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纳豆激酶(nattokinase, NK)是一种由纳豆芽孢杆菌发酵产生的丝氨酸蛋白酶,具有良好的纤溶活性。本研究从wako Nattokinase中分离纯化出高品质的纳豆芽孢杆菌,旨在探究最适宜该菌产纳豆激酶的发酵培养基氮源。研究人员选择了6种氮源对其进行发酵实验,通过连续测定发酵液的菌量、pH和纤溶活性以观察不同氮源对纳豆芽孢杆菌产纳豆激酶的影响。研究结果表明:最优氮源为乳清蛋白,在以此为氮源的培养基中发酵培养120 h后,纳豆激酶的纤溶活性高达1 757.79 U/mL。以乳清蛋白发酵培养基对纳豆芽孢杆菌进行发酵,不仅可以得到高活性的纳豆激酶,还可为纳豆激酶应用于食品、保健品领域提供思路。 相似文献
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本文从血栓栓塞疾病的形成机制为依据,综述了纳豆激酶的药理作用。介绍了国内外产纤溶酶菌株的筛选方法,纳豆激酶的分离、纯化常用技术,纳豆激酶的生理生化特性、活性测定方法。并说明了影响纳豆激酶稳定的4个因素,和增加纳豆激酶产量的有效方法。通过药食两用纳豆激酶的研究进展,希望对纳豆激酶的研究提供理论基础。 相似文献
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纳豆激酶基因的表达及纯化 总被引:5,自引:0,他引:5
利用PCR方法从分泌纳豆激酶的枯草杆菌基因组DNA中扩增得到纳豆激酶基因(NK),利用基因重组技术构建了纳豆激酶基因的表达载体pETNK。在诱导下,实现了在大肠杆菌中高效表达,经SDS-PAGE电泳分析和薄层扫描结果显示,表达的目的蛋白占菌体蛋白的21.5%。将表达产物经过DEAE-Cellulos-DE52和Sephedax-G100两个柱分离纯化,得到纯的纳豆激酶蛋白干粉,经琼脂糖-纤维蛋白平板法测出纳豆激酶干粉的溶栓活性相当于200u尿激酶。从基因工程角度研究纳豆激酶基因的克隆、表达及纯化,为用基因工程菌生产纳豆激酶奠定了基础。 相似文献
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《微生物学杂志》2017,(1)
纳豆激酶(Nattokinase)作为一种新型溶解血栓的纤维蛋白溶解酶,有着广阔的市场前景和巨大的产业化潜力。本研究从枯草芽胞杆菌(Bacillus subtilis natto)发酵液中提取、纯化纳豆激酶,依次通过硫酸铵盐析、凝胶过滤层析和疏水层析方法纯化纳豆激酶,纯化倍数为5.2,纯化率为46.3%。纯化后的纳豆激酶经SDS-PAGE电泳显示相对分子量约为28 kD a,纤维蛋白平板法显示活性为4 580 IU/mg。但纳豆激酶在枯草芽胞杆菌发酵液中的含量较低,因此在原核表达载体大肠埃希菌(Escherichia coli)BL21(DE3)中克隆并高效表达了纳豆激酶基因,其纳豆激酶产量显著提高,但酶活相对降低。 相似文献
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纳豆激酶基因工程研究进展 总被引:4,自引:0,他引:4
纳豆激酶是由纳豆芽孢杆菌(Bacillussubtillisnatto)分泌的一种具有纤溶作用的碱性丝氨酸蛋白酶。对纳豆激酶基因的克隆与表达,基因与蛋白质结构以及基因工程纳豆激酶的特性和功能进行了综述。 相似文献
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纳豆激酶基因的克隆及其在毕赤酵母中的表达 总被引:1,自引:0,他引:1
纳豆激酶纳是从日本传统食品纳豆中发现的一类具有溶栓效果的蛋白酶,由于其具有安全,高效,作用时间长,易吸收,廉价等优点,现在正成为一个开发治疗血栓类疾病药物的研究热点。从本实验室保存的一株高溶栓的纳豆杆菌N07出发,提取总基因组DNA,利用PCR手段扩增获得了纳豆激酶长为825bp的成熟肽基因片段。构建重组表达质粒pPICZaA-NK,经EcoR I、Xba I双酶切、PCR、测序验证得出重组表达质粒上的外源基因即为825bp的目的片段;将重组质粒pPICZaA-NK用内切酶Sac I线性化后电击导入毕赤酵母X33,通过含Zeocin的YPDS平板筛选获得重组酵母。重组酵母在BMMY培养基中发酵培养,用1%甲醇诱导目的蛋白表达。用纤维蛋白平板法检测发现发酵上清具有纤溶活性,经硫酸铵盐析、透析、Sephadex-G50过柱等步骤分离得到纳豆激酶蛋白,进行SDS-PAGE鉴定表明,表达的纳豆激酶蛋白分子量为27KD。以尿激酶为标准,实验所得纳豆激酶发酵上清液溶栓活性约为195U/mL。成功的将纳豆激酶成熟肽基因在毕赤酵母X33中表达,为纳豆激酶基因工程进一步研究奠定基础。 相似文献
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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。 相似文献
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国内外蝗害治理技术现状与展望 总被引:3,自引:0,他引:3
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、... 相似文献
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Synthesis and turnover of cerebrosides and phosphatidylserine of myelin and microsomal fractions of adult and developing rat brain. 总被引:5,自引:1,他引:5
