【作者】 姚大伟；

【导师】 侯加法； 杨德吉；

【作者基本信息】 南京农业大学 ， 临床兽医学， 2010， 博士

【摘要】 随着畜牧业的可持续发展,我国蛋白质饲料持续短缺,新型蛋白质饲料资源的开发变得越来越紧迫。新型蛋白质饲料的开发技术主要是利用现代生物技术将一些利用率低的饲料资源或非常规饲料资源变为新型蛋白饲料,提高其利用率。现代生物技术在开发利用新型蛋白质饲料方面主要应用发酵工程技术生产发酵饲料、酶工程技术生产酶解饲料等。无论是发酵工程技术还是酶工程技术都涉及微生物资源或微生物酶资源的开发与利用。利用发酵技术或酶技术在以羽毛粉为对象开发新型蛋白质饲料的过程中筛选各种细菌、真菌和放线菌。其中细菌以芽孢杆菌属细菌居多,该菌属细菌多数产碱性蛋白酶,已有大量的碱性蛋白酶被开发应用于各种工业生产。目前,在羽毛角蛋白降解方面还仅处于酶的分离与纯化的研究,如何采用基因工程技术提高酶的产量是当今的研究热点。本试验在本实验室前期微生物菌种资源筛选的基础上对比研究血红蛋白降解菌短小芽孢杆菌NJM4和角蛋白降解菌短小芽孢杆菌WHK产蛋白酶性质,从中选取产蛋白酶活力较高的WHK4菌株作为研究素材,对其蛋白酶进行分离纯化,并对蛋白酶的酶学性质进行了研究,深入了解该酶的特性,为今后的开发利用奠定理论基础。同时对于如何提高NJM4的产酶能力本试验采用基因重组的方式研究碱性蛋白酶基因在大肠杆菌和短小芽孢杆菌中的表达。试验分为以下6个部分：试验一、短小芽孢杆菌NJM4与WHK4生物学特性的比较研究系统地比较研究了短小芽孢杆菌—-NJM4与WHK4生物学的差异,分析两者产蛋白酶差异的原因。首先比较两者的培养、生理生化特性,然后比较分析了两者在相同培养基内对产蛋白酶活力及其对不同底物活力的影响。在基因水平上比较了两者碱性蛋白酶基因以及该基因上游启动子区域的基因序列。结果显示：两者生理生化特性无差异,在培养特性方面则有差异,WHK4的生长速度较快,在相同的培养基中24 h内产生芽孢,而NJM4在相同的时间内未形成芽孢；在产酶方面,WHK4在血红蛋白和羽毛粉发酵培养基内产蛋白酶活力高于NJM4,在营养丰富的LB培养基中产蛋白酶活力却低于NJM4。分别以不同底物测定的蛋白酶活力WHK4均高于NJM4。在基因水平上两者碱性蛋白酶基因1152bp的碱基中虽然只有一个碱基不同,但其对应的密码子所编码的氨基酸属同一个氨基酸,启动子区域49bp的基因序列完全相同。试验表明WHK4较NJM4产酶能力强,酶活力高,是产蛋白酶的优良菌株。试验二、短小芽孢杆菌WHK4以羽毛粉为底物产蛋白酶条件的优化探索WHK4以羽毛粉为底物产酶的最佳条件和最佳培养基组成。以羽毛粉发酵培养基为基础,首先采用单因子试验研究底物浓度、初始pH、接种量、外加碳源、外加氮源对WHK4产酶活力的影响。在单因子试验的基础上采用正交试验设计对底物浓度、温度、初始pH、接种量、外加(NH4)2SO4、外加麦芽糖进行优化。结果显示：WHK4最佳的产酶条件为初始pH 7.38,菌龄16 h,接种量5%,37℃。最佳的培养基组成为：1 L基础发酵培养基,40.0 g羽毛粉,10.0g(NH4)2SO4和10.0 g麦芽糖。在优化的条件下WHK424 h产蛋白酶活力为90U·mL-1。WHK4培养条件及培养基的优化为其产蛋白酶的分离纯化奠定了基础。试验三、短小芽孢杆菌WHK4蛋白酶的分离、纯化及酶学特性的研究为得到WHK4酶的纯品,更好的了解该酶的酶学特性,本试验在优化的产酶条件下首先制备粗酶液,然后采用50%饱和硫酸铵盐析,沉淀用1/10体积的PBS溶液溶解后透析除盐,经Sephdex G-100凝胶过滤层析分离出蛋白酶。对所得的蛋白酶进行SDS-PAGE酶谱分析,测定酶最适作用温度和pH,对酶学特性进行测定。结果显示：WHK4以羽毛粉为底物发酵液中含有两种蛋白酶,其中一种蛋白酶分子量约为50 KD,该酶作用最适温度为60℃,最适pH为8.5, Fe3+、Cu2+、SDS、EDTA对蛋白酶活力有较强的抑制作用,Ba2+、Ca2+、Mg2+和Fe2+以及有机溶剂DMSO、异丙醇、甘油,表面活性剂TritonX-100对蛋白酶活力几乎无影响。表明该酶属于金属蛋白酶,在耐有机溶剂生物催化剂方面有潜在的应用前景,在洗涤工业生产中也有较大的应用价值。试验四、短小芽孢杆菌NJM4碱性蛋白酶基因的表达与活性分析为提高碱性蛋白酶的产酶量,本试验研究其在大肠杆菌中的表达。采用同源克隆的方法设计碱性蛋白酶基因扩增引物,用高保真DNA聚合酶进行PCR扩增,然后将扩增的PCR产物和克隆载体pUC57经XbaⅠ和BamHⅠ(?)双酶切后连接构建pUC57-AP,转化TOP10感受态细胞,涂布LB/Amp/X-gal/IPTG培养基平板,挑选白色菌落进行PCR鉴定,然后测序分析。将PCR产物和表达载体pET-28b经NcoⅠ和BamHⅠ双酶切后连接构建pET-28b-AP,转化BL21(DE3)感受态细胞,涂布LB/Kan/酪蛋白培养基平板,挑选有水解圈的阳性克隆进行PCR鉴定,对阳性克隆用IPTG诱导4h后测定发酵液的蛋白酶活力。结果表明,克隆得到的NJM4碱性蛋白酶基因全长为1152 bp,编码383个氨基酸,成熟肽276个氨基酸。从LB/Kan/酪蛋白平板培养基筛选有透明水解圈的菌株,命名为BL21(DE3)-28b-AP。该菌株经IPTG诱导后发酵上清液蛋白酶活力为不含重组子菌株的37.3倍。成功地构建具有蛋白酶活力的高效表达的菌株为碱性蛋白酶的批量生产奠定了基础。试验五、短小芽孢杆菌NJM4原生质体转化研究探索短小芽孢杆菌原生质体的形成条件及其转化的可行性。采用溶菌酶脱壁制备原生质体,进行单因子试验,显微镜观察酶的浓度、作用温度,酶解时间对原生质体形成率的影响。在最适宜的条件下制备原生质体,在Ca2+的环境中用PEG6000诱导pUC57-AP重组质粒的转化,采用含氨苄青霉素的DM3培养基平板筛选转化子。结果显示,用含氨苄青霉素的DM3培养基平板筛选的转化子经含氨苄青霉素的液体培养基培养后其蛋白酶的活力几乎丧失。表明短小芽孢杆菌NJM4易于原生质体化,可以实现外源质粒转化,是一株潜在的基因工程受体菌。试验六、分光光度计法检测原生质体形成过程的初步研究探索芽孢杆菌原生质体形成过程中光密度的变化与原生质体形成率之间的关系。采用分光光度计测定原生质体形成过程中溶液的OD600nm,同时采用镜检计数测定原生质体形成率,分别绘制光密度变化曲线和原生质形成曲线,采用SPSS统计软件中Curve Estimation分析两者之间的相关性并进行曲线拟合。结果显示：原生质体溶液光密度的变化和原生质体的形成率之间存在显著的相关性(R2=0.985,P<0.01),随着原生质体形成率的增加溶液光密度逐渐减小,两者之间呈负相关,3次多项式曲线拟合最佳(Y=0.746-0.01t+1.83×10-4t2-1.171×10-6t3)。表明原生质体形成过程中通过动态监测原生质体溶液光密度的变化可实现原生质体形成率的定量分析。更多还原

