野生灵芝BSU01的分离鉴定、生物学特性及其木质纤维素酶活分析
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摘要
灵芝作为传统中药,具有重要的医药和经济价值。本研究以一株野生灵芝属真菌BSU01为实验材料,通过形态特征和ITS序列聚类分析对其进行了鉴定,探讨了该菌株菌丝生长的最适碳源、氮源、C/N、pH及温度等生物学特征,以及该菌株产木质纤维降解素酶的能力。结果表明,菌株BSU01的形态特征与有柄灵芝相符;且与有柄灵芝ITS序列一致性达99%,并在系统发育树上聚在一起;菌株BSU01菌丝生长最适碳源为果糖或者葡萄糖,氮源为酵母提取物,C/N比为20/1到30/1,温度为30℃,pH值为5.5;菌株BSU01的漆酶、木质素过氧化物酶和Mn依赖过氧化物酶分别为12.13 U/L、0.52 U/L和0.33 U/L;CMCase和木聚糖酶酶活分别为7.14 U/mL和1.88 U/m L。本研究结果将为该菌的进一步开发利用提供数据参考。
Ganoderma, as a traditional Chinese medicine, possesses important medical and economic value. This study took a wild Ganoderma sp., named BSU01 as test materials, identified it by morphology combined with molecular phylogenetic analysis, and evaluated the optimum carbon source of its growth, nitrogen source, C/N, p H, temperature and other biological characteristics, as well as the ability of its production of lignocellulose-degrading enzyme activity. The results showed that morphological characteristics of strain BSU01 were consistent with Ganoderma gibbosum, and the identity of their ITS sequences reached 99% and they gathered together in the phylogenetic tree.In addtion, its optimum carbon source for mycelial growth was fructose or glucose. The nitrogen source was yeast extract, and C/N ratio was from 20/1 to 30/1. The optimum temperature was found to be at 30℃. The optimum pH value was 5.5. The laccase, lignin peroxidase and Mn-dependent peroxidase of strain BSU01 were 12.13 U/L、0.52 U/L and 0.33 U/L, respectively; The CMCase and xylanase activity were 7.14 U/mL and 1.88 U/mL,respectively. All these results would provide data reference for further development and utilization of this strain.
Ganoderma, as a traditional Chinese medicine, possesses important medical and economic value. This study took a wild Ganoderma sp., named BSU01 as test materials, identified it by morphology combined with molecular phylogenetic analysis, and evaluated the optimum carbon source of its growth, nitrogen source, C/N, p H, temperature and other biological characteristics, as well as the ability of its production of lignocellulose-degrading enzyme activity. The results showed that morphological characteristics of strain BSU01 were consistent with Ganoderma gibbosum, and the identity of their ITS sequences reached 99% and they gathered together in the phylogenetic tree.In addtion, its optimum carbon source for mycelial growth was fructose or glucose. The nitrogen source was yeast extract, and C/N ratio was from 20/1 to 30/1. The optimum temperature was found to be at 30℃. The optimum pH value was 5.5. The laccase, lignin peroxidase and Mn-dependent peroxidase of strain BSU01 were 12.13 U/L、0.52 U/L and 0.33 U/L, respectively; The CMCase and xylanase activity were 7.14 U/mL and 1.88 U/mL,respectively. All these results would provide data reference for further development and utilization of this strain.
