黑曲霉木聚糖酶固体发酵及酶学特性研究
|
摘要
本文利用三点法和插片法对黑曲霉SB-02(Aspergillus Niger SB-02)菌株进行了菌落形态及菌株的观察:利用双层平板法和三点法研究了黑曲霉SB-02菌株对培养基中木聚糖的降解情况:通过摇床培养,初步测定了黑曲霉SB-02菌株所产木聚糖酶的活性大小。
同时对黑曲霉固体发酵木聚糖酶的条件进行了优化,优化因子有:碳源,氮源,培养温度,培养时间,初始pH值,初始水分,接种量,浸提剂等。结果表明:以麸皮和玉米芯质量比7:3混合后作为碳源,以0.15%的硫酸铵为无机氮源,物料与水分的比为1:1;培养温度28-30℃;培养基起始pH值为4.8;接种量为4%(107个/ml),加入0.05%的吐温-20,发酵时间为72h,加入10倍培养基干重的自来水,于40℃摇床浸提1h,得到的木聚糖酶活为650.45IU/ml。
将固体发酵得到的粗酶经过滤、离心、超滤、冻干,制得粗酶粉,研究了粗酶粉的组分和反应特性,结果表明:在所得到的粗酶粉中,含有多种降解酶组分,分别是木聚糖酶、纤维素酶,滤纸酶、淀粉酶;分别占总酶活的66.3%、17.1%、3.8%、12.7%;粗酶的最适反应温度为50℃,最适反应pH值为4.8,Fe3+对酶活具有较强的激活作用, Mg2+具有一定的抑制作用,其他金属离子无显著作用。
经过DEAE A-25葡聚糖凝胶层析发现,黑曲霉木聚糖酶中含有X-Ⅰ,X-Ⅱ,X-Ⅲ三种木聚糖酶同工酶组分,X-Ⅰ为不吸附组分;X-Ⅰ,X-Ⅱ含量较少,X-Ⅲ含量较高。经PAGE,三种组分均呈单一条带。经纯化,木聚糖酶的比活力提高了2倍左右,酶活回收率为26.3%。
The paper uses three-point method and the interpolation method to abserve the colony morphology , external hyphae and spores of the laboratory-preserved Aspergillus niger SB-02. It is studied the Aspergillus niger SB-02’s degradation of xylan on double-deck plate and determinated the activity of xylanase after 3 days liquid fermentation.
The paper focus on optimization of the solid fermentation conditions for xylanase. Optimization factors are as follows: carbon, nitrogen, temperature, incubation time, the initial pH , the initial moisture, inoculation, extraction, etc. The results show that: the bran and corn-cob are mixed at ratio 7:3 as a mixtured carbon source; the best inorganic nitrogen source is ammonium sulfate(0.15%); the ratio of the materials and the water is 1:1 ; the appropriate culture temperature is 28-30℃; the initial pH is 4.8; the appropriate inoculation is 4% (107 / ml); the content of Tween-20 is 0.05%; the liquid fermentation time is 72 hours; 10 times the medium of water, extracted for 1 hour at 40℃; the activity of xylanase is up to 650.45 U/ml.
The crude enzyme powder is made from extraction,filtration,centrifugation,ultrafiltration, freeze-dried of the solid-state fermentation. The results show that: the crude enzyme powder contains various degrading enzymes,such as xylanase, cellulase, filter enzyme, amylase; respectively,their activity is 66.3%, 17.1% and 3.8% , 12.7%; the optimum reaction temperature of the crude xylanase is 50℃and the optimum reaction PH value is 4.8; Fe3+ strongly actived the activity of the xylanase; Mg2+ has a certain inhinition ; the Other metal ions have no significant role.After dextran gel chromatography of DEAE A-25 ,we found that Aspergillus niger xylanase contains three isozyme: X-Ⅰ, X-Ⅱ, X-Ⅲ. X-Ⅲis not a adsorption component; the content of X-Ⅰ, X-Ⅱare fewer than X-Ⅲ. After PAGE ,three isozymes all showed a single band. After purification, Xylanase activity increases 2 times and enzyme recovery is 26.3%.
