嗜热脂肪酶分子克隆及酶学性质研究
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摘要
脂肪酶是具有甘油三酯水解活性的重要工业用酶,发现和表征新型脂肪酶对于促进相关工业发展具有重要意义。从嗜热菌Fervidobacterium nodosum基因组中发现基因Fond_1333编码302个氨基酸,其氨基酸序列含有脂肪酶A-X-S-X-G基序、负氧离子洞保守区域,因此推测基因Fond_1333可能为脂肪酶基因。
本文应用PCR技术成功地克隆Fond_1333基因,构建了携带pET-28a+ Fond_1333质粒的E.coli BL21 CodonPlus工程菌,并对重组蛋白质(FnL)的酶学性质进行了较为系统的研究。FnL能水解各种不同碳链长度甘油三酯和对硝基苯酚酯,最适底物分别为三丁酸甘油酯和对硝基苯酚癸酸酯;其最适反应温度为70℃,最适pH值为9.0;具有良好的生物学稳定性。另外,重组FnL的酶活力易受金属离子、抑制剂和表面活性剂等影响。研究表明FnL是一种新型嗜热脂肪酶。
Lipases, interfacially activated triacylglycerol hydrolases, have a wide existence in animal, plant and microorganism. They belong to serine hydrolase family with a triad made up of Ser, Asp and His, or Ser, Glu and His to achieve the catalytic activity. It shows a wide specificity and regioselectivity towards a variety of triglycerides. Owing to these characteristics, it is wildly applied in food industry, pharmaceuticals, chemical industry, polluted water curing, petroleum exploiting etc. However, the biological stability of lipases available could not meet the need of industry and limited its development. Therefore, it is a significant and urgent program to explore new promising lipases to cater to the fast developing industry.
Up to now, a large quantity of methods has been set up to obtain new genes and proteins, and searching potential target genes in the genomes is a simple and effective one. A huge database has been made with the sequences of a variety of genomes. The interested genes can be predicted after analyzing the sequences in the database and cloned easily by molecular technology.
In the present study, gene Fond_1333 in Fervidobacterium nodosum, a thermophilic microorganism, was selected as the objective to clone thermophilic lipase gene. Gene Fond_1333 is made up of 906 nucleotides and encodes 302 amino acids (FnL). FnL contains the conserved motif A-X-S-X-G, which is common in most of lipases, and thus is predicted as a new lipase. The theoretical mass and isoelectric point of FnL are 34841.35 Da and 5.40, respectively. Conserved domains search at NCBI revealed that FnL had the conserved active domain and the conserved domain of oxyanion hole, which were common in most lipases. The prediction of the secondary structure indicated that FnL was a typicalα/βfolding hydrolase includingα-helix,β-sheet and coil. In addition, there was a longα-helix at the C terminal of FnL, which might make a great contribution to the high thermostability. Compared with various lipases belonging to different families, FnL had a low homology and considered to be likely belonging to lipase family VI. Therefore, FnL is a new member of lipase family VI.
The thermophilic lipase gene Fond_1333 has been cloned, and overexpressed in the insoluble form in E. coli CodonPlus. Therefore, we established a simple and quick method to obtain target protein. The collected cells were first mixed with 50 mM PBS (pH 8.0), ultrasonic cell disintegrated and centrifuged at 5000 rpm for 20 minutes. The deposit part was washed with the 0.2% DOC solution for 20 minutes at room temperature, and then centrifuged at 5000 rpm for 20 minutes again. The deposit part was resuspended with the 0.25% Tween-80 solution at 60℃for 20 minutes, and then centrifuged at 12000 rpm for 20 minutes. The supernatants were collected as the crude FnL solution, and the purity was very high. Further purification step could be performed by HiTrap Q Sepharose chromatography, depending on the research purpose.
FnL could catalyze the hydrolysis of triglycerides and p-nitrophenyl esters, and its optimal substrates were tributyrin and pNP-C10, respectively. The optimal reaction temperature and pH value were 70℃and 9.0, respectively. The enzyme was stable at high temperature, and the t1/2 at 60℃was up to 100 hours.
