Trametes sp. SYBC-L3固态发酵产漆酶及其分离纯化和特性的研究
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摘要
漆酶(Laccase, EC 1.10.3.2)是一种多酚氧化酶,可以结合多个铜离子,能催化多酚类、芳胺类、羧酸类、生物色素等的氧化,也具有降解木质素的功能。因此,研究其酶的分离、纯化、酶学性质具有一定的理论和现实意义。
论文研究了菌株Trametes sp. SYBC-L3固态发酵产漆酶的培养基组成和发酵条件;探讨了漆酶分离纯化方法;分析了漆酶的酶学特性;研究了漆酶在几种染料脱色中的作用;得到如下主要结果:
(1)最适固态培养基组成为:豆粕和木屑的比例为1 : 1,麦芽糖浓度为1.0 g/L,NaNO3浓度为0.71 mol/L,Cu2+浓度为0.3 g/L,含水量为65%。最适装样量为40 g/250 mL三角瓶,30℃培养10 d。在最优培养基和培养条件下,酶活最高达91.06 U/g干曲,是优化前(10.37 U/g干曲)的8.78倍。
(2)通过硫酸铵分级沉淀、DEAE-cellulose-52离子交换层析、Sephadex G-100凝胶过滤层析三步分离纯化得到电泳纯的漆酶(Lac-T),纯化倍数19.58倍,回收率为18.54%。通过SDS-PAGE实验得出其分子量约为58.7 kDa。以DMP(2, 6-二甲氧基酚)为底物时,在最适温度和最适pH的条件下测得Lac-T的Km为1.32 mmol/L,Vmax为1742.76 U/mg。
(3)纯化后的漆酶(Lac-T)最适温度为65℃,在40℃保温1 h仍能保持80%以上的活力,温度稳定性较好;最适pH为3.5,在pH 4.0~9.0的范围内于30℃保温24 h仍能保持80%以上的活力;SO42-、Mg 2+、Cu2+对Lac-T有促进作用;CO32-、K+对Lac-T有轻微的抑制作用,Fe2+的抑制作用较明显。NO3-、Cl-、PO43-、Na+基本不影响Lac-T活力。
(4)初步研究了Trametes sp. SYBC-L3固态发酵产漆酶在偶氮染料和蒽醌染料脱色中的作用,用漆酶处理偶氮染料媒介红5G,40 min脱色率达到88%,处理蒽醌类染料弱酸蓝25和酸性蓝129,40 min脱色率分别达到76%和75%,有较好的脱色效果,显示了其在毛纺染料脱色中的潜力。
Laccase (EC 1.10.3.2), a type of polyphenol oxidases, can combine a number of copper ions. It can oxidize a wide range of substrates, such as polyphenols, carboxylic acids, biological pigments and several aromatic compounds. It also has the ability to degrade lignin. The study on laccase has a certain degree of theoretical and practical significance.
Laccase produced by Trametes sp. SYBC-L3 was studied under solid-state fermentation with different medial and fermenting conditions. Moreover, the purification, characterization and decolorizing role in several dyes of laccase were also investigated. The results were as follows:
(1) The optimal solid-state fermentation medium was soybean meal and sawdust with a ratio of 1 : 1, maltose 1.5 g/L, NaNO3 0.71 mol/L, Cu2+ 0.3 g/L, initial medium moisture 65%. The optimized loaded sample on solid-state fermentation condition was 40 g solid-state medium in 250 mL flask, 30℃,incubated for 10 d in dark. The laccase activity reached 91.06 U/g dry substrate with about 8.78 folds of the original fermenting level (10.37 U/g dry substrate).
(2) The laccase produced by strain Trametes sp. SYBC-L3 was purified via (NH4)2SO4 fractionation, DEAE-cellulose-52 ion exchange chromatography, and gel filtration with Sephadex G-100 column. Its purity and yield were 19.58 folds and 18.54% of the crude extract, respectively. The molecular mass of the purified laccase (Lac-T) was about 58.7 kDa on SDS-PAGE gel. The Km of Lac-T was 1.32 mmol/L, its Vmax was 1742.76 U/mg protein.
