摘要
漆酶是一种铜多酚氧化酶,主要存在于白腐菌中,是一种分泌至胞外的糖蛋白。漆酶可以降解木质素和氧化酚类、芳胺类化合物,有着广泛的用途。鸡腿菇是一种能高效分泌漆酶的白腐菌。
本论文首先研究了鸡腿菇在25℃、四种不同培养基中进行液体静置培养时,漆酶活力的比较。研究表明A培养基(碳源为葡萄糖,氮源为酵母浸膏)的鸡腿菇漆酶活性最高。后续实验研究了不同的金属离子和芳香族化合物对漆酶产酶的影响。结果显示添加浓度为0.8mmol/L的锰离子和3.0mmol/L的咖啡酸对鸡腿菇漆酶的诱导作用最明显。其它的化学试剂分别在不同的浓度表现出诱导作用。其中在低浓度表现出最佳诱导作用的是4-甲氧基肉桂酸、茴香酸、3,4,5-三甲氧基肉桂酸、愈创木酚、3,5-二甲氧基-4-羟基肉桂酸、香兰素、3-(4-甲氧苯基)-丙酸。而Cu~(2+)、3,4-二甲氧基苯甲醛、肉桂酸、4-羟基肉桂酸、3-(4-羟基苯基)-丙酸则在高浓度时诱导效果最好。对碳源和氮源的研究表明最佳碳源是葡萄糖,最佳氮源是蛋白胨。通过不连续聚丙酰烯酰胺凝胶电泳分析发现,不同种类、浓度的化学试剂会诱导产生不同的鸡腿菇漆酶的同工酶,由于这些同工酶具有不同的等电点所以电泳中表现出不同的谱带。漆酶最适反应pH值和温度为6.0和60℃。
本论文实验还从鸡腿菇中提取mRNA,反转录得到cDNA序列,再根据真菌漆酶蛋白质家族的保守氨基酸结构域设计一系列简并性引物,进行PCR扩增得到143bp的鸡腿菇漆酶特异性基因片段。再利用BLAST程序将该序列与NCBI数据库中的漆酶基因进行比对,证明与其它漆酶基因高度同源。再通过RACE(cDNA末端快速扩增技术)得到鸡腿菇漆酶5’端的cDNA序列,共626bp。
Laccase is a kind of copper–contained polyphenol oxidase produced mainly by white rot fungi and excreted into media in form of glycoprotein. It can degrade lignin and catalyze the oxidation of a broad number of phenolic compounds and aromatic amines. Laccase is applied in many areas. Coprinus comatus,a sort of white rot fungi produces laccase efficiently.
In this dissertation, laccase production of Coprinus comatus in four different media was studied under liquid culture at the temperature of 25℃. It was shown that the best medium was medium A which contained the glucose and yeast extract as carbon and nitrogen source respectively. The relation between the laccase production of Coprinus comatus and different metal ions and aromatic compounds was also discussed. It was shown the production of the laccase can be mostly induced by the addition of Mn2+ at concentration of 0.8 mmol/L and caffeic acid at the concentration of 3.0mmol/L. Other chemical agents contributed to the increase of the enzyme production most at different concentration. 4-methoxycinnamic acid, anisic acid, 3,4,5-trimethoxy-cinnamic acid, guaiacol, 3,5-dimethoxy-4-hydoxycinnamic acid, vanillin and 3-(4-methoxyphenyl)propionic acid induced the enzyme production most at low concentration, however, Cu2+, 3,4-dimethoxybenzaldehyde, cinnamic acid,4-hydroxycinnamic acid, and 3-(4-hydroxyphenyl)propionic acid did at high concentration. The result also showed that glucose was the best carbon source and peptone was the best nitrogen source. From the discontinuous polyacrylamide gel electrophoresis, we found different compounds at diverse concentration induced many laccase isoenzymes of Coprinus comatus displayed different bands with various pI. The optimum pH value and temperature of reaction of laccase was 6.0 and 60℃.
In this paper, extraction of total RNA from Coprinus comatus and RT technology was used to gain the cDNA sequences. A series of degenerate oligonucleotide primers were designed , coding for conservative amino acid regions in fungi laccase protein family. A PCR amplification was performed on Coprinus comatus cDNA template and a specific fragment of 143 bp was obtained. By using the program of BLAST on NCBI GenBank database, the sequence presented a high match with the laccase gene from other fungi. The cDNA sequences of 5′ends were isolated by using RACE(rapid amplification of cDNA ends) technique from Coprinus comatus.The length of sequence was 626bp.
