纤维堆囊菌So9733-1纤维素酶复合体的蛋白质组学分析与酶基因的克隆、表征

【作者】 王淑云；

【导师】 李越中；

【作者基本信息】 山东大学 ， 微生物学， 2011， 博士

【摘要】 粘细菌（myxobacteria）是一类能够进行滑行运动的革兰氏阴性杆菌,具有复杂的多细胞社会学行为,可以形成种类丰富、功能多样的生物活性物质。主要存在于土壤、食草性动物粪便、腐殖质以及树皮等组织中。根据对生物大分子的降解特性,粘细菌可以分为两大类群：溶纤维素类群和溶细菌类群。溶纤维素类群包括堆囊菌属（Sorangium）和Byssophage属。纤维堆囊菌（Sorangium celullosum）是堆囊菌属的模式种。目前关于纤维堆囊菌的研究大都集中在其新颖、丰富的活性代谢产物方面,而对于堆囊菌能够降解大分子底物这一最为独特、基本的微生物学特性,相关的研究则很少,而且没有相关的酶基因被克隆并表征。纤维堆囊菌的生长对营养的要求很低,能够在仅含有滤纸的简单无机盐平板（如CNST平板）上生长并有效地降解滤纸纤维素,另外,纤维堆囊菌对可溶性淀粉、木聚糖等复杂碳源的利用能力也较强。本实验室前期研究中,在纤维堆囊菌So9733-1菌体表面得到了一个具有内切葡聚糖酶、外切葡聚糖酶、葡萄糖苷酶、木聚糖酶等多种酶活力的大小在1500-2000 kDa的高分子量组分。我们推测此多酶组分可能组成一个大的复合体结构。为了解析此多酶复合体的组成,本论文利用蛋白质组技术（双向电泳、质谱分析、以及鸟枪法蛋白质组学）对该复合体的组分进行了分离和鉴定,并且将相关酶组分编码基因进行了克隆、表达和表征。为深入了解纤维堆囊菌多酶复合体的组织结构方式和作用机制奠定了基础。首先,我们利用经典蛋白质组学的方法（双向电泳和质谱检测）对多酶复合体的组成成分进行解析。经过条件摸索,我们成功的利用双向电泳技术将复合体的各组分分开,在凝胶图谱上,得到的蛋白点主要集中在偏酸性范围,偏碱性区域蛋白点很少。取考马斯亮蓝染色的双向电泳凝胶上的30个蛋白点进行胶内酶解和MALDI-TOF检测,结果只有1个蛋白点和来源于Ralstonia solanacearum的一个内切葡聚糖酶相匹配。我们又将上述30个蛋白点进行二级质谱鉴定（线性离子阱四级杆质谱,LTQ）。最终,有5个蛋白点（2,18,19,25,28号）得到Base peak图谱（见附录3）,而只有一个蛋白点（25号）鉴定为α-淀粉酶,得到的肽段是：R.VVVARPAPGAAIR.R。由于经典的双向电泳-质谱检测技术路线存在一些缺点（双向电泳难以分离极酸性、极碱性蛋白、疏水性蛋白等）,我们又利用了鸟枪法蛋白质组学的策略来大规模地分离和鉴定纤维堆囊菌So9733-1胞外多酶复合体的组分。最终,我们鉴定了一系列的纤维素、淀粉、甘露糖、木糖苷酶等碳水化合物的相关降解酶组分,这和纤维堆囊菌对纤维素、木聚糖、可溶性淀粉等复杂碳源的利用能力较强的特性相一致。通过对So9733-1的胞外多酶复合体的蛋白质组分析,质谱鉴定得到了肽段K.EPFEVCIDDIR.L和K.YDALAYFYHNR.S,和So ce56的一个纤维素酶基因（sce2234）编码的蛋白（YP₀01612873）相匹配。对纤维堆囊菌So ce56的所有纤维素酶基因编码蛋白分析发现,只有此蛋白含有CBM结构域。我们利用得到的肽段序列和基因sce2234的ORF序列设计引物,通过降落PCR,最终在So9733-1中得到了670 bp的纤维素酶相关基因片段。我们根据此670 bp的序列设计特异性的嵌套引物,同时利用实验室前期设计的简并引物,通过Tail-PCR技术得到了上下游的DNA序列,经过拼接,得到了完整的纤维素酶基因（命名为：celA）的ORF序列。celA基因为2,373 bp,编码含790个氨基酸残基的蛋白质,预测的分子量为84.9 kDa,理论等电点为6.36。SignalP分析表明,CelA的N-端存在一个41个氨基酸残基的信号肽。蛋白保守域分析表明,成熟CelA蛋白由N-端的CBM₄₉,一个保守的纤维素酶N-端结构域,和C-端的糖苷水解酶家族9的催化域组成。将celA ORF中编码成熟蛋白的DNA序列进行PCR扩增并连接到表达载体pET-28a（+）上,转化大肠杆菌BL21（DE3）,进行异源表达。经过不断的条件摸索,最终发现在18℃用0.5 mM的IPTG诱导20小时,菌体裂解液上清中检测到了纤维素酶活。重组蛋白的粗酶液活性分析发现,CelA最适反应温度为35℃,最适反应pH值为7.0。底物特异性分析发现,CelA主要表现为p-1,3-1,4-葡聚糖酶活性,同时具有p-],4-内切葡聚糖酶活性,p-1,3-内切葡聚糖酶,木聚糖酶活性。Ca2+在1mM的浓度下使酶活增加70%。Cu2+和Zn2+对酶活有强烈的抑制作用。我们同时利用PCR的方法扩增了celA ORF中除去N-端的信号肽和CBM结构域的基因序列,在大肠杆菌BL21（DE3）中进行异源表达,但是经IPTG诱导后,裂解液上清中检测不到任何纤维素酶活,我们推测可能CBM结构域对CelA的酶活性的发挥起重要作用。同时,我们利用蛋白质组学分析鉴定得到了肽段R.GIVPVLWDTGTDIK.R,和So ce56的sce0902基因编码的蛋白（YP₀01611539）相匹配,因此我们根据sce0902的ORF序列设计引物,以So9733-1的染色体DNA为模板,成功扩增得到了一个384 bp的DNA片断。我们根据此384 bp的序列设计特异性的嵌套引物,通过Tail-PCR技术得到了片断上下游的DNA序列,经过拼接,得到了一个完整的ORF序列,后续酶活分析发现,此基因编码淀粉酶,命名为：amyA。amyA ORF长度为1,329 bp,编码一个含442个氨基酸残基的蛋白质,预测的蛋白分子量为46.2 kDa,理论等电点为4.59。SignalP分析表明,AmyA为胞外的分泌蛋白,N末端存在一个38个氨基酸残基的信号肽。蛋白的保守域分析表明,AmyA成熟蛋白含有单一的催化结构域,同来源于糖苷水解酶家族5的纤维素酶、以及有保守的COG2730结构域的内切葡聚糖酶存在一定的相似度。将amyA ORF中编码成熟蛋白的DNA序列进行PCR扩增并连接到表达载体pET-22b（+）上,转化大肠杆菌BL21（DE3）,进行异源表达。最终通过0.5 mM的IPTG诱导,18℃继续培养20小时,在大肠杆菌的细胞裂解液上清中可检测到较高的淀粉酶活性。用镍柱亲和层析纯化裂解液上清,得到了纯化的重组蛋白。