白毒鹅膏菌漆酶的纯化、性质及偶氮染料降解研究
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摘要
论文首先从液体培养体系出发,探索不同培养条件对白毒鹅膏菌分泌漆酶的影响。我们筛选出白毒鹅膏菌的综合产酶培养基为(g/L):马铃薯200 g/L、麸皮20 g/L、KH_2PO_4 3g/L、MgSO_4 1.5g/L、V_(B1)0.01 g/L、酵母膏5 g/L。其最佳pH值范围为5.0-5.4,转速为120 rpm,温度为25℃,装液量为50 mL。按此条件进行培养,漆酶从第5d酶活力迅速提升,第8d达产酶高峰,随后产酶能力下降。
实验通过盐析、透析、DEAE-纤维素DE52离子交换层析和Sephedex G-100凝胶过滤层析等步骤,分离纯化得到电泳纯的漆酶。以邻联甲苯胺作为底物,最终漆酶被纯化了22.03倍,活力回收率为22.02%。通过SDS-聚丙烯酰胺凝胶电泳检测,漆酶的相对分子量为63kDa。
该酶具有较宽的pH值稳定性,在pH 3.6到5.2范围内能够较好地保持酶活力;该漆酶最适反应温度为20℃,在20℃到60℃范围内能够稳定的保持较长时间,但在70℃时保持5 min,且能保持20%的活力。β-巯基乙醇或L-半胱氨酸是该酶的高效抑制剂,但是EDTA或DMSO对其抑制作用不是很强。白毒鹅膏菌漆酶催化反应符合Miehaelis-Menten动力学规律。在20℃,pH 4.6的醋酸缓冲溶液中,漆酶以邻联甲苯胺作为底物时,米氏常数为66.7μmol/L。潜在的抑制剂测试中,L-半胱氨酸及2-巯基乙醇在所试浓度范围内均完全抑制漆酶的活力,SDS高浓度时对漆酶的活力高度抑制,EDTA和DMSO在所试浓度范围内对漆酶的活力微弱抑制。
利用白毒鹅膏菌漆酶可以较为彻底的降解6种染料,底物范围较广,在降解过程中,要持续通氧气(振荡)。直接黑G降解的最适pH值为5.2,而中性深黄GL降解的最适pH值为3.5。两种染料与白毒鹅膏菌漆酶作用的最适温度均为50℃。底物染料浓度为20mg/mL时,酶活力超过10 U/mL后,进一步提高酶活力对r_D基本无影响,中性深黄GL总脱色率维持在80%左右,直接黑G约维持在60%。
采用海藻酸钠包埋法和海藻酸钠-壳聚糖包埋-交联法固定化漆酶。探讨了固定化条件、固定化漆酶及游离酶的酶学性质。结果表明,包埋法和包埋-交联法固定化漆酶的最佳条件分别为海藻酸钠浓度3%、CaCl_2浓度1.5%和海藻酸钠浓度2%、CaCl_2浓度2%、壳聚糖浓度1.5%、戊二醛浓度1%。两种固定化漆酶的最适pH和最适温度相同,分别为5.0和30℃,游离酶为4.6和20℃。将固定化酶应用在偶氮染料的脱色中,包埋法脱色效果接近游离酶并且在重复进行的摇床实验中,脱色能力未降低,反应前后的酶活力均没有损失。与包埋法相比,包埋-交联法固定化漆酶机械强度更强,操作稳定性更好,使得固定化酶的重复利用率更高,但应用于染料脱色时,由于壳聚糖的吸附作用使得漆酶对于染料的降解作用降低,因此制备应用于染料降解的固定化漆酶时还应对辅料进行优化。
Firstly,In the liquid culture system,the influence on the production of laccase by Amanita verna in different fermentation conditions has been studied.Through a series of orthogonal experiments,effects of carbon source,nitrogen source and other factors on laccase production from strain Amanita verna were studied.The liquid medium used for laccase production contained(per liter)200g potato extract with 20g dextrose, 5g yeast extract,20g bran,3g KH_2PO_4,1.5g MgSO_4,pH5.0-5.4.Shaking cultivation was performed at 25℃on a rotary shaker at 120 rpm.Under the optimized fermentation process,the laccase production by strain Amanita verna was increased from the 5th day,reached the highest production on the 8th day.
The laccase was purified by a three-step procedure involving salting out,ion exchange,size exclusion chromatography.A typical procedure provided 22.03-fold purification,with a yield of 22.02%,using o-tolidine as substrate.The molecular weight of the purified laccase was 63 kDa as estimated by 12%(w/v)SDS-PAGE gel.
The enzyme's pH optimum for o-tolidine was 4.6 and optimal activity was at 20℃.After pre-incubation at different pH values for 3.5h and different temperatures for 70 min,more than 60%of the initial laccase activity was retained between pH 3.6 to 5.2 and a relative activity of 19.3%was observed at 60℃.The Km value of laccase for o-Tolidine was 66.7μmol/L at pH4.6.The effect of different chemicals on laccase activity was also tested.This laccase was strongly inhibited by L-cysteine,β-mercaptoethanol even at a low concentration,and slightly inhibited by EDTA.The combination of thermotolerance with low pH optima for aromatic compounds substrates suggests that the laccase possesses potential for use in biotechnological applications.
The laccase from Amanita verna could degradate 6 dyes efficiently under keep on ventilating condition.The laccase showed maximal decolorization rates of both dyes at 50℃and optimal decolorization rates at pH 5.5 and 3.5 for direct black G and neutral dark yellow GL,respectively.The higher the pollutant primary concentration and laccase activity,the higher decoloration rate,when the primary concentration and laccase activity were below 20mg/L and 10 U/mL,respectively.The decoloration rate of direct black G was 80%,while it was 60%to neutral dark yellow GL.
Laccase was immobilized by the sodium alginate entrapment method and the sodium alginate-chitosan entrapment-crosslinking method.The immobilization conditions and the characterizations of the immobilized and free laccase were investigated.The results showed that the optimal conditions of immobilized laccase by the entrapment method and the entrapment-crosslinking method were 3%sodium alginate,1.5%CaCl_2 and 2%sodium alginate,2%CaCl_2,1.5%chitosan,1% glutaradehyde,respectively.These two kinds of immobilized laccase exhibited the same optimal pH value and optimal action temperature which were 5.0 and 30℃, however the free enzyme's optimal pH value and optimal action temperature were 4.6 and 20℃.The ability of decolourizing azo dyes in shaky situation by sodium alginate-immobilized laccase approached to the free enzyme,and there was no loss of enzyme activity during 2 repeated batch reactions.Compare with entrapment method, entrapment-crosslinking method has a better mechanical strength and operation stability,but the decolorization rate was low because of chitosan' adsorption,so we should optimize accessories of entrapment-crosslinking immobilized laccase used for dye degradation.
