摘要
纤维素酶是公认的一种具有非专一性水解壳聚糖的代表性酶类。本实验室已从绿色木霉来源的商品纤维素酶制剂中分离纯化出一种具有水解纤维素和壳聚糖活性双功能酶。为了进一步确证纤维素酶中该双功能酶的存在,本研究采用分子生物学手段,对产纤维素酶的绿色木霉菌株的双功能酶基因进行克隆和体外重组表达,并探讨该双功能酶的基因结构与双功能性质的关系。本研究对于进一步了解双功能酶结构与功能的关系具有重要的学术意义,同时对于提高该双功能酶的活力也具有重要的应用价值。
研究了绿色木霉菌株(Trichoderma viride)在羧甲基纤维素钠(CMC)诱导下产高活性酶的发酵条件;采用柱层析系统对发酵液中的双功能酶组分进行分离纯化,从酶学性质和酶结构方面与本实验室前期从商品纤维素酶中分离出的双功能酶进行了对照比较;结果表明:在CMC诱导培养4天时发酵液中壳聚糖酶活力和纤维素酶活力同时达到最大值;所纯化的双功能酶与从商品酶中纯化的双功能酶为同一蛋白。
该双功能酶的Maldi-Tof-Tof Mass序列测定结果表明:该双功能酶的两个主要肽段的序列与GenBank里糖苷水解酶7族(GH-7)的绿色木霉产CBHⅠ(gi|295937)的氨基酸序列具有很高的同源性,推测该CCBE可能属于GH-7族。
以T.viride RNA为模板,采用RT-PCR、SMART-RACE方法克隆了双功能酶CCBE的全长cDNA序列,并进行毕赤酵母表达,结果显示:CCBE的全长cDNA序列与五个在GenBank中登录的木霉属GH-7族的CBHⅠ基因(Trichoderma.sp)具有88-100%的同源性,但与已知的壳聚糖酶和几丁质酶基因却无任何序列相似性,而重组表达产物具有强的壳聚糖酶活力,证实了绿色木霉所产CCBE属于GH-7族,两活力产自同一基因,其mRNA可能存在选择性剪接现象。
采用RT-PCR法测定了不同诱导时间绿色木霉CCBE的mRNA表达水平,同时研究了不同内含子保留的剪接子基因在重组毕赤酵母中的转录表达情况,结果表明:绿色木霉CCBE的mRNA存在依赖于诱导时间的选择性剪接现象,四种剪接基因型DC(保留有两内含子)、CCBEIN1(内含子1保留)、CCBEIN2(内含子2保留)和CC(不含内含子)的出现以及所占比例随诱导时间而变化。各剪接子在毕赤酵母体内的转录产物相同,均不含内含子;四个重组表达产物的分子量大小相同,均具有强的壳聚糖酶活力,但纤维素酶活力却截然不同:只有CCBEIN1和CC具有CMCase活力,且前者活力大小约为后者的一半,由此推测内含子的保留尤其是内含子2的保留可能通过改变各表达产物的蛋白构象从而影响其活力表达。
对CCBE不同内含子保留的剪接基因的毕赤酵母重组表达产物水解壳聚糖和CMC的酶学性质、水解产物进行研究,并采用删除突变对双功能酶不含内含子的基因型进行C-末端删除分析,探讨了基因结构与双功能性质的关系;结果表明:不同内含子保留的双功能酶基因的体外重组酶作用两底物的性质相近但并不完全一致,作用壳聚糖的最适温度和最适pH分别集中于55-60℃和5.0-5.2;而作用CMC则为55-60℃和4.2,水解方式均为外切;与从绿色木霉中纯化的CCBE基本保持一致,其中重组酶CC的性质与纯化CCBE的一致性最高。内含子1的保留通过改变重组酶部分CMC催化结构域的正确折叠而抑制其CMC降解活力,但对其底物结合位点无影响;而对于壳聚糖酶活力,其使酶与壳聚糖结合能力下降,但对其催化域的构象影响不大;内含子2的保留改变了纤维素结合结构域的正确构象,从而完全失去CMCase活力,但不影响重组酶壳聚糖酶活力的催化结构域和底物结合结构域,而是使其催化域附近的底物进入通道变小,从而使得大分子难以进入。由此证实了: CCBE作用壳聚糖的催化结构域和底物结合位点均位于CCBE的N-端及序列中上游位置,与作用CMC的截然不同,后者则位于C-端及序列中下游部位。
本论文研究验证了绿色木霉在纤维素诱导条件下会产生具有壳聚糖和纤维素降解活性的双功能酶,采用分子生物学手段确证了纤维素酶中双功能酶的存在;同时发现了绿色木霉所产的双功能酶基因具有内含子保留的选择性剪接现象,探讨了双功能酶不同内含子保留的基因结构与双功能性质的关系。
Cellulase is recognized as a typical non-specific chitosan-degrading enzyme.In our previous study, a bifunctional enzyme with chitosanolytic and cellulolytic activity (CCBE) has been purified from the commercial cellulase produced by Trichoderma viride. in order to confirm the existence of CCBE in cellulase, in this study, the gene of CCBE from cellulase hyper-producing T.viride were cloned and expressed in vitro using the molecular biological methods, and the relationship between the gene structure and bifunctional properties of CCBEwas also discussed, which is of important academic value in elucidating the relationship between structure and function of CCBE and of practical significance in improving the activity of this enzyme.
A cellulase-producing T.viride strain was chosen to determine the production condition of high active enzyme induced by CMC, showing that both of the chitosanase and cellulase activities approached maximum after induction for 4 days. The bifunctional enzyme in the culture was then purified and identified as identical to that from commercial cellulase, convincing that CCBE could be produced by T.viride with induction of CMC.
Maldi-Tof-Tof Mass analysis showed that two major peptide peaks of apparent mass 1764.000 and 1932.7490 of CCBE digested by trypsin was found high homology with cellobiohydrolaseⅠ(CBHⅠ) gi|295937from T.viride . Accordingly, CCBE was presumed as an enzyme belonging to GH-7.
Taking the total RNA of T.viride as template, the full-length cDNA sequence of CCBE was cloned by RT-PCR and SMART-RACE, and then expressed in Pichia patoris. Sequence analysis indicated that the full- length cDNA sequence of CCBE had 88-100% identity with the four CBHⅠgenes (Trichoderma. sp), while no similarity with chitosanase or chitinase genes reported in GenBank, which confirmed that the CCBE from T.viride belonged to GH-7,and its mRNA probably underwent alternative splicing .
