【作者】 周晨妍；

【导师】 王武；

【作者基本信息】 江南大学 ， 发酵工程， 2008， 博士

【摘要】 木聚糖酶（EC 3.2.1.8）以内切方式水解木聚糖分子中的β-1,4-木糖苷键,产生不同链长的寡糖和木糖,是木聚糖降解酶系中最关键的酶。它广泛存在于各种微生物中,在饲料、造纸、食品、医药以及能源工业中有着广阔的应用前景。根据宇佐美曲霉（Aspergillus usamii）E001木聚糖酶（XynⅡ）N末端15个氨基酸残基序列以及真核生物mRNA在3′端存在poly（A）等序列信息,利用RACE技术扩增和克隆了相关的基因片段,获得了XynⅡ的全长cDNA序列和DNA序列,并分析了该序列的有关信息。克隆的cDNA序列、DNA序列的Genbank登录号分别为DQ114485和DQ191144。成熟肽基因推导的氨基酸序列同其他微生物来源的若干G/11族木聚糖酶相比,具有较高的同源性,属内比对,与来源于Aspergillus niger（GenBank登录号:ANU39784）的木聚糖酶同源性最高,达89%。将XynⅡ成熟肽的cDNA序列克隆至pET-28a（+）表达载体中,在大肠杆菌BL21-CodonPlus（DE3）-RIL中进行了IPTG诱导表达,木聚糖酶的比酶活最高达49.61U/mg。进而将该基因克隆到毕赤酵母表达载体pPIC9K中,转化毕赤酵母GS115和KM71,经筛选获得阳性重组菌PXGL98（Mut⁺）和PXKL29（Mut^s）。该木聚糖酶基因在2种毕赤酵母中均实现了分泌表达,工程菌发酵条件优化后,PXGL98与PXKL29发酵液中的比酶活分别可达3139.68 U/mg和3846.83 U/mg。对木聚糖酶XynⅡ进行同源建模和序列比较,定向诱变证明在催化反应中起重要作用的氨基酸残基为Glu-79和Glu-170。研究发现第11族木聚糖酶的催化结构域在B折叠股A3和B3之间存在一个保守的氨基酸位点Asp-37,该位点与木聚糖酶的pH特性有关,据此设计了XynⅡ的D37N定向诱变。酶学性质分析显示,XynⅡ_D37N的最适pH值由4.2升高到5.3,pH稳定范围也向碱性方向偏移,由pH 3.0-7.5变为pH 3.0-9.0。这表明木聚糖酶XynⅡ的第37位Asp与最适pH值相关,为进一步的结构与功能研究提供了理论基础。在XynⅡ的“Ser/Thr”平面引入精氨酸的四点诱变（ST4）和五点诱变（ST5）,对酶的热稳定性进行改造。获得的两个突变酶在热稳定性上比野生型酶都有不同程度的提高。突变酶ST4和突变酶ST5使酶的最适温度分别由原酶的50℃提高为52℃和55℃。55℃保温15 min,ST4、ST5的残留酶活性由原酶的20%提高为65%和75%;保温1h,ST4、ST5的残留酶活性由原酶的15%提高为50%和65%。该突变酶在保持了XynⅡ优良性质的基础上,进一步提高了其热稳定性,具有更好的应用价值。更多还原

【Abstract】 Xylanase（EC 3.2.1.8） can hydrolyzeβ-1,4-glycosidic linkages of the xylan backbone to produce xylooligosaccharides and D-xylose.Hence,it is the crucial enzyme component of xylanolytic enzyme systems.It broadly exists in microorganisms and has wide commercial application in industrial processes,such as feed,paper,foodstuff,medicine and energy industries.Based on the information of N-terminal amino acid sequence of XynⅡfrom Aspergillus usamii E001,the appropriate codon usage of Aspergillus and 3’-terminal poly（A） of eukaryotic mRNA,the XynⅡcDNA was amplified by RACE method,and then the DNA sequence was amplified using specific primers of cDNA.Both sequences of cDNA and DNA were analyzed and were submitted to GenBank（Accn:DQ 114485,DQ 191144）.The amino acid sequence had higher similarity with those of G/11 family xylanases reported from other microorganisms.Compared with other Aspergillus sp.,the highest similarity was up to 89% with Aspergillus niger（GenBank Accn:ANU39784）.XynⅡcDNA fragment encoding mature peptide was inserted into the plasmid pET-28a（+） and expressed in E.coli BL21-CodonPlus（DE3）-RIL.A maximum activity of 49.6 U mg^-1 was obtained from cellular extract of E.coli BL21-CodonPlus（DE3）-RIL harboring pET-28a-xynlI.Then the mature peptide cDNA was cloned into the Pichia pastoris expression vector pPIC9K,resulting in the recombinant plasmid pPIC9K-xynⅡ.Linearized with SalⅠ,pPIC9K-xyn//was transformed into P.pastoris GS 115 and KM71,respectively. After selection,the recombinant P.pastoris PXGL98（Mut+） and PXKL29（Muts） were obtained.Both of the recombinant strains could secrete functional xylanase,and in shake-flask culture induced with methanol,the maximal enzymatic activities of PXGL98 and PXKL29 were up to 3139.68 U/rag and 3846.83 U/mg,respectively.A homology modeling of XynⅡwas constructed by SWISS-MODEL and BLAST. Mutational analysis of the xynⅡgene products showed that Glu-79 and Glu-170 were the important catalytic amino acid residues in the active site.A conserved amino acid,Asp-37 had been found in the catalytic domain betweenβ-sheet A3 and B3 in the tertiary structure,which influenced the pH properties of enzyme.Then a D37N mutation was introduced in XynⅡby site-directed mutagenesis.The mutant xylanase(XynⅡ_D37N) expressed in P pastoris were purified and its enzymatic properties were determined.The result revealed that the optimal pH of XynⅡD37N was increased from 4.2 to 5.3 and the pH stability of XynⅡ_D37N was changed from pH 3.0-7.5 to pH 3.0-9.0.The mutant xylanase XynⅡ_D37N was a good material for further research in the relationship between structure and function of xylanase.Replacing several serine and threonine residues on the Ser/Thr surface of XynⅡwith arginines effectively increased the thermostability of the enzyme.The substitution of Ser and Thr residues on the Ser/Thr surface of the enzyme with four（ST4） or five arginines（ST5） led to an increase in optimal temperature of the enzymes by 2℃and 5℃for the ST4 and ST5, respectively.The modified enzymes ST4 and ST5 showed 65%and 75%of maximal activities after incubated for 15 min at 55℃compared to only 20%activity for wild-type enzyme.After incubated for 1 h at 55℃,ST4 and ST5 showed 50%and 65%of maximal activity compared to only 15%activity for wild-type enzyme.Having the good properties of XynⅡ,the mutants with higher thermostability are potentially useful in industrial applications.更多还原