Isolation and characterization of Aeromonas sp. B-5 capable of decolorizing various dyes

Aeromonas sp. B-5, which has the ability to decolorize azo dyes, was isolated from soil. Aeromonas sp. B-5 completely decolorized 100 mg/l of Bordeaux S by reductive cleavage of azo bonds under static conditions in 24 h. Though the decolorization of Bordeaux S by Aeromonas sp. B-5 was suppressed under shaking, rapid decolorization was observed when the culture was changed to static conditions after cultivation with shaking. The indigoid dye, Acid blue 74, was decolorized by Aeromonas sp. B-5 under shaking conditions, in contrast to the decolorization of azo dyes.     

一株产低温纤维素酶放线菌的分离与产酶研究

用羧甲基纤维素钠-刚果红培养基从内蒙大青沟采集的森林土壤中分离了一株产纤维素酶的耐低温菌,命名为ND2-1,其形态和16S r DNA序列分析结果表明ND2-1为链霉菌属（Streptomyces sp.）的放线菌。ND2-1能在10³5℃范围内生长,最适生长温度为25℃,属耐低温菌。滤纸崩解实验表明,ND2-1能高效降解纤维素。ND2-1在25℃条件下,在含有2 g/L羧甲基纤维素钠（CMC-Na）的培养基中培养3 d后酶活性最高。对其酶学性质的初步研究结果表明:该菌株所产纤维素酶在p H3.0⁹.0范围内稳定,且酶活性都较高,最适反应p H为7.0;最适反应温度为30℃,K+和Cu2+对酶活有抑制作用,Mn2+能提高相对酶活性至166.7%。      

L-谷氨酸氧化酶的分离纯化及酶学性质的研究

采用硫酸铵分级沉淀、HPLC脱盐、离子交换、Superdex G-200凝胶层析等步骤从华美链霉菌(Streptomyces sp.)发酵液中分离纯化L-谷氨酸氧化酶(GLOD),经过SDS-PAGE电泳检测,样品达到电泳级纯,分子量为140ku。对纯化的GLOD研究发现,该酶的最适反应温度为50℃,最适pH值为7.0,温度稳定范围为0℃～50℃,pH稳定范围在6.0～9.0,GLOD催化L-谷氨酸的米氏常数Km为2.1×10-4mol/L,Na+、K+、Mg2+对酶活几乎没有影响,而Hg2+、Cu2+、Ag+均不同程度的抑制酶的活性。     

链霉菌Streptomyces sp. FJS31-2次级代谢产物UK-78623的分离鉴定及抑菌活性研究

该研究对卤化II型聚酮类抗生素zunyimycins产生菌链霉菌（Streptomyces sp.）FJS31-2的次级代谢产物进行挖掘。次级代谢产物经乙酸乙酯萃取浓缩后,依次采用硅胶柱层析法、薄层层析（TLC）法、高效液相色谱（HPLC）法等方法进行分离和纯化,通过质谱（MS）法和核磁共振（NMR）等分析单体化合物的结构,并采用滤纸片法对其抗菌活性进行测试。结果表明,初次从该菌发酵物中分离得到多环内酯类化合物UK-78623,其对小肠结肠炎耶尔森氏菌（Yersinia enterocolitica）具有明显抑制作用,其抑菌圈直径为25.2 mm。     

Isolation, characterization, and molecular cloning of a thermostable xylitol oxidase from Streptomyces sp. IKD472