The differential expression level of CCBE mRNA was tested during different inducing periods of T.viride, indicating that mRNA of CCBE underwent a unique alternative splicing which is dependent on the inducing time. The appearance and proportion of four splicers varied with the inducing time increasing. The four splicers were assembled artificially by GDS technique and then expressed in Pichia patoris. The transcripts of four different transformants showed sequence-identical and had no introns retaining, which means that the amino acid sequence and molecular weights of the four recombinant enzymes were identical. Activity analysis showed that all of the splicers had comparable strong chitosanase activity, while their cellulolytic activity varied. CCBEIN1 had moderate CMCase activity compared to its chitosanase activity, and the dual activities of CC were comparative, however, CCBEIN2 and DC almost lost their CMCase activity, indicating the retained introns, especially intron 2 changed the conformation of their recombinant enzymes to make them lost their CMCase activity.
The characteristics and hydrolysis products of four recombinant enzymes toward chitosan and CMC were similar but not completely uniform. The optimum temperature and pH of four enzymes toward chitosan were in a range of 55-60℃and pH 5.0-5.2, respectively, while toward CMC, both CCBEIN1 and CC exhibited optimal condtions among 55-60℃and at pH4.2; among these, the recombinant CC showed highest consistent with the purified CCBE.
The retained intron 1 in genes partially changed the folding of the catalytic domain of the recombinant enzymes on CMC,but had no effects on their CMC binding domain;while chitosan binding capacity lowered when intron 1 retained and its catalytic domain for chitosan hydrolysis varied not significantly.However, genes with intron 2 made its expressing products show narrower loop channels, while their CBD conformation also changed to lose the binding capacity of CMC, resulting in the lost of CMCase activity of CCBEIN2 and DC.From the above, it is confirmed that the catalytic and substrate-binding domains of CCBE on chitosan hydrolysis located in the upper-middle reaches of N-terminal, which is different with those of CMC, locating in the lower reaches.
In this study, it is convinced that bifunctional enzyme with chitosan- and CMC- degrading activity could be produced by T.viride with the induction of CMC.The results confirmed the existence of CCBE from cellulase and elucidated the relationship between the gene structure and bifunctional properties of CCBE.
引文
[1]周孙英,余萍,陈盛等.四种不同类型酶降解壳聚糖的效果比较[J].海峡药学,2003,15(1):58-61.
[2]邱乐泉,施杨芳,朱玮玮.假单胞菌H3壳聚糖酶的纯化及部分酶学性质研究[J].食品与发酵工业,2004,30(3):49-52.
[3]林强,马可立.利用纤维素酶催化水解壳聚糖的研究[J].日用化学工业,2003,33(1):22-25.
[4]刘羿君,蒋英,封云芳,等.特种纤维素酶催化水解壳聚糖及壳寡糖的制备研究[J].功能高分子学报,2005, 18(2):325-329.
[5]陈江燕,邬国铭.纤维素酶对壳聚糖降解作用的研究[J].广东医学院学报,2003, 21(2):105-107.
[6]周桂,何子平,邓光辉,etc.纤维素酶与淀粉酶降解壳聚糖的动力学研究[J].海洋科学,2003,27(11):59-63.
[7]刘靖,夏文水.纤维素酶中具有壳聚糖水解酶活性成分的鉴定[J].中国生物化学与分子生物学报,2005, 21(5):713-716.
[8]Liu J, Xia WS.Purification and characterization of a bifunctional enzyme with Chitosanase and cellulase activity from commercial cellulase[J]. Biochem Eng J, 2006, 30 (1):82-87.
[9]Sardar M, Roy I, Gupta MN.A smart bioconjugate of alginate and pectinase with unusual biological activity toward chitosan[J].Biotechnology Progress, 2003, 19(6):1654-1658.
[10]Kittur F S, Kumar A, Tharanathan R N, etc. Low molecular weight chitosans - preparation by depolymerization with Aspergillus niger pectinase, and characterization [J]. Carbohyd Res, 2003, 338 :1283-1290.
[11]Roy I, Sardar M, Gupta MN. Hydrolysis of chitin by pectinexTM[J]. Enzyme and Microbial Technology, 2003, 32(5):582-588.
[12]Kittura FS, Kumara ABV, Gowdab LR,et al. Chitosanolysis by a pectinase isozyme of Aspergillus niger—A non-specific activity[J]. Carbohydrate Polymers, 2003, 53(2):191-196.
[13]Shin-ya Y, Lee MY, Hinode H,etc.Effects of N-acetylation degree on N-acetylated chitosan hydrolysis with commercially available and modified pectinases[J]. Biochemical Engineering Journal,2001,7(1):85-88.
[14]Muzzarellia R, Tomasetti M.Depolymerization of chitosan with the aid of papain[J]. Enzyme Microb Technol,1994,16(2): 110-114.
[15]苏畅,夏文水,姚惠源.木瓜蛋白酶降解壳聚糖[J].无锡轻工大学学报,2002,21(3): 112-115.
[16]Terbojevich M, Cosani A,Muzzarelli RAA.Molecular parameters of chitosans depolymerized with the aid of papain.Carbohydrate Polymers,1996,29(1): 63-68.
[17]黄永春,李琳,郭祀远,等.木瓜蛋白酶对壳聚糖的降解特性[J].华南理工大学学报(自然科学版),2003,31(6):71-75.
[18]夏文水, Muzzarclli RAA.脂肪酶解聚壳聚糖及其衍生物的研究[J].无锡轻工大学学报,1996,15(1):325-332.
[19]周孙英,余萍,陈盛,等.脂肪酶催化壳聚糖降解的特性[J].福建医科大学学报,2002,36(9):302-305.
[20]王丽娟,夏文水,陈小娥,等.脂肪酶水解壳聚糖作用研究[J].食品工业科技,2004,25(5): 302-304.
[21]Jang HK, Yi JH, Kim JT, et al.Purification, characterization, and gene cloning of chitosanase from Bacillus cereus H-1[J]. Korean. J. Microb. Biotech.,2003,31: 216-223.
[22]Gao XA, Ju WT, JungWJ,et al. Purification and characterization of chitosanase from Bacillus cereus D-11[J]. Carbohydr.Polym., 2008,72(3):513-520.