Furthermore, FnL possessed good resistance against organic solvents. All the organic reagents employed, including methanol, ethanol, propanol, acetone, DMSO and DMF, did not inhibit the catalytic activity. Surprisingly, the enzyme could be activated to some extent in some of them (Propanol, Acetone, DMSO and DMF). Therefore, thermophilic lipase FnL was considered to have the potential to be applied in the synthesis of chemicals in organic medium.
The effects of pH value, metal ions, surfactants and inhibitors were also investigated in the thesis. FnL kept high catalytic ability at a relative broad pH value, from 8.0 to 10.0, but it was stable in the pH range 8.0-9.0. Metal ions, inhibitors and surfactants had significant effects on the catalytic activity of FnL. Among them, Mg2+, Na+, Ca2+, Co2+ and Triton X-100 could activate FnL. On the other hand, Zn2+, Cu2+, MoO42-, DOC, SDS, PMSF and high concentration of Tween-80 would inhibit its catalytic ability. In a word, proper conditions should be controlled when thermophilic FnL was employed in the catalysis.
In this work, FnL was successfully cloned, expressed and characterized. Our research showed that FnL was a new thermophilic lipase of high thermostability and resistance against organic reagents. Therefore, FnL was a potential target to be widely used in the biochemical industry.
本文应用PCR技术成功地克隆Fond_1333基因,构建了携带pET-28a+ Fond_1333质粒的E.coli BL21 CodonPlus工程菌,并对重组蛋白质(FnL)的酶学性质进行了较为系统的研究。FnL能水解各种不同碳链长度甘油三酯和对硝基苯酚酯,最适底物分别为三丁酸甘油酯和对硝基苯酚癸酸酯;其最适反应温度为70℃,最适pH值为9.0;具有良好的生物学稳定性。另外,重组FnL的酶活力易受金属离子、抑制剂和表面活性剂等影响。研究表明FnL是一种新型嗜热脂肪酶。
Lipases, interfacially activated triacylglycerol hydrolases, have a wide existence in animal, plant and microorganism. They belong to serine hydrolase family with a triad made up of Ser, Asp and His, or Ser, Glu and His to achieve the catalytic activity. It shows a wide specificity and regioselectivity towards a variety of triglycerides. Owing to these characteristics, it is wildly applied in food industry, pharmaceuticals, chemical industry, polluted water curing, petroleum exploiting etc. However, the biological stability of lipases available could not meet the need of industry and limited its development. Therefore, it is a significant and urgent program to explore new promising lipases to cater to the fast developing industry.
Up to now, a large quantity of methods has been set up to obtain new genes and proteins, and searching potential target genes in the genomes is a simple and effective one. A huge database has been made with the sequences of a variety of genomes. The interested genes can be predicted after analyzing the sequences in the database and cloned easily by molecular technology.
In the present study, gene Fond_1333 in Fervidobacterium nodosum, a thermophilic microorganism, was selected as the objective to clone thermophilic lipase gene. Gene Fond_1333 is made up of 906 nucleotides and encodes 302 amino acids (FnL). FnL contains the conserved motif A-X-S-X-G, which is common in most of lipases, and thus is predicted as a new lipase. The theoretical mass and isoelectric point of FnL are 34841.35 Da and 5.40, respectively. Conserved domains search at NCBI revealed that FnL had the conserved active domain and the conserved domain of oxyanion hole, which were common in most lipases. The prediction of the secondary structure indicated that FnL was a typicalα/βfolding hydrolase includingα-helix,β-sheet and coil. In addition, there was a longα-helix at the C terminal of FnL, which might make a great contribution to the high thermostability. Compared with various lipases belonging to different families, FnL had a low homology and considered to be likely belonging to lipase family VI. Therefore, FnL is a new member of lipase family VI.
The thermophilic lipase gene Fond_1333 has been cloned, and overexpressed in the insoluble form in E. coli CodonPlus. Therefore, we established a simple and quick method to obtain target protein. The collected cells were first mixed with 50 mM PBS (pH 8.0), ultrasonic cell disintegrated and centrifuged at 5000 rpm for 20 minutes. The deposit part was washed with the 0.2% DOC solution for 20 minutes at room temperature, and then centrifuged at 5000 rpm for 20 minutes again. The deposit part was resuspended with the 0.25% Tween-80 solution at 60℃for 20 minutes, and then centrifuged at 12000 rpm for 20 minutes. The supernatants were collected as the crude FnL solution, and the purity was very high. Further purification step could be performed by HiTrap Q Sepharose chromatography, depending on the research purpose.
