跳到主要內容

臺灣博碩士論文加值系統

(35.172.223.251) 您好!臺灣時間:2022/08/17 00:51
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

: 
twitterline
研究生:陳有綸
研究生(外文):Chen, Yew-loom
論文名稱:利用定向演化的方法篩選嗜鹼之木聚糖酵素及其特性分析
論文名稱(外文):Directed evolution and characterization of alkalophilic xylanases
指導教授:鄭國展
指導教授(外文):Cheng, Kuo-Joan
學位類別:博士
校院名稱:國防醫學院
系所名稱:生命科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:英文
中文關鍵詞:木聚糖酵素定向演化嗜鹼酵素
相關次數:
  • 被引用被引用:0
  • 點閱點閱:584
  • 評分評分:
  • 下載下載:38
  • 收藏至我的研究室書目清單書目收藏:0
本實驗所使用的木聚糖酵素 (xylanase) 基因係從一種生存於牛胃的真菌 —
Neocallimastix patriciarum 所分離出來. 本實驗的目的在改造此木聚糖酵素, 以使它更嗜鹼; 酵素嗜鹼表示在鹼性環境下具有較高活性. 本實驗採用定向演化 (Directed Evolution) 的觀念從事改造酵素工作; 使用的材料是xynC 之N 端具活性的區域. 嗜鹼的木聚糖酵素較適於紙漿之脫色, 其次, 木聚糖 (xylan) 在鹼性的環境下溶解度較高, 使嗜鹼木聚糖酵素更適於運用在工業用途上, 因而促使本實驗之進行.
定向演化之執行必須有許多突變基因以便人工所創造的環境可篩選最適的基因. 其突變基因庫的建立是利用易錯聚合酵素連鎖反應 (error-prone PCR) 的方法; 變株 xyn-O9D (含有 I97S點突變) 首先被篩選到, 經由分析純化酵素的酸鹼活性圖 (pH-activity profile), xyn-O9D 確實較野生株 xyn-CD/WT 嗜鹼. 然後以xyn-O9D 的基因當材料繼續製造突變株, 繼續篩選, 並成功得到另外十一個嗜鹼突變株. 它們含有的突變如下: D65N, D65E, D65G, D65A, D65V, E24V, S128F, N164D, K168N, L208F, G214V 以及 V218A.
本實驗成功將木聚糖酵素的最適酸鹼度提高三個 pH單位. 證明在此之篩選方法為可行. 此篩選方法亦可運用於它種酵素. 至於這些製造出來的突變株是否真能用於工業, 有待實驗證明. 本實驗亦對某些木聚糖酵素的其他特性, 如耐熱, 嗜熱, 耐鹼與酵素動力學作若干分析, 並試圖解釋某些現象. 至於更深入詳細的分子運作原理須由精密儀器如 X-光繞射儀作進一步分析. 本實驗所找到的嗜鹼胺基酸突變對於酵素的嗜鹼機制的研究有一定的幫助.

A xylanase gene from the ruminal fungus Neocallimastix patriciarum was cloned and expressed in Escherichia coil. The goal of the study was to make more alkalophilic xylanases by the approach of directed evolution, using the catalytic domain of xynC gene as the starting material.
The mutant xylanases derived from error-prone PCR, which carried I97S and one or two other amino acid mutations, were named as PCR-generated mutant xylanases. The pH-activity profiles of those PCR-generated mutant xylanases were compared to that of wild type. The composite mutant of xyn-CDBFV with seven substitutions (E24V, I97S, S128F, N164D, K168N, G214V and V218A) was achieved and displayed much higher alkalophilicity than the wild type. The xyn-CDBFV xylanase gene was then cloned into pPICZαA expression vector and the resultant xylanase-expression vector was used to transform Pichia pastoris cells. The composite xylanase of xyn-CDBFN ( with the substitutions of E24V, D65N, I97S, S128F, N164D, K168N, L208F, G214R and V218A ) had optimal pH values of 6.0∼9.0. The pH optima of the xyn-CD/WT xylanase had been greatly increased by 3 units in this study, using the approaches of directed evolution and site-directed mutagenesis.
