LAspartic acid

Ultrafiltration

Reactor tX—R

coryneform bacteria using electroporation (14-16) and other techniques has also been developed (17,18). Both maleate isomerase and aspartase can be overproduced in co-ryneform bacterial strain MJ-233. These recombinant cells can therefore be employed for L-aspartic acid production from maleic acid as a starting raw material.

Development of a Stable Host-Vector System. One of the important factors in the application of genetic engineering techniques for industrial use is the development of a stable host-vector system. For this purpose, we isolated various plasmids from coryneform bacteria and identified a genetic element that is important for stable plasmid maintenance (19). We then constructed a highly stable host-vector system.

Cloning and Expression of the Maleate Isomerase and As-partase Genes. Isolation of both the maleate isomerase gene and the aspartase gene from a genomic library was achieved by hybridization with DNA probes synthesized on the basis of the N-terminal amino acid sequences of each purified enzyme. Using this methodology, we cloned maleate isomerase (20) and aspartase (21) genes from the Al-caligenas faecalis and coryneform bacterial strain MJ-233, respectively, and achieved high-level expression in recombinant cells of coryneform bacterial strain MJ-233.

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