FnL could catalyze the hydrolysis of triglycerides and p-nitrophenyl esters, and its optimal substrates were tributyrin and pNP-C10, respectively. The optimal reaction temperature and pH value were 70℃and 9.0, respectively. The enzyme was stable at high temperature, and the t1/2 at 60℃was up to 100 hours.
Furthermore, FnL possessed good resistance against organic solvents. All the organic reagents employed, including methanol, ethanol, propanol, acetone, DMSO and DMF, did not inhibit the catalytic activity. Surprisingly, the enzyme could be activated to some extent in some of them (Propanol, Acetone, DMSO and DMF). Therefore, thermophilic lipase FnL was considered to have the potential to be applied in the synthesis of chemicals in organic medium.
The effects of pH value, metal ions, surfactants and inhibitors were also investigated in the thesis. FnL kept high catalytic ability at a relative broad pH value, from 8.0 to 10.0, but it was stable in the pH range 8.0-9.0. Metal ions, inhibitors and surfactants had significant effects on the catalytic activity of FnL. Among them, Mg2+, Na+, Ca2+, Co2+ and Triton X-100 could activate FnL. On the other hand, Zn2+, Cu2+, MoO42-, DOC, SDS, PMSF and high concentration of Tween-80 would inhibit its catalytic ability. In a word, proper conditions should be controlled when thermophilic FnL was employed in the catalysis.
In this work, FnL was successfully cloned, expressed and characterized. Our research showed that FnL was a new thermophilic lipase of high thermostability and resistance against organic reagents. Therefore, FnL was a potential target to be widely used in the biochemical industry.
引文
1. 《嗜极微生物》武汉大学出版社。
2. 张敏,东秀珠。“973 项目极端微生物及其功能利用的基础研究”研究进展。微生物学报 (2006) 46 (2)。
3. Siddiqui K.S., Cavicchioli R. Cold-adapted enzymes. Annual Review of Biochemistry (2006) 75: 403-33.
4. 惠明,赵坤。极端微生物及其研究进展。河南职业技术师范学院院报(2003) 31 (2): 55-58。
5. Andreas P.M. Weber et al. Metabolism and Metabolomics of Eukaryotes Living Under Extreme Conditions. International Review of Cytology (2007) 256: 1-34.
6. Rothschild L.J., Mancinelli R.L.. Life in extreme environments. Nature (2001) 409 (6823): 1092-101.
7. Edwards H.G.M.. A novel extremophile strategy studied by Raman spectroscopy. Spectrochimica Acta Part A (2007) 68: 1126–1132.
8. 王红妹。极端微生物的多样性及其应用。枣庄学院学报 (2006) 23 (2): 88-92.
9. 任红妍。嗜热微生物。生物学通报 (1995) 30 (3): 18
10. David W Hough, Micheal J Danson. Extremozymes. Current Opinion in Chemical Biology (1999) 3: 39-46.
11. Jorgevillar S.E., Edwards H.G.M., Worland M.R.. Comparative Evaluation of Raman Spectroscopy at Different wavelengths for Extremophile Exemplars. Origins of Life and Evolution of Biospheres (2005) 35: 489–506.
12. 董锡文,薛春梅,吴玉德。极端微生物及其适应机理的研究进展。微生物学杂志 (2005) 25 (1): 74-77。
13. 齐春梅,张小平,姚昕。嗜热微生物酶的嗜热机制及应用研究进展。微生物学杂志 (2004) 24 (4): 39-41。
14. 郝涤非。极端微生物及其应用。生物学教学 (2006) 3 (6): 2-3。
15. 马瑛等。嗜热微生物在废水处理中的应用。上海环境科学 (1997) 16 (8): 26-33。
16. 刘爱民,黄为一。极端酶的研究。微生物学杂志 (2004) 24 (6): 47-50。
17. 张锐,曾润颖。极端酶的研究进展。台湾海峡 (2001) 20 (3): 405-410。
18. 翁墚,冯雁。极端酶的研究进展。生物化学与生物物理进展 (2002) 29 (6): 847-850。
19. Hani Goodarzi, Marco Archetti et al. Amino acid and codon usage profiles: Adaptive changes in the frequency of amino acids and codons. Gene (2008) 407: 30–41.
