摘要
随着石化能源的日益枯竭,生物柴油作为一种清洁环保、性能优良的绿色可替代能源受到广泛的关注。脂肪酶法制备生物柴油技术由于具备反应条件温和、无污染物排放等优点,发展前景广阔。但脂肪酶价格高、在有机溶剂中易失活等缺点,限制了酶法制备生物柴油工艺的推广和工业化。开发脂肪酶表面展示技术则能较好解决上述问题。表面展示脂肪酶除可回收重复利用、增强酶的稳定性外,还能省去传统固定化酶复杂而耗时的分离纯化工作,节约成本；而且酶被固定在细胞表面,底物易于进入,产物易于脱离。此外,表面展示还可作为脂肪酶定向进化的高通量筛选手段,为后续脂肪酶的分子改造提供技术平台。本论文主要研究工作和结果摘要如下：
(1)利用生物信息学方法预测了解脂耶氏酵母脂肪酶Lip2的疏水性、柔性、二级结构、三级结构等结构特征,确定了催化三联体S162、D230、H289及阴离子氧洞T88和L163,明晰了活性中心与其N端和C端的位置关系。
(2)首次以C端锚定的Cwp2作为锚定蛋白展示了Lip2。利用免疫荧光分析及平板活性检测确定Lip2成功展示在酿酒酵母INVScl细胞表面。尽管添加了不同连接序列,但所得展示脂肪酶活性仍较低,连接序列(G4S)3可在一定程度上提高脂肪酶的活性,使其酶活从4.3 U/g干细胞提高为7.6 U/g干细胞,但仍无水解橄榄油活性,结合其三维结构预测结果,我们推测Cwp2可能在一定程度上影响了脂肪酶活性中心的构象。展示脂肪酶最适温度和最适pH分别为40°C、pH 8.0；Ca2＋离子对其酶活有明显促进作用,表面活性剂Triton X-100与Tween 80对其酶活基本没有影响,其DMSO稳定性较好。此外,展示的Lip2还表现出比游离态Lip2更好的温度稳定性。
(3)以仅469个氨基酸残基的Flo1片段作为锚定蛋白,并在其与Lip2之间加入(G4S)3链接序列,成功地展示了Lip2。免疫荧光分析和平板活性检测均确认了Lip2展示在酿酒酵母细胞表面,其水解pNPC活力最高达65.2 U/g干细胞,高于以Cwp2作为锚定蛋白的展示酶活,但仍无水解橄榄油活性。其最适温度和最适pH分别为40°C、pH 8.0；相对于以Cwp2为锚定蛋白展示的Lip2,而SDS以及DMSO稳定性有所下降。上述结果说明N端锚定的Flo1片段对表面展示Lip2的活性抑制相对较小。
(4)首次利用a凝集素小亚基Aga2作为锚定蛋白展示了Lip2。利用免疫荧光检测确定Lip2成功展示在酿酒酵母细胞表面,利用平板检测证实展示Lip2具备水解橄榄油活性。表面展示Lip2最高水解pNPC酶活可达634.9U/g干细胞,远高于目前已报道的酵母表面展示脂肪酶的酶活。其温度稳定性比以Cwp2和Flo1片段作为锚定蛋白展示的Lip2为高,尤其具备良好的甲醇、乙醇耐受性,20%的甲醇、乙醇处理0.5 h后,酶活分别为初始酶活的109.4%和98.5%,远高于游离态Lip2,更适用于生物柴油的制备。
(5)通过比较三种不同的锚定蛋白对表面展示Lip2活性的影响,初步了解了不同锚定蛋白可能对脂肪酶结构造成的影响,从而粗略总结出一般脂肪酶表面展示时锚定蛋白的选择原则,即先对脂肪酶进行结构预测了解其三维结构特征,然后选择锚定结构域远离脂肪酶活性中心的细胞壁蛋白作为锚定蛋白。遵循该原则对解脂耶氏酵母脂肪酶Lip7、Lip8以及黑曲霉脂肪酶ANL进行了表面展示研究。结果表明,展示的Lip7、Lip8表现出较高活力,分别为282.9 U/g干细胞、121.3 U/g干细胞,且温度稳定性较高,50°C温浴5h后残余酶活仍保持在初始酶活的90%以上；展示的ANL酶活为43.8 U/g干细胞,也同样具备较好的温度稳定性,50°C温浴4h仍保持95%以上的残余酶活。
With the gradual depletion of fossil oil, biodiesel, a renewable, non-toxic and biodegradable fuel, has gained more attentions in recent years for its ability to replace fossil fuels. The use of lipase instead of acid or alkaline as catalyst for biodiesel production has great market potentiality due to its environment-friendly property and mild reaction conditions. However, lipases have some disadvantages, such as high price and easily being inactivated in organic solvent, which restrict industrialization of this technology. The above problem could be solved by surface display of lipase, because surface display of lipases as a whole-cell catalyst can be recycled and the stability of lipase can be greatly improved in comparison with traditional immobilization of lipases. Compared with conventional immobilized enzymes, the whole-cell catalysts have unique advantages, such as a simpler product purification and a more cost-effective downstream processing. Furthermore, because the lipases are immobilized on the surface of cells, the substrates can be simply access to the enzyme and the products can be easily released. Additionally, surface display of lipase can also serve as an efficient high-throughput screening method for protein engineering of lipase. The main results of this study were summarized as follows:
1. Through bioinformatic analysis, we have learnt many characteristics of Yarrowia lipolytica lipase Lip2, such as hydrophobicity, flexibility, secendary structure and tertiary structure. Its catalytic triad consists of S162, D230 and H289. T88 and L163 form the oxyanion hole. Its activity centre is close to the C-terminal.