同时对黑曲霉固体发酵木聚糖酶的条件进行了优化,优化因子有:碳源,氮源,培养温度,培养时间,初始pH值,初始水分,接种量,浸提剂等。结果表明:以麸皮和玉米芯质量比7:3混合后作为碳源,以0.15%的硫酸铵为无机氮源,物料与水分的比为1:1;培养温度28-30℃;培养基起始pH值为4.8;接种量为4%(107个/ml),加入0.05%的吐温-20,发酵时间为72h,加入10倍培养基干重的自来水,于40℃摇床浸提1h,得到的木聚糖酶活为650.45IU/ml。
将固体发酵得到的粗酶经过滤、离心、超滤、冻干,制得粗酶粉,研究了粗酶粉的组分和反应特性,结果表明:在所得到的粗酶粉中,含有多种降解酶组分,分别是木聚糖酶、纤维素酶,滤纸酶、淀粉酶;分别占总酶活的66.3%、17.1%、3.8%、12.7%;粗酶的最适反应温度为50℃,最适反应pH值为4.8,Fe3+对酶活具有较强的激活作用, Mg2+具有一定的抑制作用,其他金属离子无显著作用。
经过DEAE A-25葡聚糖凝胶层析发现,黑曲霉木聚糖酶中含有X-Ⅰ,X-Ⅱ,X-Ⅲ三种木聚糖酶同工酶组分,X-Ⅰ为不吸附组分;X-Ⅰ,X-Ⅱ含量较少,X-Ⅲ含量较高。经PAGE,三种组分均呈单一条带。经纯化,木聚糖酶的比活力提高了2倍左右,酶活回收率为26.3%。
The paper uses three-point method and the interpolation method to abserve the colony morphology , external hyphae and spores of the laboratory-preserved Aspergillus niger SB-02. It is studied the Aspergillus niger SB-02’s degradation of xylan on double-deck plate and determinated the activity of xylanase after 3 days liquid fermentation.
The paper focus on optimization of the solid fermentation conditions for xylanase. Optimization factors are as follows: carbon, nitrogen, temperature, incubation time, the initial pH , the initial moisture, inoculation, extraction, etc. The results show that: the bran and corn-cob are mixed at ratio 7:3 as a mixtured carbon source; the best inorganic nitrogen source is ammonium sulfate(0.15%); the ratio of the materials and the water is 1:1 ; the appropriate culture temperature is 28-30℃; the initial pH is 4.8; the appropriate inoculation is 4% (107 / ml); the content of Tween-20 is 0.05%; the liquid fermentation time is 72 hours; 10 times the medium of water, extracted for 1 hour at 40℃; the activity of xylanase is up to 650.45 U/ml.
The crude enzyme powder is made from extraction,filtration,centrifugation,ultrafiltration, freeze-dried of the solid-state fermentation. The results show that: the crude enzyme powder contains various degrading enzymes,such as xylanase, cellulase, filter enzyme, amylase; respectively,their activity is 66.3%, 17.1% and 3.8% , 12.7%; the optimum reaction temperature of the crude xylanase is 50℃and the optimum reaction PH value is 4.8; Fe3+ strongly actived the activity of the xylanase; Mg2+ has a certain inhinition ; the Other metal ions have no significant role.After dextran gel chromatography of DEAE A-25 ,we found that Aspergillus niger xylanase contains three isozyme: X-Ⅰ, X-Ⅱ, X-Ⅲ. X-Ⅲis not a adsorption component; the content of X-Ⅰ, X-Ⅱare fewer than X-Ⅲ. After PAGE ,three isozymes all showed a single band. After purification, Xylanase activity increases 2 times and enzyme recovery is 26.3%.
引文
[1]HenrissatB,BairochA.New families in the classification of glycosyl hydrolases based on aminoacid sequ -ences imilarities.BiochemJ,1993,293(Pt3):781-788.
[2]Prade RA.Xylanases:from biology to biotechnology.Biotechnol GenetEngRev,1996,13:101-131.
