纤维素酶新型发酵工艺研究
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摘要
本文对纤维素酶传统发酵采用的固态发酵(SSF,Solid Substrate Fermentation)、液态发酵(SF,Submerged Fermentation)工艺及本文提出的固-液交替新型发酵工艺(SSSF,Solid Substrate-Submerged Fermentation)从单菌发酵、混菌发酵纤维素酶活性及纤维素酶得率3 方面进行了系统的比较研究,同时研究了两种提取剂及8 种氮源对固态发酵产物纤维素酶活性测定结果的影响。主要结果如下:
1.前人测定纤维素酶活采用缓冲液提取剂与纯水为提取剂时测定所得纤维素酶活不同。本研究得到的酶活测定结果表明,对于青霉Q1 和黑曲霉H8,用纯水浸提时的纤维素酶活性高于缓冲液提取。以水为提取剂测定纤维素酶活性时,浸提时间应控制在45min 左右,时间过长会导致酶活降低。
2.本文提出的固-液交替新型发酵工艺(SSSF)能够充分发挥传统固态(SSF)、液态(SF)发酵工艺的优点,弥补两种工艺的不足之处,提高纤维素酶活性。
① 纤维素酶产生菌单菌发酵时,采用SSSF 工艺可使黑曲霉H8 的FPA、CMC-Na 及AVI 平均酶活较SSF 工艺分别提高166.7%、264.0%及273.6%,较SF 工艺分别提高798.1%、2325.5%及547.6%。与之类似,采用SSSF 工艺可使青霉Q1 的FPA、CMC-Na及AVI 平均酶活较SSF 工艺分别提高185.2%、240.6%及257.9%,较SF 工艺分别提高1161.3%、2798.0%及419.5%。
② 纤维素酶产生菌与酵母菌混合发酵时,采用SSSF 工艺可使黑曲霉H8 的FPA、CMC-Na 及AVI 平均酶活较SSF 工艺分别提高499.1%、469.0%及459.7%,较SF 工艺分别提高12659.2%、1872.8%及6718.2%。与之类似,采用SSSF 工艺可使青霉Q1 的FPA、CMC-Na 及AVI 平均酶活较SSF 工艺分别提高805.6%、434.4%及708.0%,较SF工艺分别提高1791.2%、1382.5%及558.7%。由此可见,对纤维素酶发酵而言,本研究提出的SSSF 新型发酵工艺远优于传统的SSF 和SF 工艺。
③ 在SSSF 工艺条件下,黑曲霉H81、H82 的粗酶提取物得率分别为14.9%、18.3%,较SSF 工艺,其得率分别提高26.3%及63.4%;在SSSF 工艺条件下,青霉Q11、Q12 的粗酶提取物得率分别为15.4%、14.4%,较SSF 工艺,其得率分别提高30.5%、28.6%。所以,相对于传统的SSF 固态发酵工艺,采用本研究提出的SSSF 新型发酵工艺时,纤维素酶的产率及酶活更高。
3.混菌培养能有效提高纤维素酶活性。
①在SSSF 中,黑曲霉H8 与酵母菌混合发酵时, FPA、CMC-Na 及AVI 平均酶活分别较单菌发酵时提高3.4%、20.1%及42.0%;青霉Q1 与酵母菌混合发酵时,FPA、CMC-Na
The traditional modes of fermentation for producing cellulase including Solid Substrate Fermentation(SSF), Submerged Fermentation(SF), and a novel mode, Solid Substrate-Submerged Fermentation(SSSF), which was designed in this paper were systemataically studied from three parts of enzyme activity of single culture, mixed culture and recovery rate of cellulase. At the same time, we discussed the influence of two sorts of extractants and eight sorts of nitrogen sources on testing of cellulase activity. The main results showed:
1. In the past, the cellulase activity was different when researchers adopted buffer and pure water to extract the cellulase, respectively. However, according to the experiment, the cellulase activity of H8 and Q1 were higher when using pure water as extractant than buffer in the vitality test of cellulase. When extracting gross cellulase from the fermentation products, the time of extraction should be controlled about 45minutes, and too long will lead to the reduction of enzyme activity.
2. The SSSF is a new fermentation technology, which integrates the advantages of SSF with SF perfectly, and improved the cellulase activity in the process of fermentation.
①In the single culture, the SSSF made the FPA, CMC-Na and AVI of H8 increase averagely by 166.7 percent, 264.0 percent, and 273.6 percent, respectively, compared with SSF; and increase averagely 798.1 percent, 2325.5 percent, and 547.6 percent, respectively, compared with SF. Similarly, compared with SSF, the FPA, CMC-Na, and AVI of Q1 improved averagely 185.2 percent, 240.6 percent, and 257.9 percent, respectively if adopting SSSF; and improved averagely 1161.3 percent, 2798.0 percent, and 419.5 percent, respectively, compared with SF.