A thermophilic bacterium, Streptomyces sp. IKD472, that can oxidize xylitol was isolated from a hot spring and was found to produce xylitol oxidase. The purified enzyme was a monomeric protein with an apparent molecular weight of 43 k as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration. This novel enzyme is capable of catalyzing the oxidation of one mole of xylitol to form one mole each of xylose and hydrogen peroxide. Since the V(max)K(m) value for xylitol was two and four times higher than those for galactitol and n-sorbitol, respectively, the enzyme was designated as xylitol oxidase. The enzyme was stable in the pH range from 5.5 to 10.5 and at temperatures up to 65 degrees C. The optimal temperature and pH were 55 degrees C and pH 7.5, respectively. Xylitol oxidase bound one mole of FAD as a coenzyme per mole of protein. The amino acid sequence of the NH2 terminus and the fragments obtained by lysylendpeptidase digestion of xylitol oxidase were determined for preparation of synthetic oligonucleotides as hybridization probes. A 2.8-kb chromosomal fragment hybridizing to the probes was cloned into pUC18 in Escherichia coli. The gene consists of an open reading frame of 1245 by that encodes a protein containing 415 amino acids with a molecular weight of 44,730 but without the conserved nucleotide-binding sequence, Gly-X-Gly-X-X-Gly. The amino acid sequence has 70% identity to putative oxidoreductase from Streptomyces coelicolar, 51% to sorbitol oxidase from Streptomyces sp., and 26% to L-gulonolactone oxidase from rat in terms of the overall amino acid sequence. DNA manipulation of the cloned gene in E. coli, by alteration of a strong promoter and a synthesized ribosome-binding sequence at an appropriate position, resulted in overproduction of xylitol oxidase 100 times more than that produced in the original Streptomyces sp. IKD472. The enzyme properties of recombinant xylitol oxidase were the same as those of the authentic enzyme. Stable xylitol oxidases, which allow easier quantitative analysis of xylitol, are useful for clinical applications.     

Design of PCR primers and gene probes for the general detection of bacterial populations capable of degrading aromatic compounds via catechol cleavage pathways

For the general detection of bacterial populations capable of degrading aromatic compounds, two PCR primer sets were designed which can, respectively, amplify specific fragments from a wide variety of catechol 1,2-dioxygenase (C12O) and catechol 2,3-dioxygenase (C23O) genes. The C12O-targeting primer set (C12O primers) was designed based on the homologous regions of 11 C12O genes listed in the GenBank, while the C23O-targeting one (C23O primers) was designed based on those of 17 known C23O genes. Oligonucleotide probes (C12Op and C23Op) were also designed from the internal homologous regions to identify the amplified fragments. The specificity of the primer sets and probes was confirmed using authentic bacterial strains known to carry the C12O and/or C23O genes used for the primer and probe design. Various authentic bacterial strains carrying neither C12O nor C23O genes were used as negative controls. PCR with the C12O primers amplified DNA fragments of the expected sizes from 5 of the 6 known C12O-carrying bacterial strains tested, and positive signals were obtained from 4 of the 5 amplified fragments on Southern hybridization with the C12Op. The C23O primers amplified DNA fragments of the expected size from all the 11 tested C23O-carrying bacterial strains used for their design, while the C23Op detected positive signals in the amplified fragments from 9 strains. On the other hand, no DNA fragments were amplified from the negative controls. To evaluate the applicability of the designed primers and probes for the general detection of aromatic compound-degrading bacteria, they were applied to wild-type phenol- and/or benzoate-degrading bacteria newly isolated from a variety of environments. The C12O and/or C23O primers amplified DNA fragments of the expected sizes from 69 of the 106 wild-type strains tested, while the C12Op and/or C23Op detected positive signals in the amplified fragments from 63 strains. These results suggest that our primer and probe systems can detect a considerable proportion of bacteria which can degrade aromatic compounds via catechol cleavage pathways.     

氰戊菊酯降解菌的筛选与鉴定及其降解条件优化

采用富集培养法从喷施拟除虫菊酯类农药的菜园土壤中,分离得到一株能降解氰戊菊酯的细菌BFE-023。经生理生化和16S r DNA序列分析,将菌株BFE-023鉴定为地衣芽孢杆菌（Bacillus licheniformis）。应用Plackett-Burman实验设计确定了影响该菌株降解氰戊菊酯的主要影响因素,利用响应面分析法优化了其降解条件。在优化条件下,研究菌株BFE-023对氰戊菊酯的降解过程及其中间产物3-苯氧基苯甲酸（3-PBA）的生成规律。结果表明,培养时间和降解体系中氰戊菊酯浓度及氯化铁含量是影响其降解的主要因素,优化条件下60 h内对氰戊菊酯降解率可达到88.71%,与所建立的模型预测值（88.78%）相吻合。菌株BEF-023降解氰戊菊酯的过程中,降解中间产物3-PBA的生成量呈现先明显增加后逐渐减少的趋势,说明菌株BEF-023可能具备继续降解中间产物3-PBA的能力。      

