G. Analysis of large libraries of protein mutants using flow cytometry. Adv Protein Chem. 2000, 55: 293-315.
7. Daugherty PS, Iverson BL, Georgiou G. Flow cytometricscreening of cell-based libraries. J Immunol Methods. 2000, 243: 211-227.
8. Boder ET, Wittrup KD. Optimal screening of surface-displayed polypeptide libraries. Biotechnol Prog. 1998, 14: 55-62.