Thermostable phenylalanine dehydrogenase from a mesophilic Microbacterium sp. strain DM 86-1
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© Springer-Verlag 1998
O R I G I N A L PA P E R
Yasuhisa Asano · Masako Tanetani
Thermostable phenylalanine dehydrogenase from a mesophilic Microbacterium sp. strain DM 86-1
Received: 30 July 1997 / Accepted: 4 November 1997
Abstract Bacteria that produced NAD+-dependent phenylalanine dehydrogenase (EC 1.4.1.20) were selected among L-methionine utilizers isolated from soil. A bacterial strain showing phenylalanine dehydrogenase activity was chosen and classified in the genus Microbacterium. Phenylalanine dehydrogenase was purified from the crude extract of Microbacterium sp. strain DM 86-1 (TPU 3592) to homogeneity as judged by SDS-polyacrylamide disc gel electrophoresis. The enzyme has an isoelectric point of 5.8 and a relative molecular weight (Mr) of approximately 330,000. The enzyme is composed of eight identical subunits with an Mr of approximately 41,000. The apparent Km values for L-phenylalanine and NAD+ were calculated to be 0.10 mM and 0.20 mM, respectively. No loss of the enzyme activity was observed upon incubation at 55° C for 10 min. Key words Phenylalanine dehydrogenase · Microbacterium sp. strain DM 86-1
orimetric microdetermination of L-phenylalanine to detect phenylketonuria in the blood of neonates in Japan (Asano et al. 1987b; Naruse et al. 1992). It is important to compare similar enzymes of different origins to understand the structure and functions of enzymes. For example, the thermostability of enzymes among similar enzymes from different origins has been discussed (Rice et al. 1996; Gerlind et al. 1997). Extensive exploitation of enzymes from nature and wide knowledge of their physicochemical properties are required for successful applications (Asano et al. 1987b; Cooper et al. 1989; Gelbard et al. 1990; Nakamura et al. 1996). The enzyme can be modified rationally after its three dimensional structure has been determined (Seah et al. 1995). In this paper, as a first step in investigating the relationship between the structure and function of phenylalanine dehydrogenases, we report the occurrence, purification, and characterization of phenylalanine dehydrogenase in a mesophile Microbacterium sp. The properties of the enzyme were compared with those of other phenylalanine dehydrogenases.
Introduction We have purified and characterized phenylalanine dehydrogenase (L-phenylalanine:NAD+ oxidoreductase, deaminating; EC 1.4.1.20) from Sporosarcina ureae (Asano et al. 1987a), Bacillus sphaericus (Asano et al. 1987a, 1990; Okazaki et al. 1988), and Bacillus badius (Asano et al. 1987b; Yamada et al. 1995). The former two enzymes were obtained in crystalline forms (Asano et al. 1987a). Much attention has been paid to phenylalanine dehydrogenase because it catalyzes the enantioselective synthesis of L-phenylalanine and related L-amino acids from their keto analogs (Asano et al. 1990). Since 1992, phenylalanine dehydrogenase has been used as a reagent in the colY. Asano (Y) · M. Tanetani Biotechnology Research Center, Toyama Prefectural University, 5180 Kurokawa, Kosugi, Toyama, 939-0398 Japan Tel.
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