Isolation of l-phenylalanine dehydrogenase from Rhodococcus sp. M4 and its application for the production of l-phenylalanine

Hummel, Werner; Schütte, H. R.; Schmidt, E. T.; Wandrey, Christian; Kula, Maria‐Regina

doi:10.1007/bf00253523

Cited by 124 publications

(47 citation statements)

References 15 publications

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“…The following standard proteins were used to make a calibration curve: ferritin (molecular weight, 445,000), B. sphaericus alanine dehydro- genase (231,000), lactate dehydrogenase (145,000), bovine serum albumin (67,000), and ovalbumin (43,000). The subunit molecular weight was determined by SDS-polyacrylamide gel electrophoresis with the following marker proteins: bovine serum albumin (molecular weight, 67,000), catalase (57,500), ovalbumin (43,000), ,-lactoglobulin (18,400), and cytochrome c (13,000 T. intermedius IFO 14230 exhibited the highest specific activity of phenylalanine dehydrogenase and produced the enzyme most abundantly. This strain was used for enzyme purification.…”

Section: [4r-2h]-and [4s-2h]mentioning

confidence: 99%

“…The absence of enhancement with L-phenylalanine in enzyme production is peculiar to the T. intermediius IFO 14230 enzyme. Enzyme production in other microorganisms is significantly enhanced by addition of L-phenylalanine to basal medium containing peptone or corn steep liquor (1,3,13,19).…”

Section: [4r-2h]-and [4s-2h]mentioning

confidence: 99%

“…isolated from soil (14) and was purified from several mesophiles, Sporosarcina ureae (1, 3), Bacillus sphaericus (3), Bacillus badius (4), Rhodococcus sp. (5,13), Rhodococcus maris (17), and Nocardia sp. (9), for characterization.…”

mentioning

confidence: 99%

“…(9), for characterization. The enzyme is important for the synthesis of Lphenylalanine and its related L amino acids from the corresponding oxo analogs and ammonia (2,12,13). We have shown that leucine dehydrogenase (EC 1.4.1.9) of a thermophile is much more stable than that of a mesophile not only at high temperatures but also under various other conditions (20).…”

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Distribution, purification, and characterization of thermostable phenylalanine dehydrogenase from thermophilic actinomycetes

et al. 1991

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Phenylalanine dehydrogenase (L-phenylalanine:NAD oxidoreductase, deaminating; EC 1.4.1.-) was found in various thermophilic actinomycetes. We purified the enzyme to homogeneity from Thermoactinomyces intermedius IFO 14230 by heat treatment and by Red Sepharose 4B, DEAE-Toyopearl, Sepharose CL-4B, and Sephadex G-100 chromatographies with a 13% yield. The relative molecular weight of the native enzyme was estimated to be about 270,000 by gel filtration. The enzyme consists of six subunits identical in molecular weight (41,000) and is highly thermostable: it is not inactivated by incubation at pH 7.2 and 70°C for at least 60 min or in the range of pH 5 to 10.8 at 50°C for 10 min. The enzyme preferably acts on L-phenylalanine and its 2-oxo analog, phenylpyruvate, in the presence of NAD and NADH, respectively. Initial velocity and product inhibition studies showed that the oxidative deamination proceeds through a sequential ordered binary-ternary mechanism. The Km values for L-phenylalanine, NAD, phenylpyruvate, NADH, and ammonia were 0.22, 0.078, 0.045, 0.025, and 106 mM, respectively. The pro-S hydrogen at C-4 of the dihydronicotinamide ring of NADH was exclusively transferred to the substrate.Phenylalanine dehydrogenase (L-phenylalanine:NAD oxidoreductase, deaminating; EC 1.4.1.-) catalyzes the reversible deamination of L-phenylalanine to phenylpyruvate and ammonia in the presence of NAD. It was first found in Brevibacterium sp. isolated from soil (14) and was purified from several mesophiles, Sporosarcina ureae (1, 3), Bacillus sphaericus (3), Bacillus badius (4), Rhodococcus sp. (5, 13), Rhodococcus maris (17), and Nocardia sp. (9), for characterization. The enzyme is important for the synthesis of Lphenylalanine and its related L amino acids from the corresponding oxo analogs and ammonia (2,12,13). We have shown that leucine dehydrogenase (EC 1.4.1.9) of a thermophile is much more stable than that of a mesophile not only at high temperatures but also under various other conditions (20). The thermostable leucine dehydrogenase is advantageous to L-leucine production in a membrane reactor (25) and for analysis of substrate amino acids and oxo acids (23). The thermostable enzyme has been studied to elucidate the relationship between structure and catalytic function (18). We first found the thermostable phenylalanine dehydrogenase in a thermophilic actinomycete, Thermoactinomyces intermedius IFO 14230, and showed that it is useful for the selective determination of L-phenylalanine and phenylpyruvate (24).In this report, we describe the distribution of phenylalanine dehydrogenase in thermophiles and the purification and characterization of the enzyme from T. intermedius IFO 14230. MATERIALS AND METHODSMaterials. Standard proteins for molecular weight determination were purchased from Sigma Chemical, DEAE-

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Section: [4r-2h]-and [4s-2h]mentioning

confidence: 99%

Section: [4r-2h]-and [4s-2h]mentioning

confidence: 99%

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confidence: 99%

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Distribution, purification, and characterization of thermostable phenylalanine dehydrogenase from thermophilic actinomycetes

et al. 1991

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“…In contrast, PheDH from Rhodococcus sp. M4 shows 34-fold discrimination in favor of L-phenylalanine (3). Similarly, with the enzymes from Bacillus badius and Sporosarcina ureae, tyrosine gives only 9 and 5.4%, respectively, of the specific activity with phenylalanine (1,4).…”

