津巴布韦烟叶中淀粉酶和蛋白酶产生菌的分离及鉴定

目的:从津巴布韦烟叶中分离产蛋白酶菌和产淀粉酶能力最高的菌株,并对其进行鉴定。方法:采用淀粉富集培养基和酪蛋白富集培养基分别分离津巴布韦烟叶中的产淀粉酶和产蛋白酶菌株,通过生理生化实验和16SrRNA序列分析鉴定分离的菌株。结果:产蛋白酶菌株菌体不透明、表面有褶皱,蛋白酶酶活为52.10±0.13 U/mL;产淀粉酶菌株菌体表面呈黏状,淀粉酶酶活为3.69±0.07 U/mL;产蛋白酶与产淀粉酶的2株菌均与枯草芽孢杆菌的16S rRNA序列有100%的相似性,结合生理生化指标初步鉴定为枯草芽孢杆菌。结论:获得的2株菌在降解烟叶的蛋白质和淀粉过程中可能起重要作用。     

烟叶果胶质分解菌的选育

从烟叶叶面分离到产果胶酶真菌 18株 ,在此基础上 ,进行液体培养 ,测定酶活 ,得到一株酶活性较高的黄曲霉DPE - 0 0 5。对该菌株产酶培养基的碳、氮源进行正交法研究 ,正交实验的结果表明 ,影响该菌产酶活性的因素依次为A(麸皮 ) >B(乳糖 ) >C(果胶 ) >D(硫酸铵 ) ,其最佳组合为A2B3C3D1。最适产酶条件为 :麸皮 4 0 % ,果胶 0 3% ,乳糖 1 5 % ,(NH4 ) 2 SO4 0 5 % ,KH2 PO4 0 2 5 % ,MgSO4 ·7H2 O 0 0 5 % ,NaNO30 0 2 % ,FeSO4 ·7H2 O 0 0 0 1% ,起始pH 6 0 ,在 2 8℃摇床培养 7d产酶量达到最高。以玉溪B3F烟叶为材料 ,施加DPE - 0 0 5菌株所产酶液 ,在 5 0℃贮存 12h。经化学成分检测 ,结果表明 ,果胶质降低了 18 15 %。     

一株产高温蛋白酶嗜热菌A-2的筛选鉴定及酶学特性分析

本研究为从云南腾冲热泉中分离纯化得到一株产高温蛋白酶的菌株并对其进行驯化培养,用以探究该菌株的生长条件及酶学特性,通过选择培养基筛选能够分解脱脂奶粉产蛋白酶的菌株,应用常规方法液体培养菌体,探究温度、pH、碳源、氮源对菌株生长情况的影响,并采用福林酚法测蛋白酶活性。并提取蛋白酶液对酶的最适pH、温度以及热稳定性、pH稳定性进行研究。结果发现通过含脱脂奶粉的固体培养基筛选得到一株产蛋白酶菌株A-2,经过生理生化试验和16S rDNA鉴定知该菌种属于Aneurinibacillus属。酵母粉、葡萄糖、55℃、pH值7.5分别为菌株生长的最适氮源、碳源、温度和pH。此外该菌株所产的蛋白酶最适温度为60℃,在pH值7~9具有较好的酶活性。因此,该菌株为嗜热芽孢杆菌,所产的碱性蛋白酶具有较高的耐受温度和pH稳定性,为进一步开发利用提供参考的价值。     

烟碱降解菌的选育及改善上部烟叶品质研究

从烟叶叶面和植烟土壤中分离到降解烟碱的微生物18株,经初筛和复筛得到降解烟碱活性较高的微生物菌Nic22。对该菌株产酶培养条件进行优化研究,实验表明:Nic22降解烟碱效果受到多种因素的影响,添加适量的精制淀粉和(NH4)2SO4作为外加碳氮源可明显促进烟碱的降解,适宜的摇瓶培养温度、pH和时间分别为30℃、6.0~7.0和9h。采用Nic22菌株所产酶液对上部烟叶进行处理试验,卷烟评吸结果表明:上部烟叶经酶液处理可明显减轻杂气和刺激性,抽吸品质得到显著提高。     

“黑老虎”晾晒烟叶表面产酶细菌的鉴定及其在烟草加工上的应用

从江西"黑老虎"晾晒烟叶表面分离筛选出具备产木聚糖酶、果胶酶、纤维素酶、蛋白酶和淀粉酶能力的细菌,并将这些菌株添加到烟丝上进行陈化。结果表明:菌株促进了烟丝的陈化过程,主要体现在香气更加充实、杂气减少、刺激性减弱、余味更净,同时产生香味物质,减少有害物质的含量。其中两株菌经过生理生化实验和16S rDNA鉴定,判定为短小芽孢杆菌和解淀粉芽孢杆菌,此外对这两株菌的培养条件进行了初步优化。从烟叶表面分离筛选的产酶微生物在烟叶陈化过程起重要作用,可制备成微生物菌剂加速烟叶陈化并实现提质增香的效果。本研究以江西"黑老虎"烟叶作为研究对象,根据鉴定平板的水解圈分离筛选具有产木聚糖酶、果胶酶、纤维素酶、蛋白酶、淀粉酶能力的细菌,对陈化结果进行感官评价后发现所筛选的3株产酶细菌(82#,83#和85#)均能有效提升烟丝的品质,主要体现在香气更加充实、杂气减少、刺激性减弱、余味更净。结合形态学观察、理化试验及16S rDNA序列分析,确定3株潜在的提质增香菌均为芽孢杆菌。其中83#为短小芽孢杆菌Bacillus pumilus,而85#为解淀粉芽孢杆菌Bacillus amyloliquefaciens。研究结果表明筛选得到的菌株83#和85#具有作为提质增香菌剂的应用潜力,并为芽孢杆菌应用于烟叶陈化提供理论依据。     

