Journal
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
Volume 56, Issue 3, Pages 894-902Publisher
AMER CHEMICAL SOC
DOI: 10.1021/jf072614u
Keywords
Rhodotorula glutinis yeast cells; phenylalanine ammonia-lyase; Mn2+-dependent storage stability; enzyme induction; GC-MS determination of PAL reverse activity
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Phenylalanine ammonia-lyase (PAL; E C 4.3.1.5) reverse reaction has been exploited for the commercial production of optically pure L-phenylalanine from trans-cinnamic acid. Optimal conditions for the growth and PAL activity of Rhodotorula glutinis cells and an improved method for the synthesis Of L-phenylalanine have been reported. A major problem encountered during these studies was rapid loss of PAL activity during storage of the yeast cells, which were therefore unsuitable for long-term and repeated use. Enhancement of enzyme stability in the presence of various additives including polyhydric compounds and metal ions is described. Whole cells retained nearly 85% of the original enzyme activity for at least 12 weeks when a low concentration of Mn2+ (0.01%) was included in the storage buffer medium (50 mM Tris-HCl, pH 8.8). In contrast, <3.0% activity was present in the control within 4 weeks. Mn2+-dependent stabilization of PAL was also observed with an isolated enzyme preparation (73% retention in activity for 12 weeks) obtained by ultrasonication of R. glutinis whole cells. The data suggest that Mn2+ ions may be responsible for the specific stabilization of a more active conformation of the enzyme. In addition, enzyme stability as a function of temperature was studied, and the optimal temperature for maximal activity retention was 0-2 degrees C. The effects of various additives on the induction of PAL have also been examined. These results could have direct implications in studies on activity, inhibition, and reaction mechanism of this biotechnologically important enzyme.
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