Bacillus pumilus XJU-13的分离鉴定及其碱性脂肪酶酶学特性分析

目的:对从新疆精河地区一处工业污水中分离得到的一株产碱性脂肪酶细菌进行研究.方法:通过生理生化检测,16SrDNA序列同源性分析和G+Cmol%含量的测定对命名为XJU-13的这株菌进行鉴定.结果:该菌株可在pH 3.0～12.5的广泛酸碱泛围的营养肉汤培养基中生长.最适生长温度为37℃.基于16S rDNA序列同源性构建系统进化树分析表明与Bacillus pumilus clone B257聚在同一亚分枝,序列相似性达100%.数据证明XJU-13属于Bacillus pumilus.由于在氧化酶反应及淀粉水解实验与伯杰氏鉴定手册有差异,具不可比拟的pH耐受性,且脂肪酸含量与参考菌株差异较大,认为这是Bacillus pumilus中的一个新品系.该菌株产生的脂肪酶最适pH为10,最适温度为35℃,且在广泛pH(pH4-10)范围具稳定性.酶活可被Mg2+、K+、Ba2+、Pb+盐强烈抑制,被Ca2+、Cu2+、Al+及Fe2+盐激活.Zn2+对酶活无影响.结论:实验表明,XJU-13应属于B.pumilus.B.pumilus XJu-13中分离到的碱性脂肪酶有很好的特性及潜能,以期为工业应用提供数据.     

低温淀粉酶菌株C2的分离鉴定及其产低温淀粉酶的酶学性质

获得低温淀粉酶高产菌株,确定该菌株所产淀粉酶的酶学性质.从大黑山(大连)污泥中筛选菌株,通过菌株的形态特征、生理生化和16S rDNA序列鉴定确定其种属,对其酶学性质进行初步研究.获得1株低温淀粉酶高产菌株C2,经鉴定其为微小杆菌属,C2所产低温淀粉酶最适反应温度为25℃,酶的热稳定性比较差,最适pH为7.5,Ca2+和Fe2+对该酶有激活作用,Cu2+、Ni2+、Go2+等抑制酶活性.经薄层层析(TLC)鉴定酶解产物为葡萄糖,说明该菌株具有产生低温淀粉糖化酶的能力.菌株C2所产淀粉酶符合低温淀粉酶性质,值得进一步研究.     

中国吐鲁番两株产木聚糖酶的极端耐碱Bacillus halodurans的分类鉴定

通过生理生化实验、16S rDNA序列分析和同源性杂交,将分离到的XJU-1和XJU-80菌种进行了分类鉴定。XJU-1和XJU-80具有较宽的pH生长范围(分别是pH4·5 ~12·6和pH3·8 ~12·6) ,其G C mol%含量分别是40·5mol%和42·2mol%。16S rDNA序列分析和DNA-DNA同源杂交结果表明,XJU-1和XJU-80与Bacillushalodurans C-125和Bacillus halodurans DSM497T具有较高的同源性(99 %) ;两者之间也具有85 %的相关性,但其与Bacillus halodurans C-125和Bacillus halodurans DSM497T分别具有81·3 %和71·5 %的相关性。基于以上结果,将两株分离菌株分类为Bacillus halodurans的两个新品系。     

家蚕肠道产淀粉酶细菌的分离鉴定及其酶基因的克隆与表达

从家蚕(Bombyx mori L.)5龄幼虫肠道分离鉴定产淀粉酶细菌菌株以用作微生态制剂的研究,并对该菌α-淀粉酶基因进行克隆、序列分析及在大肠杆菌中原核表达.通过含淀粉NA培养基筛选分离得到产淀粉酶菌,通过形态学观察及16S rDNA序列分析鉴定其种属,DNS法测定酶活,并设计了淀粉酶基因引物进行克隆,构建了DZ-...     

