Paraconiothyium variable GHJ-4木质素降解酶的酶学性质

doi:10.11707/j.1001-7488.20170112

摘要/Abstract

摘要： [目的] 研究木质素降解子囊菌Paraconiothyrium variabile GHJ-4分泌的漆酶（laccase）、锰过氧化物酶（MnP）和木质素过氧化物酶（LiP）3种木质素降解酶的酶活性变化规律及其酶学性质，为利用该菌株进行木质素降解酶的工业化生产和应用提供依据。[方法] 分别以愈创木酚、2，6-二甲基苯酚和黎芦醇为底物测定laccase，MnP和LiP的酶活性，研究3种酶的最适反应温度和pH、温度和pH稳定性及金属离子对其活性的影响。[结果] GHJ-4菌株发酵18，15和21天后，分别获得laccase，MnP和LiP最高酶活为1 390.3，30.3和52.5 U·mL^-1。laccase的最适反应温度为55℃左右，最适反应pH为5.5左右，在55℃以下、pH 4.0~7.0的范围内较为稳定；MnP的最适反应温度为60℃左右，最适反应pH为5.0左右，在55℃以下、pH 4.0~9.0的范围内较为稳定；LiP的最适反应温度为40℃左右，最适反应pH为3.0左右，在40℃以下、pH 2.0~4.0的范围内较为稳定。Mg²⁺，Zn²⁺，Cu²⁺，K⁺对laccase起促进作用，Na⁺和Zn²⁺对LiP起促进作用，Mn²⁺对MnP起激活作用，而Fe³⁺，Ca²⁺，Pb²⁺，Co²⁺，Al³⁺对3种酶均起抑制作用，其中尤以5 mmol·L^-1Fe³⁺对3种酶的抑制作用最强。[结论] 温度、pH及金属离子对Paraconiothyrium variabile GHJ-4 3种木质素降解酶的酶学特性均有着不同程度的影响。

关键词: Paraconiothyrium variabile GHJ-4, 漆酶, 锰过氧化物酶, 木质素过氧化物酶, 酶学性质

Abstract: [Objective] The production and enzymological characteristics of ligninolytic enzymes, including laccase, manganese peroxidase (MnP) and lignin peroxidase (LiP) produced by Paraconiothyrium variabile GHJ-4 were investigated. This study could provide theoretical data for commercial process and application of Paraconiothyrium variabile GHJ-4 ligninolytic enzymes.[Method] Laccase, MnP and LiP activity were measured with guaiacol, 2,6-Dimethylphenol and veratryl alcohol as substrate respectively. Effects of temperature, pH value and metal irons on three ligninolytic enzymes were assayed. [Result] Laccase, MnP and LiP activity reached the peak value of 1 390.3 U·mL^-1, 30.3 U·mL^-1 and 52.5 U·mL^-1 respectively on the 18^th, 15^thand 21^stday during the fermentation. The optimum temperature and pH of laccase were 55℃ and 5.5 respectively, and the enzyme activity was stable under 55℃ and pH 4.0-7.0. The optimum temperature and pH of MnP were 60℃ and 5.0 respectively, and the enzyme activity was stable under 55℃ and pH 4.0-9.0. The optimum temperature and pH of LiP were 40℃ and 3.0 respectively, and the enzyme activity was stable under 40℃ and pH 2.0-4.0. Laccase activity was enhanced by the metal ions Mg²⁺,Zn²⁺,Cu²⁺,K⁺, LiP activity was enhanced by Na⁺ and Zn²⁺, and MnP activity was enhanced by Mn²⁺. Whereas three ligninolytic enzymes were inhibited by Fe³⁺, Ca²⁺, Pb²⁺, Co²⁺ and Al³⁺, especially the effect of Fe³⁺ was the strongest.[Conclusion] The effects of temperature, pH and metal ions on activities of ligninolytic enzymes from Paraconiothyrium variabile GHJ-4 were different.

Key words: Paraconiothyrium variabile GHJ-4, laccase, manganese peroxidase(MnP), lignin peroxidase(LiP), enzyme characterization

中图分类号:

Q933

王凤娟, 李伟庆, 牟志美, 王彦文, 刘庆信, 高绘菊. Paraconiothyium variable GHJ-4木质素降解酶的酶学性质[J]. 林业科学, 2017, 53(1): 94-100.

