【作者】 马安周；

【导师】 曲音波； 庄国强；

【作者基本信息】 山东大学 ， 微生物学， 2008， 博士

【摘要】 蛋白质在固-液界面处的吸附通常存在不可逆的吸附,这种不可逆的吸附在酶反应体系中也经常发生。在纤维素酶-纤维素水解体系中,由于纤维素的水不溶性形成了固体纤维素-液相两相系统,纤维素酶吸附和水解作用是发生在固-液界面处的。借助表面等离子共振技术,在恒温40℃的条件下观察了外切纤维二糖水解酶Ⅰ在纤维素表面的吸附特征并测定了吸附动力学数据。吸附在纤维素表面的纤维素酶CBH I在纤维素-液相界面处表现出部分不可逆的吸附特点。考虑到不可逆吸附的特点,建立了能够很好的描述纤维素酶在纤维素表面实际吸附过程的数学模型。其中模型的相关动力学参数:Kads,0.26(L·cm^-2·s^-1);Ktra,0.41(s^-1)。为了评估纤维素酶吸附对酶水解纤维素的影响,使用纤维素CF11和纤维素酶CBHⅠ反应体系来测定反应动力学动态变化。以不可逆吸附的纤维素酶CBHⅠ的表面密度同反应过程中的水解速率作图,直观的显示了它们之间负相关的特点,即随着反应过程中表面密度的增加,纤维素的水解速率呈现下降。发现纤维素酶CBHⅠ的不可逆吸附同纤维素水解速率的变化相关,提出了产生这种结果的可能机制,同时提供了通过改变纤维素酶的不可逆吸附的特点来提高纤维素酶水解效率的思路。开展了采用酶工程技术手段改善纤维素酶的特性的研究。首先考察了噬菌体展示技术应用于纤维素酶改造工程上的可行性。通过将来自瑞氏木霉的外切纤维素酶CBHⅠ和内切纤维素酶EGⅠ的cDNA基因序列克隆到噬菌粒载体pCANTAB 5E中,分别构建了重组噬菌粒载体pCANTAB-cbh I和pCANTAB-egⅠ。经IPTG诱导后在大肠杆菌TG1中得到表达,表达的纤维素酶连接着噬菌体基因3编码的尾丝蛋白PⅢ,以融合蛋白的形式展示在噬菌体M13的表面。分别测定了对外切纤维素酶和内切纤维素酶各自的水溶性底物pNPC和CMC的水解活性,表明噬菌体展示的纤维素酶依然保持酶水解活力。ELISA实验结果表明噬菌体展示的纤维素酶对滤纸纤维素有吸附活性。总之,噬菌体展示的纤维素酶依然保持它们的生物学功能,噬菌体展示系可以用在纤维素酶工程上。利用随机突变PCR技术结合噬菌体展示系统进行了纤维素酶的定向进化。通过优化随机突变PCR反应体系条件和参数,在扩增外切纤维素酶CBHⅠ的基因时,PCR反应体系中加入3%的DMSO,获得高质量的扩增效果。扩增的纤维素酶基因片段的突变频率控制在4.5～5.1/Kb水平,对应于蛋白质的突变频率发生数个氨基酸的变化,适合蛋白质的定向进化研究。优化了连接体系,克隆DNA片段同载体的摩尔比为5∶1时连接效果最佳。改进了利用KOB培养基培养TG1制备高效电转感受态的方法,获得的感受态效率达到3×10^8～9/μg质粒。基于这些工作基础,构建的外切纤维素酶CBHⅠ和内切纤维素酶EGⅠ的突变体库的库容都达到10⁶克隆子水平。利用变性剂盐酸胍处理噬菌体展示酶库,在逐渐提高筛选压力的过程中筛选掉稳定性差的展示突变体。这为利用噬菌体展示突变体酶库开展纤维素酶的定向进化研究打下了基础。为了进一步拓展新型纤维素酶的筛选范围,利用表型特征以及系统进化分析和分子生物学的方法鉴定了一株源自招远金矿酸性水样的细菌ZY-1,并分析了其在分类学上的地位。根据16S rDNA序列,菌株ZY-1在进化亲缘关系上同Acidiphilium属的一些种较近,同Acidiphilium cryptum和Acidiphilium multivorum聚在同一个亚类中。菌株ZY-1的细胞脂肪酸以直链、支链、不饱和脂肪酸的形式存在。其中,菌株ZY-1的脂肪酸主要是以饱和脂肪酸为主,但也含有一定量的不饱和脂肪酸18∶1ω9t,以及i16∶0支链脂肪酸,少量带有一个环丙烷的脂肪酸19∶0 cycloω9c。虽然菌株ZY-1的16S rDNA序列同A.cryptum和A.multivorum的相似度为99%,但是在生理生化以及形态特征上并不一致。菌株ZY-1生长温度范围28～42℃,最适生长温度约在37℃。生长的pH值范围2.0～4.0,最适生长pH在3.0附近。ZY-1的菌落呈白色,光滑,凸起,最大直径1mm。革兰氏阴性,短棒状。碳源利用分析表明在耗氧条件下能够以多种营养物为唯一碳源进行异养生活。以含有不同碳源的培养基培养时,菌株ZY-1表现出不同的菌体特征,当以葡萄糖为唯一碳源进行培养时,菌体的表面比较光滑。但是当在纤维素为唯一碳源培养时,菌体的表面分布有很多突起物。这也是首次在Acidiphilium属中发现能够利用纤维素的菌株。通过透射电镜观察发现,嗜酸菌ZY-1菌体表面在降解纤维素时生长的突起物和报道的纤维小体较为类似。另外,也分离出一株能够利用纤维素的真菌ZY-E1,经表型分析和18S rDNA鉴定表明这株真菌属于烟曲霉,同时展示了矿区极端酸性环境栖息微生物的多样性。更多还原