2. Lip2 from Y. lipolytica was displayed on the cell surface of Saccharomyces cerevisiae using Cwp2 as an anchor protein for the first time. Successful display of the lipase on the cell surface was confirmed by immunofluorescence microscopy and halo assay. The length of linker sequences was further examined to confirm whether the correct conformation of Lip2 was maintained. The results showed that the displayed Lip2 exhibited the highest activity at 7.6 U/g-dry cell when using (G4S)3 sequence as the linker, with an optimal temperature and pH at 40℃and pH 8.0. The displayed lipase did not lose any activity after being treated with 0.1% Triton X-100 and 0.1% Tween 80 for 30 min, and it retained 92% of its original activity after incubation in 10% DMSO for 30 min. It also exhibited better thermostability than free-form Lip2 as reported previously.
3. A Flol fragment consisting of 469 amino acid residues was used as an anchor protein for surface display of Lip2 in S. cerevisiae. Successful surface display of Lip2 was also confirmed by immunofluorescence microscopy and halo assay. The results showed that the highest activity of displayed Lip2 was 65.2 U/g-dry cell, much higher than that of using Cwp2 as anchor protein. Compared with the displayed Lip2 using Cwp2 as an anchor protein, the stability against SDS and DMSO was slightly decreased. The optimal temperature and pH, as well as the thermostability were consistent with that of using Cwp2 as anchor protein.
4. It is also for the first time that the a-agglutinin was used as an anchor protein for surface display of Lip2. From immunofluorescence microscopy and halo assay was successful surface display of Lip2 in S. cerevisiae confirmed. The activity of displayed Lip2 reached 634.9 U/g-dry cell, much higher than those of using Cwp2 and Flol fragment as anchor proteins. The optimal temperature and pH was 40℃and pH 8.0. Compared with the displayed Lip2 using Cwp2 and Flol fragment as anchor proteins, the thermostability and the stability against organic solvent were much improved. Especially, the displayed Lip2 remained 109.4% and 98.5% of its original acitivity after being treated with 20% methanol and ethanol, much better than free-form Lip2, which suggests that the displayed Lip2 will be more suitable for biodiesel production.
5. By comparing the results of surface display of Lip2 using three different anchor proteins, we have preliminarily learned the influence of different anchor proteins on the conformation of displayed Lip2. Based on this, we bring forward a rough principle on how to select a suitable anchor protein for surface display of a target lipase. It says that the structure feature of the target protein should first be assayed through bioinformatics and then the cell wall protein with its anchor domain far away from the activity centre of the target protein should be chosen as the anchor protein. By following this principle, we successfully displayed Y. lipolytica lipases Lip7, Lip8 and Aspergillus niger lipase (ANL) on the surface of S. cerevisiae. The displayed Lip7 and Lip8 showed high activities, which reached 282.9 U/g-dry cell and 121.3 U/g-dry cell, respectively. The thermostability of displayed Lip7 and Lip8 were much better than that of the displayed Lip2. Each displayed lipase retained above 90% of its original activity after incubation at 50℃for 5 h. However, the surface displayed ANL exhibited a relatively low activity of 43.8 U/g-dry cell. It could also remain more than 95% of its original activity after incubation 50℃for 4 h.
引文
[1]Jaeger KE, Reetz MT. Microbial lipases form versatile tools for biotechnology[J]. Trends in Biotechnology,1998,16:396-403
[2]Reis P, Holmberg K, Watzke H, et al. Lipases at interfaces:A review[J]. Advances in Colloid and Interface Science,2009,147:237-250
[3]Schmid RD, Verger R. Lipases:Interfacial enzymes with attractive applications[J]. Angewandte Chemie International Edition,1998,37:1608-1633
[4]Villeneuve P, Muderhwa JM, Graille J, Haas MJ. Customizing lipases for biocatalysis:a survey of chemical, physical and molecular biological approaches [J]. Journal of Molecular Catalysis B:Enzymatic,2000,9:113-148
[5]Jaeger KE, Eggert T. Lipases for biotechnology [J]. Current opinion in biotechnology,2002,13:390-397
[6]Hasan F, Shah AA, Hameed A. Industrial applications of microbial lipases[J].Enzyme and Microbial Technology,2006,39:235-251
[7]Jaeger KE, Dijkstra BW, Reetz MT. Bacterial biocatalysts:Molecular biology, three-dimensional structures, and biotechnological applications of lipases[J]. Annual Review of Microbiology,1999,53:315-351
[8]Arai S, Nakashima K, Tanino T, et al. Production of biodiesel fuel from soybean oil catalyzed by fungus whole-cell biocatalysts in ionic liquids[J]. Enzyme and Microbial Technology,2010,46(1):51-55