② In the mixed culture, compared with SSF, the average improvement of FPA, CMC-Na, and AVI of H8 reached 499.1 percent, 469.0 percent if using SSSF; and reached 459.7 percent; 12659.2 percent, 1872.8 percent, and 6718.2 percent, respectively, compared with SF. Likewise, the average improvement of FPA, CMC-Na, and AVI of Q1 grew up by 805.6 percent, 434.4 percent, and 708.0 percent, compared with SSF if adopting SSSF; and
increased by 1791.2 percent, 1382.5 percent, and 558.7 percent, respectively, compared with SF. So to fermentation for producing cellulase, the SSSF has more advantages over traditional SSF and SF. ③ In the SSSF, the recovery rate of extract of H81 and H82 were 14.9 percent, 18.3 percent, which increased by 26.3 percent, 63.4 percent than that of SSF, respectively. Similarly, the recovery rate of extract of Q11 and Q12 were 15.4 percent, 14.4 percent, which increasd by 30.5 percent and 28.6 percent, compared with SSF. Therefore, in contrast with the SSF, more cellulase with higher activity can be obtained from the SSSF. 3. Mixed culture can improve cellulase activity effectively. ①In the SSSF, the FPA, CMC-Na, and AVI of H8 and yeast in mixed culture improved averagely by 3.4 percent, 20.1 percent, and 42.0 percent than that in single culture; and the FPA, CMC-Na, and AVI of Q1 and yeast in mixed culture increased by 16.7 percent, 3.9 percent, and 5.8 percent, respectively, compared with single culture. ②In the SSF, the FPA, CMC-Na, and AVI of H8 and yeast in mixed culture improved averagely by 14.2 percent, 19.8 percent, and 30.1 percent than that of single culture; similarly, the FPA, CMC-Na, and AVI of Q1 and yeast in mixed culture increased averagely by 69.0 percent, 17.9 percent, and 1.4 percent, respectively, compared with single culture. ③In the SF, the FPA, CMC-Na, and AVI of H8 and yeast in mixed culture rised averagely by 40.6 percent, 27.0 percent, and 134.0 percent; and that of Q1 and yeast in mixed culture were up averagely by 26.1 percent, 67.2 percent, and 3.2 percent, respectively. 4. Making a comparison between organic nitrogen sources and inorganic nitrogen sources, the microorganism absorbs and utilizes the organic nitrogen more effectively than the inorganic nitrogen. Through SSF, we found that the cellulase activity of solid fermentation products adding organic nitrogen sources in the medium was higher than that of adding inorganic sources. To Q1, the oil residue and its acid hydrolysis can be used in fungi fermentation as a better organic nitrogen sources. To H8, dried blood and its hydrolysis are both better organic nitrogen sources. The acid hydrolysis of three kinds of organic nitrogen sources can not improve the enzymatic activity significantly so that we can add directly these nitrogen sources to the medium, and do not need to hydrolyse them。
1.前人测定纤维素酶活采用缓冲液提取剂与纯水为提取剂时测定所得纤维素酶活不同。本研究得到的酶活测定结果表明,对于青霉Q1 和黑曲霉H8,用纯水浸提时的纤维素酶活性高于缓冲液提取。以水为提取剂测定纤维素酶活性时,浸提时间应控制在45min 左右,时间过长会导致酶活降低。
2.本文提出的固-液交替新型发酵工艺(SSSF)能够充分发挥传统固态(SSF)、液态(SF)发酵工艺的优点,弥补两种工艺的不足之处,提高纤维素酶活性。
① 纤维素酶产生菌单菌发酵时,采用SSSF 工艺可使黑曲霉H8 的FPA、CMC-Na 及AVI 平均酶活较SSF 工艺分别提高166.7%、264.0%及273.6%,较SF 工艺分别提高798.1%、2325.5%及547.6%。与之类似,采用SSSF 工艺可使青霉Q1 的FPA、CMC-Na及AVI 平均酶活较SSF 工艺分别提高185.2%、240.6%及257.9%,较SF 工艺分别提高1161.3%、2798.0%及419.5%。
② 纤维素酶产生菌与酵母菌混合发酵时,采用SSSF 工艺可使黑曲霉H8 的FPA、CMC-Na 及AVI 平均酶活较SSF 工艺分别提高499.1%、469.0%及459.7%,较SF 工艺分别提高12659.2%、1872.8%及6718.2%。与之类似,采用SSSF 工艺可使青霉Q1 的FPA、CMC-Na 及AVI 平均酶活较SSF 工艺分别提高805.6%、434.4%及708.0%,较SF工艺分别提高1791.2%、1382.5%及558.7%。由此可见,对纤维素酶发酵而言,本研究提出的SSSF 新型发酵工艺远优于传统的SSF 和SF 工艺。
③ 在SSSF 工艺条件下,黑曲霉H81、H82 的粗酶提取物得率分别为14.9%、18.3%,较SSF 工艺,其得率分别提高26.3%及63.4%;在SSSF 工艺条件下,青霉Q11、Q12 的粗酶提取物得率分别为15.4%、14.4%,较SSF 工艺,其得率分别提高30.5%、28.6%。所以,相对于传统的SSF 固态发酵工艺,采用本研究提出的SSSF 新型发酵工艺时,纤维素酶的产率及酶活更高。
3.混菌培养能有效提高纤维素酶活性。
①在SSSF 中,黑曲霉H8 与酵母菌混合发酵时, FPA、CMC-Na 及AVI 平均酶活分别较单菌发酵时提高3.4%、20.1%及42.0%;青霉Q1 与酵母菌混合发酵时,FPA、CMC-Na
The traditional modes of fermentation for producing cellulase including Solid Substrate Fermentation(SSF), Submerged Fermentation(SF), and a novel mode, Solid Substrate-Submerged Fermentation(SSSF), which was designed in this paper were systemataically studied from three parts of enzyme activity of single culture, mixed culture and recovery rate of cellulase. At the same time, we discussed the influence of two sorts of extractants and eight sorts of nitrogen sources on testing of cellulase activity. The main results showed:
1. In the past, the cellulase activity was different when researchers adopted buffer and pure water to extract the cellulase, respectively. However, according to the experiment, the cellulase activity of H8 and Q1 were higher when using pure water as extractant than buffer in the vitality test of cellulase. When extracting gross cellulase from the fermentation products, the time of extraction should be controlled about 45minutes, and too long will lead to the reduction of enzyme activity.
2. The SSSF is a new fermentation technology, which integrates the advantages of SSF with SF perfectly, and improved the cellulase activity in the process of fermentation.
①In the single culture, the SSSF made the FPA, CMC-Na and AVI of H8 increase averagely by 166.7 percent, 264.0 percent, and 273.6 percent, respectively, compared with SSF; and increase averagely 798.1 percent, 2325.5 percent, and 547.6 percent, respectively, compared with SF. Similarly, compared with SSF, the FPA, CMC-Na, and AVI of Q1 improved averagely 185.2 percent, 240.6 percent, and 257.9 percent, respectively if adopting SSSF; and improved averagely 1161.3 percent, 2798.0 percent, and 419.5 percent, respectively, compared with SF.
