木质纤维素分解复合菌系WSD-5组成菌株的分离及其产酶特性

复合菌系WSD-5具有高效的分解能力和产酶能力,以探明WSD-5的协同分解机理和优化高效组合为目的,通过纯培养分离手段,获得了11株细菌和3株真菌。16S rDNA比对结果表明,细菌分别为Pseudomonas sp.、Pseudomonas aeruginosa、Achromobacter sp.、Stenotrophomonas sp.、Bacillus fusiformis、Bacillus cereus、Brevundimonas sp.、Ochrobactrum sp.、Cytophaga sp.、Benzo(a)pyrene-degrading bacter、Flavobacterium sp.的近缘种;26S rDNA比对结果表明3株真菌分别为Pseudallescheria boydii、Coprinus cinereus的近缘种。分离菌株中有4株细菌和3株真菌能在CMC平板上产生透明圈,但以糖化力法测定酶活结果只有3株真菌具有产酶能力。3株真菌的酶活动态测定结果,酶活的高峰均出现在7?14 d,并且呈现多峰变化;3株真菌的酶活种类表现为,滤纸酶活性、纤维素内切酶活性和外切酶活性均以菌株F1最高,分别达到了1.05、5.53和0.56 U/mL,β-葡萄糖甘酶活性和木聚糖酶活性以菌株FC最高,分别达到0.44和58.95 U/mL,其木聚糖酶活为F1最高值的6倍。     

纳豆激酶固态发酵的参数优化

方法:在对实验室分离的纳豆激酶产生菌进行菌株鉴定及其酶学性质研究的基础上,对影响菌株固态发酵产酶的工艺参数进行了优化研究.结果:发酵温度、发酵时间及培养基碳氮比、料水比、pH对纳豆激酶的产生都有较大影响,而接种量对纳豆激酶影响较小;在优化条件下,纳豆激酶固态发酵酶活可达到8 300U/g,为已知最高纳豆激酶单位酶活.     

纳豆激酶的原核表达、纯化及其特性分析（英文）

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。     

纳豆激酶的分离纯化研究

从实验室保存的一株高产纳豆激酶菌株出发,进行发酵产酶,确立具有纤溶活力的纳豆激酶的分离纯化工艺,主要通过硫酸铵分级盐析法和Phenyl Sepharose疏水柱层析进行分离纯化,用纤维蛋白平板法测定酶活力,用SDS-PAGE电泳验证为电泳纯,分子量为28kD。其有望开发为新型的口服溶栓药物。     

1株产几丁质酶海洋细菌Z-1的分离与鉴定

根据在胶体几丁质筛选平板上透明圈的大小,从大连海域海泥中筛选、分离出3株产几丁质酶的菌株,结合各菌株分泌的几丁质酶催化几丁质降解的产物及酶活特征,菌株Z-1为初筛3个菌株中较优良的产酶菌株。菌株Z-1产生的几丁质酶可将几丁质催化降解为4～5聚合度的几丁寡糖。电镜观察菌株Z-1为杆菌形态,提取菌株Z-1的基因组DNA,利用16S rDNA通用引物,扩增出菌株Z-1的16S rDNA序列,并与Gen-Bank其他已知菌株16S rDNA序列进行对比分析,建立了进化树,推断菌株Z-1为芽胞杆菌属,暂定名为Bacillus sp.Z-1。     

药食两用纳豆激酶的研究进展

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。     

海洋低温几丁质酶菌株筛选、鉴定及酶谱分析

以胶体几丁质为唯一碳源,从大连渤海湾的底泥样品中分离到1株高产低温几丁质酶的海洋细菌,命名为DL-06。由菌株的形态特征结合16S rDNA系统发育分析,初步确定该菌株属于交替假单胞菌(Pseudoalteromonas sp. DL-06)。该菌株经30 h摇瓶发酵后测定粗酶液几丁质酶酶活为9.184 U/mL,最适反应温度为15 ℃,60 ℃孵育1 h仍保持50%以上的酶活性,表明该低温酶具有一定热稳定性。经SDS-PAGE及酶谱分析,该菌株能够产生至少3种以上不同分子质量的几丁质酶组分。Pseudoalteromonas sp. DL-06产几丁质酶在低温下高活性与热稳定特点,使其具有潜在的工业应用价值。     

UV与DES复合诱变选育高产脂肪酶菌株

以产脂肪酶菌株BaciUus sp CS-4为出发菌株,进行了UV与硫酸二乙酯(DES)复合诱变处理.筛选出一株高酶活的目的菌株,命名为Bacillus spDE-8.其酶活为每毫升14.85U,比出发菌株提高48.2%.传代实验证明,其遗传性能稳定.     

