【作者】 何力；

【导师】 谢从新；

【作者基本信息】 华中农业大学 ， 水产养殖， 2008， 博士

【摘要】 纤维素主要存在于植物的细胞壁中,是自然界中存在最广泛的一类碳水化合物,同时它也是地球上数量最大的可再生资源。由于纤维素分子是以β-1,4糖苷键相连接而形成的直链多糖,在自然状况下难以降解成可以利用的二糖或单糖等短链低聚糖,利用微生物产生的纤维素酶将其转化为人类可以直接利用的物质,解决急需的能源、食物和化工原料等,是科学界长期研究的课题之一,这对于人类社会解决环境污染、食物短缺和能源危机等具有重大的现实意义。研究表明,自然界中许多陆生真菌和细菌能够产纤维素酶。真菌产生的纤维素酶类通常为酸性酶,最适PH在3-5之间,在碱性范围内无活性或活性很低,且多数靠固态发酵来产纤维素酶,这使得真菌纤维素酶的应用和产量受到一定的限制;细菌所产生的纤维素酶一般适应中性至偏碱性环境,由于相应的酶对天然纤维素的水解作用较弱,长期以来其开发利用没有得到足够的重视,近年,随着中性和碱性纤维素酶在棉织品水洗整理工艺及洗涤剂工业中的成功应用,细菌纤维素酶已显示出良好的使用性能和经济价值。细菌具有结构简单、繁殖快等特点,是基因工程的主要材料,其液态深层发酵具有条件容易控制,不易染杂菌,生产效率高等优势,使得从细菌中提取纤细素酶将可能以其高效性而成为一个重要的途径。目前报道的产纤维素酶细菌主要为产芽孢梭菌（Clostridum SP）、类似黄链霉菌（Xanthomonas）、嗜酸纤维素分解菌（Acidothermus Cellulolyticus）等,这些菌株大多为陆生菌株。从水体特别是从水生动物体内筛选的能产纤维素酶的细菌菌株报道甚少。自然条件下,水生植物是草食性鱼类的主要食物来源,从生理适应性的角度推断,此类鱼可能具有纤维素的分解能力,从其他陆生食草动物的研究成果来分析,这种分解能力应来自于肠道中的纤维素分解菌。常见的草食性鱼类有草鱼（Ctenopharyngodon idellus）、鳊（Parabramis pekinensis）、鲂（Megalobramaamblycephala）等,其中生长速度最快,食草量最大的是草鱼。本课题以草鱼为对象,就草食性鱼类肠道纤维素分解菌进行了相关研究。实验中,利用刚果红选择平板法,从草鱼肠道内筛选出四株能产纤维素酶,并具有较高酶活性的细菌菌株,分别测定了各菌株的内切β—葡萄糖苷酶、纤维二糖水解酶和滤纸酶的活力。从细菌形态、生理生化和16S rRNA层面上对纤维素分解菌进行了分类鉴定,结果为阿斯布肠道杆菌（Enterobacter asburiae）和枯草芽孢杆菌（bacillus subtilis）。对其中X7菌株的液态发酵产酶条件进行了研究,初步确定产酶的最适培养条件为:初始PH值为7.0-7.3,接种量为5%（v/v）,培养温度为37℃;最适培养基为:羧甲基纤维素钠（CMC-Na）1%（w/v）,蛋白胨1%（w/v）,KH₂PO₄0.1%（w/v）,NaCL0.5%（w/v）。在最适条件下,液态培养菌株,通过超声波破碎细胞壁、磁力搅拌菌体、离心过滤菌体等方法处理发酵液,检测并比较纤维素酶的活性,认为该菌株所产的酶为胞外酶。利用盐析、DEAE-Sephadex A-50离子交换层析,Sephadex G-75凝胶过滤,经过SDS-聚丙烯酰胺电泳检验,初步判定该酶为多亚基结构。对纯化后的纤维素酶的酶学性质进行了研究。以羧甲基纤维素作为底物时,该菌株所产纤维素酶的最适宜的催化温度为55℃,酶反应的温度范围较宽,在45℃-65℃的温度范围内,热稳定性良好,在80℃条件下仍残存有30%的酶活。最适宜的PH值为7.0,在4.0-7.0之间的PH值范围内,酶的活力较稳定。Mn²⁺、Mg²⁺对酶的活性有激活作用;Fe²⁺、Ca²⁺、Zn²⁺对酶的活性有抑制作用;Cu²⁺、Co²⁺有明显的毒害作用。以羧甲基纤维素钠（CMC-Na）为底物的Km值为5.3×10^-3g/ml。为了解药物对纤维素酶产生菌的生长和活性影响,以指导渔用药物的使用,进行了相应菌株的药物试验。抑菌实验表明:四种菌株对诺氟沙星、头孢哌酮、庆大霉素等药物高度敏感,对磺胺二甲嘧啶和土霉素相对不敏感。MIC和MBC的实验表明:氟苯尼考、诺氟沙星对四种菌的纤维素酶活性有较强的抑制作用;土霉素的抑制影响相对较弱;磺胺二甲嘧啶无明显影响。更多还原

