| Xylanase and mannanase, as two kinds of hemicellulase, were widely used in food, feed, papermaking, textile and medicine fields.In this study, two xylanase genes (xyn10and xyn11) from Aspergillus niger were optimized, spliced, and expressed in Pichia pastoris X33. The characteristics of recombinant Xyn10and Xyn11were assessed, and fermentation conditions for recombinant strain Ax11-151were optimized based on response surface analysis. Chimeric genes of xylanase-mannanase and mannanase-xylanase were constructed by ligase and overlap extention PCR. The characteristics of recombinant chimeras as well as the effects of different linker peptides on chimeras were also studied. The main contents contain the following aspects:1. Constitution, characterization of recombinant xylanases Xyn11and optimization of fermentation conditionAccording to Pichia pastoris’s codon usage bias, GC content, the stability of mRNA secondary structure and other factors, two xylanase genes were optimized and divided into40and24oligonucleotides approximately60mer in length with no gaps. These fragments were assemblyed into full-length genes by dual asymmetric PCR and overlap extension PCR. SDS-PAGE analysis of purified recombinant xylanases displayed protein bands of about33kDa for Xyn10and20kDa for Xyn11, which were identical to the theoritical molecular weights.The optimal temperature and pH value of recombinant Xyn10were60℃and5.0, repectively. Xyn10showed a broad stability over the pH range of3.0to13.0, while nearly lost all activity after incubation at80℃-90℃and pH2.2. The recombinant Xyn11exhibited optimal activity at50℃and pH5.0. After incubation over the pH range of1.0to12.0at room temperature for2h, Xynll maintained more than71%of its original activity. Xyn11only lost60%of its original activity at90℃for10min. The specific activities of purified Xyn10and Xyn11were2504U/mg and22253U/mg, for oat spelt xylan. Both recombinant xylanases had the highest affinities towards birchwood xylan and the greatest catalytic efficiency against beechwood xylan, and the kcat/Km of Xyn11was3401.08mL/mg/s.The strains with high-level expression of recombinant Xyn11were screened based on semimicro-DNS method.After continuous cultivation, the most stable inheritance strain, named Ax11-151with the highest activity was selected for further studies. Fermentation conditions of recombinant strain Ax11-151were studied to explore the effects of nitrogen sources, carbon sources, pH, and liquid volume on the production of recombinant Xynll. The results showed that the yield of recombinant Xyn11after48h of cultivation was increased by151%(2,883.86U/mL), compared with YPD medium.2. Construction of Xyn-Man and Man-Xyn chimerasThe xylanase gene xyn11with excellent properties and mannanase gene man26constructed in previous work were selected as parents of chimeras. The pepides of10,15and20amino acid residues consisted of (GGGGS)n (2≤n≤4) and (EAAAK)n (2≤n≤4) were used as linkers and chimeric genes were constructed with two fused directions. Twelve correct chimeric genes and three mutations were constructed by ligase and overlap extention PCR. All the fifteen chimeric genes were successfully expressed in Pichia pastoris strain X33, and exhibited both xylanase and mannanase activities. SDS-PAGE analysis of each chimera displayed high glycosylation. However, after treatment with Endo H, the chimera displayed a single brand at about57-58kDa, which was identical to the theoretical molecular weight.All the chimeras showed the similar temperature and pH properties. The relative activities of chimeras with mannanase gene sequence at N-terminal portions displayed5%-10%higher than the parent Xyn11, while a drastic decline in activities was observed between55℃-65℃and75℃-80℃, compared with parent Xyn11.Moreover, the stabilities of the chimeras with (EAAAK)n (2≤n≤4) as linkers were higher than that with (GGGGS)n (2≤n≤4) as linkers. The catalytic efficiencies (kcat/Km) of chimeras were21%-42%of parent Xyn11, toward oat spelt xylan. The catalytic efficiencies (kcat/Km) of chimeras were greater than parent Man26, toward locust bean gum. On the other hand, the catalytic efficiencies of chimeras with (GGGGS)3and (EAAAK)3as linkers were higher than with two and four repeats length (GGGGS) and (EAAAK) as linkers (except chimera ME3X).This results revealed that chimeras of XE3M and XS3M with xylanase at the N-teminal displayed higher synergistic efficiency towards luffa cylindrica than chimeras ME3X and MS3X with mannanase at the N-terminal. The synergistic efficiency of hydrolyzing luffa cylindrica of70-200mesh was higher than of20-70mesh by chimeras, and chimera XE3M showed highest synergistic efficiency towards luffa cylindrica of70-200mesh with1.57of synergy degree.In conclusion, the recombinant Xyn11displayed a broad range of working pH and temperature, pH tolerance, an excellent thermotolerance, high expression and extremely high catalytic efficiency, which had a great potential for industrial applications such as food, feed, and medicine. To some extent, this study highlights the potential for construction of bifunctional xylanase-mannanase and mannanase-xylanase, and selection of linkers and fused directions. |
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