翻译:Production of phenylalanine__ ammonia-lyase (PAL)

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Materials and methods
Culture media. The basal salts medium contained (g-l-~):
KH2PO4 (4), Na2HPO4 (2), MgSO4-7H20 (1.2), NH4CI (2.0), yeast extract (0.22), CaC12.6H20, FeCI3.6H20 (0.08), ZnSOa.7H20 (0.001), NaCI (0.5). PAL Induction Medium (PAL-I) contained ( g - l - l ) : yeast extract (10), peptone (10), NaCI (5), L-phenylalanine (5), L-isoleucine (0.5). Yeast Extract Medium (YM) contained (g.l-~): yeast extract (20), peptone (20), NaC1 (5), NH4CI (1.0), t,-phenylalanine (5). Phenylalanine Selective Medium (PS) contained basal salts medium plus 15 g.1 -~ L-phenylalanine as principal carbon source. Transcinnamic acid Selective Medium (TCS) contained basal salts, 7 g. 1- 1 NH4CI and 0.3 g. 1- I yeast extract.
Appl Microbiol Biotechnol (1987)25:406--414
Applied Microbiology Biotechnoloion of phenylalanine ammonia-lyase (PAL): isolation and evaluation of yeast strains suitable for commercial production of L-phenylalanine
Present address: Genzyme (U.K.) Ltd., Springfield Mill,
Maidstone, Kent ME14 2LE, U.K. Offprint requests to: Christopher T. Evans
C. T. Evans et al.: Isolation of PAL-containing yeast strains
Christopher T. Evans*, Kim Hanna, Dayle Conrad, Wendy Peterson, and Masanaru Misawa
Allelix Inc., 6850 Goreway Drive, Mississauga, Ontario, Canada
Summary. Phenylalanine Ammonia-Lyase (PAL)
containing microorganisms were isolated from a wide variety of natural habitats. The best 21 strains to emerge from the primary screen were screened for PAL activities in both directions using L-phenylalanine and t-cinnamate substrates. Twelve of the latter strains were compared for total cell production and PAL activity and 7 isolates were chosen for examination of the extent of PAL induction in various media. On the basis of these screens, isolate SPA 10 (identified as Rhodotorula rubra) was selected for further optimization. Growth was optimal at 28°C and pH 5.0, although cellular PAL activity was shown to be higher at sub-optimal temperatures (36°C) and pH (8.0) for growth. Synthesis of PAL was repressed when grown in the presence of various sugars and NH~- ions. Manipulation of fermentation conditions enabled PAL synthesis to occur at maximum biomass levels, upon glucose exhaustion. PAL was rapidly inactivated within cells shortly after maximum synthesis was attained: feeding of D,L-isoleucine and low concentrations of D,L-phenylalanine, and shifting of fermentation temperature conferred catalyst stability for fermentations over 100 h. These results demonstrate the suitability and superiority of isolate SPA 10 for the commercial production of L-phenylalanine from trans-cinnamic acid.
Introduction
The commercial importance of the amino acid Lphenylalanine has increased dramatically with the advent of the dipeptide sweetener, Aspartame. Traditionally, L-phenylalanine has been produced by chemical synthesis and direct fermentation (Tsuchida et al. 1975) but, recently, various biotransformation methods have been investigated to increase production of this aromatic amino acid (Calton et al. 1986). One of the better known bioconversions is that using trans-cinnamic acid and the enzyme Phenylalanine Ammonia-Lyase (PAL). The latter enzyme can be obtained from a number of plant and microbial sources; the enzyme from yeast has been extensively used and is now the basis of a large scale production process established by Genex Corp. (Hamilton et al. 1985). Although PAL has been identified as a major route to L-phenylalanine production, there are still numerous problems associated with the rates of conversion, yields, stability and reuseability of the catalyst. The latter reasons, and the declining price of trans-cinnamic feedstock, suggest that exploitation of this process is still possible to establish the most economic route to L-phenylalanine production. The present paper describes the isolation and characterization of PAL-containing yeast strains as the first part of an extensive programme aimed at manipulating the properties of the PAL bioconversion route to establish a superior process for L-phenylalanine production. Before embarking upon such a programme, it was imperative that the correct process strain be selected at the outset and that all subsequent genetic and physiological manipulations be targeted to this strain and enzyme. The isolation and properties of such a strain are described.