[23]Ogura J, Toyoda A, Kurosawa T,et al. Purification,characterization and gene analysis of cellulase (Cel8A) from Lysobacter sp.IB-9374[J]. Biosci. Biotech. Biochem., 2006,70: 2420-2428.
[24]Su CX, Wang DM, Yao LM, et al.Purification, characterization, and gene cloning of a chitosanase from Bacillus species strain S65[J].J. Agri. Food. Chem., 2006,54: 4208-4214.
[25]Tanabe T, Morinaga K, Fukamizo T.Novel chitosanase from Streptomyces griseus HUT 6037 with transglycosylation activity[J]. Biosci. Biotechnol. Biochem. , 2003, 67:354-364.
[26]Hong IP, Jang HK, Lee SY, et al.Clone and characterization of a bifunctional cellulase-chitosanase gene from Bacillus licheniformis NBL420[J]. J. Microbiol. Biotechnol. , 2003, 13: 35-42.
[27]Kurakake M, Yo-u S, Nakagawa K, et al. Properties of Chitosanase from Bacillus cereus S1[J].Current Microbiol., 2000,40:6-9.
[28]Pedraza-Reyes M. The bifunctional enzyme chitosanase-cellulase produced by the gram negative microorganism Myxobater.sp.AL-1 is highly similar to Bacillus subtilis endo- glucanases[J]. Arch.Microbiol., 1997,168: 321-327.
[29]Hedges A, Wolfe RS.Extracellular enzyme from Myxcobacter AL-1 that exhibits bothβ-1,4-glucanase and chitosanase activities[J].J.Bacteriol.1974,120(2):844-853.
[30] Seino H, Tsukuda K, Shimasue Y.Properties and action pattern of a chitosanase from Bacillus sp. PI-7s[J].Agric.Biol.Chem,1991,55(9):2421-2423.
[31]Choi YJ, Kim EJ, Piao ZY,et al. Purification and characterization of chitosanase from Bacillus sp.strain KCTC 0377BP and its application for the production of chitosan oligosaccharides[J]. Appl. Environ. Microbe., 2004, 70:4522- 4531.
[32]Ohtakara A, Ogata H.Purification and characterisation of chitosanase from Streptomyces griseus[M].In Chitin, Chitosan and Related Enzymes, ed. J. P. Zikakis, Academic Press,1984,pp147-59.
[33]Pelletier A, Sygusch J.Purification & characterization of three chitosanase activities from Bacillus megaterium Pl[J]. Appl.Environ.Microbial,1990,56(4): 844-848.
[34]Mitsutomi M, Isono M,Uchiyama A,et al. Chitosanase activity of the enzyme previous reported asβ-1,3/β-1,4-glucanase from Bacillus circulans WL-12[J].Biosci. Biotech. Biochem, 1998,62:2107-2114.
[35]Ike M, Ko Y, Yokoyama K,et al. Cellobiohydrolase I (Cel7A) from Trichoderma reesei has chitosanase activity[J]. J. Mol. Cat. B: Enzymatic., 2007,47:159-163.
[36]Xia WS, Lee DX.Purification and characterization of exo-β-d-glucosaminidase from commercial lipase. Carbohydr Polym, 2008, 74(3):544-551.
[37]刘靖,夏文水.色氨酸残基在双功能酶CCBE中的作用[J].食品科学,2007,28(9): 425-429.
[38]刘靖,夏文水.双功能酶水解壳聚糖和纤维素的活性中心研究[J].食品与机械, 2007,23(2): 31-33.
[39]刘靖,夏文水.焦碳酸二乙酯及碳二亚胺对纤维素酶-壳聚糖酶双功能酶的修饰作用[J].食品科学,2008,29(8): 460-463.
[40]刘靖.纤维素酶水解壳聚糖的特性及机理研究[D]:[博士学位论文].无锡:江南大学, 2006.
[41]刘北东.绿色木霉纤维素酶系基因克隆表达及特性研究[D]:[博士学位论文].哈尔滨:哈尔滨工业大学生命科学与工程系,2004.
[42]Beldman G,Leeuwen MFS, Rombouts FM,et al. The cellulase of Trichoderma viride[J]. Eur. J. Biochem, 1984,146(2):301-308.
[43]Watanabe,M. Endoglucanase II-T.viride. GenBank: AB021657.1.1998.12.20.
[44]Henrissat B, Bairoch A. Updating the sequence-based classification of glycosyl hydrolases[J]. Biochem. J.,1996,316:695-696.
[45]Henrissat B, Davies G. Structural and sequence-based classification of glycoside hydrolases[J]. Current Opinion in Structural Biology, 1997,7:637-644.
[46]Shoseyov O, Shani Z, Levy I. Carbohydrate binding modules: biochemical properties and novel applications [J]. Microbiol Mol Biol Rev,2006, 70(2): 283-295
[47]Bourne Y, Henrissat B. Glycoside hydrolases and glycosyl transferases: families and functional modules[J]. Curr Opin Struct Biol, 2001,11(5): 593-600
[48]Gusakov AV, Sinitsyn A P, Salanovich TN, et al. Purification, cloning and characterisation of two forms of thermostable and highly active cellobiohydrolaseI (Cel7A) produced by the industrial strain of Chrysosporium lucknowense[J]. Enzyme.Microbial Technol., 2005, 36: 57-69.
[49]王建荣,张曼夫.绿色木霉(Trichoderma viride)基因组文库构建及其CBHI基因阳性克隆筛选[J].上海农业学报,1993,9(3): 1-5.
[50]王建荣,张曼夫,黄涛.绿色木霉纤维素酶CBHII基因的结构研究[J].遗传学报, 1995,22(1): 74-80.
[51]刘北东.绿色木霉纤维素酶系基因克隆表达及特性研究[D]:[博士学位论文].哈尔滨:哈尔滨工业大学生命科学与工程系,2004.
[52]Kanokratana P, Chantasingh D, Champreda V, et al. Identification and expression of cellobiohydrolase (CBHI) gene from an endophytic fungus, Fusicoccum sp. (BCC4124) in Pichia pastoris[J].Protein Exp. Purif, 2008, 58(1): 148-153.
[53]Saloheimo A, Henrissat B, Hoffrén AM,et al.A novel, small endoglucanase gene, egl5, from Trichoderma reesei isolated by expression in yeast[J]. 1994,13(2):163-167.