② In the mixed culture, compared with SSF, the average improvement of FPA, CMC-Na, and AVI of H8 reached 499.1 percent, 469.0 percent if using SSSF; and reached 459.7 percent; 12659.2 percent, 1872.8 percent, and 6718.2 percent, respectively, compared with SF. Likewise, the average improvement of FPA, CMC-Na, and AVI of Q1 grew up by 805.6 percent, 434.4 percent, and 708.0 percent, compared with SSF if adopting SSSF; and
increased by 1791.2 percent, 1382.5 percent, and 558.7 percent, respectively, compared with SF. So to fermentation for producing cellulase, the SSSF has more advantages over traditional SSF and SF. ③ In the SSSF, the recovery rate of extract of H81 and H82 were 14.9 percent, 18.3 percent, which increased by 26.3 percent, 63.4 percent than that of SSF, respectively. Similarly, the recovery rate of extract of Q11 and Q12 were 15.4 percent, 14.4 percent, which increasd by 30.5 percent and 28.6 percent, compared with SSF. Therefore, in contrast with the SSF, more cellulase with higher activity can be obtained from the SSSF. 3. Mixed culture can improve cellulase activity effectively. ①In the SSSF, the FPA, CMC-Na, and AVI of H8 and yeast in mixed culture improved averagely by 3.4 percent, 20.1 percent, and 42.0 percent than that in single culture; and the FPA, CMC-Na, and AVI of Q1 and yeast in mixed culture increased by 16.7 percent, 3.9 percent, and 5.8 percent, respectively, compared with single culture. ②In the SSF, the FPA, CMC-Na, and AVI of H8 and yeast in mixed culture improved averagely by 14.2 percent, 19.8 percent, and 30.1 percent than that of single culture; similarly, the FPA, CMC-Na, and AVI of Q1 and yeast in mixed culture increased averagely by 69.0 percent, 17.9 percent, and 1.4 percent, respectively, compared with single culture. ③In the SF, the FPA, CMC-Na, and AVI of H8 and yeast in mixed culture rised averagely by 40.6 percent, 27.0 percent, and 134.0 percent; and that of Q1 and yeast in mixed culture were up averagely by 26.1 percent, 67.2 percent, and 3.2 percent, respectively. 4. Making a comparison between organic nitrogen sources and inorganic nitrogen sources, the microorganism absorbs and utilizes the organic nitrogen more effectively than the inorganic nitrogen. Through SSF, we found that the cellulase activity of solid fermentation products adding organic nitrogen sources in the medium was higher than that of adding inorganic sources. To Q1, the oil residue and its acid hydrolysis can be used in fungi fermentation as a better organic nitrogen sources. To H8, dried blood and its hydrolysis are both better organic nitrogen sources. The acid hydrolysis of three kinds of organic nitrogen sources can not improve the enzymatic activity significantly so that we can add directly these nitrogen sources to the medium, and do not need to hydrolyse them。
引文
1.王多仁 纤维素酶开发与应用进展[J]. 北京日化,2002,3:1～5
2.于顺鹏等.菇渣中纤维素酶及其在沼气发酵中的作用[J].南京农业大学学报,1991,14(2):65～68
3.赵永贵,韩恒芬.植物纤维素开发利用研究现状及对策[J].河南科技,1990,12:9～12
4.B.Gallo, R. Andreotti,C. Roche, Ryu and Mandels. cellulase production by a new mutant strain of Trichoderma reesei MCG 77[J]. Biotechnology Bioengeneer 1978,8:89～101
5.E. Samain, P.Debire,J.P.Touzel. High level production of a cellulase-free xylanase in glucose-limited fed-batch cultures of a thermophilic Bacillus strain[J]. Biotechnology 1997,58(2):71～78
6.I.V.Soloveva, O.N. Okunev,E.G.Kryukora Neutral cellulases of mycelial fungi:searching for producers and their characterization[J].Biochem.Microbiology 1997,33(4):388～392
7.焦瑞身主编.微生物工程[M].化学工业出版社,2003,7
8.Riesenberg D,Guthke[R]. Appl Microbiol Biotechnol,1999,51: 422
9.Lee Y-S, Chang H N[J]. Adv Biochem Eng Biotechnol, 1995,52: 27
10.Suzuki T. [J] Ferment Bioeng, 1996,82: 264
11.Nakano K, Rischke M, Sato S, et al[J]. Appl Microbiol Biotechnol, 1997,48: 597
12.Holst O, Manelius A, Krahe M, et al[J]. Comp Biochem Physiol, 1997,118A: 415
