Study On The Effects Of The Interactions Between The N-terminal3-strands Of A Xylanase Rfom Aspergillus Oryzae To Its Thermostability

Xylanases are widely used for biomass modification in animal feeding, pulpbleaching, health medicines drugs and so on. Unfortunately, most of commercializedxylanases are mesophilic at the present time, which prevent them from being applied incertain industrial processes where high temperatures are required. The maturepeptide-encoding gene Aoxyn11A of a familly11xylanase had been cloned; a heterozygousgene, AEx11A, had also been constructed through a sequence substitution in the N-terminalregion of Aoxyn11A in our laboratory. Aoxyn11A and AEx11A were expressed in Pichiapastoris (P. pastoris) GSl15respectively, and the effects of temperatures on thethermostabilities of the expressed AoXyn11A and AEx11A were analyzed. The resultsshowed AEx11A exhibited better thermostability than AoXyn11A. But the sequencesubstitution involved many amino acid residues, the role of a single factor for increasingthermostability remains elusive. On the base of the experimental results previously, thisstudy is aimed to study the mechanism for increasing thermostability in depth byintroducing into Aoxyn11A hydrogen bonds, a disulfide and an aromatic amino acid in turn.DNAMAN6.0program was employed to put up an amino acid homology alignment ofAoXyn11A with EvXyn11TS, which showed the two enzymes shares40.5%identity in theN-terminal37residues (numbered by AoXyn11A) while the identity of the following aminoacid residues is above70%. The result hinted the importance of the N-terminal region of theenzymes to their thermostabilities because the thermostability of EvXyn11TSis much betterthan that of AoXyn11A. So using AoXyn11A as female parent, the mutants Aoxyn11AC5and Aoxyn11AC5-C32were constructed respectively by introducing in turn the amino acidresidues asparagine (N), alanine (A), glutamine (Q), threonine (T), cysteine (C) andmutation T32C, which were located in the corresponding region of EvXyn11TS, toAoxyn11A. Homology modelling showed the amino acid sequence NAQTC bringshydrogen bonds (H-bonds) to the mutants and the mutation T32C brings a disulfide bridgeto Aoxyn11AC5-C32. The molecular dynamics simulations (MD) showed the mutants hadlower root-mean-square deviation (RMSD) values than that of Aoxyn11A, which indicatedthe mutants would be more thermostable than Aoxyn11A in a high-temperatureenvironment. The three genes were expressed in P. pastoris GSl15respectively, and theeffects of temperatures on the thermostabilities of the three expressed enzymes wereanalyzed. After being incubated at55℃for30min, AoXyn11A retained3%of its originalactivity while AoXyn11AC5retained12%and AoXyn11AC5–C32retained49%of their original activities respectively. The results indicated that the two mutants have a betterthermostabilities than that of AoXyn11A. The further discussion showed that while theinteractions between the N-terminal β-strands of AoXyn11A were enhanced, itsthermostability was improved correspondingly.Based on this theory, the mutation N9H was introduced into AoXyn11A. Homologymodelling showed there might be aromatic rings and H-bonds interaction formed asintroducing histidine into the N-terminal region of AoXyn11A that was favourable to theenhancement of the interaction between the N-terminal β-strands. The genes AoXynl1A andAoXynl1N9Hwere expressed in P. pastoris GSl15respectively. And the effects oftemperatures on the thermostabilities of the expressed two enzymes were analyzed. Afterbeing incubated at50℃for30min, AoXyn11A retained52%of its original activity whileAoXyn11AN9Hretained82%, which showed AoXyn11AN9Halso has a better thermostabilitythan that of AoXyn11A.In conclusion, the N-terminal region of AoXyn11A is important to its thermostability.Whether it is H-bonds or disulphide bridges or aromatic amino acids, they all can enhancethe interaction between the N-terminal β-strands of AoXynl1A and improve itsthermostability correspondingly.