20. Hefang Xie et al. Probing the Structural Basis for the Difference in Thermostability Displayed by Family10 Xylanases. Journal of Molecular Biology (2006) 360: 157–167.
21. 段杉,彭志英。食品工业新酶源。中国食品添加剂 (2001) 5: 7-11。
22. 段杉等。食品工业新酶源开发及应用。粮食与油脂 (2002) 6: 35-37。
23. 许燕滨, 杨汝德等。极端酶的稳定性及其在废水处理中的应用。化工环保 (2001) 21 (1): 21-24。
24. 林影。极端酶及其工业应用。工业微生物 (2000) 30 (2): 51-53。
25. 王丽红。极端酶在食品工业上的应用。食品工业科技 (2006) 7: 189-192。
26. 龙彪等。食品应用新酶源—极端酶。中国食品添加剂 (2005) 3: 47-50。
27. 嗜极端酶首次实际应用于油田采油。Chemical Engineering (1999) 106(2): 19。
28. 李子东等。极端微生物:一种新型的酶资源。微生物学杂志 (2004) 24:89-91。
29. 王健鑫。嗜极菌的极端酶的若干研究进展。浙江海洋学院学报 (自然科学版) (2003) 22 (2): 158-162。
30. Dolores Reyes-Duarte et al. Conversion of a Carboxylesterase into a Triacylglycerol Lipase by a Random Mutation. Angewandte Chemie-International Edition (2005) 44: 7553–7557.
31. 魏铁麒,沙长青等。微生物脂肪酶及其应用。生物技术 (1997) 7(4): 8-10。
32. Casimir C. Akoh et al. GDSL family of serine esterases/lipase. Progress in Lipid Research (2004) 43: 534-552.
33. Jean Louis Arpigny, Karl-Erich Jaeger. Bacterial lipolytic enzymes: classification and properties. Biochemistry Journal (1999) 343: 177-183.
34. Ahmed Aloulou et al. Exploring the specific features of interfacial enzymology based on lipase studies. Biochimica et Biophysica Acta (2006) 1761: 995–1013.
35. Subbulakshmi Latha Cherukuvada et al. Evidence of a Double-Lid Movement in Pseudomonas aeruginosa Lipase: Insights from Molecular DynamicsS imulations. PLoS Computational Biology (2005) 1(3): e28.
36. Stefania Brocca, Francesco Sedundo, mattia ossola, et al. Sequence of the lid affects activity and specificity of Candida rugosa lipase isoenzymes. Protein Science (2003) 12: 2312-2319.
37. Nathalie Griffon et al. Substrate specificity of lipoprotein lipase and endothelial lipase: studies of lid chimeras. Journal of Lipid Research (2006) 47: 1803-1811.
38. Anthony Levasseur et al. Tracking the connection between evolutionary and functional shifts using the fungal lipase/feruloyl esterase A family. BMC Evolutionary Biology (2006) 6: 92.
39. Peter Fojan et al. What distinguishes an esterase from a lipase: A novelstructural approach. Biochimie (2000) 82: 1033-1041.
40. 咸漠,康亦兼等。脂肪酶催化反应的研究进展。青岛化工学院学报 (2000) 21 (3)。
41. 郑毅,叶海梅等。脂肪酶活力测定研究进展。工业微生物 (2005) 35 (4)。
42. Antoni Sanchez, M.Angels Gordillo, Jose Luis Montesinos et al. On-Line Determination of the Total Lipolytic Activity in a Four-Phase System Using a Lipase Adsorption LAW. Journal of Bioscience and Bioengineering (1999) 87 (4): 500-506.
43. Ilja Ignatijev, Gintaras Valincius, Irena Svedaite, et al. Direct amperometric determination of lipase activity. Analytical Biochemistry (2005) 344: 275-277.
44. Ivanka Karadzic et al. Purification and Characterization of an Alkaline Lipase from Pseudomonas aerginosa Isolated from Putrid Mineral Cutting Oil as Component of Metalworking Fluid. Journal of Bioscience and Bioengineering (2006) 102 (2): 82-89.
