【作者】 荆海强；

【导师】 张钦宪； Anthony J.Clarke；

【作者基本信息】 郑州大学 ， 病理学与病理生理学， 2008， 博士

【摘要】 纤维素是植物细胞壁的主要结构组成物,是由β-1,4糖苷键组成的地球上最丰富的葡萄糖残基同聚物。近年来,通过纤维素的生物降解和发酵生产生物燃料和其他附加价值的产品已成为一种重要的潜在能源。纤维素的生物降解包括三种纤维素酶:葡聚糖内切酶（endo-β-1,4-glucanases,EC 3.2.1.4,）,葡聚糖外切酶,又称纤维二糖水解酶（exo-β-1,D-glucanases,Cellobiohydrolases,EC3.2.1.91）和β-葡聚糖苷酶（β-glucosidases,EC 3.2.1.21）等的共同作用,首先成为葡萄糖,最后经过发酵成为乙醇。前两类酶将不溶性的纤维素水解为可溶性的低聚糖,而β-葡聚糖苷酶又将该低聚糖分解为自由葡萄糖。这些纤维素酶基本上是一种模块化的酶,包括催化模块和一个或多个辅助模块。对于后者来讲,最常见的是糖基结合模块（Carbohydrate binding modules简称CBM）。糖基结合模块是具有特征性折叠结构的邻近的氨基酸片段,能够结合糖类基质。通过一级和二级的结构相似性,糖基激活酶数据库（http://www.cazy.org）目前列出了52族CBM。基于结构和功能的关系,对于结合在纤维素上的CBM,很多属于第2族。此族中的CBM,到目前为止,在大量的微生物体内出现,包括12种细菌,两种古细菌,12中真核生物和7种病毒。按照作用底物的特异性,CBM2分为两个亚组,CBM2a结合纤维素,CBM2b结合木聚糖（xylan）。尽管这两亚族都含有约100个氨基酸并折叠成相似的β三明质（β-sandwich）结构,其主要差异是模块的结合面。在CBM2a中,纤维素结合表面由于含有三个色氨酸残基而呈疏水性,该三个色氨酸残基对于纤维素的结合是必要的。目前的研究表明,结合模块主要表现出两方面的功能:一是促使酶在不溶纤维素体上的结合,二是减弱纤维素晶体结构中氢键网络的稳定性。然而通过荧光光漂白恢复技术对结合表面扩散的测量表明,CBM对纤维素的结合是动态的,模块在晶体纤维素表面上移动,用表面分析技术测试蛋白质在不溶性物质表面上作用,对于直观和深入地了解这种移动的过程和机理是十分必要的。在本研究中,我们克隆和表达了粪碱纤维单胞菌分泌的内切纤维素酶A（CenA）的部分基因序列,该序列编码信息肽和糖基结合模块(CBM_CenA)。同时为利用原子力显微镜（AFM）研究结合模块的动力学特性,通过结构预测和定点变异,将该结合模块中87位的苏氨酸替代为半胱氨酸(CBM_CenA T87C)使其能共价结合到AFM的探针金片上。在突变体CBM_CenA T87C的基础上,我们又重组了四个突变体(CBM_CenA T87C+W81A;CBM_CenAT87C+N114W;CBM_CenAT87C+N114Y;CBM_CenAT87C+N114F)以用来测试对纤维素的结合性质。方法1.以完整的cenA基因为模板PCR扩增基因前426个碱基对,上游引物和下游引物分别为5’-ATCCAGATCGAATTCATGTCCACCCGCAGAACC-3·和5’-AATTTACATAAGCTTTCAGCTGGTCGTCGGCACGGTGCC-3’（下划线部分分别为EcoRⅠ和HindⅢ酶切位点）。2.扩增的开放阅读框由高纯PCR产物纯化试剂盒提纯,以EcoRⅠ和HindⅢ酶切,然后与同样酶切的pET30a（+）质粒连接。3.将融合质粒转化进E.coli DH5α感受态细胞中。从转化株中分离构建体,通过凝胶电泳筛选大小正确的插入子,然后进行序列测定以确定目的核酸的插入。将获得的构建体命名为pACHJ-1。4.通过定点突变,将完整CenA的结合模块中87位的苏氨酸替代为半胱氨酸。以pACHJ-1为模板,使用Pfu Turbo DNA聚合酶,按照QuikChange定点突变的方法,正向引物为5’-GCGTCGTGCAACGGCGGCCAGGTC-3’,反向引物为5’-GCCGTTGCACGACGCGGTGCCGTTCC-3’（改变的核酸分别用黑体字标示）。PCR扩增后,在50μLPCR反应混合物中加入DpnⅠ酶,在37℃下反应至少1小时以去除原始甲基化的模板。所得质粒（pACHJ-2）转化进E.coliDH5α细胞中。通过DNA测序筛选正确的突变体。5.将pACHJ-1或pACHJ-2转化进E.coli BL21（DE3）细胞中,然后在含50μg/mL卡那霉素的LB肉汤37℃孵育。当培养液的吸光度达到0.6时,加入IPTG,使最终浓度为0.1到1mM以利于基因表达。分别在15℃,30℃,和37℃下孵育细胞并以吸光度监测细胞的生长。6.目的蛋白用Ni₂+NTA琼脂胶亲和柱和阴离子交换色谱（Source Q）进行纯化。用凝胶电泳检测纯化程度,以蛋白印迹确定目的蛋白。7.以微晶纤维素为对象,用纤维素结合测试法进行不溶性纤维素的结合检测。在pH为7.0的50mM磷酸钠缓冲液中,利用纯化的CBM2a与微晶纤维素Avicel在4℃和30℃下进行结合实验。结合8小时后,离心除去不溶的纤维素和已经结合的蛋白质,利用紫外分光光度计测量CBM2a开始的浓度和结合后上清液的浓度,CBM2a的结合量由此两浓度之差计算。8.在突变体CBM_CenA T87C的基础上,利用不同的引物,重组另外四个突变体CBM_CenAT87C+W81A;CBM_CenAT87C+N114W;CBM_CenAT87C+N114Y;CBM_CenA T87C+N114F用以结合与原子力显微镜探针上以检测对纤维素的结合动力学。结果1.编码CenA的CBM2a基因序列的克隆在cenA核酸序列的基础上,合成了合适的寡核苷酸引物用于PCR扩增。扩增产物包括编码信息肽和整个核糖结合模块的N端142个氨基酸。扩增产物被克隆进含编码His₆标签的pET30a（+）载体中,然后此构建体pACHJ-1被转化进E.coli DH5α细胞中。核酸序列经测序得到确认,可以用于后续实验。2.CBM2a的产生首先CBM2a构建体pACHJ-1被转化进E.coli BL21（DE3）细胞中进行诱导表达实验。当细胞生长至对数生长期时,用不同浓度的IPTG（0.1 mM到1mM）分别在15℃,30℃,和37℃下诱导CBM2a的表达。在12小时内的表达期间内,以SDS-PAGE和蛋白印迹法分析目的蛋白表达情况。实验表明,即使在不同浓度的IPTG下延长表达时间,pACHJ-1编码的CBM2a在15℃或37℃下的表达量也相对较少。所确定的优化诱导条件是:0.5mM IPTG在30℃下诱导2.5小时。3.CBM2a的纯化利用金属离子鏊合亲和层析法(Ni²⁺-NTA琼脂胶)分离和纯化带His₆标签的CBM2a。在pH8的缓冲溶液中将粗蛋白在树脂中上样,然后增大咪唑浓度洗脱CBM,用SDS PAGE胶确定纯度。如果细胞没有进行及时处理提取蛋白,当储存时间超过3天时,与CBM2a共同洗脱的杂蛋白会增多。