【作者】 张慧敏；

【导师】 杨严俊；

【作者基本信息】 江南大学 ， 食品科学， 2013， 博士

【摘要】 β-1,4-内切木聚糖酶(EC3.2.1.8)是一类从木聚糖主链的内部随机切割β-1,4木糖苷键的水解酶，简称木聚糖酶。近年来，随着人们对自然界半纤维素资源的开发和低聚木糖生理功能的发现，木聚糖酶获得了广泛的应用。宇佐美曲霉(Aspergillus usamii) E001可产具高催化活性的中温木聚糖酶，为了进一步提高木聚糖酶的活力，并探讨其耐热机制，本论文克隆和表达了来自A. usamii E001的糖苷水解酶11家族(GHF11)木聚糖酶AusXyn11D及AusXyn11A的基因序列。并以具高比酶活的AusXyn11A为研究对象，通过分子动力学模拟的方法指导其N端替换，以改善其热稳定性，此外，还分析了杂合木聚糖酶的耐热机制及水解产物特性。以曲霉基因组中的木聚糖酶保守序列为基础，利用多种PCR技术获得A. usamiiE001木聚糖酶基因Ausxyn11D的完整cDNA和DNA序列，其GenBank登录号分别为JQ219105和HQ724287。氨基酸序列同源性分析结果表明AusXyn11D具有GHF11木聚糖酶高度保守的氨基酸片段及催化活性中心，且与A. usamii E001中两种已知的GHF11木聚糖酶的同源性分别为58%和37%。同源建模结果显示，AusXyn11D具有GHF11木聚糖酶典型的“右手”型结构，表明AusXyn11D属于GHF11木聚糖酶。依据Ausxyn11D的cDNA序列设计引物，利用RT-PCR技术克隆其成熟肽基因，同时根据GenBank中A. usamii E001GHF11木聚糖酶AusXyn11A的基因序列，扩增得到成熟肽基因Ausxyn11A，然后，成功构建毕赤酵母(Pichia pastoris) GS115重组子GS115/Ausxyn11D及GS115/Ausxyn11A。经甲醇诱导表达后，重组木聚糖酶reAusXyn11D、reAusXyn11A的比酶活分别达到150.3U/mg和22,714U/mg； reAusXyn11D和reAusXyn11A的Topt分别为55℃和50℃，分别在50℃和45℃以下稳定，最适pH值均偏酸性，且pH稳定范围分别为3.5~6.5和4.0~8.0。但与reAusXyn11D相比，reAusXyn11A具有高比酶活的优点，因此具有重要的工业应用价值。此外，为了获得高产木聚糖酶的基因工程菌株，对GS115/Ausxyn11A的发酵条件进行优化，reAusXyn11A的酶活最高可达912.6U/mL，是优化前的2.14倍。对文献报道的耐热木聚糖酶EvXyn11TS基因序列进行毕赤酵母密码子优化，人工合成其优化基因Syxyn11，并在P. pastoris GS115中获得表达，重组木聚糖酶reSyXyn11的比酶活为363.2U/mg，Topt可高达85℃，并在80℃以下稳定，最适pH值为6.5，在pH4.5~9.0的范围内稳定，是目前最耐热的GHF11木聚糖酶之一。通过分子动力学模拟的方法分析AusXyn11A和EvXyn11TS的N端序列，确定用EvXyn11TS的Asn1-Arg38区域替换AusXyn11A中的Ser1-Ala33区域，以此构建杂合酶AEXynM。重组杂合木聚糖酶reAEXynM的比酶活为19,237U/mg，稍低于reAusXyn11A。reAEXynM的Topt为70℃，75℃以下稳定，较reAusXyn11A有显著性提高，其Tm值可高达91.6℃，虽略低于文献报道的EvXyn11TS的Tm值，但比reAusXyn11A提高了34.0℃，表明杂合酶的热稳定性大大提高了。采用分子动力学模拟的方法分析AEXynM与AusXyn11A中的差异氨基酸，并结合分子间作用力的分析，推测与AEXynM热稳定性相关的位点为Cys5、Pro9及His14，以此构建突变酶基因，获得重组突变酶reAEXynMC5T、reAEXynMP9S和reAEXynMH14N。三种突变酶的热稳定性均出现一定程度的下降，其中reAEXynMC5T的热稳定性最差，证实N端二硫键(Cys5-Cys32)的添加是AEXynM热稳定性提高的主要原因之一；而reAEXynMP9S和reAEXynMH14N则表现出相对微弱的下降，结构分析表明，Pro9可提高β-转角的刚性，而His14与Phe17可形成氢键，加固了β-折叠B1与B2之间的连接，是影响AEXynM热稳定性的重要因素。以玉米芯木聚糖及桦木木聚糖为底物，研究AEXynM的水解过程及产物，研究结果显示玉米芯木聚糖的水解产物以木二糖和木三糖为主，分别占水解产物总量的42.33%和38.76%；而桦木木聚糖的水解产物主要以木二糖为主，可占水解产物总量的58.56%；两种底物的水解液中仅有少量木糖被检出，表明其在低聚木糖制备方面具有极大的应用潜力。更多还原