[54]Graessle S, Haas H, Friedlin E,et al.Regulated system for heterologous gene expression in Penicillium chrysogenum[J].Appl Environ Microbiol, 1997,63(2):753-6. [55]Minet M, Lacroute F.Cloning and sequencing of a human cDNA coding for a multifunctional polypeptide of the purine pathway by complementation of the ade2-101 mutant in Saccharomyces cerevisiae[J]. Curr Genet, 1990,18(4):287-91.
[56]Siegei R S. Methylotroyphic yeast Pichia pastour produced in High-cell-density fermentation with high cell yields as vehicle for recombinant protein production[J]. Biotechnology and Bioegineering,1989, 34: 403-404.
[57]欧阳立明.张惠展.张嗣同.巴斯德毕赤酵母的基因表达系统研究进展[J].生物化学与生物物理进展[J].2000,27(2):151-154.
[58]张伍魁,范清林,宋礼华.毕赤酵母表达系统在外源基因表达中的研究进展及应用[J].中国生物工程杂志.2006,26(1):87-91.
[59]Lee YJ, Kim BK, Lee, BH,et al. Purification and characterization of cellulase produced by Bacillus amyoliquefaciens DL-3 utilizing rice hull[J].Bioresour. Technol, 2008, 99: 378–386.
[60]Nishida Y, Suzuki K, Kumagai Y, et al. Isolation and primary structure of a cellulase from the Japanese sea urchin Strongylocentrotus nudus[J]. Biochimie, 2007,89: 1002–1011.
[61]Collins CM, Murray PG, Denman S,et al. Molecular cloning and expression analysis of two distinctβ-glucosidase genes, bg1 and aven1, with very different biological roles from the thermophilic, saprophytic fungus Talaromyces emersonii[J]. Mycolog. Res, 2007, 111: 840-849.
[62]Bischoff KM, Liu SQ, Hughes SR. Cloning and characterization of a recombinant family 5 endoglucanase from Bacillus licheniformis strain B-41361[J]. Proc. Biochem, 2007,42:1150-1154.
[63]Takashima S, Nakamura A, Hidaka M,et al. Molecular Cloning and Expression of the Novel Fungal Beta-glucosidase Genes from Humicolagrisea and Trichoderma reesei[J]. J.Biochem,1999,125(4):728-36.
[64]Tsai CF, Qiu X, Liu JH. A Comparative Analysis of Two cDNA Clones of the Cellulase Gene Family from Anaerobic Fungus Piromyces rhizinflata[J]. Anaerobe., 2003,9(3): 131-140.
[65]Bae HJ, Turcotte G, Chamberland H,et al. A Comparative Study between an Endoglucanase IV and its Fused Protein Complex Ce15-CBM6[J]. FEMS Microbiol Letters, 2003, 227(2): 175-181.
[66]Haakana H, Miettinen-Oinonen A, Joutsjoki V, et al. Cloning of Cellulase Genes from Melanocarpus albomyces and Their Efficient Expression in Trichoderma reesei[J]. Enzyme Microbial Technol, 2004, 34(2): 159-167.
[67]Harhangi HR, Akhmanova A, Steenbakkers PJM,et al. Genomic DNA Analysis of Genes Encoding (hemi-) Cellulolytic Enzymes of the Anaerobic Fungus Piromyces sp. E2 [J]. Gene, 2003, 314:73-80.
[68]Murray PG, Collins CM, Grassick A, et al. Molecular Cloning,Transcriptional, and Expression Analysis of the First Cellulase Gene (cbh2), Encoding Cellobiohydrolase II,from the Moderately Thermophilic Fungus Talaromycese mersonii and Structure Prediction of the Gene Product[J]. Biochem Biophys Res Commun, 2003, 301(2): 280-286.
[69]Ai YC, Wilson DB.Mutation and Expression of N233C-D506C of Cellulase Cel6B from Thermobifida fusca in Escherichia coli[J]. Enzyme MicrobTechnol, 2002,30(6): 804-808.
[70]Kim P, Tae KH, Chung KJ, et al. Purification of a constitutive chitosanase produced by Bacillus sp. MET 1299 with cloning and expression of the gene[J]. FEMS Microbiol. Lett., 2004,240, 31-39.
[71]Hakamada Y, Endo K, Takizawa S, et al. Enzymatic properties,crystallization, and deduced amino acid sequence of an alkaline endoglucanase from Bacillus circulans[J]. Biochim. Biophys. Acta,2002,1570: 174-180.
[72]Kimoto H, Kusaoke H, Yamamoto I,et al. Biochemical and genetic properties of Paenibacillus glycosyl hydrolase having chitosanase activity and discoidin domain[J]. J Biol Chem,2002,277(17): 14695-14702.
[73]Adachi W, Sakihama Y, Shimizu S,et al. Crystal Structure of Family GH-8 Chitosanase with Subclass II Specificity from Bacillus sp. K17[J]. J. Mol. Biol, 2004, 343(3): 785–795.
[74]张晓娜,肖华胜.真核生物m RNA选择性剪接体检测方法研究进展[J].生命科学仪器, 2008 ,6:8-11.
[75]夏慧煜,李衍达.真核基因选择性剪接机理的初步研究[J].清华大学学报(自然科学版),2003, 43(4):30-35.
[76]林鲁萍,马飞,王义权.基因选择性剪接的生物信息学研究概况[J].遗传, 2005, 27(6):1001-1006.
[77]Early P, Rogers J, Davis M, et al.Two mRNAs can be produced from a single immunoglobulinμgene by alternative RNA processing pathways[J]. Cell,1980, 20:313-319.
[78]Rosenfeld M G, Lin C R, Amara S G, et al.Calcitonin mRNA polymorphism: peptide switching associated with alternative RNA splicing events[J]. Proc Natl Acad Sci USA, 1982 ,79(6):1717-1721.
[79]Sharp PA.Split genes and RNA splicing[J]. Cell,1994,77(6):805-815.
[80]Gelfand MS, Dubchak I, Dralyuk I, et al. ASDB: database of alternatively spliced genes [J]. Nucleic Acids Res, 1999, 27(1):301-302.
[81]Lopez AJ. Alternative splicing of pre-mRNA: developmental consequences and mechanisms of regulation [J]. Annu Rev Genet, 1998, 32:279-305.
[82]Zhang Y, Zhang J,Cheng H,et al. Identification of Alternate Splice Variants for Murine SmX5[J]. Acta Genetica Sinica , 2003 , 30 (6) : 515-520.