通过渗析将咪唑和缓冲液中其他成分去除后,样品进一步用阴离子交换色谱（Source Q）纯化。在pH为8.5时,CBM2a保留在树脂上,将NaCl用线性梯度增加到1M,发现CBM2a在约50 mM NaCl时CBM2a被洗脱。通过渗析去除NaCl,用SDS-PAGE检测CBM2a的纯度达到98%以上。利用这种制备和纯化条件,在1升培养液中通常可以得到0.9毫克的纯CBM2a。用抗-His标签的单克隆抗体通过蛋白印迹法确认了CBM2a蛋白。4.CBM2a的纤维素结合性质在本实验条件下,CBM2a开始的浓度与结合量直接相关。对于不同的起始浓度,结合系数在78%到88%之间。令人感兴趣的是,这种结合没有随温度而有变化,因为在30℃下的有相似的结合结果。CBM2a的结合在实质上是不可逆的。在4℃或30℃下,独立的CBM2a并没有随水从不溶性的纤维素上面洗脱下来,即使用5%的SDS,回收的CBM2a也不超过25%。这种情况与缺少连接桥（Pro-Thr盒）的CenA的情况相似:此蛋白的结合被认为在实际上是不可逆的。5.定点突变在对CBM2a进行三维结构预测的基础上,半胱氨酸替代87位的苏氨酸以利于该模块结合到AFM的探针上。与推测的一致,与野生型的CBM2a相比较,该突变体保留了相同的结合特性。约74%的突变体与Avicel相结合。其他四种突变体(CBM_CenAT87C+W81A;CBM_CenAT87C+N114W;CBM_CenAT87C+N114Y;CBM_CenA T87C+N114F)也成功地得到构建,表达和纯化。用本实验方法得到的纯的突变体的浓度可以满足用AFM或其他敏感分析技术测试结合特性和机理的要求。结论1.精确地克隆并表达了由粪碱纤维单胞菌分泌的纤维素内切酶A基因（cenA）的仅编码N端前导肽和糖基结合模块（CBM）的片段,并纯化了目的蛋白,建立了诱导和表达的优化条件。2.测试了CBM_Cena在不溶性纤维素上的结合性质,明确了其结合的不可逆性,随起始浓度的不同,结合率在78%和88%之间。3.通过结构预测,构建了突变体CBM_CenA T87C,该突变体与野生型的CBM2a相比较,保留了原有结合特性。4.在突变体CBM_CenA T87C的基础上,构建,表达并纯化了四个CBM_CenA突变体(CBM_CenAAT87C+W81A;CBM_CenAT87C+N114W;CBM_CenAT87C+N114Y;CBM_CenA T87C+N114F),用于原子显微镜的结合以研究其与纤维素结合的动力学性质。更多还原

【Abstract】 Cellulose,the major structural component of plant cell walls,is a homopolymer ofβ-1,4-linked glucose residues.It has attracted renewed interest as a potential source of energy through its biodegradation and fermentation to biofuels.The biodegradation of cellulose involves the concerted action of three types of enzymes, endo-β-1,4-glucanases（EC 3.2.1.4）,exo-β-1,D-glucanases,or cellobiohydrolases（EC 3.2.1.91）,andβ-glucosidases（EC 3.2.1.21）.The former two classes of enzymes function to hydrolyze insoluble cellulose into soluble oligosaccharides which then serve as substrates forβ-glucosidases to release free glucose.Many of these cellulolytic enzymes,and polysaccharide hydrolases in general,are modular proteins,being comprised of catalytic modules and one or more ancillary modules.Of the latter,the most common are the carbohydrate binding modules（CBM）.CBMs are defined as contiguous amino acid sequences with a discreet fold that possess carbohydrate-binding activity.The Carbohydrate Active Enzymes（CAZy） database（http://www.cazy.org/） currently lists 52 CBM families classified by primary and secondary structural similarities.Of the CBMs that bind cellulose,much is known about members of family 2 in terms of their structure and function relationship.To date,Family 2 CBMs are found in a large number of microorganisms involving 73 species of bacteria,2 species of archaea,12 species of eukaryotes,and seven viruses. CBM2 has been divided into two subfamilies according to substrate specificities; CBM2a binds cellulose whereas CMB2b interacts specifically with xylan.Whereas both involve approx.100 amino acid residues that fold similarly into aβ-sandwich, significant differences define the binding face of the module.In CBM2a,the cellulose-binding surface is relatively hydrophobic being comprised of three tryptophan residues which have been demonstrated to be essential