引文
Baby S.,Johnson A.J.,and Govindan B.,2015,Secondary metabolites from Ganoderma,Phytochemistry,114:66-101
Bishop K.S.,Kao C.H.J.,Xu Y.,Glucina M.P.,Paterson R.R.M.,and Ferguson L.R.,2015,From 2 000 years of Ganoderma lucidum to recent developments in nutraceuticals,Phytochemistry,114:56-65
Chai X.Y.,An S.D.,Sheng S.,and Su H.,2015,Screening of laccase-producing macrofungi and optimization of productions conditions,Xibei Nonglin Keiji Daxue(Ziran Kexxue Ban)(Jourmal of Northwest A&F University)(Natural Science Edition),43(3):205-210(柴新义,安双登,盛硕,苏海,2015,产漆酶真菌筛选及其产酶条件的优化,西北农林科技大学(自然科学版),43(3):205-210)
Chen X.D.,Zeng N.K.,and Lan J.,2010,Cultural characteristics of mycelia of Ganoderma gibbosum,Zhongguo Zhongyao Zazhi(China Journal of Chinese Material Medica),35(15):1939-1942(陈向东,曾念开,兰进,2010,药用真菌有柄树舌菌丝培养特性的研究,中国中药杂志,35(15):1939-1942)
Cilerdzic J.,Stajic M.,and Vukojevic J.,2016,Degradation of wheat straw and oak sawdust by Ganoderma applanatum,Inernational Biodeterioration&Biodegradation,114:39-44
Huang L.H.,Yang X.B.,Zhang Z.,Hu H.P.,Zhang Y.F.,and Wu Q.P.,2010,Identification of Ganoderma spawns based on ITS sequence analysis,Zhongguo Shiyongjun(Edible Fungi of China),29(1):55-57(黄龙花,杨小兵,张智,胡慧萍,张一凡,吴清平,2010,基于ITS序列分析鉴定灵芝属菌种,中国食用菌,29(1):55-57)
Jo W.S.,Cho Y.J.,Cho D.H.,Park S.D.,Yoo Y.B.,and Seok S.J.,2009,Culture conditions for the mycelial growth of Ganoderma applanatum,Mycobiology,37(2):94-102
Lan Y.F.,Wang Q.W.,Tang L.N.,and Li X.M.,2016,Comparision to mycelial growth rates of three of Ganoderma on different carbon source,nitrogen source and temperature,Zhongguo Shiyongjun(Edible Fungi of China),35(1):31-33(兰玉菲,王庆武,唐丽娜,李秀梅,2016,灵芝属3个种菌丝生长的最适碳源、氮源及温度比较,中国食用菌,35(1):31-33)
Liang Z.Q.,and Chen Z.W.,2011,Study on the biological characteristics of mycelium of Ganoderma gibbosum,Zhongguo Nongxue Tongbao(Chinese Agricultural Science Bulletin),27(18):164-167(梁志群,陈子武,2011,有柄灵芝菌丝生物学特性研究,中国农学通报,27(18):164-167)
Maganhotto de Souza Silva C.M.,Soares de Melo I.,and Roberto de Oliveira P.,2005,Ligninolytic enzyme production by Ganoderma spp.,Enzyme and Microbial Technology,37(3):324-329
Menon V.,and Rao M.,2012,Trends in bioconversion of lignocellulose:Biofuels,platform chemicals&biorefinery concept,Progress in Energy and Combustion Science,38(4):522-550
Mikiashvili N.,Wasser S.P.,Nevo E.,and Elisashvili V.,2006,Effects of carbon and nitrogen sources on Pleurotus ostreatus ligninolytic enzyme activity,World Journal of Microbiology and Biotechnology,22(9):999-1002
Ntougias S.,Baldrian P.,Ehaliotis C.,Nerud F.,Antoniou T.,MerhautováV.,and Zervakis G.I.,2012,Biodegradation and detoxification of olive mill wastewater by selected strains of the mushroom genera Ganoderma and Pleurotus,Chemosphere,88(5):620-626
Qin G.J.,Wang S.S.,Chen Q.J.,Zhang H.L.,and Zhang G.Q.,2015,Isolation,identification,and biological characteristics of a wild Amauroderma mushroom,Yingyong Yu Huanjing Shengwu Xuebao(Chinese Journal of Applied&Environmental Biology),21(3):464-469(秦改娟,王珊珊,陈青君,张昊琳,张国庆,2015,一株假芝的分离鉴定与生物学特性,应用与环境生物学报,21(3):464-469)
Praveen K.,Usha Y.U.,Viswanath B.and Reddy B.R.,2012,Kinetic properties of manganese peroxidase from the mushroom stereum ostrea and its ability to decolorize dyes,Journal of Microbiology and Biotechnology,22(11):1540-1548
