Properties of Crystalline ω-Amino Acid : Pyruvate Aminotransferase of Pseudomonas sp. F–126

ω-Amino acid: pyruvate aminotransferase, purified to homogeneity and crystallized from a Pseudomonas sp. F–126, has a molecular weight of 172,000 or 167,000±3000 as determined by the gel-filtration or sedimentation equilibrium method, respectively. The enzyme catalyzes the transamination between various ω-amino acids or amines and pyruvate which is the exclusive amino acceptor. α-Amino acids except l-α-alanine are inert as amino donor. The Michaelis constants are 3.3 mm for β-alanine, 19 mm for 2-aminoethane sulfonate and 3.3 mm for pyruvate. The enzyme has a maximum activity in the pH range of 8.5~10.5. The enzyme is stable at pH 8.0~10.0 and at up to 65°C at pH 8.0. Carbonyl reagents strongly inhibit the enzyme activity. Pyridoxal 5′-phosphate and pyridoxamine 5′-phosphate reactivate the enzyme inactivated by carbonyl reagents. The inhibition constants were determined to be 0.73 mm for d-penicillamine and 0.58 mm for d-cycloserine. Thiol reagents, chelating agents and l-α-amino acids showed no effect on the enzyme activity.     

Monoamine Transamination Catalyzed by ω-Amino Acid; Pyruvate Aminotransferase of Pseudomonas sp. F-126

ω-Amino acid: pyruvate aminotransferase of Pseudomonas sp. F-126 catalyzes stoichiometricalJy a transamination between various amines and pyruvate. Most of alkyl and aromatic monoamines served as an amino donor. The enzyme activity was affected by carbon number of straight-chain alkylmonoamines with a maximum activity at 5-carbon unit, n-amylamine. Michaelis constants for n-butylamine and pyruvate were calculated to be 66.6 mm and 5.5 mm respectively. The enzyme was active in the alkaline range with a maximum at pH 10.5 ~ 11.0, though not any activity was observed at the pH below 8.0. The optimum temperature for the reaction was at 60°C.     

Diamine Transamination Catalyzed by ω-Amino Acid : Pyruvate Aminotransferase of Pseudomonas sp. F–126

ω-Amino acid: pyruvate aminotransferase of Pseudomonas sp. F–126 catalyzes a transamination between various diamines and pyruvate, an exclusive amino acceptor. Based on a stoichiometric studies it was shown that one of the two amino groups of 1,2-diaminoethane, putrescine and cadaverine transaminated to pyruvate. The transamination between putrescine and pyruvate seemed to proceed by a ping-pong bi bi mechanism. Michaelis constants for putrescine and pyruvate were calculated to be 76.9 and 6.25 mm, respectively.     

三株Pseudomonas sp.的吡啶降解多样性分析

目的:为了探讨3株Pseudomonas sp.对吡啶降解存在多样性.方法:基于16S rRNA和ISR序列分析,对3株分离菌株进行初步鉴定,进而通过Touch -Down PCR,对3株细菌降解吡啶的多样性进行分析.结果:3株细菌XJUHX -1、XJUHX - 12和XJUHX - 16初步鉴定为Pseudomonas,3株实验菌株的部分降解基因的扩增条带有差异.结论:同属的3株 Pseudomonas在吡啶降解上存在多样性.     

Structural studies of β-hairpin peptidomimetic antibiotics that target LptD in Pseudomonas sp.

We report structural studies in aqueous solution on backbone cyclic peptides that possess potent antimicrobial activity specifically against Pseudomonas sp. The peptides target the β-barrel outer membrane protein LptD, which plays an essential role in lipopolysaccharide transport to the outer membrane. The peptide L27-11 contains a 12-residue loop (T¹W²L³K⁴K⁵R⁶R⁷W⁸K⁹K¹⁰A¹¹K¹²) linked to a DPro–LPro template. Two related peptides were also studied, one with various Lys to ornithine or diaminobutyric acid substitutions as well as a DLys⁶ (called LB-01), and another containing the same loop sequence, but linked to an LPro–DPro template (called LB-02). NMR studies and MD simulations show that L27-11 and LB-01 adopt β-hairpin structures in solution. In contrast, LB-02 is more flexible and importantly, adopts a wide variety of different backbone conformations, but not β-hairpin conformations. L27-11 and LB-01 show antimicrobial activity in the nanomolar range against Pseudomonas aeruginosa, whereas LB-02 is essentially inactive. Thus the β-hairpin structure of the peptide is important for antimicrobial activity. An alanine scan of L27-11 revealed that tryptophan side chains (W²/W⁸) displayed on opposite faces of the β-hairpin represent key groups contributing to antimicrobial activity.     

