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材料。它是由苯丙烷单元构建的聚合物。在结构中，β-芳基链接是最丰富的（约50％）。 β-淀粉的切割是木质素生物降解中最重要的过程。我们已经隔离了paucimobilis的假单胞菌，能够降低β-aryl eher链接。我们在细胞膜分数中介绍了β-静脉酶的活力。在该研究计划中，我们尝试建立一个特定的修饰过程，该过程通过基因工程构建了木质素可降解paucimobilis的基因功能。首先，我们分离了β-静酶基因，该基因包含843 bp的开放式阅读框。该基因在大肠杆菌中表达，该酶具有与paucimobilis酶相同的正常。 β-醚酶的底物特异性是β-芳基醚，在calpha-losion中包含一个羰基。在p.paucimobilis中，卡尔法脱水酶在β-酰亚胺化合物的钙含量位置上催化酒精基团的氧化。然后，我们分离了钙脱水酶基因。该基因包含一个915 bp的开放式阅读框，位于β-醚酶基因上游的1 kbp中。该基因在大肠杆菌中被染色，并且该酶具有与p.paucimobilis enzme相同的正当性。我们将另一个β-静态基因鉴于上述两个基因之间。大肠杆菌中新基因的β-醚酶活性高200多倍。这两个β-静态基因对Gultathion-s-转移酶是同源，并且在谷胱甘肽加上后，在携带β-醚酶基因的大肠杆菌中观察到了β-醚酶活性的显着加速。

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,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, S.Kubota, Y.Katayama et al.: "Characterization of the Calpha-dehydrogenase gene involved in the cleavage of beta-aryl ether by Pseudomonas paucimobilis" Biosci.Biotech.Biochem.Vol.57. 1655-1659 (1993)

E.Masai、S.Kubota、Y.Katayama 等人：“参与少动假单胞菌裂解 β-芳基醚的 Cα-脱氢酶基因的表征”Biosci.Biotech.Biochem.Vol.57。