(3) The optimal catalytic temperature of laccase (Lac-T) was 65℃, the optimal pH was 3.5. Lac-T was stable under different pH and with relative activity of about 80% at the pH range between 4.0 and 9.0, which indicated that it adapted to a wide range of pH. Lac-T could be activated by SO42-, Mg2+ and Cu2+, and inhibited by CO32- and K+, more significant inhibited by Fe2+. NO3-, Cl-, PO43-, Na+ didn’t affect the activity of Lac-T in a certain extend.
(4) The laccase produced by Trametes sp. SYBC-L3 could degrade textile dyes. Eighty eight percent of decolorization was achieved with laccase treatment on Mordant Red 5G, 76% on Weak Acid Blue 25 and 75% on Acid Blue 129 for 40 min, respectively, which indicating that the laccase had the potential ability for decolorization.
论文研究了菌株Trametes sp. SYBC-L3固态发酵产漆酶的培养基组成和发酵条件;探讨了漆酶分离纯化方法;分析了漆酶的酶学特性;研究了漆酶在几种染料脱色中的作用;得到如下主要结果:
(1)最适固态培养基组成为:豆粕和木屑的比例为1 : 1,麦芽糖浓度为1.0 g/L,NaNO3浓度为0.71 mol/L,Cu2+浓度为0.3 g/L,含水量为65%。最适装样量为40 g/250 mL三角瓶,30℃培养10 d。在最优培养基和培养条件下,酶活最高达91.06 U/g干曲,是优化前(10.37 U/g干曲)的8.78倍。
(2)通过硫酸铵分级沉淀、DEAE-cellulose-52离子交换层析、Sephadex G-100凝胶过滤层析三步分离纯化得到电泳纯的漆酶(Lac-T),纯化倍数19.58倍,回收率为18.54%。通过SDS-PAGE实验得出其分子量约为58.7 kDa。以DMP(2, 6-二甲氧基酚)为底物时,在最适温度和最适pH的条件下测得Lac-T的Km为1.32 mmol/L,Vmax为1742.76 U/mg。
(3)纯化后的漆酶(Lac-T)最适温度为65℃,在40℃保温1 h仍能保持80%以上的活力,温度稳定性较好;最适pH为3.5,在pH 4.0~9.0的范围内于30℃保温24 h仍能保持80%以上的活力;SO42-、Mg 2+、Cu2+对Lac-T有促进作用;CO32-、K+对Lac-T有轻微的抑制作用,Fe2+的抑制作用较明显。NO3-、Cl-、PO43-、Na+基本不影响Lac-T活力。
(4)初步研究了Trametes sp. SYBC-L3固态发酵产漆酶在偶氮染料和蒽醌染料脱色中的作用,用漆酶处理偶氮染料媒介红5G,40 min脱色率达到88%,处理蒽醌类染料弱酸蓝25和酸性蓝129,40 min脱色率分别达到76%和75%,有较好的脱色效果,显示了其在毛纺染料脱色中的潜力。
Laccase (EC 1.10.3.2), a type of polyphenol oxidases, can combine a number of copper ions. It can oxidize a wide range of substrates, such as polyphenols, carboxylic acids, biological pigments and several aromatic compounds. It also has the ability to degrade lignin. The study on laccase has a certain degree of theoretical and practical significance.
Laccase produced by Trametes sp. SYBC-L3 was studied under solid-state fermentation with different medial and fermenting conditions. Moreover, the purification, characterization and decolorizing role in several dyes of laccase were also investigated. The results were as follows:
(1) The optimal solid-state fermentation medium was soybean meal and sawdust with a ratio of 1 : 1, maltose 1.5 g/L, NaNO3 0.71 mol/L, Cu2+ 0.3 g/L, initial medium moisture 65%. The optimized loaded sample on solid-state fermentation condition was 40 g solid-state medium in 250 mL flask, 30℃,incubated for 10 d in dark. The laccase activity reached 91.06 U/g dry substrate with about 8.78 folds of the original fermenting level (10.37 U/g dry substrate).