实验通过盐析、透析、DEAE-纤维素DE52离子交换层析和Sephedex G-100凝胶过滤层析等步骤,分离纯化得到电泳纯的漆酶。以邻联甲苯胺作为底物,最终漆酶被纯化了22.03倍,活力回收率为22.02%。通过SDS-聚丙烯酰胺凝胶电泳检测,漆酶的相对分子量为63kDa。
该酶具有较宽的pH值稳定性,在pH 3.6到5.2范围内能够较好地保持酶活力;该漆酶最适反应温度为20℃,在20℃到60℃范围内能够稳定的保持较长时间,但在70℃时保持5 min,且能保持20%的活力。β-巯基乙醇或L-半胱氨酸是该酶的高效抑制剂,但是EDTA或DMSO对其抑制作用不是很强。白毒鹅膏菌漆酶催化反应符合Miehaelis-Menten动力学规律。在20℃,pH 4.6的醋酸缓冲溶液中,漆酶以邻联甲苯胺作为底物时,米氏常数为66.7μmol/L。潜在的抑制剂测试中,L-半胱氨酸及2-巯基乙醇在所试浓度范围内均完全抑制漆酶的活力,SDS高浓度时对漆酶的活力高度抑制,EDTA和DMSO在所试浓度范围内对漆酶的活力微弱抑制。
利用白毒鹅膏菌漆酶可以较为彻底的降解6种染料,底物范围较广,在降解过程中,要持续通氧气(振荡)。直接黑G降解的最适pH值为5.2,而中性深黄GL降解的最适pH值为3.5。两种染料与白毒鹅膏菌漆酶作用的最适温度均为50℃。底物染料浓度为20mg/mL时,酶活力超过10 U/mL后,进一步提高酶活力对r_D基本无影响,中性深黄GL总脱色率维持在80%左右,直接黑G约维持在60%。
采用海藻酸钠包埋法和海藻酸钠-壳聚糖包埋-交联法固定化漆酶。探讨了固定化条件、固定化漆酶及游离酶的酶学性质。结果表明,包埋法和包埋-交联法固定化漆酶的最佳条件分别为海藻酸钠浓度3%、CaCl_2浓度1.5%和海藻酸钠浓度2%、CaCl_2浓度2%、壳聚糖浓度1.5%、戊二醛浓度1%。两种固定化漆酶的最适pH和最适温度相同,分别为5.0和30℃,游离酶为4.6和20℃。将固定化酶应用在偶氮染料的脱色中,包埋法脱色效果接近游离酶并且在重复进行的摇床实验中,脱色能力未降低,反应前后的酶活力均没有损失。与包埋法相比,包埋-交联法固定化漆酶机械强度更强,操作稳定性更好,使得固定化酶的重复利用率更高,但应用于染料脱色时,由于壳聚糖的吸附作用使得漆酶对于染料的降解作用降低,因此制备应用于染料降解的固定化漆酶时还应对辅料进行优化。
Firstly,In the liquid culture system,the influence on the production of laccase by Amanita verna in different fermentation conditions has been studied.Through a series of orthogonal experiments,effects of carbon source,nitrogen source and other factors on laccase production from strain Amanita verna were studied.The liquid medium used for laccase production contained(per liter)200g potato extract with 20g dextrose, 5g yeast extract,20g bran,3g KH_2PO_4,1.5g MgSO_4,pH5.0-5.4.Shaking cultivation was performed at 25℃on a rotary shaker at 120 rpm.Under the optimized fermentation process,the laccase production by strain Amanita verna was increased from the 5th day,reached the highest production on the 8th day.
The laccase was purified by a three-step procedure involving salting out,ion exchange,size exclusion chromatography.A typical procedure provided 22.03-fold purification,with a yield of 22.02%,using o-tolidine as substrate.The molecular weight of the purified laccase was 63 kDa as estimated by 12%(w/v)SDS-PAGE gel.
The enzyme's pH optimum for o-tolidine was 4.6 and optimal activity was at 20℃.After pre-incubation at different pH values for 3.5h and different temperatures for 70 min,more than 60%of the initial laccase activity was retained between pH 3.6 to 5.2 and a relative activity of 19.3%was observed at 60℃.The Km value of laccase for o-Tolidine was 66.7μmol/L at pH4.6.The effect of different chemicals on laccase activity was also tested.This laccase was strongly inhibited by L-cysteine,β-mercaptoethanol even at a low concentration,and slightly inhibited by EDTA.The combination of thermotolerance with low pH optima for aromatic compounds substrates suggests that the laccase possesses potential for use in biotechnological applications.
The laccase from Amanita verna could degradate 6 dyes efficiently under keep on ventilating condition.The laccase showed maximal decolorization rates of both dyes at 50℃and optimal decolorization rates at pH 5.5 and 3.5 for direct black G and neutral dark yellow GL,respectively.The higher the pollutant primary concentration and laccase activity,the higher decoloration rate,when the primary concentration and laccase activity were below 20mg/L and 10 U/mL,respectively.The decoloration rate of direct black G was 80%,while it was 60%to neutral dark yellow GL.
Laccase was immobilized by the sodium alginate entrapment method and the sodium alginate-chitosan entrapment-crosslinking method.The immobilization conditions and the characterizations of the immobilized and free laccase were investigated.The results showed that the optimal conditions of immobilized laccase by the entrapment method and the entrapment-crosslinking method were 3%sodium alginate,1.5%CaCl_2 and 2%sodium alginate,2%CaCl_2,1.5%chitosan,1% glutaradehyde,respectively.These two kinds of immobilized laccase exhibited the same optimal pH value and optimal action temperature which were 5.0 and 30℃, however the free enzyme's optimal pH value and optimal action temperature were 4.6 and 20℃.The ability of decolourizing azo dyes in shaky situation by sodium alginate-immobilized laccase approached to the free enzyme,and there was no loss of enzyme activity during 2 repeated batch reactions.Compare with entrapment method, entrapment-crosslinking method has a better mechanical strength and operation stability,but the decolorization rate was low because of chitosan' adsorption,so we should optimize accessories of entrapment-crosslinking immobilized laccase used for dye degradation.
