【作者】 苏贝；

【导师】 于文功；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2009， 硕士

【摘要】 纤维素是D-葡萄糖以β-1,4-糖苷键组成的线性大分子,它是植物细胞壁的主要组成成分并且是自然界中含量最高的多糖类物质。纤维素应用广泛,特别是作为可再生的碳源和能源,已成为基础和应用研究的热点。纤维素酶是一类能够将纤维素降解为葡萄糖的多组分酶系的总称,属于糖苷水解酶,传统上被分为3类组分:内切葡聚糖酶(endo-1,4-β-D-glucanase, EG, EC 3.2.1.4)、外切葡聚糖酶(exo-l,4 -β- D- glucanase, EC 3.2.1.91)、β-葡萄糖苷酶(β-l,4-D -glucosidase,EC 3.2.1.21)。这三种酶协同作用,分解纤维素产生寡糖和纤维二糖,最终水解产物为葡萄糖。纤维素酶的研究不仅在糖生物学领域具有重要理论意义,而且纤维素酶降解纤维素较传统的理化方法经济、有效,在纺织、日用化工、食品发酵、畜牧饲料等领域都有广泛的应用。本文利用硫酸铵沉淀、离子交换层析、凝胶过滤层析技术,从海洋细菌Cellulophaga sp. QY201发酵液上清中分离纯化到一种内切葡聚糖酶,命名为CelA。其比活力为59.8 U/mg,纯化倍数为33.2倍,回收率为3.2%,SDS-PAGE显示为单一条带,分子量为35.0 kDa。酶学性质研究表明,其最适反应温度和pH分别为50℃和pH6.0;该酶在0-40℃之间和pH4.0-6.6范围内稳定;在0℃,CelA仍保持最大酶活的50%-55%;将其在40℃保存3小时后,活力几乎没有丧失,24小时后仍保持酶活的88%; Na+、K+、Ni2+能促进酶活性, Cu2+、Mg2+、Fe2+、Ba2+、Zn2+、Ca2+、Fe3+、SDS能抑制酶的活性,但很多离子对其作用并不明显;将CelA在蛋白酶K终浓度为0.1mg/mL的体系中于37℃温浴30min,仍保存酶活的95%。底物特异性分析结果表明,该酶主要降解CMCNa,对whatman滤纸、木聚糖、微晶纤维素有微弱的降解作用,对CMC、水杨素、淀粉无降解作用;TLC分析其最小降解底物为纤维四糖,降解纤维寡糖的产物主要为纤维二糖和纤维三糖,还伴有少量的葡萄糖产生。为了克隆celA基因,我们对纯化的野生酶进行了氨基酸测序,根据其N-端和中间3段肽段的氨基酸序列设计简并引物,利用简并PCR和反向PCR从海洋细菌Cellulophaga sp.QY201的基因组DNA中克隆到CelA的编码基因celA。分析表明其开放阅读框为1080bp,编码359个氨基酸,理论分子量为40.799 kDa, N-端存在一个51个氨基酸组成的信号肽序列;推测其成熟蛋白由308个氨基酸组成,理论分子量和等电点分别为35.08kDa和5.0,其理论分子量与野生酶的SDS-PAGE结果完全一致;氨基酸序列分析发现其属于糖苷水解酶家族5。将celA基因连入pET24a (+)表达载体,并在E.coli BL21 (DE3)表达菌株中进行了表达。将菌体离心后溶于20mmolpH=4.6的Na2HPO4-柠檬酸缓冲液,超声破碎后,Ni离子亲和层析分离纯化重组酶。SDS-PAGE分析结果表明,纯化后的酶为单一条带,达到电泳纯,其分子量约为36kDa,与理论分子量一致。重组酶的酶学性质与野生酶基本一致。综上所述,本论文从海洋细菌Cellulophaga sp. QY201的发酵液上清中分离到一种中性内切葡聚糖酶CelA,该酶在0℃仍具有活性,且温度稳定性好;利用多种PCR技术克隆了其编码基因,并在大肠杆菌中实现了高效表达。该酶的发现不仅丰富了纤维素酶的多样性,而且有望用于纺织行业和日用化工行业,具有重要的理论意义和广泛应用前景。更多还原

