【作者】 王谦；

【导师】 刘建新； 孙建义；

【作者基本信息】 浙江大学 ， 动物营养与饲料科学， 2012， 博士

【摘要】 木聚糖是谷物类饲料中一种主要的抗营养因子,通常需要添加内切木聚糖酶(EC3.2.1.8)破坏木聚糖长链结构,释放出短链木寡糖,在木糖苷酶(EC3.2.1.37)的协同作用下,木聚糖可最终被水解成木糖。近年来,内切木聚糖酶已被广泛应用于饲料、食品和制浆造纸工业等,此外以木糖为底物生产燃料乙醇的研究也具有广阔前景。然而,目前木聚糖酶仍存在酶的催化活性不高、酶学特性不能满足工业加工或生产应用的需求或酶的生产成本较高等不足,亟需开发兼具高活性且酶学特性优良的理想木聚糖酶基因及其高效表达宿主。本研究克隆了糖苷水解酶家族11的4种内切木聚糖酶基因和1种木糖苷酶基因,以其中的优良基因为基础对其进行分子改良,构建了相应的高拷贝重组酵母表达宿主,在摇瓶中发酵表达内切木聚糖酶和木糖苷酶,并对底物形态和水解特性进行分析。主要结果如下：1内切木聚糖酶与木糖苷酶基因的克隆及原核表达本研究克隆了家族11黑曲霉(Aspergillus niger xylanase, anx)、枯草芽孢杆菌(Bacillus subtilis xylanase, bsx)、褐色高温单孢菌(Thermomonospora fusca xylanse, tfx)和杂合内切木聚糖酶(hybrid xylanse, atx)基因以及枯草芽孢杆菌来源的木糖苷酶(B. subtilis xylosidase, xylo)基因,并在大肠杆菌中进行表达。酶学特性分析发现,所有木聚糖酶均在酸性条件下较为稳定。其中,ATx和Tfx兼具高酶活性和良好的热稳定性,且Tfx具备良好的抗抑制剂特性和一个家族11木聚糖酶少有的碳水化合物结合域(carbohydrate binding module, CBM)。因此,atx和tfx可作为后续分子改良的基因材料。2atx的分子改良及重组酵母菌的构建基于非理性设计思路,采用定向进化提高atx的催化活性。定向进化策略包括易错PCR (error-prone PCR, EP-PCR)产生随机突变,在微孔板上筛选木聚糖酶活性提高的优势突变体。一轮EP-PCR后共筛选1530个转化子得到一个酶活性提高的突变体基因FSI-A124。(?)将atx和FSI-A124分别转化毕赤酵母(Pichia past or is) GS115,在PGAP启动子和α交配因子信号肽的控制下分泌表达木聚糖酶,96h发酵上清液中比活性分别为954和1556U/mmg。突变酶YFSI-A124的Km为4.25mg/ml,较之野生酶YATx下降4.7%,催化转化数Kcat则提高44%。然而,酶学特性分析显示,YFSI-A124热稳定性略有下降。氨基酸比对和三维结构分析发现,L49P替换使得酶的催化区域变得柔和,突变酶与底物木聚糖的亲和能力增强,酶的催化效率提升,另一方面氢键作用、相邻氨基酸之间的范德华力和表面疏水作用减弱,导致酶的热稳定性下降。3tfx的分子改良综合非理性设计和理性设计的优势,采用定向进化和定点突变相结合的技术,提高tfx的催化活性和热稳定性。首先利用两轮EP-PCR随机突变,经微孔板筛选约7000个突变体,获得6个木聚糖酶活性提高的优势突变体。并对其中4处可能与酶的催化活性或热稳定性相关的氨基酸残基进行序列饱和突变。将优势突变体用于DNA shuffling和StEP进行优势重组,经筛选3000个转化子共获得3个木聚糖酶活性提高的优势突变体。之后,将S144C点突变引入G3DS2,使分子内产生一个新的二硫键,获得兼具高活性和良好热稳定性的突变基因G4SM/(S62T/S144C/N198D/A217V)。G4SM1的特异性比活为2036±45.8U/mg,是野生酶Tfx的2.12倍,而Km值为1.84mg/ml,较之野生酶显著下降(P<0.05),而Kcat/Km提高76%,提示酶与底物的亲和能力增强,且酶的催化效率提高。三维结构分析显示,推测位于催化区的两处氨基酸替换S62T和S144C有助于提高酶的催化活性和稳定性。4构建包含高拷贝G4SM1基因的重组酵母菌采用两种不同的方法构建包含高拷贝G4SM1及其催化区G4SM1CBM基因的重组酵母。一是采用高浓度抗生素压力法筛选酵母转化子,将电击后的转化子分别涂布于含500、1000、1500和2000μg/ml zeocin的YPD平板,在1500和2000μg/ml zeocin的平板上挑取阳性转化子进行拷贝数鉴定,Southern blot和qPCR分析显示,28号菌株为包含两拷贝G4SM1的重组酵母菌,命名为33-1。研究发现,Tfx中包含一段没有催化活性的CBM2,为了减少高拷贝外源基因对宿主的压力,将Tfx中不影响其其催化活性的CBM2截去,采用一对同尾酶BamH I和BglⅡ(?)将表达盒头尾相连,构建一个包含四拷贝表达盒的穿梭载体pGAPZaA/G4SM1_CBM (4),再将该载体转化P. pastoris SMD1168获得包含四拷贝G4SM1_CBM基因的重组酵母33-2, Southern blot和qPCR进一步验证其正确性。5共表达内切木聚糖酶和木糖苷酶的重组酵母菌的构建及摇瓶发酵、底物形态和水解特性分析利用BamH Ⅰ和BglⅡ(?)将pGAPZαA/G4SM1_CBM (4)与包含木糖苷酶基因的pPICZαA/xylo相连接,获得共表达内切木聚糖酶和木糖苷酶的重组载体paAX-GAPGC(4),转化P. pastoris SMD1168获得共表达宿主33-3。将菌株33-1和33-2在YPD培养基中分泌表达内切木聚糖酶,培养120h后发酵上清液中总蛋白含量分别为0.49g/l和0.65g/l。菌株33-3能在YPD培养基中仅表达内切木聚糖酶,同时也能在甲醇的诱导下共表达内切木聚糖酶和木糖苷酶,120h发酵上清液中总蛋白含量达1.22g/l。底物形态和水解特性分析显示,未处理的桦木木聚糖结构致密、表面粗糙,多糖分子粘连程度高,经内切木聚糖酶水解后,长链结构被破坏,分子呈现较小的颗粒状态,表面形态趋于平整。水解24h后,木二糖和木三糖的浓度分别为0.954±0.093mg/ml和0.360±0.022mg/ml,分别占总水解产物的62.6%和23.6%。在内切木聚糖酶和木糖苷酶的协同作用下,木聚糖可彻底水解成木糖,水解24h后,木糖的浓度为1.198±0.016mg/ml,占总水解产物的69.1%。综上所述,本研究采用理性设计与非理性设计相结合的方法改良内切木聚糖酶基因tfx,构建的高拷贝重组酵母33-1和33-2、共表达重组酵母33-3摇瓶发酵蛋白表达量较高,可在发酵罐中进行高密度发酵进一步提高蛋白表达水平,在饲料工业和生物能源应用具有良好的应用前景。更多还原

【Abstract】 Xylan is a main anti-nutritional factor in cereals. Xylanases (EC3.2.1.8) are glycosidases that break long sugar chain of xylan into xylooligosaccharides (XOs), the synergic effects of both endo-xylanase and exo-xylosidase (EC3.2.1.37) catalyze xylan into D-xylose. In the past decade, xylanases have been widely used in paper pulp, food and feed industries. Xylose has been of tremendeous interests to produce bio-ethanol. Therefore, it is desirable for xylanase to exhibit high catalytic activity and thermostability to handle industrial tasks. The current