[9]Kobayashi S. Recent developments in lipase-catalyzed synthesis of polyesters [J]. Macromolecular Rapid Communications,2009,30(4-5):237-266
[10]Takahashi S, Ueda M, Tanaka A. Function of the prosequence for in vivo folding and secretion of active Rhizopus oryzae lipase in Saccharomyces cerevisiae[J]. Applied Microbiology and Biotechnology,2001,55:454-462
[11]Kugimiya W, Otani Y, Hashimoto Y, et al. Molecular cloning and nucleotide sequence of the lipase gene from Pseudomonas fragi[J]. Biochemical and Biophysical Research Communications,1986,141:185-190
[12]Chuang YC, Chiou SF, Su JH, et al. Molecular analysis and expression of the extracellular lipase of Aeromonas hydrophila MCC-2[J]. Microbiology,1997,143: 803-12
[13]张搏,杨江科,苏华武,闫云君.脂肪酶产生菌的筛选、鉴定及其产酶条件优化[J].生物技术,2007,17：23-26
[14]Kugimiya W, Otani Y, Hashimoto Y, et al. Molecular cloning and nucleotide sequence of the lipase gene from Pseudomonas fragi[J]. Biochemical and Biophysical Research Communications,1986,141:185-190
[15]Minning S, Schmidt-Dannert C, Schmid RD. Functional expression of Rhizopus oryzae lipase in Pichia pastoris:high-level production and some properties[J]. Journal of Biotechnology,1998,66:147-156
[16]Pleiss J, Fischer M, Peiker M, et al. Lipase engineering database:Understanding and exploiting sequence-structure-function relationships[J]. Journal of Molecular Catalysis B:Enzymatic,2000,10:491-508
[17]Fischer M, Thai QK, Grieb M, et al. DWARF-a data warehouse system for analyzing protein families[J]. BMC Bioinformatics,2006,7:495-505
[18]Nardini M, Dijkstra BW. a/(3 hydrolase fold enzymes:the family keeps growing[J]. Current Opinion in Structural Biology,1999,9:732-737
[19]van Pouderoyen G, Eggert T, Jaeger KE, et al. The crystal structure of Bacillus subtilis lipase:A minimal α/β hydrolase fold enzyme[J]. Journal of Molecular Biology,2001,309:215-226
[20]Nardini M, Lang DA, Liebeton K, et al. Crystal structure of Pseudomonas aeruginosa lipase in the open conformation[J]. The Journal of Biological Chemistry,2000,275(40):31219-31255
[21]Brady L, Brzozowski AM, Derewenda ZS, et al. A serine protease triad forms the catalytic centre of a triacylglycerol lipase[J]. Nature,1990,343:767-770
[22]Schrag JD, Li Y, Wu S, et al. Ser-His-Glu triad forms the catalytic site of the lipase from Geotrichum candidum[J]. Nature,1991,351:761-764
[23]Grochulski P, Li Y, Schrag JD, et al. Insights into interfacial activation from an open structure of Candida rugosa lipase[J]. The Journal of Biological Chemistry, 1993,268:12843-12847
[24]Pleiss J, Fischer M, Schmid RD. Anatomy of lipase binding sites:the scissile fatty acid binding site[J]. Chemistry and Physics of Lipids,1998,93:67-80
[25]Jaeger KE, Ransac S, Dijkstra BW, et al. Bacterial lipase[J]. FEMS Microbiology Reviews,1994,15:29-63
[26]Verger R. Interfacial activation of lipases:facts and artifacts [J]. Trends in Biotechnology,1997,15(1):32-38
[27]Brzozowski AM, Derewenda U, Derewenda ZS, et al. A model for interfacial activation in lipases from the structure of a fungal lipase-inhibitor complex[J]. Nature,1991,351:491-494
[28]Noble MEM, Cleasby A, Johnson LN, et al. Analysis of the structure of Pseudomonas glumae lipase[J]. Protein Engineering,1994,7(4):559-562
[29]Uppenberg J, Hansen MT, Patkar S, et al. The sequence, crystal structure determination and refinement of two crystal forms of lipase B from Candida antarctica[J]. Structure,1994,2(4):293-308
[30]Ferrato F, Carriere F, Sarda L, et al. A critical reevaluation of the phenomenon of interfacial activation[J]. Methods Enzymology,1997,286:327-347
[31]Xu XB. Production of specific-structured triacylglycerols by lipase-catalyzed reactions:a review[J]. European Journal of Lipid Science and Technology,2000, 102(4):287-303
[32]Wang Y, Srivastava KC, Shen G-J, et al. Thermostable alkaline lipase from a newly isolated thermophilic Bacillus strain A30-1 (ATCC 53841)[J]. Journal of Fermentation and Bioengineering,1995,79:433-438
[33]Gilbert EJ, Cornish A, Jones CW. Purification and properties of extracellular lipase from Pseudomonas aeruginosa EF2[J]. Journal of General Microbiology, 1991,137:2223-2229
[34]Gilbert EJ, Drozd JW, Jones CW. Physiological regulation and optimization of lipase activity in Pseudomonas aeruginosa EF2[J]. Journal of General Microbiology,1991,137:2215-2221
[35]Misset O, Gerritse G, Jaeger KE, et al. The structure function relationship of the lipases from Pseudomonas aeruginosa and Bacillus subtilis[J]. Protein Engineering,1994,7:523-529
[36]Lesuisse E, Schanck K, Colson C. Purification and preliminary characterization of the extracellular lipase of Bacillus subtilis 168, an extremely basic pH-tolerant enzyme[J]. European Journal of Biochemistry,1993,216:155-160
[37]Dharmsthiti S, Luchai S. Production, purification and characterization of thermophilic lipase from Bacillus sp. THL027[J]. FEMS Microbiology Letters, 1999,179:241-246