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Single Amino Acid Substitution in Bacillus sphaericus Phenylalanine Dehydrogenase Dramatically Increases Its Discrimination between Phenylalanine and Tyrosine Substrates

et al. 2002

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Homology-based modeling of phenylalanine dehydrogenases (PheDHs) from various sources, using the structures of homologous enzymes Clostridium symbiosum glutamate dehydrogenase and Bacillus sphaericus leucine dehydrogenase as a guide, revealed that an asparagine residue at position 145 of B. sphaericus PheDH was replaced by valine or alanine in PheDHs from other sources. This difference was proposed to be the basis for the poor discrimination by the B. sphaericus enzyme between the substrates L-phenylalanine and L-tyrosine. Residue 145 of this enzyme was altered, by site-specific mutagenesis, to hydrophobic residues alanine, valine, leucine, and isoleucine, respectively. The resultant mutants showed a high discrimination, above 50-fold, between L-phenylalanine and L-tyrosine. This higher specificity toward L-phenylalanine was due to K m values for L-phenylalanine lowered more than 20-fold compared to the values for L-tyrosine. The greater specificity for L-phenylalanine in the wild-type Bacillus badius enzyme, which has a valine residue in the corresponding position, was also found to be largely due to a lower K m for this substrate. Activities were also measured with a range of six amino acids with aliphatic, nonpolar side chains, and with the corresponding oxoacids, and in all cases the specificity constants for these substrates were increased in the mutant enzymes. As with phenylalanine, these increases are mainly attributable to large decreases in K m values.The label phenylalanine dehydrogenase (PheDH 1 ) (EC 1.4.1.20) denotes a class of enzymes that catalyze the reversible NAD + -dependent oxidative deamination of a broad range of hydrophobic amino acid substrates, with particular preference for aromatic side chains. The enzyme from Bacillus sphaericus is unusual within this class since its activities toward L-phenylalanine and L-tyrosine are similar (1, 2). In contrast, PheDH from Rhodococcus sp. M4 shows 34-fold discrimination in favor of L-phenylalanine (3). Similarly, with the enzymes from Bacillus badius and Sporosarcina ureae, tyrosine gives only 9 and 5.4%, respectively, of the specific activity with phenylalanine (1, 4). It is of interest to understand the structural basis for this difference in discrimination between aromatic amino acid substrates that differ by only a single hydroxyl substituent. Determining the molecular details of substrate recognition in the family of amino acid dehydrogenases has also practical relevance, as these enzymes are useful as biocatalysts for stereospecific synthesis of amino acids, and as diagnostic reagents for a variety of inborn errors of amino acid metabolism. Indeed, the thermostability of PheDH from B. sphaericus would recommend its use as a diagnostic reagent. However, major interference from tyrosine makes it unsuitable for measuring serum L-phenylalanine levels in the diagnosis of phenylketonuria (PKU) (2).The rational design of enzymes with altered substrate specificities for use in chemical synthesis or clinical diagnosis is a major goal of our ...

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Enzyme engineering aspects of biocatalysis: Cofactor regeneration as example

Kragl

Kruse

Hummel

et al. 2000

Biotechnol. Bioeng.

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112

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Reaction engineering is an important tool in the case of cofactor depending enzyme‐catalyzed reactions. It allows the establishment of conditions resulting in lower product specific cofactor costs as compared with product‐specific enzyme costs. This is shown for the stereospecific reduction of carbonyl compounds yielding chiral amino acids and alcohols. In continuous processes, cofactor costs can be reduced if the cofactor can be retained within the bioreactor or recycled into it after separation of the product. In case of readily water‐soluble substrates it is even possible to recycle the cofactor during a single pass through a continuously operated reactor more than 4000 times because normally very low cofactor concentrations are sufficient to saturate the enzymes involved. L‐tert‐Leucine has been produced by reductive amination with a space‐time yield of up to 366 g L−1 d−1 in a single continuously operated enzyme membrane reactor and a two‐stage cascade. Total turnover number of the cofactor NAD+ increased to 4230. (S)‐1‐Phenyl‐2‐propanol was obtained by reduction of the corresponding ketone in an membrane reactor with integrated extraction of the product. A new alcohol dehydrogenase from Rhodococcus erythropolis was used. A space‐time yield of 63 g L−1 d−1 and a total turnover number of 1350 have been reached. L‐Leucine has been produced using polymer‐enlarged NADH. The total turnover number was 80,000 at a space‐time yield of 214 g L−1 d−1. © 1996 John Wiley & Sons, Inc.

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Isolation of l-phenylalanine dehydrogenase from Rhodococcus sp. M4 and its application for the production of l-phenylalanine

Cited by 124 publications

References 15 publications

Distribution, purification, and characterization of thermostable phenylalanine dehydrogenase from thermophilic actinomycetes

Distribution, purification, and characterization of thermostable phenylalanine dehydrogenase from thermophilic actinomycetes

Single Amino Acid Substitution in Bacillus sphaericus Phenylalanine Dehydrogenase Dramatically Increases Its Discrimination between Phenylalanine and Tyrosine Substrates

Enzyme engineering aspects of biocatalysis: Cofactor regeneration as example

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