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。     

一株产α-高温淀粉酶的地衣芽孢杆菌的分离和筛选

从西藏当雄温泉附近的土壤中用锥虫蓝平板,55℃培养,筛选到一株分泌高温淀粉酶的菌株,经生理生化初步鉴定后,克隆16s rDNA基因测序,提交GenBank比对后,鉴定为地衣芽孢杆菌(Bacillus licheninformis),命名为B.licheninformis LT.通过对B.licheninformis LT的生长条件和产酶条件的研究表明:该菌高温特性良好,最高可以在65℃生长,产酶最适温度50℃;可以在pH 3.O～pH 11.0的LB培养基中生长,最佳产酶pH 10.0,LB培养基加入淀粉诱导,发酵液酶活可达80 U.对该菌所产α-高温淀粉酶的性质研究表明:酶的最适反应温度为95℃,100℃处理60 min发酵液酶活力没有明显下降,最适酶作用pH 10.0,添加1g/L的钙离子具有激活作用.     

产α-淀粉酶菌株的分离、鉴定及酶学性质研究

目的:筛选高产α-淀粉酶菌株,为工业生产α-淀粉酶提供储备菌株。方法:利用碘液显色法和摇瓶发酵法,从土壤中筛选产α-淀粉酶菌株;通过菌落形态、菌体特征观察和16S rDNA序列比对对菌种进行鉴定;发酵粗酶液经硫酸铵沉淀、透析脱盐后,对其酶学性质进行初步研究。结果:从土壤中筛选到一株高产α-淀粉酶菌株,枯草芽孢杆菌Bacillus subtilis XL-15。该菌株所产α-淀粉酶的最适反应温度为50℃,最适作用pH为6.5;Ca2 和Mn2 对酶有激活作用,而Cu2 、Zn2 和EDTA对酶有抑制作用;酶的动力学研究测出米氏常数Km值为1.726mg/mL。结论:该菌株是产α-淀粉酶的较好材料,且具有一定的应用前景。     

一株高产淀粉酶的解淀粉芽胞杆菌的分离鉴定及其产酶条件优化

目的从土壤中分离产淀粉酶相对较高的菌株,并对其产酶条件进行优化,以期获得更经济、易得的高活力淀粉酶。方法用平板稀释涂布法分别从温州及其周边地区多个淀粉含量高的土壤、污水中采集的样品中筛选产淀粉酶菌株,通过菌落形态、生理生化反应和16S rDNA序列比对对菌株进行鉴定;并从碳源、氮源、离子浓度及紫外诱变等产酶条件对高产淀粉酶菌株进行优化;利用碘-淀粉法等对粗酶液酶学性质进行初步研究。结果从采集到的26个样品中共筛选到19株酶活较高、产酶稳定的野生型菌株。其中编号为Z19的菌株酶活最高,达到351.78 U/mL。经形态和生理生化鉴定,结合16S rDNA序列分析,将该菌株鉴定为解淀粉芽胞杆菌。对菌株Z19的液体发酵条件研究表明,最佳条件为玉米粉1.0%,明胶1.5%,淀粉含量0.5%,NaCl 0.5%,pH7.0,发酵温度37℃,72 h,酶活达到1 360.92 U/mL;在固体培养基上,直接用麸皮,接种量为0.1%,含水量为60%,pH7.0,120 h时,酶活达到1 504.2 U/mL。其最适酶反应pH为6.5,反应温度55~60℃,底物浓度为0.5%。结论从土壤中分离得到了产淀粉酶相对较高的解淀粉芽胞杆菌Z19菌株,产酶条件适用于工业生产,具有一定的应用前景。     

一株产漆酶细菌的分离鉴定及酶学性质研究

[目的]分离获得产漆酶的细菌菌株,研究漆酶的酶学性质并应用于染料脱色.[方法]利用含铜的富集培养基筛选产漆酶细菌;通过形态特征、生理生化试验及16SrDNA序列分析等方法进行鉴定;以丁香醛连氮为底物测定漆酶的酶学性质;通过测定染料在最大吸收波长下吸光值的变化评价漆酶对染料的脱色效果.[结果]从森林土壤中筛选到一株漆酶高产菌株LS05,初步鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens);菌株LS05的芽孢漆酶以丁香醛连氮为底物的最适pH为6.6,最适温度为70℃;该酶具有较好的稳定性,经70℃处理10h或在pH 9.0条件下放置10d后可保留活性.对抑制剂SDS和EDTA具有一定的抗性,在碱性条件下可有效脱色不同的工业染料,RB亮蓝、活性黑和靛红1h内的脱色率达93％以上.[结论]Bacillus amyloliquefaciens LS05的芽孢漆酶在高温和碱性条件下稳定性强,相对于真菌漆酶具有更好的工业应用特性,可有效用于工业染料废水的处理.     