(2) The laccase produced by strain Trametes sp. SYBC-L3 was purified via (NH4)2SO4 fractionation, DEAE-cellulose-52 ion exchange chromatography, and gel filtration with Sephadex G-100 column. Its purity and yield were 19.58 folds and 18.54% of the crude extract, respectively. The molecular mass of the purified laccase (Lac-T) was about 58.7 kDa on SDS-PAGE gel. The Km of Lac-T was 1.32 mmol/L, its Vmax was 1742.76 U/mg protein.
(3) The optimal catalytic temperature of laccase (Lac-T) was 65℃, the optimal pH was 3.5. Lac-T was stable under different pH and with relative activity of about 80% at the pH range between 4.0 and 9.0, which indicated that it adapted to a wide range of pH. Lac-T could be activated by SO42-, Mg2+ and Cu2+, and inhibited by CO32- and K+, more significant inhibited by Fe2+. NO3-, Cl-, PO43-, Na+ didn’t affect the activity of Lac-T in a certain extend.
(4) The laccase produced by Trametes sp. SYBC-L3 could degrade textile dyes. Eighty eight percent of decolorization was achieved with laccase treatment on Mordant Red 5G, 76% on Weak Acid Blue 25 and 75% on Acid Blue 129 for 40 min, respectively, which indicating that the laccase had the potential ability for decolorization.
引文
1. Karhuen E, Niku-Paavola M L. A novel combination of prosthetic groups in a fungal laccase; PQQ and two copper atoms [J]. FEBS Letters, 1990, 267(1): 6-8.
2.施晓燕. Phellinus sp. SYBC-L2固态发酵产漆酶条件的优化及其酶学性质的研究[D]: [硕士学位论文].无锡:江南大学生物工程学院. 2007.
3. Sakurai T, Suzuki S. Versicolorectrosopy of cucumber ascorbate oxidase and fungal laccase [C]. In: Messerschmidt. A Multi-copper Oxidases. Singapore: World Sciencitific Press, 1997, 225-250.
4. Yoshinori N. Purification and characteristization of laccase from white rot fungus trametes sanguine M85-2 [J]. Ferment Bioeng, 1995, 80(1): 91-93
5. Chefdtzb B, Chen Y. Purification and characterization of laccase from Chaetomium thermophiolium and its role in humification [J]. Applenviron Micro, 1998, 64(9): 3175-3179.
6. Christopher F. The structure and function of fungal laccase [J]. Microbiology, 1994, 140(1): 19-24.
7.励建荣,李丹.漆酶在食品工业中的应用[J].现代食品科技2006, 22(4): 262-268.
8.堵国成,赵政,陈坚.真菌漆酶的酶活测定及其在织物染料生物脱色中的应用[J].江南大学学报(自然科学版), 2003, 2 (1): 83-85, 90.
9. Szklarz G D, Antibus R K. Production of phenol oxidases and peroxidases by wood-rotting fungi[J]. Mycologia, 1989, 81(2): 234-240.
10. Reinhammar B. Copper proteins and copper enzymes (Ⅲ) [M]. Lontie R Ed: Boca CRC Press, 1984.
11.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(5): 9-11, 33.
12.张力,韩大勇,邵喜霞.白腐真菌木质素降解酶系研究进展[J].中国牧业通讯, 2008(23): 6-8, 45.
13.陈利萍,陈竹君,董伟敏.日本茄F1离体快速繁殖[J ].植物生理学通讯, 1996 , 32 (1) , 26
14.王首锋,梁海曼.升汞和次氯酸钠对黄瓜种子萌发及幼苗生长的影响[J].植物生理学通讯, 1996, 32 (2) , 117-120.
15. Kojima Y, Tsukada Y, Kawai Y, et al. Cloning, sequence analysis, and expression of ligninolytic phenoloxidase genes of the white-rot basidiomycete Coriolus hirsutus[J]. Biol. Chem, 1990, 265: 15223-15230.