The study describes an useful and powerful screening method to find alkalophilic mutant xylanases; the study also presents a man-made mutant xylanase that was much better for the application of bio-bleaching than the wild type. The real usefulness of xyn-CDBFV and Pichia-xyn-CDBFV xylanases for bio-bleaching remains to be evaluated. The amino acid substitutions found by the screening method supply a potential basis for the study of the mechanism of alkalophilicity of xylanases and other enzymes.

Abstract in Chinese 1
Abstract 3
Introduction 7
Materials and Methods 18
1. The xylanase C gene from Neocallimastix patriciarum 18
2. Construction of an expressing vector consisting of the catalytic domain of
xynC gene 18
3. Expression and purification of xyn-CD/WT and the mutant xylanases 20
4. Error-prone PCR and construction of the libraries of variant xyn-CD/WT xylanase gene 21
5. Screening for alkalophilic xylanase variants 21
6. Two step selection process 22
7. Site-directed mutagenesis 22
8. Xylanase activity and pH-activity profile assays 23
9. Assay of resistance of the xylanases to the alkaline environment 24
10. Assay of thermostability and temperature optima of the xylanases 24
11. Kinetic study 24
12. Expression of xyn-CDBFV xylanase gene in Pichia pastoris 25
Results 27
1. The pH-activity profile of xyn-CD/WT xylanase 27
2. The pH-activity profile of xyn-O9D xylanase derived from xyn-CD/WT 27
3. The pH-activity profiles of the six non-D65 alkalophilic mutant xylanases derived from xyn-O9D using error-prone PCR 28
4. The pH-activity profiles of the five D65 alkalophilic mutant xylanases derived from xyn-O9D xylanase using error-prone PCR 29
5. The pH-activity profiles of the two D65 mutant xylanases derived from xyn-O9D using site-directed mutagenesis 31
6. The pH-activity profiles of those composite mutant xylanases made by using site-directed mutagenesis 32
7. The specific activities of those D65 mutant xylanases 34
8. The specific activities of those non-D65 mutant xylanases 34
9. The temperature-activity profiles of xyn-CD/WT and some of those non-D65 mutant xylanases 35
10. The thermostability of xyn-CD/WT, xyn-CDBV and xyn-CDBFV 36
11. xyn-CDBFV mutant xylanase is alkaline-stable and alkaline-activated 36
12. The shift of the pH-activity profile of xyn-CDBFV measured at 62℃, as compared to that at 47℃ 36
13. The shift of temperature-activity profile of xyn-CDBFV when measured at pH 9.0 as compared to that at pH 6.0 37
14. The expression of xyn-CDBFV gene in Pichia pastoris cells, and the volume
and mass specific activities of Pichia-xyn-CDBFV 37
15. The pH-activity profiles of Pichia-xyn-CDBFV, which were measured at 47℃ and 62℃ 38
16. The temperature-activity profiles of Pichia-xyn-CDBFV when measured at pH 6.0 and 9.0 39
17. The thermostability of Pichia-xyn-CDBFV 39
Discussion 40
1. The screening method used in this study to select alkalophilic mutant xylanases was useful 40
2. The xyn-CDBFV xylanase has higher potential than xyn-CD/WT for industrial applications 41
3. To interpret how these amino acid substitutions of those non-D65 and xyn-CDBFV mutant xylanases help increase the alkalophilicity 41
4. To interpret how these amino acid substitutions of those D65 mutant xylanases help improve the alkalophilicity 43
5. Why the specific activities of D65 mutant xylanases are much lower than that of xyn-CD/WT and xyn-O9D? 46
6. The kinetic data of these D65 and non-D65 mutant xylanases 47
7. To interpret why the temperature optima and thermostability of some of the non-D65 xylanases are increased 48
8. The properties of alkaline-activation and alkaline-stability of xyn-CDBFV make it better for pulp and paper industry 49
9. Pichia-xyn-CDBFV is better than xyn-CDBFV for pulp and paper industry in at least three ways 49
10. The shifts of pH-activity and temperature-activity profiles of xyn-CDBFV and Pichia-xyn-CDBFV when measured at different pH and temperature 50
References 52

REFERENCES
1. Timell, T.E. (1967) Recent progress in the chemistry of wood hemicelluloses. Wood Sci. Technol. 1, 45-70.
2. Aspinall, G.O. (1959) Structural chemistry of the hemicelluloses. Adv. Carbohydr. Chem. 14, 429-468.
3. Timell, T.E. (1965) Wood hemicelluloses: Part II. Carbohydr. Chem. 20, 409-483.
4. Kulkarni, N., Shendye, A., Rao, M. (1999) Molecular and biotechnological aspects of xylanases. FEMS Microbio. Rev. 23, 411-456.