Purification and Some Properties of Two Cα-dehydrogenases Oxidizing Dimeric Lignin Model Compounds from Pseudomonas sp. TMY1009

Two NAD-dependent dehydrogenases which oxidize secondary alcoholic groups at the C_α position of dimeric lignin model compounds were purified from Pseudomonas sp. TMY1009. These enzymes have been designated C_α-dehydrogenase I and II (DH-I and DH-II). DH-II was purified to electrophoretic homogeneity. The molecular weight of DH-II, which is composed of four identical subunits, is 125,000. DH-I was partially purified and the molecular weight of DH-I is 94,000. Both DH-I and DH-II are active for three kinds of dimeric lignin model compounds related to major lignin substructures, although their specificities and affinities are different.     

The Mode of Action of Endo α-1,4 Polygalactosaminidase from Pseudomonas sp. 881 on Galactosaminooligosaccharides

The mode of action of the endo (α-1,4 polygalactosaminidase from Pseudomonas sp. 881 on galactosaminooligosaccharides (GOSs) was studied. The enzyme could hydrolyze (α-1,4 polygalactosamine to GOSs by the endo-split manner. Tetraose and longer GOSs were hydrolyzed to galactosaminobiose and galactosaminotriose as the final products. Galactosaminomonomer (galactosamine) could not be produced as an enzymatic product. From the dependency of kinetic parameters on the chain lengths of the substrates, it was suggested that the enzyme has 8 subsites. A catalytic site of the enzyme is located between the third and the fourth sites from the non-reducing end, since the main product from GOSs was galactosaminotriose, and galactosaminotetraitol remained in the hydrolyzate of galactosaminoheptaitol digestion. The enzyme showed transglycosylating activity on GOS4.     

Purification and Some Properties of the Endo α-1,4 Polygalactosaminidase from Pseudomonas sp.

The endo α-1,4 polygalactosaminidase from Pseudomonas sp. 881 was purified from the culture nitrate by ethanol precipitation and sequential column chromatographies on CM-Sephadex C-25, Sephadex G-50 and Phenyl-Sepharose CL-4B. The purified enzyme was electrophoretically homogeneous and its molecular weight and isoelectric point were 31,000 and 6.7, respectively. The optimum pH and temperature for hydrolysis of polygalactosamine were 5.0 and 55°C, respectively. The enzyme was stable up to 45°C for 15min and from pH 4.0 to 7.6 at 37°C for 1 hr.

The Km value was 0.05% α-1,4 polygalactosamine and the V was 0.154μmol reducing sugar (galactosamine)/min/μg protein. This polygalactosaminidase was inhibited by Sn²⁺ , Fe²⁺ , Fe³⁺ , Hg²⁺, Cu²⁺ ions and SDS. The enzyme did not hydrolyze oligo galactosamines (n < tetramer) or N-acetyl-polygalactosamines. It acted only on oligo galactosamine (n > trimer) and polygalactosamine endogeneously so far tested.     

Structure of the Pyoverdin PVD 2908 – a new Pyoverdin from Pseudomonas sp. 2908

An unknown siderophore (pyoverdin) was isolated from the strain Pseudomonas sp. 2908. The structure of the pyoverdin – called PVD 2908 – was elucidated by spectroscopic methods and degradation studies. Some other siderophores were identified by LC/ESI-MS-screening based on the knowledge of PVD 2908.     

Pseudomonas sp.引起欧洲鳗Li烂鳃病的初步研究

欧洲鳗鲡［Ａｎｇｕｉｌｌａ 　ａｎｇｕｉｌｌａ（Ｌ．）］养殖过程中,细菌性疾病危害严重,其中烂鳃病周年发生且发病率高。欧洲鳗烂鳃病常与寄生虫病、真菌病并发,因而给治疗带来了困难。鳗鲡烂鳃病病原主要有柱状屈桡杆菌、噬纤维菌等［１－３］。本文报告由 Ｐｓｅｕｄｏｍｏｎａｓ　ｓｐ．引起的欧洲鳗鲡烂鳃病。１材料与方法１．１病原菌分离取无寄生虫与真菌感染,鳃丝溃烂的濒死病鳗,用无菌生理盐水冲洗体表三遍,解剖取出鳃,经无菌生理盐水充分冲洗后,捣烂鳃丝,接种环取样于普通平板培养基划线,３０℃培养２４ｈ后,挑取形…     