【Abstract】 Along with the sustainable development of animal husbandry, protein feeds is continuing shortage in our country, so exploitation of the new type protein feed resources become more and more urgent. The technology of the new type protein feed exploitation is to utilize modern biotechnoliques to take some low available feeds or uncommon feeds into the new type protein feeds, in order to enhance their availability. The application of the modern biotechniques is to produce fermentated feeds with fermentation engineering and enzymolysis feeds with enzyme engineering. No matter fermentation engineering or enzyme engineering is involved in the development and utilization of the microbial resources or microbial enzyme resources. A number of microorganisms such as bacteria, fungi, actinobacteria.have been isolated from exploitation of the new type feather protein feeds, Most of them belong to Bacillus which produces the alkaline protease. A number of alkaline proteases have been commercially exploited to assist protein degradation in various industrial processes. Many researchs are still only in the separation and purification of protease in aspect of degradation of feather keratin. How to improve the production of protease by technique for gene engineering is hot issue in present research. the comparative study of protease production characteristics between hemoglobin-degrading bacteria NJM4 and keratin-degrading bacteria WHK4 based on the previous research in our laboratory. WHK4 was selected for separating the protease and investigating the characteristics of the protease because of the high protease production. In order to improve the protease production of NJM4, the expression of alkaline protease in Escherichia. coli and Bacillus pumilus was studied by genetic recombination. The details are divided into six parts as follows:ExperimentⅠThe comparison of biological characteristics between Bacillus pumilus NJM4 and Bacillus pumilus WHK4 Comparison of biological characteristics between Bacillus pumilus NJM4 and Bacillus pumilus WHK4 were studied systematically, and the differences of protease production betweent them were analyzed. Firstly, the cultural, physiological and biochemical characteristics were compared. Then, the difference of protease production in the same medium and protease activity using the same protein as substrate were compared. The sequences of the alkaline protease gene and the promoter gene of alkaline protease were aligned in gene level. The results showed that there were no differences of physiological and biochemical characteristics between them. However, the growth velocity of WHK4 was faster than the NJM4. The