[38]Finkelstein AE, Strawich ES, Sonnino S. Characterization and partial purification of a lipase from Pseudomonas aeruginosa[J]. Biochimica et Biophysica Acta, 1970,206:380-391
[39]Kordel M, Hofmann B, Schaumburg D, et al. Extracellular lipase of Pseudomonas sp. strain ATCC 21808:purification, characterization, crystallization and preliminary X-ray diffraction data[J]. Journal of Bacteriology,1991,173: 4836-4841
[40]Horiuti Y, Imamura S. Purification of lipase from Chromobacterium viscosum by chromatography on palmitoyl cellulose[J]. The Journal of Biochemistry,1977,81: 1639-1649
[41]Angultra J, Rodrigue Z, Aparicio LB, et al. Purification, gene cloning, amino acid sequence analysis and expression of an extracellular lipase from an Aeromonas hydrophila human isolate[J]. Applied and Environmental Microbiology,1993,59: 2411-2417
[42]Muraoka T, Ando T, Okuda H. Purification and properties of a novel lipase from Staphylococcus aureus 226[J]. Journal of Biochemistry,1982,92:1933-1939
[43]Davranov K. Microbial lipases in biotechnology [J]. Applied Biochemistry and Microbiology,1994,30:527-534
[44]Hassing GS. Partial purification and some properties of a lipase from Corynebacterium acne[J]. Biochimica et Biophysica Acta,1971,242:331
[45]Sugihara A, Tani T, Tominaga Y. Purification and characterization of a novel thermostable lipase from Bacillus sp.[J]. Journal of Biochemistry,1991,109: 211-216
[46]Lanser AC, Manthey LK, Hou CT. (2002) Regioselectivity of new bacterial lipases determined by hydrolysis of triolein[J]. Current Microbiology,2002,44: 336-340
[47]Yamamoto K, Fujiwara N. The hydrolysis of castor oil using a lipase from Pseudomonas sp. FB-24:positional and substrate specificity of the enzyme and optimum reaction conditions[J]. Bioscience, Biotechnology and Biochemistry, 1995,59:1262-1266
[48]Kaieda M, Samukaw T, Matsumoto T, et al. Biodiesel fuel production from plant oil catalyzed by Rhizopus oryzae lipase in a water-containing system without organic solvent[J]. Journal of Bioscience and Bioengineering,1999,88:627-631
[49]Zheng LY, Zhang SQ, Zhao LF, et al. Resolution of N-(2-ethyl-6-methylphenyl) alanine via free and immobilized lipase from Pseudomonas cepacia[J]. Journal of Molecular Catalysis B:Enzymatic,2006,38:119-125
[50]da Silva VCF, Contesini FJ, de Oliveira Carvalho P. Enantioselective behavior of lipases from Aspergillus niger immobilized in different supports[J]. Journal of Industrial Microbiology and Biotechnology,2009,36:949-954
[51]Bossaert WD, de Vos DE, Van Rhijn WM, et al. Mesoporous sulfonic acids as selective heterogeneous catalysts for the synthesis of monoglycerides[J]. Journal of Catalysis,1999,182(1):156-164
[52]Liu QT, Clench MR, Kinderlerer JL. Monoacylglycerols derived from butter oil by Penicillium roquefortii in suspension cultures[J]. Journal of the Science of Food and Agriculture,2002,82(5):553-558
[53]Stevenson DE, Stanley RA, Furneaux RH. Glycerolysis of tallow with immobilised lipase[J]. Biotechnology Letters,1993,15:1043-1048
[54]Shimadaa Y, Sugiharaa A, Maruyama K, et al. Production of structured lipid containing docosahexaenoic and caprylic acids using immobilized Rhizopus delemar lipase[J]. Journal of Fermentation and Bioengineering,1996,81(4): 299-303
[55]Wainer IW. The therapeutic promise of single enantiomers:introduction[J]. Human Psychopharmacology:Clinical and Experimental,2001,16(32):S73-S77
[56]Morrone R, Nicolosi G, Patti A, et al. Resolution of racemic flurbiprofen by lipase-mediated esterification in organic solvent[J]. Tetrahedron:Asymmetry, 1995,6(7):1773-1778
[57]Muralidhar RV, Chirumamilla RR, Ramachandran VN, et al. Resolution of (RS)-proglumide using lipase from Candida cylindracea[J].Bioorganic & Medicinal Chemistry,2002,10(5):1471-1475
[58]Yang JK, Guo DY, Yan YJ. Cloning, expression and characterization of a novel thermal stable and short-chain alcohol tolerant lipase from Burkholderia cepacia strain G63[J]. Journal of Molecular Catalysis B:Enzymatic,2007,45(3-4):91-96
[59]Hernandez-Martina E, Otero C. Different enzyme requirements for the synthesis of biodiesel:Novozym 435 and Lipozyme TL IM[J]. Bioresource Technology,2008, 99(2):277-286
[60]Smith GP. Filamentous Fusion phage:novel expression vector that display cloned antigens on the viron surface. Science,1985,228(4705):1315-1317
[61]Scott JK, Smith GP. Searching for peptide ligands with 811 epitop library. Science, 1990,249(4967):386-390
[62]Benhar I. Biotechnological applications of phage and cell display[J]. Biotechnology Advances,2001,19:1-33
[63]Wernerus H, Stahl S. Biotechnological applications for surface-engineered bacteria[J]. Biotechnology and Applied Biochemistry,2004,40:209-228
[64]Sergeeva A, Kolonin MG, Molldrem JJ, et al. Display technologies:Application for the discovery of drug and gene delivery agents[J]. Advanced Drug Delivery Reviews,2006,58:1622-1654