深海细菌Pseudomonas sp.IOFA1醇脱氢酶酶学性质研究

[目的]对来自印度洋深海的一株假单胞菌醇脱氢酶(Adh)进行序列分析和酶活性质分析。[方法]首先以同源比对和进化树聚类为手段分析该酶序列信息。其次,在E.coli宿主中进行重组表达和镍柱亲和纯化后,对重组醇脱氢酶的酶活性质进行进一步地研究。[结果]结果显示Adh蛋白与其它物种已知醇脱氢酶的氨基酸序列最高相似性为81%,分属于第三类醇脱氢酶蛋白。酶学性质分析表明,重组酶Adh的最适作用温度为42℃,表现出良好的中低温适应性;最适p H值为5.0,在p H 4~6时具有较高的活性,表明Adh为酸性醇脱氢酶。Zn~(~(2+))、Na+在终浓度为0.5 mmol/L时对Adh有明显的激活作用,尤其是Zn~(2+)可使Adh的酶活显著提高11%。[结论]实现了adh基因在大肠杆菌的高效表达,为Adh的应用提供了理论依据。     

Low-specificity l-threonine aldolase of Pseudomonas sp. NCIMB 10558: purification, characterization and its application to β-hydroxy-α-amino acid synthesis

Low-specificity l-threonine aldolase, catalyzing the reversible cleavage/condensation reaction between l-threonine/l-allo-threonine and glycine plus acetaldehyde, was purified to homogeneity from Pseudomonas sp. NCIMB 10558. The enzyme has an apparent molecular mass of approximately 145 kDa and consists of four identical subunits with a molecular mass of 38 kDa. The enzyme, requiring pyridoxal- 5′-phosphate as a coenzyme, is strictly l-specific at the α position, whereas it can not distinguish between threo and erythro forms at the β position. Besides the reversible cleavage/condensation of threonine, the enzyme also catalyzes the reversible interconversion between glycine plus various aldehydes and l-β-hydroxy-α-amino acids, including l-β-(3,4-dihydroxyphenyl)serine, l-β-(3,4-met‐hylenedioxyphenyl)serine and l-β-phenylserine, providing a new route for the industrial production of these important amino acids. Received: 10 November 1997 / Received revision: 7 January 1998 / Accepted 30 January 1998     

Expression of the Alcaligenes eutrophus poly(β-hydroxybutyric acid)-synthetic pathway in Pseudomonas sp.

The 12.5-kb EcoRI restriction fragment PP1 of Alcaligenes eutrophus strain H16, which encodes for -ketothiolase, NADP-dependent acetoacetyl-CoA reductase and poly(-hydroxybutyric acid)-synthase was mobilized to six different species of the genus Pseudomonas belonging to the rRNA homology group I. Pseudomonas aeruginosa, P. fluorescens, P. putida, P. oleovorans, P. stutzeri and P. syringae, which are unable to synthesize and accumulate poly(-hydroxybutyric acid), PHB, were employed as recipients. Whereas the A. eutrophus PHB-synthetic enzymes were only marginally expressed in P. stutzeri, they were readily expressed in the other species. For example, the specific activity of PHB-synthase was 1.8 U/g protein in transconjugants of P. stutzeri but was between 21 and 77 U/mg protein in transconjugants of the other species. All recombinant strains harboring plasmid pVK101::PP1 except those of P. stutzeri accumulated PHB; the PHB content of the cells grown on gluconate under nitrogen limitation varied between 8 and 24.3% of the cellular dry mass.Abbreviations PHB poly(-hydroxybutyric acid) - PHA poly(hydroxyalkanoic acid)     

Isolation of Pseudomonas sp. Strain HB01 Which Degrades the Persistent Brominated Flame Retardant γ-Hexabromocyclododecane

In this study, we isolated Pseudomonas sp. strain HB01 from a soil sample contaminated with brominated pollutants, and found that it degraded γ-hexabromocyclododecane (γ-HBCD). The strain degraded 81% of 1 mM γ-HBCD within 5 d of culture. Furthermore, it demonstrated biodegradation of structurally related (bromo) alkanes. To the best of our knowledge, this is the first report that outlines the isolation of a bacterial strain that degrades γ-HBCD.     

Characterization of a Novel Cr6+ Reducing Pseudomonas sp. with Plant Growth–Promoting Potential

The isolate RNP4 obtained from a long-term tannery waste contaminated soil was characterized and presumptively identified as Pseudomonas sp. The strain RNP4 tolerated concentrations up to 450 mg Cr⁶⁺/L on a Luria-Bartani (LB) agar medium and reduced a substantial amount of Cr⁶⁺ to Cr³⁺ in the LB liquid medium. The ability of performing multifarious activities in tandem suggested the uniqueness of isolate RNP4. The strain produced a substantial amount of indole acetic acid (IAA) in tryptophan-supplemented medium. The strain also exhibited the production of siderophore and solubilization of phosphorus in mineral salt medium and SRS1 medium, respectively. Concurrent production of IAA and siderophore and the solubilization of phosphorus revealed its plant growth promotion potential. Furthermore, the strain was able to promote the growth of black gram, Indian mustard, and pearl millet in the presence of Cr⁶⁺. Thus, the innate capability of this novel isolate for parallel bioremediation and plant growth promotion has significance in the management of environmental and agricultural problems.     