一株中温淀粉酶菌株的分离鉴定及其产酶条件分析

利用固体淀粉筛选培养基,从安阳市郊区面粉厂附近的土壤里分离筛选出1株产淀粉酶的菌株,编号为MF-3-2.经过菌株形态、革兰氏染色、16S rDNA鉴定及系统进化树分析,初步确定其为枯草芽孢杆菌(Bacillus subtilis).摇瓶培养后对其酶学性质研究发现,该菌株淀粉酶的最适温度为65℃,最适pH值为6.0,在pH值4.8～6.0范围内仍能残余70％以上的酶活力.该菌株的最适生长温度为40℃,最适生长pH值为6.5.产酶条件优化结果表明:最适碳源为马铃薯淀粉,最适氮源为豆粕粉,最适碳氮比为1∶15,发酵温度30℃,发酵pH值6.0,装液量10％,种龄10h,接种量5％,转速200 r/min,48 h达到产酶高峰.通过发酵产酶条件优化,其淀粉酶活性达到86.8 U/mL,是优化前的35倍.另外,在酸性条件下还具有较好的活性.因此,该菌株的淀粉酶具有潜在的工业应用前景.     

两株具有抗癌活性内生细菌的分离及分类

从西双版纳植物样品爵床[Rostellularia procumbens(L.) Nees]中分离到2株具有较强抗癌活性的内生细菌YIM 56077和YIM 56081。通过对其进行表型特征、生长及生理生化特性、细胞化学组份以及系统进化分析,发现这2株菌与B.flexus IFO15715T的亲缘关系最近,但它们在生长、生理生化等特征上表现明显差异,是芽胞杆菌属的2个具有开发潜力的新菌株。     

Biodegradation of 3-Nitrobenzoate by <Emphasis Type="Italic">Bacillus flexus</Emphasis> strain XJU-4

Bacillus flexus strain XJU-4 utilized 3-nitrobenzoate at 12 mM as a sole source of carbon and energy. This strain also utilized 4-nitrobenzoate, 2-nitrotoluene and nitrobenzene as growth substrates. The optimum conditions for degradation of 3-nitrobenzoate by the organism were found to be at pH 7.0 and temperature 30°C. Metabolite analysis, growth and enzymatic studies have revealed that the organism degraded 3-nitrobenzoate by oxidative mechanism through protocatechuate with the release of nitrite. The cells grown on 3-nitrobenzoate utilized protocatechuate but not 3-hydroxybenzoate, 3-aminobenzoate, 4-hydroxy-3-nitrobenzoate and 4-nitrocatechol. The cell-free extract of Bacillus flexus strain XJU-4 grown on 3-nitrobenzoate contained the activity of protocatechuate 2,3-dioxygenase, which suggest that protocatechuate was further degraded by a novel 2,3-dioxygenative meta-cleavage pathway.     

Newly isolated Bacillus clausii GMBAE 42: an alkaline protease producer capable to grow under higly alkaline conditions