[83]Connor VO, Genin A, Davis S,et al. Differential Amplification of Intron-containing Transcripts Reveals Long Term Potentiation-associated Up-regulation of Specific Pde10A Phosphodiesterase Splice Variants[J]. J Biol Chem, 2004, 279(16): 15841-15849.
[84]Beck ET, Blair CD, Black WC,et al.Alternative splicing generates multiple transcripts of the Inhibitor of Apoptosis Protein 1 in Aedes and Culex spp. Mosquitoes[J]. Insect Biochem Mol Biol,2007,37(11): 1222-1233.
[85]许先国,吴俊杰,洪小珍,等.鉴定9个新的RHD基因mRNA可变剪接体[J].遗传, 2006, 28(10): 1213-1218.
[86]Baba Y, Shimonaka A, Koga J,et al.Alternative Splicing Produces Two Endoglucanases with One or Two Carbohydrate-Binding Modules in Mucor circinelloides [J]. J. Bacteriol, 2005, 187(9):3045-3051.
[87]Lin S, Cheng D, Liu MS,et al. Human acyl-CoA:cholesterol acyltransferase-1 in the endoplasmic reticulum contains seven transmembrane domains[J]. J Biol Chem. 1999, 274(33):23276-23285.
[88]李杨,苏乔,安利佳.基因组的“沙漠区域”内含子及其在植物基因工程中的应用[J].分子植物育种,2004,2(4):569-573.
[89]Salgueiro S, Pignocchi C, Parry MAJ. Intorn-mediated gusA expression in tritordeum and wheat resulting from particle bombardment[J]. Plant Mol. Biol., 2000, 42: 615-622.
[90]Birch PRJ, Sims PFG, Broda P.Substrate-Dependent Differential Splicing of Introns in the Regions Encoding the Cellulose Binding Domains of Two Exocellobiohydrolase I-Like Genes in Phanerochaete chrysosporium[J] .Applied Environ. Microbial,1995, 16(10): 3741-3744.
[91]Baba Y, Shimonaka A, Jinichiro Koga J,et al.Alternative Splicing Produces Two Endoglucanases with One or Two Carbohydrate-Binding Modules in Mucor circinelloides[J]. J.Bacteriol, 2005, 187(9):3045–3051.
[92]Costanzo S, Ospina-Giraldo MD, Deahl KL,et al.Alternate intron processing of family 5 endoglucanase transcripts from the genus Phytophthora[J]. Curr Genet, 2007,52:115–123.
[93]Gusakov AV, Sinitsyn A P, Salanovich TN, et al. Purification, cloning and characterisation of two forms of thermostable and highly active cellobiohydrolaseI (Cel7A) produced by the industrial strain of Chrysosporium lucknowense[J]. Enzyme.Microbial Technol., 2005, 36: 57-69.
[94]Curach NC, Te’O VSJ, Gibbs MD,et al.Isolation, characterization and expression of the hex1 gene from Trichoderma reesei[J]. Gene, 2004,331:133-140.
[95]Yamada T,Hiramatsu S,Songsri P,et al. Alternative Expression of a Chitosanase Gene Produces Two Different Proteins in Cells Infected with Chlorella Virus CVK2[J]. Virology, 1997,230: 361-368.
[96]许先国,吴俊杰等.鉴定9个新的RHD基因mRNA可变剪接体[J].遗传, 2006, 28 (10) : 1212- 1218.
[97]Lopez A. Alternative splicing of pre-mRNA:developmental consequences and mech anisms of regulation[J]. Ann. Rev. Genet,1998,32:279 - 305.
[98]Modrek B, Lee C. A genomic view of alternative splicing [J]. Nature Genet, 2002, 30:13 -19.
[99]Shobhit G, Dorothea Z, Bernhard K, et al. Strengths and weaknesses of EST- basedprediction of tissue- specific alternative splicing [J] . BMC Genomics, 2004, 5:72.
[100]Xu Q, Modrek B, Lee C,et al.Genome- wide detection of tissue- specific alternative splicing in the human transcriptome[J]. Nucleic Acids Research, 2002,30(17): 3754- 66.
[1]Hong SP, Kim DS. Chitosanolytic characteristics of cellulases from Trichoderma viride and Trichoderma reesei[J]. Korean J Food Sci. Technol., 1998,30:245-252.
[2]Hedges A,Wolfe RS.Extracellular enzyme from Myxcobacter AL-1 that exhibits bothβ-1,4-glucanase and chitosanase activities[J]. J.Bacteriol,1974,120(2): 844-853.
[3]Ohtakara A, Ogat H. Purification and characterisation of chitosanase from Streptomyces griseus[M]. In Chitin, Chitosan and Related Enzymes, ed. J. P. Zikakis,cademic Press,1984, pp147-59.
[4]Pelletier A, Sygusch J.Purification & characterization of three chitosanase activities from Bacillus megaterium Pl[J]. Appl.Environ.Microbial,1990,56(4): 844-848.
[5]Kurakake M, Yo-u S, Nakagawa K,et al. Properties of Chitosanase from Bacillus cereus S1[J]. Current Microbiology,2000,40:6-9.
[6]Mitsutomi M, Isono M, Uchiyama A,et al.Chitosanase activity of the enzyme previous reported asβ-1,3/β-1,4-glucanase from Bacillus circulans WL-12[J]. Biosci. Biotech. Biochem,1998,62(11): 2107-2114.
[7]Ike M, Ko Y, Yokoyama K, et al. Cellobiohydrolase I (Cel7A) from Trichoderma reesei has chitosanase activity[J]. J. Mol. Cat. B: Enzymatic.,2007, 47:159-163.
[8]Ogura J, Toyoda A, Kurosawa T, et al. Purification,characterization and gene analysis of cellulase (Cel8A) from Lysobacter sp.IB-9374[J]. Biosci. Biotech. Biochem., 2006,70: 2420-2428.
[9]Hong IP, Jang HK, Lee SY, et al. Clone and characterization of a bifunctional cellulase-chitosanase gene from Bacillus licheniformis NBL420[J]. J. Microbiol .Biotechnol., 2003, 13:35-42.
[10]Liu J, Xia WS.Purification and characterization of a bifunctional enzyme with Chitosanase and cellulase activity from commercial cellulase[J]. Biochem.Eng. J,2006,30 (1):82-87.
[11]Tanabe T, Morinaga K, Fukamizo T.Novel chitosanase from Streptomyces griseus HUT 6037 with transglycosylation activity[J]. Biosci. Biotech. Biochem. , 2003, 67: 354-364.
