Phylogenetic Analysis and Detection on the Major Ligninolytic Enzymes System of Grifola frondosa

YIN Li-wei; CHI Yu-jie; WANG Xue-tong

Volume 23 Issue 4

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Phylogenetic Analysis and Detection on the Major Ligninolytic Enzymes System of Grifola frondosa

1.
School of Forestry , Northeast Forestry University , Harbin 150040, Heilongjiang, China

Abstract

ITS sequence of Grifola frondosa strain qx2 was amplified and sequenced (GenBank accession number:GU584099), a phylogenetic analysis was performed on G. frondosa qx2 and its related white-rot fungi. The results showed that G. frondosa and G. sordulenta had the closest genetic relationship, and G. frondosa was closer to other several white-rot fungi. The strain was stilly cultured at 25 ℃ under 4 different kinds of LNAS (Low Nitrogen Asparagine Succinic acid) culture solution, the extracellular enzyme solutions were sampled at different interval, OD values of the solutions, representing manganese peroxidase (MnP), Laccase and lignin peroxidase (LiP) activities, were measured spectrophotometrically by monitoring the oxidation of 2,6-DMP at 470 nm, ABTS at 420 nm and veratryl alcohol(VA) at 310 nm, the lignin-degrading enzymes of the fungus and the relationships between enzyme activities and medium composition and substrates were gained. Results indicated that G. frondosa could produce MnP and Laccase simultaneously, but no Lip. Substrates wood sawdust could slightly enhanced MnP and Laccase activities. The biggest MnP and Laccase activity were 2.96 U·L-1 and 4.49 U·L-1 when no substrates were added, and 8.06 U·L-1 and 9.85 U·L-1 when substrates added.
- Grifola frondosa,
- ITS sequences,
- ligninolytic enzymes,
- manganese peroxidase,
- Laccase

References

[1]	Alic M, Akileswaran L, Gold M H. Characterization of the gene encoding manganese peroxidase isozyme 3 from Phanerochaete chrysosporium[J]. Biochimica et Biophysica Acta, 1997, 1338:1-7
[2]	Tello M, Corsini G, Larrondo L F, et al. Characterization of three new manganese peroxidase genes from the ligninolytic basidiomycete Ceriporiopsis subvermispora[J]. Biochimica et Biophysica Acta, 2000, 1490:137-144
[3]	Irie T, Honda Y, Ha H C, et al. Isolation of cDNA and genomic fragments encoding the major manganese peroxidase isozyme from the white rot basidiomycete Pleurotus ostreatus[J]. J Wood Sci, 2000, 46:230-233
[4]	Johansson T, Nyman P O, Cullen D. Differential regulation of mnp2, a new manganese peroxidase encoding gene from the ligninolytic fungus Trametes versicolor PRL 572[J]. Applied and Environmental Microbiology, 2002, 68:2077-2080
[5]	Hildén K, Martinez A T, Hatakka A, et al. The two manganese peroxidases Pr-MnP2 and Pr-MnP3 of Phlebia radiata, a lignin-degrading basidiomycete, are phylogenetically and structurally divergent[J]. Fungal Genetics and Biology, 2005,42:403-419
[6]	孙迅. 粗毛栓菌的木质纤维素降解酶及其基因克隆 . 成都:四川大学, 2004
[7]	官敏. 好食脉孢菌产锰过氧化物酶培养条件的优化、酶的纯化以及酶学性质的分析 . 广州:暨南大学, 2006
[8]	闫洪波. 偏肿拟栓菌锰过氧化物酶cDNA基因克隆及在毕赤酵母中的表达 . 哈尔滨:东北林业大学, 2009
[9]	张银波, 姜琼, 江木兰, 等. 金针菇漆酶基因的克隆及其在毕赤酵母中的表达研究[J] . 微生物学报,2004 , 44(6):775-779
[10]	谌斌, 唐雪明, 沈微. 粗糙脉孢菌漆酶基因的克隆及在毕赤酵母中的初步表达[J] .食品与生物技术学报, 2006, 25(4):43-54
[11]	Cui F J, Tao W Y, Xu Z H, et al. Structural analysis of anti-tumor heteropolysaccharide GFPS1b from the cultured mycelia of Grifola frondosa GF9801 [J]. Bioresource Technology, 2007, 98:395-401
[12]	Lee B C, Bae J T, Pyo H B, et al. Biological activities of the polysaccharides produced from submerged culture of the edible Basidiomycete Grifola frondosa [J]. Enzyme and Microbial Technology, 2003, 32: 574–581
[13]	王海燕. 灰树花GPD启动子及药效相关基因的克隆 . 海口: 华南热带农业大学, 2004
[14]	崔凤杰. 灰树花深层发酵条件优化及其菌丝体抗肿瘤糖肽的研究 . 无锡: 江南大学, 2006
[15]	池玉杰, 李俊涛, 刘智会. 灰树花的培养特性与液体菌种栽培技术[J]. 东北林业大学学报, 2007, 35(3) : 49-52, 58
[16]	Shen Q, Geiser D M, Royse D J . Molecular phylogenetic analysis of Grifola frondosa (maitake) reveals a species partition separating eastern North American and Asian isolates [J]. Mycologia, 2002, 94(3) : 472–482
[17]	张美彦, 尚晓冬, 李玉, 等. ITS2RFLP及SRAP标记在灰树花种质评价中的应用[J] . 食用菌学报, 2009,16 (3) : 5-10
[18]	Maeda Y, Kajiwara S, Ohtaguchi K. Manganese peroxidase gene of the perennial mushroom Elfvingia applanata:cloning and evaluation of its relationship with lignin degradation[J] . Biotechnology Letters, 2001, 23: 103-109
[19]	Lankinen P, Hilden K, Aro N, et al. Manganese peroxidase of Agaricus bisporus: grain bran-promoted production and gene characterization[J]. Appl Microbiol Biotechnol, 2005, 66:401-407
[20]	Nagai M, Sakamoto Y, Nakade K, et al. Isolation and characterization of the gene encoding a manganese peroxidase from Lentinula edodes[J]. Mycoscience, 2007, 48: 125-130
[21]	Kirk T K, Schultz E, Conors W J, et al. Influence of culture parameters on lignin melabolism by Phanerochaete chrysosporium[J]. Arch Microbiol, 1978, 117: 277-285
[22]	Hatakka A, Uusi-r A K. Degradation of ¹⁴C-labelled poplar wood lignin by selected white-rot fungi[J]. Eur J Appl Microbiol Biotechnol, 198 , 17:235-242
[23]	Eggert C, Temp U, Eriksson K E. The ligninolytic system of the white-rot fungus Pycnoporus cinnabarinus: purification and characterization of the laccase [J]. Applied and Environmental Microbiology, 1996, 62 : 1151-1158
[24]	WariishiI H, Valli K, Gold M H. Manganese (II) oxidation by manganese peroxidase from the basidiomycete Phanerochaete chrysosporium kinetic mechanism and role of chelators[J]. Journal of Biological Chemistry, 1992, 267(33) : 23688-23695
[25]	Tien M, Kirk T K. Lignin peroxidase of Phanerochaete chrysosporium[J]. Methods in Enzymology, 1988, 161: 238-249
[26]	Chi Y J, Hatakka A, Maijala P. Can co-culturing of two white-rot fungi increase lignin degradation and the production of lignin-degrading enzymes?[J]. International Biodeterioration and Biodegradation, 2007, 59(1): 32-39
[27]	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[J] . 林业科学, 2009, 45(12):154-158

