跳到主要內容

臺灣博碩士論文加值系統

(44.201.97.224) 您好!臺灣時間:2024/04/18 03:30
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

我願授權國圖
: 
twitterline
研究生:鄢志仲
研究生(外文):Chih-Chung Yen
論文名稱:利用directedevolution置換假絲酵母(Lip4)之受質結合位置以改變其受質選擇性之研究
論文名稱(外文):Altering the substrate specificity of Candida rugosa LIP4 by directed evolution of substrate binding site
指導教授:莊慧文蕭介夫蕭介夫引用關係
指導教授(外文):Huey-wen ChuangJei-Fu Shaw
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:農業生物技術研究所
學門:農業科學學門
學類:農業技術學類
論文種類:學術論文
論文出版年:2004
畢業學年度:94
語文別:中文
中文關鍵詞:directed evolution受質結合位置假絲酵母脂肪酶
相關次數:
  • 被引用被引用:2
  • 點閱點閱:225
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
假絲酵母菌 Candida rugosa 是一種能在菌體外產生脂肪酶(lipase)的真菌,該脂肪酶為一種重要之工業用酵素,並已被廣泛應用於生物技術領域。例如,水解產生不同碳鏈之脂肪酸及合成不同碳鏈之脂類,目前已知該酵素的組成包含了至少五種同功酶 LIP1~LIP5,且該五種同功酶之胺基酸序列相似度極高,且受質專一性均不相同。本實驗著力於 LIP4 之 directed evolution 以改變其受質專一性,並提高其受質應用性。
本實驗利用 Discovery Studio 蛋白質結構模擬程式將已解析之LIP1 及 LIP3 之 3D 立體結構,以及胺基酸序列為模板(template)模擬出 LIP4 的蛋白質模擬(protein modeling),並以之與受質進行結合模擬得到 LIP4 之 substrate-binding site 與受質結合之相對位置,進而推測出 LIP4 在 A296、V344 及 H448 位置,對受質專一性有決定性之影響。
利用 error-prone PCR的方式,針對 LIP4 之 A296、V344 及 H448 之受質結合區胺基酸進行隨機突變,再以電破法(electroporation)的方式轉型至酵母菌(Pichia pastoris)而後以高專一性之selection plates 培養篩選分別獲得比 LIP4更具有水解能力之突變株,經由序列分析得到 A296I、V344Q、V344H 及 H448S之突變脂肪酶。
生化特性及應用分析突變脂肪酶 A296I、V344Q、V344H 及 H448S 對不同受質之催化活性相較於 wild-type lipase 有顯著之差異。A296I具有較廣泛之最適反應溫度(37-50℃);應用分析上對於短碳鏈(C4-C8)之 p-nitrophenyl esters 及 triacylglycerols 有較佳之水解活性,而 H448S 則對於長碳鏈(C16)之 triacylglycerols 有較佳的水解活性。
在膽固醇酯解(cholesterol esterase)活性測定方面, A296I 對中碳鏈受質(C12)之水解能力較 wide-type 有增加六倍的,V344Q亦增加達三倍。
Candida rugosa (formerly Candida cylindracea) lipase (CRL)is a very important industrial enzyme that is widely used in biotechnological applications such as production of fatty acids, synthesis of various esters, Lipase comprises at least five isozymes (LIP1~LIP5) which share a similar amino acid sequence but with different specificities for substrates. The focus of this experiment was to use directed evolution to create thousands of mutant enzymes from a wild type version in order to search for variants that have an increased specificity of substrates.
In this experiment, Discovery Studio, a program simulating protein structure, was used to analyze the 3D structure of LIP1 and LIP3, and the amino acid sequence was also used as a template to simulate protein modeling of LIP4. By combining the 3D structure and protein modeling with substrates to conduct an integrated simulation, the relative position joining substrates and LIP4’s substrate-binding site was deduced the analysis showed that the amino acids of LIP4 at A296, V344, and H448 play decisive roles in changing its substrate specificity.
