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

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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.