| Phenylalanine hydroxylase(Phenylalanine hydroxylase, PAH, EC 1.14.16.1) is an aromatic amino acid hydroxylase(Aromatic amino acid hydroxylase, AAAH), which catalyzes phenylalanine to tyrosine. Phenylalanine hydroxylase from Chromobacterium violaceum(CvPAH) is a potential medicine for phenylketonuria(PKU), which is highly similar to single subunit structure of human phenylalanine hydroxylase(hPAH), shows similar catalytic properties and higher activity at physiological temperature and pH. However, the application of CvPAH is mainly limited by the low thermostability and storage stability. Improving CvPAH thermal stability and storage stability can lay a solid foundation for its application in the field of functional foods.In this study, the effect of the N-terminal flexible region on enzyme property was researched and thermal stability of CvPAH was improved by molecular modification. Furthermore, the effects of additives on the thermostability of PAH were tested. The research results were reported as follows:(1) As indicated by gaussian network model, the N-terminal 10 amino acids of CvPAH is a large flexible segment. In order to study the impact of the segment on the thermal stability of CvPAH, the sequence was truncated or recombined to generate CvPAH, N- 7, N- 8, N- 9, N- 10 and CvPAH+T7 tag. The optimum reaction temperature of the mutant enzyme was 40 ℃. Compared with other enzymes, thermal stability of N- 9 and CvPAH+T7 tag were increased. On the other hand, the optimum pH study showed that the optimum pH of CvPAH+T7 tag shifted from 8.5 to 7.5. Among the modified enzymes, the recombinant enzyme CvPAH+T7 tag had higher activity, 562.7 U·mg-1 at pH 7.5 and 37 ℃, which was significantly increased compared to the activity of the original enzyme(377.23 U·mg-1).(2) In proteins of thermophilic bacteria, Gly is tend to be replaced by Ala and Lys is tend to be replaced by Arg to adapt the high environmental temperature. All the Gly on CvPAH were mutated to Ala and Lys were mutated to Arg. The results revealed that half-life of K94 R and G221 A mutants at 50 ℃ were 26.2 min and 16.8 min, which were increased by 1.9-times and 0.9-times in contrast to the recombinant CvPAH(9.0 min), respectively. The residual activity of K94R/G221 A mutant was improved to 65.6% after keeping at 50 ℃ for 1 h, which was 6.6 time higher than the recombinant CvPAH(8.6%). Circular dichroism(CD) spectroscopy revealed that Tm values of the recombinant Cv PAH, K94 R, G221 A and K94R/G221 A were 51.5 ℃, 53.8 ℃, 53.1 ℃ and 54.8 ℃, respectively.(3) The effects of several additives on the thermostability of the recombinant CvPAH were tested. Glycerol trehalose, raffinose and mannitol could improve the thermal stability of the recombinant CvPAH. An optimal combination additive of 2.5 mmol·L-1 mannitol, 1 mmol·L-1 raffinose and 1.5 mmol·L-1 trehalose was obtained using the orthogonal experiment. With the optimal combination additive, residual activity of the recombinant CvPAH could reach as high as 99.3%. Both 10% glycerol and the optimal combination additive could significantly improve the storage stability of the recombinant CvPAH at 4 ℃ and 20 ℃. With 10% glycerol or combination additive, the half-life of the recombinant CvPAH at 37 ℃ were increased to 129.9 h and 237.5 h, respectively, which were 2.8 times and 4.0 times higher than that without protective additive. |
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