for binding to cellulose.Current studies showed that the CBMs appear to aid in both the adsorption of the enzymes to the insoluble cellulose substrate and the destabilization of the hydrogen-bonding network within the crystalline substrate.However,surface diffusion measurements using fluorescence recovery techniques indicated that this interaction of the CBM is dynamic,involving mobility of the module along the surface of the crystalline cellulose.So it is very important to understand this mobility and binding mechanism of proteins at insoluble surfaces with surface analysis techniques.Herein,we describe the cloning and expression of the partial gene sequence of cellulase A（CenA） from Cellulomnas fimi encoding its leader sequence and CBM2a.To facilitate covalent binding of the isolated CBM2a to AFM probes with the appropriate orientation for binding to cellulose,a mutants form of the CBM was engineered and produced involving the site-specific replacement of Thr87 with Cys.Based on CBM_CenA T87C,four mutants(CBM_CenAT87C+W81A; CBM_CenAT87C+N114W;CBM_CenAT87C+N114Y;CBM_CenA T87C+N114F) were engineered to test the character of binding to cellulose.Methods1.The complete cenA gene was cloned previously from C.fimi genomic DNA was used as the template DNA for PCR amplification of first 426 base pairs of cenA using the following upstream 5’-ATCCAGATCGAATTCATGTCCACCCGCAGAACC-3’,and downstream 5’-AATTTACATAAGCTTTCAGCTGGTCGTCGGCACGGTGCC-3’ primers, respectively（EcoRⅠand HindⅢrestriction sites,respectively,are underlined）.2.Amplified ORFs were cleaned using High Pure PCR Product Purification kit, digested with EcoRⅠand HindⅢand ligated with appropriately digested pET30a（+） plasmid DNA.3.Recombinant plasmids were transformed into competent E.coli DH5α. Individual constructs were isolated from transformants,screened for the correct size of insert by agarose gel electrophoresis,and they were sequenced to confirm nucleotide identity.The resulting isolated construct was named pACHJ-1.4.Thr87 was targeted for replacement with Cys by site directed mutagenesis of the truncated and cloned cenA gene to facilitate binding of the module to AFM probes.Mutagenesis was performed using pACHJ-1 as template for Pfu Turbo DNA polymerase according to the QuikChange Site-Directed Mutagenesis with the forward and reverse primers 5’-GCGTCGTGCAACGGCGGCCAGGTC-3’ and 5’-GCCGTTGCACGACGCGGTGCCGTTCC-3’,respectively,where the boldface denotes the changed nucleotides.Following PCR,DpnⅠwas added to a 50μl PCR reaction mixture and incubated for at least 1 h at 37℃to remove the original,methylated template.The resulting plasmid（pACHJ-2） was used to transform E.coli DH5αand clones were screened for the correct mutations by DNA sequencing.5.E.coli BL21（DE3） freshly transformed with either pACHJ-1 or pACHJ-2 was inoculated into LB broth supplemented with 50μg/mL Kan and incubated at 37℃.When the cultures reached mid-exponential phase（absorbance approx.0.6）, freshly prepared 0.1-1 mM IPTG（final concentration） was added to induce gene expression.Incubation of cells at either 15℃,30℃,or 37℃was continued for 12 h and growth was monitored by absorbance measurements.6.The target protein was purified by a combination of affinity using Ni₂+NTA-agarose and anion exchange chromatographies using Source Q, respectively.The mutants were confirmed by SDS-PAGE and Western blot analysis with an anti-His₆ antibody. 