Purification and characterization of phenylalanine dehydrogenase from thermotolerant Bacillus badius BC1

Abstract

Thermotolerant bacteria producing NAD+-dependent phenylalanine dehydrogenase were screened and identified as Bacillus badius in the previous study. Two strains were obtained and strain BCl. having the highest enzyme activity, was further studied. The optimal conditions for enzyme production was 24 hours of cultivation in 1% peptone medium. pH 6.5 containing 0.8% L-phenylalanine at 37℃. The enzyme was purified to homogeneity by 40-50% saturated ammonium sulfate precipitation, DEAE-Toyopearl, first Butyl-Toyopearl and second Butyl-Toyopearl column chromatography with 20 % yield and 160.7 purification fold. The relative molecular weight of the native enzyme was estimated to be about 358,000 by gel filtration and consisted of 8 subunits identical in molecular weight (44,500). The enzyme showed high substrate specificity in the oxidative deamination on L-phenylalanine while that of the reductive amination was on phenylpyruvate. 3- Acetylpyridine-NAD+ gave 1.65 times higher activity than its natural coenzyme. NAD+. The optimum pH for the oxidative deamination and reductive amination were 10.7 and 8.3, respectively whereas optimum temperature were 50 and 45℃, respectively. The enzyme was stable over a pH range from 6.0 to ll.0. No loss of the enzyme activity was observed upon, incubation at 40℃ for 2 hours and 50% of the activity was retained after incubation at the same temperature for 30 hours. The enzyme reaction was inactivated by HgCl2, AgN03 and FeSO4. Dphenylalanine. D-tryptophan, D-methionine and o-fluoro-DL-phenylalanine significant inhibited the oxidative deamination of L-phenylalanine. The enzyme was chemically modified with a series of group specific reagents to identify essential amino acid residues. Incubation of the enzyme with 10 mM of N-bromosuccinimide (NBS), chloramine T (CT), diethylpyrocarbonate (DEPC) and 2,4,6-trinitrobenzenesulfonic acid (TNBS) which were specific for tryptophan, methionine, histidine and lysine, respectively, led to complete loss of enzyme activity. In addition 10 mM of phenylglyoxal (PG) which was specific for arginine reduced the enzyme activity to about 10%, while N-acetylimidazolc (NAI), dithiothreitol (DTT) and phenylmethylsulfonyl fluoride (PMSF), the modifiers of tyrosine, cysteine and serine, respectively, did not affect the enzyme activity. The apparent Km values for L-phenylalanine, NAD+. NADH. phenylpyruvate and ammonium were calculated to be 0.59 mM, 0.28 mM, 0.07 mM, 0.33 mM and 200 mM. respectively. Initial velocity and product inhibition studies showed that the oxidative deamination proceeded through a sequential ordered binary-ternary mechanism, in which the sequence of substrate binding to the enzyme was NAD+ and L-phenylalanine and then the sequence of product release was ammonia, phenylpyruvate and NADH, respectively.