Gene cloning and characterization of L-Lysine 6-Dehydrogenase from Achromobacter denitrificans for L-Pipecolic acid production

Abstract

NAD+-dependent lysine 6-dehydrogenase (Lys 6-DH, EC 1.4.1.18) catalyzes the irreversible oxidative deamination of L-lysine to form ammonia and piperidiene-6-carboxylate. A Lys 6-DH producing bacterium strain K-1, screened from soil sample, was identified as Achromobacter denitrificans. Lys 6-DH from A. denitrificans K-1 was purified 175 fold with a 34% yield. The amino acid sequences of internal peptide fragments of Lys 6-DH were determined and used for degenerated primer design. The partial nucleotide sequence of the lysine 6-dehydrogenase gene (lys 6-dh) was investigated by PCR technique. Using inverse PCR, the whole lys 6-dh gene was obtained. The gene was cloned and expressed in E. coli BL 21(DE3) using expression vector, pET-17b. The optimum condition for lys 6-dh gene expression was induction with 0.2 mM IPTG for 4 hours. The specific activity of crude recombinant enzyme was 63 fold higher than that of the enzyme from A. denitrificans. After daily subculture for 80 days, the lys 6-dh gene expression in E. coli BL 21(DE3) remained 100% of that of the parent. Recombinant enzyme was purified 2.8 fold with a 47% yield. The enzyme had a molecular mass about 240,000 Da with 6 identical subunits. The enzyme had high substrate specificity on L-lysine and NAD+. The optimum pH and temperature were 9.3 and 50oC, respectively. The enzyme was stable over a pH range from 7.5 to 8.0. The apparent Km value for L-lysine and NAD+ of dimeric and hexameric form of this enzyme were 11.11, 0.138, 8.62 and 0.092 mM, respectively. For L-pipecolic acid production, the pyrroline-5-carboxylate reductase gene (p5cr) from Bacillus cereus ATCC 11778, encoding pyrroline-5-carboxylate reductase catalyses piperidiene-6-carboxylate to L-pipecolic acid, was co-existed with the lys 6-dh gene and transformed into E. coli BL21(DE3) using pET-17b. The optimum condition for both genes expression was induction with 0.1 mM IPTG for 3 hours. The specific activity of lysine 6-dehydrogenase and pyrroline-5-carboxylate reductase form crude enzyme were 25 and 11 folds higher than those of the enzyme from A. denitrificans and B. cereus, respectively. The highest production of L-pipecolic acid was obtained when E. coli cells was treated by xylene for 5 min. to increase cell permeability and then incubated in the reaction mixture consisited of 200 mM L-ysine in 200 mM Tris-HCl buffer, pH 9.0 for 24 hours. The amount of L-pipecolic acid production was 77 mM (9.9 g/l).