Wang Fengjuan, Li Weiqing, Mu Zhimei, Wang Yanwen, Liu Qingxin, Gao Huiju. Enzymological Characteristics of Ligninolytic Enzyme from Paraconiothyrium variabile GHJ-4[J]. Scientia Silvae Sinicae, 2017, 53(1): 94-100.

参考文献

白娜. 2010. 哈茨木霉WRF-2木质素降解酶系高产菌株培育及其对玉米秸秆生物降解研究.杨凌:西北农林科技大学硕士学位论文.
(Bai N. 2010. Cultivation of high ligninilytic enzyme of Trichoderma harzianum WRF-2 and study on biodergradation of corn straw. Yangling: MS thesis of Northwest A&F University. [in Chinese])
程晓滨. 2007. 裂褶菌F17锰过氧化物酶的分离纯化及其对偶氮染料脱色的研究.合肥:安徽大学硕士学位论文.
(Cheng X B. 2007. Purification of a new manganese peroxidase of the white-rot fungus Schizophyllum sp. F17, and decolorization of azo dyes by the enzyme. Hefei: MS thesis of Anhui University. [in Chinese])
池玉杰, 闫洪波. 2009. 红平菇木质素降解酶系统漆酶、锰过氧化物酶及木质素过氧化物酶的检测. 林业科学, 45(12):154-158.
(Chi Y J, Yan H B. 2009. Detection on laccase, manganese peroxidase and lignin peroxidase in ligninolytic enzymes of Pleurotus djamor. Scientia Silvae Sinicae, 45(12):154-158. [in Chinese])
崔艳红,韩庆功,胡志明,等.2012.木质层孔菌诱导漆酶及部分酶学性质研究.广东农业科学,(12):114-116.
(Cui Y H, Han Q G, Hu Z M, et al.2012.Research on laccase induced by Fomes ligmosus and its enzymatic properties.Guangdong Agricultural Sciences,(12):114-116.[in Chinese])
董旭杰, 曹福祥, 陈静, 等. 2007. 3种白腐菌木质素降解酶的比较.中南林业科技大学学报, 27(3):131-135.
(Dong X J, Cao F X, Chen J, et al. 2007. Comparison of lignin degradation enzymes produced by three white-rot fungi. Journal of Central South University of Forestry & Technology, 27(3): 131-135. [in Chinese])
董学卫,朱启忠,吕新萍,等.2007. 白毒鹅膏菌胞外漆酶的部分酶学性质研究.石河子大学学报: 自然科学版, 25(2):132-136.
(Dong X W, Zhu Q Z, Lü X P, et al. 2007.Studies on partial character of extracellular laccase from Amanita verna. Journal of Shihezi University: Natural Science, 25(2):132-136. [in Chinese])
段传人, 朱丽平, 姚月良. 2009. 三种白腐菌及其组合菌种木质素降解酶比较研究. 菌物学报, 28(4):577-583.
(Duan C R, Zhu L P, Yao Y L. 2009. Comparative studies on lignin degradation enzymes produced by three species of white-rot fungi and combination of the strains. Mycosystema, 28(4): 577-583. [in Chinese])
金春德, 王进, 毛胜凤, 等. 2009. 漆酶活化产生的活性氧类自由基与竹粉板性能的关系. 北京林业大学学报, 31(2):119-121.
(Jin C D, Wang J, Mao S F, et al. 2009.Treatment of bamboo flour by reactive oxygen species free radicals: Effects on panel properties. Journal of Beijing Forestry University, 31(2):119-121. [in Chinese])
金剑, 康文丽, 生吉萍, 等. 2010. 云芝(Coriolus versicolor)木质素过氧化物酶(LiP)酶学性质分析. 食品科学, 31(17):224-227.
(Jin J, Kang W L, Sheng J P, et al. 2010. Enzymological Characteristics of Lignin Peroxidase (LiP) from Coriolus versicolor. Food Science, 31(17):224-227. [in Chinese])
孔令营,郭道森,赵博光,等. 2010. 荧光假单胞菌 GcM5-1A胞外木质素过氧化物酶的初步纯化及性质研究.北京林业大学学报, 32(3):112-116.
(Kong L Y, Guo D S, Zhao B G. et al. 2010. Preliminary purification and characterization of extracellular lignin peroxidase from Pseudomonas fluorescens GcM5-1A. Journal of Beijing Forestry University, 32(3):112—116. [in Chinese])