study was designed to clone four xylanase genes from family11and one xylosidase gene, modify the gene with high catalytic activity and good properties, and construct recombinant yeast containing high-copy xyalanse genes. After that, xylanase and xylosidase were heterologously co-expressed in flask medium. The secreted proteins were further subjected to analyze substrate morphology and hydrolytic products of birchwood xylan. The main results were as follow:1Cloning and expression of endo-xylanse and xylosidase in E. coliFour family11xylanase genes (Aspergillus niger xylanase, anx; Bacillus subtilis xylanase, bsx; Thermomonospora fusca xylanase, tfx; hybrid xylanase, atx) and B. subtilis xylosidase (xylo) gene were cloned and expressed in E. coli. All xylanases were stable under acidy conditions. Among them, both ATx and Tfx showed high acitivity and good thermostability. Furthermore, Tfx was observed to be capable to endure xylanase inhibior and contain a carbohydrate binding module (CBM), which was seldom found in family11xylanses. With the context, xylanase genes atx and tfx were employed for further protein enginnering.2Molecular modification of atx and construction of the recombinant yeastA directed evolution strategy comprising of error-prone PCR (EP-PCR) and high-throughput screening based on micro-plate was employed to improve the catalytic activity of atx, according to the non-rational degisn. A total of1530clones were screened to obtain the mutant FSI-A124with most improved xylanase activity. The wild type gene atx and the mutant FSI-A124were transformed into Pichia pastoris GS115to target the PGAP locus of yeast genome by electroporation. The recombinant xylanases driven by the Saccharomyces cerevisiae a-mating factor were secreted into culture medium. After incubation in YPD medium for96h, the specific activities of YATx and YFSI-A124reached954and1556U/mg, respectively. The Km of YFSI-A124was4.25mg/ml,4.7%lower than that of YATx, while the turnover number kcat was44%higher. Nevertheless, YFSI-A124was less thermally stable than YATx. Amino acid alignment and three-dimensional strucute revealed that a single substitution L49P was formed within the sequence of YFSI-A124. The mutagenesis possibly made the catalytic module of YFSI-A124more flexible than wild type. Therfore, the pocket cavity was supposed to have good affinity to substrate and catalytic efficiency. The decrease of thermostability was attributed to decline of hydrogen bond, van der Waals force and hydrophobic interactions with other residues nearby the catalytic center.3Molecular modification of tfxBoth directed evolution and site-directed mutagenesis were employed to improve the catalytic activity and thermostability of tfx. Two round EP-PCR and high-throughput screening based on micro-plate were initially used to screen7000clones for six mutants with improved activity. Four mutation sites possibly relative to catalytic acticvity and enzymatic properties were optimized using site-saturation mutagenesis. After that, all mutants obtained were subjected to DNA shuffling and StEP. A total of3000clones were screened to obtain three mutants with improved activity. A single substitution S144C was further introduced into G3DS2to obtain the mutant G4SM1(S62T/S144C/N198D/A217V) with most improved activity and thermostability. The specific activity of G4SM1was2036±45.8U/mg,2.12times wild type, while the Km value (1.84mg/ml) was27.8%lower (P<0.05). Furthermore, the kcat/Km ratio of G4SM1was improved by76%, indicating that the affinity to substrate and catalytic effiency were dramatically enhanced. Three-dimensional structure analysis revealed that the substitutions S62T and S144C, located at the catalytic domain, were responsible for the improved activity and thermostability.4Construction of recombinant yeast containg high-copy G4SM1 The construction of recombinant yeast containg high-copy genes were achieved using two different methods. The YPD plates with high concentrations of zeocin were used to obtain high-zeocin resistant transformants. After electroporation, the transformed cells were spread onto YPD plates supplemented with500,1000,1500and2000μg/ml zeocin. The positive clones of1500and2000μg/ml zeocin plates were picked and subjected to evaluation of gene copy number. Southern blot and qPCR showed that the No.28strain (designated as33-1) was a recombinant yeast containing two-copy integrated G4SM1. It was suggested by both previous study and our result, CBM2of Tfx was non-essential for catalysis. With the context, CBM2was removed from Tfx and the catalytic domain was used for construction of high-copy yeast. A pair of isocaudarners (BamHⅠ and BglⅡ) was employed to establish a shuttle vector pGAPZαA/G4SM1_CBM (4), which contains four tandemly arranged expression cassettes. The resultant plasmid was transformed into P. pastoris SMD1168and the gene copy number of the strain (also named as33-2) was confirmed by Southern blot and qPCR.5Construction of a recombinant yeast co-expressing endo-xylanase and xylosidase, fermentation in flask medium, and characterization of xylan morphology and its hydrolytic productsThe co-expression pladmid was constructed by ligating pGAPZaA/G4SM1_CBM(4) and pPICZaA/xylo using BamHⅠ and BglⅡ. The resultant plasmid was transformed into P. pastoris SMD1168to obtain the co-expression strain33-3. Both33-1and33-2were cultured in YPD medium for120h. The recombinant protein concentrations secredted into supernatant were0.49and0.65g/l. The co-expression strain33-3could either secrete endo-xylanase only in YPD medium or secrete both endo-xylansae and xylosidasae when methanol was supplemented. After incubation for120h, total protein concentration in culture supernatant was1.22g/1/. The kurtosis surface of xylan substrate was observed to be noticeably altered. The untreated birchwood xylan exhibited well-arranged polysaccharide molecules and a sharp kurtosis surface, and polysaccharides were observed to be viscous. After hydrolysis of xylan by endo-xylanase, endo-xylanase broke the ligation between polysaccharides into small particles, the concentrations of xylobiose and xylotriose were0.954±0.093and0.360±0.022mg/ml, or62.6%and23.6%of total XOs. Furthermore, the synergic effects of endo-xylanase and β-xylosidase were capable to convert xylan into xylose. After hydrolysis for24h, the concentration of xylose was1.198±0.016mg/ml,69.1%of total XOs. In summary, endo-xylanase gene tfx was modified using rational and non-rational design. The mutant gene was subjected to construct two high-copy yeasts33-1and33-2, and a co-expression yeast33-3. The proteins concentrations in culture medium of these strains were high, and could be further improved by high-density fermentation in fermenter. The strains are potential candidates for application in feed and biofuel industries.更多还原