Biochemical characterization and application analysis of mutant lines obtained by error-prone PCR showed that the catalytic activity of the directed evolution strains A296I, V344Q, V344H, and H448S have remarkable differences in substrate specificity from that of wild-type. In application analysis of hydrolytic activity with short-chain (C4-C8) p-nitrophenyl esters and triacylglycerols, H448S shows a higher activity on long-chain (C16) triacylglycerols than the wild type LIP4.Where as A296I and V344Q, which hydrolyze the medium-chain (C12) cholesterol ester, exhibit 6-fold and 3-fold in enhancements activity, respectively. The catalytic efficiency of these mutant enzymes with specific substrates was also found to be superior than that of wild type LIP4.
目 錄
中文摘要 i
英文摘要 iii
目錄 v
圖表目錄 viii
緒論 1
一、脂肪酶的生化特性 1
二、脂肪酶的結構特性 2
1. 一級胺基酸序列 2
2. 三級結構之特徵 2
3. 活化部位之催化三元體 3
4. 含氧陰離子中間產物穩定區( oxyanion hole ) 3
5. 口蓋狀結構 ( lid ) 與界面活化現象 4
三、脂肪酶之催化反應機制 4
四、脂肪酶的應用性 5
五、Candida rugosa 脂肪酶 8
1. 脂肪酶為一種多型性同功脂肪�@ ( multipe isoenzymes ) ……….. 8
2. C. rugosa 脂肪酶基因群族 ( lipase gene family ) 9
3. 遺傳工程蛋白質之研究 9
4. Candida rugosa lipase LIP1 之三級結構 10
六、前人研究 12
七、研究緣起 13
材料與方法 15
一、菌種與培養基 15
二、質體 DNA 的製備與error-prone PCR 16
1. 質體 16
2. 質體 DNA 的製備 16
3. Error-prone PCR 17
4.限制酶( restriction enzyme ) 的使用 17
5.瓊脂醣膠電泳 ( agarose gel electrophoresis ) 18
6. DNA之回收 ( Gel extraction ) 18
7. DNA 接合 ( ligation ) 19
8.質體轉形 ( transformation ) 19
9.篩選 ( screening ) 20
三、Candida rugosa mLIP4 表現載體 ( expression vector ) 之構築 20
四、Pichia pastoris 的質體轉型 21
五、Genomic DNA 定序分析(Genomic DNA sequencing) 23
六、蛋白質的表現與純化 23
七、蛋白質電泳分析 24
八、脂肪酶生化特性之測定 27
(1) 脂肪酶活性測定 28
1-1 Esterase activity assay 28
1-2 Lipase activity assay 28
1-3 Cholesterol esterase activity assay 29
(2) 最適 pH 值 30
(3) 最適作用溫度 30
(4) 脂肪酶熱穩定性分析 31
結果 32
一、yeast 表現系統的選擇 32
二、Candida rugosa 脂肪�@表現載體的構築 32
三、重組蛋白質的表現 33
四、重組蛋白質生化性質分析 34
1. 最適反應 pH 值 ( Optimal pH ) 34
2. 最適反應溫度 ( Optimal temperature ) 35
3. 熱穩定性分析 ( Thermal stability ) 36
4. 酯解酶活性測定 ( Esterases activity ) 37
5. 脂肪酶活性測定 ( Lipases activity ) 38
6. 膽固醇酯解酶活性測定 ( Cholesterol esterase activity ) 40
討論 43
一、在 P. pastoris 系統中表現 43
二、LIP4 wild-type、A296I、V344Q、V344H 及 H448S 最適 pH 值比較 45
三、LIP4 wild-type、A296I、V344Q、V344H 及 H448S 最適反應溫度比較 45
四、LIP4 wild-type、A296I、V344Q、V344H 及 H448S 熱穩定性比較 47
五、就 LIP4 wild-type、A296I、V344Q、V344H 及 H448S 對水解催化功能的影響 47
1. 對於 esterase 活性功能比較 ( soluble substrate ) 48
2. 對於 lipase 活性功能比較 ( insoluble substrate ) 50
3. 對於 cholesterol esterase 活性功能比較 52
參考文獻 54
附錄……………………………………………………………………103
參 考 文 獻
1. Brockman, H. L., W. E. Momson, and T. Tsujita. 1988. Lipid-lipid complexes: properties and effects on lipase binding to surfaces. JAOCS 65(6):891-896.