5. Viikari, L., Kantelinen, A., Sundquist, J. and Linko, M. (1994) Xylanases in bleaching: From an idea to the industry. FEMS Micrbio. Rev. 13, 335-350.
6. Beck, C.I. and Scoot, D. (1974) Enzymes in foods ﹣for better or worse. Adv. Chem. Ser. 138, 1-17.
7. Biely, P. (1985) Microbial xylanolytic systems. Trends Biotechnol. 3, 288-290.
8. McCleary, B.V. (1986) Enzymatic modification of plant polysaccharides. Int. J. Macromol. 8, 349-354.
9. Wong, K.K.Y. and Saddler, N.J. (1992) Applications of hemicellulases in the food, feed, and pulp and paper industries,. In: Hemicelluloses and Hemicellulases (Coughlen, P.P. and Hazlewood, G.P., Eds.), pp. 127-143. Portland Press, London.
10. Chauvt, J.M., Comtat, J. and Noe, p. (1987) Assistance in bleaching of never-tired pulps by the use of xylanases consequences on pulp properties. Proceedings of 4th International Congress on Wood and Pulping Industry, Paris, April 27-30, pp. 325-327.
11. Jager, A., Sinner, M., Purkarthofer, H., Esterbaure, H. and Ditzelmuller, G. (1992) Biobleaching with xylanse from a thermophilic fungus. In: Biotechnology in the pulp and paper industry (Kuwahara, M. and Shimada, M., Eds.) pp. 115-121. UNI, Tokyo.
12. Kirk, T.K. and Jeffries, T.W. (1996) Enzymes for pulp and paper processing. Chapter 1: Roles for microbial enzymes in pulp and paper processing, 1996 American Chemical Society, pp. 3-14.
13. Viikari, L., Ranua, M., Kantelinen, A., Sundquist, J. and Linko, M. (1986) Biotechnology in the pulp and paper industry, Stockholm, Sweden, pp. 67-69.
14. Bajpai, P. (1999) Application of enzymes in the pulp and paper industry. Biotechnol. Prog. 15, 147-157.
15. Paice, M.G., Bernier, R.Jr. and Jurasek, L. (1988) Viscosity-enhancing bleaching of hardwood kraft pulp with xylanase from a cloned gene. Biotechnol. Bioeng. 32, 235-239.
16. Clark, T.A., McDonald, A.G., Senior, D.J. and Mayers, P.R. (1990) Mannanase and xylanase treatment of softwood chemical pulps: effects on pulp properties and bleachability. In: Biotechnology in Pulp and Paper Manufacture (Kirk, T.K. and Chang, H.M. Eds.), pp. 153-167. Butterworth-Heinenmann, Boston, M.A.
17. du Manoir, J.R., Hamilton, J., Senior, D.J., Bernier, R.L., Grant, J.E. and Mooser, L.E. (1991) Biobleaching of kraft pulps with cellulase free xylanase. Proc. Int. Pulp Bleaching Conf., Stockholm, pp. 123-138.
18. Shoham, Y., Schwartz, Z., Khasin, A., Gat, O., Zosim, Z. and Rosenberg, E. (1992) Delignification of wood pulp by a thermostable xylanase from Bacillus stearotherphilus strain T-6. Biodegradation. 3, 207-218.
19. Morris, D.D., Gibbs, M.D., Chin, C.W.J., Koh, M.H., Wong, K.K., Allison, R.W., Nelson, P.J. and Bergquist, P.L. (1998) Cloning of the xynB gene from Dictyoglomus thermophilum Rt46B.1 and action of the gene product on kraft pulp. Applied and Environmental Microbiology. May, pp. 1759-1765.