[12]Pedraza-Reyes M.The bifunctional enzyme chitosanase-cellulase produced by the gram negative microorganism Myxobater.sp.AL-1 is highly similar to Bacillus subtilis endo- glucanases[J]. Arch.Microbiol., 1997,168: 321-327.
[13]吴静,汪天虹.丝状真菌瑞氏木霉生产重组蛋白的分子生物学研究进展.生物工程进展, 1999, 19(2):8-12.
[14]范秀容,沈萍.微生物学实验[M].第二版.北京:高等教育出版社,1989:133-136.
[15]Mandels M, Reese ET. Induction of cellulase in Trichoderma viride as influenced by carbon sources and metals[J].J.Bacteriol.,1957,73:269-278.
[16]蒋挺大.壳聚糖[M].北京:化学工业出版社,2001,259-263.
[17]Wang W, Bo S, Qin W. Determination of the Mark– Houwink equation for chitosans with different degrees of deacetylation [J]. Int J Biol Macromol, 1991, 13(10):281-285.
[18]刘北东.绿色木霉纤维素酶系基因克隆表达及特性研究.[D]:[博士学位论文].哈尔滨:哈尔滨工业大学,2004.
[19]Miller GL.Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar[J].Anal. Chem.,1959,31(3):426-428.
[20]李华,高丽.葡萄浆果中β-葡萄糖苷酶活性测定条件的研究.酿酒科技, 2007(8): 146-149.
[21]Lowry O H, Rose N J, Fan A L, et al., Protein measurement with the folin phenol reagent [J]. J. Biol. Chem., 1951,193:265-276.
[22]汪家政,范明.蛋白质技术手册[M].北京:科学出版社,2001.
[23]刘靖.纤维素酶水解壳聚糖的特性及机理研究[D]:[博士学位论文].无锡:江南大学, 2006.
[24]Laemmli UK.Cleavage the head of bacteriophage T4 [J]. Nature,1970, 227: 680-685.
[25]夏其昌主编.蛋白质化学研究技术与进展[M].北京:科学出版社,1999.
[26]Lineweaver H, Burk D. The determination of enzyme dissociation constants[J]. J Am Chem Soc, 1934, 56: 658-666
[27]Kotsira VP, Clonis YD. Chemical Modification of Barley Root Oxalate Oxidase Shows the Presence of a Lysine, a Carboxylate, and Disulfides, Essential for Enzyme Activity[J]. Arch. Biochem. Biophys, 1998,356: 117-126.
[28]林强,马可立.利用纤维素酶催化水解壳聚糖的研究[J].日用化学工业,2003,33(1): 22-25.
[29]刘羿君,蒋英,封云芳,等.特种纤维素酶催化水解壳聚糖及壳寡糖的制备研究[J].功能高分子学报,2005,18(2): 325-329.
[30]陈江燕,邬国铭.纤维素酶对壳聚糖降解作用的研究[J].广东医学院学报,2003,21(2): 105-107.
[31]周桂,何子平,邓光辉,等.纤维素酶与淀粉酶降解壳聚糖的动力学研究[J].海洋科学,2003,27(11): 59-63.
[32]Qin CQ,Zhou B, Zeng LT, et al.The physicochemical properties and antitumor activity of cellulase-treated chitosan[J].Food chem.,2004,84(1): 107-115.
[33]Zhang H,Du YG,Yu XG,et al.Preparation of chitooligosaccharide from chitosan by a complex enzyme[J].Carbohydr Res,1999,320(3-4): 257-260.
[34]夏其昌,曾嵘.蛋白质化学与蛋白质组学[M].北京:科学出版社,2004,6-7.
[35]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. 138-140
[36]郭勇.酶工程原理与技术[M].北京高等教育出版社,2005,205-206.
[37]李曙光,白雪芳,杜煜光.壳寡糖的分离分析及诱抗活性分析[J].中国海洋药物. 2002, 6:1-3.
[38]陈江燕,邬国铭.纤维素酶对壳聚糖降解作用的研究[J].广东医学院学报,2003, 21(2):105-107.
[39]陈晶,付华,陈益.质谱在肽和蛋白质序列分析中的应用[J].有机化学,2002, 22(2): 81-90.
[40]Alcalde M, Plou FJ, Boada MP, et al.Chemical modification of carboxylic residues in a cyclodextrin glucanotransferase and its implication in the hydrolysis/ transglycosylation ratio of theα-amylase family[J]. J. Mol. Catalys. B: Enzymatic,2003,26:57–67.
[41]Petibois C, Gouspillou G, Wehbe K. Analysis of type I and IV collagens by FT-IR spectroscopy and imaging for a molecular investigation of skeletal muscle connective tissue[J].Anal Bioanal Chem, 2006), 386:1961–1966.
[42]谢孟峡,刘媛.红外光谱酰胺Ⅲ带用于蛋白质二级结构的测定研究.高等学校化学学报,2003, 24(02):197-198.
[1]刘北东.绿色木霉纤维素酶系基因克隆表达及特性研究[D]:[博士学位论文].哈尔滨:哈尔滨工业大学生命科学与工程系,2004.
[2]Beldman G,Leeuwen MFS, Rombouts FM,et al. The cellulase of Trichoderma viride[J]. Eur. J. Biochem, 1984,146(2):301-308.
[3]Watanabe,M. Endoglucanase II-T.viride. GenBank: AB021657.1.1998.12.20.
[4]刘靖.纤维素酶水解壳聚糖的特性及机理研究[D]:[博士学位论文].无锡:江南大学, 2006.
[5]刘靖,夏文水.纤维素酶中具有壳聚糖水解酶活性成分的鉴定[J].中国生物化学与分子生物学报,2005, 21 (5): 713-716.
[6]Liu J, Xia WS. Purification and characterization of a bifunctional enzyme with Chitosanase and cellulase activity from commercial cellulase[J]. Biochem. Eng. J,2006,30:82-87.
[7]刘靖,夏文水.纤维素酶中双功能酶水解壳聚糖作用方式的研究[J].食品研究与开发, 2006,32(5):32-36.
[8]刘靖,夏文水.色氨酸残基在双功能酶CCBE中的作用[J].食品科学, 2007, 28(9):425-429,
[9]刘靖,夏文水.双功能酶水解壳聚糖和纤维素的活性中心研究[J].食品与机械, 2007,23(2):31-33.
