Abstract
BackgroundXylanases 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.ResultsA 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 EvXyn11TS, 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 (Tm) 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 (AExynMC5T, AExynMP9S, and AExynMH14N) 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 (Cys5, Pro9, and His14) in the replaced N-terminus contributed mainly to the high thermostability of AEXynM.ConclusionsThis work highly enhanced the thermostability of AuXyn11A by N-terminus replacement, and further verified, by site-directed mutagenesis, that Cys5, Pro9, and His14 contributed mainly to the improved thermostability. It will provide an effective strategy for improving the thermostabilities of other enzymes.
Highlights
Xylanases have drawn much attention owing to possessing great potential in various industrial applications
Most wild-type xylanases are poor in thermostability, which prevented them from being used in bioprocesses where high temperatures were required to improve the availability and solubility of substrates, and to reduce the viscosity and microbial contamination of the reaction solution [6]
Based on the analytical result that the rigidity of a protein was positively related to its thermostability [18], the designed hybrid xylanase AEXynM was predicted to be more thermostable than the wild-type xylanase AuXyn11A
Summary
Xylanases have drawn much attention owing to possessing great potential in various industrial applications. Xylanase, used in bioethanol production from lignocellulosic materials, could promote the hydrolysis of cellulose by decomposing xylan which restricts the access of cellulase to the cellulose surface [5]. As far as we know, some domains or local regions affecting protein thermostability, such as salt bridge, hydrogen bond, charged surface residue, disulfide bridge, and N- or C-terminus, have been revealed [9,10]. Among those factors, the importance of the Nterminus in maintaining xylanase thermostability was highlighted by some researchers [11,12]
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