引文
1.钱亚鹏,钱世均.真菌漆酶极其应用[J].生物工程进展,2001,21(5):23-28.
2.Niku-Paavola ML,Raanua M,Soumakkia,et al.Effect of lignin-modifying enzymes of pulp[J].Bioresour Techno 1,1994,50(1):73.
3.Falcon MA et al.Isolation of microorganisms with lignin transformation potential from soil of Tenerife island[J].Soil Biol.Biochem.,1995(27):121-126
4.Hatakka A,Biodegradation of lignin.In:Hofrichter M,Steinbuchel A eds.Biopolymers.Vol.1-Lignin,humic substances and coal[J].Weinheim,Germany:Wiley-VCH.2001(1):129-180
5.Banerjee UC,Vohra RM.Production of laccase by Curvularia sp[J].Folia Microbiol.,1991(36):343-346.
6.Junghanns C,Moeder M,Krauss G,Martin C,Schlosser D.Degradation of the xenoestrogen nonylphenol by aquatic fungi and their laccases[J].Microbiology,2005(151):45-57.
7.Ikeda R,Sugita T,Jacobson ES,Shinoda T.Effects of melanin upon susceptibility of Cryptococcus to antifungals[J].Microbiol.Immunol.,2003(47):271-277.
8.Jimenez M,Gonzalez AE,Martinez M J,Martinez AT,D'ale BE.Screening of yeasts isolated from decayed wood for lignocellulose degrading enzyme activities [J].Mycol.Res.,1991(95):1299-1302.
9.D'Souza TM,Boominathan K & Reddy CA.Isolation of laccase gene-specific sequences from white rot and.brown rot fungi by PCR[J].Appl.Environ.Microbiol.,1996(62):3739-3744.
10.Lee KH,Wi SG.Singh AP,Kim YS.Micromorphological characteristics of decayed wood and laccase produced by the brown-rot fungus Coniophora puteana[J].J Wood Sci.2004(50):281-284.
11.甘景镐.生漆的化学[M].北京:科学出版社,1984,73-117
12.Yaropolov A I,Skorobogat'ko O V,Vartanov S S,et al.Laccase:properties,catalytic mechanism and applicability[J].Appl.Biochem.Biotechnol.,1994(49):257-280
13.Givaudan A,Effosse A,Faure D et al.Polyphenol oxidase in Azospirillum Iipoferum isolated from rice rhizosphere:evidence for laccase activity in nonmotile strains of Azospirillum lipoferum[J].FEMS Microbiol.Lett.,1993(108):205-210
14.Diamantidis G,Effosse A,Potier Pet al.Purification and characterization of the first bacterial laccase in the rhizospheric bacterium Azospirillum lipoferum[J].Soil Biol.Biochem.,2000(32):919-927
15.Hullo MF,Moszer I,Danchin A et al.CotA of Bacillus substilis is a copper-dependent laccase[J].J Bacteriol.2001(183):5426-5430
16.Martins LO,Soares CM,Pereira MM et al.Molecular and biochemical characterization of a highly stable bacterial laccase that occurs as a structural component of the Bacillus subtilis endospore coat[J].J Biol.Chem.,2002(277):18849-18859
17.Van Waasbergen LG,Hildebrand M,Tebo BM.Identification and characterization of a gene cluster involved in manganese oxidation by spores of a marine Bacillus sp.strain SG-1[J].J Bacteriol.1996(178):3517-3530
18.张敏,肖亚中,龚为民.真菌漆酶的结构和功能[J].生物学杂志,2003,(5):6-8
19.王国栋,陈晓亚.漆酶的性质、功能、催化机理和应用[J].植物学通报,2003(20):69-75.
20.王海磊,李宗义.三种重要木质素降解酶研究进展[J].生物学杂志,2003(20):9-11
21.宋安东,王艳婕,王松江.漆酶的分子生物学研究及其应用[J].信阳师范学院学报,2004(17):244-248
22.万云洋,杜予民.漆酶结构与催化机理.化学结构在线预览版,http://www.hxtb.org
23.季立才,胡培植等.漆酶催化氧化反应研究进展[J].林产化学与工业,1997(1):79-84
24.Hakulinen N,Kiiskinen LL,Kruus K,Saloheimo M,Paananen A,Koivula A,Rouvinen J.Crystal structure of a laccase from Melanocarpus albomyces with an intact trinuclear copper site[J].Nat.Struct.Biol.,2002(8):601-605
25.Ducros V,Brzozowski AM,Wilson KS,Ostergaard P,Schneider P,Svendson A,Davies GJ.Structure of the laccase from Coprinus cinereus at 1.68 A resolution:evidence for different 'type 2 Cu-depleted' isoforms[J].Acta Crystallogr D Biol.Crystallogr.,2001(57):333-336.
26.Archibald FS.,Bourbonnais R.,Jurasek L.,Paice M.G.and Reid I.D..Kraft pulp bleaching and delignification by Trametes versicolor[J].J.Biotechnol.,1997(53):215-236.
27.Anastasia P.,Litvintseva,Joan M.Henson.Cloning,Characterization,and Transcription of Three Laccase Genes from Gaeumannomyces graminis var.tritici,the Take-All Fungus[J].Appl.Environ.Microbiol.,2002,68(3):1305-1311
28.Boghos Dedeyan,Agnieszka Klonowska,Simone Tagger,Thierry Tron,Gilles Iacazio,Gerard Gil,and Jean Le Petit.Biochemical and Molecular Characterization of a Laccase from Marasmius quercophilus[J].Appl.Environ.Microbiol.,2000(3):925-929
29.王光辉,季立才.漆树漆酶的催化氧化作用:3,5-二叔丁基-1,2-苯二酚的催化氧化[J].武汉大学学报(自科版),1989(4):106-108
30.Aktas N.,Cicek H et al.Reaction kinetics for laccase-catalyzed polymerization of 1-naphthol[J].Bioresour.Technol.,2001(1):29-36.
31.A.A.Leontievsky,N.M.Myasoedova,B.P.Baskunov,C.S.Evans,L.A.Golovleva.Transformation of 2,4,6- trichlorophenol by the white rot fungi Panus tigrinus and Coriolus Versicolor[J].Biodegradation,2000(11):331-340
32.季立才,胡培植,徐文中.漆树漆酶催化2,6-二甲基酚的氧化反应[J].高等学校化学学报,1992(7):940-944.
