木质素氧化酶系高产复合真菌培养体系构建

doi:10.11707/j.1001-7488.LYKX20230074

摘要/Abstract

摘要：

目的: 通过菌株初筛、单因素培养条件比较以及复合真菌培养体系下木质素氧化酶系酶活性的变化规律研究，获取最优产酶效率培养体系，为生物制浆预处理提供基础和科学依据。方法: 采集颐和园树木上的真菌子实体作为菌种来源，通过生长速度和愈创木酚培养基初筛得到生长快且漆酶活性高的白腐菌菌株，测定菌株木质素氧化酶系漆酶、锰过氧化物酶和木质素过氧化物酶的分泌规律。基于单因素试验探究不同碳源、氮源、pH、添加剂、金属离子、温度等条件对菌株分泌木质素氧化酶系的影响，根据产酶规律选择不同菌株构建1∶1复合真菌培养体系，分析其产酶规律和产酶效率。结果: 初筛得到1株生长快速、高漆酶活性的菌株，经鉴定为白腐菌粗毛栓孔菌（Trametes hirsute）。单因素试验结果显示，葡萄糖和酵母浸粉分别是T. hirsute产酶的最优碳源和氮源；单独添加Cu²⁺的产酶效率优于同时添加Cu²⁺和Mn²⁺，产酶量随金属离子浓度增大呈先升后降趋势，0.25 mmol·L^?1 Cu²⁺添加效果最佳；产酶量随温度升高也呈先升后降趋势，在38 ℃、pH=6.5且添加0.5 mmol·L^?1藜芦醇和0.5 mmol·L^?1对苯二胺条件下对T. hirsute产酶均有促进作用。在培养条件优化基础上，构建T. hirsute与密粘褶菌（Gloeophyllum trabeum）复合真菌培养体系，其产酶能力进一步提高，该体系的木质素氧化酶系3种酶最大总产酶量达1 505.81 U·L^?1，相比对照组提升约63.61%，其中漆酶活性相比对照组提高84.37%，且提前1天达到产酶峰值。结论: 通过初筛获取高漆酶活性白腐菌株T. hirsute，优化产酶条件可显著提升其木质素氧化酶系产酶量，在培养条件优化基础上构建的T. hirsute与G. trabeum复合真菌培养体系能够进一步提升其产酶能力，并促使漆酶提前1天达到产酶峰值，在生物预处理方面展现出巨大应用潜力。

关键词: 粗毛栓孔菌, 木质素氧化酶系, 复合真菌培养体系, 生物预处理, 漆酶

Abstract:

Objective: This study aims to obtain the optimal ligninolytic enzymes production system through the preliminary screening of fungal strains, determination of the optimal culture conditions and construction of the compound fungal culture system, which provides basic and scientific basis for pretreatment of bio-pulping. Method: The fungal fruiting bodies on the trees of the Summer Palace were collected as the source of strains. The white rot fungi strains with fast growth rate and high laccase activity were screened by growth rate and guaiacol culture medium. Then, the secretion of laccase (Lac), manganese- peroxidase (MnP) and lignin-peroxidase (LiP) were measured. The effects of different carbon sources, nitrogen sources, pH, additives, metal ions, temperature on the secretion of ligninolytic enzyme system were studied by single factor experiment. According to the enzyme production, different strains were selected to construct the 1∶1 compound fungal culture system, and the enzyme production and enzyme production efficiency were studied. Result: A strain with rapid growth and high laccase activity was obtained and identified as white rot fungus Trametes hirsute. The results of single factor test showed that glucose and yeast extract were the optimal carbon and nitrogen sources for T. hirsute ligninolytic enzymes production, respectively. The ligninolytic enzymes production efficiency of adding Cu^{2 +} alone was better than that of adding both Cu²⁺ and Mn²⁺. The ligninolytic enzymes production increased first and then decreased with the increase of metal ion concentration, and 0.25 mmol·L^?1 Cu^{2 +} was the best. At 38 ℃, pH=6.5 and 0.5 mmol·L^?1 veratrol and 0.5 mmol·L^?1 p-phenylenediamine, the lignin oxidase production of T. hirsute was promoted. Based on the optimization of culture conditions, a composite fungal culture system of T. hirsute and Gloeophyllum trabeum was constructed, and its enzyme production ability was further improved. The maximum total lignin oxidase production of the cultures reached 1 505.81 U·L^?1, which was about 63.61% higher than that of the control group. The laccase activity was 84.37% higher than that of the control group, and total ligninolytic enzymes production peak was reached one day in advance. Conclusion: High laccase activity fungus T. hirsute was obtained by preliminary screening. Its ligninolytic enzyme system was significantly increased by optimizing the enzyme production conditions. Based on the optimized medium, the T. hirsute and G. trabeum compound fungal culture system further improved the total lignin oxidase production and promoted laccase to reach the peak enzyme production one day in advance. The research provides a basis for the efficient utilization of lignocellulose with biological pretreatment.

