Biodegradation of Phthalate Isomers by Pseudomonas aeruginosa PP4, Pseudomonas sp. PPD and Acinetobacter lwoffii ISP4

Pseudomonas aeruginosa PP4, Pseudomonas sp. PPD and Acinetobacter lwoffii ISP4 capable of utilizing phthalate isomers were isolated from the soil using enrichment culture technique. The strain ISP4 metabolizes isophthalate, while PPD and PP4 utilizes all three phthalate isomers (ortho-, iso- and tere-) as the sole carbon source. ISP4 utilizes isophthalate (0.1%) more rapidly (doubling time, 0.9 h) compared to PPD (4.64 h), PP4 (7.91 h) and other reported strains so far. The metabolic pathways in these isolates were initiated by dihydroxylation of phthalate isomers. Phthalate is hydroxylated to 3,4-dihydro-3,4-dihydroxyphthalate and 4,5-dihydro-4,5-dihydroxyphthalate in strains PP4 and PPD, respectively; while terephthalate is hydroxylated to 2-hydro-1,2-dihydroxyterephthalate. All three strains hydroxylate isophthalate to 4-hydro-3,4-dihydroxyisophthalate. The generated dihydroxyphthalates were subsequently metabolized to 3,4-dihydroxybenzoate (3,4-DHB) which was further metabolized by ortho ring-cleavage pathway. PP4 and PPD cells grown on phthalate, isophthalate or terephthalate showed respiration on respective phthalate isomer and the activity of corresponding ring-hydroxylating dioxygenase, suggesting the carbon source specific induction of three different ring-hydroxylating dioxygenases. We report, for the first time, the activity of isophthalate dioxygenase and its reductase component in the cell-free extracts. The enzyme showed maximum activity with reduced nicotinamide adenine dinucleotide (NADH) in the pH range 8–8.5. Cells grown on glucose failed to respire on phthalate isomers and 3,4-DHB and showed significantly low activities of the enzymes suggesting that the enzymes are inducible.     

类产碱假单胞菌谷氨酸脱氢酶的提纯、鉴定及某些特性的初步研究

从类产碱假单胞菌纯化出电泳纯的谷氨酸脱氢酶,用聚丙烯酰胺梯度凝胶电泳和ＳＤＳ－聚丙烯酰胺凝胶电泳测得分子量为２９０ ｋＤ,亚基分子量为４７ ｋＤ,提示该酶为六聚体．该酶对ＮＡＤＰ（Ｈ）和底物均具有高度专一性,对谷氨酸、α－酮戊二酸及ＮＡＤＰ＋ 的Ｋｍ 值分别为：２８ ｍ ｍ ｏｌ／Ｌ、１．２ｍ ｍ ｏｌ／Ｌ及０．０６３ ｍ ｍ ｏｌ／Ｌ．用Ｈｉｌｌ作图法求得酶对ＮＨ＋４ 和ＮＡＤＰＨ 的［Ｓ］０．５分别为２４ ｍ ｍ ｏｌ／Ｌ和０．０３７ ｍ ｍ ｏｌ／Ｌ．最适反应温度为５０℃,催化氨化反应和脱氨反应的最适ｐＨ 分别为８．０和８．８,在热稳定性方面不及嗜热细菌的谷氨酸脱氢酶稳定．提纯的谷氨酸脱氢酶在低温（４℃）条件下,可在Ｔｒｉｓ－ＨＣｌ缓冲液中贮存半年以上,活力无明显下降,冷冻则可导致纯酶液迅速失活．氮源对菌体谷氨酸脱氢酶水平有显著影响．     