33.Tetsuya E,Hiroyuki U,Yoshiko T.,Takayuki.Degradation of Bisphenol A by purified Laccase from Trametes villosa[J].Biochem.Biophys.Res.Commun.,2001(284):704-706
34.Fahraeus G,Lijunggren H.Substrates specificity of a purified fungal laccase.Biochim Biophys.Acta,1961(46):22
35.季立才,王光辉,胡培植.漆树漆酶的新底物[J].1990(3):189-193
36.刘尚旭,赖寒.木质素降解酶的分子生物学研究进展[J].重庆教育学院学报,2001(3):64-67
37.Christopher F.The structure and function of fungal laccase[J].Microbiology,1994(1):19
38.Ducros,V Brzozowski AM,Wilson KS,et al.Crystal structure of the type-2 Cu depleted Laccase from coprinus cinereus at 2.2 A resolution.Nat.struct.Biol,1998(5):310-316
39.Selinheimo E,Kruus K,Buchert J,Hopia A,Autio K.Effects of laccase,xylanase and their combination on the rheological properties of wheat doughs.J Cereal Sci 2006(43):152-159.
40.Minussi RC,Pastore GM,Dura N.Potential applications of laccase in the food industry[J].Trends Food Sci.Technol.,2002(13):205-216.
41.Camarero S,Garcia O,Vidal T,Colom J,del Rio JC,Gutierrez A,et al.Efficient bleaching of non-wood high-quality paper pulp using laccase-mediator system[J].EnzymeMicrob.Technol.,2004(35):113-120.
42.Huttermann A,Mai C,Kharazipour.Modification of lignin for the production of new compounded materials[J].Appl.Microbiol.Biotechnol.,2001(55):387-394.
43.Riu J,Schon seeI,Barcelo D.Determination of sulfonated azo dyes in groundwater and industrial effluents by automated solid-phase extraction followed by capillary electrophoresis/mass spectrometry[J].J Mass.Spectrom.,1998(33):653-663.
44.宋美静.纸浆氯漂废水的处理[J].纤维素科学与技术,1999(2):22-25.
45.堵国成,赵政,陈坚.真菌漆酶的活性测定及其在织物染料生物脱色中的应用[J].江南大学学报,2003(1):83-90.
46.Roy JJ,Abraham TE,Abhijith KS,Sujith kumar PV,Thakur MS.Biosensor for the determination of phenols based on Cross-Linked Enzyme Crystals(CLEC)of laccase[J].Biosens.Bioelectron.,2005(21):206-211.
47.Nyanhongo GS,Rodriguez Couto,S,Gtibitz GM.Coupling of 2,4,6-trinitrotoluene(TNT)metabolites onto humic monomers by a new laccase from Trametes modesta[J].Chemosphere,2006(3):359-370
48.Mustafa R,Muniglia L,Rovel B,Girardin M.Phenolic colorants obtained by enzymatic synthesis using a fungal laccase in a hydroorganic biphasic system[J].Food Res.Int.,2005(38):995-1000.
49.Golz-Berner K.Walzel B.Zastrow L.Doucet O(04.03.2004).Cosmetic and dermatological preparation containing copperbinding proteins for skin lightening.Int Pat Appl WO2004017931.
50.Ishikawa Y,Esker T,Leder A.Chemical economics handbook:dyes.Menlo Park (CA):SRI Chemical and Health Business Services;2000.
51.Acuner,E.,Dilek,F.B.Treatment of tectilon yellow 2G by Chlorella vulgaris[J].Process Biochem.,2004(39):623-631.
52.Golka,K.,Kopps,S.,Myslak,Z.W.Carcinogenicity of azo colorants:influence of solubility and bioavailability[J].Toxicol.Lett.,2004(151):203-210.
53.Robinson T,McMullan G,Marchant R,Nigam P.Remediation of dyes in textile effluent:a critical review on current treatment technologies with a proposed alternative[J].Bioresour Technol.2001(77):247-255.
54.Willmott N,Guthrie J,Nelson G.The biotechnology approach to colour removal from textile effluent[J].J Soc.Dyers Colour.,1998(114):38-41.
55.Zissi U,Lyberatos G.Partial degradation of p-aminoazobenzene by a defined mixed culture of Bacillus subtilis and Stenotrophomonas maltophilia.Biotechnol.Bioeng.,2001(72):49-54.
56.Elthabeth R,Michael AP and Rafeel VD,Current Microbiology,1999.38.27-32
57.Michael AP,Rosa R,Pauel T et al.Appli.Environ,Microbiol.,1999,(9):3805-3809
58.Duran,N.et al.Applications of laccases and tyrosinases(phenoloxidases)immobilized on different supports:a review.Enzyme Microb.Technol.,2002(31):907-931
59.李冬生,康旭,杨红.漆酶固定化及其性质研究[J].广东食品工业科技,2004,20(2):6
60.H Xiao,J Huang,C LiuI,D Jiang.Immobilization of laccase on amine-terminated magnetic nano-composite by glutaraldehyde crosslinking method[J].Transactions of Nonferrous Metals Society of China,2006(16):414-418
61.C Mousty,L Vieille,S Cosnier.Laccase immobilization in redox active layered double hydroxides:A reagentless amperometric biosensor[J].Biosens.Bioelectron.,2007(8):1733-1738
62.Paolo Brandi,Alessandro D'Annibale,Carlo Galli,Patrizia Gentili' and Ana Sofia Nunes Pontes.In search for practical advantages from the immobilisation of an enzyme:the case of laccase[J].J.Mol.Catal.B:Enzym.,2006(1-3):61-69
63.王军涛,黄俊,刘诚,李斌.CoTAPc-Fe_3O_4纳米复合固定化漆酶研究[J].武汉理工大学学报,2007(1):1-3
64.史伯安.漆酚-(8-羟基)喹啉-Cu(Ⅱ)高分子配合物固定化漆树酶的研究[J].化学与生物工程,2005(6):15-17
65.Young L and Jian Yu.Ligninase-catalyzed decolorization synthetic dyes[J].Water Res.1997(31):1187-1193.
66.Bourbonnais,R.,Paice,M.G.Oxidation of non-phenolic substrates:an expanded role of laccase in lignin biodegradation[J].FEBS Lett.1990(267):99-102.
67.Call,H.P.,Mu"cke,I.History,overview and applications of mediated ligninolytic systems,especially laccase-mediator-systems(Lignozym process)[J].J.Biotechnol.1997(53):163-202.