Key words: Trametes hirsute, ligninolytic enzyme system, compound fungal culture system, biological pretreatment, laccase

中图分类号:

S782.33

熊怡心,王爽,马星霞,孙志勤. 木质素氧化酶系高产复合真菌培养体系构建[J]. 林业科学, 2024, 60(10): 133-142.

Yixin Xiong,Shuang Wang,Xingxia Ma,Zhiqin Sun. Construction of Compound Fungal Culture System for High-Yield Ligninolytic Enzymes[J]. Scientia Silvae Sinicae, 2024, 60(10): 133-142.

图/表 11

图1

图2

图3

图4

图5

图6

图7

图8

图9

表1

图10

参考文献 0

	蔡绍祥, 黄燕萍, 李朱锋, 等. 木材化学成分对细胞壁纵向黏弹性的影响. 森林工程, 2022, 38 (3): 54- 62.
	Cai S X, Huang Y P, Li Z F, et al. Effect of wood chemical composition on longitudinal viscoelastic properties of cell walls. Forest Engineering, 2022, 38 (3): 54- 62.
	陈　晨, 程　旭, 王立朝, 等. 改性纳米复合防腐剂对木材耐腐性能的研究. 森林工程, 2022, 38 (6): 61- 68.
	Chen C, Cheng X, Wang L C, et al. Study on wood decay resistance of modified nanocomposite preservatives. Forest Engineering, 2022, 38 (6): 61- 68.
	池玉杰, 闫洪波. 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, 2009, 45 (12): 154- 158. doi: 10.11707/j.1001-7488.20091227
	Chi Y J, Yan H B. Detection on laccase, manganese peroxidase and lignin peroxidase in ligninolytic enzymes of Pleurotus djamor. Scientia Silvae Sinicae, 2009, 45 (12): 154- 158. doi: 10.11707/j.1001-7488.20091227
	方　旋, 温敬伟, 陈　粤, 等. 南越国宫署遗址出土木质水槽原位保存环境下的真菌多样性. 林业科学, 2021, 57 (7): 131- 141. doi: 10.11707/j.1001-7488.20210714
	Fang X, Wen J W, Chen Y, et al. Fungal diversity of wooden flume unearthed from Nanyue National Palace site under in situ preservation environment. Scientia Silvae Sinicae, 2021, 57 (7): 131- 141. doi: 10.11707/j.1001-7488.20210714
	郭　梅, 蒲军, 梁　鹏, 等. 白腐菌Trametes versicolor产漆酶发酵条件的优化. 食品研究与开发, 2006, 27 (6): 9- 12. doi: 10.3969/j.issn.1005-6521.2006.06.004
	Guo M, Pu J, Liang P, et al. Studies on fermentation conditions for laccase production from white-rot fungi Trametes versicolor. Food Research and Development, 2006, 27 (6): 9- 12. doi: 10.3969/j.issn.1005-6521.2006.06.004
	荚　荣, 汤必奎, 张晓宾, 等. 藜芦醇和吐温80对白腐菌产木质素降解酶的影响及在偶氮染料脱色中的作用. 生物工程学报, 2004, 20 (2): 302- 305. doi: 10.3321/j.issn:1000-3061.2004.02.029
	Jia R, Tang B K, Zhang X B, et al. Effects of veratryl alcohol and tween 80 on ligninase production and its roles in decolorization of azo dyes by white-rot basidiomycete PM2. Chinese Journal of Biotechnology, 2004, 20 (2): 302- 305. doi: 10.3321/j.issn:1000-3061.2004.02.029
	李翠珍, 文湘华. 白腐真菌F2的生长及产木质素降解酶特性的研究. 环境科学学报, 2005, 25 (2): 226- 231. doi: 10.3321/j.issn:0253-2468.2005.02.017
	Li C Z, Wen X H. Characterization of growth and ligninolytic enzymes production of a white rot fungus F2. Acta Scientiae Circumstantiae, 2005, 25 (2): 226- 231. doi: 10.3321/j.issn:0253-2468.2005.02.017
	李慧蓉. 2005. 白腐真菌生物学和生物技术. 北京: 化学工业出版社.
	Li H R. 2005. Biology and biotechnology of white rot fungi. Beijing: Chemical Industry Press. ［in Chinese］
	李旭东, 荚　荣, 程晓滨, 等. 锰过氧化物酶的固态发酵及其对染料的脱色作用. 环境科学学报, 2008, 28 (3): 490- 495. doi: 10.3321/j.issn:0253-2468.2008.03.013
	Li X D, Jia R, Cheng X B, et al. Solid-state fermentation of MnP and decolorization of dyes. Acta Scientiae Circumstantiae, 2008, 28 (3): 490- 495. doi: 10.3321/j.issn:0253-2468.2008.03.013