天山一号冰川底部沉积层产蛋白酶耐低温菌株的筛选及其系统发育

[目的]通过对天山一号冰川底部沉积层耐低温菌的分离和其中产蛋白酶菌株的筛选,了解冰川微生物生理多样性和系统发育多样性,为高效低温蛋白酶生物技术的研发奠定基础.[方法]采用稀浓度的R2A、TSB平板涂布分离可培养细菌,通过脱脂乳选择性培养基筛选产蛋白酶的耐低温菌株.对分离菌株表型特征、生理生化特性、最适生长温度、耐盐性、产酶性能进行了比较,结合16S rRNA基因序列同源性分析确定产蛋白酶菌株的多样性和系统进化地位,通过BOX-PCR指纹技术分析16S rRNA基因序列高相似度的近缘菌株的遗传差异.[结果]从125株分离物中筛选到27株产蛋白酶的耐低温菌株,其中21株为适冷菌,仅6株菌为专性嗜冷菌,革兰氏阴性菌居多,假单胞菌属(Pseudomonas)菌株占40.7％.产酶菌株隶属于5个系统发育类群、9个属,其中γ-Proteobacteria、Actinobacteria、CFB(Cytophaga-Flexibacter-Bacteroides)为优势类群.[结论]天山1号冰川底部沉积层冻土中产蛋白酶的耐低温细菌多样性较丰富,本研究筛选得到的同属近缘种群较多,其产酶性状存在差异,适合开展微生物种群的生物地理学研究.     

复合酶产生菌的筛选及其在烟叶醇化中的应用

从进口烟叶表面分离筛选出5株产复合酶的菌株,其中菌株HY-2能产多种复合酶,且活性相对较高,通过生理生化及16SrDNA序列分析,鉴定该菌株为解淀粉芽孢杆菌（Bacillus amyloliquefaciens）。利用该菌株配制成的生物制剂（编号MR—HY）,分别采用回潮控温控湿及直接添加的方式在烟叶醇化中进行应用。结果表明,采用回潮控温控湿方式,生物制剂能在短时间内快速作用烟叶中组分,可使得烟叶糖氮比、糖碱比更趋于协调,品质得到一定的提升;采用直接添加方式也能有效的加速烟叶醇化过程,同时烟叶的香气量增加,香气质感变好,杂气及刺激降低。可见,通过添加该菌株配制的生物制剂能有效的改善烟叶醇化过程,有较好的应用空间。     

产弹性蛋白酶菌种的筛选及发酵条件优化研究

从合肥肉联厂附近的土壤和污水中分离得到19株产弹性蛋白酶菌株,初步鉴定该菌株属于假单胞菌属.经过发酵复筛有三株产酶能力超过15u/mL.实验对菌株最佳产酶发酵条件进行了优化2%干酪素、0.5%葡萄糖、0.4%酵母膏、0.2% K2HPO4、0.01% MgSO4·7H2O;起始pH值7.0;最适发酵温度为30℃;装液量为25mL/250mL;该菌株在28h左右产弹性蛋白酶的量达18u/mL.     

产碱性蛋白酶细菌Bs08菌株的分离及其产酶特性的研究

在产蛋白酶细菌生态分布及产酶条件研究的基础上,对产碱性蛋白酶细菌进行了分离和筛选。从780株细菌中获得一株产酶活性较高的菌株。研究了该菌株的产酶特性,以及影响其产酶量的主要因素。     

醇化烟叶酵母菌分离鉴定及其生物学特性研究

采用涂布培养法和富集培养法对醇化烟叶样品中酵母菌进行了分离,富集培养法分离到23株,涂布培养法分离到6株。29株酵母菌的生物学特性研究表明,其中果胶酶、蛋白酶、淀粉酶、脂酶、纤维素酶、酚类利用、木质素利用、尼古丁降解、发酵产物气味等试验呈阳性的菌株数分别是1、10、0、3、10、22、1、21和29株。利用ATB系统,将ATY1、ATY8和ATY27等3株酵母菌分别鉴定为黏红酵母(Rhodotorula glutinis)、罗伦隐球酵母(Cryptococcus laurentii)和粗壮假丝酵母(Candida valida),各菌株鉴定结果的符合率分别为99.4%、98.2%和89.9%。研究结果表明,醇化烟叶中酵母菌种群数量较小,但酵母菌的生物学特性有利于烟叶醇化,值得进一步深入研究。     

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、...     

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.     

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.     

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

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。     

Kinetics and thermodynamics of peroxidase- and laccase-catalyzed oxidation of N-substituted phenothiazines and phenoxazines

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 ×10⁷M⁻¹s⁻¹to 2.6×10⁸M⁻¹s⁻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 1.8×10⁵M⁻¹s⁻¹ to 2.0×10⁷M⁻¹s⁻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 1.6×10⁸M⁻¹s⁻¹ and a reorganization energy of 0.30 eV. The respective parameters for laccase were 5.0×10⁷M⁻¹s⁻¹ and 0.29 eV. Received: 20 September 1999 / Accepted: 24 February 2000