68.王宜磊,邓郑旭.采绒革盖菌CV-8漆酶活性的初步研究[J].微生物学杂志.1998(4):60-62.
69.Ross IK.The role.of laccase in carpophore initiation in Coprinus congregatus[J].J Gen Microbiol,1982(128):2755-2762
70.Bradford M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J],Anal Biochem.,1976(72):248-254
71.胡景江,王俊明,张高伟,曹支敏.皱马鞍菌液体培养过程中胞外酶及还原糖的动态变化[J].西北农林科技大学学报(自然科学版),2005(11):94-98
72.Han MJ,Choi HT,Song HG.Purification and characterization of laccase from the white rot fungus Trametes versicolor[J].J Microbiol 2005(43):555-560
73.Wang HX,Ng TB.Purification of a laccase from fruiting bodies of the mushroom Pleurotus eryngii[J].Appl Microbiol Biotechnol,2006(69):521-525.
74.刘友勋,郜金荣,黄娟.白腐菌在两种固体基质上的漆酶产量及同工酶活性比较研究[J].氨基酸和生物资源,2007(3):33-35
75.W Liu,Y Chao,S Liu,et al.Molecular cloning and characterization of a laccase gene from the basidiomycete from lignosus and expression in Pichia pastoris[J].Appl.Microbiol.Biotechnol.,2003(2):174-181.
76.Y Xiao,X Tu,J Wang,et al.Purification,molecular characterization and reactivity aromatic compound of a laccase from basidomycete Trametes sp.Strain AH 28-2[J].Appl.Microbiol.Biotechnol.,2003(6):700-707.
77.康从宝,刘巧,李清心,等.白腐菌产漆酶的纯化及部分酶学性质[J].中国生物化学与分子生物学报,2002(5):638-642.
78.李杨,段新源,刘稳,等.杂色云芝组成型漆酶Ⅰ的纯化和底物专一性[J].中国生物化学与分子生物学报,2002(6):737-740.
79.秦小琼,傅庭治,曹幼琴,等.红栓菌胞外漆酶的诱导、纯化及部分性质研究[J].微生物学报,1996(5):360-366.
80.朱启忠等编著.生物化学技术指南.新疆科技卫生出版社.1995,新疆
81.陈钧辉等主编.生物化学实验(第三版).科学出版社,2005.7,北京
82.Jung H,Xu F,Li K.Purification and characterization of laccase from wood-degrading fungus Trichophyton rubrum LKY-7[J].Enzyme Microb.Technol.,2002(30):161-168
83.Jaouani A,Guillen F,Penninckx M J,Martinez AT,Martinez MJ.Role of Pycnoporus coccineus laccase in the degradation of aromatic compounds in olive oil mill wastewater[J].Enzyme.Microb.Technol.,2005(36):478-486
84.Iyer G,Chattoo B.Purification and characterization of laccase from the dee blast fungus,Magnaporthe grisea[J].FEMS Microbiol.Lett.,2003(227):121-126.
85.Baldrian P.Purification and characterization of laccase from the white-rot fungus Daedalea quercina and decolorization of synthetic dyes by the enzyme[J].Appl.Microbiol.Biotechnol.,2004(63):560-563
86.Nagai M,Sato T,Watanabe H,Saito K,Kawata M,Enei H.Purification and characterization of an extracellular laccase from the edible mushroom Lentinula edodes,and decolorization of chemically different dyes[J].Appl.Microbiol.Biotechnol.,2002(60):327-335
87.Eggert C,Temp U,Eriksson K-EL.The lignolytic system of the white rot fungus Pycnoporus cinnabarinus:Purification and characterization of the laccase.Appl.Environ.Microbiol.,1996(62):1151-1158
88.Vasdev K,Dhawan S,Kapoor RK,Kuhad RC.Biochemical characterization and molecular evidence of a laccase from the bird's nest fungus Cyathus bulleri[J].Fungal Genet.Biol.,2005(42):684-693
89.Zhang H,Hong YZ,Xiao YZ,Yuan J,Tu XM,Zhang XQ.Efficient production of laccases by Trametes sp.AH28-2 in cocultivation with a Trichoderma strain[J].Appl.Microbiol.Biotechnol.,2006(73):89-94
90.Meyer U.Biodegradation of synthetic organic colorants[J].FEMS Symp.1981(12):371-385.
91.Holme,I.Ecological Aspects of Color Chemistry:Developments in the Chemistry and Technology of Organic Dyes,edited by J.Griffiths,1984:111-128.Oxford:Society for Chemical Industry.
92.Robinson T,McMullan G,Marchant R,Nigam P.Remediation of dyes in textile effluent:a critical review on current treatment technologies with a proposed alternative[J].Bioresour Technol,2001(77):247-255.
93.Piontek,K.et al.Crystal structure of a laccase from the fungus Trametes versicolor at 1.90A resolution containing a full complement of coppers[J].J.Biol.Chem.2002(277):37663-37669
94.Claus,H.Laccases:structure,reactions,distribution[J].Micron.,2004(35):93-96
95.龙胜亚,肖海燕,周菊英,黄俊等.磁性壳聚糖微球的制备及其用作漆酶固定化载体[J].应用化学,2005(22):1122-1126
96.陈騊声,居乃琥,陈石根.固定化酶理论与应用[M].北京:轻工业出版社,1987
97.陈万成,蒋笃孝,宋龄瑛,欧仕益.壳聚糖/海藻酸钠微胶囊固定化磷脂酶A_1(PLA_1)的研究[J],食品科技.2004(11):14-17.
98.蒋挺大.甲壳素[M].北京:化学工业出版社,2003
99.孟颢华.沪酿3.042米曲霉的纯化复壮[J].中国调味品,2001(7):7-9.
100.何青,向洋.米曲霉酶活力与孢子特征关系的研究[J].中国酿造,1993(5):33-37.
101.阚建全.食品化学[M].北京:科学出版社,2002.
102.Ida Genta,Monica Costantini,Annalia Asti,Bice Conti,Luisa Montanari.Influence of glutaraldehyde on drug release and mucoadhesive properties of chitosan microspheres[J].Carbohydr.Polym.,1998(36):81-88
103.Wu FJ,Flach CR,Seaton BA,et al.Stability of annexin Ⅴ in ternary complexes with Ca~(2+)and anoinic phospholipids:IR studies of monolayer and buik phases[J].Biochem.,1999,38(2):792.
