Production Of L-phenylalanine Research Articles

Production of L-phenylalanine by a mutant of Brevibacterium lactofermentum 2256.

Excellent L-phenylalanine producers were screened from among tyrosine, methionine doubly auxotrophic and antimetabolite resistant mutants derived from a L-glutamic acid-producing bacterium; Brevibacterium lactofermentum 2256. A selected mutant, Brevibacterium lactofermentum No. 123, produced 21.7mg/ml of L-phenylalanine in 72 hr when 13% glucose was supplied as a carbon source. The accumulation of L-phenylalanine by strain No. 123 became 5.17-fold higher than that by parental strain No. 808. This yield was the highest among those so far reported. The addition of fumaric acid to the culture medium promoted the L-phenylalanine production. Accumulation of other amino acids in the culture broth was negligible compared with that of the main product, L-phenylalanine.

Enzymatic production of L-phenylalanine from trans-cinnamic acid by Endomyces lindneri.

Enzymatic production of L-phenylalanine from trans-cinnamic acid by Endomyces lindneri.

L-Phenylalanine dehydrogenase from Brevibacterium sp. for production of l-phenylalanine by reductive amination of phenylpyruvate

For production of l-phenylalanine the reductive amination of phenylpyruvate, catalyzed by phenylalanine-dehydrogenase was examined. To reach high levels and a sufficient stability of the inducible intracellular enzyme, growth conditions of Brevibacterium sp. are optimized. For continuous production of l-phenylalanine in an enzyme membrane reactor, the kinetic parameters of the partially purified enzyme are determined. In continuous production a space time yield of 37.4 g l-Phe l-1 d-1 can be reached. By means of the measured kinetic parameters and simultaneous calculation of the mass balances of all reaction components the behaviour of the reactor can be simulated. For certain conditions the multi-enzyme-system shows multiple steady-states.

Enzymatic production of l-phenylalanine from acetamidocinnamic acid: breeding of an acetamidocinnamate amidohydrolase-hyperproducing mutant

To increase the productivity of l-phenylalanine from acetamidocinnamic acid, we screened bacteria containing high acetamidocinnamate amidohydrolase activity, and strain S-5 containing high activity was isolated from soil. The bacteria were identified as Corynebacterium sp. S-5. When strain S-5 was cultured in a medium containing acetamidocinnamic acid as the sole carbon source or enzyme inducer, the formation of acetamidocinnamate amidohydrolase was observed. This was controlled by catabolite repression. When the strain was cultured in a medium containing glucose and acetamidocinnamic acid as the sole nitrogen source, it showed low acetamidocinnamate amidohydrolase activity and an increased doubling time. To obtain acetamidocinnamate amidohydrolase-hyperproducing strain, we enriched cells growing faster than strain S-5 in a medium containing glucose and acetamidocinnamic acid by continuous culture of mutagenized cells. Mutant C-23 had 12-fold the enzyme production and 3-fold the growth rate of the wild-type strain in a medium containing glucose. Acetamidocinnamate amidohydrolase formation in the mutant did not require acetamidocinnamic acid as enzyme inducer and was resistant to catabolite repression.

L-PHENYLALANINE PRODUCTION IN CONTINUOUS CULTURE USING A HYPERPRODUCING MUTANT OF ESCHERICHIA COLI K-12

Abstract Continuous production of L-phenylalanine was studied using a hyperproducing regulatory mutant strain of Escherichia coli K-12. From chemostat studies it was established that phosphate was the most efficient growth-limiting nutrient in phenylalanine production. Under these conditions (medium containing 1300 mg/1 K2HPO4) a maximal phenylalanine concentration of 16.4 g/1 was achieved at a dilution rate of 0.03 hr−1. It was also found that the specific production rates and production yields were reduced at higher cell densities presumably due to partial inhibition at the high phenylalanine concentrations. By comparison with other microorganisms used for commercial phenylalanine production, a process using E. coli offers the advantages of higher productivities together with the use of defined media which may be more suitable for efficient product recovery.

Production of L-phenylalanine from acetamidocinnamic acid usingBacillus sphaericus andParacoccus denitrificans

To establish an efficient production method for L-phenylalanine (L-Phe), the possibility of L-Phe production from acetamidocinnamic acid by a two-step enzyme reaction was investigated.Bacillus sphaericus N-7 (FERM-P no. 6746) was selected as an enzyme source for the acylase used to form phenylpyruvic acid from aceta midocinnamic acid.Paracoccus denitrificans IFO 12442 was selected as an aminotransferase source for the conversion of phenylpyruvic acid to L-Phe. By using both cell types, 0.46M L-Phe was produced in a molar yield of 92% from 0.5M acetamidocinnamic acid.

Pilot-scale production of l-phenylalanine from d-glucose

Effects of inoculum size and total sugar content on both l-phenylalanine productivity and titre have been investigated using a tyrosine auxotrophic regulatory mutant of Escherichia coli. Fermentations were carried out in a 500 litre pilot fermenter with intermittent feeding of d-glucose plus phosphate. It was found that the productivity was not greatly affected by inoculum size. However, the l-phenylalanine titre was significantly affected by total sugar content. Relatively high productivities of up to 0.35–0.40 g l-phenylalanine l −1 h −1 have been achieved at l-phenylalanine titres of 14–15 g l −1.