	王　东, 林兰英, 傅　峰. 木材多尺度结构差异对其破坏影响的研究进展. 林业科学, 2020, 56 (8): 141- 147. doi: 10.11707/j.1001-7488.20200816
	Wang D, Lin L Y, Fu F. The effects of multiscale structure differences on wood fracture: a review. Scientia Silvae Sinicae, 2020, 56 (8): 141- 147. doi: 10.11707/j.1001-7488.20200816
	王海宽, 路福平. 培养条件对甲醇毕赤酵母异源表达木质素过氧化物酶的影响. 天津科技大学学报, 2007, 22 (1): 33- 36. doi: 10.3969/j.issn.1672-6510.2007.01.009
	Wang H K, Lu F P. Effect of cultivation condition on heterologous expression of lignin peroxidase in Pichia methanolica. Journal of Tianjin University of Science & Technology, 2007, 22 (1): 33- 36. doi: 10.3969/j.issn.1672-6510.2007.01.009
	王宜磊, 朱　陶, 邓振旭. 愈创木酚法快速筛选漆酶产生菌. 生物技术, 2007, 17 (2): 40- 42. doi: 10.3969/j.issn.1004-311X.2007.02.014
	Wang Y L, Zhu T, Deng Z X. Using O-methoxyphenol to fast screen laccase produced fungus. Biotechnology, 2007, 17 (2): 40- 42. doi: 10.3969/j.issn.1004-311X.2007.02.014
	吴柯军, 闫绍鹏, 卢　宏, 等. 不同木质底物诱导下白囊耙齿菌胞外木质纤维素酶活性和胞内蛋白质组的差异. 林业科学, 2016, 52 (8): 157- 166.
	Wu K J, Yan S P, Lu H, et al. Difference in the activity of extracellular lignocellulolytic enzymes and the intracellular proteome of Irpex lacteus induced by different wood substrates. Scientia Silvae Sinicae, 2016, 52 (8): 157- 166.
	Biko O D V, Viljoen-Bloom M, van Zyl W H. Microbial lignin peroxidases: applications, production challenges and future perspectives. Enzyme and Microbial Technology, 2020, 141, 109669. doi: 10.1016/j.enzmictec.2020.109669
	Birhanlı E, Ali Noma S A, Boran F, et al. Design of laccase–metal–organic framework hybrid constructs for biocatalytic removal of textile dyes. Chemosphere, 2022, 292, 133382. doi: 10.1016/j.chemosphere.2021.133382
	Chi Y J, Hatakka A, Maijala P. 2007. Can co-culturing of two white-rot fungi increase lignin degradation and the production of lignin-degrading enzymes? International Biodeterioration & Biodegradation, 59(1): 32−39.
	Dashtban M, Schraft H, Qin W S. Fungal bioconversion of lignocellulosic residues; opportunities & perspectives. International Journal of Biological Sciences, 2009, 5 (6): 578- 595.
	Do N H, Pham H H, Le T M, et al. The novel method to reduce the silica content in lignin recovered from black liquor originating from rice straw. Scientific Reports, 2020, 10 (1): 21263. doi: 10.1038/s41598-020-77867-5
	Fonseca M I, Fariña J I, Castrillo M L, et al. Biopulping of wood chips with Phlebia brevispora BAFC 633 reduces lignin content and improves pulp quality. International Biodeterioration & Biodegradation, 2014, 90, 29- 35.
	Karkera K, Pendse A, Aruna K. Studies on biosurfactant production by Pseudomonas aeruginosa R2 isolated from oil contaminated soil sample. Asian Journal of Biological Sciences, 2012, 7 (2): 123- 129.
	Kundu P, Manna B, Majumder S, et al. Species-wide metabolic interaction network for understanding natural lignocellulose digestion in termite gut microbiota. Scientific Reports, 2019, 9 (1): 16329. doi: 10.1038/s41598-019-52843-w