[65]Jiang J, Abu-Shilbayeh L, Rao VB. Display of a PorA peptide from Neisseria meningitidis on the bacteriophage T4 capsid surface [J]. Infection and Immunity, 1997,65:4770-4777
[66]Kuwabara I, Maruyama H, Mikawa YG, Zuberi RI, Liu FT, Maruyama IN. Efficient epitope mapping by bacteriophage lambda surface display [J]. Nature Biotechnology,1997,15:74-78
[67]Mikawa YG, Maruyama IN, Brenner S. Surface display of proteins on bacteriophage lambda heads[J]. Journal of Molecular Biology,1996,262:21-30
[68]Houshmand H, Froman G, Magnusson G. Use of bacteriophage T7 displayed peptides for determination of monoclonal antibody specificity and biosensor analysis of the binding reaction[J]. Analytical Biochemistry,1999,268:363-370
[69]Heal KG, Hill HR, Stockley PG, et al. Expression and immunogenicity of a liver stage malaria epitope presented as a foreign peptide on the surface of RNA free MS2 bacteriophage capsids[J]. Vaccine,1999,18:251-8
[70]Fernandez-Gacio A, Uguen M, Fastrez J. Phage display as a tool for the directed evolution of enzymes[J]. Trends in Biotechnology,2003,21(9):408-414
[71]Hoischen C, Fritsche C, Gumpert J, et al. Novel bacterial membrane surface display system using cell wall-less L-form of Proteus mirabilis and Escherichia coli[J]. Applied and Environmental Microbiology,2002,68(2):525-531
[72]Bingle WH, Nomellini JF, Smit J. (1997) Cell-surface display of a Pseudomonas aeruginosa strain K pilin peptide within the paracrystalline S-layer of Caulobacter crescentus[J]. Molecular Microbiology,1997,26:277-288
[73]Kim JH, Park IS, Kim BG. Development and characterization of membrane surface display system using molecular chaperon, prsA, of Bacillus subtilis[J]. Biochemical and Biophysical Research Communications,2005,334:1248-1253
[74]Lofblom J, Wernerus H, Stahl S. Fine affinity discrimination by normalized fluorescence activated cell sorting in staphylococcal surface display [J]. FEMS Microbiology Letters,2005,248:189-198
[75]Avall-Jaaskelainen S, Lindholm A, and Palva A. Surface display of the receptor-binding region of the Lactobacillus brevis S-layer protein in Lactococcus lactis provides nonadhesive lactococci with the ability to adhere to intestinal epithelial Cells[J]. Applied and Environmental Microbiology,2003,69(4): 2230-2236
[76]Desvaux M, Dumas E, Chafsey I, et al. Protein cell surface display in Gram-positive bacteria:from single protein tomacromolecular protein structure[J]. FEMS Microbiology Letters,2006,256:1-15
[77]Schreuder MP, Brekelmans S, Van Den EH, et al. Targeting of a heterologous protein to the cell wall of Saccharomyces cerevisiae[J]. Yeast,1993,9(4):399-409
[78]Mergler M, Wolf K, Zimmermann M. Development of a bisphenol A-adsorbing yeast by surface display of the Kluyveromyces yellow enzyme on Pichia pastoris[J]. Applied Microbiology and Biotechnology,2004,63:418-421
[79]Kim SY, Sohn JH, Pyun YR, et al. A cell surface display system using novel GPI-anchored proteins in Hansenula polymorpha[J]. Yeast,2002,19:1153-1163
[80]Uccelletti D, De Jaco A, Farina F, et al. Cell surface expression of a GPI-anchored form of mouse acetylcholinesterase in KlpmrlΔ cells of Kluyveromyces lactis[J]. Biochemical and Biophysical Research Communications,2002,298:559-565
[81]Yue LX, Chi ZM, Wang L, et al. Construction of a new plasmid for surface display on cells of Yarrowia lipolytica[J]. Journal of Microbiological Methods, 2008,72:116-123
[82]Tanino T, Fukuda H, Kondo Akihiko. Construction of a Pichia pastoris cell-surface display system using Flolp anchor system[J]. Biotechnology Progress, 2006,22:989-993
[83]Aguilar-Uscanga B, Francois JM. A study of the yeast cell wall composition and structure in response to growth conditions and mode of cultivation[J]. Letters in Applied Microbiology,2003,37:268-274
[84]Klis FM, Boorsma A, De Groot PWJ. Cell wall construction in Saccharomyces cerevisiae[J]. Yeast,2006,23:185-202
[85]Lipke PN, Ovalle R. Cell wall architecture in yeast:new structure and new challenges[J]. Journal of Bacteriology,1998,180(15):3735-3740
[86]Kato M, Kuzuhara Y, Maeda H, et al. Analysis of a processing system for proteases using yeast cell surface engineering:conversion of precursor of proteinase A to active proteinase A[J]. Applied Genetics and Molecular Biotechnology,2006,72:1229-1237
[87]Mo AY, Park SM, Kim YS, et al. Expression of fungal phytase on the cell surface of Saccharomyces cerevisiae[J]. Biotechnology and Bioprocess Engineering,2005, 10:576-581
[88]Breinig F, Diehl B, Rau S, et al. Cell surface expression of bacterial esterase A by Saccharomyces cerevisiae and its enhancement by constitutive activation of the cellular unfolded protein response[J]. Applied and Environmental Microbiology, 2006,72(11):7140-7147
[89]Sato N, Matsumoto T, Ueda M, et al. Long anchor using Flol protein enhances reactivity of cell surface-displayed glucoamylase to polymer substrates[J]. Applied Microbiology and Biotechnology,2002,60:469-474