李阿敏, 李国庆, 常艳, 等. 2015. 白腐菌降解木质素的研究进展. 食药用菌, 23(2):95-101.
(Li A M, Li G Q, Chang Y, et al. 2015. Research progress on lignin degradation by white rot fungi. Edible and medicinal mushrooms, 23(2):95-101. [in Chinese])
宋安东, 张百良, 吴坤, 等. 2005. 杂色云芝产木质纤维素酶及对稻草秸秆的降解. 过程工程学报, 5(4):414-419.
(Song A D, Zhang B L, Wu K, et al. 2005. Production of lignocellulolytic enzymes and rice straw biodegradation by Coriolus versicolor. The Chinese Journal of Process Engineering, 5(4):414-419. [in Chinese])
苏小军, 熊兴耀,谭兴和,等. 2009. 黑曲霉AF-1固态发酵生产生淀粉酶的条件优化.湖南农业大学学报:自然科学版, 35(2):208-212.
(Su X J, Xiong X Y, Tan X H, et al. 2009. Optimization of raw-starch-hydrolysing amylase production by Aspergillus niger AF-1 in solid state fermentation. Journal of Hunan Agricultural University:Natural Sciences, 35(2):208-212. [in Chinese])
王娟, 宗学刚. 2010. 漆酶处理三倍体毛白杨APMP及对其中残余木素作用的研究. 造纸化学品, 22(1):15-20.
(Wang J, Zong X G. 2010. Treatment of APMP of triploid of Populus tomentosa with laccase and its reaction with residual lignin of APA.Paper Chemicals, 22(1):15-20. [in Chinese])
王敏. 2011. 白腐菌降解木质素研究进展. 衡水学院学报, 13(1):51-53.
(Wang M. 2011. Progress of research on white rot fungi degradating lignin. Journal of Hengshui University,13(1):51-53. [in Chinese])
王维乐. 2011. Paraconiothyrium variabile GHJ-4漆酶的发酵、分离纯化及其酶学性质的研究. 泰安:山东农业大学硕士学位论文.
(Wang W L. 2011. Fermentation, purification and characterization of laccase from Paraconiothyrium variabile GHJ-4. Tai'an: MS thesis of Shandong Agricultural University. [in Chinese])
吴会广. 2008. Rhizoctonia sp. SYBC-M3产锰过氧化物酶发酵条件优化及酶学性质研究. 无锡:江南大学硕士学位论文.
(Wu H G. 2008. Optimization of Rhizoctonia sp. SYBC-M3 fermentation for manganese peroxidase production and the enzyme characterization. Wuxi: MS thesis of Jiangnan University. [in Chinese])
肖楚,刘佳,许修宏. 2011. 黑木耳漆酶酶学性质的研究.中国农学通报, 27(25):158-161.
(Xiao C, Liu J, Xu X H. 2011. Studies on enzymatic properties of laccase from Auricularia. Chinese Agricultural Science Bulletin, 27(25):158-161. [in Chinese])
徐淑霞,张跃灵,张世敏,等. 2007. 黄孢原毛平革菌过氧化物酶的分离、纯化和酶学特性研究. 农业环境科学学报, 26(1):295-300.
(Xu S X, Zhang Y L, Zhang S M. et al. 2007. Purification and properties of peroxidase from Phanerochaete chrysosporium. Journal of Agro-Environment Science, 26(1):295-300. [in Chinese])
杨暖. 2009. 绿色糖单孢菌木质素过氧化物酶发酵工艺、纯化及酶学性质研究. 北京:北京林业大学硕士学位论文.
(Yang N.2009. Studies on fermentation conditions, purification and characteristics of lignin peroxidases from Saccharomonospora viridis. Beijing: MS thesis of Beijing Forestry University. [in Chinese])
尤纪雪, 陈星星, 李雨楠, 等.2008. 漆酶-木聚糖酶体系直接降解木质素的研究. 纤维素科学与技术,16(2):12-17.
(You J X, Chen X X, Li Y N. et al. 2008. Study on direct degradation of lignin with laccase/xylanase system. Journal of Cellulose Science and Technology, 16(2):12-17. [in Chinese])
喻云梅,刘赟,翁恩琪,等.2005.白腐真菌木质素降解酶的产生及其调控机制研究进展.安全与环境学报,5(2):82-86.
