Expression, Characterization And Molecular Modification Of Phenylalanine Hydroxylase From Chromobacterium Violaceum

Phenylalanine hydroxylase (PAH), a non-heme iron enzyme, is responsible for thephenylalanine conversion to tyrosine. Its malfunction causes phenylketonuria (PKU).Addition of PAH in food could decrease the content of phenylalanine, which is useful forPKU patient. PAH from Chromobacterium violaceum (CvPAH) has potential pharmaceuticalvalue due to its relative structural simplicity and structural similarity and the closer propertyto the catalytic domain of human PAH. However, there is no systematic study on PAHcharacterization so far. Moreover, the low thermal stability of CvPAH narrows down itsapplication.The CvPAH was cloned and expressed in Escherichia coli in this study. Thecharacterization of CvPAH was further studied systematically. Then molecular modificationwas used to improve the thermal stability of CvPAH. Main results of this study were asfollows:We cloned the pah gene from C. violaceum and constructed recombinant expressionvector pET24a-pah. Finally, the E. coli BL21(DE3)/pET24a-pah expression strain wasconstructed. High expression level of recombinant PAH was obtained under the optimalinduction conditions: with final IPTG concentration of0.6mmol·L-1, at24℃for24h.The specific activity of recombination protein was503.2U·mg-1after purification. Thestudies of enzymatic properties showed that the enzyme displayed maximum activity at40℃,and its half-life was15min at50℃. The optimal pH was pH7.5, and the enzyme activity wasstable at the range of pH6-8. Under the conditions of37℃and pH7.5, its Kmwas1.5mmol·L-1, Vmaxwas0.5mmol·min-1, kcatwas5.05s-1, and the catalytic efficiency (kcat/Km)was3.37L (mmol·s)-1.To improve the thermal stability of CvPAH, several CvPAH mutants were designedbased on an analysis of harmonic dynamics. The amino acids with high flexibility weresubstituted by praline (P), a rigid amino acid. Besides, disulfide bonds were constructed byinvoving cysteine (C) mutations at specific sites: K16C/W121C, H41C/R76C, V151C/F247C,A168C/Y262C and K239C/G274C in CvPAH. Among these mutants, the D267P and CvPAH(K239C/G274C) mutants showed2.5-fold higher thermal stabilities than the wild-type afterincubation at50℃for10min. The melting temperature detected by circular dichroism (CD)of the mutants showed5℃higher than the wild-type. Meanwhile, the catalytic efficiency (kcat/Km) of the mutants could reach a comparative level of the wild-type. Therefore, effectivethermal stable PAH mutants were constructed in this study. These results will be useful forfurther studies and industrial applications of PAH.