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

Summaryl-Phenylalanine dehydrogenase [l-phenylalanine: NAD+-oxidoreductase (deaminating)] of Rhodococcus sp. strain M4 was studied emphasizing its application for the production of l-phenylalanine. A high enzyme level (30,000 U·l-1, 25–30 U·mg-1 in the crude extract) could be reached during aerob degradation of l-phenylalanine (10 g·l-1) under optimized growth coditions. A partial purification of the intracellular enzyme by liquid-liquid extraction, and DEAE-cellulose led to a specific activity of more than 1300 U·mg-1. The continuous production of l-phenylalanine in an enzyme-membrane-reactor for 350h resulted in a space-time yield of 456 g·l-1·d-1 with a mean substrate conversion of 95%. Consumption of phenylalanine dehydrogenase was 1,500 U·kg Phe-1.

[1]  K. Aida,et al.  ON THE ENZYMATIC PREPARATION OF L-PHENYLALANINE. , 1959 .

[2]  C. Wandrey,et al.  Enzymatic production of l-phenylalanine from the racemic mixture of d,l-phenyllactate , 1987, Applied Microbiology and Biotechnology.

[3]  M. Kula,et al.  Process design for large-scale purification of formate dehydrogenase fromcandida boidinii by affinity partition , 1986 .

[4]  Y. Asano,et al.  Crystallization of Phenylalanine Dehydrogenase from Sporosarcina ureae , 1985 .

[5]  H. Hustedt,et al.  Purification of enzymes by liquid-liquid extraction , 1982 .

[6]  T. Tosa,et al.  L-Phenylalanine formation from acetamidocinnamic acid by newly isolated bacteria , 1984, Applied Microbiology and Biotechnology.

[7]  Christian Wandrey,et al.  Continuous enzymatic transformation in an enzyme membrane reactor with simultaneous NAD(H) regeneration , 1981, Biotechnology and bioengineering.

[8]  M. Kula,et al.  Isolation and characterization of a bacterium possessing L-phenylalanine dehydrogenase activity , 2004, Archives of Microbiology.

[9]  C. Wandrey,et al.  On-line Determination of Reaction Rate Versus Substrate Concentration for an Enzymatically Catalysed Reaction by Means of a Microcomputer System , 1980 .

[10]  S. Kinoshita,et al.  Microbial Production of l-Phenylalanine from n-Alkanes , 1970 .

[11]  K. Sigler,et al.  Methods in industrial microbiology , 1983 .

[12]  C. Cooney,et al.  Selective production of phenylalanine from phenylpyruvate using growing cells ofCorynebacteriumglutamicum , 1985, Biotechnology Letters.

[13]  C. Wandrey,et al.  Continuous production of L‐phenylalanine by transamination , 1987, Biotechnology and bioengineering.

[14]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[15]  C. Wandrey,et al.  l-Phenylalanine dehydrogenase from Brevibacterium sp. for production of l-phenylalanine by reductive amination of phenylpyruvate , 1986, Applied Microbiology and Biotechnology.