(Yu Y M, Liu Y, Wang E Q, et al. 2005. Review on the production and regulation mechanism of ligninolytic enzymes of the white rot fungi.Journal of Safety and Environment,5(2):82-86.[in Chinese])
张连慧, 刘卫晓, 葛克山, 等. 2005. 变色栓菌产锰过氧化物酶的条件优化. 微生物学通报, 32(5):98-102.
(Zhang L H, Liu W X, Ge K S, et al. 2005. The optimization of manganese peroxidase production by Trametes versicolor. Journal of Microbiology, 32(5):98-102. [in Chinese])
张莉. 2009. 白腐菌(Trametes pubesces MB89)漆酶酶学性质及其对酚类化合物的降解特性研究. 杨凌:西北农林科技大学博士学位论文.
(Zhang L. 2009. Characterstics of laccase from white rot rungus (Trametes pubesces MB89) and its use in phenolic compunds degradation.Yangling: PhD thesis of Northwest A&F University. [in Chinese])
张文婷. 2011. Trametes trogii WT-1木质素降解作用的研究. 泰安:山东农业大学博士学位论文.
(Zhang W T. 2011. Study on the lignin decomposition of Trametes trogii WT-1. Tai'an: PhD thesis of Shandong Agricultural University. [in Chinese])
赵晓燕, 邢增涛, 邵毅, 等.2012. 灰树花漆酶酶学性质的初步研究.天然产物研究与开发, 24:824-827.
(Zhao X Y, Xing Z T, Shao Y, et al. 2012. Preliminary study on enzyme property of Grifola frondosa extracellular laccase. Nat Prod Res Dev, 24:824-827. [in Chinese])
周菲. 2011. 产漆酶木霉ZF-2分离、发酵与初步应用研究. 泰安:山东农业大学硕士学位论文.
(Zhou F. 2011. Isolating, fermentation and primary application by laccase from Trichoderma harzianum ZF-2. Tai'an:MS thesis of Shandong Agricultural University. [in Chinese])
Abrahão M C, Gugliotta A M, Silva R D, et al. 2008. Ligninolytic activity from newly isolated basidiomycete strains and effect of these enzymes on the azo dye orange II decolourisation. Ann Microbiol, 58:427-432.
Adler E. 1977. Lignin chemistry—past, present and future. Wood Sci Technol,11(3): 169-218.
Gao H J, Wang Y W, Zhang W T, et al. 2011. Isolation, identification and application in lignin degradation of an ascomycete GHJ-4. Afr J Biotechnol, 10(20):4166-4174.
Gao H J, Chu X, Wang Y W, et al. 2013. Media optimization for laccase production by Trichoderma harzianum ZF-2 using response surface methodology. Microbiol Biotechnol,23(12):1757-1764.
Glenn J K, Gold M H. 1983. Decolorization of several polymeric dyes by the lignin-degrading basidiomycete Phanerochaete chrysosporium. Appl Environ Microbiol, 45(6):1741-1747.
Hatakka A. 1994. Lignin-modifying enzymes from selected white-rot fungi: production and role in lignin degradation. FEMS Microbiol Rev, 13(3):125-135.
Leonowicz A, Cho N S, Luterek J, et al. 2001. Fungal laccase: properties and activity on lignin. J Basic Microbiol, 41(3/4):185-227.
Orth A B, Royse D J, Tien M. 1993. Ubiquity of lignin-degrading peroxidases among various wood-degrading fungi. Appl Environ Microbiol, 59(12):4017-4023.
Wariishi H, Valli K, Gold M H. 1992. Manganese II oxidation by manganese peroxidase from the basidiomycete Phanerochaete chrysosporium-kinetic mechanism and role of chelators. Journal of Biological Chemistry, 267(33):23688-23695.

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