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Background  Xylanases have drawn much attention owing to possessing great potential in various industrial applications. However, the applicability of xylanases, exemplified by the production of bioethanol and xylooligosaccharides (XOSs), was bottlenecked by their low stabilities at higher temperatures. The main purpose of this work was to improve the thermostability of AuXyn11A, a mesophilic glycoside hydrolase (GH) family 11 xylanase from  Aspergillus usamii  E001, by N-terminus replacement.    Results  A hybrid xylanase with high thermostability, named AEXynM, was predicted by computational methods, and constructed by substituting the N-terminal 33 amino acids of AuXyn11A with the corresponding 38 ones of  Ev Xyn11 TS , a hyperthermostable family 11 xylanase. Two AuXyn11A- and AEXynM-encoding genes,  Auxyn11A  and  AExynM , were then highly expressed in  Pichia pastoris  GS115, respectively. The specific activities of two recombinant xylanases (reAuXyn11A and reAEXynM) were 10,437 and 9,529 U mg -1 . The temperature optimum and stability of reAEXynM reached 70 and 75°C, respectively, much higher than those (50 and 45°C) of reAuXyn11A. The melting temperature ( T  m ) of reAEXynM, measured using the Protein Thermal Shift (PTS) method, increased by 34.0°C as compared with that of reAuXyn11A. Analyzed by HPLC, xylobiose and xylotriose as the major hydrolytic products were excised from corncob xylan by reAEXynM. Additionally, three single mutant genes from  AExynM  ( AExynM  C5T ,  AExynM  P9S , and  AExynM  H14N ) were constructed by site-directed mutagenesis as designed theoretically, and expressed in  P. pastoris  GS115, respectively. The thermostabilities of three recombinant mutants clearly decreased as compared with that of reAEXynM, which demonstrated that the three amino acids (Cys 5 , Pro 9 , and His 14 ) in the replaced N-terminus contributed mainly to the high thermostability of AEXynM.    Conclusions  This work highly enhanced the thermostability of AuXyn11A by N-terminus replacement, and further verified, by site-directed mutagenesis, that Cys 5 , Pro 9 , and His 14  contributed mainly to the improved thermostability. It will provide an effective strategy for improving the thermostabilities of other enzymes.

Determinants for the improved thermostability of a mesophilic family 11 xylanase predicted by computational methods