104.朱启忠,赵亮云,赵宏,韩晓弟.壳聚糖对酸性染料的吸附性能[J].资源开发与市场2006(2):101-102
2.Niku-Paavola ML,Raanua M,Soumakkia,et al.Effect of lignin-modifying enzymes of pulp[J].Bioresour Techno 1,1994,50(1):73.
3.Falcon MA et al.Isolation of microorganisms with lignin transformation potential from soil of Tenerife island[J].Soil Biol.Biochem.,1995(27):121-126
4.Hatakka A,Biodegradation of lignin.In:Hofrichter M,Steinbuchel A eds.Biopolymers.Vol.1-Lignin,humic substances and coal[J].Weinheim,Germany:Wiley-VCH.2001(1):129-180
5.Banerjee UC,Vohra RM.Production of laccase by Curvularia sp[J].Folia Microbiol.,1991(36):343-346.
6.Junghanns C,Moeder M,Krauss G,Martin C,Schlosser D.Degradation of the xenoestrogen nonylphenol by aquatic fungi and their laccases[J].Microbiology,2005(151):45-57.
7.Ikeda R,Sugita T,Jacobson ES,Shinoda T.Effects of melanin upon susceptibility of Cryptococcus to antifungals[J].Microbiol.Immunol.,2003(47):271-277.
8.Jimenez M,Gonzalez AE,Martinez M J,Martinez AT,D'ale BE.Screening of yeasts isolated from decayed wood for lignocellulose degrading enzyme activities [J].Mycol.Res.,1991(95):1299-1302.
9.D'Souza TM,Boominathan K & Reddy CA.Isolation of laccase gene-specific sequences from white rot and.brown rot fungi by PCR[J].Appl.Environ.Microbiol.,1996(62):3739-3744.
10.Lee KH,Wi SG.Singh AP,Kim YS.Micromorphological characteristics of decayed wood and laccase produced by the brown-rot fungus Coniophora puteana[J].J Wood Sci.2004(50):281-284.
11.甘景镐.生漆的化学[M].北京:科学出版社,1984,73-117
12.Yaropolov A I,Skorobogat'ko O V,Vartanov S S,et al.Laccase:properties,catalytic mechanism and applicability[J].Appl.Biochem.Biotechnol.,1994(49):257-280
13.Givaudan A,Effosse A,Faure D et al.Polyphenol oxidase in Azospirillum Iipoferum isolated from rice rhizosphere:evidence for laccase activity in nonmotile strains of Azospirillum lipoferum[J].FEMS Microbiol.Lett.,1993(108):205-210
14.Diamantidis G,Effosse A,Potier Pet al.Purification and characterization of the first bacterial laccase in the rhizospheric bacterium Azospirillum lipoferum[J].Soil Biol.Biochem.,2000(32):919-927
15.Hullo MF,Moszer I,Danchin A et al.CotA of Bacillus substilis is a copper-dependent laccase[J].J Bacteriol.2001(183):5426-5430
16.Martins LO,Soares CM,Pereira MM et al.Molecular and biochemical characterization of a highly stable bacterial laccase that occurs as a structural component of the Bacillus subtilis endospore coat[J].J Biol.Chem.,2002(277):18849-18859
17.Van Waasbergen LG,Hildebrand M,Tebo BM.Identification and characterization of a gene cluster involved in manganese oxidation by spores of a marine Bacillus sp.strain SG-1[J].J Bacteriol.1996(178):3517-3530
18.张敏,肖亚中,龚为民.真菌漆酶的结构和功能[J].生物学杂志,2003,(5):6-8
19.王国栋,陈晓亚.漆酶的性质、功能、催化机理和应用[J].植物学通报,2003(20):69-75.
20.王海磊,李宗义.三种重要木质素降解酶研究进展[J].生物学杂志,2003(20):9-11
21.宋安东,王艳婕,王松江.漆酶的分子生物学研究及其应用[J].信阳师范学院学报,2004(17):244-248
22.万云洋,杜予民.漆酶结构与催化机理.化学结构在线预览版,http://www.hxtb.org
23.季立才,胡培植等.漆酶催化氧化反应研究进展[J].林产化学与工业,1997(1):79-84
24.Hakulinen N,Kiiskinen LL,Kruus K,Saloheimo M,Paananen A,Koivula A,Rouvinen J.Crystal structure of a laccase from Melanocarpus albomyces with an intact trinuclear copper site[J].Nat.Struct.Biol.,2002(8):601-605
25.Ducros V,Brzozowski AM,Wilson KS,Ostergaard P,Schneider P,Svendson A,Davies GJ.Structure of the laccase from Coprinus cinereus at 1.68 A resolution:evidence for different 'type 2 Cu-depleted' isoforms[J].Acta Crystallogr D Biol.Crystallogr.,2001(57):333-336.
26.Archibald FS.,Bourbonnais R.,Jurasek L.,Paice M.G.and Reid I.D..Kraft pulp bleaching and delignification by Trametes versicolor[J].J.Biotechnol.,1997(53):215-236.
27.Anastasia P.,Litvintseva,Joan M.Henson.Cloning,Characterization,and Transcription of Three Laccase Genes from Gaeumannomyces graminis var.tritici,the Take-All Fungus[J].Appl.Environ.Microbiol.,2002,68(3):1305-1311
28.Boghos Dedeyan,Agnieszka Klonowska,Simone Tagger,Thierry Tron,Gilles Iacazio,Gerard Gil,and Jean Le Petit.Biochemical and Molecular Characterization of a Laccase from Marasmius quercophilus[J].Appl.Environ.Microbiol.,2000(3):925-929
29.王光辉,季立才.漆树漆酶的催化氧化作用:3,5-二叔丁基-1,2-苯二酚的催化氧化[J].武汉大学学报(自科版),1989(4):106-108
30.Aktas N.,Cicek H et al.Reaction kinetics for laccase-catalyzed polymerization of 1-naphthol[J].Bioresour.Technol.,2001(1):29-36.
31.A.A.Leontievsky,N.M.Myasoedova,B.P.Baskunov,C.S.Evans,L.A.Golovleva.Transformation of 2,4,6- trichlorophenol by the white rot fungi Panus tigrinus and Coriolus Versicolor[J].Biodegradation,2000(11):331-340
32.季立才,胡培植,徐文中.漆树漆酶催化2,6-二甲基酚的氧化反应[J].高等学校化学学报,1992(7):940-944.