[10]刘靖,夏文水.焦碳酸二乙酯及碳二亚胺对纤维素酶-壳聚糖酶双功能酶的修饰作用[J].食品科学, 2008,29(8):460-463.
[11]Mandels M, Reese ET.Induction of cellulase in Trichoderma viride as influenced by carbon sources and metals[J].J.Bacteriol,1957,73:269-278.
[12]吴志红,汪天虹,黄卫,等.简便易行的丝状真菌染色体DNA提取法[J].菌物系统. 2001,20(4):575~577.
[13]Matz MV. Amplification of representative cDNA pools from microscopic amounts of animal tissue. In: Ying, S.Y. (Ed.), Generation of cDNA Libraries: Methods and Protocols[M]. Edited by Humana Press, Totowa NJ, 2003,pp.103-116.
[14]Matz MV, Alieva NO, Chenchik A,et al.Amplifi cation of cDNA Ends Using PCR Suppression Effect and Step-Out PCR. In: Ying, S.Y. (Ed.), Generation of cDNA Libraries: Methods and Protocols[M]. Edited by Humana Press, Totowa NJ,2003,pp.41-49.
[15]Sambrook J,Russell DW. Molecular cloning: a laboratory manual[M]. 3rd ed. New York: Cold Spring Harbor Laboratory, 2001.
[16]www.invitrogen.com. Multi-Copy Pichia Expression Kit. For the Isolation and Expression of Recombinant Proteins from Pichia pastoris Strains Containing Multiple Copies of a Particular Gene. Catalog no. K1750-01.
[17]Lowry OH, Rose NJ, Fan AL, et al.Protein measurement with the folin phenol reagent [J]. J. Biol. Chem., 1951,193:265-276.
[18]Laemmli UK, Cleavage of structural proteins during the assembly of the head of bacteriophage T4 [J]. Nature, 1970,227:680-685.
[19]夏其昌.蛋白质化学研究技术与进展[M].北京:科学出版社,1999.
[20]李成文.现代免疫化学技术[M].上海:科学技术出版社,1992, 182~185.
[21]Baldrian P.Fungal laccases-occurrence and properties[J].FEMS microbial Rev,2006, 20:215-242.
[22]田耕,刘炯,晖蓝翎. NCBI网站及GenBank数据库概述[J].国外医学分子生物学分册, 2000, 22(5): 317-320
[23]马东晖,李小洁,马辉文.国际互联网上的NCBI分子生物学数据库简介[J].微生物学通报, 1999, 26(2): 150-153.
[24]王禄山,高培基.生物信息学应用技术[M].北京:化学化工出版社. 2008,51.
[25]N?lting B.The three-dimensional structure of proteins.In: Methods in Modern Biophysics [M]. Berlin. 2005, pp, 1-22.
[26]胡笳,郭燕婷,李艳.蛋白质翻译后修饰研究进展[J].科学通报,2005,50(11):106-1072.
[27].Cassey P J. Protein lopidation in cell signaling[J].Science, 1995, 268:221-225.
[28]Comer F I, Hart GW. O-Glycosylation of Nuclear and Cytosolic Proteins[J]. J Biol.Chem, 2000, 275(38):29179-29182 .
[29]Ike M, Ko Y, Yokoyama K, et al.Cellobiohydrolase I (Cel7A) from Trichoderma reesei has chitosanase activity[J]. J. Mol. Cat. B: Enzymatic., 2007, 47:159-163.
[30]Pedraza M.The bifunctional enzyme chitosanase-cellulase produced by the gram negative microorganism Myxobater.sp.AL-1 is highly similar to Bacillus subtilis endo-glucanases[J]. Arch. Microbiol, 1997,168:321-327.
[31]Tanabe T, Morinaga K, Fukamizo T, et al.Novel chitosanase from Streptomyces griseus HUT 6037 with transglycosylation activity[J]. Biosci Biotechnol Biochem, 2003, 67: 354- 364.
[32]Gao XA, Ju WT, Jung WJ, et al. Purification and characterization of chitosanase from Bacillus cereus D-11[J]. Carbohydr.Polym, 2008,72:513-520.
[33]Ogura J, Toyoda A, Kurosawa T,et al. Chong AL, Chohnan S, &Masaki TPurification, characterization and gene analysis of cellulase (Cel8A) from Lysobacter sp.IB-9374[J]. Biosci. Biotech. Biochem., 2006,70:2420-2428.
[34]Turner PC, McLennan AG, Bates AD,et al.White. Instant notes molecular biology[M].2nd. Oxford : BIOS Sciencetific Pub. Ltd. 2000.
[35]Faustino NA, Cooper TA. Pre-mRNA splicing and human disease[J]. Gene Develop, 2005, 17: 419-437.
[36]Galy V, Gadal O, Fromont-Racine M,et al. Nuclear retention of unspliced mRNAs in yeast is mediated by perinuclear Mlp1[J].Cell. 2004,116:63-73.
[37]Hang GW, Song HD, Chen Z. Molecular mechanism of mRNA alternative splicing[J]. Acta genetica sinica.2004, 31:102-107.
[38]王璋.食品酶学[M].北京:轻工业出版社,1990.
[1]张晓娜,肖华胜.真核生物m RNA选择性剪接体检测方法研究进展[J].生命科学仪器, 2008 ,6:8-11.
[2]夏慧煜,李衍达.真核基因选择性剪接机理的初步研究[J].清华大学学报(自然科学版),2003, 43(4):30-35.
[3]林鲁萍,马飞,王义权.基因选择性剪接的生物信息学研究概况[J].遗传, 2005, 27(6):1001-1006.
[4]Early P, Rogers J, Davis M, et al.Two mRNAs can be produced from a single immunoglobulinμgene by alternative RNA processing pathways[J]. Cell,1980, 20:313-319.
[5]Rosenfeld M G, Lin C R, Amara S G, et al.Calcitonin mRNA polymorphism: peptide switching associated with alternative RNA splicing events[J]. Proc Natl Acad Sci US A,1982,79(6):1717-1721.
[6]Sharp PA.Split genes and RNA splicing[J]. Cell,1994,77(6):805-815.
[7]Gelfand MS, Dubchak I, Dralyuk I, et al. ASDB: database of alternatively spliced genes [J]. Nucleic Acids Res, 1999, 27(1):301-302.
[8]Lopez AJ. Alternative splicing of pre-mRNA: developmental consequences and mechanisms of regulation [J]. Annu Rev Genet, 1998, 32:279-305.