重组蛋白的酶活表征发现,AmyA最适反应温度为45℃,最适反应pH值为6.5。AmyA具有较高的淀粉酶活性,经过12小时的作用,同时表现出了p-1,3-1,4-葡聚糖酶、β-1,4内切葡聚糖酶活性、木聚糖酶活性、以及海带多糖降解活性。AmyA对金属离子或化学试剂很敏感,K+、Ca2+和Mg2+在1 mM的浓度下可使酶活稍有降低。1mM的Cu2+、Zn2+和EDTA对酶活有强烈的抑制作用。实验室前期研究得到了422bp的木聚糖酶基因片段（DQ354922）,异源表达此片断并制备多克隆抗体,免疫印迹和免疫荧光定位分析显示此酶可能是多酶复合体的一个组分并定位在细胞表面。本文利用Tail-PCR技术,成功地克隆了此木聚糖酶基因（xynA）完整的ORF序列,经过异源表达和性质分析,发现此酶是一个适冷的木聚糖酶,而且热稳定性很低。xynA的ORF长度为1,209bp,基因的G+C百分含量为52.27%,远低于报道的粘细菌DNA的G+C百分含量（67-72%）,因此我们推测此基因可能是由其他基因G+C百分含量低的细菌转移进纤维堆囊菌So9733-1。xynA基因编码一个含402个氨基酸残基的蛋白质,预测的蛋白分子量为45.8 kDa,理论等电点为4.83。SignalP分析表明,蛋白的N末端存在一个41个氨基酸残基的信号肽。蛋白保守域分析表明,XynA只含有单一的糖苷水解酶家族10的催化域。三维结构预测分析发现XynA的催化域是一个典型的（p/α）8折叠结构。PCR扩增xynA ORF中编码成熟蛋白的序列,在大肠杆菌BL21（DE3）中进行异源表达。经过不断的条件摸索,最终发现在18℃用1mM的IPTG诱导20小时,重组蛋白以胞内可溶形式表达。经过镍柱亲和层析和透析,得到了电泳纯的有活性的重组蛋白XynA。重组蛋白酶活表征分析发现,XynA最适反应温度为30℃,在0℃和5℃时分别保持13.7%和33.3%的相对活性。50℃作用20 min,酶活性丧失80%。因此,XynA为一个典型的适冷木聚糖酶,热稳定性低。在30℃,酶对三种木聚糖底物beechwood xylan, birchwood xylan和oat spelt xylan的Km值分别为30.74,32.77 and 41.87 mg/ml。XynA的最适反应pH值为7.0。Ca2+能够极大地促进酶活。XynA降解木聚糖和木寡糖主要生成木糖和木二糖。XynA作为一个适冷的木聚糖酶,可以应用到一些需要低温的生产过程中,特别是食品产业。同时,我们利用纯化的木聚糖酶XynA,制备了多克隆抗体,为后续分析此酶的细胞定位等奠定了基础。对纤维堆囊菌So9733-1多酶复合体的三个酶组分（CelA, AmyA, XynA）的基因的克隆和重组蛋白分析,为进一步深化研究多酶复合体的组织形式和功能奠定了良好的基础。更多还原

【Abstract】 Myxobacteria are Gram-negative gliding bacteria, which exhibit complicated multicellular social behavior and produce large numbers of bioactive compounds that have unique structures and diverse functions. The principal habitats of myxobacteria are soil, dung, decaying plant material, and the bark of living and dead trees. Based on their degradation ability for biomacromolecular, myxobacteria can be divided into two groups:cellulolytic myxobacteria and bacteriolytic myxobacteria.Within cellulolytic group, there are two genuses named Sorangium and Byssophage, in which Sorangium cellulosum is the type species of Sorangium.Studies on S. cellulosum have mainly been focused on its excellent production ability of diverse and novel bioactive secondary metabolites, whereas the degradation ability on macromolecules, the unique and basic property of S. cellulosum, have received less attentions and none of the involving enzymes has been characterized so far. S. cellulosum can grow on simple inorganic salt medium （e.g. CNST medium） with sterilized filter paper as the sole carbon source and can degrade complex polysaccharides such as cellulose, solulable starch and xylan efficiently.Our previous studies showed that a high-molecular weight component about 1500-2000 kDa with multi-enzymatic activities （e.g. endo-glucanase, exo-glucanase, xylanase, Glucosidase et al.） was linked to the cellular surfaces and might organize into a large complex.In this paper, we isolated and identified the components of the multi-enzyme