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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. 相似文献
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L.A. Devriese J.I. Cruz Colque P. De Herdt F. Haesebrouck 《Journal of applied microbiology》1992,73(5):421-425
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. 相似文献
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. 相似文献
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Molecular characterisation of species and genotypes of Cryptosporidium and Giardia and assessment of zoonotic transmission 总被引:4,自引:0,他引:4
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. 相似文献
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白术同源四倍体的诱导和鉴定及其与二倍体过氧化物酶的比较 总被引:18,自引:0,他引:18
以白术(Atractylodes macrooephala Koidz.)二倍体组培苗为材料,对其四倍体诱导方法进行研究,共获得45个白术同源四倍体株系,为优良株系的选育提供了材料。此外,还分析比较了其中8个白术四倍体株系与二倍体的过氧化物酶同工酶(POD)的酶谱差异,发现四倍体各株系过氧化物酶同工酶谱比二倍体的均多了Rf0.310的谱带,且总过氧化物酶比活力也发生了很大改变,对探讨白术四倍体优良株系的生理生化机理具有一定的参考价值。 相似文献
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Juozas Kulys Kastis Krikstopaitis Arturas Ziemys 《Journal of biological inorganic chemistry》2000,5(3):333-340
N -substituted phenothiazines (PTs) and phenoxazines (POs) catalyzed by fungal Coprinus cinereus peroxidase and Polyporus pinsitus laccase were investigated at pH 4–10. In the case of peroxidase, an apparent bimolecular rate constant (expressed as k
cat/K
m) varied from 1 ×107 M−1 s−1to 2.6×108 M−1 s−1 at pH 7.0. The constants for PO oxidation were higher in comparison to PT. pH dependence revealed two or three ionizable
groups with pK
a values of 4.9–5.7 and 7.7–9.7 that significantly affected the activity of peroxidase. Single-turnover experiments showed
that the limiting step of PT oxidation was reduction of compound II and second-order rate constants were obtained which were
consistent with the constants at steady-state conditions. Laccase-catalyzed PT and PO oxidation rates were lower; apparent
bimolecular rate constants varied from 1.8×105 M−1 s−1 to 2.0×107 M−1 s−1 at pH 5.3. PO constants were higher in comparison to PT, as was the case with peroxidase. The dependence of the apparent
bimolecular constants of compound II or copper type 1 reduction, in the case of peroxidase or laccase, respectively, was analyzed
in the framework of the Marcus outer-sphere electron-transfer theory. Peroxidase-catalyzed reactions with PT, as well as PO,
fitted the same hyperbolic dependence with a maximal oxidation rate of 1.6×108 M−1 s−1 and a reorganization energy of 0.30 eV. The respective parameters for laccase were 5.0×107 M−1 s−1 and 0.29 eV.
Received: 20 September 1999 / Accepted: 24 February 2000 相似文献