一株产高酶活性碱性磷酸酶解淀粉芽孢杆菌的分离及其phoD碱性磷酸酶基因的克隆与表达

[背景]碱性磷酸酶作为工具酶被广泛应用于各个领域,在免疫学检测方面应用较多的是PhoA家族的碱性磷酸酶,尚无关于PhoD家族的碱性磷酸酶在免疫学检测方面的研究。[目的]筛选出一株产高酶活性PhoD家族碱性磷酸酶的细菌,并将其phoD基因进行克隆表达,研究PhoD的酶学性质,为PhoD家族的碱性磷酸酶在免疫学检测方面的应用奠定一定的基础。[方法]采取有机质丰富的土样在有机磷平板中进行细菌分离,以4-硝基苯磷酸二钠盐（4-nitrophenyl phosphate disodium salt hexahydrate,p-NPP）为底物测定有机磷平板中单菌落的酶活性,选取酶活性高的菌株作为目的菌株,克隆其phoD基因。[结果]筛选到一株产碱性磷酸酶酶活性高的菌株S2-4,通过16S rRNA基因序列同源性比较分析,鉴定该菌株为解淀粉芽孢杆菌,克隆了其phoD基因并进行诱导表达。研究了纯化后PhoD的酶学性质,PhoD的最适反应温度为70℃;最适反应pH为9.8;PhoD最适Ca²⁺浓度为3 mmol/L,Mg²⁺对PhoD的酶活性有抑制作用,K     

一株产甘露聚糖酶菌株的分离鉴定及酶的纯化与性质

【目的】筛选性能良好的产碱性甘露聚糖酶的菌株,对菌株进行多项分类鉴定,分离纯化所产甘露聚糖酶并进行性质研究。【方法】利用碱性魔芋粉培养基分离纯化产甘露聚糖酶的嗜碱菌,通过形态特征观察、生理生化测定、16S rRNA序列分析等实验确定菌株的分类地位。利用硫酸铵沉淀、阴离子交换层析和分子筛层析得到电泳纯的酶,分析了酶的最适温度、最适pH、温度和pH稳定性、NaCl以及金属离子等的耐受性。【结果】从我国内蒙古碱湖样品中分离得到一株产碱性甘露聚糖酶的菌株HMTS15,经过多项分类鉴定显示其是与Bacillus agaradhaerens DSM 8721不同的新菌株。菌株HMTS15所产的甘露聚糖酶反应的最适pH为10.0,最适温度75℃。【结论】多项分类结果鉴定菌株为Bacillus agaradhaerens HMTS15。该菌株产生的碱性甘露聚糖酶与同类其他来源的酶相比具有更好的热稳定性和pH适应性,有进一步的研究价值。     

高浓度氯苯优势降解菌的筛选及其降解酶的纯化

[目的]分离纯化出一株高浓度氯苯优势降解菌株,对其所产氯苯降解酶进行分离与纯化,为该菌株及其氯苯降解酶的研究提供理论参考.[方法]利用梯度富集培养技术和无菌滤纸片平板法分离菌株,通过形态特征及16S rRNA基因序列分析初步鉴定菌株,用气相色谱法测定培养液中氯苯浓度,以单位细胞氯苯降解率评价菌株对氯苯的降解能力,以氯苯降解率表示氯苯降解酶的活性.取纯化菌株的发酵酶液制备粗酶液,经硫酸铵梯度盐析、透析脱盐、DE-52离子交换层析、G-100凝胶层析和透析浓缩后,进行SDS-PAGE凝胶电泳检验酶的纯度并测定酶的分子量.[结果]从氯苯长期驯化的成熟期活性污泥中筛选到一株以氯苯为唯一碳源和能源的氯苯优势降解细菌LW13,该菌株在以2000 mg/L氯苯为唯一碳源的无机盐培养基中仍能正常生长,其单位细胞氯苯降解率可达1.37 ×10-10.扫描电镜观察到该菌株细胞大小约为2.3 ×0.8μm,长有数根端生鞭毛.16S rRNA基因序列相似性比较表明该菌株与Lysinibacillus fusiformis(溶藻菌)的相似性达95.5％.所纯化的氯苯降解酶为胞外酶,带正电荷,其分子大小约为57 kDa.整个纯化过程中酶纯化倍数化达8.0倍,酶活回收率达52.51％,酶量回收率达6.57％.纯化后的氯苯降解酶在30℃-55℃和pH在6.0-8.0之间都保持较高的酶活性,其最适反应温度和pH分别在40℃和pH8.0左右.[结论]所分离的氯苯优势降解菌属于Lysinibacillus属菌株,该菌株能有效降解高浓度(500-2000 mg/L)氯苯废水,通过逐级分离纯化,可获得氯苯降解酶纯酶,纯化指标符合分离纯化基本规律,纯化效果较为理想.     