氰戊菊酯降解菌的筛选与鉴定及其降解条件优化

采用富集培养法从喷施拟除虫菊酯类农药的菜园土壤中,分离得到一株能降解氰戊菊酯的细菌BFE-023。经生理生化和16S r DNA序列分析,将菌株BFE-023鉴定为地衣芽孢杆菌(Bacillus licheniformis)。应用Plackett-Burman实验设计确定了影响该菌株降解氰戊菊酯的主要影响因素,利用响应面分析法优化了其降解条件。在优化条件下,研究菌株BFE-023对氰戊菊酯的降解过程及其中间产物3-苯氧基苯甲酸(3-PBA)的生成规律。结果表明,培养时间和降解体系中氰戊菊酯浓度及氯化铁含量是影响其降解的主要因素,优化条件下60 h内对氰戊菊酯降解率可达到88.71%,与所建立的模型预测值(88.78%)相吻合。菌株BEF-023降解氰戊菊酯的过程中,降解中间产物3-PBA的生成量呈现先明显增加后逐渐减少的趋势,说明菌株BEF-023可能具备继续降解中间产物3-PBA的能力。     

降烟碱细菌-烟草节杆菌K9的分离及鉴定

采用烟碱为唯一碳源的选择性培养基,对烟草、植烟土壤及烟草污水中的细菌进行选择性分离,共分离得到97株能够分解利用烟碱的细菌,其中菌株K9分解能力最强。经生理生化分析以及16SrDNA序列同源性分析,鉴定为烟草节杆菌(Arthrobacternicotianae)。为微生物降烟碱的研究及应用提供了理论依据。     

链霉菌 CSDX076次级代谢产物Pseurotin A的分离及结构鉴定

对从赤水丹霞山土壤样品中分离得到的链霉菌(Streptomyces sp.)CSDX076进行液体发酵。利用乙酸乙酯萃取,硅胶柱层析、薄层层析和反相中压柱层析对发酵液中的次级代谢产物进行分离、纯化;通过核磁共振法对纯化的分离产物进行结构鉴定。结果表明,该研究从链霉菌属中成功分离得到次级代谢产物Pseurotin A。采用滤纸片法对Pseurotin A进行抗菌活性测试,Pseurotin A对金黄色葡萄球菌具有抑制作用,其抑菌圈直径为5.0 mm。     

玉米赤霉烯酮的分离鉴定及其降解特性研究

采用富集培养的方法从麦粒中筛选到一株玉米赤霉烯酮(ZEN)降解菌7D3-2,该菌株对ZEN的降解能力高达95%;通过对7D3-2形态、生理生化特性及16S r DNA、gyr A基因系统进化发育地位分析,该菌株被鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。研究了初始p H、培养温度、接种量及ZEN初始质量浓度等环境条件对7D3-2降解ZEN的影响,结果表明:7D3-2降解ZEN的最适温度为30℃,最适p H为7.0;当接种量小于10%时,7D3-2对ZEN的降解率与接种量成正比;ZEN质量浓度在1.0~25.0 mg/L范围内,7D3-2对ZEN的降解差异不显著,当ZEN质量浓度大于25.0 mg/L时,ZEN降解率与ZEN初始质量浓度成反比,但是ZEN被降解的绝对量与其ZEN初始质量浓度呈正相关;降解物质的定域实验结果显示:7D3-2中降解ZEN的物质主要位于细胞外。7D3-2不仅对培养液中的ZEN具有降解效果,也能很好地降解玉米粉中的ZEN。本研究结果不仅可以丰富ZEN降解菌种质资源库,在粮食和饲料的ZEN生物脱毒方面还具有广阔的应用前景。     