7.The ability of purified CBM2a to bind to micro-crystalline cellulose was tested using Avicel in 50 mM sodium phosphate buffer,pH 7.0 at 4℃.The amount of CBM2a bound was determined by difference using UV absorbance spectroscopy to measure protein concentrations in both the starting material and supernatants following incubation for 8 h and removal of the insoluble cellulose and adsorbed protein by centrifugation.8.Based on CBM_CenA T87C,four mutants were engineered using proper primers, respectively,to attach to AFM involving to test the character of binding to cellulose.(CBM_CenAT87C+W81A;CBM_CenAT87C+N114W; CBM_CenAT87C+N114Y;CBM_CenA T87C+N114F)Results1.Cloning of gene sequence encoding CBM2a from CenABased on the nucleotide sequence of cenA,appropriate oligonucleotide primers were synthesized for PCR amplification of the N-terminal 142 amino acids of the encoded protein which would include its signal pcptide,the entire CBM2a.The amplified product was cloned into pET30a（+） in frame with coding for a cleavable N-terminal His₆ tag,and the construct,pACHJ-1,was transformed into E.coli DH5α. The nucleotide sequence was confirmed by DNA sequencing prior to subsequent use.2.Production of CBM2aInitially,the CBM2a construct pACHJ-1 was transformed into E.coli BL21 （DE3） for induction-expression trials.Cells were grown to mid-exponential phase and then induced for cbm2a expression with varying concentrations of IPTG（0.1 mM to 1 mM） and incubation at either 15℃,30℃,or 37℃.Continuing growth was monitored over a 12 h period by both turbidity measurements while CBM2a production was followed by SDS-PAGE and Western immunoblot analysis. Relatively poor expression of cbm2a from pACHJ-1 was observed when induction was conducted at either 15℃or 37℃,even for extended periods of time with varying IPTG concentrations.Optimum induction conditions were established to involve the addition of 0.5 mM IPTG for 2.5 h at 30℃.3.Purification of CBM2aImmobilized metal ion affinity chromatography with Ni²⁺-NTA agarose was used to isolate and purify His₆-tagged CBM2a.Crude protein samples were applied to the resin at pH 8 and after an initial wash,a step gradient of increasing imidazole served to elute the CBM with minimal contamination as determined by SDS-PAGE.The extent of the contaminating protons co-eluting with CMB2a was observed to increase if cell pellets were not used immediately but instead stored for extended periods of time（more than 3 days）.After removal of the imidazole and other buffer components by