[3]苏纯阳,程学慧,刘涛,等.新一代饲用酶制剂——细菌性木聚糖酶[J].饲料广角,2004,(12):41-43.
[4]KoEP,AkatsukaH,Moriyama Hetal.Site directed mutagenes is at aspartate and glutamate residues of xylanase from Bacillus pumillus.BiochecJ,1992,288:117-121.
[5]Moreau A,Roberge M,Manin Cetal.Identification of two acidic residues involved in the catalysis of xylanase from Streptomyces lividans.BiochemJ,1994,302:291-295.
[6]Kulkrni N,Shendye A,Rao M.Molecular and biotechnological aspects of xylanases.FEMS Microbiol Rev,1999,23(4):441-456.
[7]Argos P,Rossmann M G,Grau U M,etal.Thermalstability and protein structure [J].Biochemistry,1979,18: 5698.
[8]Matthews B,Nicholson H,Becktel W.Enhanced protein thermostability from site directed mutations that decrese the entropy of unfolding[J].ProNatl Acad Sci USA,1987,84:6663-6667.
[9]Chan M K,Mukund S,Kletzin A,etal.Structure of a hyperthermopHilic tung stopper in enzyme,aldehyde ferrdoxin oxidoreductase[J].Science,1995,267:1467-1469.
[10]Sakon J ,Adney W S ,Himmel M E ,etal.Crystal structure of thermostable family 5 endocellulase E1 from Acidothermuscellulolyticus in complex with cellotetraose[J].Biochemistry,1996,35:10648-10660.
[11]Ishikawa K ,Okumura M, Katayanagi K,etal.Crystal structure of ribonuclease H from Thermusthermop Hilus H B8 refined at 2.8 resolution[J].J Mol Biol,1993,230:529-542.
[12]Nicholsn H ,Anederson D E ,Daopin S ,etal .Analysis of the interaction between charged sidechains and the α-helix dipole using designed thermostable mutans of pHage T4 lysozyme[J].Biochemistry,1991, 30:9816-9828.
[13]Daggett V ,Levitt M.Protein unfolding pathways explored through molecular dynamics simultions[J]. J Mol Biol,1993,232:600-619.
[14]Kumar S ,Tssai CJ,Nussinov R .Factors enhancing protein thermostability .Protein Eng,2000,13(3): 179-191.
[15]Lehmann M ,Wyss M.Engineering proteins for thermostability :the use of sequence aligaments versus rational design and directed evolution .Curr Opin Biotechnol,2001,12(4):371-375.
[16]杨浩萌,等.通过 N 端替换提高木聚糖酶的热稳定性[J].生物工程学报,2006,22(1):26~32.
[17]刘亮伟,张革新,贺铁明,等.F/10 和 G/11 木聚糖酶家族的不同热稳定机制[J].无锡轻工大学学报,2005,24(1):52~58.
[18]杨浩萌,姚斌,等.木聚糖酶 XYNB 分子中折叠股 B1 和 B2 间的疏水作用对酶热稳定性的影响.生物工程学报[J].2005,21(3):414~419.
[19]杨浩萌,姚斌,范云六.木聚糖酶分子结构与重要酶学性质关系的研究进展.生物工程学报[J].2005,21(1):6~11.
[20]Paula S Pereira,Henlena Paveia,MariaCosta Ferreira,etal.Molecular Biotechnology,2003,24:257-281.
[21]孙雷,朱孝霖,李环,等.木聚糖酶分离纯化技术[J].生物技术通报.2005,5:51~54.
[22]李彩霞,刘书钗,房桂干.木聚糖酶酶活测定方法的探讨[J].2002,20(6):95~96.
[23]呼和,王德勋,等.用酶联吸附法测定木聚糖酶活力的研究[J].畜牧与饲料科学.2005,2:5~9.
[24]Nicolas P,Dieter K,Francois S,etal.Increased xylanase yield in Streptomyces lividans:Dependence on number of ribosome binding sites.Nature Biotechnology,1996,14:756-759.
[25]Ruiz Arribas A,Fernandez Abalos J M,Sanchez P,etal.Overproduction,purification,and biochemical characterization of a xylanase(Xysl)from Streptomyces halsted I I J M8.Appl Environ Microbiol,1997,61 (6):2414-2419.