13.Hartbrich A, Schnitz G, Weuster-Botz D, et al[J]. Biotech. Bioeng, 1996,51: 624
14.Bermejo L L, Welker N E, Papoutsakis E T[J]. App Environ Microbiol, 1998, 64: 1079
15.Kamoshita Y, Ohashi R, Suzuki T.[J] Ferment Bioeng, 1998, 85(4): 422
16.Amos B, Al-Rubeai M, Emery A N.[J] Enzyme Microb Technol, 1994, 16: 688
17.邹崇达,李友荣.[J] 中国抗生素杂志,1992,17(1):6
18.储炬,李友荣.[J] 华东理工大学学报,1994,20(5):605
19.Kobayashi T, Minami T.[J] Kemikaru Enjiniarilgu, 1988, 33: 965
20.徐凤彩主编.酶工程[M].中国农业出版社,2000,10
21.J. Lenz, M. H?fer, J.-B. Krasenbrink etc. A survey of computational and physical methods applied to solid-state fermentation[J]. Appl Microbiol Biotechnol, 2004,65: 9～17
22.Kiran Mazumdar-Shaw and Shrikumar Suryanarayan. Commercialization of a Novel Fermentation Concept. Advances in Biochemical Engineering/Biotechnology, 2003, 85: 29～42
23.Mandels.M and E.T.Reese. lnduction of cellulase in Trichoderma viride as influenced by carbon sources and metals[J].Bacterial.1977,73:269
24.Jacob,F.and J.Monod.Genetic regulatory mechanisms in the synthesis of proteins,[J]. Mol.Biol.1987,3:318
25.Nisizawa,T., Susuki.H and Nisizawa,K. Catabolite repression of cellulase Formation in Trichoderma viride[J]. Biochem 1972,71(6):999
26.Nisizawa, T., Suzuki,H., Nakayama,M. and Nisizawa,K.. Inductive Formation of Cellulase by sophorose in Trichoderma viride T[J]. Biochem. 1971,70(3):375
27.Pardee, A. B. and prestidge, L. S. The initial kinetics of enzyme induction[J]. Biochem Biophys.Acta,1981,49:77
28.王沁等.黑曲霉W25 菌株纤维素酶合成调控问题初探[J].工业微生物,1995,25(3):13～15
29.Wisiman.A.Enzyme induction Basic Life Sciences[J], ed.by D.V. Parke Plenum Press, London and New York, P.I 1975,6
30.Mandels.M.,F.W.parrish and E.T. Reese.Sophorose as an inducer of cellulase in Trichoderma viride[J].Bacterial. 1972,83:400
31.Reese,E. T., and Masuire.lncrease in cellulase yields by addition of surfactants to cellobiose cultures of Trichoderma viride Devel[J].lndust.Microbiol 1971,12:212
32.勇强,陈艳萍等.添加纤维二糖,淀粉水解液对纤维素酶制备的影响[J].南京林业大学学报,1998,22(2):53～56
33.张海,刘涛.纤维素水解产物对木霉A10 纤维素酶的抑制作用[J].西北大学学报(自科版),1995,25(1):51～54
34.朱雨生,谭常.木霉纤维素酶的诱导形成及其调节(Ⅲ)葡萄糖母液和槐豆荚根提取液对纤维素酶诱导效应的分析[J].植物生理学报,1978,4(1):19～26
35.Bisaria, v.s.,and glose T. K, Biodegradation of cellulosic materials microoganisrns. enzymes,substrates and products[J]. Inzyme uicrob. Technology 1981,3:90～104
36.朱雨生,谭常.木霉纤维素酶的诱导形成及其调节(Ⅰ)槐糖对木霉EA3-867 洗涤菌丝体纤维素酶形成的诱导作用及降解物阻遏现象[J].植物生理学报,1978,4(1):1～17
37.Spielman, S. and Dunn, R. Interactions between enzyme forming synthesis during adaptation[J]. J.Gen. Physiol 1978,31:153
38.Pardee, A. B. and prestidge, L. S. The initial kinetics of enzyme induction[J]. Biochem Biophys.Acta,1981,49:77
39.Zahner,H. and W.K. Maas. Biology of Antibiotics[J]. Springer-verlag New York, Heidelberg, Berlin. 1972:86
40.朱雨生,谭常.木霉纤维素酶的诱导形成及其调节(Ⅰ)槐糖对木霉EA3-867 纤维素酶形成的诱导作用[J].微生物学报,1978,18(4):320～331
41.Crombrugghe, B. D., R. L. Perlan, H.E. Varmus and Ⅰ. pastun Regulatio of inducible enzyme synthesis in qschrichia coli by cyclic adenosin 3′, 5′-monophosbhate[J]. Biol.chem..1969,244:5828
42.Makman, R. S and E. W. Sutherland,Adenosine 3′,5′-phosphate in E. coli[J]. Biochem. Chern 1985,240:1309
43.Perlman, R. L., B. D. Crombrugghe and I. Pastqn, cyclic Amp regulates catabolite and transient repression in E. coli[J]. Nature, 1979,223:810
44.Child. J. S.et al.Determination of cellulase activity using hydroxyethyl cellulose as substracts. Can[J]. Biochem,1973,51:39
45.王冬,曲音波等.L-山梨糖提高木霉纤维素酶合成速率机制的研究[J].真菌学报,1995,14(2):143～147
46.Hulme.M.A.. Viscosimetric determination of cmcase activity In method in qnzymology[J],1988,160:130
47.Merivuor.H.,Sands.J.A..Montenecourt. Effects of tunicamycin on secretion and enzyatic activities of cellulase from Trichoderma reesei[J].Appl.Microbiology.Biotechnol.1985,23(1):60～66
48.杨艳红, 郑一敏, 王伯初等,多菌种固体共发酵生物软化稻壳的研究[J]. 微生物学通报,2004,31(4):14～18
49.李艳,黄威滨. 里氏木霉与黑曲霉共同培养固态发酵生产饲用酶的研究[J]. 粮食与饲料工业, 2002,9:29～31
50.陈庆森,刘剑虹,潘建阳等. 利用多菌种共发酵技术转化玉米秸秆的研究[J]. 食品与发酵工业,1999,25(5):1～6
