高效石油降解菌的筛选及其降解性能研究

从长期被石油污染的土壤中驯化筛选、分离出2株高效石油降解菌Y-7和Y-9,通过形态学特征的观察和生理生化试验对其进行初步鉴定,鉴定结果分别为假单胞菌属(Pseudomonas sp.)和芽孢杆菌属(Bacillus sp.)。同时,研究并分析了不同pH、温度、初始石油浓度、接种量、吐温80等条件对菌体生长和石油降解率的影响。结果表明,在试验条件下,2株优势菌在初始pH为7左右,对石油的降解率可分别高达68.7%,74.5%,偏酸或偏碱的环境均不利于菌体的生长;培养温度对2株菌体生长和石油降解率的影响较大,最佳温度为35℃,降解率达到最大,分别为73.1%和69.6%;石油初始浓度大于0.4g/L时,Y-7降油率从69%降到49%,Y-9基本变化不大,控制石油物质浓度在0.4g/L,有利于对石油的生物处理;最佳接种量为2mL/L;吐温80对石油的降解促进作用有待进一步分析与研究。     

一株苯酚降解菌的鉴定及其降解特性

【目的】鉴定从某化工厂附近土样中分离到的一株耐高浓度苯酚的菌株T10,通过优化菌株的培养条件提高菌株对苯酚的降解率。【方法】根据菌株的形态、生理生化鉴定及16S rDNA测序分析确定其种属,以液体摇瓶培养菌株T10对苯酚的降解率为指标,对菌株的生长条件进行优化。【结果】菌株T10属恶臭假单胞菌(Pseudomonas putida)。添加葡萄糖、蛋白胨能有效缩短T10菌的生长周期,并使苯酚的降解率提高1.7倍。在菌体初始接种浓度为10%、温度为30°C、转速为180 r/min条件下,对初始苯酚浓度、pH和装液量的响应面优化结果如下:初始苯酚浓度3 000 mg/L、pH 7.5和装液量80 mL/250 mL,苯酚去除率最高可达到87.56%。【结论】T10菌能够耐受较高浓度的含酚废水,并且对苯酚有较强的降解能力,为下一步利用生物法处理含酚废水提供科学依据。     

一株苯酚降解菌的分离与鉴定

目的：筛选能高效降解苯酚的微生物,并进行初步鉴定。方法：从某焦化厂排水沟采集污泥,通过逐步驯化筛选苯酚降解菌株;利用形态观察、生理生化检测、16SrDNA序列分析进行初步鉴定。结果：筛选获得1株苯酚降解菌JDM-2—1,该菌能够以苯酚为惟一碳源,耐酚能力高达2200mg／L,在30℃和pH7．0条件下,42h内能将800mg／L的苯酚彻底降解;初步鉴定其为球形芽孢杆菌（Bacillus sphaericus）。结论：菌株JDM-2-1是一株高效降解苯酚的球形芽孢杆菌。     

对硝基苯酚降解菌P3的分离、降解特性及基因工程菌的构建

分离到一株假单胞菌 (Pseudomonassp .)P3 ,该菌能够以对硝基苯酚为唯一碳源和氮源进行生长。在有外加氮源的条件下 ,P3降解对硝基苯酚并在培养液中积累亚硝酸根。P3有比较广泛的底物适应性 ,对多种芳香族化合物都有降解能力。不同金属离子对P3降解对硝基苯酚有不同的作用。葡萄糖的存在对P3降解对硝基苯酚无明显促进作用 ,而微量酵母粉可以大大促进P3对硝基苯酚的降解。以P3为受体菌 ,通过接合转移的手段将甲基对硫磷水解酶基因mpd克隆至P3菌中 ,获得了表达甲基对硫磷水解酶活性的基因工程菌PM ,PM能够以甲基对硫磷为唯一碳源进行生长。工程菌PM具有较高的甲基对硫磷降解活性及稳定性     

聚丁二酸丁二醇酯降解菌株的筛选及其降解性能研究

从石油污染土壤中筛选到1株具有聚丁二酸丁二醇酯降解能力的菌株PBS1302,经菌体形态特征、菌落培养特征、生理生化鉴定和16S r DNA基因序列分析,初步鉴定为铜绿假单胞菌(Pseudomonas aeruginosa)。该菌株在培养温度37℃,培养基起始p H 6.8的条件下经6 d的培养,对聚丁二酸丁二醇酯薄膜的降解率可达36.9%。经电子显微镜观察,与降解前相比,聚丁二酸丁二醇酯薄膜表面变得粗糙,出现明显的蚀刻痕迹。     

