Enzyme activity evaluation of organic solvent‐treated phenylalanine ammonia lyase

Abstract

The direct one-step synthesis of L-phenylalanine methyl ester in an organic-aqueous biphasic system using phenylalanine ammonia lyase (E.C.4.3.1.5, PAL) containing Rhodotorula glutinis yeast whole cells was reported earlier. We report here further optimization of this biotransformation using isolated PAL, when the lyophilized enzyme is treated with different water miscible and water immiscible organic solvents. Use of isolated PAL enzyme is advantageous in overcoming diffusion barriers encountered when using PAL containing R.glutinis whole cells, and resulted in increased product yield due to better interaction of enzyme with the substrate. Among the water miscible solvents, ethanol treated and methanol-treated enzymes supported maximum PAL forward and reverse activities; respectively. In the water immiscible solvents category, heptane-treated enzyme exhibited maximal activity for both PAL forward and reverse reactions. PAL activity obtained with enzyme specimens treated with methanol, ethanol, and heptane varied in the range of 91–99% of that observed in aqueous buffer medium for the forward reaction; and 89–95% for the reverse reaction. n-butanol,acetone, and benzene were found to have a inhibitory effect on PAL enzyme, in that, it resulted in only 31–33% activity of that obtained with aqueous solution. Raman spectroscopy was used to monitor amide I and II bands which are sensitive to changes in the secondary structure of proteins. No changes in structure could be detected from the analyses of AI and AII bands of PAL spectra. This data obtained for PAL, a tetramer, could be significant in predicting how solvent interactions affect the structure and function of multimeric proteins and enzymes in nonaqueous media.

Rhodotorula glutinis Phenylalanine/Tyrosine Ammonia Lyase Enzyme Catalyzed Synthesis of the Methyl Ester of para-Hydroxycinnamic Acid and its Potential Antibacterial Activity

“…There was no pronounced effect on PTAL enzyme activity when the speed of agitating the reaction mixture during the incubation period was increased above 50 rpm ( Table 1 ). These results correspond well with almost all studies on effect of agitation on PAL protein using L -Phe, L -PM, t -CA, and t -CM substrates; optimal enzymatic conversions have been obtained at ∼50–100 rpm ( Yamada et al, 1981 ; Evans et al, 1987a , b ; D’Cunha et al, 1994 , 1996 ; Quinn et al, 2011 ).…”

Immobilization of Cross-Linked Phenylalanine Ammonia Lyase Aggregates in Microporous Silica Gel

“…Phenylalanine ammonia-lyase (E.C.4.3.1.5-PAL) belongs to the family of lyases [16] , [17] . It has been used chiefly in the manufacture of L-phenylalanine by reversing the enzyme reaction with high concentration of trans-cinnamic acids and ammonia at an elevated pH.…”

Expanding the organic toolbox: a guide to integrating biocatalysis in synthesis

“…For example, lyophilisation in the presence of kosmotropic salts can allow for recovery of enzyme activity upon solubilization in organic solvent. 14 While the main thrust of research in this area dates back to the 1980's and 1990's, developments in this area continue to be of interest, as illustrated by specific, recent reports on the activity of a phenylalanine ammonia lyase 15 or of a feruloyl esterase for breakdown of lignocellulosic biomass. 16 The choice of co-solvent has an important impact not only on enzyme activity and reagent solubilisation, but also on reaction 'greenness'; an excellent source of information is the 'GSK solvent selection guide', which considers the environmental sustainability of over 100 solvents, in the context of manufacturing active pharmaceutical ingredients.…”