51.杨树林,吴旻,宁长发等.利用农业废弃物微生物共发酵制取蛋白质的研究[J].中山大学学报,2003,6(42):115～117
52.张冬艳, 张瑞兰, 张 通. 绿色木酶与产朊假丝酵母混合发酵生产纤维素酶和单细胞蛋白[J].内蒙古工业大学学报,2003,22(3):180～185
53.张英华,梁静思,龙广宇等.用木素木霉和凤尾菇菌两菌交替发酵法提高纤维素酶活力研究[J].菌物系统,1999,18(2):184～191
54.R.Dueas, R.P.Tengerdy, M.Gutierrez-Correa. Cellulase production by mixed fungi in solid-substrate fermentation of bagasse[J]. World Journal of Microbiology and Biotechnology(Historical Archive),1995,11(3): 333～337
55.Z.Wu and Y.Y. Lee. Inhibition of the enzymatic hydrolysis of cellulose by ethanol[J]. Biotechnology Letter,1997,19(10): 977～979
56.Muhannad I. Massadeh, Wan Mohtar Wan Yusoff, Othman Omar etc. Synergism of cellulase enzymes in mixed culture solid substrate fermentation[J]. Biotechnology Letters,2001,23: 1771～1774
57.Reese.E.T., J.E. Lola and F.W.Parrish Modified substrates and modified products as inducers of carbohydrases[J]. Bacterial 1969,100:1151
58.Shu Chen and Morris Wayman. Use of sorbose to enhance celluiase activity in a Trichoderma reesei cellulase system produced on wheat hydrolysate[J]. Biotechnology Techniques(Historical Archive), 1993,7(5): 345～350
59.刘德海,安明理,王红云等. 固态发酵复合酶培养条件的优化及工艺研究[J]. 饲料工业,2004,25(6): 56～58
60.费笛波,冯观泉,叶玲玲等. 黑曲霉6042液体发酵生产饲用复合酶的工艺[J]. 浙江农业学报,1996,8(6): 357～361
61.张继泉,王瑞明,孙玉英等.里氏木霉生产纤维素酶的研究进展[J].饲料工业,2003,24(1):9～13
62.王兰芬.纤维素酶的作用机理及开发应用[J].酿酒科技,1997,6:16
63.张树政主编. 酶制剂工业[M]. 科学出版社. 1984,7
64.Ghose TK(1987) Measurement of cellulase activities[J]. Pure Appl. Chem. 59:257～268
65. Katalin Urbanszki, Gyorgy Szakacs, Robert P.Tengerdy. Standardization of the filter paper activity assay for solid substrate fermentation[J]. Biotechnology Letters,2000, 22:65～69
66.施曾辉,刘尔教.酶固体生产和应用中的有关问题[J].中国调味品,1997,10:6～10
67.勇强.分批添料半连续发酵制备纤维素酶[J].林产化学与工业,1998,18(2):73～77
68.余晓斌,具润谟.分批与流加发酵法生产纤维素酶的研究[J].食品与发酵工业,1999,25(1): 16～19
69.夏黎明,代淑梅.应用固定化里氏木霉糖化玉米秸秆纤维素的研究[J].微生物学报,1998,38(2):114～119
70.龙鸿,朱玉环,辛孝贵.纤维素酶对小麦、水稻和高梁茎秆作用研究[J].沈阳农业大学学报,1996,12(27):94～97
71.T.K.Ghose and A.N.Pathak 纤维素酶的应用[J].应用微生物,1974,4:48～50
72.Toyama,N and K. Ogawa. Sugar production from agricultural Woody Wastes by saccharification with Trichoderma viride cellulase Bioengineering.Symp.[J] 1975,5:225
73.王燕.纤维素酶与饲料生产[J].黑龙江畜牧兽医,1999,7:22～23
74.王泉林等.纤维素酶饲用复合酶制剂的生产和应用研究(续)[J].饲料博览.1996,2:32～33
75.尹清强等.复合酶制剂对猪增重的影响[J].饲料研究,1992,3:5
76.尹清强.纤维素酶制剂对奶年产奶量和饲料效率的影响[J].吉林畜牧兽医,1991,6:1
77.高大威.酶制剂在饲料中的应用[J].饲料工业,1992,13(1):28
78.朱玉环,陈祖洁.纤维素酶在反刍动物饲料中的应用研究[J].沈阳农业大学学报,1996,27(专辑):64～69
79.瞿明仁,晏向华,黎观红.饲用真菌纤维素酶研究与应用[J].饲料工业,1999,20(12):30～32
80.尹清强等.纤维素酶对兔增重肠绒毛结构胃内容物及睾丸内无机元素含量的影响[J].黑龙江畜牧兽医,1992,1:14
81.程丽娟,薛泉宏主编.微生物学实验技术[M].世界图书出版公司,2000,4
82.曲音波,高培基,王祖农.青霉的纤维素抗降解物阻遏突变株的选育[J].真菌学报,1984,(4):238～243
83.高培基.纤维素酶活力测定方法研究进展[J].工业微生物,1985,153(6):5～8
84.宋荣钊,何泳生.纤维素生物量的转化及预处理方法[J].江西科学,1989,7(4):60～64
85.周正红,高孔荣等.纤维素酶在食品发酵工业中的应用及前景[J].暨南大学学报(自科与医学版),1998,19(5):125～130
86.加滕久明等.酶在果蔬加工中的应用[J].应用微生物,1986,2:19
87.朱知难.纤维素酶生产与应用[J].广东科技,1995,9～10:5～6
88.邱立友,朱德育.黑曲霉酶学特性及其在洒精生产中的应用[J].河南农业大学学报,1993,27(3):291～295
89.王贵权等.纤维素酶治疗牛消化道疾病150 例[J].黑龙江畜牧盖医,1991,7:31
90.只兴有.纤维素酶治疗奶牛低酸度酒精阳性乳[J].黑龙江畜牧兽医,1991,6:33
91.杜秉海,曲音波等.纤维废渣固态酒精发酵及纤维素-淀粉共发酵的研究[J].食品与发酵工业,1995,5:15～20
92.王沁,赵学慧.纤维素酶水解稻草生产单细胞蛋白(SCP):Ⅱ稻草酶解液生产SCP 的研究[J].微生物学杂志,1993,13(2):8～12
93.张遐耘,张文悦.黄酒糟纤维素酶处理和单细胞蛋白生产的研究[J].粮食与饲料工业,1998,7:24～25
94.Mandels,M., sternberg,D., and Andreotti,R.E. Symp. On qnzymatic Hydrolysis of cellulose Aulanko[J],Finland,ed.M. Bailey,1975,3:12～14
95.外山信男等.纤维素酶研究进展(综述)[J].发酵工业杂志,1969,47(11):714
96.王兰芬.纤维素酶的作用机理及开发应用[J].酿酒科技,1997,6:16
97.瞿明仁,晏向华,黎观红.饲用真菌纤维素酶研究与应用[J].饲料工业,1999,20(12):30～32
98.T.K.Ghose and A.N.Pathak 纤维素酶的应用[J].应用微生物,1974,4:48～50
99.于顺鹏等.菇渣中纤维素酶及其在沼气发酵中的作用[J].南京农业大学学报,1991,14(2):65～68
100.Eveleigh,D.E. Cellulase a perspective pbil Trans.R.Soc.Lona[A]. 1987,321～435
101.Faterstam L. T. and Pettersson L.G.[J] FEBS letter 1980,119:97～100
102.Gallo,B.J. et al. Biotechnology and Bioengineering. Symposium Biotechnology in qnergy production and conserration published by Tohn wiley & Sons[J]. 1978,8:89～101