[26]Rose S H,vanZyl W H.Constitutive expression of the Trichodermareesei beta1,4entoglucanase gene (xyn2) and the beta 1,4entogucanase gene(egl)in Aspergillus niger in molasses and defined glucose media .Appl Microbiol Biotechnol,2002,58(4):461-468.
[27]Zhang H L,Yao B,Wang Y R,etal.Expression of xylanase gene xyn from Streptomyces olivace oviridis Alin E scherichiacoli and Pichiapastoris.Chinese J Biotechnol,2003,19(1):76-80.
[28]Herbers K,Wilke I ,Sonnewakl U.A themostable xylanase from Clostridium thermocellum expressed at high levels in the apoplast of thansgenic tobacco has no detrimentao effects and is easilypurified.Bio/Te -chnology,1995,13:63~66.
[39]Sun J,Kawazu T,Kimura T,Karita S,Sakka K,Ohmiya K.High expression of the xylanase B gene from Clostridium stercerarium in tobacco cells,Journal of Fermentation and Bioengineering,1997,84(3):219~ 223.
[30]杨培龙,姚斌,等. 在烟草中表达的高比活性木聚糖酶 XynB[J].作物学报. 2006,32(6): 176~181.
[31]Cottrell M,Moore J,Kirchman D.Chitinases from uncultured marine microorganisms.Appl.Environ. Microbiol,1999,65:2553-2557.
[32]Anwar S,Peter L B.A gene encoding a novel extremely thermostable1,4xylanase isolated dire-ctly from an environmen DNA sample.ExremopHiles,2003,7:63-70.
[33]张桂敏,庄永红,刘婷,等.土壤微生物 DNA 木聚糖酶基因多样性的研究[J].土壤学报.2006,43(2):295~299.
[34]李春华,李翔,马立新.酸性木聚糖酶基因的克隆及其在毕赤酵母中的分泌表达[J].微生物学通报.2005,32(6):89~95.
[35]朱惠莉,黎锡流.糖化酶的固定化研究进展[J].食品与发酵工业,2001,27(6):75-78.
[36]入江利夫,小西哲哉,田子山保典,等.Trichodermaviride(reesei)变异株K 10 34 の生产するキシラナゼの固定化[J].发酵工学会志,1991,69(3):151～157.
[37]Abdel Naby M A.etal.Immobilization of Aspergillus niger NRC107 xylanase and beta xylosidase,and properties of the immobilized enzymes[J].Appl.Biochem Biotechnol,1993,38(1-2):69-81.
[38]Tyagi R etal.Immobilization of Aspergillusniger xylanase on magneticlatex beads[J].Biotechnol Appl Biochem,1995,21:217-222.
[39]Meryam Sardaraetal.Simultaneous purification and immobilization of Aspergillusniger xylanase on the reversibly soluble polymereudragit TML 100[J].Enzyme and Microbial Technology,2000,27(9):672-679.
[40]朱启忠.青霉菌胞外半纤维素酶的固定化研究[J].微生物学杂志,2000,20(2):56～57.
[41]朱启忠.壳聚糖固定化半纤维素酶的研究[J].生物化学与生物物理进展,2000,27(3):274～277.
[42]蔡敬民,吴克,张洁,等.脱乙酰壳聚糖固定化宇佐美曲霉木聚糖酶及固定化酶的性质和应用[J].中国生物化学与分子生物学报,2002,18(5):548～522.
[43]张剑,范芳. 固定化木聚糖酶的比较研究[J].酿酒科技,2007,2:35-37.
[44]Jurasek,L.Paice and M.Pulp. paer and biotechnology [J].Chemtechnology,1986,16:360-365.
[45]陈永平,廖建和,王志贤.木薯淀粉、纤维素接枝共聚物的合成及生物降解性能的研究[J].热带农业科学,1993(3):19-24.
[46]高洁,李军训,肖军,等.黑曲霉ASP-12固态发酵木聚糖酶培养条件研究[J].饲料工业,2006, 27(6):16-18.
