Fundamental studies on effective utilization of wood biomass

木材生物质有效利用的基础研究

• 批准号: 05660379
• 负责人: ASADA Yasuhiko
• 金额: $ 1.02万
• 依托单位: Kagawa University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05660379/
• 关键词: Lignin; Basidiomycete; Pleurotus ostreatus; Manganese; Peroxidase; Wood biomass; 遺伝子工学

A lignin-degrading basidiomycete, Pleurotus ostreatus produces manganese-dependent peroxidase (MnP) which is considered to be one of ligninolytic enzymes. I have investigated on this enzyme enzymologically and genetically, and obtained the following results.1.P.ostreatus produced much higher amount of MnP in the medium containg Mn^<2+> than in the medium without added Mn^<2+>, indicating that MnP is induced by Mn^<2+>.2.MnP has been purified to homogeneity and characterized.3.A cDNA and genomic DNA encoding MnP were cloned and characterized. The sequnce analyzes of MnP and its cDNA revealed that a mature form of MnP consists of 332 amino acid residues preceeded by a signal peptide of 29 amino acid residues. Consistent with the peroxidase mechanism of MnP,the proximal histidine, the distal histidine, and the distal arginine are all conserved and regions flanking these residues display homology with other peroxidases. A single potential N-glycosylation site with the general sequence Asn-X-Ser/Thr is present in the deduced MnP sequence. Comparison of the sequences of cDNA and genomic DNA revealed that the protein encoding sequence of the MnP gene is interrupted by 15 introns which conform to the universal G-T/A-G splicing rule observed for the 3' and 5' intron boudaries. The putative eukaryotic regulatory sequences, "TATA" and "CAAT", are present in the 5' non-coding region of the MnP gene.4.To express the MnP-encoding cDNA in the fungus, Aspergillus oryzae a MnP expression plasmid (pMAR5-MnP) was constructed, and it was introduced into the A.oryzae strain. The MnP cDNA was expressed in A.oryzae under the alpha-amylase-encoding amybeta promoter. The MnP mRNA was produced in A.oryzae transformants. No active MnP protein, however, could be detected.

平菇是一种木质素降解担子菌，产生锰依赖性过氧化物酶 (MnP)，该酶被认为是木质素分解酶之一。本人对该酶进行了酶学和遗传学研究，得到如下结果：1.P.ostreatus在含有Mn^<2+>的培养基中产生的MnP量远高于不添加Mn^<2+>的培养基，表明MnP是由Mn^<2+>诱导的。2.MnP已被纯化至均质并进行表征。3.A cDNA和基因组DNA 编码 MnP 的基因被克隆并表征。 MnP及其cDNA的序列分析表明，成熟形式的MnP由332个氨基酸残基组成，前面是29个氨基酸残基的信号肽。与 MnP 的过氧化物酶机制一致，近端组氨酸、远端组氨酸和远端精氨酸都是保守的，并且这些残基侧翼的区域与其他过氧化物酶表现出同源性。推导的 MnP 序列中存在一个具有通用序列 Asn-X-Ser/Thr 的潜在 N-糖基化位点。 cDNA和基因组DNA序列的比较表明，MnP基因的蛋白质编码序列被15个内含子打断，这符合3'和5'内含子边界观察到的通用G-T/A-G剪接规则。假定的真核调控序列“TATA”和“CAAT”存在于MnP基因的5'非编码区。4.为了在真菌米曲霉中表达MnP编码cDNA，构建了MnP表达质粒(pMAR5-MnP)，并将其导入米曲霉菌株中。 MnP cDNA 在米曲霉中在编码α-淀粉酶的amybeta 启动子下表达。 MnP mRNA 在米曲霉转化体中产生。然而，没有检测到活性 MnP 蛋白。

项目成果

Y.Asada,M.Ichizaki,A.Watanabe,M.Kuwahara: "Purification and dharacterization of alcohol oxidase of a legnin-degrading basidiomycete,Phanerochaete chrysosporium" Tech.Bull Fac.Agr.Kagawa Univ.47(発表予定). (1995)

Y.Asada、M.Ichizaki、A.Watanabe、M.Kuwahara：“豆素降解担子菌、Phanerochaete chrysosporium 的醇氧化酶的纯化和表征”Tech.Bull Fac.Agr.Kakawa Univ.47（待提交）。 1995）

Y.Kimura, Y.Asada, M.Kuwahara: "Immunological reaction usiing antiserum for lignin peroxidase of Bjerkandera adusta" Mokuzai Gakkaishi. 40-6. 661-665 (1994)

Y.Kimura、Y.Asada、M.Kuwahara：“使用 Bjerkandera adusta 木质素过氧化物酶抗血清的免疫反应”Mokuzai Gakkaishi。

Y.Kimura,Y.Asada,M.Kuwahara: "Immunological reaction using antiserum for lignin peroxidase of bjerkandeara adusta" Mokuzai Gakkaishi. 40. 661-665 (1994)

Y.Kimura、Y.Asada、M.Kuwahara：“使用 bjerkandeara adusta 木质素过氧化物酶抗血清的免疫反应”Mokuzai Gakkaishi。

H.Sawai-Hatanaka, T.Ashikari, Y.Tanaka, Y.Asada, T.Nakayama, H.Minakata, N.Kunishita, K.Fukuyama, H.Matsubara, H.Yamada, Y.Shibano, T.Amachi: "Cloning, sequencing, and heterologous expression of a gene coding for Arthromyces ramosus peroxidase" Biosci.Bio

H.Sawai-Hatanaka、T.Ashikari、Y.Tanaka、Y.Asada、T.Nakayama、H.Minakata、N.Kunishita、K.Fukuyama、H.Matsubara、H.Yamada、Y.Shibano、T.Amachi：

H.Sawai-Hatanaka et.al.: "Cloneng and sequencing,and heterologous expression of a gene coding for Arthromyces ramosus peroxidase" Biosci.Biotech.Biochem.(発表予定). (1995)

H. Sawai-Hatanaka 等人：“编码节肢菌过氧化物酶的基因的克隆、测序和异源表达”Biosci.Biotech.Biochem（待提交）。