spore of WHK4 was formted in 24 h in the same medium with NJM4,but the NJM4 did not. In aspect of protease production, the protease production of WHK4 was higher than NJM4 in hemoglobin and feather meal fermentation medium and lower than NJM4 in LB medium. The protease activity of WHK4 was always higher than NJM4 using different protein as substrate. There was only one base difference in gene level. It was suggest that WHK4 is a Superior strain for protease production.ExperimentⅡOptimization of protease production by Bacillus pumilus WHK4 using the feather meal as subtrateTo obtain the maximum protease production the optimal conditions were studied. The effects of feather meal concentration, initial pH, inoculum size, the additional carbon source and nitrogen sources on protease activity were studied by single-factor test. Then the feather meal concentration, temperature, initial pH, inoculum size, the additional Ammonium Sulfate and maltose were optimized by orthogonal test based on the single-factor test. The optimal conditions were achieved at initial pH 7.38, inoculum size 5% with 16 h old inoculum, incubation temperature 37℃, and the medium composed of 40 g feather meal,10.0 g (NH4)2SO4,10.0 g maltos in 1000mL based fermentation medium. Maximum protease production attained at the optimal conditions was (90U·mL-1) after 24 h cultivation. Those conditions have established the base for further seperation and purification of protease of WHK4ExperimentⅢStudy on seperation, purification and enzymological characteristics of protease in Bacillus pumilus WHK4The aim of the experiment was to isolate and investigate the protease of Bacillus pumilus. The crude enzyme was achieved in optimal medium at optimal conditions. The protease was purified from crude enzyme by 50% Ammonium Sulfate, Sephdex G-100 chromatography and analyzed by SDS-PAGE and zymography. The effect of protease activity on temperature and pH was detected and the characteristics of protease were investigated. It showed that there are two proteases in crude enzyme. One protease with molecular weights of 50 KD showed an optimal activity at 60℃and pH8.5. The protease activity was inhibited in presence of Fe3+, Cu2+, SDS and EDTA identifying it as a metalloprotease. There was no effect on protease activity in presence of Ba2+, Ca2+, Mg2+, Fe2+, DMSO, isopropanol, glycerine and Triton X-100. It was suggest that the protease obtained will be potentially applied in organic solvent tolerant biocatalysis and detergent industry.Experiment IV Genetic expression and bioactivity of recombinant protein of alkaline protease in Bacillus pumilus NJM4The aim of this experiment is to express the alkaline protease in E. coli. Primers were designed by homology-based cloning to clone the alkaline protease gene use pfu DNA Polymerase. The