2. Sarda, L. and Desnuelle, P. 1958. Bischem. Biophys. Acta. 30:513-521.
3. Macrae, S. R. 1983. Lipsdr-vstslyzed interesterication of oils and fats J. Am. Oil Chem. Soc. 60:243-246.
4. Vadehra, D. V. 1974. Staphylococcus lipase. Lipids. 9:158-165.
5. Okumara, S., M. Iwai, Y. Tominaga. 1984. Synthesis of ester oligomer by Aspergillius niger lipase. Agric. Biol. Chem. 48(1):2805-2808.
6. Linfield, W. M., Barauskas, R. A., Sivieri, L., Serota, S. and Stevenson, R. W., Sr. 1984. Enzymatic fat hydrolysis and synthesis. JAOCS 61:191-195.
7. Jensen, R. G. 1974. Characteristics of the lipase from the mold Geotrichum candidum: A review. Lipids. 9:149-157.
8. Miller, C., H. Ausrin, L. Posorske, and J. Gonzlez. 1988. Characteristics of an immobilized lipase for the commercial systhesis of esters. JAOCS. 65(6):927-931.
9. Kugimiya, W., Otani, Y., Hashimoto, Y. and Takagi, Y. 1986. Molecular cloning and nucleotide sequence of the lipase gene from Pseudomonas fragi. Biochem. Biophys. Res. Comm. 141:185-190.
10. Lee, C. Y., and Iandolo, J. J. 1986. lysogenic conversion of Staphylococcus lipase is caused by insertion of the bacteriophage L54a genome into the lipase structural gene. J. Bacteriol. 166:385-391.
11. Uppenberg, J., Hansen, M. T., Patkar, S. and Jones, T. A. 1994. Sturcture. 2:293-308.
12. Antonian, E. 1988. Recent advences in the purification, characterization and structure determination of lipase. LIPIDS. 23(12):1101-1106.
13. Polgar, L. 1992. Structure relationship between lipase and peptedase of the prolyl oligoprprtidase family. FEBS. 311(3):281-284.
14. Brady, L., Brzozowski, A. M., Derewenda, Z. S., Dodson, E., Dodson, G., Tolley, S., Turkenburg, J. P., Christiansen, L., Jensen, B. H., Norskov, L., Thim, L. and Menge, U. 1990. A serin protease triad forms the catalytic center of a triacylglycerol lipase. Nature. 343(33):747-770.
15. Winkler, F. K., D’Arcy, A. and Hunziker, W. 1990. Structure of human pancreatic lipase. Nature. 343:771-774.
16. Schrag, J. D., Y. Li, S. Wu and M. Cygler. 1991. Ser-His-Glu triad forms the catalytic site of lipase from Geotrichum candudum, Nature. 351(27):71-764.
17. Sussman, J. L., Harel, M., Frolow, F., Oefner, C., Goldman, A., Toker, L. and Silman, I. 1991. Science. 253:872-879.
18. Liao, D. I., Breddam, K., Sweet, R. M., Bullock, T. and Remington, S. J. 1992 Refined atomic model of wheat serine carboxypetidase II at 2.2-Å resolution. Biochemistry. 31:9796-9812.
19. Pathak, D., Ngai, K. L., and ollis, D. 1998. J. Mol. Biol. 204:435-445.
20. Noble, M. E. W., A. Cleasby, L. N. Johnson, M. R. Egmond, and L. G. J. Frenken. 1993. The crystal structure of triacylglycerol lipase from Pseudomonas glumae reveal a partially redundant catalytic aspartate. FEBS. 331(1,2):123-128.