16. Saloheimo M, Niku-paavola M L, Knowles KC. Isolation and structural analysis of the laccase gene from the lignin-degrading fungus Phlebia radiate [J]. GenMicrobiol, 1991, 137: 1537-1544.
17. Coll P M, Tabernero C, Santamaria R, et al. Characterization and structural analysis of the laccase I gene from the newly isolated ligninolytic Ba-sidiomycete PM1(CECT 2971)[J]. Appl Environ Microbiol, 1993, 59: 4129-4135.
18. Anastasia P L, Litvintseva J M. Cloning, haracterization, and transcription of three laccase genes from gaeumannomyces graminis vartritici, the take-all fungus [J]. Applied and Environmental Microbiology, 2002, 68(3): 1305-1311.
19.梁帅,周德明,冯友仁.白腐真菌漆酶的研究进展及应用前景[J].安徽农业科学, 2008, 36(4): 1317-1319.
20. Santos M, Souza A, Rosa A, et al. Thermal denaturation: is solid-state fermentation really a goodtechnology for the production of enzyme [J]. Bioresource Technology, 2004, 93: 261-268.
21.姚跃飞,曾柏全.现代固态发酵技术在视频加工业中的应用[J].食品与机械, 2005, 21(6): 89-92.
22. Krishna C. Solid-state fermentation systems an overview [J]. Critical Reviews in Biotechnol, 2005, 25 (1-2): 1-30.
23.汪小峰,闰云君.固态发酵产微生物脂肪酶[J].中国生物工程杂志, 2009, 29(1): 105-110.
24. Couto R, Sanroman A. Application of solid-state fermentation to food industry a review [J]. Journal of Food Engineering, 2006, 76(3): 291-302.
25.孙芹英,葛春梅,张洁.灵芝固态发酵产漆酶及对秸秆木质素的降解[J].合肥学院学报, 2007, 17(3): 6-9.
26.付时雨,余惠生.贝壳状革耳菌在固体及液体培养过程漆酶同工酶的产生研究[J].纤维素科学与技术, 1998, 6(3): 32-37.
27.王彩华,余惠生,贝壳状革耳菌和黄孢平革菌固体培养酶系比较[J].微生物学报, 1999, 39(2): 127-131.
28. Galliano H, Gas G, Boudet A. Lignin degradation by Rigidoporus lignosus involves synergisticaction of two oxidizing enzymes: Mn peroxidase and laccase [J]. FEMS Microbiology Letters, 1990, 67(3):
295-299.
29.胡平平,付时雨,余惠生.固体培养条件下外加营养及微量元素对贝壳状革耳菌酶系产生的影响[J].纤维素科学与技术, 2001, 9(4): 44-50.
30. Gose G, Marta P, Susana C, et al. Sarcoma M A. Chestnut shell and barley bran as potential substrates for laccase production by Coriolopsis rigida under solid-state conditions [J]. Journal of Food Engineering, 2005, 68: 315-319.
31. Juan M, Anne L, Laurence C, et al. Laccase production by Pycnoporus cinnabarinus grownon sugarcane bagasse: Influence of ethanol vapours as inducer[J]. Process Biochemistry, 2005, 40: 3365-3371.
32.彭丹,谢更新,曾光明.黄孢原毛平革菌固态发酵产漆酶的研究及应用[J].环境科学, 2008, 29(12): 3568-3573.
33.施晓燕,蔡宇杰,王志新.产漆酶准进筛选及其固态发酵条件的初步研究[J].食品与药品, 2009, 11(01): 11-14.
34.田亚平.生化分离技术[M]:北京:化学工业出版社, 2006, 114-122.
35. Shuttl L, Bollag M. Soluble and immobilized laccase as catalysts for the transformation of substituted phenols [J]. Enzyme Microb Technol. 1986, 8(3): 171
36.魏华丽,史安石,石淑兰.漆酶及其应用[J].黑龙江造纸, 2004(4): 38-39, 50.
37.高恩丽,仲伟佳,付小兰.云芝菌固态发酵产漆酶及其对二氯酚脱氯作用的研究[J].食品与发酵工业, 2008, 34(6): 32-36.