引文
1.王金南.发展生态工业是解决工业污染的重要途径.中国环境报,2001,。
2.冀珍芳, 石淑兰.木质素的微生物降解.广西轻工业,2002,12(1): 4-5。
3.Pirkko Karhunen, Petteri Rummakko, Jussi Sipil? and G?sta Brunow., Dibenzodioxocins: A novel type of linkage in softwood lignins. Tetrahedron Lett. 1995, 36: 4501-4504.
4.张甲耀, 罗宇煊.木质素降解酶及其调控机理研究的进展.上海环境科学,1998,11: 45-48。
5.Kirk T.K, Farrell R.L.Enzymatic “combustion”:the microbial degradation of lignin. Ann. Rev. Microbiol,1987,41: 465-505.
6.黄清荣,卜庆梅, 缪静.鸡腿菇高产新技术研究.中国食用菌, 2001,04(15):63-68。
6.Yaropolov A.I,Skorobogatko O.V and Vartanov S.et al.Laccase: properties, catalytic mechanism and applicability,Applied Biochemistry and Biotechnology, 1994,49: 257.
7.肖亚中,张敏,龚为民.真菌漆酶的结构与功能.微生物学报,2000,04(6):33-38
8.卢雪梅,刘紫鹃,高培基.木质素生物降解的化学反应机制.林产化学与工业, 1996,02:13-18。
9.Rayner A.D.M. and Boddy L, Fungal decomposition of wood.FEMS.Microbio.Lett, 1988,56:587.
10.Andrzej Lionowicz, Anna Matuszewska, Jolanta Luterek and Dirk Ziegenhagen.Biodegradation of lignin by White Rot Fungi, Fungal Genetics and Biology,1999,27: 175-185.
11.段新源,卢雪梅,王 蔚,高培基.黄孢原毛平革菌合成漆酶能力的研究.生物工程学报, 2002,37(1):87-91
12.Eriksson K.E.L., Blanchette R.A. and Ander P.Microbial and enzymatic degradation of wood and wood components. Springer-Verlag, Berlin, Germany, 1990,16:407.
13.Dey S., Maiti T.K. and Bhattacharyya B.C.Production of some extracellular enzymes by a lignin peroxidase-producing brown rot fungus, Polyporus ostreiformis, and its comparative abilities for lignin degradation and dye decolorization. Appl. Environ.Microbiol,1994,60: 4216-4218.
14.Blanchette R.A., Cease K.R. and Abad A.R.An evaluation of different forms of deterioration found in archaeological wood. Int. Biodeter,1991,28:3-22.
15.Robert A Blanchette.A review of microbial deterioration found in archaeological wood from different environments.International Biodeterioration & Biodegradation,2000,46(3): 189-204.
16.Marie Baucher, Claire Halpin, Michel Petit-Conil and Wout Boerjan.Lignin: Genetic Engineering and Impact on Pulping. Biochemistry and Molecular Biology, 2003,38(4):305-350.
17.吴坤,张世敏,朱显峰.木质素生物降解研究进展.河南农业大学学报,2000,34(4):349-354.
18.王佳玲,余惠生.付时雨白腐菌漆酶的研究进展. 微生物学通报,1998,25(4):233-235.
19.Harald Willershausen, Alexander J?ger and Helmut Graf.Ligninase production of Phanerochaete chrysosporium by immobilization in bioreactors. Biotechnology, 1987,6(3): 239-243
20.Young L. and Jian Yu.Ligninase-catalyzed decolorization synthetic dyes. Water Res,1997,5(31):1187-1193.
21. Francisco Guillen , María Jesus Martínez , Carmen Munoz and Angel T. Martínez. Quinone Redox.Cycling in the Ligninolytic FungusPleurotus eryngiiLeading to Extracellular Production of Superoxide Anion Radical . Archives of Biochemistry and Biophysics,1997,339(1):190-199.
22. Yoshi H.Chemistry of Lacquer(Urusht)part1.J Chem Soc,1983,43:472-486
23.Thurston CF.The structure and function of fungal laccase Microbiology , 1994,140,19-26
24.GivaudanA,EffosseA,FaureD,et al.Polyphenoloxidase from Azospirillum lipoferum isolated from the rhizosphere:evidence for aLaccase in non-motile strains of Azospirillum lipoferum.FEMS Microbiol.Lett, 1993,108,205-210.