33.Tetsuya E,Hiroyuki U,Yoshiko T.,Takayuki.Degradation of Bisphenol A by purified Laccase from Trametes villosa[J].Biochem.Biophys.Res.Commun.,2001(284):704-706
34.Fahraeus G,Lijunggren H.Substrates specificity of a purified fungal laccase.Biochim Biophys.Acta,1961(46):22
35.季立才,王光辉,胡培植.漆树漆酶的新底物[J].1990(3):189-193
36.刘尚旭,赖寒.木质素降解酶的分子生物学研究进展[J].重庆教育学院学报,2001(3):64-67
37.Christopher F.The structure and function of fungal laccase[J].Microbiology,1994(1):19
38.Ducros,V Brzozowski AM,Wilson KS,et al.Crystal structure of the type-2 Cu depleted Laccase from coprinus cinereus at 2.2 A resolution.Nat.struct.Biol,1998(5):310-316
39.Selinheimo E,Kruus K,Buchert J,Hopia A,Autio K.Effects of laccase,xylanase and their combination on the rheological properties of wheat doughs.J Cereal Sci 2006(43):152-159.
40.Minussi RC,Pastore GM,Dura N.Potential applications of laccase in the food industry[J].Trends Food Sci.Technol.,2002(13):205-216.
41.Camarero S,Garcia O,Vidal T,Colom J,del Rio JC,Gutierrez A,et al.Efficient bleaching of non-wood high-quality paper pulp using laccase-mediator system[J].EnzymeMicrob.Technol.,2004(35):113-120.
42.Huttermann A,Mai C,Kharazipour.Modification of lignin for the production of new compounded materials[J].Appl.Microbiol.Biotechnol.,2001(55):387-394.
43.Riu J,Schon seeI,Barcelo D.Determination of sulfonated azo dyes in groundwater and industrial effluents by automated solid-phase extraction followed by capillary electrophoresis/mass spectrometry[J].J Mass.Spectrom.,1998(33):653-663.
44.宋美静.纸浆氯漂废水的处理[J].纤维素科学与技术,1999(2):22-25.
45.堵国成,赵政,陈坚.真菌漆酶的活性测定及其在织物染料生物脱色中的应用[J].江南大学学报,2003(1):83-90.
46.Roy JJ,Abraham TE,Abhijith KS,Sujith kumar PV,Thakur MS.Biosensor for the determination of phenols based on Cross-Linked Enzyme Crystals(CLEC)of laccase[J].Biosens.Bioelectron.,2005(21):206-211.
47.Nyanhongo GS,Rodriguez Couto,S,Gtibitz GM.Coupling of 2,4,6-trinitrotoluene(TNT)metabolites onto humic monomers by a new laccase from Trametes modesta[J].Chemosphere,2006(3):359-370
48.Mustafa R,Muniglia L,Rovel B,Girardin M.Phenolic colorants obtained by enzymatic synthesis using a fungal laccase in a hydroorganic biphasic system[J].Food Res.Int.,2005(38):995-1000.
49.Golz-Berner K.Walzel B.Zastrow L.Doucet O(04.03.2004).Cosmetic and dermatological preparation containing copperbinding proteins for skin lightening.Int Pat Appl WO2004017931.
50.Ishikawa Y,Esker T,Leder A.Chemical economics handbook:dyes.Menlo Park (CA):SRI Chemical and Health Business Services;2000.
51.Acuner,E.,Dilek,F.B.Treatment of tectilon yellow 2G by Chlorella vulgaris[J].Process Biochem.,2004(39):623-631.
52.Golka,K.,Kopps,S.,Myslak,Z.W.Carcinogenicity of azo colorants:influence of solubility and bioavailability[J].Toxicol.Lett.,2004(151):203-210.
53.Robinson T,McMullan G,Marchant R,Nigam P.Remediation of dyes in textile effluent:a critical review on current treatment technologies with a proposed alternative[J].Bioresour Technol.2001(77):247-255.
54.Willmott N,Guthrie J,Nelson G.The biotechnology approach to colour removal from textile effluent[J].J Soc.Dyers Colour.,1998(114):38-41.
55.Zissi U,Lyberatos G.Partial degradation of p-aminoazobenzene by a defined mixed culture of Bacillus subtilis and Stenotrophomonas maltophilia.Biotechnol.Bioeng.,2001(72):49-54.
56.Elthabeth R,Michael AP and Rafeel VD,Current Microbiology,1999.38.27-32
57.Michael AP,Rosa R,Pauel T et al.Appli.Environ,Microbiol.,1999,(9):3805-3809
58.Duran,N.et al.Applications of laccases and tyrosinases(phenoloxidases)immobilized on different supports:a review.Enzyme Microb.Technol.,2002(31):907-931
59.李冬生,康旭,杨红.漆酶固定化及其性质研究[J].广东食品工业科技,2004,20(2):6
60.H Xiao,J Huang,C LiuI,D Jiang.Immobilization of laccase on amine-terminated magnetic nano-composite by glutaraldehyde crosslinking method[J].Transactions of Nonferrous Metals Society of China,2006(16):414-418
61.C Mousty,L Vieille,S Cosnier.Laccase immobilization in redox active layered double hydroxides:A reagentless amperometric biosensor[J].Biosens.Bioelectron.,2007(8):1733-1738
62.Paolo Brandi,Alessandro D'Annibale,Carlo Galli,Patrizia Gentili' and Ana Sofia Nunes Pontes.In search for practical advantages from the immobilisation of an enzyme:the case of laccase[J].J.Mol.Catal.B:Enzym.,2006(1-3):61-69
63.王军涛,黄俊,刘诚,李斌.CoTAPc-Fe_3O_4纳米复合固定化漆酶研究[J].武汉理工大学学报,2007(1):1-3
64.史伯安.漆酚-(8-羟基)喹啉-Cu(Ⅱ)高分子配合物固定化漆树酶的研究[J].化学与生物工程,2005(6):15-17
65.Young L and Jian Yu.Ligninase-catalyzed decolorization synthetic dyes[J].Water Res.1997(31):1187-1193.
66.Bourbonnais,R.,Paice,M.G.Oxidation of non-phenolic substrates:an expanded role of laccase in lignin biodegradation[J].FEBS Lett.1990(267):99-102.
67.Call,H.P.,Mu"cke,I.History,overview and applications of mediated ligninolytic systems,especially laccase-mediator-systems(Lignozym process)[J].J.Biotechnol.1997(53):163-202.
68.王宜磊,邓郑旭.采绒革盖菌CV-8漆酶活性的初步研究[J].微生物学杂志.1998(4):60-62.