103.Gama F. M., Mota. M.. Enzymatic hydrolysis of cellulose(Ⅰ) relationship between kinetics and physical-chemical parameters[J]. Biocatalysis Biotranstorm 1997,15(3):221～236
104.Gama-F.M.,Mota.M..Enzymatic hydrolysis of cellulose( Ⅱ) x-ray Photoelection spectroscopy studies on cellulase adsorption[J]. Biocatalysis Biotranstorm 1997,15(3):237～250
105.Gilligan W., Reese E.T. Canada.[J] Jomicrobiolosy 1954,1:90～107
106.Grohmann.K., Himmel. M., Rivard.C. Chemical-mechanical Methods for the enhanced utilization of straw[J]. Biotechnology Bioengeering 1984,14:137～157
107.Hulme.M.A.. Viscosimetric determination of cmcase activity In method in qnzymology[J],1988,160:130
108.Selby. K. et al.[J]Advance in chomictry 1969,95:34～50
109.Spiegelman.Nuclear and cytoplasmic factors controlling enzymatic constitution[J].Cold spring Harbor Symp.Quant. Biol. 1997,Ⅱ,256
110.Spielman, S. and Dunn, R. Interactions between enzyme forming synthesis during adaptation[J]. Gen. Physiol 1978,31:153
111.Voloch,M,Landisch.M.R,Cantarella.M. Reparation and Cellodextrins Using Sulfuric acid[J]. Biotechnology.Bioengeneery, 1984,26(5):557～559
112.Vranska M. and Hiely P. Carbohydr.[J] Research 1992,227:19～27
113.Van Tibenrqh H. and Claeyssens M.[J] FEBS Letter 1985,187:283～288
114.White A.R.and Brown R.M. The elevendays international symposium on wood pulping chemistry[J]. SPCI Stockholm 1981,5:44～46
2.于顺鹏等.菇渣中纤维素酶及其在沼气发酵中的作用[J].南京农业大学学报,1991,14(2):65～68
3.赵永贵,韩恒芬.植物纤维素开发利用研究现状及对策[J].河南科技,1990,12:9～12
4.B.Gallo, R. Andreotti,C. Roche, Ryu and Mandels. cellulase production by a new mutant strain of Trichoderma reesei MCG 77[J]. Biotechnology Bioengeneer 1978,8:89～101
5.E. Samain, P.Debire,J.P.Touzel. High level production of a cellulase-free xylanase in glucose-limited fed-batch cultures of a thermophilic Bacillus strain[J]. Biotechnology 1997,58(2):71～78
6.I.V.Soloveva, O.N. Okunev,E.G.Kryukora Neutral cellulases of mycelial fungi:searching for producers and their characterization[J].Biochem.Microbiology 1997,33(4):388～392
7.焦瑞身主编.微生物工程[M].化学工业出版社,2003,7
8.Riesenberg D,Guthke[R]. Appl Microbiol Biotechnol,1999,51: 422
9.Lee Y-S, Chang H N[J]. Adv Biochem Eng Biotechnol, 1995,52: 27
10.Suzuki T. [J] Ferment Bioeng, 1996,82: 264
11.Nakano K, Rischke M, Sato S, et al[J]. Appl Microbiol Biotechnol, 1997,48: 597
12.Holst O, Manelius A, Krahe M, et al[J]. Comp Biochem Physiol, 1997,118A: 415
13.Hartbrich A, Schnitz G, Weuster-Botz D, et al[J]. Biotech. Bioeng, 1996,51: 624
14.Bermejo L L, Welker N E, Papoutsakis E T[J]. App Environ Microbiol, 1998, 64: 1079
15.Kamoshita Y, Ohashi R, Suzuki T.[J] Ferment Bioeng, 1998, 85(4): 422
16.Amos B, Al-Rubeai M, Emery A N.[J] Enzyme Microb Technol, 1994, 16: 688
17.邹崇达,李友荣.[J] 中国抗生素杂志,1992,17(1):6
18.储炬,李友荣.[J] 华东理工大学学报,1994,20(5):605
19.Kobayashi T, Minami T.[J] Kemikaru Enjiniarilgu, 1988, 33: 965
20.徐凤彩主编.酶工程[M].中国农业出版社,2000,10
21.J. Lenz, M. H?fer, J.-B. Krasenbrink etc. A survey of computational and physical methods applied to solid-state fermentation[J]. Appl Microbiol Biotechnol, 2004,65: 9～17
22.Kiran Mazumdar-Shaw and Shrikumar Suryanarayan. Commercialization of a Novel Fermentation Concept. Advances in Biochemical Engineering/Biotechnology, 2003, 85: 29～42
23.Mandels.M and E.T.Reese. lnduction of cellulase in Trichoderma viride as influenced by carbon sources and metals[J].Bacterial.1977,73:269
24.Jacob,F.and J.Monod.Genetic regulatory mechanisms in the synthesis of proteins,[J]. Mol.Biol.1987,3:318
25.Nisizawa,T., Susuki.H and Nisizawa,K. Catabolite repression of cellulase Formation in Trichoderma viride[J]. Biochem 1972,71(6):999
26.Nisizawa, T., Suzuki,H., Nakayama,M. and Nisizawa,K.. Inductive Formation of Cellulase by sophorose in Trichoderma viride T[J]. Biochem. 1971,70(3):375
27.Pardee, A. B. and prestidge, L. S. The initial kinetics of enzyme induction[J]. Biochem Biophys.Acta,1981,49:77