25.Claudiae.The ligninolytic system of the white rot fungus Pycnoporuscinnabar- -inus.Purification and characterization of the laccase[J]ApplEnviron Microbiol,1996,62:920-924
26.Palmierig.Copper induction of laccase isoenzymes in the lignin olytic fungus Pleurotus ostreatus[J].ApplEnviron Microbiol,1999,66:920-924.
27.李光日,余惠生,付时雨.漆酶催化活性中心结构及其应用的研究进展,纤维素科学与技术,2000,8(2):42-47.
28.季立才,胡培植等.漆酶催化氧化反应研究进展.林产化学与工业,1997,17(1): 79-84
29.Anastasiap,Litvintseva,Joanm,etal.Cloning,characterization,and transcript- -tion of three laccase genes Gaeumannomycesgraminisvar from tritici,the take all fungus [J]. Appl Envir Microbiol,2002,68(3):1305-1311.
30.Dedeyanb,Klonowskaa,Taggers,etal.Biochemical and molecular characterization of a laccase from Marasmius quercophilus[J].ApplEnvironMicrobiol, 2000,66(3):925-929.
31.钞亚鹏,钱世均.真菌漆酶及应用.生物工程进展,2001,21(5):23-28
32.宋安东,王艳婕, 王松江. 漆酶的分子生物学研究及其应用. 信阳师范学院学报,2004(17)244-248
33. 张敏,肖亚中.白腐真菌AH28-2菌株发酵合成漆酶的初步研究,微生物学通报2001,03(6):43-48。
34.胡平平,付时雨.漆酶催化活性中心结构及其特征研究进展[J].林产化学与工业,2001,21(3):69-75
35.CHEFDTZB,BENNY,YONAC,etal.Purification and characterization of laccase from chaetomium thermophiolium and its role in humification [J].Appl Environ Micro, 1998, 64 (9):3175-3179
36. V. Ducros, A.M. Brzozowski, K.S.Wilson, P. Ostergaard, P. Schneider, A. Svendson and G. J. Davies.the structure of laccase from Coprinus cinereus at 1.6A resolution: evidence for different type 2 Cu-depleted isoforms. Acta Crystallogr, 2001,57: 333~336.
37.MesserschmidtA.etal.Mol.Biol, 1992,224,179-205.
38.ColeAP,RootDE,MukheijeeP,etal.Science, 1996,273,1848-1850.
39.FukushimaY,KirkTK.Laccase component of the Ceriporiopsis subvermisp or a lignin-degrading system.Applied and Environmental Microbiology,1995,61:872-876
40. Wood DA.Production,purification and properties of extracellular laccase of Agaricus bisporu.Journal of General Microbiology,1980,117:327-338
41. 阎宏涛,邓延倬,曾云鹗.漆酶、铜蓝蛋白的脉冲激光光声法测定[J].生物化学与生物物理进展,1989,16(4):306-309
42.缪 静,姜竹茂.漆酶的最新研究进展, 烟台师范学院学报, 2001,17(2):146-150
43.丁少军.云芝漆酶的培养和分离纯化的研究,纤维素科学与技术, 1998,6(3):16-20.
44. Clemmer J D.On-line enzyme activity determination using the stopped-flow technique: to laccase activity in pulp mill waster treatment[J]Applied Microbiology and Biotechology, 1997,48(2):307
45.望天志,李卫莲,万洪文.微量热法测定漆酶的活性[J].自然杂志, 1997,19(6):361
46.王宜磊,杨道彩,云芝胞外漆酶活性研究, 菏泽师专学报, 1998,20(4):57-59
47.朱显峰,杨生玉,沈永红.漆酶检测方法的对比研究,河南大学学报, 2002,32(3):41-43
48.王宜磊,刘兴坦.彩绒革盖菌漆酶产酶条件的初步研究,微生物学杂志, 1999,12(8): 51-56。
49.John A.Buswell,YiJin Cai,Shu-ting Chang.Effect of nutrient nitrogen and manganese on manganese peroxidase and laccase production by Lentinula (Lentinus) edodes ,FEMS Microbiology Letters, 1995,128(12):81-88.
