导　　师： 龚月生;杨明明

学科专业： 090502

授予学位： 硕士

作　　者： ;

机构地区： 西北农林科技大学

摘　　要： 纤维素是地球上最丰富的可再生资源,主要由绿色植物通过光合作用合成。由于单胃动物胃肠道缺乏纤维素酶,使得纤维素利用率较低,造成了严重的饲料浪费和环境污染。纤维素酶是指降解纤维素生成葡萄糖的一组酶的总称,通过协同作用,将纤维素降解为纤维二糖和寡糖,最终水解为葡萄糖。现阶段微生物来源的纤维素酶酶活较低,达不到工业大规模厂生产的要求,而且在畜牧生产中,制粒过程的高温对酶活的影响极大,因此获得耐热的纤维素酶基因并利用基因工程手段改造使之高效表达,作为一种切实可行的提高纤维素酶酶活的方法而广受关注。 枯草芽孢杆菌/(Bacillus subtilis/)作为表达系统,近些年来研究较深入。枯草芽孢杆菌遗传背景清晰简单；可以直接分泌胞外蛋白；与传统的大肠杆菌表达系统相比,没有致病性,本实验室已经构造一套枯草芽孢杆菌信号肽高效筛选表达系统,用于饲料酶制剂的原核表达。 1.本试验采用刚果红-稀释平板法从堆肥中筛选纤维分解菌,DNS法测定其纤维素酶酶活,并检测其热稳定性,获得一株产耐热纤维素酶菌株CY15-10,平板形态观察和革兰氏染色结果显示该菌株为革兰氏阳性菌,PCR扩增其16SrDNA并测序,该菌与多粘类芽孢杆菌/(Paenibacillus polymyxa/)同源性最高,为99/%,综上所述,确定该菌为多粘类芽孢杆菌{Paenibacillus polymyxa/)。 2.为了确定细菌最佳的生长和产酶条件,本试验设计了5个独立的单因子试验,通过确定每一个因素的最佳水平而得到最佳的组合,结果表明,菌株CY15-10在37℃、pH6.0,以羧甲基纤维素钠为碳源、氮源为牛肉膏的培养环境下,培养36h,达到产酶的高峰；设计三个单因子试验,确定该纤维素酶的最适温度,热稳定性,最适反应pH。该纤维素酶最适反应温度为55℃、最佳反应pH为6.0；75℃下处理10分钟粗酶液仍残留酶活70/%以上。 3.通过NCBI公布的该菌属的纤维素酶基因而设计特异性引物,从CY15-10基因组DNA中PCR扩增到纤维素酶基因,经过与NCBI已报道的数据库比对,该基因序列与报道过的Paenibacillus polymyxa SC2的纤维素酶基因片段同源性高达99/%。该基因全长1482bp,为一个完整的阅读框,起始密码为ATG,终止密码为TAA,连续编码493个氨基酸,信号肽预测结果表明其前36个氨基酸为信号肽,该基因表达蛋白大小为51kDa左右。 4.重新设计引物在纤维素酶基因两端引入酶切位点BamH I和Sac I,将该纤维素酶基因连接到实验室构建的大肠-枯草穿梭载体pPS上,并转化到枯草芽孢杆菌WB700中诱导表达,结果表明该重组菌在37℃,3/%麦芽糖诱导下24h酶活达2.4U//mL,比野生菌酶活提高了1倍。 Cellulose is the most abundant renewable resources on Earth, mainly synthesized by green plants through photosynthesis. For the lack of cellulose in gastrointestinal Tract of Monogastric animals, Feed nutrients can not make fully use by animals, which results in a waste of feed source and serious environmental pollution. Cellulase, referring to a group of enzymes that degrade cellulose to glucose,The enzymes degrades cellulose to cellobiose and oligosaccharides,and ultimately these intermediates will be degraded to monosaccharides.the present cellulose produced by microorganism can't reach the industrial production requests because of the low enzyme activity.High temperature caused great damage to the cellulase /(?/) while feed pelletization.Therefort as a practical approach,to screen a thermostable cellulose gene and make it express efficiently using gene modification techniques has attracted great attention. Bacillus subtilis as a widely used expression system was made further study.The system has a clear genetic background and can secret extracell proteins directly.Compared to traditional expressing system,E coli,it has no pathogenicity.a high efficiency signal peptide screending and expressing system was construced in Bacillus subtilis in our lab,filter expression system,which can be used to produce feed enzymes. 1. In this study, a hyperthermophilic cellulase-producing strain CY15-10was isolated from decayed wood and compost through the Congo red medium plate method.Based on morphology,biochemical and physiological characterization,16S rRNA sequence analysis and construction of phylogenetic tree, the homology99/%same with the16S rDNA sequence of Paenibacillus polymyxa. In conclusion, strain CY15-10was identified as Paenibacillus polymyxa. 2. A cellulose gene was amplified by specific primers designed according to the cellulose gene of Paenibacillus polymyxa published on NCBI,the total genome DNA of CY15-10as the template.The fragment was inserted into pMD19-T and sequenced,thehomology between the gene sequence and reported sequence reached99/%through BLAST online.The gene had1482bp and an interal ORF,coding493amino acids. The signal peptide prediction results showed that the first36amino acids was the signal peptide,the cellulose protein was approximately51kDa tested by SDS-PAGE and zymogram analysis. 3. In order to determine the optimal bacterial growth and enzyme production conditions,5unattached single factor experiments were at temperature37℃,pH6.0and36h cultivation;the optimum carbon source w.as CMC-Na,while the nitrogen source was beef cream.The best enzyme activity conditions were at temperature55℃,pH6.0; The enzyme performed good thermal stability:it can tolerant to75℃for10min,70/%of enzyme activity remained. 4. Two resitriction enzyme sites Bam H I and Sac I were introduced in the primers C2up//down,the cellulose gene was ligated to expressing vector pPS,which was digested by the same restriction enzymes,the integrating vector was transfonned into Bacillus subtilis WB700and the gene was induced expression extracellularly by3/%maltose,the supernatant enzyme activity was2.4U//mL and was increased by double compared to its wild type.

关 键 词： 细菌 纤维素酶 耐高温 表达载体 枯草芽孢杆菌

分 类 号： [TQ925]

领　　域： [轻工技术与工程]