45. 侯萍。几种新的基因克隆技术简介。生命科学 (1997) 9 (1): 26-28。
46. 程滨。生物信息学在新基因克隆中的应用。安徽农业技术师范学院学报(2000) 14 (3): 45-46。
47. 李鑫,章涛。新基因的克隆策略和方法。海峡药学 (2004) 16 (3): 16-20。
48. Brigitte Pohn et al. Micro-colony array based high throughput platform for enzyme library screening. Journal of Biotechnology (2007) 129: 162-170.
49. Patel B.K.C., Morgan H.W., Daniel R.M.. Fervidobacterium nodosum gen.nov. and spec.nov., a new chemoorganotrophic, caldoactive, anaerobic bacterium. Archives Microbiology (1995) 141: 63-69.
50. Ryu H.S. et al. New cold-adapted lipase from Photobacterium lipolyticum sp. nov. that is closely related to filamentous fungal lipases. Applied Microbiology and Biotechnology (2006) 70: 321–326.
51. Thean Chor Leow et al. High Level Expression of Thermostable Lipase from Geobacillus sp. Strain T1. Bioscience Biotechnology and Biochemistry (2004) 68 (1): 96-103.
52. Savvas C. Makrides. Strategies for Achieving High-Level Expression of Genes in Escherichia coli. Microbiological Reviews (1996) 512-538.
53. Overexpression and characterization of a lipase Bacillus subtilis. Protein Expression and Purification (2005).
54. Liming Ge, Peter Rudolph. Simultaneous Introduction of Multiple Mutations Using Overlap Extension PCR. BioTechniques (1997) 22: 28-30.
55. HaniGoodarzi, Marco Archetti et al. Amino acid and codon usage profiles: Adaptive changes in the frequency of amino acids and codons. Gene (2008) 407: 30-41.
56. SaHyun Hong, Hiroyuki Horiuchi, Akinori Ohta. Identification and molecular cloning of a gene of a gene encoding Phospholipase A2 (plaA) from Aspergillus nidulans. Biochinica et Biophysica Acta (2005) 1735: 222-229.
2. 张敏,东秀珠。“973 项目极端微生物及其功能利用的基础研究”研究进展。微生物学报 (2006) 46 (2)。
3. Siddiqui K.S., Cavicchioli R. Cold-adapted enzymes. Annual Review of Biochemistry (2006) 75: 403-33.
4. 惠明,赵坤。极端微生物及其研究进展。河南职业技术师范学院院报(2003) 31 (2): 55-58。
5. Andreas P.M. Weber et al. Metabolism and Metabolomics of Eukaryotes Living Under Extreme Conditions. International Review of Cytology (2007) 256: 1-34.
6. Rothschild L.J., Mancinelli R.L.. Life in extreme environments. Nature (2001) 409 (6823): 1092-101.
7. Edwards H.G.M.. A novel extremophile strategy studied by Raman spectroscopy. Spectrochimica Acta Part A (2007) 68: 1126–1132.
8. 王红妹。极端微生物的多样性及其应用。枣庄学院学报 (2006) 23 (2): 88-92.
9. 任红妍。嗜热微生物。生物学通报 (1995) 30 (3): 18
10. David W Hough, Micheal J Danson. Extremozymes. Current Opinion in Chemical Biology (1999) 3: 39-46.
11. Jorgevillar S.E., Edwards H.G.M., Worland M.R.. Comparative Evaluation of Raman Spectroscopy at Different wavelengths for Extremophile Exemplars. Origins of Life and Evolution of Biospheres (2005) 35: 489–506.
12. 董锡文,薛春梅,吴玉德。极端微生物及其适应机理的研究进展。微生物学杂志 (2005) 25 (1): 74-77。
13. 齐春梅,张小平,姚昕。嗜热微生物酶的嗜热机制及应用研究进展。微生物学杂志 (2004) 24 (4): 39-41。
14. 郝涤非。极端微生物及其应用。生物学教学 (2006) 3 (6): 2-3。
15. 马瑛等。嗜热微生物在废水处理中的应用。上海环境科学 (1997) 16 (8): 26-33。
16. 刘爱民,黄为一。极端酶的研究。微生物学杂志 (2004) 24 (6): 47-50。
17. 张锐,曾润颖。极端酶的研究进展。台湾海峡 (2001) 20 (3): 405-410。
18. 翁墚,冯雁。极端酶的研究进展。生物化学与生物物理进展 (2002) 29 (6): 847-850。
19. Hani Goodarzi, Marco Archetti et al. Amino acid and codon usage profiles: Adaptive changes in the frequency of amino acids and codons. Gene (2008) 407: 30–41.