谷氨酸生产菌S9114中的谷氨酸脱氢酶的研究

谷氨酸脱氢酶 (GDH)是谷氨酸生物合成的关键酶 ,谷氨酸棒杆菌S91 1 4是目前我国味精工业应用最广泛的生产菌种 ,其谷氨酸脱氢酶的研究尚未见报道。分离纯化该菌中的谷氨酸脱氢酶 ,研究其辅酶组成 ,对揭示谷氨酸脱氢酶的分子结构和性质 ,提高谷氨酸产率很有必要。将培养至对数期中期的细胞离心收集并用含适量DTT、ED TA的Tris_HCl缓冲液 (pH 7 5 )洗涤 ,用Frenchpressurecellpress破碎 ,离心去除菌体碎片得无细胞抽提液。然后使用 KTA_10 0快速纯化系统经DEAE_纤维素柱、疏水柱 (HIC)、G_2 0 0凝胶过滤柱层析得到纯化大约 70倍的以NAD PH为辅酶的GDH和部分纯化的以NADH辅酶的GDH。这两个酶分别对NADPH、NADH高度专一 ,不能相互代替。经HPLC和SDS_PAGE测得前一种酶的分子量和亚基分子量分别为 188kD和 32kD ,表明该酶为具有相同亚基的六聚体。酶活性测定使用HITACHIU_30 0 0分光光度计利用NAD(P)H在 340nm氧化的初速度进行。蛋白质含量测定利用Bradford方法进行 ,并以牛血清白蛋白为标准蛋白。纯化结果表明S91 1 4中确实存在两种GDH ,其中以NADH为辅酶的GDH尚未见报道。和某些具有两种GDH的微生物一样 ,S91 1 4可能也是以NADPH为辅酶的GDH参与谷氨酸的合成代谢 ,以NADH为辅酶的GDH参与谷氨酸的分解代谢。     

Homology Modeling and Molecular Docking Studies of Glutamate Dehydrogenase (GDH) from Cyanobacterium Synechocystis sp. PCC 6803

Glutamate dehydrogenase (GDH), which is present in most bacteria and eukaryotes’ mitochondria, plays an important role in amino acid metabolism. In g     

Purification and Kinetic Characterization of Glucose-6-phosphate Dehydrogenase from Dictyostelium discoideum

Reimers, J. M., Huang, Q., Albe, K. R., and Wright, B. E. 1993. Purification and kinetic characterization of glucose-6-phosphate dehydrogenase from Dictyostelium discoideum. Experimental Mycology 17, 1-6. Glucose-6-phosphate dehydrogenase from Dictyostelium discoideum was purified 650-fold and kinetically characterized. The enzyme catalyzed the conversion of G6P + NADP to 6PG + NADPH stoichiometrically and irreversibly in vitro . The purified enzyme is specific for NADP. Michaelis constants for G6P and NADP were 0.040 and 0.011 mM, respectively. NADPH was found to be a competitive inhibitor with respect to NADP with a K_i of 0.006 mM and a noncompetitive inhibitor with respect to G6P. The data from initial velocity and product inhibition studies were consistent with a sequential mechanism.     

Purification and Characterization of a Soluble and a Particulate Glutamate Dehydrogenase from Rat Brain

Glutamate dehydrogenase (GDH) activity was determined in high-speed fractions (100,000 g for 60 min) obtained from whole rat brain homogenates after removal of a low-speed pellet (480 g for 10 min). Approximately 60% of the high-speed GDH activity was particulate (associated with membrane) and the remaining was soluble (probably of mitochondrial matrix origin). Most of the particulate GDH activity resisted extraction by several commonly used detergents, high concentration of salt, and sonication; however, it was largely extractable with the cationic detergent cetyltrimethylammonium bromide (CTAB) in hypotonic buffer solution. The two GDH activities were purified using a combination of hydrophobic interaction, ion exchange, and hydroxyapatite chromatography. Throughout these purification steps the two activities showed similar behavior. Kinetic studies indicated similar Km values for the two GDH fractions for the substrates alpha-ketoglutarate, ammonia, and glutamate; however, there were small but significant differences in Km values for NADH and NADPH. Although the allosteric stimulation by ADP and L-leucine and inhibition by diethylstilbestrol was comparable, the two GDH components differed significantly in their susceptibility to GTP inhibition in the presence of 1 mM ADP, with apparent Ki values of 18.5 and 9.0 microM GTP for the soluble and particulate fractions, respectively. The Hill plot coefficient, binding constant, and cooperativity index for the GTP inhibition were also significantly different, indicating that the two GDH activities differ in their allosteric sites. In addition, enzyme activities of the two purified proteins exhibited a significant difference in thermal stability when inactivated at 45 degrees C and pH 7.4 in 50 mM phosphate buffer.     