[47]Mandel M.Cellulase production by a new mutant strain of Trichodermu reesei McG77.Biotech BioengSymp,1978(8):89-1011.
[48]高炬,等.榆耳发酵液中新半萜-榆耳三醇的结构[J].药学学报,1992,27(1):33-36.
[49]沈萍,范秀荣,李广武.微生物学实验(第三版)[M].北京:高等教育出版社,
[50]Khan,A.W.etal.:Enzyme Microb Technol[J].1986,8:373-377.
[51]张龙翔,张庭芳,李令媛.生化实验方法和技术(第二版)[M].北京:高等教育出版社,1997:1.
[52]汤鸣强.黑曲霉 SL2-111 固体发酵生产果胶脂酶的研究[J].生物技术,2006,16(2):65-68.
[53]张建新,李学梅,姜丽娜,等.黑曲霉固态发酵木聚糖酶中试条件的研究[J].微生物学杂志,2003,23(4):20-22.
[54]曾艳,刘铁汉,周培瑾,等.嗜碱菌(Bacillus sp.)ZABW6 的木聚糖酶的分离纯化及其性质[J].微生物学报,2004,44(1):75-78.
[55]张红莲,姚斌,王亚茹,等.具有高比活性的新木聚糖酶 XYNB 的酶学性质研究及其编码基因的克隆和表达[J].科学通报,2003,48(4):364-368.
[56]司丽芳,任洪涛,杨自军,等.EM 发酵鸡类与秸秆粉制作饲料的最佳比例[J].河南科技大学学报(农学版),2003,23(2):33-36.
[57]Raimbault M.,Alazard D.Culture method to study fungnal growth in solid fermentation[J].Eur.J.Appl. Microbiol.Biotechnol.1980,9:199-209.
[58]吴克,蔡敬民,张洁,等.黑曲霉 A3 木聚糖酶的纯化和性质[J].安徽机电学院学报.1998,13(3).
[2]Prade RA.Xylanases:from biology to biotechnology.Biotechnol GenetEngRev,1996,13:101-131.
[3]苏纯阳,程学慧,刘涛,等.新一代饲用酶制剂——细菌性木聚糖酶[J].饲料广角,2004,(12):41-43.
[4]KoEP,AkatsukaH,Moriyama Hetal.Site directed mutagenes is at aspartate and glutamate residues of xylanase from Bacillus pumillus.BiochecJ,1992,288:117-121.
[5]Moreau A,Roberge M,Manin Cetal.Identification of two acidic residues involved in the catalysis of xylanase from Streptomyces lividans.BiochemJ,1994,302:291-295.
[6]Kulkrni N,Shendye A,Rao M.Molecular and biotechnological aspects of xylanases.FEMS Microbiol Rev,1999,23(4):441-456.
[7]Argos P,Rossmann M G,Grau U M,etal.Thermalstability and protein structure [J].Biochemistry,1979,18: 5698.
[8]Matthews B,Nicholson H,Becktel W.Enhanced protein thermostability from site directed mutations that decrese the entropy of unfolding[J].ProNatl Acad Sci USA,1987,84:6663-6667.
[9]Chan M K,Mukund S,Kletzin A,etal.Structure of a hyperthermopHilic tung stopper in enzyme,aldehyde ferrdoxin oxidoreductase[J].Science,1995,267:1467-1469.
[10]Sakon J ,Adney W S ,Himmel M E ,etal.Crystal structure of thermostable family 5 endocellulase E1 from Acidothermuscellulolyticus in complex with cellotetraose[J].Biochemistry,1996,35:10648-10660.
[11]Ishikawa K ,Okumura M, Katayanagi K,etal.Crystal structure of ribonuclease H from Thermusthermop Hilus H B8 refined at 2.8 resolution[J].J Mol Biol,1993,230:529-542.
[12]Nicholsn H ,Anederson D E ,Daopin S ,etal .Analysis of the interaction between charged sidechains and the α-helix dipole using designed thermostable mutans of pHage T4 lysozyme[J].Biochemistry,1991, 30:9816-9828.
