嗜碱性芽孢杆菌Bacillus sp.AC-2产纤维素酶的研究
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摘要
由细菌产生的纤维素酶其最适反应pH值通常为中性到碱性，近年来，这类酶制剂在纸浆脱油墨和洗涤剂等工业领域中得到了成功的应用，对于这类酶制剂的研究具有重要的学术意义和实际应用价值。
本文选用嗜碱性芽孢杆菌Bacillus sp.AC-2发酵生产纤维素酶，在摇瓶条件下对Bacillus sp.AC-2的产酶工艺进行了研究，包括种子培养时间、接种量、初始pH值、培养温度、碳源、C／N、氮源、无机盐和表面活性剂等。采用优化后的培养基及培养条件，发酵44小时，Bacillus sp.AC-2所产纤维素酶在pH7.0和pH9.5条件下分别为111.25U／mL和76.04U／mL。
进一步在3.7L发酵罐中进行产酶研究。确立了适宜的通气量和搅拌速度分别为0.2vvm和200rpm；在最佳发酵条件下，中性纤维素酶和碱性纤维素酶产量达到了136.77U／mL和93.48U／mL。这一结果已达到同类文献报道的先进水平，显示了良好的应用前景。
对Bacillus sp.AC-2纤维素酶的酶学性质研究表明：该酶除了主要具有内切-β-葡聚糖酶(CMC酶)活性外，还有一定的淀粉酶活力和少量的滤纸酶活力，未测出纤维二糖酶活力。Bacillus sp.AC-2纤维素酶的最佳反应温度和pH值分别为60℃和7.0；在pH6.0～10.0、温度低于70℃条件下比较稳定；不同的金属离子对酶活具有一定的激活或者抑制作用。
Bacillus sp.AC-2纤维素酶在纸浆酶法脱墨方面展示了良好的应用前景，经它处理过的纸浆不仅白度好，而且对纤维的破坏程度也小；酶用量以100U／g为适宜；吐温80和适当的机械作用可以提高酶法脱墨效果。初步实验表明，Bacillussp.AC-2纤维素酶在洗涤剂工业中具有巨大的应用潜力，当酶的添加量为2％、水洗时间1小时，该酶能有效地提高洗衣粉对污布的水洗能力。
芽孢杆菌所产的纤维素酶具有重要的工业应用价值，目前对这类酶制剂的工业化生产国内尚属空白。本论文的研究结果在学术上和应用中都具有重要意义。
Cellulase production using an alkalophilic strain Bacillus sp. AC-2, the enzymatic characteristics and the applications in pulp deinking & textile biofinishing were studied in the thesis.
Fermentation conditions for the cellulase producing were studied in shaking flasks by Bacillus sp. AC-2, including inoculum age, inoculum volume, initial pH value, fermentation temperature, carbon sources, ratio of C/N, nitrogen sources and surface active agent, etc. Under the optimal conditions, the CMCase activities at pH7.0 and pH9.5 reached 111.25 U/mL and 76.04 u/ml respectively in 44 hours.
The cellulase production was carried out in a 3.7L fermentor. It was found that the optimal fermentation conditions were 0.2vvm and 200rpm. A high cellulase activity was obtained (136.77 U/mL at pH 7.0 and 93.48U/mL at pH9.5).
The studies on the enzymatic characteristic showed that Bacillus sp. AC-2 cellulase mainly had CMCase activity with a little activity of amylase and FPA. The enzyme was stable under pH 6.0-10.0 and below 70℃; The optimum temperature and pH value were 60℃ and 7.0, but it also showed a high activity at pH9.5. Metal ions had different effects to the enzymatic activity.
The cellulase from Bacillus sp. AC-2 showed a good application potential in waste paper deinldng and detergent industry. The research work in this dissertation was meaningful both in theoretical research and practical application.