complex using proteome technique, and we proceded to clone and characterization the enzymes in the complex. The work will help us to understand the organization strategy and polysaccharides degrading mechanism of the complex.First, we analyze the components of the multi-enzyme complex utilizing classical proteome technique:Two-dimensional polyacrylamide gel electrophoresis （2D-PAGE） and mass spectrometry （MS）. We successfully separate the proteins in the complex by 2D-PAGE. It was found that the isoelectric point （pI） of proteins are mainly on the acid side, and on the alkaline side, there are rare proteins.30 protein spots were excised from coomassie brilliant blue-stained gel, conducted in-gel digestion, and then analyzed using MALDI-TOF. Among the available mass spectrums of the 21 proteins, only one matched the endo-glucanase from Ralstonia solanacearum.We proceed to excise the 30 protein spots mentioned above, and then analyzed using tandem mass spectrometry （linear trap quadrupole, LTQ）. Finally, base peak chromatograms of 5 proteins were obtained, and one protein was identified to beα-amylase with the peptide sequence of R.VVVARPAPGAAIR.R.Considering the limitations of the classical proteome technique （extremely acid, alkaline, or hydrophobic protein etc. can’t be separated by 2D-PAGE）, we used shot gun proteomics to globally isolate and identify the components of the multi-enzyme complex from So9733-1. Finally, a number of cellulase, amylase, mannanase, and xylosidase were identified, and it is correspondent with the phenomenon that Sorangium cellulosum can efficiently degrade complex polysaccharides such as cellulose, solulable starch and xylan.By shot gun proteomics analysis of the multi-enzyme complex, two peptides （K.EPFEVCIDDIR.L and K.YDALAYFYHNR.S） were identified to match the cellulase （YP₀01612873, coded by gene sce2234） from So ce56. Among all the predicted cellulases of So ce56, only this protein has CBM domain. Using the sequnces of the two peptides and gene sce2234, we designed PCR primers and obtained a 670-bp cellulase-related fragment by Touch-down PCR. The nested insertion-specific primers were designed based on the 670-bp fragment, and the flanking regions of the fragment were obtained using thermal asymmetric interlaced （TAIL）-PCR. By assembling the sequences, the full-length of the ORF （named celA） was obtained.The gene celA consist of 2,373 bp encoding a 790-amino-acid protein with a calculated molecular mass of 84.9 kDa and a theoretical isoelectric point of 6.63. A 41-residue signal peptide was present at the N terminus of the protein predicted by SignalP. The mature CelA protein contained CBM₄₉ at N-terminus, a conserved Cellulase N-domain and a catalytic