28.王沁等.黑曲霉W25 菌株纤维素酶合成调控问题初探[J].工业微生物,1995,25(3):13～15
29.Wisiman.A.Enzyme induction Basic Life Sciences[J], ed.by D.V. Parke Plenum Press, London and New York, P.I 1975,6
30.Mandels.M.,F.W.parrish and E.T. Reese.Sophorose as an inducer of cellulase in Trichoderma viride[J].Bacterial. 1972,83:400
31.Reese,E. T., and Masuire.lncrease in cellulase yields by addition of surfactants to cellobiose cultures of Trichoderma viride Devel[J].lndust.Microbiol 1971,12:212
32.勇强,陈艳萍等.添加纤维二糖,淀粉水解液对纤维素酶制备的影响[J].南京林业大学学报,1998,22(2):53～56
33.张海,刘涛.纤维素水解产物对木霉A10 纤维素酶的抑制作用[J].西北大学学报(自科版),1995,25(1):51～54
34.朱雨生,谭常.木霉纤维素酶的诱导形成及其调节(Ⅲ)葡萄糖母液和槐豆荚根提取液对纤维素酶诱导效应的分析[J].植物生理学报,1978,4(1):19～26
35.Bisaria, v.s.,and glose T. K, Biodegradation of cellulosic materials microoganisrns. enzymes,substrates and products[J]. Inzyme uicrob. Technology 1981,3:90～104
36.朱雨生,谭常.木霉纤维素酶的诱导形成及其调节(Ⅰ)槐糖对木霉EA3-867 洗涤菌丝体纤维素酶形成的诱导作用及降解物阻遏现象[J].植物生理学报,1978,4(1):1～17
37.Spielman, S. and Dunn, R. Interactions between enzyme forming synthesis during adaptation[J]. J.Gen. Physiol 1978,31:153
38.Pardee, A. B. and prestidge, L. S. The initial kinetics of enzyme induction[J]. Biochem Biophys.Acta,1981,49:77
39.Zahner,H. and W.K. Maas. Biology of Antibiotics[J]. Springer-verlag New York, Heidelberg, Berlin. 1972:86
40.朱雨生,谭常.木霉纤维素酶的诱导形成及其调节(Ⅰ)槐糖对木霉EA3-867 纤维素酶形成的诱导作用[J].微生物学报,1978,18(4):320～331
41.Crombrugghe, B. D., R. L. Perlan, H.E. Varmus and Ⅰ. pastun Regulatio of inducible enzyme synthesis in qschrichia coli by cyclic adenosin 3′, 5′-monophosbhate[J]. Biol.chem..1969,244:5828
42.Makman, R. S and E. W. Sutherland,Adenosine 3′,5′-phosphate in E. coli[J]. Biochem. Chern 1985,240:1309
43.Perlman, R. L., B. D. Crombrugghe and I. Pastqn, cyclic Amp regulates catabolite and transient repression in E. coli[J]. Nature, 1979,223:810
44.Child. J. S.et al.Determination of cellulase activity using hydroxyethyl cellulose as substracts. Can[J]. Biochem,1973,51:39
45.王冬,曲音波等.L-山梨糖提高木霉纤维素酶合成速率机制的研究[J].真菌学报,1995,14(2):143～147
46.Hulme.M.A.. Viscosimetric determination of cmcase activity In method in qnzymology[J],1988,160:130
47.Merivuor.H.,Sands.J.A..Montenecourt. Effects of tunicamycin on secretion and enzyatic activities of cellulase from Trichoderma reesei[J].Appl.Microbiology.Biotechnol.1985,23(1):60～66
48.杨艳红, 郑一敏, 王伯初等,多菌种固体共发酵生物软化稻壳的研究[J]. 微生物学通报,2004,31(4):14～18
49.李艳,黄威滨. 里氏木霉与黑曲霉共同培养固态发酵生产饲用酶的研究[J]. 粮食与饲料工业, 2002,9:29～31
50.陈庆森,刘剑虹,潘建阳等. 利用多菌种共发酵技术转化玉米秸秆的研究[J]. 食品与发酵工业,1999,25(5):1～6
51.杨树林,吴旻,宁长发等.利用农业废弃物微生物共发酵制取蛋白质的研究[J].中山大学学报,2003,6(42):115～117
52.张冬艳, 张瑞兰, 张 通. 绿色木酶与产朊假丝酵母混合发酵生产纤维素酶和单细胞蛋白[J].内蒙古工业大学学报,2003,22(3):180～185
53.张英华,梁静思,龙广宇等.用木素木霉和凤尾菇菌两菌交替发酵法提高纤维素酶活力研究[J].菌物系统,1999,18(2):184～191
54.R.Dueas, R.P.Tengerdy, M.Gutierrez-Correa. Cellulase production by mixed fungi in solid-substrate fermentation of bagasse[J]. World Journal of Microbiology and Biotechnology(Historical Archive),1995,11(3): 333～337
55.Z.Wu and Y.Y. Lee. Inhibition of the enzymatic hydrolysis of cellulose by ethanol[J]. Biotechnology Letter,1997,19(10): 977～979
56.Muhannad I. Massadeh, Wan Mohtar Wan Yusoff, Othman Omar etc. Synergism of cellulase enzymes in mixed culture solid substrate fermentation[J]. Biotechnology Letters,2001,23: 1771～1774
57.Reese.E.T., J.E. Lola and F.W.Parrish Modified substrates and modified products as inducers of carbohydrases[J]. Bacterial 1969,100:1151
58.Shu Chen and Morris Wayman. Use of sorbose to enhance celluiase activity in a Trichoderma reesei cellulase system produced on wheat hydrolysate[J]. Biotechnology Techniques(Historical Archive), 1993,7(5): 345～350
59.刘德海,安明理,王红云等. 固态发酵复合酶培养条件的优化及工艺研究[J]. 饲料工业,2004,25(6): 56～58
60.费笛波,冯观泉,叶玲玲等. 黑曲霉6042液体发酵生产饲用复合酶的工艺[J]. 浙江农业学报,1996,8(6): 357～361
61.张继泉,王瑞明,孙玉英等.里氏木霉生产纤维素酶的研究进展[J].饲料工业,2003,24(1):9～13
62.王兰芬.纤维素酶的作用机理及开发应用[J].酿酒科技,1997,6:16
63.张树政主编. 酶制剂工业[M]. 科学出版社. 1984,7
64.Ghose TK(1987) Measurement of cellulase activities[J]. Pure Appl. Chem. 59:257～268