铜绿假单胞菌PIC—N萘降解基因的研究

铜绿假单胞菌ＰＩＣ－Ｎ对萘、邻苯二甲酸、水杨酸等有较强的氧化能力。发现该菌株以萘为底物可诱导产生芳香烃分解酶系。菌株中存在一个５７．４ｋｂ的质粒,经限制性内切酶ＨｉｎｄⅢ处理可产生７个片段,用限制性内切酶ＥｃｏＲ Ⅰ处理可产生８个片段。将以限制性内切酶ＨｉｎｄⅢ部分酶切的片段克隆至大肠杆菌持ｐＭＦＹ４３上,获得２９个克隆株。通过对含有菌株ＰＩＣ－Ｎ质粒ＨｉｎｄⅢ片段的７个重组闰进行限制酶分析,绘出     

分泌型重组铜绿假单胞菌外毒素A基因工程菌的发酵研究

铜绿假单胞菌外毒素A(P .aeruginosaExotoxinA ,PEA)是该菌最重要的致病、致死性物质。目前对PEA已经进行了深入的理论研究 ,PEA的结构、生物学活性以及医学应用前景都已比较明确 ,从而推动了PEA的生产和纯化研究。PEA在天然情况下产量极低 ,通常每毫升培养物中PEA的产量在纳克水平以下。大量制备高纯度PEA的工作有赖于高效表达性工程菌株的建立。本研究将构建好的 pMAL PEA原核分泌型表达载体转化大肠杆菌BL2 1,利用摇瓶发酵研究PEA工程菌的最优表达条件 ,摇瓶发酵的最优培养条件为 :温度 30℃ ,摇床转速16 0r/min ,在含 0 …     

一株苯酚降解菌的筛选及其降解特性的初步研究

苯酚是一种严重污染物,目前的化学降解方法存在众多弊端,生物处理方法越来越受到重视。从胜利油田河口采油厂的飞雁滩油田土壤样品中分离,得到一株能够利用并降解苯酚的菌株P2。该菌株能够在以苯酚为唯一碳源和能源的培养基上生长,经BIOLOG细菌自动鉴定系统及16SrDNA鉴定,该菌株为类产碱假单胞菌（Pseudomonas pseudoalcaligenes）。通过苯酚羟化酶特异性引物的设计,从该菌株扩增出苯酚羟化酶大亚基（LmPH）基因,该基因片段编码对苯酚有催化活性的多肽。苯酚降解实验证实,该菌能在30℃192h内完全降解500mg／L的苯酚,Cu^2＋严重抑制该菌株对苯酚的降解,但碱性环境有利于其对苯酚的降解。     

一株苯酚高效降解菌的分离及其分解能力的初步研究

为了寻找能高效降解苯酚的微生物 ,从武汉市某化工厂周围的下水道污泥中 ,筛选分离出一种具有很高苯酚降解能力的菌株PheD1。通过生理生化及外观鉴定[1～ 3] ,将其初步鉴定为假单胞菌 (Pseudomonassp .)。经驯化后发现 ,该菌生长的迟缓期随苯酚浓度的增大而延长 ,但比同类报道的苯酚降解菌要短 ;在 35℃对数生长期时的苯酚降解率超过同类报道[6 ] ,6 0 0mg·L- 1 苯酚浓度的完全降解时间在 2 4h之内 ,比同类报道[4～ 6 ] 苯酚降解菌的分解能力要高。该菌为好氧菌 ,在空气充足的条件下可提高降解能力。对该菌的继续研究可使其在苯酚的生物降解及污水处理等实际运用中起到重要的作用。     

同源重组法构建多功能农药降解基因工程菌研究

构建遗传稳定的多功能农药降解基因工程菌可以为农药污染的生物修复提供良好的菌种资源,然而,构建遗传稳定且不带入外源抗性的基因工程菌是一个难点。通过以受体菌的16S rDNA为同源重组指导序列、sacB基因为双交换正筛选标记构建同源重组载体,二亲结合的方法将甲基对硫磷水解酶基因（mpd）整合到呋喃丹降解菌Sphingomonas sp.CDS1染色体的16S rDNA位点,分别成功构建了含1个和2个mpd基因插入到rDNA位点且不带入外源抗性的基因工程菌株CDSmpd和CDS-2mpd。同源重组单交换的效率为3.7×10^－7～6.8×10^－7。通过PCR和Southern杂交的方法验证了同源重组事件。基因工程菌遗传稳定,能同时降解甲基对硫磷和呋喃丹。甲基对硫磷水解酶（MPH）的比活在各生长时期均高于原始出发菌株,比活最高达6.22 mu/μg。     