从黑茶中分离和筛选产单宁酶菌株

茶叶中富含单宁化合物。从分离自黑茶的真菌菌株中,筛选高产单宁酶的菌株;进而分离纯化单宁酶,分析单宁酶对茶汤的转溶效果。从不同产地的3个黑茶样品中,共分离获得44个真菌分离物;经初步鉴定,这些真菌分离物以曲霉属（Aspergillus）、青霉属（Penicillium）和散囊菌属（Eurotium）的真菌居多。以单宁酸为底物的鉴别培养基初筛表明,其中26个真菌分离物在鉴别平板上产生透明圈,显示单宁水解酶活性;通过固体发酵复筛,筛选到1株产单宁酶活性较高的菌株,初步鉴定为青霉属（Penicillium）菌株,命名为青霉MP-24菌株。青霉MP-24可以以茶叶、茶梗和麸皮等农副产品作为原料固体发酵产生单宁酶。以麸皮为原料的发酵产物经过硫酸铵分级沉淀、DEAE阴离子交换层析和葡聚糖G-150凝胶层析等分离纯化步骤,得到分子量为70 kDa的单一蛋白质条带,单宁酶活力达到603.68 U/mg。纯化获得的单宁酶对茶汤有良好的转溶效果。研究结果表明,在黑茶相关微生物中含有丰富的产单宁酶菌株,是工业酶制剂的重要资源。     

不同形态的磷源对球形棕囊藻生长及碱性磷酸酶的影响

研究了不同浓度和形态的无机磷(DIP)和有机磷(DOP)对球形棕囊藻细胞生长、培养基中磷浓度的变化以及碱性磷酸酶活性的变化的影响。在缺磷和大分子DOP(卵磷脂)作为磷源的情况下,碱性磷酸酶活性迅速提高,在第9天达到了最大值,其活性分别为19.11U和18.28U,显然在缺磷胁迫下和大分子DOP的利用过程中碱性磷酸酶起着重要作用。DIP(磷酸二氢钾)和小分子DOP(β-甘油磷酸钠)都容易直接被球形棕囊藻吸收利用,碱性磷酸酶活性变化不明显。     

大鼠脂多糖结合蛋白的分离纯化及对脂多糖的调节作用

经硫酸铵沉淀、Bio Rex70树脂的阳离子交换层析和MonoQ预装柱的阴离子交换层析 ,从大鼠急性期血清中分离纯化了脂多糖结合蛋白 .SDS PAGE为单一条带 ,分子量 60kD ,所纯化的大鼠脂多糖结合蛋白可明显增强脂多糖与单核巨噬细胞的结合 .脂多糖结合蛋白对脂多糖诱导肺泡巨噬细胞中肿瘤坏死因子α(TNF α)mRNA表达的调节作用具有明显的剂量依赖性 .     

苦荞籽粒芦丁降解酶的纯化、酶学性质及部分一级结构分析

芦丁降解酶催化芦丁降解为水溶性降低的槲皮素,是形成苦荞苦味的主要因素。研究以榆6-21苦荞籽粒粉为材料,通过硫酸铵分级沉淀、疏水层析、阳离子交换层析、凝胶过滤层析分离纯化芦丁降解酶,SDS-PAGE显示纯化的芦丁降解酶为分子量66 kDa的单一条带。芦丁降解酶的最适pH值为5.0,最适温度为50℃。对其催化特性研究表明,该酶的K_m值为0.27 mmol/L,V_(max)值为39.68 U/mg。Cu~(2+)、Zn~(2+)、Mn~(2+)和EDTA对其有不同程度的抑制作用。纯化的芦丁降解酶可耐受50%(V/V)的甲醇。Edman降解法测定其N端序列为TVSRSSFPDGFLFGL。MALDI-TOF-MS(基质辅助激光解吸电离飞行时间质谱)二级质谱获得了其15个内肽序列。该研究为从转录组数据中确定芦丁降解酶的候选基因及深入研究芦丁降解酶的生物学功能奠定了基础。     