腌渍食品亚硝酸盐降解菌的分离、鉴定及其降解特性

从腌渍豆酱、香肠、腌渍萝卜中分离到5株乳酸菌,根据形态和生理生化特征等对分离到的菌株进行初步鉴定,并研究了分离菌株的生理学性质和降解特性.初步鉴定表明,L1,L3,L4和L5菌株为明串珠菌,L2菌株为链球菌.L2菌株最适生长温度为30 ℃,L1,L3,L4和L5菌株最适生长温度为25 ℃,5个菌株的最适生长pH值均为6.5,在4%～6% NaCl溶液中均能正常生长繁殖.L2菌株在30 ℃时对亚硝酸钠的降解率最高,其他菌株在25 ℃时最高;5个菌株均在pH=3.5时对亚硝酸钠的降解率最高.在适宜pH值和培养温度下,L3和L5菌株对亚硝酸钠的降解率超过93%,具有较大的应用价值.     

Isolation and characterization of long-chain-alkane degrading Bacillus thermoleovorans from deep subterranean petroleum reservoirs

Two extremely thermophilic alkane-degrading bacterial strains, B23 and H41, were respectively isolated from deep subterranean petroleum reservoirs in the Minami-aga (Niigata) and Yabase (Akita) oil fields. Both strains were able to grow at temperatures ranging from 50 to 80 degrees C, with optimal growth at 70 degrees C for B23 and 65 degrees C for H41. From 16S rRNA gene sequence analysis and physiological characterization, both strains were identified as Bacillus thermoleovorans (identities of 99.5% and 99.6% to strain DSM 5366, and 98.3% and 98.7% to the type strain LEH-1(TS), respectively). Strains B23 and H41 effectively (60%) degraded n-alkanes longer than C12 and C15, respectively, at 70 degrees C, while strain LEH-1(TS) degraded undecane (C11) most effectively. When B23 and H41 were cultivated in the presence of heptadecane, heptadecanoate and pentadecanoate were specifically accumulated in the cells. These results strongly suggest that the two strains degraded n-alkanes by a terminal oxidation pathway, followed by a beta-oxidation pathway.     

Isolation of a thermophilic poly-L-lactide degrading bacterium from compost and its enzymatic characterization

Thermophilic poly-L-lactide-degrading bacteria were isolated from a garbage fermentor. One of the isolates, strain PL21, was identified as Bacillus smithii based on its physiological properties, sugar assimilation pattern, and partial 16S rDNA sequence. The degradation activity of poly-L-lactide exibited by the culture fluid was parallel to the esterase activity, and the purified enzyme was active against various fatty acid esters and poly-L-lactide, at 60 degrees C and pH 5.     

产琼胶酶菌株Agarivorans sp.FG15的筛选鉴定与酶学性质

从中国南海热带海洋环境中分离纯化出产琼胶酶菌株FG15,并以16S rDNA进行分子鉴定,分析其酶学性质。FG15为革兰氏阴性球菌,对硫酸链霉素,氨苄青霉素,羧苄青霉素和氯霉素都不敏感。通过16S rDNA序列分析和BLAST同源性比对发现:FG15菌株的16S rDNA序列与船蛆杆菌(Teredinibacter turnerae),噬琼胶菌属(Agarivorans sp.)对应序列同源性最高,为95%。利用MEGA 5.0软件构建系统发育树,FG15与噬琼胶菌属(Agarivorans sp.)亲缘关系最近,同源性高达99%。因此,可以初步鉴定FG15为噬琼胶菌属(Agarivorans sp.)。酶学性质的研究表明:FG15所产琼胶酶的最适温度为36℃,在20~45℃之间热稳定性较好;最适pH为7.5,在pH 7.0~8.0之间酸碱稳定性较好;产酶主要为胞外酶;琼脂酶的动力学参数米氏常数Km为4.978mg/mL,最大反应速率Vmax为10.33μmol/(L·min)。     