	Levin L, Villalba L, Da Re V, et al. Comparative studies of loblolly pine biodegradation and enzyme production by Argentinean white rot fungi focused on biopulping processes. Process Biochemistry, 2007, 42 (6): 995- 1002. doi: 10.1016/j.procbio.2007.03.008
	Lin C W, Lai C Y, Liu S H, et al. Enhancing bioelectricity generation and removal of copper in microbial fuel cells with a laccase-catalyzed biocathode. Journal of Cleaner Production, 2021, 298, 126726. doi: 10.1016/j.jclepro.2021.126726
	Lubbers R J M, Dilokpimol A, Visser J, et al. A comparison between the homocyclic aromatic metabolic pathways from plant-derived compounds by bacteria and fungi. Biotechnology Advances, 2019, 37 (7): 107396. doi: 10.1016/j.biotechadv.2019.05.002
	Mboowa D. A review of the traditional pulping methods and the recent improvements in the pulping processes. Biomass Conversion and Biorefinery, 2024, 14 (1): 1- 12. doi: 10.1007/s13399-020-01243-6
	Nagpal R, Bhardwaj N K, Mishra O P, et al. Cleaner bio-pulping approach for the production of better strength rice straw paper. Journal of Cleaner Production, 2021, 318, 128539. doi: 10.1016/j.jclepro.2021.128539
	Nakazawa T, Morimoto R, Wu H L, et al. Dominant effects of gat1 mutations on the ligninolytic activity of the white-rot fungus Pleurotus ostreatus. Fungal Biology, 2019, 123 (3): 209- 217. doi: 10.1016/j.funbio.2018.12.007
	Singh P, Sulaiman O, Hashim R, et al. Biopulping of lignocellulosic material using different fungal species: a review. Reviews in Environmental Science and Bio/Technology, 2010, 9 (2): 141- 151. doi: 10.1007/s11157-010-9200-0
	Sun S N, Sun S L, Cao X F, et al. The role of pretreatment in improving the enzymatic hydrolysis of lignocellulosic materials. Bioresource Technology, 2016, 199, 49- 58. doi: 10.1016/j.biortech.2015.08.061
	Taha M, Shahsavari E, Al-Hothaly K, et al. Enhanced biological straw saccharification through coculturing of lignocellulose-degrading microorganisms. Applied Biochemistry and Biotechnology, 2015, 175 (8): 3709- 3728. doi: 10.1007/s12010-015-1539-9
	Wang J H, Li L L, Xu H M, et al. Construction of a fungal consortium for effective degradation of rice straw lignin and potential application in bio-pulping. Bioresource Technology, 2022, 344, 126168. doi: 10.1016/j.biortech.2021.126168
	Xu X Y, Shen X T, Yuan X J, et al. Metabolomics investigation of an association of induced features and corresponding fungus during the co-culture of Trametes versicolor and Ganoderma applanatum. Frontiers in Microbiology, 2018, 8, 2647. doi: 10.3389/fmicb.2017.02647
	Yao L, Zhu L P, Xu X Y, et al. Discovery of novel xylosides in co-culture of basidiomycetes Trametes versicolor and Ganoderma applanatum by integrated metabolomics and bioinformatics. Scientific Reports, 2016, 6, 33237. doi: 10.1038/srep33237
	Zanellati A, Spina F, Bonaterra M, et al. Screening and evaluation of phenols and furans degrading fungi for the biological pretreatment of lignocellulosic biomass. International Biodeterioration & Biodegradation, 2021, 161, 105246.