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

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Phylogenetic Analysis and Detection on the Major Ligninolytic Enzymes System of Grifola frondosa

1. School of Forestry , Northeast Forestry University , Harbin 150040, Heilongjiang, China

Abstract: ITS sequence of Grifola frondosa strain qx2 was amplified and sequenced (GenBank accession number:GU584099), a phylogenetic analysis was performed on G. frondosa qx2 and its related white-rot fungi. The results showed that G. frondosa and G. sordulenta had the closest genetic relationship, and G. frondosa was closer to other several white-rot fungi. The strain was stilly cultured at 25 ℃ under 4 different kinds of LNAS (Low Nitrogen Asparagine Succinic acid) culture solution, the extracellular enzyme solutions were sampled at different interval, OD values of the solutions, representing manganese peroxidase (MnP), Laccase and lignin peroxidase (LiP) activities, were measured spectrophotometrically by monitoring the oxidation of 2,6-DMP at 470 nm, ABTS at 420 nm and veratryl alcohol(VA) at 310 nm, the lignin-degrading enzymes of the fungus and the relationships between enzyme activities and medium composition and substrates were gained. Results indicated that G. frondosa could produce MnP and Laccase simultaneously, but no Lip. Substrates wood sawdust could slightly enhanced MnP and Laccase activities. The biggest MnP and Laccase activity were 2.96 U·L-1 and 4.49 U·L-1 when no substrates were added, and 8.06 U·L-1 and 9.85 U·L-1 when substrates added.

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Reference (27)