domain of the glycoside hydrolase family 9. The ORF encoding the mature cellulase was subcloned into pET-28a（+） and expressed in Escherichia coli BL21 （DE3）. After induction with 0.5 mM IPTG at 18℃for 20 hr, the recombinant CelA was expressed in the supernatant of the cell lysates with cellulase activity.Characterization analysis suggested that the optimum pH and temperature for the crude enzyme were 7.0 and 35℃, respectively. Crude CelA exibit activities ofβ-1,3-1,4-glucanase,β-1,4-glucanase,β-1,3-glucanase, and xylanase. Ca2+ enhanced CelA activity by about 70% at 1 mM concentration and Cu2+, Zn2+ strongly inhibited the CelA activity.We also amplified the ORF coding the mature cellulase without the domain CBM₄₉, and expressed in Escherichia coli BL21 （DE3）. After induction with IPTG, the supernatant of the cell lysates had no cellulase activity. We presumed that CBM₄₉ was essential for the enzyme to function properly.In the proteomics analysis of the multi-enzyme complex, a peptide （R.GIVPVLWDTGTDIK.R） was obtained to match the protein （YP₀01611539, coded by gene sce0902） from So ce56. Based on the sequnce of the gene sce0902, a 384-bp DNA fragment was amplified from So9733-1 by Touch-down PCR. To obtain the whole sequence of the gene （named amyA）, the TAIL-PCR technique was employed to amplify the 5’and 3’flanking sequences of the fragment.Finally, the full-length of the gene amyA containing 1,329 bp was obtained. The gene encoded a 442-amino acid protein and a putative 38-amino acid signal peptide. The estimated molecular weight of Amy was 46.2 kDa and the theoretical isoelectric point was 4.59. The mature protein contained a single catalytic domain similar to proteins from the glycoside hydrolase family 5 and the endoglucanases of COG2730. The gene coding the mature AmyA was ligated into the expression vector pET-22b（+）, and expressed in Escherichia coli BL21 （DE3）. After induction with 0.5 mM IPTG at 18℃for 20 hr, the recombinant protein was expressed in cell lysate supernatant with amylase activity.The recombinant protein was purified to electrophoretic homogeneity by Ni-affinity chromatography and subsequently characterized. The recombinant AmyA had optimum activity at pH 6.5 and 40℃. AmyA exibit high activity of amylase, and after incubation for 12 hr, activities ofβ-1,3-1,4-glucanase,β-1,4-glucanase, (3-1,3-glucanase, and xylanase were detected. AmyA was sensitive to mental irons and chemicals:K+, Ca2+ and Mg2+ at 1 mM concentration can slightly inhibit the activity while Cu2+, Zn2+ and EDTA at 1 mM concentration strongly inhibited the enzyme activity.A 422-bp xylanase fragment was obtained in our previous research. Using the antibodies of the fragment, the xylanase was