【作者】 刘明启；

【导师】 孙建义；

【作者基本信息】 浙江大学 ， 动物营养与饲料科学， 2007， 博士

【摘要】 为了提高木聚糖酶的热稳定性、催化活性和底物的结合水解性能,本研究以G／11家族黑曲霉木聚糖酶A(Aspergillus niger xylanase A, AnxA)和褐色高温单孢菌木聚糖酶A(Thermomonospora fusca xylanase A, TfxA)为亲本,设计出两种杂合木聚糖酶基因,并系统研究了毕赤酵母表达的杂合木聚糖酶及其亲本酶的酶学性质。主要研究结果如下：1杂合木聚糖酶基因atx的分子设计和表达为了提高AnxA的热稳定性和催化活性,其催化结构域N-末端序列被TfxA中相应序列所取代,获得的杂合木聚糖酶基因atx在大肠杆菌中的表达产物EATx主要以包涵体形式存在。atx在毕赤酵母中获得表达,其表达产物PATx和AnxA具有相同的催化活性中心。经0.25%甲醇诱导培养96h,基因工程菌pPATx86发酵上清液中木聚糖酶PATx活性为633.0 U／mg,分别是酵母表达黑曲霉木聚糖酶A (reAnxA)和酵母表达褐色高温单孢木聚糖酶A (reTfxA)的3.6倍和5.4倍。因此,通过N-末端序列替换显著提高了AnxA的催化活性。进一步分析表明,IPATx、reAnxA和reTfxA均无纤维素酶活性。2杂合木聚糖酶PATx及其亲本酶的酶学性质PATx的最适温度和最适pH分别为60℃和pH5.0；在70℃、pH5.0条件下处理2 min,残余酶活为72%；在pH3.0.9.0,25℃条件下处理1h,残余酶活均在80%以上。reAnxA的最适温度和最适pH分别是50℃和pH5.0；在70℃、pH5.0条件下处理2 min,残余酶活为42%；在pH3.0-8.0,25℃条件下处理1h,残余酶活均在80%以上。reTfxA的最适温度和最适pH分别是60℃和pH6.0；在70℃、pH6.0条件下处理2 min,残余酶活为70%；在pH5.0-9.0,25℃条件下处理1h,残余酶活均在80%以上。SDS-PAGE结果表明,IPATx的相对分子量为20.78 kDa,与理论推算的分子量(21.04 kDa)相吻合。杂合木聚糖酶PATx具有较好的pH稳定性。通过与TfxA的N-末端序列替换显著提高了AnxA的热稳定性,表明reTfxA的N-末端序列与酶蛋白的热稳定性密切相关。小麦木聚糖酶抑制剂TAXI-I对PATx、reAnxA和reTfxA均具有抑制活性。相同酶活的PATx、reAnxA和reTfxA分别与TAXI-I在20℃下作用30 min,其残余酶活分别为35.0%、18.0%和85.3%。表明,TfxA的N-末端序列替换提高了AnxA对小麦木聚糖酶抑制剂的抗性。采用高效液相色谱(HPLC)法,对杂合木聚糖酶PATx及其亲本酶水解桦木木聚糖和小麦麸皮不溶木聚糖产物的分析表明,杂合木聚糖酶PATx水解桦木木聚糖和小麦麸皮不溶木聚糖产物分别为木糖至木五糖、木糖至木六糖,其主要产物均为木三糖；reAnxA水解桦木木聚糖和小麦麸皮不溶木聚糖产物分别为木糖至木五糖、木糖至木六糖,其主要产物均为木三糖；而reTfxA水解桦木木聚糖和小麦麸皮不溶木聚糖产物均为木糖至木六糖,其主要产物均为木二糖。以低聚木糖为底物对杂合木聚糖酶PATx及其亲本酶的水解模式研究表明,杂合木聚糖酶PATx为内切型木聚糖酶,其对木二糖和木三糖没有水解活性,能够水解木四糖、木五糖和木六糖,最小水解单元为木四糖；reAnxA和reTfxA均能水解木二糖至木六糖,二者最小水解单元均为木二糖；且PATx、reAnxA和reTfxA均具有转糖苷化活性。3杂合木聚糖酶基因atxx的分子设计和表达为了提高杂合木聚糖酶PATx对纤维素类底物的结合和水解能力,将TfxA的连接序列和木聚糖结合域(XBD)融合在PATx的C-末端,获得的杂合木聚糖酶基因atxx在毕赤酵母中获得分泌表达,表达产物为PATxX。经0.25%甲醇诱导培养96h,基因工程菌pPATxXl发酵上清液中PATxX的木聚糖酶活性和纤维素酶活性分别为452.1 U／mg和19.3 U／mg。4杂合木聚糖酶PATxX的酶学性质PATxX木聚糖酶的最适温度和最适pH分别为60℃和pH5.0,与杂合木聚糖酶PATx相同。PATxX在70℃、pH7.0下处理2 min,残余酶活为66%；在pH4.0-9.0,25℃下处理1h,残余酶活均不低于80%。PATxX木聚糖酶与小麦木聚糖酶抑制剂作用后其残余酶活为41.0%。杂合木聚糖酶PATxX水解桦木木聚糖产物为木糖至木五糖,主要产物为木三糖；PATxX水解小麦麸皮不溶木聚糖产物为木糖至木六糖,主要产物为木六糖,其次为木二糖和木三糖。IPATxX为内切型木聚糖酶,对木二糖至木六糖均具有水解活性,最小水解单元为木二糖,并具有转糖苷化活性。25℃,结合3h后,PATxX和reTfxA对1%微晶纤维素的结合率分别为3.7%和7.6%,而PATx和reAnxA无结合微晶纤维素的能力。PATx、reAnxA和reTfxA均无纤维素酶活性。PATxX纤维素酶的最适温度和最适pH分别为60℃和pH6.0。SDS-PAGE结果表明，PATxX的相对分子量为33.01 kDa,与理论推算的分子量(32.15 kDa)相吻合。通过与TfxA的连接序列和木聚糖结合域融合,使PATx具备了结合水解纤维素的能力。更多还原

【Abstract】 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.更多还原