21. Derewenda, Z. S. and U. Derewenda. 1991. Relationships among serine hydrolase: evidence for a common structure motif on triaclglyceride lipases, Biochem Cell Biol. 69:842-851.
22. Derewenda, Z. S. and U. Derewenda. 1993. News from the interface: the molecular structure of triacylglycerude lipase. TIBS. 18:20-25.
23. Misset, O. 1994. The structure-function relationship of the lipase from Pseudomonas aerugosa and Bacillus subtilis. Prot. End. 7:523-529.
24. Schrag, J. D., Y. Li, S. Wu and M. Cygler. 1991. Ser-His-Glu triad forms the catalytic site of lipase from Geotrichum candudum, Nature. 351(27):71-764.
25. Ollis, D. L., Cheah, E., Cygler, M., Dijkstra, B., Frolow, F., Franken, S. J. L., Verschueren, K. H. C. and Goldman, A. 1992. The α/β hydrolase fold. Protein Engeineering. 5(3):197-211.
26. Warshel, A., Naray-Szabo, G., Sussman, F., and Hwang, J. K. 1989. how do serine proteases really work ? Biochemistry. 28,3629-3637.
27. Desnuelle, p. 1972. The lipase. p.575-616. In P. D. Boyer (ed.), The enzyme, 3rd ed, vol 7. Academic Press, New York.
28. Verger, R. 1997. Interfacial actvation of lipase: facts and artifacts. Trenda Biotechnol. 15:32-38.
29. Louwrier, A., G. J. Drtina, and A. M. Klibanov. 1996. On the issue of interfacial activation of lipase in nonaqueous media. Biotechnol. Bioeng. 50:1-5.
30. Uppenberg, J., M. T. Hansen, S. Patkar, and T. A. Jones. 1994. The sequence, crystal structure determination and refinement of two crystal forms of lipase B from Candida Antarctica. Structure. 2:239-308.
31. Chapus, C. and M. Semeriva, C. Bovier-Lapierre, and P. Desnuelle. 1976. Mechanism of pancreatic lipase action. 1. Interfacial activation of pancreatic lipase. Biochemistry 15(23):4980-4987.
32. Chapus, C. and M. Semeriva. 1976. Mechanism of pancreatic lipase action 2. Catalytic properties of modified lipase. Biochemistry 15(23):4988-4991.
33. Dziezak, J. D. 1986. Enzyme modification of dairy products. Food Technol. 4:114-120.
34. Henderson, R. J., Burkow, I. C. and Millar, R. M. 1993. Hydrolysis of fish oils containing polymers of triacylglycerols by pancreatic lipase in vitro. LIPIDS 28(4):313-319.
35. Yadwad, V. B., Ward, O. P. and Noronha, L. C. 1991. Application of lipase to concentrate the Docosahexaenoic acid (DHA) fraction of fish oil. Biotechnology and Bioengineering. 38:956-959.
36. Haraldsson, G. G., Hoskuldsson, P. A., Sigurdsson, S. T., Thrsteinsson, F. and Gudbjarnason, S. 1989. The prepaaration of triglycerides highly enriched with ω-3 polyunstaturated fatty acids via lipase catalyzed interesterification. Tatrahedron lett. 30(13):1671-1674.
37. Yadwad, V. B., Ward, O. P. and Noronha, L. C. 1991. Application of lipase to concentrate the Docosahexaenoic acid (DHA) fraction of fish oil. Biotechnology and Bioengineering. 38:956-959.
38. Shaw, J. F. and Lo, S. 1994. Production of propylene glycol fatty acid monoesters by lipase-catalyzed reactions in organic solvents. J. Am. Oil Chem. Soc. 71(7):715-719.
39. Kloosterman, M., Elferink, V. M., van lersel, J., Roskman, J. H., Meijer, E. M. Hulshof, L. A. and Sheldon R. A. 1998. Lipase in the proparatin of β-blockers. TIBTECH 6:251-256.