[90]Verstrepen KJ, Derdelinckx G, Verachtert H, et al. Yeast flocculation:what brewers should know[J]. Applied Microbiology and Biotechnology,2003, 61:197-205
[91]Matsumoto T, Fukuda H, Ueda M, et al. Construction of yeast strains with high cell surface lipase activity by using novel display systems based on the Flolp flocculation functional domain[J]. Applied and Environmental Microbiology,2002, 68(9):4517-4522
[92]Ecker M, Deutzmann R, Lehle L, et al. Pir proteins of Saccharomyces cerevisiae are attached to β-1,3-glucan by a new protein-carbohydrate linkage[J]. The Journal of Biological Chemistry,2006,281(17):11523-11529
[93]Zhao H, Shen ZM, Kahn PC, et al. Interaction of α-agglutinin and a-agglutinin, Saccharomyces cerevisiae sexual cell adhesion molecules[J]. Journal of Bacteriology,2001,183(9):2874-2880
[94]Tanino T, Fukuda H, Kondo A. Construction of a Pichia pastoris cell-surface display system using Flolp anchor system[J]. Biotechnology Progress,2006,22: 989-993
[95]Wang QJ, Li L, Chen M. Construction of a Novel Pichia pastoris cell-surface display system based on the cell wall protein Pirl [J]. Current Microbiology,2008, 56:352-357
[96]Hoogenboom HR, de Bruinea AP, Hufton SE, et al. Antibody phage display technology and its applications [J]. Immunotechnology,1998,4(1):1-20
[97]Tong AHY, Drees B, Nardelli G, et al. A combined experimental and computational strategy to define protein interaction networks for peptide recognition modules[J]. Science,2002,295:321-324
[98]McKenzie KM, Videlock EJ, Splittgerber U, et al. Simultaneous identification of multiple protein targets by using complementary-DNA phage display and a natural-product-mimetic probe[J], Angewandte Chemie International Edition,2004, 43:4052-4055.
[99]Kang SM, Rhee JK, Kim EJ, et al. Bacterial cell surface display for epitope mapping of hepatitis c virus core antigen[J]. FEMS Microbiology Letters,2003, 26(2):347-353
[100]Shibasaki S, Maeda H, Ueda M. Molecular display technology using yeast-Arming Technology[J]. Analytical Sciences,2009,25:41-49
[101]Fernandez-Gacio A, Uguen M, Fastrez J. Phage display as a tool for the directed evolution of enzymes[J]. Trends in Biotechnology,2003,21(9):403-414
[102]Kondo A, Shigechi H, Abe M, et al. High-level ethanol production from starch by a flocculent Saccharomyces cerevisiae strain displaying cell-surface glucoamylase[J]. Applied Microbiology and Biotechnology,2002,58:291-296
[103]Shigechi H, Fujita Y, Koh J, et al. Energy-saving direct ethanol production from low-temperature-cooked corn starch using a cell-surface engineered yeast strain co-displaying glucoamylase and a-amylase[J]. Biochemical Engineering Journal, 2004,18:149-153
[104]Shigechi H, Koh J, Fujita Y, et al. Direct production of ethanol from Rraw corn starch via fermentation by use of a novel surface-engineered yeast strain codisplaying glucoamylase and a-amylase[J]. Applied and Environmental Microbiology,2004,70(8):5037-5040
[105]Haan RD, Rose SH, Lynd LR, et al. Hydrolysis and fermentation of amorphous cellulose by recombinant Saccharomyces cerevisiae[J]. Metabolic Engineering, 2007,9:87-94
[106]Grange DCL, Pretorius IS, Claeyssens M, et al. Degradation of xylan to D-xylose by recombinant Saccharomyces cerevisiae coexpressing the Aspergillus niger a-xylosidase (xlnD) and the Trichoderma reesei xylanase Ⅱ(xyn2) Genes[J]. Applied and Environmental Microbiology,2001,67(12):5512-5519
[107]Katahira S, Fujita Y, Mizuike A, et al. Construction of a xylan-fermenting yeast strain through codisplay of xylanolytic enzymes on the surface of xylose-utilizing Saccharomyces cerevisiae cells[J]. Applied and Environmental Microbiology, 2004,70(9):5407-5414
[108]Shibasaki S, Ueda M, Ye K, et al. Creation of cell surface-engineered yeast which can emit different fluorescence in response to the glucose concentration[J]. Applied Microbiology and Biotechnology,2001,57:528-533
[109]Shibasaki S, Ninomiya Y, Ueda M, et al. Intelligent yeast strain with the ability of self-monitoring of the concentrations of intra-and extracellular phosphate or ammonium ion by emission of fluorescence from the cell surface[J]. Applied Microbiology and Biotechnology,2001,57:702-707
[110]Vinopal S, Ruml T, Kotrba P. Biosorption of Cd2+ and Zn2+ by cell surface-engineered Saccharomyces cerevisiae[J]. International Biodeterioration & Biodegradation,2007,60(2):96-102
[111]Wang A, Mulchandani A, Chen W. Specific adhesion to cellulose and hydmlysis of organophosphate nerve agents by a genetically engineered Escherichia coli strain with a surface-expressed cellulose-binding domain and organophosphorus hydrolase[J]. Applied and Environmental Microbiology,2002,68(4):1684-1689
[112]Takayama K, Suye S, Kuroda K, et al. Surface display of organophosphorus hydrolase on Saccharomyces cerevisiae[J]. Biotechnology Progress,2006,22: 939-943