69.Ross IK.The role.of laccase in carpophore initiation in Coprinus congregatus[J].J Gen Microbiol,1982(128):2755-2762
70.Bradford M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J],Anal Biochem.,1976(72):248-254
71.胡景江,王俊明,张高伟,曹支敏.皱马鞍菌液体培养过程中胞外酶及还原糖的动态变化[J].西北农林科技大学学报(自然科学版),2005(11):94-98
72.Han MJ,Choi HT,Song HG.Purification and characterization of laccase from the white rot fungus Trametes versicolor[J].J Microbiol 2005(43):555-560
73.Wang HX,Ng TB.Purification of a laccase from fruiting bodies of the mushroom Pleurotus eryngii[J].Appl Microbiol Biotechnol,2006(69):521-525.
74.刘友勋,郜金荣,黄娟.白腐菌在两种固体基质上的漆酶产量及同工酶活性比较研究[J].氨基酸和生物资源,2007(3):33-35
75.W Liu,Y Chao,S Liu,et al.Molecular cloning and characterization of a laccase gene from the basidiomycete from lignosus and expression in Pichia pastoris[J].Appl.Microbiol.Biotechnol.,2003(2):174-181.
76.Y Xiao,X Tu,J Wang,et al.Purification,molecular characterization and reactivity aromatic compound of a laccase from basidomycete Trametes sp.Strain AH 28-2[J].Appl.Microbiol.Biotechnol.,2003(6):700-707.
77.康从宝,刘巧,李清心,等.白腐菌产漆酶的纯化及部分酶学性质[J].中国生物化学与分子生物学报,2002(5):638-642.
78.李杨,段新源,刘稳,等.杂色云芝组成型漆酶Ⅰ的纯化和底物专一性[J].中国生物化学与分子生物学报,2002(6):737-740.
79.秦小琼,傅庭治,曹幼琴,等.红栓菌胞外漆酶的诱导、纯化及部分性质研究[J].微生物学报,1996(5):360-366.
80.朱启忠等编著.生物化学技术指南.新疆科技卫生出版社.1995,新疆
81.陈钧辉等主编.生物化学实验(第三版).科学出版社,2005.7,北京
82.Jung H,Xu F,Li K.Purification and characterization of laccase from wood-degrading fungus Trichophyton rubrum LKY-7[J].Enzyme Microb.Technol.,2002(30):161-168
83.Jaouani A,Guillen F,Penninckx M J,Martinez AT,Martinez MJ.Role of Pycnoporus coccineus laccase in the degradation of aromatic compounds in olive oil mill wastewater[J].Enzyme.Microb.Technol.,2005(36):478-486
84.Iyer G,Chattoo B.Purification and characterization of laccase from the dee blast fungus,Magnaporthe grisea[J].FEMS Microbiol.Lett.,2003(227):121-126.
85.Baldrian P.Purification and characterization of laccase from the white-rot fungus Daedalea quercina and decolorization of synthetic dyes by the enzyme[J].Appl.Microbiol.Biotechnol.,2004(63):560-563
86.Nagai M,Sato T,Watanabe H,Saito K,Kawata M,Enei H.Purification and characterization of an extracellular laccase from the edible mushroom Lentinula edodes,and decolorization of chemically different dyes[J].Appl.Microbiol.Biotechnol.,2002(60):327-335
87.Eggert C,Temp U,Eriksson K-EL.The lignolytic system of the white rot fungus Pycnoporus cinnabarinus:Purification and characterization of the laccase.Appl.Environ.Microbiol.,1996(62):1151-1158
88.Vasdev K,Dhawan S,Kapoor RK,Kuhad RC.Biochemical characterization and molecular evidence of a laccase from the bird's nest fungus Cyathus bulleri[J].Fungal Genet.Biol.,2005(42):684-693
89.Zhang H,Hong YZ,Xiao YZ,Yuan J,Tu XM,Zhang XQ.Efficient production of laccases by Trametes sp.AH28-2 in cocultivation with a Trichoderma strain[J].Appl.Microbiol.Biotechnol.,2006(73):89-94
90.Meyer U.Biodegradation of synthetic organic colorants[J].FEMS Symp.1981(12):371-385.
91.Holme,I.Ecological Aspects of Color Chemistry:Developments in the Chemistry and Technology of Organic Dyes,edited by J.Griffiths,1984:111-128.Oxford:Society for Chemical Industry.
92.Robinson T,McMullan G,Marchant R,Nigam P.Remediation of dyes in textile effluent:a critical review on current treatment technologies with a proposed alternative[J].Bioresour Technol,2001(77):247-255.
93.Piontek,K.et al.Crystal structure of a laccase from the fungus Trametes versicolor at 1.90A resolution containing a full complement of coppers[J].J.Biol.Chem.2002(277):37663-37669
94.Claus,H.Laccases:structure,reactions,distribution[J].Micron.,2004(35):93-96
95.龙胜亚,肖海燕,周菊英,黄俊等.磁性壳聚糖微球的制备及其用作漆酶固定化载体[J].应用化学,2005(22):1122-1126
96.陈騊声,居乃琥,陈石根.固定化酶理论与应用[M].北京:轻工业出版社,1987
97.陈万成,蒋笃孝,宋龄瑛,欧仕益.壳聚糖/海藻酸钠微胶囊固定化磷脂酶A_1(PLA_1)的研究[J],食品科技.2004(11):14-17.
98.蒋挺大.甲壳素[M].北京:化学工业出版社,2003
99.孟颢华.沪酿3.042米曲霉的纯化复壮[J].中国调味品,2001(7):7-9.
100.何青,向洋.米曲霉酶活力与孢子特征关系的研究[J].中国酿造,1993(5):33-37.
101.阚建全.食品化学[M].北京:科学出版社,2002.
102.Ida Genta,Monica Costantini,Annalia Asti,Bice Conti,Luisa Montanari.Influence of glutaraldehyde on drug release and mucoadhesive properties of chitosan microspheres[J].Carbohydr.Polym.,1998(36):81-88
103.Wu FJ,Flach CR,Seaton BA,et al.Stability of annexin Ⅴ in ternary complexes with Ca~(2+)and anoinic phospholipids:IR studies of monolayer and buik phases[J].Biochem.,1999,38(2):792.
104.朱启忠,赵亮云,赵宏,韩晓弟.壳聚糖对酸性染料的吸附性能[J].资源开发与市场2006(2):101-102