本文选用嗜碱性芽孢杆菌Bacillus sp.AC-2发酵生产纤维素酶，在摇瓶条件下对Bacillus sp.AC-2的产酶工艺进行了研究，包括种子培养时间、接种量、初始pH值、培养温度、碳源、C／N、氮源、无机盐和表面活性剂等。采用优化后的培养基及培养条件，发酵44小时，Bacillus sp.AC-2所产纤维素酶在pH7.0和pH9.5条件下分别为111.25U／mL和76.04U／mL。
进一步在3.7L发酵罐中进行产酶研究。确立了适宜的通气量和搅拌速度分别为0.2vvm和200rpm；在最佳发酵条件下，中性纤维素酶和碱性纤维素酶产量达到了136.77U／mL和93.48U／mL。这一结果已达到同类文献报道的先进水平，显示了良好的应用前景。
对Bacillus sp.AC-2纤维素酶的酶学性质研究表明：该酶除了主要具有内切-β-葡聚糖酶(CMC酶)活性外，还有一定的淀粉酶活力和少量的滤纸酶活力，未测出纤维二糖酶活力。Bacillus sp.AC-2纤维素酶的最佳反应温度和pH值分别为60℃和7.0；在pH6.0～10.0、温度低于70℃条件下比较稳定；不同的金属离子对酶活具有一定的激活或者抑制作用。
Bacillus sp.AC-2纤维素酶在纸浆酶法脱墨方面展示了良好的应用前景，经它处理过的纸浆不仅白度好，而且对纤维的破坏程度也小；酶用量以100U／g为适宜；吐温80和适当的机械作用可以提高酶法脱墨效果。初步实验表明，Bacillussp.AC-2纤维素酶在洗涤剂工业中具有巨大的应用潜力，当酶的添加量为2％、水洗时间1小时，该酶能有效地提高洗衣粉对污布的水洗能力。
芽孢杆菌所产的纤维素酶具有重要的工业应用价值，目前对这类酶制剂的工业化生产国内尚属空白。本论文的研究结果在学术上和应用中都具有重要意义。
Cellulase production using an alkalophilic strain Bacillus sp. AC-2, the enzymatic characteristics and the applications in pulp deinking & textile biofinishing were studied in the thesis.
Fermentation conditions for the cellulase producing were studied in shaking flasks by Bacillus sp. AC-2, including inoculum age, inoculum volume, initial pH value, fermentation temperature, carbon sources, ratio of C/N, nitrogen sources and surface active agent, etc. Under the optimal conditions, the CMCase activities at pH7.0 and pH9.5 reached 111.25 U/mL and 76.04 u/ml respectively in 44 hours.
The cellulase production was carried out in a 3.7L fermentor. It was found that the optimal fermentation conditions were 0.2vvm and 200rpm. A high cellulase activity was obtained (136.77 U/mL at pH 7.0 and 93.48U/mL at pH9.5).
The studies on the enzymatic characteristic showed that Bacillus sp. AC-2 cellulase mainly had CMCase activity with a little activity of amylase and FPA. The enzyme was stable under pH 6.0-10.0 and below 70℃; The optimum temperature and pH value were 60℃ and 7.0, but it also showed a high activity at pH9.5. Metal ions had different effects to the enzymatic activity.
The cellulase from Bacillus sp. AC-2 showed a good application potential in waste paper deinldng and detergent industry. The research work in this dissertation was meaningful both in theoretical research and practical application.
引文
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3. Wilson,et al., Bioresource Technology., 1989,59:5～7
4. Wood T. M., S. I. Mccrea, C. A. Wilson, K. M. Bhat, L. A. Gow, in Biochemistry and Genetics of Cellulase Degradation, J. P. Aubert, P. Beguin, and J. Millet, eds.,New York, Academic Press, 1988, pp. 31～52
5. Nieves R. A., M. E. Himmel, R. J. Tidd, R. Ellis, Appl. Environ. Microbiol., 1990,56:1103～1108
6. Sattler W., H. Esterbauer, O. Glatter, W. Steiner, Biotech. Bioeng., 1989, 33:1221～1234
7. Reuvinen J., T. Bergfers, T. Teeri, J. K. C. Knowles, T. A. Jones, Science, 1990,249:380～386
8. Woodward J., M. Lima, M. E. Lee, Biochem. J., 1988, 255:895～899
9．张树政，酶制剂工业，，科学出版社，1984：595～624
10. Claeyssens M., P. Tomme, Biochemistry, Genetics, Physiology and Application, C. P. Kubicek, etal., eds, London: The Royal Society of Chemistry, pp.1～11
11. Wood,T.M., Biotechnol. Bioeng. Symp., 1975(5):111～137