Purification and Characterization of the Coniferyl Aldehyde Dehydrogenase from Pseudomonas sp. Strain HR199 and Molecular Characterization of the Gene

The coniferyl aldehyde dehydrogenase (CALDH) of Pseudomonas sp. strain HR199 (DSM7063), which catalyzes the NAD⁺-dependent oxidation of coniferyl aldehyde to ferulic acid and which is induced during growth with eugenol as the carbon source, was purified and characterized. The native protein exhibited an apparent molecular mass of 86,000 ± 5,000 Da, and the subunit mass was 49.5 ± 2.5 kDa, indicating an α₂ structure of the native enzyme. The optimal oxidation of coniferyl aldehyde to ferulic acid was obtained at a pH of 8.8 and a temperature of 26°C. The K_m values for coniferyl aldehyde and NAD⁺ were about 7 to 12 μM and 334 μM, respectively. The enzyme also accepted other aromatic aldehydes as substrates, whereas aliphatic aldehydes were not accepted. The NH₂-terminal amino acid sequence of CALDH was determined in order to clone the encoding gene (calB). The corresponding nucleotide sequence was localized on a 9.4-kbp EcoRI fragment (E94), which was subcloned from a Pseudomonas sp. strain HR199 genomic library in the cosmid pVK100. The partial sequencing of this fragment revealed an open reading frame of 1,446 bp encoding a protein with a relative molecular weight of 51,822. The deduced amino acid sequence, which is reported for the first time for a structural gene of a CALDH, exhibited up to 38.5% amino acid identity (60% similarity) to NAD⁺-dependent aldehyde dehydrogenases from different sources.     

Purification and Characterization of the NAD-Dependent Glutamate Dehydrogenase in the Ectomycorrhizal FungusLaccaria bicolor(Maire) Orton

The NAD-dependent glutamate dehydrogenase (GDH) (EC 1.4.1.2) fromLaccaria bicolorwas purified 410-fold to apparent electrophoretic homogeneity with a 40% recovery through a three-step procedure involving ammonium sulfate precipitation, anion-exchange chromatography on DEAE–Trisacryl, and gel filtration. The molecular weight of the native enzyme determined by gel filtration was 470 kDa, whereas sodium dodecyl sulfate–polyacrylamide gel electrophoresis gave rise to a single band of 116 kDa, suggesting that the enzyme is composed of four identical subunits. The enzyme was specific for NAD(H). The pH optima were 7.4 and 8.8 for the amination and deamination reactions, respectively. The enzyme was found to be highly unstable, with virtually no activity after 20 days at −75°C, 4 days at 4°C, and 1 h at 50°C. The addition of ammonium sulfate improved greatly the stability of the enzyme and full activity was still observed after several months at −75°C. NAD-GDH activity was stimulated by Ca²⁺and Mg²⁺but strongly inhibited by Cu²⁺and slightly by the nucleotides AMP, ADP, and ATP. The Michaelis constants for NAD, NADH, 2-oxoglutarate, and ammonium were 282 μM, 89 μM, 1.35 mM, and 37 mM, respectively. The enzyme had a negative cooperativity for glutamate (Hill number of 0.3), and itsK_mvalue increased from 0.24 to 3.6 mM when the glutamate concentration exceeded 1 mM. These affinity constants of the substrates, compared with those of the NADP-GDH of the fungus, suggest that the NAD-GDH is mainly involved in the catabolism of glutamate, while the NADP-GDH is involved in the catalysis of this amino acid.     

Antimicrobial Substance Produced by Pseudomonas aeruginosa Isolated from Slaughterhouse Sediment: Physicochemical Characterization,Purification, and Identification

International Journal of Peptide Research and Therapeutics - This study aimed to produce the novel bacteriocin from Pseudomonas aeruginosa 43. The bacteriocin was purified through 80% ammonium...     