50. 吴涓,肖亚中.分光光度法测定蜜环菌胞外漆酶,厦门大学学报, 2000,04(6): 13-18。
51.兰瑞芳,林少琴.杏鲍菇漆酶的生物学特性,食用菌学报, 2002,02(12):37-42。
52. 朱启忠.毛栓菌胞外漆酶活初步研究性,微生物学杂志,2000,01(10):43-49
53. Shicheng Chen, Dengbo Ma,Wei Ge and John A.Buswell.Induction of laccase activitity in the edible straq mushroom, Volvariella volvacea, FEMS Microbiology Letters, 2003,218:143-148.
54.Laura-Leena Kiiskinen,Hetti Palonen and Markus Linder.Laccase from Melanocarpus albomyces binds effectively to cellulose, FEMS Microbiology Letters, 2004,576:251-255.
55.秦小琼,陈嘉翔,于家鸾.红栓菌的漆酶研究,中国造纸学报,1997,12(3):48-51
56. 尹艳丽 , 张晓昱 . 不同摇瓶条件对侧耳菌生长及漆酶分泌的影响 , 生物技术 , 2004,14(5):72-76
57.Hublik G, Schinner F.Characterization and immobilization of the laccase from Pleurotus ostreatrtus and its use for the continuouse limination of phenolic pollutants[J].Enzyme and Microbiol Techol, 2000,27:330-336
58.陈石良,谷文英,陶文沂,液体培养灰树花碳源营养的研究,食用菌, 1999,21(6):3-5.
59.赵华,齐刚,代彦,白腐菌液体深层培养条件的研究,生物技术, 2003,13(6):21-24.
60.肖亚中, 张敏,王芳.野生革耳菌组成型漆酶的生成条件及性质研究 ,食用菌,2000,08:67-72。
61.Eggert C, Erikson K,The lignin olytic system of the white rot fungus cnoporus cinnabarinus,purification and chacterization of the laccase,Appl Environ Microbiol, 1996,62:1151-1158
62. 张敏.蜜环菌胞外漆酶的合成、纯化及性质研究, 生物工程学报,2006,18(4):457-462
63.Hou Hongman,Zhou Jiti,WangJing.Enhancement of laccaseproduction by Pleurotus ostreatus and its use for the decoloriza-tion of anthraquinone dye,ProcessBiochemistry , 2004,39:1415-1419
64.刘尚旭,董佳里,张义正.糙皮侧耳Ax3产漆酶条件及部分酶学性质研究, 四川大学学报 2004,41(1):160-163
65.王宜磊,毕红卫.多孔菌漆酶性质研究,淄博学院学报, 2002,4(4):12-14
66.康从宝,刘巧,李清心.白腐菌产漆酶的纯化及部分酶学性质,中国生物化学与分子生物学报, 2002,15(8):638-642
67.Dedeyan B,Klonowska A,Tagger S,et al.Biochemical and molecular characterization of a laccase from Marasmius quarcophilus[J],Appl Environ.Microbiol, 2000,66(3):925-929
68.叶汉玲,王传槐, .John A. Buswell.新鲜香菇子实体漆酶的纯化与性质[J],南京林业大学学报, 1997,21(2):50-53
69.刘淑珍, 钱世钧.真菌漆酶及其应用,生物工程进展, 2001,21(5):23-28
70.Nina H, Laura-Leena K, Kristiina K, et al(2002): Crystal structure of a laccase from Melanocarpus albomyces with an inact trinuclear copper site, Nat Struct Biol,9(8):601-605
71.Palmieri G,Giardina P,Bianaco C,et al(1997): A novel white laccase from Pleurotus ostreatrtus, J Biol Chem,272(50):1301-1307
72.Shin KS, Lee YJ.Purification and characterization of a new member of the laccase family from the white-rot basidiomycete Coriolus hirsutus,Arch. Biochem Biophys, 2000,384:109-115
73.Bogdanovskaya VA,Kuznetsova LN, Tarasevich MR,Bioelectrocatalytic and enzymic acyivity of laccase in water-ethanol solutions, Russian Journal of Electrochemistry, 2002,38(10):1192-1200
74.FrohmanMA,DushMK and Martin GR.Rapid production of full-length .cDNAs from rare transcripts Amplificationusingasinglegene-specific. oligonucleotide primer. Proc Natl Acad Sci USA,1988,85:8998-9002.