基因工程抗体融合蛋白的构建

抗体融合蛋白可以具有抗体的特性和所融合的功能蛋白的活性,可广泛用于免疫治疗、免疫诊断、抗体纯化及抗体和抗原的分析定量等,特别可用于免疫导向药物的制备。基因工程抗体融合蛋白比传统的化学交联的抗体融合蛋白具有更多的优越性。本文就基因工程抗体融合蛋白的构建和性质做一综述 。     

Genetically engineered poly-gamma-glutamate producer from Bacillus subtilis ISW1214

The pgsBCA-gene disruptant from Bacillus subtilis ISW1214, i.e., MA41, does not produce poly-gamma-glutamate (PGA). We newly constructed an MA41 recombinant bearing the plasmid-borne PGA synthetic system, in which PGA production was strictly controlled by the use of xylose. Unlike the parent strain, ISW1214, the genetically engineered strain produced abundant PGA in both L-glutamate-rich and D-glutamate-rich media.     

Effect of Phenol on Bacillus subtilis Spores at Elevated Temperatures

The nature of the recovery medium is shown to influence the number of Bacillus subtilis spores which, after exposure to 2.5 or 5% phenol at high temperatures, can produce a visible colony. Higher survivor counts were obtained in nutrient agar containing L-alanine and D-glucose than in plain nutrient agar.     

Selectivity in Protein Degradation during Sporulation of Bacillus subtilis

The breakdown of cellular protein was investigated in Bacillus subtilis labeled with glycine-2-³H or L-phenylalanine-U-¹⁴C at different stages of vegetative growth and sporulation. In cells labeled with l-phenylalanine-U-¹⁴C, multiple protein turnover was observed. However, in cells labeled with glycine-2-³H, the patterns of protein turnover were quite different in the stages of growth and sporulation; proteins which were labeled at the early stationary phase were degraded rapidly, but those labeled at the late sporulation stage were hardly degraded. It was found that glycine incorporated into cells at the late sporulation stage was mainly utilized for biosynthesis of the spore coat protein. These data suggest that the spore coat protein which contains relatively large amounts of glycine is little subject to further degradation.     

Degradation of Extracytoplasmic Catalysts for Protein Folding in Bacillus subtilis

The general protein secretion pathway of Bacillus subtilis has a high capacity for protein export from the cytoplasm, which is exploited in the biotechnological production of a wide range of enzymes. These exported proteins pass the membrane in an unfolded state, and accordingly, they have to fold into their active and protease-resistant conformations once membrane passage is completed. The lipoprotein PrsA and the membrane proteins HtrA and HtrB facilitate the extracytoplasmic folding and quality control of exported proteins. Among the native exported proteins of B. subtilis are at least 10 proteases that have previously been implicated in the degradation of heterologous secreted proteins. Recently, we have shown that these proteases also degrade many native membrane proteins, lipoproteins, and secreted proteins. The present studies were therefore aimed at assessing to what extent these proteases also degrade extracytoplasmic catalysts for protein folding. To this end, we employed a collection of markerless protease mutant strains that lack up to 10 different extracytoplasmic proteases. The results show that PrsA, HtrA, and HtrB are indeed substrates of multiple extracytoplasmic proteases. Thus, improved protein secretion by multiple-protease-mutant strains may be related to both reduced proteolysis and improved posttranslocational protein folding and quality control.     

Degradation of ornithine transcarbamylase in sporulating Bacillus subtilis cells.

When Bacillus subtilis cells grew and sporulated on glucose-nutrient broth, ornithine transcarbamylase (OTCase) was synthesized in the early stationary phase and then inactivated. The loss of OTCase activity was much slower in a mutant that was deficient in a major intracellular serine protease (ISP). Immunochemical analysis showed that synthesis of OTCase decreased to a low, but detectable, level during its inactivation and that loss of activity was paralleled by loss of cross-reactive protein. Because the antibodies were capable of detecting denatured and fragmented forms of OTCase, we conclude that inactivation involved or was rapidly followed by degradation in vivo. Native OTCase was not degraded in crude extracts or when purified ISP and OTCase were incubated together under a variety of conditions. Synthesis of OTCase was not shut off normally in the ISP-deficient mutant. When the effects of continued synthesis were minimized, OTCase was degraded only slightly slower in the mutant than in its parent. Thus, the mutant had unanticipated pleiotropic characteristics, and it was unlikely that ISP played a major role in the degradation of OTCase in vivo.