内生菌Pseudomonas sp. G5 phzIR基因的克隆与表达

假单胞菌菌株G5是分离自香菜(Coriandrum sativumL.)茎内的一株内生菌,经BIOLOG系统分析其底物利用图谱,初步鉴定为桔黄假单胞菌Pseudomonas aurantiaca。大量研究已表明许多革兰氏阴性细菌应用群体感应系统,通过感应扩散性小信号分子―乙酰基高丝氨酸内酯(N-acyl homoserine lactones,AHLs),以种群密度依赖的方式调控基因表达,控制植物相关细菌的多种表型。本研究组合应用AHLs检测菌株Chromobacterium violaceum CV026和薄层层析分析,初步检测出菌株G5可产生几种可检测水平的AHLs信号分子,其中以N-hexanoyl-homoserine lactone(C6-HSL,HHL)为主,迁移率Rf值为0.4。进一步克隆和测序了该菌株中由PhzI和PhzR组成的群体感应quorumsensing系统的编码基因phzIR,并在大肠杆菌中异源表达了AHLs信号分子合成酶基因phzI。序列和系统进化分析表明它们与假单胞菌属其他的phzIR基因有高度同源性和进化上的保守性。     

Pseudomonas sp.F-12发酵优化及转化合成半胱氨酸的研究

通过不同发酵策略对Pseudomonas sp.F-12以DL-2-氨基-△2-噻唑啉-4-羧酸(DL-ATC)为底物生产L-半胱氨酸的过程进行优化,以分批发酵和指数流加发酵的形式进行生产,将发酵过程中的DL-ATC浓度分别控制在5 g/L和15 g/L。结果表明15 g/L DL-ATC分批发酵生产L-半胱氨酸得到最高酶活为149.1 U/mL,比活为70.9 U/mg DCW;采用15 g/L DL-ATC两阶段指数流加发酵,可使最高酶活达到313.3 U/mL,提高为分批发酵的2.1倍。同时在两阶段发酵过程中,能减少DL-ATC用量为5 g/L。两阶段发酵过程采用不同流加策略,证明菌体得率对酶活水平有显著影响。首次考察了Pseudomonas sp.F-12全静息细胞转化合成L-半胱氨酸的主要影响因素。结果表明Pseudomonas sp.F-12的最适转化pH为8.0,最适转化温度为30℃。加入甲苯等有机溶剂改变细胞膜通透性,使得半胱氨酸产量比对照组提高7.16倍,最高摩尔转化率达到93.3%。不同金属离子对转化影响不同,0.1 mmol/L Fe2+对转化反应有促进作用,0.1 mmol/L Co2+、Zn2+、Ni2+等重金属离子有抑制作用。研究结果为微生物法发酵及全细胞转化合成半胱氨酸的后续工业化奠定了基础。     

Pseudomonas sp. RT-1低温脂肪酶的纯化及酶学特性

Pseudomonas sp. RT-1是从低温环境下分离的低温脂肪酶产生菌,对该菌产生的胞外脂肪酶(PL-1)进行纯化,并对其酶学特性进行初步研究。Pseudomonas sp. RT-1的发酵上清液经60%(NH4)2SO4沉淀、12~14000截留相对分子质量(MWCO)透析袋透析、Sephadex G75分子筛和超滤浓缩后,得到了电泳纯的P-L1。SDS-PAGE电泳估算其表观相对分子质量为4.43×104。对其酶学特性研究表明:PL-1是低温碱性脂肪酶且对有机溶剂的耐受性较好。10~40℃内有较好的催化活性,最适作用温度为18℃;0~50℃该酶的稳定性较好,当温度超过50℃时则容易失活;最适作用pH为10.2,且pH在9~11时较稳定;该酶对有机溶剂的耐受性较好,10mmol/L的Ca2+、K+、Na+和Fe3+对PL1的酶活力有促进作用,其中Ca2+促进作用最大,提高了146.07%,而10mmol/L的Cu2+、Co2+、Mn2+、Mg2+、Zn2+、Ba2+和Al3+对酶活力具有不同程度的抑制作用,其中Al3+抑制作用最强,抑制了98.55%;PL-1对C链长度小于或等于12的短链脂肪酸形成的甘油三酯具有较强的水解能力;1mmol/L的去氧胆酸盐(desoxycholate)和0.01%的Triton X100对酶活力具有提高作用,分别提高了30.74%和11.83%;0.01%的SDS和Tween-80、1mmol/L的EDTA和尿素对酶活都有抑制作用,其中EDTA的抑制作用最大,抑制了80%。