40. Gillis, A. 1988. Research descovers new roles for lipase. J. Am. Oil Chem. Soc. 65(6):840-851.
41. 蕭介夫 (2000)固定化脂肪酶(微生物)在油脂工業之應用。生物固定化技術與產業應用
42. Brookes, I. K., Lilly, M. D. and Drozed, J. W. 1987. Use of immobillized Bacillus subtillis for the stereospecfic hydrolysis of d,1-methyl acetate. Enzyme Microb. Technol. 9:217-220.
43. Gillies, B., Yamazaki, H. and Armstrong, D. W. 1987. Production of flavor esters by immobillized lipase. Biotechmol. Lett. 9(10):709-714.
44. Iwai, M., Okumura, S. and Tsujisaka, Y. 1980. Synthesis of terpene alcohol esters by lipase. Agric. Biol. Chem. 44(11):2731-2731.
45. Okumara, S., M. Iwai, Y. Tominaga. 1984. Synthesis of ester oligomer by Aspergillius niger lipase. Agric. Biol. Chem. 48(1):2805-2808.
46. Okumara, S., M. Iwai, Y. Tominaga. 1984. Synthesis of ester oligomer by Aspergillius niger lipase. Agric. Biol. Chem. 48(1):2805-2808.
47. Ergan, F., Trani, M. and Andre, G. 1990. Production of glycerides from glycerol and fatty acid by immobillized lipase in non-aqueous media. Biotechnol. Bioeng. 35:195-200.
48. Shaw, J. F., Wang, D. L. and Wang, Y. T. 1991. Lipase-catalysed ethanolysis and isopropanolysis of triglyceride with long-chain fatty acid. Enzyme Microb. Technol.13:544-546.
49. Akoh, C. C. 1994. Enzymatic synthesis of caetylated glucose fatty acid esters in organic solvent. J. Am. Oil Chem. Soc. 71(3):319-323.
50. Shaw, J. F., Wang, D. L. and Wang, Y. T. 1991. Lipase-catalysed ethanolysis and isopropanolysis of triglyceride with long-chain fatty acid. Enzyme Microb. Technol.13:544-546.
51. Mukherjee, K. D. and Kiewitt, I. 1988. Preparation of esters resembling nature waxes by lipase-catalyzed reations. J. Agric. Food Chem. 36:1333-1336.
52. Garcia, T., Martinez, M. and Aracil, J. 1993. Enzymatic synthesis of an analogue of jojoba oil: Optimization by staistical andlysis. Enzyme Microb. Technol. 15:607-611.
53. Macrae, A. R. 1983. Lipase-catalyzed interesterification of oils and fats. J. Am. Oil Chem. Soc. 60:243-246.
54. Shaw, J. F., R. C. Chang, F. F. Wang, and Y. J. Wang. 1990. Lipolytic activitiea of lipase immobilized on six support materials. Biotechnol. Bioeng. 35:132-137.
55. Vulfson, E. N. 1994. industrial applications of lipase, p. 271-288. In P. Woolley and S. B. Petersen (ed.), Lipases-Their structure, biochemistry and application. Cambridge University Press, Cambridge, UK.
56. Wang, Y. J., J. Y. Sheu, F. F. Wang, and J. F. Shaw. 1988. The lipase catalyzed oil hydrolysis in the absence of added emulsifier. Biotechnol. Bioeng. 31:628-633.
57. Benjamin, S., nad A. Pandey. 1998. Candida rugosa lipase: molecular biology and versatility in biotechnology. Yeast. 14:1069-1087.
58. Bojarski, J., J. Oxelbark, C. Andersson, and S. Allenmark. 1993. Enantioselective lipase-catalyzed ester hydrolysis: effects on rates and enantioselectivity from a variation of the ester structure. Chirality. 5:154-158.
59. Longhi, S., F. Fusetti, R. Grandori, M. Lotti, M. Vanoni, and L. Alberghina. 1992. Cloning and nucleotide sequence of two Lip genes from Candida cylindracea. Biochim. Biophys. Acta. 1131:227-232.