confirmed to be organized in the purified cell-bound cellulolytic enzyme complex by Western blotting and located on the cell surface protuberances by immunofluorescent staining. We cloned the xylanase gene （xynA） by Tail-PCR based on the 422-bp xylanase fragment. Characterization analysis suggested that the purified recombinant XynA was a cold-active xylanase with low thermostability.The gene was composed of 1,209 bp and had only 52.27% G+C content, which is much lower than that of myxobacterial DNA reported （67-72%）. It is supposed that xynA may originate from other bacterial species with lower G+C content than that of S. cellulosum. The ORF encoded a predicted protein of 402 amino acids with a calculated molecular mass of 45.8 kDa and a theoretical isoelectric point of 4.83. SignalP analysis suggested that a 41-residue signal peptide was present at the N terminus of the protein. The mature XynA protein contained a single catalytic domain of the glycoside hydrolase family 10. Furthermore, homology modeling using SwissMode programs showed a typical （β/α）8 structure in the XynA catalytic domain.The xylanase gene fragment coding the mature xylanase, was subcloned into pET-22b（+） and expressed in Escherichia coli BL21 （DE3）. After induction with 1 mM IPTG at 18℃for 20 hr, the recombinant XynA was expressed in the supernatant of the cell lysates. The recombinant protein was purified by Ni-affinity chromatography.The optimum temperature for the XynA was 30℃, and XynA exhibited 33.3% activity at 5℃and 13.7% activity at 0℃. Approximate 80% activity was lost after 20-min preincubation at 50℃. These results indicated that XynA was a cold-active xylanase with low thermostability. At 30℃, the Km values of XynA on beechwood xylan, birchwood xylan and oat spelt xylanwere 30.74,32.77 and 41.87 mg/ml, respectively. The purified XynA displayed optimum activity at pH 7.0. The activity of XynA was enhanced by the presence of Ca2+. The recombinant XynA hydrolyzed beechwood xylan, birchwood xylan and xylooligosaccharides （xylotriose, xylotetraose, and xylopentose） to produce primarily xylose and xylobiose. As a typical cold-active xylanase, XynA is most active at low and intermediate temperatures and could offer advantages over the currently used xylanases in many of the low to moderate temperature processes, in particular in the food industry.Multi-clone antibody was produced with the purified XynA as the antigen, which could be used to analyse the location of the protein in So9733-1.As the cellulase CelA, amylase Amy A, and xylanase XynA are components of the multi-enzyme complex, molecular cloning and characterization of the enzymes will help us to reaveal the organization and function of the complex.更多还原