20. Hefang Xie et al. Probing the Structural Basis for the Difference in Thermostability Displayed by Family10 Xylanases. Journal of Molecular Biology (2006) 360: 157–167.
21. 段杉,彭志英。食品工业新酶源。中国食品添加剂 (2001) 5: 7-11。
22. 段杉等。食品工业新酶源开发及应用。粮食与油脂 (2002) 6: 35-37。
23. 许燕滨, 杨汝德等。极端酶的稳定性及其在废水处理中的应用。化工环保 (2001) 21 (1): 21-24。
24. 林影。极端酶及其工业应用。工业微生物 (2000) 30 (2): 51-53。
25. 王丽红。极端酶在食品工业上的应用。食品工业科技 (2006) 7: 189-192。
26. 龙彪等。食品应用新酶源—极端酶。中国食品添加剂 (2005) 3: 47-50。
27. 嗜极端酶首次实际应用于油田采油。Chemical Engineering (1999) 106(2): 19。
28. 李子东等。极端微生物:一种新型的酶资源。微生物学杂志 (2004) 24:89-91。
29. 王健鑫。嗜极菌的极端酶的若干研究进展。浙江海洋学院学报 (自然科学版) (2003) 22 (2): 158-162。
30. Dolores Reyes-Duarte et al. Conversion of a Carboxylesterase into a Triacylglycerol Lipase by a Random Mutation. Angewandte Chemie-International Edition (2005) 44: 7553–7557.
31. 魏铁麒,沙长青等。微生物脂肪酶及其应用。生物技术 (1997) 7(4): 8-10。
32. Casimir C. Akoh et al. GDSL family of serine esterases/lipase. Progress in Lipid Research (2004) 43: 534-552.
33. Jean Louis Arpigny, Karl-Erich Jaeger. Bacterial lipolytic enzymes: classification and properties. Biochemistry Journal (1999) 343: 177-183.
34. Ahmed Aloulou et al. Exploring the specific features of interfacial enzymology based on lipase studies. Biochimica et Biophysica Acta (2006) 1761: 995–1013.
35. Subbulakshmi Latha Cherukuvada et al. Evidence of a Double-Lid Movement in Pseudomonas aeruginosa Lipase: Insights from Molecular DynamicsS imulations. PLoS Computational Biology (2005) 1(3): e28.
36. Stefania Brocca, Francesco Sedundo, mattia ossola, et al. Sequence of the lid affects activity and specificity of Candida rugosa lipase isoenzymes. Protein Science (2003) 12: 2312-2319.
37. Nathalie Griffon et al. Substrate specificity of lipoprotein lipase and endothelial lipase: studies of lid chimeras. Journal of Lipid Research (2006) 47: 1803-1811.
38. Anthony Levasseur et al. Tracking the connection between evolutionary and functional shifts using the fungal lipase/feruloyl esterase A family. BMC Evolutionary Biology (2006) 6: 92.
39. Peter Fojan et al. What distinguishes an esterase from a lipase: A novelstructural approach. Biochimie (2000) 82: 1033-1041.
40. 咸漠,康亦兼等。脂肪酶催化反应的研究进展。青岛化工学院学报 (2000) 21 (3)。
41. 郑毅,叶海梅等。脂肪酶活力测定研究进展。工业微生物 (2005) 35 (4)。
42. Antoni Sanchez, M.Angels Gordillo, Jose Luis Montesinos et al. On-Line Determination of the Total Lipolytic Activity in a Four-Phase System Using a Lipase Adsorption LAW. Journal of Bioscience and Bioengineering (1999) 87 (4): 500-506.
43. Ilja Ignatijev, Gintaras Valincius, Irena Svedaite, et al. Direct amperometric determination of lipase activity. Analytical Biochemistry (2005) 344: 275-277.
44. Ivanka Karadzic et al. Purification and Characterization of an Alkaline Lipase from Pseudomonas aerginosa Isolated from Putrid Mineral Cutting Oil as Component of Metalworking Fluid. Journal of Bioscience and Bioengineering (2006) 102 (2): 82-89.
45. 侯萍。几种新的基因克隆技术简介。生命科学 (1997) 9 (1): 26-28。
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