60. Lotti, M., R. Grandori, F. Fusetti, S. Longhi, S. Brocca, A. Tramontano, and L. Alberghina. 1993. Molecular cloning and analysis of Candida cylindracea lipase sequences. Gene. 124:45-55.
61. Kawaguchi, Y., H. Honda, J. Taniguchi-Morimura, and S. Iwasaki. 1989. The codon CUG is read as serine in an asporogenic yeast Candida cylindracea. Nature. 341:164-166.
62. Longhi, S., F. Fusetti, R. Grandori, M. Lotti, M. Vanoni, and L. Alberghina. 1992. Cloning and nucleotide sequence of two Lip genes from Candida cylindracea. Biochim. Biophys. Acta. 1131:227-232.
63. Lotti, M., R. Grandori, F. Fusetti, S. Longhi, S. Brocca, A. Tramontano, and L. Alberghina. 1993. Molecular cloning and analysis of Candida cylindracea lipase sequences. Gene. 124:45-55.
64. Antonian, E. 1988. Recent advances in the purification, characterization and structure determination of lipase. Lipids. 23:1101-1106.
65. Kawaguchi, Y., H. Honda, J. Taniguchi-Morimura, and S. Iwasaki. 1989. The codon CUG is read as serine in an asporogenic yeast Candida cylindracea. Nature. 341:164-166.
66. Takeshi, O., Suzuki, T., Miki, M., Osawa, S., Ueda, T., Watanabe, K., Nakasse, T. (1993) Non-universal decoding of the leucine codon CUG in several Candida rugosa species. Nucleic Acid Research. 21, 4039-4045.
67. Grochulski P., Y. Li, J. D. Schrag, F. Bouthillier, P. Smith, D. Harrison, B. Rubin, and M. Cygler. 1993. Insights into interfacial actbvation from an open structure of Candida rugosa lipase. J. Biol. Chem. 268:12843-12847.
68. Grochulski P., Y. Li, J. D. Schrag, and M. Cygler. 1994. Two conformational states of Candida rugosa lipase. Protein Sci. 3:82-91.
69. Grochulski P., F. Bouthillier, R. J. Kazlauskas, J. D. Scharg, E. Ziomek, and M. Cygler. 1994. Analogs of reaction intermediates identify a unique substrate binding site in Candida rugosa lipase. Biochemistry. 33:3494-3500.
70. Cygler, M., P. Grochulski, R. J. Kazlauskas, J. D. Schrag, F. Bouthillier, B. Rubin, A. N. Serreqi, and A. K. Gupta. 1994. A structural basis for the chiral preferences of lipases. J. Am. Chem. Soc. 116:3180-3186.
71. Ghosh, D., Z. Wawrzak, V. Z. Pletnev, N. Li. R. Kaiser, W. Pangborn, H. Jornvall, M. Erman, W. L. Duax. 1995. Structure of uncomplexed and linoleate-bound Candida cylindracea cholesterol esterase. Structure. 3:279-288.
72. Lopez, N., Pernas, M., Pastrana, L., Sanchez, A., Valero, F., Rua, M. 2004 . Reactivity of pure Candida rugosa lipase isoenzymatic ( lip1 , lip2 and lip3 ) in aqueous and organic media. Influence of the isoenzyme profile on the lipase performance in organic media. Biotechnol. Prog. 20: 65-73.
73. Miroslaw Cygler, Joseph D. Schrag. 1999. Structure and conformational flexibility of Candida rugosa lipase. Biochimica et Biophysica Acta. 1441:205-214.
74. Grochulski P., F. Bouthillier, R. J. Kazlauskas, J. D. Scharg, E. Ziomek, and M. Cygler. 1994. Analogs of reaction intermediates identify a unique substrate binding site in Candida rugosa lipase. Biochemistry. 33:3494-3500.
75. Cygler, M., P. Grochulski, R. J. Kazlauskas, J. D. Schrag, F. Bouthillier, B. Rubin, A. N. Serreqi, and A. K. Gupta. 1994. A structural basis for the chiral preferences of lipase. J. Am. Chem. Soc. 116:3180-3186.