dialysis,the CBM2a preparation was further purified by anion-exchange chromatography on Source Q.The CBM2a was retained by the resin at pH 8.5 and it was recovered in 50 mM NaCl after the application of a linear gradient to 1 M NaCl.After removal of the NaCl by dialysis,these preparations of CBM2a were found to be purified to greater than 98%homogeneity,as judged by SDS PAGE and densitometry.This production and purification protocol routinely provided an average of 0.9 mg purified CBM2a per L of cell culture.Western immunoblot analysis using and anti-hexa-His tag polyclonal antibody confirmed the identity of the engineered CBM2a.4.Cellulose binding activity of CBM2a.Under the conditions,a direct relationship was observed between the amount of CBM2a applied to the cellulose and that adsorbed.Depending upon initial protein concentrations,the efficiency of binding was found to be between 78%and 88%. Interestingly,this binding was not observed to be dependent on temperature as similar results were obtained after incubation at 30℃.The binding of CBM2a was found to be virtually irreversible.The isolated CBM2a did not elute from the insoluble cellulose in water at either 4℃or 30℃. Even elution with 5%SDS led to the recovery of no more than 25%of the adsorbed module.This situation was thus analogous to that observed involving CenA derivative that lacked a complete linker peptide（Pro-Thr box）;binding of this protein was considered to be ’quassi-irreversible’. 5.Site-directed mutagenesisBased on the predicted three-dimensional structure of CBM2a,Thr87 was replaced with Cys to facilitate binding of the module to AFM probes.As expected,the Thr87→Cys mutant CBM2a retained the same binding capacity to Avicel as compared to wild type CBM2a.Thus,74%of the applied mutant CBM2a was retained by Avicel.Other four mutants(CBM_CenAT87C+W81A;CBM_CenAT87C+ N114W;CBM_CenAT87C+N114Y;CBM_CenAT87C+N114F) were successfully constructed,expressed and purified.Sufficient concentrations of the mutant module were obtained in homogeneous form by this protocol to permit its further characterization and delineation of its mechanism of action by AFM and other sensitive analytical techniques.Conclusions1.The gene encoding CBM2a and leader sequence of CenA from Cellulomonas fimi was first cloned and expressed.The target protein was purified.Optimum induction and expression conditions were established.2.The binding of CBM_CenA to insoluble cellulose is virtually irreversible,depending upon initial protein concentrations,the efficiency of binding was found to be between 78%and 88%.This binding was not observed to be dependent on temperature.3.The Thr87→Cys mutant CBM2a retained the same binding capacity to Avicel as compared to wild type CBM2a.4.Based on CBM_CenAT87C,four mutants(CBM_CenAT87C+W81A; CBM_CenAT87C+N114W;CBM_CenAT87C+N114Y;CBM_CenA T87C+N114F) were constructed,expressed and purified to test the character of binding to cellulose by Atomic Force Microscope.更多还原