产碱性木聚糖酶菌株的筛选及酶学性质

从青海盐碱湖土壤中筛选到25株产碱性木聚糖酶的菌株,其中编号为QH14的菌株产酶量达648.79U/mL,纯化后比活可达1148.56 U/mg。16 SrDNA鉴定表明菌株QH14属于短小芽孢杆菌,命名为Bacillus sp.QH14。从该菌株的基因组中克隆获得了碱性木聚糖酶编码基因XynQH14,并在大肠杆菌E.coliBL21(DE3)中获得重组表达。通过Ni-NTA亲和层析分离纯化后的重组QH14木聚糖酶比活达700.47 U/mg。该碱性木聚糖酶的酶促反应最适温度为60℃,最适pH为9.2;55℃处理1h仍保持50%的活力;在pH7.0~11条件下37℃处理酶液24 h后均保持80%以上的活力,且在pH11缓冲溶液中50℃处理24 h仍保持31.02%的酶活,显示了该碱性木聚糖酶较好的热稳定性和碱稳定,提示该碱性木聚糖酶在制浆造纸、纺织等行业的应用潜力。     

产碱性木聚糖酶菌株的筛选及酶学性质

从青海盐碱湖土壤中筛选到25株产碱性木聚糖酶的菌株, 其中编号为QH14的菌株产酶量达648.79 U/mL, 纯化后比活可达1148.56 U/mg。16 SrDNA鉴定表明菌株QH14属于短小芽孢杆菌, 命名为Bacillus sp. QH14。从该菌株的基因组中克隆获得了碱性木聚糖酶编码基因XynQH14, 并在大肠杆菌E.coliBL21(DE3)中获得重组表达。通过Ni-NTA亲和层析分离纯化后的重组QH14木聚糖酶比活达700.47 U/mg。该碱性木聚糖酶的酶促反应最适温度为60℃, 最适pH为9.2; 55℃处理1h仍保持50%的活力; 在pH7.0~11条件下37℃处理酶液24 h后均保持80%以上的活力, 且在pH11缓冲溶液中50℃处理24 h仍保持31.02%的酶活, 显示了该碱性木聚糖酶较好的热稳定性和碱稳定, 提示该碱性木聚糖酶在制浆造纸、纺织等行业的应用潜力。     

InhA-like protease secreted by Bacillus sp. S17110 inhabited in turban shell

A strain producing a potent protease was isolated from turban shell. The strain was identified as Bacillus sp. S17110 based on phylogenetic analysis. The enzyme was purified from culture supernatant of Bacillus sp. S17110 to homogeneity by ammonium sulfate precipitation, SP-Sepharose, and DEAE-Sepharose anion exchange chromatography. Protease activity of the purified protein against casein was found to be stable at pH 7 to pH 10 and around 50 degrees . Approximately 70% of proteolytic activity of the enzyme was detected either in the presence of 100 mM SDS or Tween 20. The enzyme activity was enhanced in the presence of Ca2+, Zn2+, Mg2+, but was inhibited by EDTA, indicating that it requires metal for its activity. The purified enzyme was found to be a monomeric protein with a molecular mass of 75 kDa, as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography. The purified enzyme was analyzed through peptide fingerprint mass spectra generated from matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) and a BLAST search, and identified as immune inhibitor A (inhA) deduced from nucleotide sequence of B. cereus G9241. Since InhA was identified as protease that cleave antibacterial proteins found in insect, inhA-like protease purified from Bacillus sp. S17110 might be pathogenic to sea invertebrates.     