条斑紫菜蛋白酶解产物中α-葡萄糖苷酶抑制剂纯化及特性

以α-葡萄糖苷酶抑制活性为指标,优选出1398中性蛋白酶、碱性蛋白酶和氨肽酶在一定条件下复配酶解条斑紫菜蛋白制备α-葡萄糖苷酶抑制剂。酶解液加入乙醇至终浓度为60%以沉淀去除多糖,上清液为α-葡萄糖苷酶抑制剂粗品。该粗品利用SP Sepharose High Performance阳离子交换层析、Sephadex G-10凝胶层析和Mono Q阴离子交换层析进行分离纯化,获得一种肽类α-葡萄糖苷酶抑制剂(LGI)。LGI经反向高效液相层析测定纯度为62.4%,基本特性分析显示其具较好的温度和pH稳定性,对α-葡萄糖苷酶半抑制浓度IC50值为97.36μg/mL,属于一种非竞争性抑制剂。     

一种来源于青霉的新的α-半乳糖苷酶的分离纯化及其酶学性质

从丝状真菌中筛选到一株产α-半乳糖苷酶的菌株F63,对该菌株进行了形态观察和18SrDNA序列分析,该菌株属于青霉属。采用硫酸铵沉淀、阴离子交换层析和分子筛层析等方法分离纯化了该菌株的一种α-半乳糖苷酶。经过聚丙烯酰胺凝胶电泳,此酶蛋白的分子量约为82kDa。该α-半乳糖苷酶反应的最适pH为5.0,最适温度为45℃。此α-半乳糖苷酶的热稳定性在40℃以下,pH稳定性为pH5.0-6.0。与已报道的α-半乳糖苷酶的活性都受到Ag 的强烈抑制不同的是,该α-半乳糖苷酶受Ag 的抑制作用不显著。以pNPG为底物的Km值为1.4mmol/L和Vmax=1.556mmol/L.min-1.mg-1。该酶可以有效降解蜜二糖、棉子糖和水苏糖,但不能降解末端含α-半乳糖苷键的多糖。通过利用质谱技术对纯化的α-半乳糖苷酶进行鉴定以及内肽的N端测序证明该蛋白为一种新的α-半乳糖苷酶。     

海洋微生物有机磷降解酶的纯化与性质研究

从长期受有机磷农药污染的海水中分离得到1株能高效降解农药的芽胞杆菌M-1,通过离子交换层析、凝胶过滤层析等方法从发酵液中分离纯化了有机磷农药降解酶,SDS-PAGE测得该酶的分子质量约为45 kD。酶反应最适pH为7.5,最适反应温度为30℃,30℃下保温30 min,酶活力基本不变,高于30℃酶活力则迅速下降;K 、Na 、Ca2 、Mn2 对酶活性有促进作用,Hg2 、Zn2 和Cu2 等对酶有抑制作用。     

壳聚糖酶的克隆表达与壳寡糖的制备分析

目的:克隆壳聚糖酶基因于大肠杆菌中实现高表达,制备壳寡糖。方法:以枯草芽孢杆菌总DNA为模板扩增壳聚糖酶基因(CSN),克隆至载体pET23a(+)上,转化菌株BL21(DE3)。重组子经0.5 mmol/L IPTG诱导后,SDS-PAGE和质谱检测与鉴定重组酶。酶纯化后水解壳聚糖,薄层色谱分析其水解产物。结果:质谱证明壳聚糖酶(31.5kDa)成功表达,表达量占菌体总蛋白的45%左右。纯化后重组酶浓度为900 mg/L,纯度95%、回收率85%,酶活力为10 000 U/mg。壳聚糖降解产物为壳二糖至壳四糖。结论:原核表达载体pET23a(+)-CSN构建正确,壳聚糖酶表达量与活性高,适用于水解壳聚糖制备壳寡糖。     

国内外蝗害治理技术现状与展望

本文首先概述了国内外蝗虫发生与为害的态势,总结了现阶段我国蝗虫发生与为害的主要特点:即农田飞蝗暴发频繁而且严重,草原土蝗的发生时常造成严重的经济损失,而且侵入城市干扰市民生活,我国与周边国家之间蝗虫过境迁移频繁,使用化学农药污染环境和农产品;分析了国内外蝗虫防治对策与技术的发展现状,重点介绍了应急防治和可持续治理对策、...     

