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

木质素是生物圈中含量最丰富的芳香物质。它是一种由苯丙烷基团构成的聚合物。在该结构中，β-芳醚键含量最多(约50%)。β-芳醚的裂解是木质素生物降解过程中最重要的过程。我们已经分离到能够降解β-芳基Eher连接的少见假单胞菌。在本研究项目中，我们试图通过基因工程的方法，建立一种利用木质素降解少见杆菌的基因功能构建的特异性修饰过程。首先，我们克隆了含有一个843个碱基的开放阅读框的β-乙醚酶基因。该基因在大肠杆菌中得到了表达，并具有与少见葡萄球菌相同的性质。β-醚酶的底物专一性是一种β-芳基醚，它在Calpha位置上含有一个羰基。在P.paucimobilis中，Calpha-脱氢酶催化β-芳醚化合物Calpha位上的醇基的氧化。然后，我们克隆了Calpha-脱氢酶基因。该基因含有一个915个碱基的开放阅读框，位于β-醚酶基因上游1个碱基上。该基因在大肠杆菌中表达，该酶具有与少见P.paucimobilis酶相同的性质。我们鉴定了另一个β-醚酶基因，位于上述两个基因之间。在大肠杆菌中表达的新基因的β-乙醚酶活性是稀有芽孢杆菌的200多倍。这两个β-乙醚酶基因是谷胱甘肽-S-转移酶的同源基因，加入谷胱甘肽后，在携带该基因的大肠杆菌中，β-乙醚酶的活性显著提高。

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.