12. Tibeurgh H,Tomme P, Claeyssens M,et al. FEBS Lett. 1985,204(2):223～227
13. Rouvinen J, Bergfors T, Teeri T. Science.1990.249(4967):380～386
14. Juy M, Alzari P M, et al. Nature.1992,357(6373):89～91
15. Spezio M, Wilson D B, Karplus P A. Biochemisty,1993,32(38):9906～9916
16. Diven C,Stahlberg J, Reinikainen T, et al.Science. 1994.265(5171:524～528
17. Meinke A, Damude H G, Tomme P, et al. J Bio Chem,1991,270(9):4383～4386
18. Sinnott M L. Chem Rev,1990,90(12):1171～1192
19. Mitsuishi Y, nitisinprasert S, Saloheimo M, et al. FEBS Lett, 1990,275 (1): 135～138
20. Macarron R, Beeumen J, Henrissat B,et al. FEBS Lett,1993,316(2):137～140
21. Trimbur D E,Warren RA,Withers S G. J Bio Chem,1992,267(15):10248～10251
22. Tull D, Withers S G, Gilkes N R, et al. J Biol Chem, 1991,266(24): 15621～15625
23. Wang Q,Tull D,Meinke A,et al. J Biol Chem, 1993,268 (19): 14096～14102
24. Tomme P, Warren R A, Gilkes N R. Adv Microb Physiol, 1995,37(1): 1～81
25. Kraulis P J, Jones T A. Proteins, 1987,2(1): 188～201
26. Xu G Y, Ong E,Gilkes N, et al. Biochemistry,1995,34(21):6993～7009
27. Linder M, Mattine M L, Kontteli M,et al. Protein Sci,1995,4(6): 1056～1064
28. Linder M,Lindeberg G, Reinikainen T, et al. FEBS Lett,1995,372(1):96～98
29. Henrissat B. Biochem J,1991,280(2):309～316
30. Henrissat B..FEBS Lett,1995,362(2):281～285
31. Jenkins J. Proc Natl Acad Sci USA, 1995,92(15):7090～7094
32. Dominguez R. Nat Struct Biol,1995,2(2):569～576
33. Sulzenbacher G. Biochemistry, 1996,35(48): 15280～15287
34．阎伯旭，齐飞，等．生物化学与生物物理进展，1999，26(3)：233～237
35. Himmel M. E., etal., Handbook on Biotechnol: Production and Utilization (Edited by Wyman C. E.), Taylor & Fancis, 1996, 143～161
36. Nidetzky B., S. Steiner, M. Hayn, M. Claeyssens, Biochem. J., 1994, 29:705～711
37. Beguin P., Annual Reviews in Microbiology, 1990, 44:219～248
38. Tomme P., V. Heriban, and M. Claeyssens, Biotechnology Letters, 1990, 12:525～530
39．荒开基夫．日本酿造协会杂志，1998，83(3)：177～182
40. Esterbauer H,et al.Bioresources Technology,1991,36:51～57
41. Enari T W, et al.CRC Critical Reviews in Biotechnology,1987,5(1):67～72
42. Ito S,Shikata S,Ozaki K,et al. Agric.Biol.Chem.1989,53(5):1275～1281
43. Kawai S,Okoshi H,Ozaki K, et al. Agric.Biol.Chem.1988,52(6):1425～1431
44. 9999
45. 严福红．武汉轻工设计，1999， 4:1～6
46. Ghose T. K., Pure & Appl. Chem., 1987, 59:257～268
47. Sharrock K. R., J. Biochem. Biophys. Methods, 1988, 17:81～106
48. Deshpande M. V., K. E. Eriksson, L. G. Pettersson, Anal. Biochem., 1984, 138:481～487
49. Manning K., J. Biochem. Biotech. Methods, 1981, 5:189～202
50．公开特许公报，昭58，224686。
51. Koki Horikoshi. NippoNogei kagaku Kaishi,63(8):7～8
52．伊藤进，佐藤朋一．公开特许公报，昭63，109771：381～393
53．川合修次，押野一志．公开特性公报，昭64，37285：495～504
54. Shikata S,Saeki K, Okoshi H. Agric Biol Chem,1990,54(1): 91～96.
55．川合修次，押野一志．公开特性公报，昭64，37286：505～512
56. Grajal-Martin M J, Simon C J, Mue hlbauer F J. Molecular Plant Pathology, 1993,83 (6):612～614
57．川合修次，押野一志．公开特性公报，昭63，273474：417～424
58．宋桂经，王冬，等．微生物学报．1995，35(1)：38～44
59．王冬，宋桂经，等．生物化学与生物物理进展，1994，21(3)：237～241
60．王丹敏，宋桂经，等．微生物学通报，1997，24(5)：278～281
61．田新玉，王欣．微生物学通报，1997，24(4)：195～198
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