Purification and characterization of leucine dehydrogenase from Alcaligenes faecalis

Abstract

Leucine dehydrogenase producing bacteria were screened from soil sample and the isolate which produced the highest enzyme activity was identified as Alcaligenes faecalis. Optimum condition for L-Leucine dehydrogenase production was cultivation in 1% peptone medium pH 7.2 at 37ํC for 24 hours. After the enzyme was purified to homogeneity by DEAE-Toyopearl, Butyl-Toyopearl and Hitrap Q chromatography columns, % recovery and purification fold were 16.4 and 66.4, respectively. The enzyme had the molecular weight of 536,000 daltons and consisted of 10 identical subunits of 52,000 daltons. Substrats specificty of the enzyme on L-valine and L-isoleucine were 1.33 and 1.13 fold of that on L-leucine in oxidative deamination. In reductive amination, substrate specificity on [alpha]-ketoisovalerate, [alpha]-ketovalerate and [alpha]-keto-[beta]-methylvalerate were 2.0, 1.35 and 1.23 fold of that on [alpha]-ketoisocaprorate. The coenzyme specificity on 3-acetylepyridine adinine dinucleotide was about 4.78 fold higher than NAD[supercript +]. The optimum pH for oxidative deamination and reductive amination were 10.8 and 8.8 where as optimum temperatures were 45 ํC, and 55 ํC respectively. The enzyme retained its full activity upon the incubation at 45 ํC for 8 hours. The enzyme activity was completely inhibited by HgCI [subscript2] at final concentration of 1 mM. Chemical modification of the enzyme at tryptophan, methionine and lysine residues by incubation with 10 mM of group-specific reagents: N-bromosuccinimide, chloramine T and 2,4,6- trinitrobenzenesulfonic acid, respectively, led to completely loss of enzyme activity. The apparent K[subscript m] values for L-leucine, L-isoleuine, L-valine, NAD[superscript +], NADH, [alpha]-ketoisocaproate and ammonia were 4.2, 4.3, 14.0, 0.44, 0.02, 3.33 and 100 mM, respectively. The steady state kinetic studies including product inhibition on the enzyme reaction indicated that the oxidative deamination proceeds through a sequential ordered binary-ternary-ternary mechanism in which NAD[superscript +] binds first to the enzyme followed by L-leucine and products are released in the order of ammonia, [alpha]-ketoisocaproate and NADH, respectively. N-terminal sequence was M E I F N Y M E Q A D Y E Q L V I X Q D and internal amino acid sequence of the enzyme were, PGPXGPAGSKG (or V) EPGPAGPXG, T (or L, Y, V) LPGLAGTXG and RDNIPSYVAADRLAEERIRVA.