The development of a new method for specific cleavage of lignin beta-ether linkage by genetic engineering.

通过基因工程开发一种特异性裂解木质素β-醚键的新方法。

• 批准号: 04454088
• 负责人: KATAYAMA Yoshihiro
• 金额: $ 4.22万
• 依托单位: Tokyo University of Agriculture & Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04454088/
• 关键词: Lignin biodegradation; beta-Arylether linkage; beta-Etherase; Calpha-Dehydrogenase; DNA sequence; Gene function; Glutathione-S-transferase; Glutahione; β-エーテラーゼ; β-エーテル結合; 遣伝子機能; 塩基配列; 酵素機能領域; NAD-結合部位

Lignin is the most abundant aromatic material in the biosphere. It is a polymer constructed with phenylpropanoid units. In the structure, beta-arylether linkage is the most aboundant (approximately 50 %). Cleavage of beta-arylether is the most important process in lignin biodegradation. We already isolated Pseudomonas paucimobilis which was able to degrade beta-aryl eher linkage. And we deteced beta-etherase acivity in the cellular membrane fraction.In this research program, we try to establish a specific modification process constructed with gene functions of lignin degradable P.paucimobilis by genetic engineering. At first, we isolated the beta-etherase gene which contains an open reading frame of 843 bp. This gene was expressed in Escherichia coli, and the enzyme had the same properies as the P.paucimobilis enzyme. The substrate specificity of beta-etherase is a beta-aryl ether that contains a carbonyl group at the Calpha-position. In P.paucimobilis, Calpha-dehydrogenase catalyzes the oxidation of alcohol group at the Calpha-position of beta-arylether compound. Then, we isolated the Calpha-dehydrogenase gene. This gene contains an open reading frame of 915 bp and located in 1 kbp upstream of the beta-etherase gene. This gene was wxpressed in E.coli, and the enzyme had the same properies as the P.paucimobilis enzme.We idenified another beta-etherase gene, which lies between two genes described above. The beta-etherase activity of the new gene expresed in E.coli was more than 200 times as high as that of P.paucimoblis. These two beta-etherase genes are homologus to gultathion-S-transferase, and upon addtion of glutahione a remarkable acceleration of beta-etherase activity was observed in the E.coli carrying the beta-etherase gene.

木质素是生物圈中最丰富的芳香物质。它是一种由苯基丙烷单元构成的聚合物。在结构中，-芳醚键是最丰富的（约50%）。-芳醚的裂解是木质素生物降解过程中最重要的过程。我们已经分离出了能降解-芳基醚键的少动假单胞菌。我们检测了细胞膜部分的乙醚酶活性。在本研究项目中，我们试图通过基因工程的方法建立一个具有木质素可降解的paucimobilis基因功能的特异性修饰过程。首先，我们分离到β -醚酶基因，该基因包含一个843 bp的开放阅读框。该基因在大肠杆菌中表达，酶具有与ppaucimobilis酶相同的特性。醚酶的底物特异性是在α位置含有羰基的-芳基醚。在paucimobilis中，calpha脱氢酶催化-芳醚化合物calpha位置的醇基氧化。然后，我们分离了calpha脱氢酶基因。该基因包含一个915 bp的开放阅读框，位于β -醚酶基因上游1 kbp处。该基因在大肠杆菌中表达，该酶具有与P.paucimobilis酶相同的特性。我们确定了另一个β -醚酶基因，它位于上述两个基因之间。在大肠杆菌中表达的新基因的β -醚酶活性比paucimmoblis高200倍以上。这两种β -醚酶基因与谷胱甘肽- s -转移酶同源，在携带β -醚酶基因的大肠杆菌中，添加谷胱甘肽可显著加速β -醚酶的活性。

项目成果

E.Masai,Y.Katayama,他4名: "A bacterial enzyme degrading the model lignin compound β-etherase is a member of the glutathione-S-transferase superfamily" FEBS LETTERS. 323. 135-140 (1993)

E. Masai、Y. Katayama 和其他 4 人：“降解模型木质素化合物 β-醚酶的细菌酶是谷胱甘肽-S-转移酶超家族的成员”FEBS LETTERS 323. 135-140 (1993)。

E.Masai,S.Kubota,Y.Katayama他3名: "Characterization of the Cα-Dehydrogenase Gene Involved in the Cleavage of β-Aryl Ether by Pseudomonas paucimobilis." Bioscience Biotechnology Biochemistry. 57. 1655-1659 (1993)

E. Masai、S. Kubota、Y. Katayama 和其他 3 人：“参与少动假单胞菌裂解 β-芳基醚的 Cα-脱氢酶基因的表征。生物科学生物技术生物化学”，57。1655-1659 (1993)。

E.Masai, Y.Katayama, et al.: "A bacterial enzyme degrading the model lignin compound beta-etherase is a member of the glutathione-S-transferase superfamily" FEBS LETTERS. Vol.323. 135-140 (1993)

E.Masai、Y.Katayama 等人：“降解模型木质素化合物 β-醚酶的细菌酶是谷胱甘肽-S-转移酶超家族的成员”FEBS LETTERS。

E.Masai,Y.Katayama,他4名: "A bacterial enzyme degrading the model lignin compound β-etherase is a member of the glutathinoe-S-transferase superfamily" FEBS LETTERS. 323. 135-140 (1993)

E. Masai、Y. Katayama 和其他 4 人：“降解模型木质素化合物 β-醚酶的细菌酶是谷胱甘肽-S-转移酶超家族的成员”FEBS LETTERS 323. 135-140 (1993)。

E.Masai,S.Kubota,Y.Katayama 他3名: "Characterization of the Cα-Dehydrogenase Gene Involved in the Cleavage of β-Aryl Ether by Pseudomonas paucimobilis." Bioscience Biotechnology Biochemistry. 57. 1655-1659 (1993)

E. Masai、S. Kubota、Y. Katayama 和其他 3 人：“参与少动假单胞菌裂解 β-芳基醚的 Cα-脱氢酶基因的表征。生物科学生物技术生物化学”，57。1655-1659 (1993)。