Xylanase Gene Mutation And Expression In Pichia Pastoris

Xylan is a major component of hemicellulose, which widely presents in the plant cell wall and is the big renewable resources after cellulose in nature. A variety of feed grains such as wheat, barley and oats contain much xylan, it affects the utilization and absorption of nutrients, so the selection of suitable xylanase to degrade xylan is essential. However, the natural enzyme produced by microbes is mostly mixed enzymes with lower yields and activity.Using genetic engineering techniques to obtain high dynamic, yield and stable xylanase resources has become hot research point.The target xylanase gene was amplified by RT-PCR, which was then cloned into PMD19-T vector and sequenced to determine and compare xylanase gene sequence published in GenBank. The result revealed that the xylanase gene had 100% homology with the published sequence from Aspergillus niger strian C71 (JF693944.1). The coding region of the gene did not contain a signal peptide sequence and intron sequences, encoded 225 amino acids. The protein molecular weight was 24.13 kDa, and protein isoelectric point was 5.23. The eukaryotic expression vector (pGAPZαA-xylanase gene) was constructed successfully, and then transformed into Pichia pastoris by electroporation. The recombinant yeast secreted considerable protein bands detected by electrophoresis, it was confirmed that xylanase gene was successfully expressed in Pichia pastoris.The xylanase activity of recombinant Pichia pastoris could reach 0.1531 U/mL after 24 h incubation when 1% xylan was replaced by 2% wheat bran. Six mutants of xylanase genes were obtained by error-prone PCR, which were mutant 1 (D117N), mutant 4 (L34V), mutant 5 (T43A), mutant 6 (T116I), mutant 7 (K179T), mutant 8 (F53L, G107S). After building six pGAPZαA-xylanase mutant gene expression vectors,30 strains of recombinant Pichia pastoris with mutant genes were obtained,5 strains for each mutent gene. After determining xylanase activity, mutant strain 1-2 was found to produce highest anount of xylanase with 0.1480 U/mL after 12 h incubation, which was higher than the natural gene (0.1360 U/mL) and other mutent genes (P<0.05).After comparing the xylanase characters between the natural gene and mutant gene, it was found that the optimum temperature and pH of both xylanases were 40℃ and 5.50 (P<0.05). It was confirmed that adding 0.2% Tween 80 could significantly increase xylanase activity by about 30%(P<0.05). The further enzymolysis of mash wheat showed that the natural xylanase from Aspergillus niger could significantly increase arabinose and xylose production; however, its ability for xylose production is lower than the mutant xylanase (P<0.05). Compared with the expressed natural xylanase, the mutant xylanase could significantly reduce glucose production (P<0.05), but increase xylose production, indicating that the mutant xylanase had better ability to degrade xylan (P<0.05). In conclusion, even though the mutant xylanase had the same enzyme biochemical characters as the natural enzyme, the mutant enzyme had the higher ability to degrade xylan. In addition 0.2% addition of Tween 80 was able to increase xylanase production for recombinant Pichia pastoris.