38.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.
39.付时雨,詹怀宇,余惠生.介体系统漂白尾叶桉硫酸盐浆的初步研究[J].中国造纸, 2000(2): 8-12.
40.付时雨,余惠生,王佳玲.漆酶-助剂体系催化氧化具有α-苄基氢的木素模型物[J].纤维素科学与技术, 1999, 7(1): 45-50.
41.季立才,胡培植.漆酶催化氧化反应研究进展[J].林产化学与工业, 1997, 17(1): 79-83.
42.李光日,余惠生,付时雨.漆酶催化活性中心结构及应用的研究进展[J].纤维素科学与技术, 2000, 8(2): 42-48.
43. Robinson T, Chandran B, Nigam P. Studies on the production of enzymes by white-rot fungi for the decolourisation of textile dyes [J]. Enzyme and Microbial Technology, 2001, 29: 575-579.
44. Campos R, Kanelbauer A, Robra K H, et al. Indigo degradation with purified laccases from Trametes hirsute and Sclerotium rolfsii [J]. Journal of Biltechnology, 2001, 89: 131-139.
45.候红漫,周集体,陈丽.白腐菌漆酶特性及异生芳香化合物的降解[J].林产化学与工业, 2003, 23(1): 89-94.
46. Yaropolov I. Laccase properties, catalytic mechanism and applicability [J]. Applied Biochemistry and Biotechnology, 1994, 49(3): 257-278.
47. Wong Y. Laccase-catalyzed decolorization of synthetic dyes[J]. Water Research, 1999, 33(16): 3512-3520
48.郭梅,路福平,刘敏尧.基因工程菌漆酶对蒽醌染料的脱色研究[J].环境科学与技术2009, 32(4): 133-136.
49.曹治云,郑腾,谢必峰等.漆酶在生物检测中的应用进展[J].传感器技术, 2004, 23(8): 1-4.
50.董辉.汽车用传感器[M].北京:北京理工大学出版社, 2000, 101-148.
51.丁元涛,吴晖.漆酶及其在食品工业中的应用[J].食品工业, 2004(3): 26-27.
52.卢仁杰,谢国豪.无水乙醇法提纯粗茶皂素初探[J].上饶师专学报, 1996, 46-48.
53. Huang P, Du M. Flow-injection stopped-flow spectrofluorimetric kinetic determination of total ascorbic acid based on an enzyme-linked coupled reaction[J]. A nalytical Chemical Acta, 1995, 309(1-3): 271.
54.周攀登,付时雨.漆酶催化对苯基苯酚的聚合[J].高分子学报, 2004(4): 614-616.
55.李慧荣,王安松,黄民生.黄孢原毛平革菌对固体介质中染料的降解反应[J].农业生态环境, 2004, 20(3): 60-65, 69.
2.施晓燕. Phellinus sp. SYBC-L2固态发酵产漆酶条件的优化及其酶学性质的研究[D]: [硕士学位论文].无锡:江南大学生物工程学院. 2007.
3. Sakurai T, Suzuki S. Versicolorectrosopy of cucumber ascorbate oxidase and fungal laccase [C]. In: Messerschmidt. A Multi-copper Oxidases. Singapore: World Sciencitific Press, 1997, 225-250.
4. Yoshinori N. Purification and characteristization of laccase from white rot fungus trametes sanguine M85-2 [J]. Ferment Bioeng, 1995, 80(1): 91-93
5. Chefdtzb B, Chen Y. Purification and characterization of laccase from Chaetomium thermophiolium and its role in humification [J]. Applenviron Micro, 1998, 64(9): 3175-3179.
6. Christopher F. The structure and function of fungal laccase [J]. Microbiology, 1994, 140(1): 19-24.
7.励建荣,李丹.漆酶在食品工业中的应用[J].现代食品科技2006, 22(4): 262-268.
8.堵国成,赵政,陈坚.真菌漆酶的酶活测定及其在织物染料生物脱色中的应用[J].江南大学学报(自然科学版), 2003, 2 (1): 83-85, 90.