[13]Daggett V ,Levitt M.Protein unfolding pathways explored through molecular dynamics simultions[J]. J Mol Biol,1993,232:600-619.
[14]Kumar S ,Tssai CJ,Nussinov R .Factors enhancing protein thermostability .Protein Eng,2000,13(3): 179-191.
[15]Lehmann M ,Wyss M.Engineering proteins for thermostability :the use of sequence aligaments versus rational design and directed evolution .Curr Opin Biotechnol,2001,12(4):371-375.
[16]杨浩萌,等.通过 N 端替换提高木聚糖酶的热稳定性[J].生物工程学报,2006,22(1):26~32.
[17]刘亮伟,张革新,贺铁明,等.F/10 和 G/11 木聚糖酶家族的不同热稳定机制[J].无锡轻工大学学报,2005,24(1):52~58.
[18]杨浩萌,姚斌,等.木聚糖酶 XYNB 分子中折叠股 B1 和 B2 间的疏水作用对酶热稳定性的影响.生物工程学报[J].2005,21(3):414~419.
[19]杨浩萌,姚斌,范云六.木聚糖酶分子结构与重要酶学性质关系的研究进展.生物工程学报[J].2005,21(1):6~11.
[20]Paula S Pereira,Henlena Paveia,MariaCosta Ferreira,etal.Molecular Biotechnology,2003,24:257-281.
[21]孙雷,朱孝霖,李环,等.木聚糖酶分离纯化技术[J].生物技术通报.2005,5:51~54.
[22]李彩霞,刘书钗,房桂干.木聚糖酶酶活测定方法的探讨[J].2002,20(6):95~96.
[23]呼和,王德勋,等.用酶联吸附法测定木聚糖酶活力的研究[J].畜牧与饲料科学.2005,2:5~9.
[24]Nicolas P,Dieter K,Francois S,etal.Increased xylanase yield in Streptomyces lividans:Dependence on number of ribosome binding sites.Nature Biotechnology,1996,14:756-759.
[25]Ruiz Arribas A,Fernandez Abalos J M,Sanchez P,etal.Overproduction,purification,and biochemical characterization of a xylanase(Xysl)from Streptomyces halsted I I J M8.Appl Environ Microbiol,1997,61 (6):2414-2419.
[26]Rose S H,vanZyl W H.Constitutive expression of the Trichodermareesei beta1,4entoglucanase gene (xyn2) and the beta 1,4entogucanase gene(egl)in Aspergillus niger in molasses and defined glucose media .Appl Microbiol Biotechnol,2002,58(4):461-468.
[27]Zhang H L,Yao B,Wang Y R,etal.Expression of xylanase gene xyn from Streptomyces olivace oviridis Alin E scherichiacoli and Pichiapastoris.Chinese J Biotechnol,2003,19(1):76-80.
[28]Herbers K,Wilke I ,Sonnewakl U.A themostable xylanase from Clostridium thermocellum expressed at high levels in the apoplast of thansgenic tobacco has no detrimentao effects and is easilypurified.Bio/Te -chnology,1995,13:63~66.
[39]Sun J,Kawazu T,Kimura T,Karita S,Sakka K,Ohmiya K.High expression of the xylanase B gene from Clostridium stercerarium in tobacco cells,Journal of Fermentation and Bioengineering,1997,84(3):219~ 223.
[30]杨培龙,姚斌,等. 在烟草中表达的高比活性木聚糖酶 XynB[J].作物学报. 2006,32(6): 176~181.
[31]Cottrell M,Moore J,Kirchman D.Chitinases from uncultured marine microorganisms.Appl.Environ. Microbiol,1999,65:2553-2557.
[32]Anwar S,Peter L B.A gene encoding a novel extremely thermostable1,4xylanase isolated dire-ctly from an environmen DNA sample.ExremopHiles,2003,7:63-70.
[33]张桂敏,庄永红,刘婷,等.土壤微生物 DNA 木聚糖酶基因多样性的研究[J].土壤学报.2006,43(2):295~299.