76. Ghosh, D., Z. Wawrzak, V. Z. Pletnev, N. Li. R. Kaiser, W. Pangborn, H. Jornvall, M. Erman, W. L. Duax. 1995. Structure of uncomplexed and linoleate-bound Candida cylindracea cholesterol esterase. Structure. 3:279-288.
77. Tang, S. J., Sun, K. H., Sun, G..H., Chang, T. Y., Lee, G.. C. 2000 Recombination expression of the Candida rugosa lip4 lipase in Escherichia coli. Protion Expr. Purif. 20, 308-313.
78. Tang, S. J., Sun, K. H., Sun, G.. H., Chang, T. Y., Wu, W. L.,Lee, G. C. 2003 A transformation system for the non-universal CUG(Ser) codon usage species Candida rugosa. Journal of Microbiological Methods. 52, 231-238.
79. Tang, S. J., Shaw, J. F., Sun, K. H., Sun, G.. H., Chang, T. Y., Lin, C. K., Lo, Y. C., Lee, G.. C. 2001 Recombination expression and characterization of the Candida rogasa lip4 lipase in Pichia pastoris: comparison of glycosylation, activity,and stability. Arch. Biochem. Biophys. 387, 93-98.
80. Kazlauskas, R. 2005 Enzymes in focus. Nature 436, 1096-1097.
81. 李立群(2002)假絲酵母(Lip2)重組基因之蛋白質表現、生化特性及應用。國立台灣海洋大學水產生物技術研究所碩士論文
82. Lee, G. C., Lee, L. C., Sava, V., Shaw, J. F. (2002) Multiple mutagenesis of non-universal serine codons of the Candida rugosa lip2 gene and biochemical characterization of purified recombinant LIP2 lipase overexpressed in Pichia pastoris. J. Biochem. 366, 603-611.
83. Mancheno, J. M., Ochoa, M. J. M.B., Rua, M. L., Hermoso, J. 2003 Structural insights into the lipase/esterase behavior in the Candida rugosa lipases family: crystal structure of the lipase 2 isoenzyme at 1.97 A resolution. J. Mol. Biol. 332, 1059-1069.
84. Sambrook, J., Fritsch, E. F., and Maniatis, T. 1989. in Molecular cloning. A laboratory manul. Cold Spring Harbor Laboratory Press, New York.
85. Hiroaki, I., Nojima, H., and Okayama, H. (1990) High efficiency transformation of Escherichia coli with plasmids. Gene. 96:23-28.
86. Sekar, V. (1987) A rapid screening procedure for the identification of recombinant bacterial clones. Bio-Techeniq. 5(1):11-13.
87. Siedel, J., W. Rollinger, P. Roschlau, and J. Ziegenhorn. 1985. Total cholesterol, end point and kinetic method, P. 134-148. In H. U. Bergmeyer (ed.), Methods of Enzymatic Analysis, 3rd Ed., Vol. 8. VCH, Weinheim, Germany.
88. Allain C. C., L. S. Poon, C. S. G. Chan, W. Richmona, and P. C. Fu. 1974. Enzymatic determination of total serum cholesterol. Clin. Chem. 20:470-475.
89. Miroslaw Cygler, Joseph D. Schrag. 1999. Structure and conformational flexibility of Candida rugosa lipase. Biochimica et Biophysica Acta. 1441:205-214.
90. Ghosh, D., Z. Wawrzak, V. Z. Pletnev, N. Li. R. Kaiser, W. Pangborn, H. Jornvall, M. Erman, W. L. Duax. 1995. Structure of uncomplexed and linoleate-bound Candida cylindracea cholesterol esterase. Structure. 3:279-288.
91. 李冠群 (1999)Candida rugosa 脂肪酶之基因調控於蛋白質工程。國立陽明大學生命科學院生物化學研究所博士論文。
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
無相關期刊