[9]Zhang Y, Zhang J,Cheng H,et al. Identification of Alternate Splice Variants for Murine SmX5[J]. Acta Genetica Sinica , 2003 , 30 (6) : 515-520.
[10]Connor V O, Genin A, Sabrina Davis S,et al. Differential Amplification of Intron- containing Transcripts Reveals Long Term Potentiation-associated Up-regulation of Specific Pde10A Phosphodiesterase Splice Variants[J]. J Biol Chem, 2004, 279(16): 15841-15849.
[11]Beck ET, Blair CD, Black WC,et al.Alternative splicing generates multiple transcripts of the Inhibitor of Apoptosis Protein 1 in Aedes and Culex spp. Mosquitoes[J]. Insect Biochem Mol Biol,2007,37(11): 1222–1233.
[12]许先国,吴俊杰,洪小珍,等.鉴定9个新的RHD基因mRNA可变剪接体[J].遗传, 2006, 28(10): 1213~1218.
[13]Baba Y, Shimonaka A, Koga J,et al.Alternative Splicing Produces Two Endoglucanases with One or Two Carbohydrate-Binding Modules in Mucor circinelloides [J]. J. Bacteriol, 2005, 187(9):3045-3051.
[14]Lin S, Cheng D, Liu MS,et al. Human acyl-CoA:cholesterol acyltransferase-1 in the endoplasmic reticulum contains seven transmembrane domains[J]. J Biol Chem. 1999, 274(33): 23276-23285.
[15]李杨,苏乔,安利佳.基因组的“沙漠区域”内含子及其在植物基因工程中的应用[J].分子植物育种,2004,2(4):569-573.
[16]Salgueiro S, Pignocchi C, Parry MAJ. Intorn-mediated gusA expression in tritordeum and 90wheat resulting from particle bombardment[J]. Plant Mol. Biol., 2000, 42: 615-622.
[17]Birch P R J, Sims P F G, Broda P.Substrate-Dependent Differential Splicing of Introns in the Regions Encoding the Cellulose Binding Domains of Two Exocellobiohydrolase I-Like Genes in Phanerochaete chrysosporium[J].Applied Environ. Microbial,1995, 16(10): 3741-3744.
[18]Baba Y, Shimonaka A, Jinichiro Koga J,et al.Alternative Splicing Produces Two Endoglucanases with One or Two Carbohydrate-Binding Modules in Mucor circinelloides[J].J.Bacteriol, 2005, 187(9):3045-3051.
[19]Costanzo S, Ospina-Giraldo MD, Deahl KL,et al.Alternate intron processing of family 5 endoglucanase transcripts from the genus Phytophthora[J]. Curr Genet, 2007,52:115-123.
[20]Gusakov AV, Sinitsyn A P, Salanovich TN, et al. Purification, cloning and characterisation of two forms of thermostable and highly active cellobiohydrolaseI (Cel7A) produced by the industrial strain of Chrysosporium lucknowense[J]. Enzyme.Microbial Technol., 2005, 36: 57-69.
[21]Curach NC, Te’O VSJ, Gibbs MD,et al.Isolation, characterization and expression of the hex1 gene from Trichoderma reesei[J]. Gene, 2004,331:133-140.
[22]Yamada T,Hiramatsu S,Songsri P,et al. Alternative Expression of a Chitosanase Gene Produces Two Different Proteins in Cells Infected with Chlorella Virus CVK2[J]. VIROLOGY, 1997,230: 361-368.
[23]An XP., Lu J, Huang JD, et al. Rapid Assembly of Multiple-Exon cDNA Directly from Genomic DNA[J].PLoS ONE,2007,2(11): e1179.doi:10.1371/journal.pone. 0001179.
[24]Sambrook J,Russell DW. Molecular cloning: a laboratory manual [M]. 3rd ed. New York: Cold Spring Harbor Laboratory, 2001.
[25]Lowry OH, Rose NJ, Fan AL, et al., Protein measurement with the folin phenol reagent [J]. J. Biol. Chem., 1951,193:265-276.
[26]姚晓敏,马涵慧,王灿华,等.真核生物pre-mRNA剪接的研究进展[J].上海交通大学学报&农业科学版,2005,23(1):96-101,110.
[27]刘进元,李文君,王薛林,等译.分子生物学快速精要中文版[M].2nd.Turner PC etc (eds).北京:科学出版社,2001.
[28]Tanaka A, Mita1 S, Ohta S, et al. Enhancement of foreign gene expression by a dicot Intron in rice but not in tobacco is correlated with an increased level of mRNA and an efficient splicing of the intron[J]. Nucleic.,Acids.,Res., 1990,18(23): 6767-6770.
[29]Kim KN, Guiltinan MJ. Identification of cis-acting elements important for expression of the starch-branching enzyme I gene in maize endosperm[J]. Plant Physiology.,1999, 121: 225-236.
[30]Siegei R S. Methylotroyphic yeast Pichia pastour produced in High-cell-density fermentation with high cell yields as vehicle for recombinant protein production[J]. Biotechnol Bioengi, 1989, 34: 403-404.
[31]欧阳立明,张惠展,张嗣同.巴斯德毕赤酵母的基因表达系统研究进展[J].生物化学与生物物理进展,2000,27(2):151-154.
[32]张伍魁,范清林,宋礼华.毕赤酵母表达系统在外源基因表达中的研究进展及应用[J].中国生物工程杂志,2006,26(1):87-91.
[33]汪天虹,王春卉,高培基.纤维素酶纤维素吸附区的结构与功能[J].生物工程进展, 2000, 20(2): 37-40.
[34]汪天虹,邹玉霞,石屹峰,等.微紫青霉CBHⅠ酶纤维素结合结构域在大肠杆菌中的分泌型表达及性质研究[J].中国生物化学与分子生物学报,2000,16(5):644-649.
[35]Levy I, Shoseyov O.Cellulose-binding domains: Biotechnological applications[J]. Biotechnology Advances, 2002, 20(3-4): 191-213.
[36]Takashima S, Hidaka M, Nakamura A, et al.Correlation between cellulose binding and activity of cellulose-binding domain mutants of Humicola grisea cellobiohydrolase I [J]. FEBS Letters, 2007, 581(30): 5891-5896.
[37]王建荣,张曼夫,黄涛.绿色木霉纤维素酶CBHII基因的结构研究[J].遗传学报, 1995,22(1):74-80.