一株纤维蛋白溶解酶产生菌的鉴定及其产酶条件初步研究

从亚洲传统发酵食品--虾酱中筛选到一株产纤维蛋白溶解酶能力较强的菌株,通过形态和常规生理生化性质鉴定,发现该菌株与芽孢杆菌属细菌的特征很相近,结合16S rDNA序列分析,构建系统发育树,确定其分类地位,由中国典型培养物保藏中心定名为Bacillus sp.nov.SK006(CCTCC No.M 205071),并优化了发酵培养基组成及培养条件,本研究为该菌株的深入研究和广泛应用提供了理论依据.     

一株产纤溶酶菌株的分离鉴定及其纤溶组分分析

【目的】筛选性能良好的产纤溶酶菌株,对菌株进行多项分类鉴定,分析其纤溶酶系的组成特征及纤溶能力。【方法】通过酪蛋白培养基初筛,琼脂-纤维蛋白双层平板复筛,从海泥、土壤等环境中筛选纤维蛋白降解菌,以尿激酶为标准测定纤溶酶活性。通过形态学、生理生化特征研究,结合16S rDNA基因序列分析菌株种类及系统分类地位。通过SDS-PAGE和纤维蛋白酶谱法分析胞外纤溶酶系的组成特征。【结果】筛选到一株能降解纤维蛋白的细菌CNY16,鉴定其为沙福芽孢杆菌(Bacillus safensis)。该酶为胞外酶,SDS-PAGE和纤维蛋白酶谱结果表明该纤溶酶系有至少两种分子量大小不同的纤溶酶,分别约33 kD和23 kD。能有效溶解血块中纤维蛋白,并且对红细胞无降解作用。【结论】细菌CNY16是一株新的纤溶酶产生菌,纤溶酶活性及稳定性较好,具有潜在开发价值。为获取新型纤溶酶提供了一种新的菌源。     

一株东祁连山耐低温纤维素降解菌株的分离、鉴定及产酶特性

从东祁连山高寒草甸土壤中分离得到一株纤维素酶高产菌株【3-2。根据菌体形态观察、革兰氏染色反应及16S rDNA序列测定以及序列同源性比较,确定鉴定该菌为芽孢杆菌属的成员(Bacillus sp.)。对该菌的产纤维素酶条件研究结果表明:该菌在5℃~45℃、初始pH 4.0~9.0的环境下均能产纤维素酶。在初始pH为8.0、盐浓度为2.0%~3.0%之间、培养温度为20℃培养条件下最适产酶。在5℃时,相对酶活仍能保持60%。     

Purification of a bacteriolytic enzyme from Bacillus sp. active against Streptococcus mutans

Bacillus sp. strain YU-1006, which was isolated from soil, produced a bacteriolytic enzyme which was active against Streptococcus mutans KCTC 3283. The enzyme was purified 60 fold with a 6% yield by CM-cellulose column chromatography and CM-Sephadex G-50 column chromatography. Lytic activity was stable in the range of pH 6.0~9.0 up to 42°e active enzyme is a 24,000 dalton monomer as estimated by SDS-PAGE.     

片烟产果胶酶细菌的鉴定及酶活测定

以果胶为碳源, 对津巴布韦片烟烟叶表面产果胶酶细菌进行分离, 采用16S rDNA限制性酶切片段长度多态性分析(ARDRA)和测序方法, 结合形态学、生理生化实验, 对所分离产果胶酶菌株进行鉴定, 同时研究培养时间、温度、起始pH、接种量对菌株产酶的影响。结果表明, 从津巴布韦片烟烟叶表面分离得到的产果胶酶菌株主要为芽孢杆菌属的枯草芽孢杆菌Bacillus subtilis和产碱菌属的粪产碱菌Alcaligenes faecalis。在所分离的菌株中, 枯草芽孢杆菌T10酶活力最高, 以6%的接种量, 在温度为35 °C、起始pH为7.5条件下培养48?56 h, 其果胶酶酶活为571 U/mg, 聚半乳糖醛酸裂解酶酶活为297 U/mg。     

Purification and characterization of β-Mannanase from Reinekea sp. KIT-YO10 with transglycosylation activity