The fermentation process for l-phenylalanine production using an auxotrophic regulatory mutant of Escherichia coli

A process for l-phenylalanine production was studied using a tyrosine auxotrophic regulatory mutant of Escherichia coli, resistant to both β-2-thienyl-dl-alanine and p-fluoro-dl-phenylalanine. Fermentations were carried out in a 30-1 fermentor with intermittent feeding of glucose plus phosphate. The mutant accumulated l-phenylalanine in the fermentation broth up to 15 g/l at pH 7.0 and 33°C. Column chromatography on a strong cation exchanger was employed as the most effective step in the purification of l-phenyl-alanine from the broth. This step brought about 4-fold concentration of the product with 96% recovery.

L-phenylalanine production by auxotrophic regulatory mutants ofEscherichia coli—L-phenylalanine production by mutants ofE. coli

Various regulatory mutants ofEscherichia coli have been isolated using phenylalanine and tyrosine analogues. It has been found that the growth of wild type strain ofE. coli W3110 was strongly inhibited by phenylalanine analogues. Regulatory mutants resistant to phenylalanine analogue could accumulate b-phenylalanine at concentrations of 5–6 g/l. However, L-phenylalanine accumulation was increased significantly up to 11.4 g/1 using a tyrosine auxotrophic mutant resistant to phenylalanine analogue such as β-2-thienyl-DL-alanine.

L-Phenylalanine Production by Analog-resistant Mutants of Corynebacterium glutamicum

Mutants resistant to phenylalanine- or tyrosine-analogs were isolated from a wild type strain of Corynebacterium glutamicum ATCC 13032 and a tyrosine auxotroph TL-3 by mutagenic treatment with N-methyl-N’-nitro-N-nitroso guanidine (NTG) and screened for l-phenylalanine production. The growth of C. glutamicum ATCC 13032 was strongly inhibited by a phenylalanine analog, p-fluorophenylalanine (PFP), at a concentration of 1 mg/ml. C. glutamicum TL-3 was inhibited by PFP and another phenylalanine analog, β-2-thienylalanine (TA), at a concentration of 50 μg/ml and 400 μg/ml respectively. A prototrophic mutant resistant to PFP (4 mg/ml) produced l-phenylalanine at a concentration of 5.5 mg/ml and a trace amount of l-tyrosine in a cane molasses medium containing 10% of sugar calculated as glucose. A tyrosine auxotrophic mutant resistant to PFP (100 μg/ml) and p-aminophenyl-alanine (PAP) (1 mg/ml), 31–PAP–20–22, produced L-phenylalanine at a concentration of 9.5 mg/ml in the molasses medium. l-Phenylalanine production in these mutants was inhibited by l-tyrosine and stimulated by l-tryptophan.

L-Phenylalanine Production by Analog-resistant Mutants ofCorynebacterium glutamicum

Mutants resistant to phenylalanine- or tyrosine-analogs were isolated from a wild type strain of Corynebacterium glutamicum ATCC 13032 and a tyrosine auxotroph TL-3 by mutagenic treatment with N-methyl-N’-nitro-N-nitroso guanidine (NTG) and screened for l-phenylalanine production. The growth of C. glutamicum ATCC 13032 was strongly inhibited by a phenylalanine analog, p-fluorophenylalanine (PFP), at a concentration of 1 mg/ml. C. glutamicum TL-3 was inhibited by PFP and another phenylalanine analog, β-2-thienylalanine (TA), at a concentration of 50 μg/ml and 400 μg/ml respectively. A prototrophic mutant resistant to PFP (4 mg/ml) produced l-phenylalanine at a concentration of 5.5 mg/ml and a trace amount of l-tyrosine in a cane molasses medium containing 10% of sugar calculated as glucose. A tyrosine auxotrophic mutant resistant to PFP (100 μg/ml) and p-aminophenyl-alanine (PAP) (1 mg/ml), 31–PAP–20–22, produced L-phenylalanine at a concentration of 9.5 mg/ml in the molasses medium. l-Phenylalanine produc...

Microbial Production ofl-Phenylalanine fromn-Alkanes

A tyrosine auxotroph derived from a hydrocarbon utilizing bacterium, Corynebacterium sp. KY 4309, was found to accumulate a large amount of l-phenylalanine in the broth. The cultural conditions for l-phenylalanine production were studied. The pH value during cultivations exhibited a remakable effect on l-phenylalanine production. The addition of l-tryptophan enhanced the l-phenylalanine accumulation. Shikimic acid and phenylpyruvic acid are possible precursors of phenylalanine biosynthesis in this bacterium. Production of l-phenylalanine attained to a level of 10 mg per ml for 68 hr under optimal conditions.

Microbial Production of L-Phenylalanine from n-Alkanes

Microbial Production of L-Phenylalanine from n-Alkanes

Amino Acid Isomerization in the Production of l-Phenylalanine from d-Phenylalanine by Bacteria1

To establish an advantageous method for the production of l-amino acids, microbial isomerization of d- and dl-amino acids to l-amino acids was studied. Screening experiments on a number of microorganisms showed that cell suspensions of Pseudomonas fluorescens and P. miyamizu were capable of isomerizing d- and dl-phenylalanines to l-phenylalanine. Various conditions suitable for isomerization by these organisms were investigated. Cells grown in a medium containing d-phenylalanine showed highest isomerization activity, and almost completely converted d- or dl-phenylalanine into l-phenylalanine within 24 to 48 hr of incubation. Enzymatic studies on this isomerizing system suggested that the isomerization of d- or dl-phenylalanine is not catalyzed by a single enzyme, "amino acid isomerase," but the conversion proceeds by a two step system as follows: d-pheylalanine is oxidized to phenylpyruvic acid by d-amino acid oxidase, and the acid is converted to l-phenylalanine by transamination or reductive amination.