【Abstract】 In this study the work focused on three points:study on adsorption and hydrolysis reaction of cellulase during cellulose hydrolysis process; directed evolution of cellulase;isolation of novel cellulase from environment.First,study on adsorption and hydrolysis of cellulase during hydrolysis process.Protein adsorption onto solid substrates usually takes place in an irreversible fashion and this irreversible adsorption also occurs in some enzymatic reactions.In fact,the adsorption and hydrolysis of cellulose by cellulases occurred at the solid-liquid interface in the biphasic system on account of the insolublecellulose in liquid.In this study the adsorption characteristic of cellobiohydrolaseⅠonto cellulose was monitored by surface plasmon response.The results show that once cellulase adsorbes onto cellulose,it’s desorption is not easy and partially irrevisible adsorption can be observed.Taking into account the irreversible adsorption,the developed model properly exhibits the actual adsorption behavior of cellulase.Several simulated parameters in the equation such as Kads(0.26L·cm^-2·s^-1)and Ktra 0.41 (s^-1)were given.To evaluate the influence of adsorption on cellulose enzymatic hydrolysis,the reaction dynamics on pure cellulose were determined.A plot of the hydrolysis rate against the surface density of irreversibly adsorbed cellobiohydrolaseⅠ,revealed an inverse relationship in which an apparent decrease in the hydrolysis rate was observed with increasing surface density.Taken together,results presented here should be useful for modifying the binding characteristics of CBHⅠand making them more effective in cellulose hydrolysis.Second,directed evolution of cellulase.To test whether the phage display technology could be applied in cellulase engineering,the phagemids harboring the genes encoding the mature forms of CBHⅠand endoglucanaseⅠfrom filamentous fungus Trichoderma reesei, respectively,were constructed.CBHⅠand EGⅠfused to the phage coat protein encoded by the g3 gene were displayed on phage M13.The phage-bound cellulases retained their activities as determined by hydrolysis of the corresponding substrates.Also,their binding abilities to insoluble cellulose substrate were confirmed by an ELISA method. Overall,these results demonstrate that cellulases can be displayed on phage surface while maintaining their biological function,implicating phage display to be a potentially useful technology platform for directed evolution of improved cellulases.The cbhⅠDNA can be properly obtained with the addition of 3% DMSO in the random mutant PCR system.The mutant frequency of PCR production per Kb fell into 4.5～5.1/Kb,which is optimum to make the corresponding mutant protein have a variety with several amnion acids, was controlled by optimizing parameters of random mutant PCR.The optimal molar ratio of DNA and vector is 5 to 1 used in the ligation system.Transformation efficiency of 3×10^8～9/μg supercoiled pUCmt- 18 with E.coli TG1 can be obtained,when electrocompetent cell prepared by developed KOB medium.The primary library size of CBHⅠand EGⅠapproximately reached 10⁶ members,respectively,when used the above mentioned optimal mutant PCR system,molar ratio in ligation and elcetrocomepent TG1.Subsequently the phage libraries were subjected to denaturing conditions to exert selection pressure on protein stability before panning in the primary library treated by low concentration of denatured reagent GdnHC1.Third,isolation of novel cellulases from environment.A bacterial strain,ZY-1,which was isolated from acid stream of gold mine in Zhao Yuan city,was subjected to a polyphasic taxonomic study using phenotypic characterization and phylogenetic and genetic methods. Phylogenetic analysis based on 16S rDNA sequences showed that strain ZY-1 forms an evolutionary lineage within the radiation enclosing Acidiphilium species and,in particular,a coherent cluster with Acidiphilium cryptum and Acidiphilium multivorum.Strain ZY-1 had a cellular fatty acid profile containing straight-chain,branched, unsaturated and 9-cyclo fatty acids.The major fatty acid was saturated fatty acid.Moderate amounts of branched-chain fatty acids（i16:0）and fatty acid with a cyclopropane（19:0 cycloω9c）were present. Importantly,the unsaturated fatty acid（18:1ω9t）existed substantially with lower amounts.The 16S rDNA of strain ZY-1 has a high similarity to that of the relative strains of A.cryptum and A.multivorum.However, there exist obvious differences in physiological characteristics and morphological distinctiveness.The range of growth temperature of strain ZY-1 was from 28 to 42℃,which reveals that the strain is a mesophilic bacterium,and the optimal growth temperature is around 37℃.Strain ZY-1 is an acidophile growing optimally at approx,pH 2～4 and the optimum growth pH value is around 3.0.Colonies of ZY-1 are smooth,shiny,convex,and white and have a maximum diameter of 1 mm.The cells display short rods and Gram-negative.The organisms grew heterotrophically under aerobic conditions and they did use a broad range of organic substrate as the sole carbonsource.Phenotypic distinctiveness was shown when strain ZY-1 was cultured in medium with various nutrients as the sole carbon source.The surface of the strain ZY-1 cells cultured with glucose as the sole carbon source was smooth observed by transmission electron microscope,while protrusions can be observed on the cell surface when cured with the sole carbon source of cellulose.This is the first report that the strain in Acidiphilium genus has the ability to use of cellulose.Importantly, these appendixes of strain ZY-1 cultured in cellulose medium were similar to the feature of cellosome.In addition,a fungal strain named ZY-E1,which can digest cellulose,was isolated and identified.Strain ZY-E1 belongs to Aspergillus fumigatus based on the phenotypic characteristic and the sequence of 18S rDNA.The bacterial and the fungal strains were isolated which also displayed the diversity of microbe community inhabiting mostly acid environment.更多还原