20. Baraznenok, V.A., Becker, E.G., Ankudimova, N.V. and Okunev, N.N. (1999) Characterization of neutral xylanases from Chaetomium cellulolyticum and their biobleaching effect on eucalyptus pulp. Enzyme Microb. Technol. 25, 651-659.
21. Georis, J., Giannotta, F., Buyl, E.D., Granier, B. and Frere, J-M. (2000) Purification and properties of three endo-β-1,4-xylanases produced by Streptomyces sp. strain S38 which differ in their ability to enhance the bleaching of kraft pulps. Enzyme Microb. Technol. 26, 178-186.
22. Bim, M. and Franco, T.T. (2000) Extraction in aqueous two-phase systems of alkaline xylanase produced by Bacillus pumilus and its application in kraft pulp bleaching. J. Chromatography B. 743, 349-356.
23. Yllner, S., Ostberg, K. and Stockman, L. (1957) Svensk Papp. 60, 795.
24. Axelsson, S., Croon, I. And Enstrom, B. (1962) Svensk Papperstidn. 65, 693.
25. Meller, A. (1965) Holzforschung. 19, 118.
26. Gierrer, J. and Wannstrom, S. (1984) Holzforschung. 38, 181.
27. Viikari, L., Suurnakki, A. and Buchert, J. (1996) Enzymes for Pulp and Paper Processing. Chapter 2: Enzyme-aided bleaching of kraft pulps: fundamental mechanisms and practical applications. 1996 American Chemical Society. pp. 15-24.
28. Tamblyn Lee, J.M., Hu, Y., Zhu, H., Cheng, K.J., Krell, P.J. and Forsberg C.W. (1993) Cloning of a xylanase gene from the riminal fungus Neocallimastix patriciarum 27 and its expression in Escherichia coli. C. J. Microbio. 39, 134-139.
29. Liu, J.H., Selinger, B.L., Tsai, C.H. and Cheng, K.J. (1999) Characterization of a Neocallimastix patriciarum xylanase gene and its product. C. J. Microbio. 45, 970-974.
30. Gruber, K., Klintschar, G., Hayn, M., Schlacher, A., Steiner, W. and Kratky, C. (1998) Thermophilic xylanase from Thermomyces lanuginosus: high-resolution X-ray structure and modeling studies. Biochemistry. 37, 13475-13485.
31. Georis, J., Esteves, F.D., Lamotte-Brasseur, J., Bougnet, V., Devreese, B., Giannotta, F., Granier, B. and Frere, J.-M. (2000) An additional aromatic interaction improves the thermostability and thermophilicity of a mesophilic family 11 xylanase: structural basis and molecular study. Protein Sci. 9, 466-475.
32. Koshland, D.E. (1953) Stereochemistry and the mechanism of enzymatic reactions. Biol. Rev. Camb. Philos. Soc. 28, 416-436.
33. Sinnott, M.L. (1990) Catalytic mechanisms of enzymic glycosyl transfer. Chem Rev. 90, 1171-1202.
34. Davies, G. and Henrissat, B. (1995) Structures and mechanisms of glycosyl hydrolases. Structure 3, 853-859.
35. Bayer, E.A., Shimon, L.J.W., Shoham, Y. and Lamed, R. (1998) Cellulosomes-structure and ultrastructure. J. Struct. Biol. 124, 221-234.
36. Joshi, M.D., Sidhu, G., Pot, I., Brayer, G.D., Withers, S.G. and Mclntosh, L.P. (2000) Hydrogen bonding and catalysis: a novel explanation for how a single amino acid substitution can change the pH optimum of a glycosidase. J. Mol. Biol. 299, 255-279.
37. Okazaki, W., Akiba, T., Horikoshi, K. and Akahoshi, R. (1984) Production and properties of two types of xylanases from alkalophilic thermophilic Bacillus sp. Appl. Microbiol. Biotechnol. 19, 335-340.
38. Dey, D., Hinge, J., Shendye, A. and Rao, M. (1992) Purification and properties of extracellular endoxylanases from alkalophilic thermophilic Bacillus sp. C. J. Microbio. 38, 436-442.
39. Nakamura, S., Wakabayashi, K., Nakai, R., Aono, R. and Horikoshi, K. (1993) Purification and some properties of an alkaline xylanase from alkalophilic Bacillus sp. 41M-1. Appl. Environm. Microbiol. 59, 2311-2316.