[113]Rizos K, Lattemann CT, Bumann D, et al. Efficacious surface exposure of antigenic UreA fragments from Helicobacter pylori in Salmonella vaccine strains[J]. Infection and Immunity,2003,71(11):6320-6328
[114]Danielsen S, Eklund M, Deussen HJ, et al. In vitro selection of enzymatically active lipase variants from phage libraries using a mechanism-based inhibitor[J]. Gene,2001,272(1-2):267-274
[115]Droge MJ, Ruggeberg CJ, van der Sloot AM, at al. Binding of phage displayed Bacillus subtilis lipase A to a phosphonate suicide inhibitor [J]. Journal of Biotechnology,2003,101(1):19-28
[116]Droge MJ, Boersma YL, van Pouderoyen G, et al. Directed evolution of Bacillus subtilis lipase A by use of enantiomeric phosphonate inhibitors:crystal structures and phage display selection[J]. ChemBioChem,2006,7(1):149-157
[117]Lee SH, Choi JI, Park SJ, et al. Display of bacterial lipase on the Escherichia coli cell surface by using FadL as an anchoring motif and use of the enzyme in enantioselective biocatalysis[J]. Applied and Environmental Microbiology,2004, 70(9):5074-5080
[118]Lee SH, Choia JH, Parka SH, et al. Enantioselective resolution of racemic compounds by cell surface displayed lipase[J]. Enzyme and Microbial Technology, 2004,35(5):429-436
[119]Lee SH, Choi JI, Han MJ, et al. Display of lipase on the cell surface of Escherichia coli using OprF as an anchor and its application to enantioselective resolution in organic solvent[J]. Biotechnology and Bioengineering,2005,90(2):223-230
[120]Lee SH, Lee SY, Park BC. Cell surface display of lipase in Pseudomonas putida KT2442 using OprF as an anchoring motif and its biocatalytic applications[J]. Applied and Environmental Microbiology,2005,71(12):8581-8586
[121]Jung HC, Ko S, Ju SJ, et al. Bacterial cell surface display of lipase and its randomly mutated library facilitates high-throughput screening of mutants showing higher specific activities[J]. Journal of Molecular Catalysis B:Enzymatic, 2003,26(3):177-184
[122]Jung HC, Kwon SJ, Pan JG. Display of a thermostable lipase on the surface of a solvent-resistant bacterium, Pseudomonas putida GM730, and its applications in whole-cell biocatalysis[J]. BMC Biotechnology,2006,6:23
[123]Becker S, Theile S, Heppeler N, et al. A generic system for the Escherichia coli cell-surface display of lipolytic enzymes[J]. FEBS Letters,2005,579(5): 1177-1182
[124]Narita J, Okano K, Tateno T, et al. Display of active enzymes on the cell surface of Escherichia coli using PgsA anchor protein and their application to bioconversion[J]. Applied Microbiology and Biotechnology,2006,70(5):564-572
[125]Tsuchiya A, Kobayashi G, Yamamoto H, et al. Production of a recombinant lipase artificially localized on the Bacillus subtilis cell surface[J]. FEMS Microbiology Letters,1999,176(2):373-378
[126]Kobayashi G, Toida J, Akamatsu T, et al. Accumulation of an artificial cell wall-binding lipase by Bacillus subtilis wprA and/or sigD mutants [J]. FEMS Microbiology Letters,2000,188(2):165-169
[127]Schreuder MP, Mooren ATA, Toschka HY, et al. Immobilizing proteins on the yeast cells[J]. Trends Biotechnology,1996,14:115-120
[128]Washida M, Takahashi S, Ueda M, et al. Spacer-mediated display of active lipase on the yeast cell surface. Applied Microbiology and Biotechnology,2001,56(5-6): 681-686
[129]Kato M, Fuchimoto J, Tanino T, et al. Preparation of a whole-cell biocatalyst of mutated Candida antarctica lipase B (mCALB) by a yeast molecular display system and its practical properties [J]. Applied Microbiology and Biotechnology, 2007,75(3):549-555
[130]Tanino T, Aoki T, Chung WY, et al. Improvement of a Candida antarctica lipase B-displaying yeast whole-cell biocatalyst and its application to the polyester synthesis reaction[J]. Applied Microbiology and Biotechnology,2009,82:59-66
[131]Matsumoto T, Fukuda H, Ueda M, et al. Construction of yeast strains with high cell surface lipase activity by using novel display systems based on the Flolp flocculation functional domain[J]. Applied and Environmental Microbiology,2002, 68(9):4517-4522
[132]Tanino T, Fukuda H, Kondo A. Construction of a Pichia pastoris cell-surface display system using Flolp anchor system[J]. Biotechnology Progress,2006,22(4): 989-993
[133]Jiang ZB, Song HT, Gupta N, et al. Cell surface display of functionally active lipases from Yarrowia lipolytica in Pichia pastoris[J]. Protein Expression and Purification,2007,56(1):35-39
[134]He XL, Chen BQ, Tan TW. Enzymatic synthesis of 2-ethylhexyl esters of fatty acids by immobilized lipase from Candida sp.99-125[J]. Journal of Molecular Catalysis B:Enzymatic,2002,18 (4-6):333-339
[135]Guieysse D, Sandoval G, Faure L, et al. New efficient lipase from Yarrowia lipolytica for the resolution of 2-bromo-arylacetic acid esters[J]. Tetrahedron: Asymmetry,2004,15 (22):3539-3543
