| Recently, with the development of biomass resources and agricultural by-products andthe deeply studies of XOS physiological functions, xylanases have been among the mostintensively studied as a key enzymes for the degradation of bio-resources.Endo-β-1,4-xylanase (EC3.2.1.8) can randomly catalyze the hydrolysis of internalβ-1,4-D-xylosidic linkages of xylans to produce xylooligosaccharides and small amounts ofxylose. As a environmentally effective biocatalyst, xylanase has been widely used by animalfeed, pulp and paper, bio fuel, medical and pharmaceutical, food and so on. Although therewere a lot of reports about the cloning and expression of xylanases, their applications are stilllimited by their poor thermostability in the extreme environment.To obtain a thermostable xylanase, we cloned the full-length cDNA and partial DNAsequences of GH10family xylanase AusXyn10A from A. usamii E001, the maturepeptide-encoding gene of AusXyn10A was firstly expressed in Pichia pastoris GS115andEscherichia coli BL21, respectively, and then engineering the thermostability by computeraided rational/semi-rational design through N/C terminus substitution, disulfide design andmutation. The main results are listed as follows:(1) The full-length cDNA and DNA sequences of AusXyn10A gene were amplified by3′,5′rapid amplification of cDNA ends (RACE) and hairpin structure-mediated PCR techniquesdesigned in this work; The cDNA includes80bp5′non-coding region,171bp3′non-codingregion and an984bp open reading frame (ORF) which codes for a327aa protein; Thecomplete DNA of the Ausxyn10A gene is2,255bp in length, harboring510bp5′promoterregions,171bp3′flanking regulatory regions, nine short introns ranging from52to62bp anda984-bp open reading frame; Bioinformatics analysis indicated that the ORF of AusXyn10Aencodes a19-aa signal peptide, a6-aa propeptide, and a302-aa mature peptide.(2) The Ausxyn10A was integrated into P. pastoris GS115genome by pPIC9KMvecotorwhich designed in this work, and one strain labeled as GSX10A4-14having the highestrecombinant xylanase activity of100.8U mL-1was chosen from P. pastoris transformants.The expression conditions of GSX10A4-14was optimized. As a result, the activity ofreAusXyn10A reached368.6U mL-1which was3.25times as high as that expressed using thestandard protocol under the optimized conditions (inoculum size initial8%, pH value7.0,methanol concentration2.5%, induction period120h and induction temperature30℃). Afterpurified, The reAusXyn10A displayed the highest activities at50℃, and stable at45℃. ThepH optima was at5.5, and stable at a pH range of4.5~8.5. Its activity was not significantlyaffected by metal ions tested and EDTA, but inhibited by Mn2+. The kinetic parameters, Kmand Kcat, of the reAusXyn10A towards birchwood xylan were2.25mg mL-1and3,419.4s-1,respectively. The optimized hydrolysis conditions of corncob xylan by reAusXyn10A were asfollows:50g L-1corncob xylan,300U g-1xylanase dosage and10h hydrolytic time. Underthis condition, the content of reducing sugars was reached15.45g L-1. TLC analysis verifiedthat the AusXyn10A yielded a major product of xylotriose.(3) As the result of sequence alignment of TmxB (a thermophilic GH family10xylanase from Thermotoga maritima MSB8) and AusXyn10A, a mutant xylanase AusXynCRC1waspredicted by substituting a segment A300NA302at C terminus of AusXyn10A with theKEVLEKKIEER of TmxB and then expressed in P. pastoris GS115. The results demonstratedthat consistent with the reAusXyn10A, reAusXynCRC1also displayed the highest activity at50℃, while the t1/250was droped from40min to5min. A mutanted xylanase namedAusXynCRC2was constructed by H13L and A12K mutate based on the3-D structurecompared results. The results demonstrated that compared with reAusXynCRC1, the t1/250ofreAusXynCRC2was extended from5min to8min. Moreover, the activity was also increasedby38%compared with reAusXyn10A.(4) Based on molecular dynamics simulation and comparison of B-factor values, amutant xylanase AusXynCRN1was predicted by substituting a segment NRLTTGKNA at Nterminus of AusXyn10A with the corresponding one from Y25TLTKDSKTP34of TaXyn10, athermophilic GH family10xylanase from Thermoascus aurantiacus. Consistent with thereAusXyn10A, AusXynCRN1also displayed the highest activity at50℃, while, its t1/255wasextended from6.9min to8min. Based3-D structure compared result, another mutantxylanase reAusXynCRN2was designed by mutating S286G and H288F. The temperatureoptimum of recombinant AusXynCRN2was10℃higher than that of reAusXyn10A. Its t1/255was10.4-fold longer than that of reAusXyn10A. As compared with reAusXyn10A,reAusXynCRN2displayed a significant increase in enzyme activity value.(5) Based on molecular dynamics simulation and Disulfide by Design V1.20computedresults, two mutant xylanase D7C/G42C and S246C/A297C were designed and thenexpressed in P. pastoris GS115, respectively. The results demonstrated that the temperatureoptimum of reS246C/A297C was5℃higher than that of reAusXyn10A. Its t1/255wasapproximately2-fold longer than that of reAusXyn10A. However, the temperature optimumand thermostability of reD7C/G42C were all decreased compared with reAusXyn10A.(6) The AusXyn10A was successfully heterogeneous expressed in E. coli BL21throughpET-28a. The enzymatic property measured by PCR instrument and microplatespectrophotometer demonstrated that the AusXyn10A displayed the highest activity at44℃.The pH optima was at5.5, and stable at a pH range of6.6~7.8. Based on multiple sequencesalignment and comparison of B-factor values, three sites (R59, S278and S280) were seletedand implemented by saturation mutagenesis or site-directed mutagenesis. After transformationand high throughput screening, four mutants included S280V, S278A, N111S andR59C/A35G were screen out. The results demonstrated that the temperature optimum of allfour mutates were2℃higher than that of AusXyn10A. The T5020of four mutants were alsoincreased. Based on the analysis results of all mutates, the reXynCRN2-S246C/A297C wasconstructed by combing N terminus substitution (with mutates S286G/H288F) and mutatesD7C/G42C. The t1/260of reXynCRN2-S246C/A297C was19min and1.6-fold longer than thatof reAusXynCRN2, demonstrating that the thermostability of reXynCRN2-S246C/A297Cwas further improved compared with reAusXynCRN2. |
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