Marine bacterium Reinekea sp. KIT-YO10 was isolated from the seashore of Kanazawa Port in Japan as a seaweed-degrading bacterium. Homology between KIT-YO10 16S rDNA and the 16S rDNA of Reinekea blandensis and Reinekea marinisedimentorum was 96.4 and 95.4%, respectively. Endo-1,4-β-D-mannanase (β-mannanase, EC 3.2.1.78) from Reinekea sp. KIT-YO10 was purified 29.4-fold to a 21% yield using anion exchange chromatography. The purified enzyme had a molecular mass of 44.3?kDa, as estimated by SDS-PAGE. Furthermore, the purified enzyme displayed high specificity for konjac glucomannan, with no secondary agarase and arginase activity detected. Hydrolysis of konjac glucomannan and locust bean gum yielded oligosaccharides, compatible with an endo mode of substrate depolymerization. The purified enzyme possessed transglycosylation activity when mannooligosaccharides (mannotriose or mannotetraose) were used as substrates. Optimal pH and temperature were determined to be 8.0 and 70?°C, respectively. It showed thermostability at temperatures from 20 to 50?°C and alkaline stability up to pH 10.0. The current enzyme was thermostable and thermophile compared to the β-mannanase of other marine bacteria.     

Molecular and biochemical characterization of a new endoinulinase producing bacterial strain of Bacillus safensis AS-08

Microbial inulinases are an important class of industrial enzymes, which are used for the production of fructooligosaccharides and high-fructose syrup. Endoinulinase producing bacterial strains were isolated from soil samples taken from the vicinity of Asparagus sp. root tubers. All the bacterial strains were screened for inulinase activity. The primary screening was carried out based on hydrolytic zone on agar plates containing inulin-based medium and Lugol’s iodine solution. Thus 30 inulinase producing bacterial strains were isolated. Out of 30 strains, 5 bacterial strains were found endoinulolytic, whereas 25 were exoinulolytic on the basis of action pattern of the enzyme. In tertiary screening, the bacterial isolate AS-08 was found to be most efficient for inulinase activity. Morphological, biochemical and physiological characteristics of the bacterial isolate AS-08 confirmed it as Bacillus sp. Furthermore, species-specific identification by 16S rDNA sequencing and phylogenetic analysis revealed the isolate as Bacillus safensis. Bacillus pumilus SH-B30 was found to be the nearest homolog. The strain showed maximum inulinase activity (12.56 U/mL) after 20 h of incubation at 37°C.     

1株血红蛋白分解菌的分离鉴定及其蛋白酶活力测定

为了获得能够有效降解利用屠宰场废弃血液的功能菌株,以日喀则地区屠宰场废弃血液堆积土壤样品为材料,将样品稀释涂布接种在血平板上进行分离,挑取水解圈最大的菌落进行平板划线纯化。对分离菌株进行形态学、生化反应试验、16S rDNA序列鉴定并测定其蛋白酶活性。筛选出1株能够高效降解血红蛋白的菌株命名为NwMCC01910042,该分离菌株为革兰阳性杆菌,V-P(Voges-Proskauer)试验阳性,枸橼酸盐利用、淀粉水解、明胶液化、16S rDNA序列系统进化分析显示NwMCC01910042菌株与Bacillus licheniformis ATCC 14580株的序列相似性为99.79%,与Bacillus licheniformis MSL3076株的序列相似性为99.30%,为地衣芽胞杆菌(Bacillus licheniformis),该菌株的16S rDNA序列已提交至GenBank,准入编号为MN 176417,其蛋白酶活力为188.63 U/mL。利用微生物降解生产氨基酸有机肥的关键是筛选蛋白酶的高产菌株,NwMCC01910042株菌有望作为将废弃血污降解为氨基酸的候选功能菌株。     

Isolation of a psychrotrophic Azospirillum sp. and characterization of its extracellular protease

A novel psychrotrophic bacterium secreting a protease was isolated from a mountain soil in Korea. On the basis of a 16S rDNA sequence analysis and physiological properties, the isolate was identified as an Azospirillum sp. The protease purified from the culture supernatant was a monomer in its native form with an apparent molecular mass of 48.6 kDa on SDS-PAGE. The protease was active in a broad pH range around 8.5 and at temperatures up to 40 degrees C and stable at temperatures below 30 degrees C for 3 days. The proteolytic activity was inhibited by iodoacetamide and EDTA. The Mg2+ ion did not activate the enzyme much but reversed the inhibition by EDTA, suggesting that the protease belongs to a cysteine protease stabilized by the Mg2+ ion.