【Abstract】 Cellulose is composed of D-glucose residues linked together to form linear chains viaβ-1,4-glycosidic linkages. It is the major polymeric component of plant cell wall and also the most abundant polysaccharide in nature. The value of cellulose as a renewable source of carbon and energy has made cellulose hydrolysis the subject of scientific research and industrial interest. Cellulase is a generic term of enzymes that can hydrolyze cellulose into glucose.Traditionally it has been classified into three categories, namely, endoglucanase, exoglucanase andβ-glucosidase. These enzymes can cooperate to degrade cellulose to cellooligosaccharide and cellobiose, and eventually to glucose. Degradation of cellulose by cellulase is more economical and effective than the traditionally physical and chemical methods, so it has a wide-range application in areas such as textile industry, commodity industry, food fermentation, laundry detergent and livestock feeding.An endoglucanase was purified 33.2-fold with a recovery yield of 3.2% from culture supernatants of Cellulophaga sp. QY201 to homogeneity using a combination of ammonium sulfate precipitation, DEAE-Sepharose FF anion-exchange chromatography and Superdex 75 gel filtration chromatography. The purified enzyme, which was named CelA, gave a specific activity of 59.8U/mg and a single band on SDS-PAGE with a molecular mass of 35.0 kDa.CelA was most active at 50℃and pH6.0, and stable below 40℃and over a range of pH4.0-6.6. The enzyme retained 50-55% of its maximum activity at 0℃. CelA could withstand 3 h at 40℃without irreversible loss of enzymatic activity and retain 88% of its activity at 40℃for 24h. The activity of the enzyme was enhanced in the presence of Na+、K+、Ni2+. Other compounds (Cu2+、Mg2+、Fe2+、Ba2+、Zn2+、Ca2+、 Fe3+、SDS) inhibited the activity of the enzyme,but the inhibition is not significant. Analysis also showed that CelA could retain 95% of its activity after incubated with Proteinase K (0.1mg/mL) at 37℃for 30 min. Analysis of the substrate specificity showed that CelA exhibited high activity on CMCNa, but limited activity on whatman filter paper, xylan, microcrystalline cellulose and no activity on CMC, salicin and starch. The digestion products of various cellooligosaccharides by CelA were submitted to thin-layer chromatography (TLC), which indicated that CelA produced mainly cellobiose and cellotriose along with glucose as a minor product, and the minimal hydrolysis substrate of CelA was cellotetraose.To clone the celA gene, the purified CelA protein was sequenced, and ten N- terminal amino acids and 3 internal peptides were obtained. The gene encoding CelA was cloned using a strategy combining degenerate PCR and inverse PCR. Analysis showed that gene celA contained an open reading frame of 1080bp. The predicted product was a protein of 359 amino acids with a theoretical molecular mass of 40.799 kDa. And there was a signal peptide of 51 amino acids at the N-terminal of the precursor of CelA. The deduced molecular weight and isoelectric point of mature protein were 35.08 kDa and 5.0, respectively, which was consistent with SDS-PAGE result of the native enzyme. Amino acid sequence-based classification revealed that it belonged to glycosyl hydrolase family 5. In order to obtain sufficient amount of the enzyme, the celA gene was subcloned into pET24a (+)/E.coli BL21 (DE3) expression system. The recombinant enzyme was purified by Ni-affinity chromatography after ultrasonic cell crasher in 20 mmol Na2HPO4-citric acid buffer (pH4.6). SDS-PAGE analysis yielded a single band with an apparent molecular mass of 36 kDa, which was close to the predicted molecular mass. And the recombinant enzyme shared uniform enzymatic characteristics with the native enzyme.In conclusion, an neutral endoglucanase possessing activity at 0℃and high thermal stability, was purified from the culture supernatants of marine bacterium Cellulophaga sp. QY201. Its gene was cloned and expressed in pET24a (+)/E.coli BL21 (DE3) system. The discovery of CelA not only enriches the diversity of endoglucanases but also has great practical significance in industrial application areas such as textile industry and commodity industry.更多还原