链霉菌Streptomyces sp.FJS31-2产卤化二型聚酮类化合物的发酵条件优化

以产zunyimycin A及BE-24566B类卤化天然化合物的Streptomyces sp. FJS31-2为研究对象,分别利用高分辨质谱（HRMS）和高效液相色谱（HPLC）为检测手段,对不同的碳源、氮源组合的培养基在不同的培养时间和萃取条件下获得的目标化合物进行定性和产量的分析。在基础培养基内添加10 g/L无水乙醇浸泡24 h后的腐殖酸,培养17 d,采取乙酸乙酯萃取可以使目标化合物产量提高。     

Isolation and characterization of a soluble and thermostable phosphite dehydrogenase from Ralstonia sp. strain 4506

Phosphite dehydrogenase (PtxD), which catalyzes the nearly irreversible oxidation of phosphite to phosphate with the concomitant reduction of NAD(+) to NADH, has great potential for NADH regeneration in industrial biocatalysts. Here, we isolated a soil bacterium, Ralstonia sp. strain 4506, that grew at 45°C on a minimal medium containing phosphite as the sole source of phosphorus. A recombinant PtxD of Ralstonia sp. (PtxD(R4506)) appeared in the soluble fraction in Escherichia coli. The purified PtxD(R4506) showed 6.7-fold greater catalytic efficiency (V(max)/K(m)) than the first characterized PtxD of Pseudomonas stutzeri (PtxD(PS)). Moreover, the purified PtxD(R4506) showed maximum activity at 50°C, and its half-life of thermal inactivation at 45°C was 80.5h, which is approximately 3,450-fold greater than that of PtxD(PS). Therefore, we concluded that PtxD(R4506), which shows high catalytic efficiency, solubility, and thermostability, would be useful for NADH regeneration applications.     

Isolation and characterization of alpha 2-macroglobulin from mastitis milk.

Whey samples were screened for the presence of the proteinase inhibitor alpha 2-macroglobulin (alpha 2M). From an enzymic test, alpha 2M levels in normal whey varied in the range 0.49-0.84% of the serum level, whereas in mastitis whey the activity was markedly increased, reaching values between 0.91 and 138.5% (median 7.2%) of standard serum level. In mastitis milk samples but not in normal milk alpha 2M was also detected by double immunodiffusion and Western blotting. The proteinase inhibitor was purified from a mastitis milk sample with high alpha 2M activity (138.5% of serum level). In SDS-PAGE, native-PAGE and in double immunodiffusion analysis the inhibitor appeared indistinguishable from plasma-derived alpha 2M. The alpha 2M preparation from mastitis whey migrated essentially as native alpha 2M, representing the 'slow' form of the molecule. Treatment with trypsin transformed the alpha 2M preparation to the electrophoretic 'fast' form, but treatment with methylamine had only a minor effect. The receptor recognition sites were not exposed on the isolated alpha 2M molecule but could be readily exposed by treatment of the proteinase inhibitor with trypsin, which further proved that the isolated alpha 2M was in the entire native, functionally active state.     

海洋链霉菌GB-2抗细菌物质的溶解性质和分离纯化

本实验主要对海洋链霉菌株GB-2产生的抗菌物质的溶解性进行了研究,并将其分离纯化.通过对GB-2的发酵液中抗菌物质的溶解性测定,可推知是一种极性较大的水溶性物质.超滤实验发现,抗细菌组分能通过截留分子量(COMW)≤1kD的超滤膜.Sephadcx LH-20色谱柱分离后,经抗菌活性检测发现两个活性峰,其中第1个峰对蜡样芽孢杆菌和大肠杆菌的抑菌活性最强.将第1个峰的收集液进一步用高效液相色谱(HPLC)纯化,在图谱上出现两个峰,其中第2个峰具有抑菌活性,表明该物质得到了纯化.