试验组 Test	Cu²⁺ 0.25 mmol·L^?1	VA 0.5 mmol·L^?1	对苯二胺 P-phenylenediamine 0.5 mmol·L^?1	复合培养 Compound culture	Lac/ (U·L^?1)	MnP/ (U·L^?1)	LiP/ (U·L^?1)	总酶活性 Total enzyme activity/ (U·L^?1)
对照组Control	—	—	—	—	608.21	122.92	189.22	920.35
1	+	+	—	—	788.29	131.49	227.65	1 147.43
2	+	—	+	—	897.85	126.30	216.76	1 240.91
3	+	+	+	—	966.50	114.58	234.65	1 315.73
4	+	+	—	+	1 043.08	134.65	246.59	1 424.32
5	+	—	+	+	1 114.71	129.25	241.84	1 485.80
6	+	+	+	+	1 121.32	124.81	259.68	1 505.81

[1]	饶瑾,王慧,NayebareKakwara Prosper,王婕,姜俊,杨秀树,刘庭菘,孙芳利. 漆酶催化碘化竹材的防腐性能[J]. 林业科学, 2021, 57(2): 160-167.
[2]	饶瑾,杨胜祥,吴华平,杨秀树,孙芳利. 漆酶催化碘处理竹材防霉性能[J]. 林业科学, 2020, 56(2): 148-155.
[3]	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.
[4]	石亚攀, 池玉杰, 于存. 偏肿革裥菌产漆酶活性的诱导[J]. 林业科学, 2016, 52(12): 150-155.
[5]	高冬妮, 范晓旭, 赵丹. 产漆酶半知真菌Myrothecium verrucariaNF-08菌株的分离及产酶研究[J]. 林业科学, 2015, 51(1): 80-87.
[6]	尹立伟, 池玉杰. 猴头菌菌株CB1的系统发育分析与木质素降解酶的检测[J]. 林业科学, 2013, 49(6): 129-134.
[7]	郭明, 燕冰宇, 王春鹏, 周建钟. 纤维素基质固载酶材料制备及固定化漆酶性能[J]. 林业科学, 2013, 49(11): 122-128.
[8]	王乐;陈辉;胡霞;马超. 秦岭细粘束孢产漆酶的培养条件[J]. 林业科学, 2012, 48(5): 164-167.
[9]	池玉杰闫洪波. 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池[J]. 林业科学, 2009, 12(12): 154-158.
[10]	徐春燕马富英王锦锦张晓昱. 生物预处理竹子对纤维酶糖化的影响[J]. 林业科学, 2008, 44(10): 168-172.
[11]	段新芳曹远林曹永建周冠武陈永圣朱家琪赵保路. 漆酶活化处理对木材自由基变化的影响[J]. 林业科学, 2007, 43(4): 134-136.
[12]	朱家琪史广兴. 酶活化处理条件及其对松木纤维胶合性能的影响初探[J]. 林业科学, 2004, 40(4): 153-156.