[1]	Alic M, Akileswaran L, Gold M H. Characterization of the gene encoding manganese peroxidase isozyme 3 from Phanerochaete chrysosporium[J]. Biochimica et Biophysica Acta, 1997, 1338:1-7
[2]	Tello M, Corsini G, Larrondo L F, et al. Characterization of three new manganese peroxidase genes from the ligninolytic basidiomycete Ceriporiopsis subvermispora[J]. Biochimica et Biophysica Acta, 2000, 1490:137-144
[3]	Irie T, Honda Y, Ha H C, et al. Isolation of cDNA and genomic fragments encoding the major manganese peroxidase isozyme from the white rot basidiomycete Pleurotus ostreatus[J]. J Wood Sci, 2000, 46:230-233
[4]	Johansson T, Nyman P O, Cullen D. Differential regulation of mnp2, a new manganese peroxidase encoding gene from the ligninolytic fungus Trametes versicolor PRL 572[J]. Applied and Environmental Microbiology, 2002, 68:2077-2080
[5]	Hildén K, Martinez A T, Hatakka A, et al. The two manganese peroxidases Pr-MnP2 and Pr-MnP3 of Phlebia radiata, a lignin-degrading basidiomycete, are phylogenetically and structurally divergent[J]. Fungal Genetics and Biology, 2005,42:403-419
[6]	孙迅. 粗毛栓菌的木质纤维素降解酶及其基因克隆 . 成都:四川大学, 2004
[7]	官敏. 好食脉孢菌产锰过氧化物酶培养条件的优化、酶的纯化以及酶学性质的分析 . 广州:暨南大学, 2006
[8]	闫洪波. 偏肿拟栓菌锰过氧化物酶cDNA基因克隆及在毕赤酵母中的表达 . 哈尔滨:东北林业大学, 2009
[9]	张银波, 姜琼, 江木兰, 等. 金针菇漆酶基因的克隆及其在毕赤酵母中的表达研究[J] . 微生物学报,2004 , 44(6):775-779
[10]	谌斌, 唐雪明, 沈微. 粗糙脉孢菌漆酶基因的克隆及在毕赤酵母中的初步表达[J] .食品与生物技术学报, 2006, 25(4):43-54
[11]	Cui F J, Tao W Y, Xu Z H, et al. Structural analysis of anti-tumor heteropolysaccharide GFPS1b from the cultured mycelia of Grifola frondosa GF9801 [J]. Bioresource Technology, 2007, 98:395-401
[12]	Lee B C, Bae J T, Pyo H B, et al. Biological activities of the polysaccharides produced from submerged culture of the edible Basidiomycete Grifola frondosa [J]. Enzyme and Microbial Technology, 2003, 32: 574–581
[13]	王海燕. 灰树花GPD启动子及药效相关基因的克隆 . 海口: 华南热带农业大学, 2004
[14]	崔凤杰. 灰树花深层发酵条件优化及其菌丝体抗肿瘤糖肽的研究 . 无锡: 江南大学, 2006
[15]	池玉杰, 李俊涛, 刘智会. 灰树花的培养特性与液体菌种栽培技术[J]. 东北林业大学学报, 2007, 35(3) : 49-52, 58
[16]	Shen Q, Geiser D M, Royse D J . Molecular phylogenetic analysis of Grifola frondosa (maitake) reveals a species partition separating eastern North American and Asian isolates [J]. Mycologia, 2002, 94(3) : 472–482
[17]	张美彦, 尚晓冬, 李玉, 等. ITS2RFLP及SRAP标记在灰树花种质评价中的应用[J] . 食用菌学报, 2009,16 (3) : 5-10
[18]	Maeda Y, Kajiwara S, Ohtaguchi K. Manganese peroxidase gene of the perennial mushroom Elfvingia applanata:cloning and evaluation of its relationship with lignin degradation[J] . Biotechnology Letters, 2001, 23: 103-109
[19]	Lankinen P, Hilden K, Aro N, et al. Manganese peroxidase of Agaricus bisporus: grain bran-promoted production and gene characterization[J]. Appl Microbiol Biotechnol, 2005, 66:401-407
[20]	Nagai M, Sakamoto Y, Nakade K, et al. Isolation and characterization of the gene encoding a manganese peroxidase from Lentinula edodes[J]. Mycoscience, 2007, 48: 125-130
[21]	Kirk T K, Schultz E, Conors W J, et al. Influence of culture parameters on lignin melabolism by Phanerochaete chrysosporium[J]. Arch Microbiol, 1978, 117: 277-285
[22]	Hatakka A, Uusi-r A K. Degradation of ¹⁴C-labelled poplar wood lignin by selected white-rot fungi[J]. Eur J Appl Microbiol Biotechnol, 198 , 17:235-242
[23]	Eggert C, Temp U, Eriksson K E. The ligninolytic system of the white-rot fungus Pycnoporus cinnabarinus: purification and characterization of the laccase [J]. Applied and Environmental Microbiology, 1996, 62 : 1151-1158
[24]	WariishiI H, Valli K, Gold M H. Manganese (II) oxidation by manganese peroxidase from the basidiomycete Phanerochaete chrysosporium kinetic mechanism and role of chelators[J]. Journal of Biological Chemistry, 1992, 267(33) : 23688-23695
[25]	Tien M, Kirk T K. Lignin peroxidase of Phanerochaete chrysosporium[J]. Methods in Enzymology, 1988, 161: 238-249
[26]	Chi Y J, Hatakka A, Maijala P. Can co-culturing of two white-rot fungi increase lignin degradation and the production of lignin-degrading enzymes?[J]. International Biodeterioration and Biodegradation, 2007, 59(1): 32-39
[27]	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[J] . 林业科学, 2009, 45(12):154-158

Phylogenetic Analysis and Detection on the Major Ligninolytic Enzymes System of Grifola frondosa

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通讯作者: 陈斌, bchen63@163.com

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Phylogenetic Analysis and Detection on the Major Ligninolytic Enzymes System of Grifola frondosa

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