Improvement Of The Thermostability Of GHF11Xylanase From Aspergillus Usamii By Computer Aided Design

Xylanase (EC3.2.1.8), abbreviated from β-1,4-endoxylanase, can catalyze the hydrolysisof internal β-1,4-D-xylosidic linkages of xylans. Recently, with the development ofhemicellulose and xylooligosaccharides, xylanases have been applied in many industryprocesses. Aspergillus usamii E001can produce a series of mesophilic xylanases with highspecific activities. To further improve the activity of xylanase, and study its thermotolerantmechanism, we cloned and expressed two genes encoding glycoside hydrolase family11(GHF11) xylanases from A. usamii E001(AusXyn11D and AusXyn11A), respectively. Then,to improve the thermostability of AusXyn11A, its N-terminus replacement was predicted bycomputer aided design using molecular dynamics (MD) simulation. In addtion, thethermotolerant mechanism and hydrolytic product of the hybrid xylanase were analyzed.Based on the conserved peptide segments in four putative xylanase sequences fromAspergillus sp., the full-length cDNA of Ausxyn11D, a gene that encodes a xylanase of A.usamii E001, was obtained, and then its DNA sequence was amplified by PCR. The cDNAand DNA sequences were deposited in the GenBank database under accession no. JQ219105and HQ724287, respectively. Multiple homology alignment of amino acid sequences verifiedthat AusXyn11D contained the motifs and catalytic residues that were strictly conservedamong all GHF11xylanases. The similarities of the primary structure of AusXyn11D withtwo GHF11xylanases from A. usamii E001were58and37%, respectively. Homologymodeling revealed that the3-D structure of AusXyn11D conformed to the GHF11xylanaseoverall crystal one, resembling the shape of a partially closed right hand. All these featuresverified that AusXyn11D was a member of GHF11.According to the cDNA sequence of Ausxyn11D, its mature peptide-encoding gene wasamplified by RT-PCR. At the same time, a gene (Ausxyn11A) coding for the mature peptide ofAusXyn11A was also cloned based on the information of its cDNA sequence in GenBank.After that, Pichia pastoris GS115transformants (GS115/Ausxyn11D and GS115/Ausxyn11A)were successfully constructed. After induction by menthanol, the specific activities ofrecombinant xylanases (reAusXyn11D and reAusXyn11A) were150.3and22,714U/mg,respectively. The reAusXyn11D and reAusXyn11A displayed their highest activities at55and50℃, and they were stable at50and45℃, respectively. Their pH optima were acidic, and thereAusXyn11D was stable at a pH range of3.5-6.5, while reAusXyn11A displayed pH stabilityat a broad range of4.0-8.0. The specific activity of reAusXyn11A was much higher than thatof reAusXyn11D, which would make reAusXyn11A a good candidate for industrialapplication. Furthermore, the expression conditions of GS115/Ausxyn11A were optimized. Asa result, the activity of reAusXyn11A reached912.6U/mL which was2.14times as high asthat expressed using the standard protocol.A codon-optimized gene, Syxyn11, which encodes a thermostable xylanase (EvXyn11TS)was synthesized and expressed in P. pastoris GS115. The specific activity of reSyXyn11was363.2U/mg. The reSyXyn11displayed pH optimum at6.5and pH stability at a broad rangeof4.5-9.0. Its temperature optimum and stability were85and80℃, respectively. The reSyXyn11was one of the most thermostable GHF11xylanases.A hybrid xylanase (AEXynM) was predicted by MD simulation, and constructed bysubstituting the N-terminal33amino acids of AusXyn11A with the corresponding38ones ofEvXyn11TS. As a result, the specific activity of reAEXynM was19,237U/mg, slightly lowerthan that of reAusXyn11A. The temperature optimum and stability of reAEXynM reached70and75℃, respectively, much higher than those of reAusXyn11A. The melting temperature(Tm) of reAEXynM was slightly lower than that of EvXyn11TS, but increased by34.0℃ascompared with that of reAusXyn11A. All the results verified that the thermostability ofAusXyn11A was obviously enhanced by N-terminus replacement.Based on MD simulation and intramolecular interaction analysis, three amino acids(Cys5, Pro9and His14) in the replaced N-terminus were considered to be responsible for thehigh thermostability of AEXynM. As a result, three recombinant mutants derived fromAEXynM (reAEXynMC5T, reAEXynMP9Sand reAEXynMH14N) were obtained by site-directedmutagenesis, respectively. The thermostabilities of three mutants decreased obviouslycompared with that of reAEXynM. Among that, the mutation of C5T caused the mostsignificant decrease in thermostability which confirmed that a unique disulfide bridge(Cys5-Cys32) may confer the high thermostability on AEXynM. Besides that, the Pro9inβ-turn and a hydrogen bond between His14and Phe17could be important to the thermostabilityof AEXynM.The hydrolytic time-course of AEXynM verified that the hydrolysis velocity of corncobsxylan was much slower than that of birchwood xylan. Xylobiose and xylotriose as the majorhydrolytic products were excised from corncob xylan by AEXynM, and their contentsreached42.33and38.76%, respectively. However, xylobiose as the major hydrolytic productwas released from birchwood xylan by AEXynM with content of58.56%. A trace of xylosewas detected during the hydrolysis, suggesting that AEXynM would be suitable for theproduction of xylooligosaccharides.