Abstract:

Three mutations (D148E/H149R, Q67E/N68R and D191E) of neutral phytase from Bacillus amyloliquefaciens
DSM 1061 have designed in our previous study. Enzymatic properties such as thermostability and catalytic efficiency were
studied in this paper. The structural changes were analyzed to explore the relationship with properties of mutant phytases.
The results showed that the half-life of D191E was 4.3 min longer than that of wild-type phytase at 85 ℃, but its catalytic
efficiency reduced to 48.9% of wild-type phytase. The catalytic efficiency of mutant D148E/H149R was increased to 229%
of wild-type phytase although the half-life was similar. The catalytic efficiency of Q67E/N68R was 93% of wild-type
phytase and the half-life was extended by 0.7 min. Circular dichroism showed that the catalytic efficiency of D148E/H149R
was improved most obviously while the content of α-helix decreased from 11.79% to 2.90%, and the content of random coil
was increased. The structure of Q67E/N68R changed little and its properties were closed to those of wild-type phytase. The
α-helical content of D191E increased to 24.59% while the catalytic efficiency decreased a lot. Using homologous models to
analyze the factors which may affect the thermal stability of phytases, we found that the B-factor value of D191 was high so
that it was unfavorable to the stability of phytase. The hydrogen bonds around mutation residues also changed. Homologous
model analysis will provide the theoretical basis for further study of phytase.

Key words: neutral phytase, site-directed mutagenesis, catalytic efficiency, circular dichroism