40. Nakamura, S., Nakai, R., Wakabayashi, K., Ishiguro, Y., Aono, R and Horikoshi, K. (1994) Thermophilic alkaline xylanase from newly isolated alkalophilic and thermophilic Bacillus sp. strain TAR-1. Biosci. Biotech. Biochem. 58, 78-81.
41. Beg, Q.K., Kapoore, M., Mahajan, L. and Hoondal, G.S. (2001) Microbial xylanases and their industrial applications: a review. Appl. Microbiol. Biotechnol. 56, 326-338.
42. Berrin, J.-G., Williamson, G., Puigserver, A., Chaix, J.-C., McLauchlan, R.W. and Juge, N. (2000) High-level production of recombinant fungal endo-β-1,4-xylanase in the methylotrophic yeast Pichia pastoris. Protein Expres. Purifi. 19, 179-187.
43. Russell, A.J. and Fersht, A.R. (1987) Rational modification of enzyme catalysis by engineering surface charge. Nature. 328, 496-500.
44. Cha, J. and Batt, C.A. (1998) Lowering the pH optimum of D-xylose isomerase: the effect of mutations of the negatively charged residues. Mol. Cells. 8, 374-382.
45. Arase, A., Yomo, T., Urabe, I., Hata, Y., Katsube, Y and Okada, H. (1993) Stabilization of xylanase by random mutagenesis. FEBS Letters. 316, 123-127.
46. Nishiha, Y., Harada, N., Teshima, S., Yamashita, M., Fujii, I., Hirayama, N. and Murooka, Y. (1997) Improvement of thermal stability of Streptomyces cholesterol oxidase by random mutagenesis and a structural interpretation. Protein Eng. 10, 231-235.
47. Giver, L., Gershenson, A., Freskgard, P.-O. and Arnold, F.H. (1998) Dircted evolution of a thermostable esterase. Proc. Natl. Acad. Sci. USA. 95, 12809-12813.
48. Zhao, H. and Arnold, F.H. (1999) Directed evolution converts subtilisin E into a functional equivalent of thermitase. Protein Eng. 12, 47-53.
49. Song, J.K. and Rhee, J.S. (2000) Simultaneous enhancement of thermostability and catalytic activity of phospholipase A1 by evolutionary molecular engineering. 66, 890-894.
50. Shibuya, H., Kaneko, S. and Hayashi, K. (2000) Enhancement of the thermostability and hydrolytic activity of xylanase by random gene shuffling. Biochem. J. 349, 651-656.
51. Fanutti, C., Ponyi, T., Black, G.W., Hazlewood, G.P. and Gilbert, H.J. (1995) The conserved noncatalytic 40-residue sequence in cellulases and hemicellulases from anaerobic fungi functions as a protein docking domain. J. Biol. Chem. 270, 29314-29322.
52. Teather, R.M. and Wood, P.J., (1982) Use of Congo red-polysaccharides interactions in enumeration and characterization of cellulytic bacteria from the bovine rumen. Appl. Environ. Microbiol. 43, 777-780.
53. Lige, B., Shengwu, M. and van Huystee, R.B. (2001) The effects of the site-directed removal of N-glycosylation from cationic peanut peroxidase on its function. Ach. Biochem. Biophy. 386, 17-24.
54. Tull, D., Gottschalk, T.E., Svendsen, I., Kramhoft, B., Phillipson, B.A., Bisgard-Frantzen, H., Olsen, O. and Svensson, B. (2001) Extensive N-glycosylation reduces the thermal stability of a recombinant alkalophilic Bacillus α-amylase produced in Pichia pastoris. Protein Expres. Purif. 21, 13-23.
55. Cesar, T. and Mrsa, V. (1996) Purification and properties of the xylanase produced by Thermomyces lanuginosus. Enzyme Microb. Technol. 19, 289-296.
56. Gilkes, N. R., Henrissat, J. A., Kilburn, D. G., Miller, R. C. and Warren, R. A. J. (1991). Domains in microbial β-1,4-glucanase: sequence conservation , function and enzyme families. Microbiol. Rev. 55, 303-315.

QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top