【Abstract】 Xylanase (EC3.2.1.8), abbreviated from β-1,4-endoxylanase, can catalyze the hydrolysisof internal β-1,4-D-xylosidic linkages of xylans. Recently, with the development ofhemicellulose and xylooligosaccharides, xylanases have been applied in many industryprocesses. Aspergillus usamii E001can produce a series of mesophilic xylanases with highspecific activities. To further improve the activity of xylanase, and study its thermotolerantmechanism, we cloned and expressed two genes encoding glycoside hydrolase family11(GHF11) xylanases from A. usamii E001(AusXyn11D and AusXyn11A), respectively. Then,to improve the thermostability of AusXyn11A, its N-terminus replacement was predicted bycomputer aided design using molecular dynamics (MD) simulation. In addtion, thethermotolerant mechanism and hydrolytic product of the hybrid xylanase were analyzed.Based on the conserved peptide segments in four putative xylanase sequences fromAspergillus sp., the full-length cDNA of Ausxyn11D, a gene that encodes a xylanase of A.usamii E001, was obtained, and then its DNA sequence was amplified by PCR. The cDNAand DNA sequences were deposited in the GenBank database under accession no. JQ219105and HQ724287, respectively. Multiple homology alignment of amino acid sequences verifiedthat AusXyn11D contained the motifs and catalytic residues that were strictly conservedamong all GHF11xylanases. The similarities of the primary structure of AusXyn11D withtwo GHF11xylanases from A. usamii E001were58and37%, respectively. Homologymodeling revealed that the3-D structure of AusXyn11D conformed to the GHF11xylanaseoverall crystal one, resembling the shape of a partially closed right hand. All these featuresverified that AusXyn11D was a member of GHF11.According to the cDNA sequence of Ausxyn11D, its mature peptide-encoding gene wasamplified by RT-PCR. At the same time, a gene (Ausxyn11A) coding for the mature peptide ofAusXyn11A was also cloned based on the information of its cDNA sequence in GenBank.After that, Pichia pastoris GS115transformants (GS115/Ausxyn11D and GS115/Ausxyn11A)were successfully constructed. After induction by menthanol, the specific activities ofrecombinant xylanases (reAusXyn11D and reAusXyn11A) were150.3and22,714U/mg,respectively. The reAusXyn11D and reAusXyn11A displayed their highest activities at55and50℃, and they were stable at50and45℃, respectively. Their pH optima were acidic, and thereAusXyn11D was stable at a pH range of3.5-6.5, while reAusXyn11A displayed pH stabilityat a broad range of4.0-8.0. The specific activity of reAusXyn11A was much higher than thatof reAusXyn11D, which would make reAusXyn11A a good candidate for industrialapplication. Furthermore, the expression conditions of GS115/Ausxyn11A were optimized. Asa result, the activity of reAusXyn11A reached912.6U/mL which was2.14times as high asthat expressed using the standard protocol.A codon-optimized gene, Syxyn11, which encodes a thermostable xylanase (EvXyn11TS)was synthesized and expressed in P. pastoris GS115. The specific activity of reSyXyn11was363.2U/mg. The reSyXyn11displayed pH optimum at6.5and pH stability at a broad rangeof4.5-9.0. Its temperature optimum and stability were85and80℃, respectively. The reSyXyn11was one of the most thermostable GHF11xylanases.A hybrid xylanase (AEXynM) was predicted by MD simulation, and constructed bysubstituting the N-terminal33amino acids of AusXyn11A with the corresponding38ones ofEvXyn11TS. As a result, the specific activity of reAEXynM was19,237U/mg, slightly lowerthan that of reAusXyn11A. The temperature optimum and stability of reAEXynM reached70and75℃, respectively, much higher than those of reAusXyn11A. The melting temperature(Tm) of reAEXynM was slightly lower than that of EvXyn11TS, but increased by34.0℃ascompared with that of reAusXyn11A. All the results verified that the thermostability ofAusXyn11A was obviously enhanced by N-terminus replacement.Based on MD simulation and intramolecular interaction analysis, three amino acids(Cys5, Pro9and His14) in the replaced N-terminus were considered to be responsible for thehigh thermostability of AEXynM. As a result, three recombinant mutants derived fromAEXynM (reAEXynMC5T, reAEXynMP9Sand reAEXynMH14N) were obtained by site-directedmutagenesis, respectively. The thermostabilities of three mutants decreased obviouslycompared with that of reAEXynM. Among that, the mutation of C5T caused the mostsignificant decrease in thermostability which confirmed that a unique disulfide bridge(Cys5-Cys32) may confer the high thermostability on AEXynM. Besides that, the Pro9inβ-turn and a hydrogen bond between His14and Phe17could be important to the thermostabilityof AEXynM.The hydrolytic time-course of AEXynM verified that the hydrolysis velocity of corncobsxylan was much slower than that of birchwood xylan. Xylobiose and xylotriose as the majorhydrolytic products were excised from corncob xylan by AEXynM, and their contentsreached42.33and38.76%, respectively. However, xylobiose as the major hydrolytic productwas released from birchwood xylan by AEXynM with content of58.56%. A trace of xylosewas detected during the hydrolysis, suggesting that AEXynM would be suitable for theproduction of xylooligosaccharides.更多还原