【作者】 唐婧；

【导师】 周泽扬；

【作者基本信息】 西南大学 ， 微生物学， 2008， 硕士

【摘要】 纤维素是自然界中含量最丰富的生物量。据估计,全球每年通过光合作用产生近千亿吨的纤维素类物质。但目前人们对纤维素的利用还非常有限,大量的天然纤维素资源仍因无法分解利用而丢弃。同时纤维素的结构特性也使得用纤维素酶直接降解纤维素的研究具有很大困难,这也为纤维素的高效性生物降解研究带来了很大难题。有鉴于此,研究高效降解纤维素的微生物菌株具有重要的意义。在天然纤维素降解过程中,微生物产生的纤维素降解酶具有决定性作用。纤维素降解酶包括三类:从纤维素长链的内部随即切割的内切型葡聚糖酶、从其非还原性末端依次切下两个糖基的外切型葡聚糖酶、以及从纤维素寡糖（主要是二聚体）的非还原端依次切下一个糖基的外切型β-葡萄糖苷酶。目前的研究认为,β-葡萄糖苷酶在纤维素的降解速率中起到关键性调控作用。提高β-葡萄糖苷酶的活力能够提高纤维素酶的降解效率,这对于纤维素的降解利用具有重要意义。本研究以获得新型β-葡萄糖苷酶基因为目标,首先筛选分离到一株β-葡萄糖苷酶的高产菌株,随后围绕菌株的分类鉴定、β-葡萄糖苷酶基因克隆、酶家族分类和酶学性质等方面进行了一系列的探讨研究。一、纤维素酶产生菌的筛选用CMC_Na平板筛选方法,从花腐败残余物中筛选出五株纤维素酶活性较高的菌株。然后结合β-葡萄糖苷酶平板筛选法,从中选出一株β-葡萄糖苷酶活性最强菌株,编号SWU-27。经初步发酵,提取菌株SWU-27胞外粗酶液,测定该菌株产胞外型内切葡聚糖酶、外切葡聚糖酶及β-葡萄糖苷酶酶活力,测定的活性结果分别为10.663U/mL、15.910U/mL和20.107U/mL,其中β-葡萄糖苷酶酶活力最高,且占总活力的43.07%。二、纤维素酶产生菌SWU-27的分类鉴定通过菌体个体形态特征、菌落特征和生理生化试验结果,并结合16S rDNA序列分析,对该菌株进行了分类鉴定。SWU-27菌体为杆状、无鞭毛、不能运动、无芽孢、无伴孢晶体、革兰氏染色呈阴性。对其12项生理生化指标进行了测定,结果与克雷伯氏杆菌属的特征相符。同时,该菌株的16S rDNA序列与Klebsiella sp.strain zlmy的165 rDNA序列有99.0%的同源性。且系统进化树表明,此菌株和克雷伯氏杆菌属的细菌具有较进的遗传距离。因此,综合各项指标的鉴定结果,我们将SWU-27菌株归属于克雷伯氏杆菌属,并将其命名为Klebsiella sp.SWU-27。三、β-葡萄糖苷酶基因的克隆及分析提取SWU-27菌株全基因组DNA,以大肠杆菌为表达宿主菌,通过鸟枪法,构建全基因组文库,从该菌株的基因组中克隆到一个新的β-葡萄糖苷酶基因nglu02。nglu02基因含有一个1230个碱基的ORF,编码409个氨基酸,预测的pI值为8.90。HCA位点同源性分析及系统发育树分析表明,该基因属于纤维素酶I家族,与Ruminococcus flavefaciens celA的进化距离较近。nglu02氨基酸序列保守区和非保守区中分别存在谷氨酸和天冬氨酸等活性氨基酸残基。四、β-葡萄糖苷酶酶学性质初探以同批发酵培养提取上清液为酶液,探索不同条件下该β-葡萄糖苷酶的活力。测定研究结果显示:该β-葡萄糖苷酶反应的最适pH为10.0左右,在pH11.0仍保持80%的酶活力。该酶的活性在中性和碱性环境下较稳定,但在酸性环境下较容易失活。反应的最适温度为30℃左右,在5℃-70℃之间能保持50%以上的酶活力,这说明该酶具有较好的温度稳定性。金属离子和表面活性剂等对该酶具有不同程度的抑制影响能力。其中酶对K⁺、Na⁺、Cu²⁺、Fe³⁺、EDTA和脲等具有较好的耐受能力,而Ca²⁺、Mg²⁺、Zn²⁺对酶具有显著的抑制能力。上述结果表明,本研究获得的β-葡萄糖苷酶具有耐热、耐碱及耐部分金属离子的特点,这使得该酶在碱性纤维素酶制剂、棉织品的水洗整理及洗涤剂工业中具有良好的应用潜力。更多还原

