A comparison between three-dimensional structures of a wild-type xylanase AoXyn11A and a hybrid xylanase
AEx11A revealed that a disulfide bridge (Cys5-Cys32) and an N-glycosylation site (Asn42) were imported into AEx11A by
N-terminal substitution of AoXyn11A with EvXyn11TS. Two mutant genes AEx11A
C5T and AEx11A
N42Q were constructed
by mutating Cys5- and Asn42-encoding codons of AEx11A into Thr5- and Gln42-encoding ones, and heterologously expressed
in Pichia pastoris GS115, respectively. The temperature optimum of the recombinant AEx11AC5T (reAEx11AC5T)
was decreased to 60°C from 80°C of reAEx11A, while its thermal inactivation half-lives at 70 and 80°C shortened to 3
and 1 min from 197 and 25 min of reAEx11A, respectively. However, there was no obvious alteration between reAEx11A
and reAEx11AC5T in pH characteristics and kinetic parameters. Furthermore, both reAEx11AN42Q and reAEx11A displayed
no significant difference in all enzymatic properties tested, except for the apparent molecular weight. We concluded
based on this study that the disulfide bridge of AEx11A was vital to its high thermostability, but the Nglycosylation
had no effect on.
Keywords: Disulfide bridge, N-glycosylation, site-directed mutagenesis, thermostability, xylanase.
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