DNA fragment was cloned into pUC57 clone vector by double digest with XbaI and BamHI, respectively, ligated together by T4 DNA ligase, Recombinant plasmid was named pUC57-AP and transformed into E.coli TOP 10. The positive clones with white color were selected from LB/Amp/X-gal/IPTG plate and identification by PCR. Sequencing of pUC57-AP was conducted using both the forward and reverse primers. The DNA fragment was cloned into pET-28b expression vector by double digest with Ncol and BamHI, respectively, ligated together by T4 DNA ligase, Recombinant plasmid was named pET-28b and transformed into E.coli BL21 (DE3). The positive clones with hydrolytic zone were selected from LB/Kan/casein plate and identification by PCR. The protease activity of the positive clone in medium was detected after inducing with IPTG. An 1152 bp length alkaline protease gene which encoded 383 amino acids was successfully cloned. A strain expressing the protease activity was named with BL21 (DE3)-28b-AP. The protease activity by BL21 (DE3)-28b-AP in medium was 37.3 times as large as the initial strain (BL21 (DE3)). The highly expression genetic engineering microorganism laid the groundwork for production of alkaline protease.Experiment V Study on the protoplast transformation of Bacillus pumilus NJM4 The conditions of protoplast formation and feasibility of protoplast transformation by plamid in Bacillus pumilus NJM4 was studied. The protoplast was prepared using lysozyme solution to remove the cell wall. The effects of lysozyme concentration, enzymolysis time and enzymolysis temperature on the formation ratio of protoplast were studied according to microscopy. The pUC57-AP was transformed into NJM4 by the induction by PEG 6000 with the presence of Ca2+ in reaction. DM3 medium (containing ampicillin 5μg·mL-1) was used for selected transformants. Amicillin resistance strains without protease activity were selected from DM3 medium. It was suggested that Bacillus pumilus NJM4 is a kind of potential genetic engineering recipient.Experiment VI Preliminary Study on Process of Protoplast Formation Monitored by SpectrophotometerTo explore correlation between optical density and protoplast formation rates in process of bacillus protoplast formation. OD600nm were evaluated by spectrophotometer in process of protoplast formation, meanwhile protoplast formation rates were determined under microscope. Optical density curves and protoplast formation curve were drawn, respectively and analyzed correlation of them by statistical software(SPSS). It was shown that there were highly significant correlations between optical density and protoplast formation rates. It was suggested that quantitative analysis of protoplast formation rates could carried out by monitored the optical density of protoplast solution.更多还原