【Abstract】 Cellulose exists in the plant cell wall and is one of the most extensive carbohydrate in nature and is the largest renewable resources in the earth.It is hard to degrade to available short chain oligosaccharide such as disaccharide and monosaccharide due to its straight chain polyose throughβ-1,4- linkage that occurs in the cell wall of superior plants in strong association with hemicelluloses and lignin.So it has great realistic meaning for human being to solve environment pollution,food shortage and energy crisis that using cellulase produced by microorganism transform cellulose to energy,chemical and food.The research about cellulase has been performed for several decades.The reports showed that the fungi were the dominant contributor to producing cellulase strains both at home and abroad at present study.The cellulase is acid enzyme whose optimum pH value was 3-5 and the activity disappeared or markedly decreased in alkaline condition.The application range and yield were limited due to cellulase producing by solid state fermentation.In the research of cellulose producing condition,little attention has been put on cellulase producing by bacteria which adapt to the neutral or partial alkaline condition and its enzyme preparation has weak hydrolytic action on natural cellulose for a long time. Bacteria cellulose enzyme preparation showed good operational performance and huge economic value on the application of cotton fabrics washing processes and detergent industry over the past decade.Bacteria are also main materials of gene engineering which shows simple structure and rapid reproduction characteristic.Gene research has recently made great progress.The liquid deep layer fermentation of bacteria has the advantage of easy-controlled operating conditions,unease contaminated bacteria and higher production efficiency etc,and therefore cellulase extracted directly from bacteria has become an important and effective enzyme abstraction approach.The bacteria producing cellulose reported at present mostly were terrestrial strains such as Clostridum SP,Xanthomonas, and Acidothermus Cellulolyticus etc.Present research dealt with screen cellulose producing bacteria from water environment especially in aquatic animal is very limited.Water plants are the major source of herbivorous fish under natural condition.These fishes may have cellulose decomposed capacity in the view of physiological adaptation. Cellulose decomposing bacteria have the ability in intestine of terricolous herbivore.We presumed that some enzyme existed in the intestine of herbivorous fish have similar capacity.Grass carp（Ctenopharyngodon idellus）,white bream（Parabramis pekinensis） and black bream（Megalobrama amblycephala） are common herbivorous,of which, grass carp is the quickest growth rate and the highest intensity grazing species.The study aimed to cellulose decomposing bacteria in grass carp.The activities of bacterial strains and cellulase in the intestine of grass carp were analyzed using congo red choice plate method.Filter paper and absorbent cotton as substrate and the concentration of glucose measured by the colorimetry of DNS,FPase, CMCase and absorbent cotton enzyme activity of liquid culture filtrates were measured. Based on analyses of bacterial form,physiology and biochemistry and 16S rRNA gene sequence,it was confirmed that the strain belonged to Enterobacter asburiae and bacillus subtilis.The optimal condition of strains X7 for solid state fermentation was pH=7.0-7.3, inoculation amount 5%（v/v）,culture temperature37℃and the optimum medium for shoots rooting was 1%sodium carboxymethyl cellulose,1%peptone,0.1%KH₂PO₄, 0.5%NaCl.The strain was cultivated by liquid still condition in optimal growth conditions.The bacteria was smashed by ultrasonic crashing instrument,and broth was magnetic stirred and centrifugal filtered.The activity of cellulose was examined and compared.The enzyme was a kind of extracellular enzyme.It indentified preliminary multi-subunit structure by salt-out,DEAE-Sephadex A-50,Sephadex G-75 and polyacrylamide gel electrophoresis.The enzymatic properties of cellulose after purification was studied:the optimal temperature and pH were 55 and 7.0.The enzyme was rather stable in the range of pH 4.0-7.0 and temperature 45-65℃.The enzyme activity was activated by Mn2⁺,Mg2⁺ and inhibited by Fe2⁺,Ca2⁺.and Zn2⁺.Cu2⁺ and Co2⁺ had obvious toxicity to the activity. The Km value with sodium carboxymethyl cellulose as a substrate was 5.3×10^-3 g/ml.Corresponding medicine test was carried out in order to understand its influence on the growth and activity of bacteria producing cellulase.Results of sensitivity to antibiotics showed that the strains were very sensitive to norfloxacin,cefoperazone and gentamicin,but insensitive to sulfamethazine and oxytetracycline.The experiments of MIC and MBC indicated that florfenicol and norfloxacin had strong inhibitory effects on enzyme activity of four strains.The inhibition of oxytetracycline was relatively weak and that of sulfamethazine was inactive.更多还原