Salmon D.N.X.,Spier M.R.,Soccol C.R.,Vandenberghe L.P.D.S.,Weingartner Montibeller V.,Bier M.C.J.,and Faraco V.,2014,Analysis of inducers of xylanase and cellulase activities production by Ganoderma applanatum LPB MR-56,Fungal Biology,118(8):655-662
Sergentani A.G.,Gonou-Zagou Z.,Kapsanaki-Gotsi E.,and Hatzinikolaou D.G.,2016,Lignocellulose degradation potential of basidiomycota from thrace(NE Greece),International Biodeterioration&Biodegradation,114:268-277
Sitarz A.K.,Mikkelsen J.D.,H?jrup P.,and Meyer A.S.,2013,Identification of a laccase from Ganoderma lucidum CBS229.93 having potential for enhancing cellulase catalyzed lignocellulose degradation,Enzyme and Microbial Technology,53(6):378-385
Su C.L.,Tang C.H.,Zhang J.S.,and Pan Y.J.,2006,The genetic relationships of isolates of Ganoderma inferred from partialβ-tubulin gene sequences,Junwu Xuebao(Mycosystema),25(3):439-445(苏春丽,唐传红,张劲松,潘迎捷,2006,基于β-微管蛋白基因部分序列探讨灵芝属菌株的亲缘关系,菌物学报,25(3):439-445)
Wang X.C.,Xi R.J.,Li Y.,Wang D.M.,and Yao Y.J.,2012,The species identity of the widely cultivated Ganoderma,'G.lucidum'(Ling-zhi),in China,PLoS One,7(7):e40857
Zhang G.G.,Lu W.P.,Lin L.Z.,and Zou J.M.,2015,Isolation identification,mycelial cultivation chatacteristics and artificial cultivation of a wild Ganoderma gibbosum strain,Beifang Yuanyi(Northern Horticulture),(22):153-157(张国广,卢文朋,林溜章,邹金美,2015,一株野生有柄树舌的鉴定、菌丝培养特性及驯化栽培,北方园艺,(22):153-157)
Zhou L.W.,Cao Y.,Wu S.H.,Vlasák J.,Li D.W.,Li M.J.,and Dai Y.C.,2015,Global diversity of the Ganoderma lucidum complex(Ganodermataceae,Polyporales)inferred from morphology and multilocus phylogeny,Phytochemistry,114:7-15
Zhou X.W.,Cong W.R.,Su K.Q.,and Zhang Y.M.,2013,Ligninolytic enzymes from Ganoderma spp:current status and potential applications,Critical Reviews in Microbiology,39(4):416-426
Bishop K.S.,Kao C.H.J.,Xu Y.,Glucina M.P.,Paterson R.R.M.,and Ferguson L.R.,2015,From 2 000 years of Ganoderma lucidum to recent developments in nutraceuticals,Phytochemistry,114:56-65
Chai X.Y.,An S.D.,Sheng S.,and Su H.,2015,Screening of laccase-producing macrofungi and optimization of productions conditions,Xibei Nonglin Keiji Daxue(Ziran Kexxue Ban)(Jourmal of Northwest A&F University)(Natural Science Edition),43(3):205-210(柴新义,安双登,盛硕,苏海,2015,产漆酶真菌筛选及其产酶条件的优化,西北农林科技大学(自然科学版),43(3):205-210)
Chen X.D.,Zeng N.K.,and Lan J.,2010,Cultural characteristics of mycelia of Ganoderma gibbosum,Zhongguo Zhongyao Zazhi(China Journal of Chinese Material Medica),35(15):1939-1942(陈向东,曾念开,兰进,2010,药用真菌有柄树舌菌丝培养特性的研究,中国中药杂志,35(15):1939-1942)
Cilerdzic J.,Stajic M.,and Vukojevic J.,2016,Degradation of wheat straw and oak sawdust by Ganoderma applanatum,Inernational Biodeterioration&Biodegradation,114:39-44
Huang L.H.,Yang X.B.,Zhang Z.,Hu H.P.,Zhang Y.F.,and Wu Q.P.,2010,Identification of Ganoderma spawns based on ITS sequence analysis,Zhongguo Shiyongjun(Edible Fungi of China),29(1):55-57(黄龙花,杨小兵,张智,胡慧萍,张一凡,吴清平,2010,基于ITS序列分析鉴定灵芝属菌种,中国食用菌,29(1):55-57)
Jo W.S.,Cho Y.J.,Cho D.H.,Park S.D.,Yoo Y.B.,and Seok S.J.,2009,Culture conditions for the mycelial growth of Ganoderma applanatum,Mycobiology,37(2):94-102
Lan Y.F.,Wang Q.W.,Tang L.N.,and Li X.M.,2016,Comparision to mycelial growth rates of three of Ganoderma on different carbon source,nitrogen source and temperature,Zhongguo Shiyongjun(Edible Fungi of China),35(1):31-33(兰玉菲,王庆武,唐丽娜,李秀梅,2016,灵芝属3个种菌丝生长的最适碳源、氮源及温度比较,中国食用菌,35(1):31-33)