65. Katalin Urbanszki, Gyorgy Szakacs, Robert P.Tengerdy. Standardization of the filter paper activity assay for solid substrate fermentation[J]. Biotechnology Letters,2000, 22:65～69
66.施曾辉,刘尔教.酶固体生产和应用中的有关问题[J].中国调味品,1997,10:6～10
67.勇强.分批添料半连续发酵制备纤维素酶[J].林产化学与工业,1998,18(2):73～77
68.余晓斌,具润谟.分批与流加发酵法生产纤维素酶的研究[J].食品与发酵工业,1999,25(1): 16～19
69.夏黎明,代淑梅.应用固定化里氏木霉糖化玉米秸秆纤维素的研究[J].微生物学报,1998,38(2):114～119
70.龙鸿,朱玉环,辛孝贵.纤维素酶对小麦、水稻和高梁茎秆作用研究[J].沈阳农业大学学报,1996,12(27):94～97
71.T.K.Ghose and A.N.Pathak 纤维素酶的应用[J].应用微生物,1974,4:48～50
72.Toyama,N and K. Ogawa. Sugar production from agricultural Woody Wastes by saccharification with Trichoderma viride cellulase Bioengineering.Symp.[J] 1975,5:225
73.王燕.纤维素酶与饲料生产[J].黑龙江畜牧兽医,1999,7:22～23
74.王泉林等.纤维素酶饲用复合酶制剂的生产和应用研究(续)[J].饲料博览.1996,2:32～33
75.尹清强等.复合酶制剂对猪增重的影响[J].饲料研究,1992,3:5
76.尹清强.纤维素酶制剂对奶年产奶量和饲料效率的影响[J].吉林畜牧兽医,1991,6:1
77.高大威.酶制剂在饲料中的应用[J].饲料工业,1992,13(1):28
78.朱玉环,陈祖洁.纤维素酶在反刍动物饲料中的应用研究[J].沈阳农业大学学报,1996,27(专辑):64～69
79.瞿明仁,晏向华,黎观红.饲用真菌纤维素酶研究与应用[J].饲料工业,1999,20(12):30～32
80.尹清强等.纤维素酶对兔增重肠绒毛结构胃内容物及睾丸内无机元素含量的影响[J].黑龙江畜牧兽医,1992,1:14
81.程丽娟,薛泉宏主编.微生物学实验技术[M].世界图书出版公司,2000,4
82.曲音波,高培基,王祖农.青霉的纤维素抗降解物阻遏突变株的选育[J].真菌学报,1984,(4):238～243
83.高培基.纤维素酶活力测定方法研究进展[J].工业微生物,1985,153(6):5～8
84.宋荣钊,何泳生.纤维素生物量的转化及预处理方法[J].江西科学,1989,7(4):60～64
85.周正红,高孔荣等.纤维素酶在食品发酵工业中的应用及前景[J].暨南大学学报(自科与医学版),1998,19(5):125～130
86.加滕久明等.酶在果蔬加工中的应用[J].应用微生物,1986,2:19
87.朱知难.纤维素酶生产与应用[J].广东科技,1995,9～10:5～6
88.邱立友,朱德育.黑曲霉酶学特性及其在洒精生产中的应用[J].河南农业大学学报,1993,27(3):291～295
89.王贵权等.纤维素酶治疗牛消化道疾病150 例[J].黑龙江畜牧盖医,1991,7:31
90.只兴有.纤维素酶治疗奶牛低酸度酒精阳性乳[J].黑龙江畜牧兽医,1991,6:33
91.杜秉海,曲音波等.纤维废渣固态酒精发酵及纤维素-淀粉共发酵的研究[J].食品与发酵工业,1995,5:15～20
92.王沁,赵学慧.纤维素酶水解稻草生产单细胞蛋白(SCP):Ⅱ稻草酶解液生产SCP 的研究[J].微生物学杂志,1993,13(2):8～12
93.张遐耘,张文悦.黄酒糟纤维素酶处理和单细胞蛋白生产的研究[J].粮食与饲料工业,1998,7:24～25
94.Mandels,M., sternberg,D., and Andreotti,R.E. Symp. On qnzymatic Hydrolysis of cellulose Aulanko[J],Finland,ed.M. Bailey,1975,3:12～14
95.外山信男等.纤维素酶研究进展(综述)[J].发酵工业杂志,1969,47(11):714
96.王兰芬.纤维素酶的作用机理及开发应用[J].酿酒科技,1997,6:16
97.瞿明仁,晏向华,黎观红.饲用真菌纤维素酶研究与应用[J].饲料工业,1999,20(12):30～32
98.T.K.Ghose and A.N.Pathak 纤维素酶的应用[J].应用微生物,1974,4:48～50
99.于顺鹏等.菇渣中纤维素酶及其在沼气发酵中的作用[J].南京农业大学学报,1991,14(2):65～68
100.Eveleigh,D.E. Cellulase a perspective pbil Trans.R.Soc.Lona[A]. 1987,321～435
101.Faterstam L. T. and Pettersson L.G.[J] FEBS letter 1980,119:97～100
102.Gallo,B.J. et al. Biotechnology and Bioengineering. Symposium Biotechnology in qnergy production and conserration published by Tohn wiley & Sons[J]. 1978,8:89～101
103.Gama F. M., Mota. M.. Enzymatic hydrolysis of cellulose(Ⅰ) relationship between kinetics and physical-chemical parameters[J]. Biocatalysis Biotranstorm 1997,15(3):221～236
104.Gama-F.M.,Mota.M..Enzymatic hydrolysis of cellulose( Ⅱ) x-ray Photoelection spectroscopy studies on cellulase adsorption[J]. Biocatalysis Biotranstorm 1997,15(3):237～250
105.Gilligan W., Reese E.T. Canada.[J] Jomicrobiolosy 1954,1:90～107
106.Grohmann.K., Himmel. M., Rivard.C. Chemical-mechanical Methods for the enhanced utilization of straw[J]. Biotechnology Bioengeering 1984,14:137～157
107.Hulme.M.A.. Viscosimetric determination of cmcase activity In method in qnzymology[J],1988,160:130
108.Selby. K. et al.[J]Advance in chomictry 1969,95:34～50
109.Spiegelman.Nuclear and cytoplasmic factors controlling enzymatic constitution[J].Cold spring Harbor Symp.Quant. Biol. 1997,Ⅱ,256
110.Spielman, S. and Dunn, R. Interactions between enzyme forming synthesis during adaptation[J]. Gen. Physiol 1978,31:153
111.Voloch,M,Landisch.M.R,Cantarella.M. Reparation and Cellodextrins Using Sulfuric acid[J]. Biotechnology.Bioengeneery, 1984,26(5):557～559
112.Vranska M. and Hiely P. Carbohydr.[J] Research 1992,227:19～27
113.Van Tibenrqh H. and Claeyssens M.[J] FEBS Letter 1985,187:283～288
114.White A.R.and Brown R.M. The elevendays international symposium on wood pulping chemistry[J]. SPCI Stockholm 1981,5:44～46