Masanaru Misawa

Biotransformation of trans-cinnamic acid to l-phenylalanine: Optimization of reaction conditions using whole yeast cells

The biotransformation of trans-cinnamic acid to l-phenylalanine has been studied using phenylalanine ammonia-lyase (PAL)-containing yeasts. Maximum conversion rates were observed using concentrations of trans-cinnamate between 30 and 60 mm and NH4OH between 3.5 and 4.5 m. Concentrations of trans-cinnamate >70 mm were inhibitory, with little reaction occurring at substrate concentrations >200 mm. The pH optimum for conversion varied significantly from one yeast to another, pH 10 being optimal for Rhodotorula rubra (code SPA 10) and Rhodotorula glutinis (SPA 20) but resulting in 40% inhibition with cells of Sporidiobolus roseus. Temperatures <34°C had very little effect on the rate of conversion, although at 40°C over 40% inhibition of the R. glutinis (strain code SPA 20) catalyst was observed. The concentration of cells used in the bioreactor varied according to the substrate concentration employed: reactors loaded with ≈50 g l−1 cell dry weight of fully induced cells were found to be optimal for relatively high trans-cinnamate feedstock concentrations (⩾16 g l−1). l-Glutamate was found to stimulate significantly the production of l-phenylalanine, but urea was ineffective. At substrate concentrations of 1, 2 and 3% trans-cinnamate, conversion yields of 100, 69 and 47%, respectively, were obtained. The presence of chloride ions was shown to markedly inhibit the transformation; using 2% trans-cinnamate in (NH4)2SO4 solution an 87% conversion yield was attained, whilst in the presence of NH4OH and 1 m KCl conversions of <20% were achieved.

Novel stabilization of phenylalanine ammonia-lyase catalyst during bioconversion of trans-cinnamic acid to L-phenylalanine

SummaryProduction of l-phenylalanine from trans-cinnamic acid using isolate SPA10 cells was reduced to 26% of that observed initially when cells were reacted a second time with fresh substrate mixture. The stability (reuseability) of Phenylalanine Ammonia-Lyase (PAL) containing cells was significantly influenced by both the trans-cinnamate concentration and initial reaction pH. Using 2% t-cinnamate, l-phenylalanine production was 7-fold greater after 3 successive runs at pH 9.0 than at the optimum of pH 10.2. Cells reacted in the presence of 5% t-cinnamate were relatively unstable. Permeabilising agents, such as toluene and xylene, stimulated l-phenylalanine production but also enhanced instability of the catalyst. Several effectors were shown to stimulate the initial rate of the PAL bioconversion, but only sorbitol, alginate, glutaraldehyde, polyethylene glycol and glycerol conferred any significant degree of stability. Sparging of cultures and bioreactors with various gases revealed that oxygen enhanced PAL inactivation, CO2 had little effect and nitrogen conferred remarkable stability on PAL activity for several weeks in culture medium. The presence of chloride ions (from HCl) and aeration of substrate mixtures resulted in poor reuseability of catalyst. A combination of H2SO4 substitution for HCl and N2-sparging resulted in excellent initial conversions and good catalyst stability at 26°C but less at 30°C. The inclusion of 1.5 M sorbitol in reaction mixtures maintained PAL stability over several successive incubations.

Production of phenylalanine ammonia-lyase (PAL): isolation and evaluation of yeast strains suitable for commercial production of l-phenylalanine

SummaryPhenylalanine 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 NH4+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.

Biotransformation of phenylpyruvic acid to L-phenylalanine using a strain of Pseudomonas fluorescens ATCC 11250 with high transaminase activity

The rate of l-phenylalanine production from phenylpyruvic acid by whole cells of Pseudomonas fluorescens strain ATCC 11250 was greater than 3 g·l-1 h-1. Synthesis of transaminase was constitutive but activity was greatest in medium containing d- or l- phenylalanine as sole nitrogen source. Maximum conversion was observed at 34–40° C and at alkaline pH, with over six times initial rate of conversion at pH 12 than at pH 5. The optimum catalyst (cell) concentration was between 10–20 mg ml-1 dry weight. The initial rate of conversion was directly proportional to phenylpyruvate concentration, up to 4%, but the conversion yield steadily decreased between 2% and 4% substrate concentration. The rate of conversion, as expected, increased as the concentration of glutamate increased. Whole cells were still capable of over 63% conversion after 40 days providing reactions were supplemented with pyridoxal phosphate. Immobilisation of cells in calcium alginate and operation of a packed bed bioreactor enabled the continuous production of l-phenylalanine in concentrations greater than 15 g·l-1 after 60 days operation.

Effect of glycerol, polyethylene glycol and glutaraldehyde on stability of phenylalanine ammonia-lyase activity in yeast

SummaryThe polyhydric alcohols, glycerol and sorbitol, significantly increased the rate ofl-phenylalanine production from trans-cinnamic acid using whole cells ofRhodotorula rubra. Chloride ions and oxygen prevented the stimulatory effect of the polyhydric alcohols. Furthermore, the severe inhibition, of the biotransformation by high trans-cinnamic acid concentrations was alleviated in the presence of glycerol, and sorbitol. The rate of conversion could be manipulated still further, even with high trnas-cinnamic acid concentrations, by elevating the reaction pH to, 12 in the presence of polyhydric alcohol. When cells were also treated first with glutaradehyde (0.1% v/v) and then polyethylene glycol (15% v/v), although neither compound stimulated the actual rate of bioconversion, the reaction was markedly stabilised and gave a 73% yield after 28 days of continuous operation.

Selective precipitation of L-phenylalanine using copper ions and its application in the bioconversion of phenylpyruvic acid

Abstract l -Phenylalanine can be selectively precipitated from a biotransformation reaction mixture as an insoluble diphenylalanine-copper complex. During the bioconversion of phenylpyruvic acid to l -phenylalanine, none of the substrates or co-products reacted with copper ions to form insoluble salts. Using immobilized cells of Pseudomonas fluorescens, l -phenylalanine produced from phenylpyruvate was precipitated in situ using copper acetate and the reaction continued by supplying fresh substrate. After 30 h, 36.5 grams of partially pure l phenylalanine were recovered from 1 liter by treatment with hydrogen sulphide. A recycle packed bed reactor was set up with the l -phenylalanine (product) in the reactor effluent continuously precipitated with copper ions. After 102h operation, 103.7 g of partially pure l -phenylalanine was recovered from the pooled precipitates. The merits of this efficient recovery method are discussed .