[34]李春华,李翔,马立新.酸性木聚糖酶基因的克隆及其在毕赤酵母中的分泌表达[J].微生物学通报.2005,32(6):89~95.
[35]朱惠莉,黎锡流.糖化酶的固定化研究进展[J].食品与发酵工业,2001,27(6):75-78.
[36]入江利夫,小西哲哉,田子山保典,等.Trichodermaviride(reesei)变异株K 10 34 の生产するキシラナゼの固定化[J].发酵工学会志,1991,69(3):151～157.
[37]Abdel Naby M A.etal.Immobilization of Aspergillus niger NRC107 xylanase and beta xylosidase,and properties of the immobilized enzymes[J].Appl.Biochem Biotechnol,1993,38(1-2):69-81.
[38]Tyagi R etal.Immobilization of Aspergillusniger xylanase on magneticlatex beads[J].Biotechnol Appl Biochem,1995,21:217-222.
[39]Meryam Sardaraetal.Simultaneous purification and immobilization of Aspergillusniger xylanase on the reversibly soluble polymereudragit TML 100[J].Enzyme and Microbial Technology,2000,27(9):672-679.
[40]朱启忠.青霉菌胞外半纤维素酶的固定化研究[J].微生物学杂志,2000,20(2):56～57.
[41]朱启忠.壳聚糖固定化半纤维素酶的研究[J].生物化学与生物物理进展,2000,27(3):274～277.
[42]蔡敬民,吴克,张洁,等.脱乙酰壳聚糖固定化宇佐美曲霉木聚糖酶及固定化酶的性质和应用[J].中国生物化学与分子生物学报,2002,18(5):548～522.
[43]张剑,范芳. 固定化木聚糖酶的比较研究[J].酿酒科技,2007,2:35-37.
[44]Jurasek,L.Paice and M.Pulp. paer and biotechnology [J].Chemtechnology,1986,16:360-365.
[45]陈永平,廖建和,王志贤.木薯淀粉、纤维素接枝共聚物的合成及生物降解性能的研究[J].热带农业科学,1993(3):19-24.
[46]高洁,李军训,肖军,等.黑曲霉ASP-12固态发酵木聚糖酶培养条件研究[J].饲料工业,2006, 27(6):16-18.
[47]Mandel M.Cellulase production by a new mutant strain of Trichodermu reesei McG77.Biotech BioengSymp,1978(8):89-1011.
[48]高炬,等.榆耳发酵液中新半萜-榆耳三醇的结构[J].药学学报,1992,27(1):33-36.
[49]沈萍,范秀荣,李广武.微生物学实验(第三版)[M].北京:高等教育出版社,
[50]Khan,A.W.etal.:Enzyme Microb Technol[J].1986,8:373-377.
[51]张龙翔,张庭芳,李令媛.生化实验方法和技术(第二版)[M].北京:高等教育出版社,1997:1.
[52]汤鸣强.黑曲霉 SL2-111 固体发酵生产果胶脂酶的研究[J].生物技术,2006,16(2):65-68.
[53]张建新,李学梅,姜丽娜,等.黑曲霉固态发酵木聚糖酶中试条件的研究[J].微生物学杂志,2003,23(4):20-22.
[54]曾艳,刘铁汉,周培瑾,等.嗜碱菌(Bacillus sp.)ZABW6 的木聚糖酶的分离纯化及其性质[J].微生物学报,2004,44(1):75-78.
[55]张红莲,姚斌,王亚茹,等.具有高比活性的新木聚糖酶 XYNB 的酶学性质研究及其编码基因的克隆和表达[J].科学通报,2003,48(4):364-368.
[56]司丽芳,任洪涛,杨自军,等.EM 发酵鸡类与秸秆粉制作饲料的最佳比例[J].河南科技大学学报(农学版),2003,23(2):33-36.
[57]Raimbault M.,Alazard D.Culture method to study fungnal growth in solid fermentation[J].Eur.J.Appl. Microbiol.Biotechnol.1980,9:199-209.
[58]吴克,蔡敬民,张洁,等.黑曲霉 A3 木聚糖酶的纯化和性质[J].安徽机电学院学报.1998,13(3).