75.CULLEN D. Recent advances on the molecular genetic fugi[J].J Biotechnol,1997,53:273-289
76.YAVERD,OVERJEROM,XUF,et al Molecular characterization of laccase gene from the Basidiomycete cinereus and heterologous express of the laccase lcc1[J]Appl EnironMicrobiol,1999,65(11):4943-4948
77.BERKA R,SCHNEDER P,GOLIGHTLY E,et al.characterization of the gene encoding on extracellar laccase of Mecsliopthora thermophila and analysis of the recombinant enzyme expressed in Aspergillus oryzae[J].Appl Eniron Microbiol,1997,63(8): 3151-3157
78.王佳玲.黄孢原毛平革菌漆酶的研究进展[J].微生物学通报,1998,25(4):233-235
79.COLL P M,TABERNEROC SANTAMARIA R,et al.characterization and structural analysis of the laccase I gene from the newly isolated lignin Olytic basidiomycete PM1[J]Appl Environ Microbiol,1993,59(12):4129-4135
80.ANASTASIA P, LITVINTSEVA, JOAN M,et al cloning characterization and transcription of three laccase genes from Gaeumannomyces graminis var tritici the take-all fungus[J].Appl Envir Microbiol,2002,68(3):1305-1311
81.D′SOUZA TM,BOOMINA THAN K,REDDYCA,Isolation of laccase gene-specific sequences from white rot and brown rot fugi byPCR[J].Appl Environ Microbiol,1996,62(10):3739-3744
82.赵敏,杨谦,宋小双.真菌漆酶分子生物学研究进展. 林产化学与工业,2005,25(1): 115-120
83.HUNOLSTEIN C V, VALENTIP, VISCA P,et al.production of laccase A and B by amutant syrain of Trametes vesicolor[J].J Gen Appl Microbiol,1986,32(1):185-191
84. CULLEN D.Recent advances on the molecular genetic fungi[J].J Biotechol,1997, 53:273-289
85.BERKA R SCHNEDER P,GOLIGHTLY E,et al.characterization of the gene encoding on extracellar laccase of Mecsliop thora thermophila and analysis of recombinant enzyme expressed in Aspergrillus oryzae[J].Appl Environ Microbiol,1997,63(8):3151-3157
86. GhindilisAL,GavrilovaVP,andYoropolovAI,BiosensorsandBioelctrics 1992,7:127-131.
87. M Smith,Thurston CF,and Wood DA,inMultiple-copperOxidaes. 1996
88. Szeponik J,Moller B,Pfeiffer D etal.Biosensors and Bioelectric,1997,12(9-10):947-952.
89. C. G. Bauer, A. Kuehn, N. Gajovic, O. Skorobogatko, P. J. Holt, N. C. Bruce, A. Makower, C. R. Lowe and F. W.Scheller.New enzyme sensors for morphine and codeine base on morphine dehydrogenase and laccase. FreseniusJ. Anal. Chem,1999,364: 179-183.
90.季立才,胡培植.漆酶的结构、功能及其应用,氨基酸和生物资源,1996,18(1):25-29
91.许颖,兰进,真菌漆酶的研究进展,食用菌学报, 2005,12(1):57-64
92.杨梅, 邓象斌,鸡腿菇菌丝深层培养研究,食用菌,2001,3:29-34。
93.王宜磊,刘兴坦.鸡腿菇漆酶产酶条件研究,山东菏泽师范专科学校学报,2000,8: 23-28。
94.肖亚中,王军,王怡平.蜜环菌胞外漆酶的合成、纯化及性质研究,生物工程学报,2004,18(4):457-462
95.王宜磊.不同真菌漆酶的性质研究.生物技术,2003,23(2):89-94
96.王佳玲,余惠生,黄秀渝. 碳源和 Mn2+对白腐菌 Panus conchatus 产木素降解酶的影响,纤维素科学与技术,1998,6(3):27-31
97付时雨.周攀登.真菌漆酶及其催化对苯基苯酚聚合条件的研究.化学通报,2005,3:226-229
98.韩晓磊.严莲荷.漆酶活性及其活性影响因素综述.生物与化学工程,2005,7:10-13
99.赵敏,钱程.白腐菌木素氧化酶系的检测及其漆酶诱导产生的研究.中国造纸学报,2005,20(2):101-105
100.S. Chen, W. Ge, and JA Buswell Biochemical and molecular characterization of a laccase from the edible straw mushroom, Volvariella volvacea FEBS J., January 15, 2004; 271(2): 318 - 328.