Enhancement Of The Thermostability And Catalytic Activity And The Cellulose Binding And Hydrolyzing Ability Of Xylanase

To enhance the thermostability, catalytic activity, and cellulose binding ability of xylanase, two hybrid xylanases were constructed using G/11 family Thermomonospora fusca xylanase A (TfxA) and Aspergillus niger xylanase A (AnxA) as parents in this study. Hybrid xylanases and their parents were expressed in Pichia pastoris and characterized. The main results were listed as following:1 Construction and expression of hybrid xylanase gene atxThe amino acid sequences of the catalytic domain of the TfxA and AnxA showed that they belonged to family 11 and shared high identity. However, further study showed that the homology of their N-terminuses was relatively low and the second structure elements of their N-terminus were different from each other. It was supposed that the N-terminus of TfxA contributed to its significant thermostability. Hybrid xylanase gene, atx, was constructed by substituting 5'end segment of the TfxA for its corresponding region of the AnxA.The atx was expressed in E. coli BL21 and its product, named as EATx, was expressed as inclusion body. The atx was expressed in Pichia pastoris and the product, PATx, was secreted into culture. After 96-h 0.25% methanol induction, the xylanase activity of PATx in culture supernatant of pPATx86 transformant reached its peak,633.0 U/mg, which was 3.6 and 5.4 times as high as those of recombinant Aspergillus niger xylanase A (reAnxA) and recombinant Thermomonospora fusca xylanase A (reTfxA), respectively. The PATx, reAnxA, and reTfxA showed no activity on CMC-Na.2 Enzymatic properties of PATx and its parentsThe optimum temperature and pH of the PATx were 60℃and pH5.0, respectively. When treated at 70℃, pH 5.0 for 2 min, its residual activity was 72% which was higher than that of reAnxA and similar to that of reTfxA. The PATx was very stable over a broader pH range and much less affected by acid/base conditions. Over 80% of PATx activity was retained after treatment of the enzyme by preincubation over a pH range of 3.0-9.0 for 1 h at 25℃.The optimum temperature and pH of the reAnxA were 50℃and pH5.0, respectively. When treated at 70℃, pH 5.0 for 2 min, the residual activity of reAnxA was 42%. Over 80% of reAnxA activity was kept after treatment of the enzyme by preincubation over a pH range of 3.0-8.0 for 1 h at 25℃. The optimum temperature and pH of the reTfxA were 60℃and pH6.0, respectively. When treated at 70℃, pH 6.0 for 2 min, the residual activity of reTfxA was 70%. Over 80% of reTfxA activity was retained after treatment of the enzyme by preincubation over a pH range of 5.0-9.0 for 1 h at 25℃. SDS-PAGE analysis showed that the molecular mass of PATx was 20.78 kDa, which was in good agreement with the molecular mass of enzyme deduced from the DNA sequence,21.04 kDa.The thermal stability and catalytic activity of AnxA were improved by N-terminus replacement with the corresponding region of TfxA. The results implied that the N-terminus of TfxA contributed to the thermostability of enzymes.PATx and its parents were partly inhibited by TAXI-I, the Triticum aestivum xylanase inhibitor from wheat. After incubation with TAXI-I at 20℃for 30 min, the residual xylanase activity of PATx, reAnxA, and reTfxA were 35.0%,18.0%, and 85.3%, respectively. The ability of AnxA resistant to TAXI-I was enhanced.The hydrolysis products from various xylans by PATx and its parents were determined and quantified by HPLC. Hydrolysis products from birchwood xylan and wheat bran insoluble xylan by PATx were xylose to xylopentaose (X-X5) and xylose to xylohexaose (X-X6), respectively. Xylotriose (X3) was the main product among them. The hydrolysis products from birchwood xylan and wheat bran insoluble xylan by reAnxA were X-X5 and X-X6, respectively. X3 was the major product. The hydrolysis products from birchwood xylan and wheat bran insoluble xylan by reTfxA were X-X6, and xylobiose (X2) was the main product.The mode of action of PATx and its parents were evaluated using xylooligosaccharides (X2-X6) as substrate. PATx might be an endo-acting xylanase and showed no activity on X2 and X3, but exhibited activity on X4, X5, and X6. X4 might be the minimum oligomer hydrolyzed by PATx. X2-X6 could be hydrolyzed by reAnxA and reTfxA, respectively. X2 was the minimum oligomer hydrolyzed by them. All of PATx, reAnxA, and reTfxA had transglycosylation activity.3 Construction and expression of hybrid xylanase gene atxxTo improve the cellulose binding ability of PATx, its C-terminus was fused with the linker sequence and xylan binding domain from TfxA. The obtained hybrid gene atxx was expressed in P. pastoris and the hybrid enzyme PATxX was obtained. After 96-h 0.25% methanol induction, xylanase and cellulase activity of the PATxX in the culture supernatant of pPATxXl transformant were 452.1 U/mg and 19.3 U/mg, respectively.4 Enzymatic properties of hybrid xylanase PATxXThe optimum temperature and pH of the PATxX xylanase were 60℃and pH5.0, respectively. When treated at 70℃, pH 7.0 for 2 min, the residual xylanase activity of PATxX was 66% which was lower than that of PATx but higher than that of reAnxA. Over 80% of PATxX xylanase activity was maintained after treatment of the enzyme by preincubation over a pH range of 4.0-9.0 for 1 h at 25℃. After incubation with TAXI-I, the residual xylanase activity of PATxX was 41.0%. SDS-PAGE analysis revealed that the molecular mass of PATxX was 33.01 kDa, which was in good agreement with the molecular mass of enzyme deduced from the DNA sequence,32.15 kDa.The hydrolysis products released by PATxX from different xylans and xylooligosaccharides were determined and quantified by HPLC. Hydrolysis products released by PATxX from birchwood xylan were X-X5 with X3 as main product. The degradation products from wheat bran insoluble xylan by PATxX were X-X6, and X6 was the main product. PATxX might be an endo-acting xylanase and showed hydrolytic activity on X2-X6. X2 might be the minimum oligomer hydrolyzed by PATxX which displayed transglycosylation activity.After incubation with microcrystal cellulose at 25℃for 3h,3.7% PATxX and 7.6% reTfxA were bound, respectively. PATx and reAnxA showed no cellulose binding ability. The optimum temperature and pH of the PATxX cellulase were 60℃and pH6.0, respectively. The PATx obtained cellulose binding and hydrolyzing ability by fusing with linker sequence and xylan binding domain from TfxA, which suggested that the linker sequcence and xylan binding domain from TfxA played important role in binding and hydrolyzing cellulose substrate.