AIMS: The isolation and identification of new Bacillus sp. capable of growing under highly alkaline conditions as alkaline protease producers. METHODS AND RESULTS: A Bacillus strain capable of growing under highly alkaline conditions was isolated from compost. The strain is a Gram-positive, spore-forming, motile, aerobic, catalase- and oxidase-positive, alkaliphilic bacterium and designated as GMBAE 42. Good growth of the strain was observed at pH 10. The strain was identified as Bacillus clausii according to the physiological properties, cellular fatty acid composition, G + C content of genomic DNA and 16S rRNA gene sequence analyses. The result of 16S rRNA sequence analyses placed this bacterium in a cluster with B. clausii. The G + C content of the genomic DNA of the isolate GMBAE 42 was found to be 49 mol%. The crude extracellular alkaline protease produced by the isolate showed maximal activity at pH 11.0 and 60 degrees C. CONCLUSIONS: The results suggest that isolated strain GMBAE 42 is a new type of B. clausii capable of growing at pH 10.0 and produce extracellular alkaline protease very active at pH 11.0. SIGNIFICANCE AND IMPACT OF THE STUDY: Isolated strain could be used in commercial alkaline protease production and its enzyme can be considered as a candidate as an additive for commercial detergents.     

中国新分离碱性耐热芽孢杆菌的木聚糖酶产酶特征

目的：检测由中国新分离碱性耐热芽孢杆菌[1]产生的粗木聚糖酶的酶活。方法：通过DNS法测定粗木聚糖酶的酶活。结果：实验表明,以橡树木聚糖为底物培养的新分离菌株在30-50℃处理2h酶活不丧失。其中,XJU-1菌株在60、70和80℃时粗酶酶活分别丧失是最初酶活的1.54%、19.09%和72.59%;而XJU-80的粗酶酶活分别是3.59%、26.43%和72.59%。两个菌株产生的粗木聚糖酶的最适pH是7.5-8.0。将该粗酶在pH 7.0-9.0（50℃）处理24h后,酶活几乎均降低最初酶活的18%。结论：由XJU-1和XJU-80产生的木聚糖酶是生化领域有用的嗜碱耐热酶。     

Identification of facultatively alkaliphilic Bacillus sp. strain YN-2000 and its fatty acid composition and cell-surface aspects depending on culture pH

Facultatively alkaliphilic Bacillus sp. strain YN-2000 was isolated from an indigo ball. Although the strain has been extensively investigated as a representative strain of alkaliphilic bacillus, its taxonomic position is not yet known. Morphological, biochemical, and physiological characteristics and chemotaxonomic properties indicated that the strain was closely related to Bacillus cohnii; this was confirmed by the high homology of the 16S rRNA sequence and the construction of a phylogenetic tree on the basis of the 16S rRNA sequence and DNA–DNA relatedness data. Strain YN-2000 contained a larger amount of unsaturated fatty acids compared with Bacillus subtilis and the obligate alkaliphile, Bacillus alcalophilus, regardless of its culture pH. When the cells were grown at pH 10, the unsaturated fatty acid content and anteiso-/iso-branched fatty acid ratio became lower than those at pH 7. This result suggests that membrane fluidity decreases when the cells are grown at pH 10 compared to those of pH 7. In the cells of strain YN-2000 grown at pH 10, the cell-surface aspect was rougher, the cell shape was longer, and the cell-surface layer was thicker compared with those of the cells grown at pH 7. The cell-surface structural change might be related to adaptation to an alkaline environment. Received: April 6, 2000 / Accepted: May 8, 2000     

Caldimonas taiwanensis sp. nov., a amylase producing bacterium isolated from a hot spring

During screening for amylase-producing bacteria, a strain designated OnlT was isolated from a hot spring located in Pingtung area, which is near the very southern part of Taiwan. Cells of this organism were Gram-negative rods motile by means of a single polar flagellum. Optimum conditions for growth were 55 degrees C and pH 7. Strain On1(T) grew well in minimal medium containing starch as the sole carbon source, and its extracellular products expressed amylase activity. The 16S rRNA gene sequence analysis indicates that strain On1(T) is a member of beta-Proteobacteria. On the basis of a phylogenetic analysis of 16S rDNA sequences, DNA-DNA similarity data, physiological and biochemical characteristics, as well as fatty acid compositions, the organism belonged to the genus Caldimonas and represented a novel species within this genus. The predominant cellular fatty acids of strain On1(T) were 16:0 (about 30%), 18:1 omega 7c (about 20%) and summed feature 3 (16:1 omega 7c or 15:0 iso 2OH or both [about 31%]). Its DNA base ratio was 65.9 mol% G + C. We propose to classify strain On1(T) (= BCRC 17405T = LMG 22827T) as Caldimonas taiwanensis sp. nov.     