9. Szklarz G D, Antibus R K. Production of phenol oxidases and peroxidases by wood-rotting fungi[J]. Mycologia, 1989, 81(2): 234-240.
10. Reinhammar B. Copper proteins and copper enzymes (Ⅲ) [M]. Lontie R Ed: Boca CRC Press, 1984.
11.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(5): 9-11, 33.
12.张力,韩大勇,邵喜霞.白腐真菌木质素降解酶系研究进展[J].中国牧业通讯, 2008(23): 6-8, 45.
13.陈利萍,陈竹君,董伟敏.日本茄F1离体快速繁殖[J ].植物生理学通讯, 1996 , 32 (1) , 26
14.王首锋,梁海曼.升汞和次氯酸钠对黄瓜种子萌发及幼苗生长的影响[J].植物生理学通讯, 1996, 32 (2) , 117-120.
15. Kojima Y, Tsukada Y, Kawai Y, et al. Cloning, sequence analysis, and expression of ligninolytic phenoloxidase genes of the white-rot basidiomycete Coriolus hirsutus[J]. Biol. Chem, 1990, 265: 15223-15230.
16. Saloheimo M, Niku-paavola M L, Knowles KC. Isolation and structural analysis of the laccase gene from the lignin-degrading fungus Phlebia radiate [J]. GenMicrobiol, 1991, 137: 1537-1544.
17. Coll P M, Tabernero C, Santamaria R, et al. Characterization and structural analysis of the laccase I gene from the newly isolated ligninolytic Ba-sidiomycete PM1(CECT 2971)[J]. Appl Environ Microbiol, 1993, 59: 4129-4135.
18. Anastasia P L, Litvintseva J M. Cloning, haracterization, and transcription of three laccase genes from gaeumannomyces graminis vartritici, the take-all fungus [J]. Applied and Environmental Microbiology, 2002, 68(3): 1305-1311.
19.梁帅,周德明,冯友仁.白腐真菌漆酶的研究进展及应用前景[J].安徽农业科学, 2008, 36(4): 1317-1319.
20. Santos M, Souza A, Rosa A, et al. Thermal denaturation: is solid-state fermentation really a goodtechnology for the production of enzyme [J]. Bioresource Technology, 2004, 93: 261-268.
21.姚跃飞,曾柏全.现代固态发酵技术在视频加工业中的应用[J].食品与机械, 2005, 21(6): 89-92.
22. Krishna C. Solid-state fermentation systems an overview [J]. Critical Reviews in Biotechnol, 2005, 25 (1-2): 1-30.
23.汪小峰,闰云君.固态发酵产微生物脂肪酶[J].中国生物工程杂志, 2009, 29(1): 105-110.
24. Couto R, Sanroman A. Application of solid-state fermentation to food industry a review [J]. Journal of Food Engineering, 2006, 76(3): 291-302.
25.孙芹英,葛春梅,张洁.灵芝固态发酵产漆酶及对秸秆木质素的降解[J].合肥学院学报, 2007, 17(3): 6-9.
26.付时雨,余惠生.贝壳状革耳菌在固体及液体培养过程漆酶同工酶的产生研究[J].纤维素科学与技术, 1998, 6(3): 32-37.
27.王彩华,余惠生,贝壳状革耳菌和黄孢平革菌固体培养酶系比较[J].微生物学报, 1999, 39(2): 127-131.
28. Galliano H, Gas G, Boudet A. Lignin degradation by Rigidoporus lignosus involves synergisticaction of two oxidizing enzymes: Mn peroxidase and laccase [J]. FEMS Microbiology Letters, 1990, 67(3):
295-299.
29.胡平平,付时雨,余惠生.固体培养条件下外加营养及微量元素对贝壳状革耳菌酶系产生的影响[J].纤维素科学与技术, 2001, 9(4): 44-50.
30. Gose G, Marta P, Susana C, et al. Sarcoma M A. Chestnut shell and barley bran as potential substrates for laccase production by Coriolopsis rigida under solid-state conditions [J]. Journal of Food Engineering, 2005, 68: 315-319.