Liang Z.Q.,and Chen Z.W.,2011,Study on the biological characteristics of mycelium of Ganoderma gibbosum,Zhongguo Nongxue Tongbao(Chinese Agricultural Science Bulletin),27(18):164-167(梁志群,陈子武,2011,有柄灵芝菌丝生物学特性研究,中国农学通报,27(18):164-167)
Maganhotto de Souza Silva C.M.,Soares de Melo I.,and Roberto de Oliveira P.,2005,Ligninolytic enzyme production by Ganoderma spp.,Enzyme and Microbial Technology,37(3):324-329
Menon V.,and Rao M.,2012,Trends in bioconversion of lignocellulose:Biofuels,platform chemicals&biorefinery concept,Progress in Energy and Combustion Science,38(4):522-550
Mikiashvili N.,Wasser S.P.,Nevo E.,and Elisashvili V.,2006,Effects of carbon and nitrogen sources on Pleurotus ostreatus ligninolytic enzyme activity,World Journal of Microbiology and Biotechnology,22(9):999-1002
Ntougias S.,Baldrian P.,Ehaliotis C.,Nerud F.,Antoniou T.,MerhautováV.,and Zervakis G.I.,2012,Biodegradation and detoxification of olive mill wastewater by selected strains of the mushroom genera Ganoderma and Pleurotus,Chemosphere,88(5):620-626
Qin G.J.,Wang S.S.,Chen Q.J.,Zhang H.L.,and Zhang G.Q.,2015,Isolation,identification,and biological characteristics of a wild Amauroderma mushroom,Yingyong Yu Huanjing Shengwu Xuebao(Chinese Journal of Applied&Environmental Biology),21(3):464-469(秦改娟,王珊珊,陈青君,张昊琳,张国庆,2015,一株假芝的分离鉴定与生物学特性,应用与环境生物学报,21(3):464-469)
Praveen K.,Usha Y.U.,Viswanath B.and Reddy B.R.,2012,Kinetic properties of manganese peroxidase from the mushroom stereum ostrea and its ability to decolorize dyes,Journal of Microbiology and Biotechnology,22(11):1540-1548
Salmon D.N.X.,Spier M.R.,Soccol C.R.,Vandenberghe L.P.D.S.,Weingartner Montibeller V.,Bier M.C.J.,and Faraco V.,2014,Analysis of inducers of xylanase and cellulase activities production by Ganoderma applanatum LPB MR-56,Fungal Biology,118(8):655-662
Sergentani A.G.,Gonou-Zagou Z.,Kapsanaki-Gotsi E.,and Hatzinikolaou D.G.,2016,Lignocellulose degradation potential of basidiomycota from thrace(NE Greece),International Biodeterioration&Biodegradation,114:268-277
Sitarz A.K.,Mikkelsen J.D.,H?jrup P.,and Meyer A.S.,2013,Identification of a laccase from Ganoderma lucidum CBS229.93 having potential for enhancing cellulase catalyzed lignocellulose degradation,Enzyme and Microbial Technology,53(6):378-385
Su C.L.,Tang C.H.,Zhang J.S.,and Pan Y.J.,2006,The genetic relationships of isolates of Ganoderma inferred from partialβ-tubulin gene sequences,Junwu Xuebao(Mycosystema),25(3):439-445(苏春丽,唐传红,张劲松,潘迎捷,2006,基于β-微管蛋白基因部分序列探讨灵芝属菌株的亲缘关系,菌物学报,25(3):439-445)
Wang X.C.,Xi R.J.,Li Y.,Wang D.M.,and Yao Y.J.,2012,The species identity of the widely cultivated Ganoderma,'G.lucidum'(Ling-zhi),in China,PLoS One,7(7):e40857
Zhang G.G.,Lu W.P.,Lin L.Z.,and Zou J.M.,2015,Isolation identification,mycelial cultivation chatacteristics and artificial cultivation of a wild Ganoderma gibbosum strain,Beifang Yuanyi(Northern Horticulture),(22):153-157(张国广,卢文朋,林溜章,邹金美,2015,一株野生有柄树舌的鉴定、菌丝培养特性及驯化栽培,北方园艺,(22):153-157)
Zhou L.W.,Cao Y.,Wu S.H.,Vlasák J.,Li D.W.,Li M.J.,and Dai Y.C.,2015,Global diversity of the Ganoderma lucidum complex(Ganodermataceae,Polyporales)inferred from morphology and multilocus phylogeny,Phytochemistry,114:7-15
Zhou X.W.,Cong W.R.,Su K.Q.,and Zhang Y.M.,2013,Ligninolytic enzymes from Ganoderma spp:current status and potential applications,Critical Reviews in Microbiology,39(4):416-426