[136]Deng L, Tan TW, Wang F, et al. Enzymatic production of fatty acid alkyl esters with a lipase preparation from Candida sp.99-125 [J]. European Journal of Lipid Science and Technology,2003,105 (12):727-734
[137]Fickers P, Benetti PH, Wache Y, et al. Hydrophobic substrate utilisation by the yeast Yarrowia lipolytica, and its potential applications[J]. FEMS Yeast Research, 2005,5 (6-7):527-543
[138]Frishman D, Argos P. Incorporation of non-local interactions in protein secondary structure prediction from the amino acid sequence[J]. Protein Engineering,1996, 9(2):133-142
[139]Geourjon C, Deleage G. SOPMA:significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments[J]. Bioinformatics,1995,11(6):681-684
[140]Rost B, Sander C, Schneider R. PHD-an automatic mail server for protein secondary structure prediction[J]. Bioinformatics,1993,10(1):53-60
[141]张海霞,唐焕文,张立震,等.蛋白质二级结构预测方法的评价[J].计算机与应用化学,2003,(3)：735-739
[142]李明,苏显中,于敏,等.蛋白质结构预测研究进展[J].生物技术,2009,19(3)：87-90
[143]Breinig F, Schmitt MJ. Spacer-elongated cell wall fusion proteins improve cell surface expression in the yeast Saccharomyces cerevisiae[J]. Applied Microbiology and Biotechnology,2002,58:637-644
[144]Yu MG, Qin SW, Tan TW. Purification and characterization of the extracellular lipase Lip2 from Yarrowia lipolytica[J].Process Biochemistry,2007,42 (3): 384-391
[145]Van der Vaart JM, te Biesebeke R, Chapman JW, et al. Comparison of cell wall proteins of Saccharomyces cerevisiae as anchors for cell surface expression of heterologous proteins[J]. Applied and Environmental Microbiology,1997,63: 615-620
[146]Ryckaert S, Martens V, De Vusser K, et al. Development of a S. cerevisiae whole cell biocatalystfor in vitro sialylation of oligosaccharides[J]. Journal of Biotechnology,2005,119:379-388
[147]Teunissen AWRH, Holub E, Van Der Huchtt J, et al. Sequence of the open reading frame of the FLOl gene from Saccharomyces cerevisiae[J]. Yeast,1993,9: 423-427
[148]Boder ET, Wittrup KD. Yeast surface display for screening combinatorial polypeptide libraries[J]. Nature biotechnology,1997,15:553-557
[149]Abe H, Ohba M, Shimma Y, et al. Yeast cells harboring human α-1,3-fucosyltransferase at the cell surface engineered using Pir, a cell wall-anchored protein[J]. FEMS Yeast Research,2004,4:417-425
[150]Straver MH, Smit Q, Kijne JW. Purification and partial characterization of a flocculin from brewer's yeast[J]. Applied and Enviromental Microbiology,1994, 60:2754-2758
[151]Kobayashi O, Hayashi N, Kuroki R, et al. Region of Flo1 Proteins Responsible for Sugar Recognition[J]. Journal of Bacteriology,1998,180(24):6503-6510
[152]Sato N, Matsmnoto T, Ueda M, et al. Long anchor using Flo1 protein enhances reactivity of cell surface-displayed glucoamylase to polymer substrates [J]. Applied Microbiology and Biotechnology,2002,60:469-474
[153]Pepper LR, Cho YK, Boder ET, et al. A Decade of Yeast Surface Display Technology:Where Are We Now?[J]. Combinatorial Chemistry & High Throughput Screening,2008,11:127-134
[154]Fickers P, Fudalej F, Le Dalla MT, et al. Identification and characterisation of LIP7 and LIP8 genes encoding two extracellular triacylglycerol lipases in the yeast Yarrowia lipolytica[J]. Fungal Genetics and Biology,2005,42:264-274
[155]Kwon DY, Hong YJ, Yoon SH. Enantiomeric synthesis of (S)-2-methylbutanoic acid methyl ester, apple flavor, using lipases in organic solvent [J]. Journal of Agricultural and Food Chemistry,2000,48,524-530
[156]Contesini FJ, Carvalho PO. Esterification of (RS)-Ibuprofen by native and commercial lipases in a two-phase system containing ionic liquids[J]. Tetrahedron: Asymmetry,2006,17:2069-2073
[157]Da Silva VCF, Contesini FJ, Carvalho PD. Enantioselective behavior of lipases from Aspergillus niger immobilized in different supports[J]. Journal of industrial microbiology,2009,36:949-954
[158]Da Silva VCF, Contesini FJ, Carvalho PD. Characterization and catalytic activity of free and immobilized lipase from Aspergillus niger:a comparative study[J]. Journal of Brazilian Chemical Society,2008,19:1468-1474
[159]Shu ZY, Yan YJ, Yang JK, et al. Aspergillus niger lipase:gene cloning, over-expression in Escherichia coli and in vitro refolding[J]. Biotechnology Letters,2007,29:1875-1879
[160]舒正玉,杨江科,闫云君.黑曲霉F044脂肪酶的分离纯化及酶学性质研究[J].生物工程学报,2007,23：96-100
[161]Hatzinikolaou DG, Macris JB, Christakopoulos, et al. Production and partial characterisation of extracellular lipase from Aspergillus niger[J]. Biotechnology Letters,1996,18:547-552
[162]Namboodiri VMH, Chattopadhyaya R. Purification and biochemical characterization of a novel thermostable lipase from Aspergillus niger[J]. Lipids, 2000,35,495-502
[163]Pokorny D, Cimerman A, Steiner W. Aspergillus niger lipases:induction, isolation and characterization of two lipases from a MZKI Al 16 strain[J]. Journal of Molecular Catalysis B:Enzymatic,1997,2:215-222