31. Juan M, Anne L, Laurence C, et al. Laccase production by Pycnoporus cinnabarinus grownon sugarcane bagasse: Influence of ethanol vapours as inducer[J]. Process Biochemistry, 2005, 40: 3365-3371.
32.彭丹,谢更新,曾光明.黄孢原毛平革菌固态发酵产漆酶的研究及应用[J].环境科学, 2008, 29(12): 3568-3573.
33.施晓燕,蔡宇杰,王志新.产漆酶准进筛选及其固态发酵条件的初步研究[J].食品与药品, 2009, 11(01): 11-14.
34.田亚平.生化分离技术[M]:北京:化学工业出版社, 2006, 114-122.
35. Shuttl L, Bollag M. Soluble and immobilized laccase as catalysts for the transformation of substituted phenols [J]. Enzyme Microb Technol. 1986, 8(3): 171
36.魏华丽,史安石,石淑兰.漆酶及其应用[J].黑龙江造纸, 2004(4): 38-39, 50.
37.高恩丽,仲伟佳,付小兰.云芝菌固态发酵产漆酶及其对二氯酚脱氯作用的研究[J].食品与发酵工业, 2008, 34(6): 32-36.
38.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.
39.付时雨,詹怀宇,余惠生.介体系统漂白尾叶桉硫酸盐浆的初步研究[J].中国造纸, 2000(2): 8-12.
40.付时雨,余惠生,王佳玲.漆酶-助剂体系催化氧化具有α-苄基氢的木素模型物[J].纤维素科学与技术, 1999, 7(1): 45-50.
41.季立才,胡培植.漆酶催化氧化反应研究进展[J].林产化学与工业, 1997, 17(1): 79-83.
42.李光日,余惠生,付时雨.漆酶催化活性中心结构及应用的研究进展[J].纤维素科学与技术, 2000, 8(2): 42-48.
43. Robinson T, Chandran B, Nigam P. Studies on the production of enzymes by white-rot fungi for the decolourisation of textile dyes [J]. Enzyme and Microbial Technology, 2001, 29: 575-579.
44. Campos R, Kanelbauer A, Robra K H, et al. Indigo degradation with purified laccases from Trametes hirsute and Sclerotium rolfsii [J]. Journal of Biltechnology, 2001, 89: 131-139.
45.候红漫,周集体,陈丽.白腐菌漆酶特性及异生芳香化合物的降解[J].林产化学与工业, 2003, 23(1): 89-94.
46. Yaropolov I. Laccase properties, catalytic mechanism and applicability [J]. Applied Biochemistry and Biotechnology, 1994, 49(3): 257-278.
47. Wong Y. Laccase-catalyzed decolorization of synthetic dyes[J]. Water Research, 1999, 33(16): 3512-3520
48.郭梅,路福平,刘敏尧.基因工程菌漆酶对蒽醌染料的脱色研究[J].环境科学与技术2009, 32(4): 133-136.
49.曹治云,郑腾,谢必峰等.漆酶在生物检测中的应用进展[J].传感器技术, 2004, 23(8): 1-4.
50.董辉.汽车用传感器[M].北京:北京理工大学出版社, 2000, 101-148.
51.丁元涛,吴晖.漆酶及其在食品工业中的应用[J].食品工业, 2004(3): 26-27.
52.卢仁杰,谢国豪.无水乙醇法提纯粗茶皂素初探[J].上饶师专学报, 1996, 46-48.
53. Huang P, Du M. Flow-injection stopped-flow spectrofluorimetric kinetic determination of total ascorbic acid based on an enzyme-linked coupled reaction[J]. A nalytical Chemical Acta, 1995, 309(1-3): 271.
54.周攀登,付时雨.漆酶催化对苯基苯酚的聚合[J].高分子学报, 2004(4): 614-616.
55.李慧荣,王安松,黄民生.黄孢原毛平革菌对固体介质中染料的降解反应[J].农业生态环境, 2004, 20(3): 60-65, 69.