Purification and properties of a low-molecular-weight, high-alkaline pectate lyase from an alkaliphilic strain of Bacillus

A low-molecular-weight, high-alkaline pectate lyase (pectate transeliminase, EC 4.2.2.2) was found in an alkaline culture of Bacillus sp. strain KSM-P15, purified to homogeneity, and crystallized. The enzyme had a relative molecular weight of approximately 20,300 as measured by sedimentation equilibrium, with a sedimentation coefficient (s20,w0) of 1.73 S. It was a basic protein with an isoelectric point of pH 10.3, and the alpha-helical content was only 6.6%. In the presence of Ca2+ ions, the enzyme degraded polygalacturonic acid in a random manner to yield 4,5-unsaturated oligo-galacturonides and had its optimal activity around pH 10.5 and 50-55 degrees C. It also had a protopectinase-like activity on cotton fibers. The N-terminal amino acid sequences of the intact protein (28 amino acids) and its two lysyl endopeptidase-cleaved peptide fragments (8 and 12 amino acids) had very low sequence similarity with pectate lyases reported to date. These results strongly suggest that the pectate lyase of Bacillus sp. strain KSM-P15 may be a novel enzyme and belongs in a new family.     

Endozoicomonas elysicola gen. nov., sp. nov., a gamma-proteobacterium isolated from the sea slug Elysia ornata

A Gram-negative, strictly aerobic, rod-formed bacterium, strain MKT110(T), was isolated from a mollusk, the sea slug Elysia ornata collected in seawater off the coast of Izu-Miyake Island, Japan at a depth of 15m. Comparative 16S rRNA gene sequences analysis indicated that the isolate MKT110(T) constituted a novel lineage in gamma-proteobacteria related to the genera Zooshikella, Oceanospirillum, Microbulbifer, Marinobacter, Saccharospirillum and Pseudomonas. The strain MKT110(T) was closely related to the clones from marine sponge Halichondria okadai (AB054136, AB054161) and the coral Pocillopora damicornis (AY700600, AY700601). The phylogenetic tree based on the 16S rRNA gene sequences showed that MKT110(T) and four clones formed a sub-lineage related to the genus Zooshikella, with a bootstrap value of 100%. MKT110(T) required salt for its growth and was mesophilic. The bacterium contained 16:1omega7c, 16:0 and 14:0 as major cellular fatty acids, and 3-OH 14:0, 3-OH 10:0 and 3-OH 12:0 as major hydroxy fatty acids. The DNA base composition of the isolate was 50.4 mol% G+C. The major quinone was Q-9. The bacterium is distinguished from currently recognized bacterial genera based on phylogenetic and phenotypic features and should be classified in a novel genus for which the name Endozoicomonas elysicola gen. nov., sp. nov. is proposed. (type strain MKT110(T)=IAM 15107(T)=KCTC 12372(T); GenBank accession no. AB196667).     

Effect of Metal Cations and pH on the Antibacterial Activity and Uptake of Long Chain Fatty Acids

S ummary . The bactericidal activity of long chain saturated fatty acids was antagonized by alkaline earth metals. The activity of linoleic acid was less effectively antagonized but was more sensitive to reversal by ferric and stannous ions. With increasing pH value the bactericidal activity of lauric acid decreased but that of the longer chain saturated acids increased. Both Gram positive and Gram negative bacteria reversibly adsorbed fatty acids. Uptake increased with decreasing pH value and increasing chain length. Although adsorbed to a lesser extent, the intrinsic activity of linoleic acid was greater than lauric acid. The uptake appeared to be non-specific and governed by the physicochemical properties of both the acids and the bacterial cell surfaces. Sensitivity to the fatty acids increased with decreasing pH value. Protoplasts of Bacillus megaterium adsorbed fatty acids to a greater extent than whole cells. Resistance of the Gram negative Pseudomonas phaseolicola was not due to non-adsorption of the fatty acids.