颧骨颧弓骨折的分类及诊治

颧骨颧弓骨折是最常见的面中部骨折之一,迄今为止,在其分类及诊治的选择上仍然存在一定争议,本文对目前颧骨颧弓骨折的分类及诊治的现状和进展作一综述,以期对临床工作提供参考。     

Molecular characterisation of species and genotypes of Cryptosporidium and Giardia and assessment of zoonotic transmission

The molecular characterisation of species and genotypes of Cryptosporidium and Giardia is essential for accurately identifying organisms and assessing zoonotic transmission. Results of recent molecular epidemiological studies strongly suggest that zoonotic transmission plays an important role in cryptosporidiosis epidemiology. In such cases the most prevalent zoonotic species is Cryptosporidium parvum. Genotyping and subtyping data suggest that zoonotic transmission is not as prevalent in the epidemiology of giardiasis. Molecular characterisation of Cryptosporidium and Giardia is a relatively recent application that is evolving as new genes are found that increase the accuracy of identification while discovering a greater diversity of species and yet unnamed taxa within these two important genera. As molecular data accumulate, our understanding of the role of zoonotic transmission in epidemiology and clinical manifestations is becoming clearer.     

Synthesis and turnover of cerebrosides and phosphatidylserine of myelin and microsomal fractions of adult and developing rat brain.

The synthesis and turnover of cerebrosides and phospholipids was followed in microsomal and myelin fractions of developing and adult rat brains after an intracerebral injection of [U-14C]serine. The kinetics of incorporation of radioactivity into microsomal and myelin cerebrosides indicate the possibility of a precursor-product relationship between cerebrosides of these membranes. The specific radioactivity of myelin cerebrosides was corrected for the deposition of newly formed cerebrosides in myelin. Multiphasic curves were obtained for the decline in specific radioactivity of myelin and microsomal cerebrosides, suggesting different cerebroside pools in these membranes. The half-life of the fast turning-over pool of cerebrosides of myelin was 7 and 22 days for the developing and adult rat brain respectively. The half-life of the slowly turning-over pool of myelin cerebrosides was about 145 days for both groups of animals. The half-life of the rapidly turning-over microsomal cerebrosides was calculated to be 20 and 40 h for the developing and adult animals respectively. The half-life of the intermediate and slowly turning-over microsomal cerebrosides was 11 and 60 days respectively, for both groups of animals. The amount of incorporation of radioactivity into microsomal cerebrosides from L-serine was greatly decreased in the adult animals, and greater amounts of the precursor were directed towards the synthesis of phosphatidylserine. In the developing animals, considerable amounts of cerebrosides were synthesized from L-serine, besides phosphatidylserine. The time-course of incorporation indicated that a precursor-product relationship exists between microsomal and myelin phosphatidylserine. The half-life of microsomal phosphatidylserine was calculated to be about 8 h for the fast turning-over pool in both groups of animals.     

Identification and composition of the tonsillar and anal enterococcal and streptococcal flora of dogs and cats

Enterococcus faecalis was the most frequently isolated enterococcal species from anal swabs and tonsils of dogs and cats, although in the anal samples from dogs Ent. hirae was found almost as often as Ent. faecalis. Most Ent.faecium strains from dog tonsils differed from those associated with humans and other animals in that they fermented sorbitol. Typical Ent. avium as well as atypical Ent. avium -like strains were seen in dogs, while the related species Ent. raffinosus was associated with cat tonsils. Enterococcus cecorum also occurred mainly in cats. Certain atypical strains, presumptively identified as Ent. cecorum , shared characteristics with Ent. columbae.
The most frequent streptococcal species in tonsils of cats and dogs were Streptococcus suis and Strep. canis. Streptococcus canis and Strep. bovis predominated in anal swabs. The canine Strep. suis differed from the common porcine strains in fermenting mannitol.
Forty-seven of the 288 isolates examined could not be identified or related to known species. The characteristics of two groups of these bacteria, provisionally called 'Ton 31 group' and 'O7 group' are described.