【Abstract】 Cellulose is the most abundant biomass in the world.It is estimated that annually about 10¹¹tons of cellulose produced by plants’photosynthesis.However,due to its complicated and its insolubility structure,most of cellulose can’t be used and is discarded by human.Researches on the powerful cellulases and the high efficient degradation of the cellulose have been slow,mainly due to its intricated structure.So, the screening and research of high efficient cellulolytic microorganisms is significant.In the biological degradation of cellulose,cellulases secreted by microorganism play an important role.Cellulose can be degraded to glucose through the synergistical hydrolysis of three classes of cellulase,including endo-β-1,4-glucanase, cellobiohydrolase andβ-glucosidase.The former two hydrolyze cellulose polymers into oligomers,mainly dimer cellobiose.β-glucosidase is a key component of the cellulase system.It further hydrolyzes cellobiose into glucose.In addition,β-glucosidase can regulate the velocity of the cellulose degradation.Therefore,high efficientβ-glucosidase have a profit of the high velocity of the cellulose degradation.This thesis was based on the significance of cellulase andβ-glucosidase,and screened and researched the strain which produced the cellulase to include the high efficientβ-glucosidase.In this paper,I report the study on anβ-glucosidase-producing bacterium, including its isolation,identification,and cloning of anβ-glucosidase gene.Partially characterization of theβ-glucosidase will also be reported.1.Isolation of a cellulases-producing bacteriumTo get a cellulase-high-yielding bacterium,a CMC_Naplate screening method was performed,and five cellulase-producing strains were obtained.Then,using the screeningβ-glucosidase plate method,one strain,termed as SWU-27,was selected for further study because of its high activity ofβ-glucosidase.The secretion of this strain exhibited activities of endoglucanase,cellubiohydrolase andβ-glucosidase,among which the highest activity was theβ-glucosidase.The activity of endoglucanase, cellubiohydrolase andβ-glucosidase is 10.663U/mL,15.910U/mL and 20.107 U/mL, respectively.The activity of theβ-glucosidase accounts for 43.07%of the total activities.2.Identification of the strain SWU-27According to the characteristics of morphology,cultivation,physiology and 16S rDNA sequence,we can identify the strain.SWU-27 was gram-negative rod bacteria, without spore,without flagellum and immobility.12 physiological and biochemical characteristics have been identified,and the identification results accord with those of the Klebsiella sp.strain.Bioinformatics analysis of 16S rDNA gene indicated that 99%identity was shared between SWU-27 strain and Klebsiella sp.strain zlmy.And the analysis on phylogenesis was performed,and the result showed that SWU-27 strain was high homology with many Klebsiella sp.strain.According to the identification results, the strain SWU-27 was identified as Klebsiella sp.And the strain SWU-27 was named Klebsiella sp.SWU-27.3.Cloning and analysis of theβ-glucosidase geneBy the shotgun cloning method and with Escherichia coli as the host cell,I isolated a novelβ-glucosidase gene,named as nglu02,from strain SWU-27.The open reading frame（ORF）of the nglu02.was 1,230 bp,encoding a peptide of 409 amino acid residues with a predicted isoelectric point value（pI）of 8.90.The hydrophobic cluster analysis and phylogenetic assay revealed that this gene belonged to cellulase family I and showed high homology to Ruminococcus flavefaciens celA.In the conservative and nonconservative fields of the polypeptide,activity residues Glu and Asp were also found.4.Partially study on the characters of theβ-glucosidaseTheβ-glucosidase was found in the culture supernatant of the same fermentation broth,and its properties were studied with the culture supernatant.The optimum pH of the enzyme activity was pH 10.0.When the pH was 11.0,about 80%enzyme activity remained.And the enzyme was stable form pH7.0 to pH11.0.The optimal temperature for the enzyme activity was 30℃,and the remain enzyme was 50%in the rang of 5℃-70℃.Different metal ions and surfactant show different effects on the enzyme activity. The enzyme was notably inhibited by 1 mM Ca²⁺,Mg²⁺and Zn²⁺,but was not so obviously effect by 1 mM K⁺,Na⁺,Cu²⁺,Fe³⁺,EDTA and Urea.Finally,theβ-glucosidase was stable in the high temperature and the alkaline environment,and endures some metal ions and surfactant,which means that this enzyme has great potential in the cellulase production,the textile biofinishing and detergent industry.更多还原