Thermostable NADP+-Dependent Medium-Chain Alcohol Dehydrogenase from Acinetobacter sp. Strain M-1: Purification and Characterization and Gene Expression in Escherichia coli

NADPH-dependent alkylaldehyde reducing enzyme, which was greatly induced by n-hexadecane, from Acinetobacter sp. strain M-1 was purified and characterized. The purified enzyme had molecular masses of 40 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 160 kDa as determined by gel filtration chromatography. The enzyme, which was shown to be highly thermostable, was most active toward n-heptanal and could use n-alkylaldehydes ranging from C₂ to C₁₄ and several substituted benzaldehydes, including the industrially important compounds cinnamyl aldehyde and anisaldehyde, as substrates. The alrA gene coding for this enzyme was cloned, and its nucleotide sequence was determined. The deduced amino acid sequence encoded by the alrA gene exhibited homology to the amino acid sequences of zinc-containing alcohol dehydrogenases from various sources. The gene could be highly expressed in Escherichia coli, and the product was purified to homogeneity by simpler procedures from the recombinant than from the original host. Our results show that this enzyme can be used for industrial bioconversion of useful alcohols and aldehydes.     

Purification and Characterization of a Novel Mannitol Dehydrogenase from a Newly Isolated Strain of Candida magnoliae

Mannitol biosynthesis in Candida magnoliae HH-01 (KCCM-10252), a yeast strain that is currently used for the industrial production of mannitol, is catalyzed by mannitol dehydrogenase (MDH) (EC 1.1.1.138). In this study, NAD(P)H-dependent MDH was purified to homogeneity from C. magnoliae HH-01 by ion-exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography. The relative molecular masses of C. magnoliae MDH, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography, were 35 and 142 kDa, respectively, indicating that the enzyme is a tetramer. This enzyme catalyzed both fructose reduction and mannitol oxidation. The pH and temperature optima for fructose reduction and mannitol oxidation were 7.5 and 37°C and 10.0 and 40°C, respectively. C. magnoliae MDH showed high substrate specificity and high catalytic efficiency (k_cat = 823 s⁻¹, K_m = 28.0 mM, and k_cat/K_m = 29.4 mM⁻¹ s⁻¹) for fructose, which may explain the high mannitol production observed in this strain. Initial velocity and product inhibition studies suggest that the reaction proceeds via a sequential ordered Bi Bi mechanism, and C. magnoliae MDH is specific for transferring the 4-pro-S hydrogen of NADPH, which is typical of a short-chain dehydrogenase reductase (SDR). The internal amino acid sequences of C. magnoliae MDH showed a significant homology with SDRs from various sources, indicating that the C. magnoliae MDH is an NAD(P)H-dependent tetrameric SDR. Although MDHs have been purified and characterized from several other sources, C. magnoliae MDH is distinguished from other MDHs by its high substrate specificity and catalytic efficiency for fructose only, which makes C. magnoliae MDH the ideal choice for industrial applications, including enzymatic synthesis of mannitol and salt-tolerant plants.     

Purification and Characterization of Cellobiose Dehydrogenase from the Plant Pathogen Sclerotium (Athelia) rolfsii

Cellobiose dehydrogenase (CDH) is an extracellular hemoflavoenzyme produced by several wood-degrading fungi. In the presence of a suitable electron acceptor, e.g., 2,6-dichloro-indophenol (DCIP), cytochrome c, or metal ions, CDH oxidizes cellobiose to cellobionolactone. The phytopathogenic fungus Sclerotium rolfsii (teleomorph: Athelia rolfsii) strain CBS 191.62 produces remarkably high levels of CDH activity when grown on a cellulose-containing medium. Of the 7,500 U of extracellular enzyme activity formed per liter, less than 10% can be attributed to the proteolytic product cellobiose:quinone oxidoreductase. As with CDH from wood-rotting fungi, the intact, monomeric enzyme from S. rolfsii contains one heme b and one flavin adenine dinucleotide cofactor per molecule. It has a molecular size of 101 kDa, of which 15% is glycosylation, and a pI value of 4.2. The preferred substrates are cellobiose and cellooligosaccharides; additionally, β-lactose, thiocellobiose, and xylobiose are efficiently oxidized. Cytochrome c (equine) and the azino-di-(3-ethyl-benzthiazolin-6-sulfonic acid) cation radical were the best electron acceptors, while DCIP, 1,4-benzoquinone, phenothiazine dyes such as methylene blue, phenoxazine dyes such as Meldola's blue, and ferricyanide were also excellent acceptors. In addition, electrons can be transferred to oxygen. Limited in vitro proteolysis with papain resulted in the formation of several protein fragments that are active with DCIP but not with cytochrome c. Such a flavin-containing fragment, with a mass of 75 kDa and a pI of 5.1 and lacking the heme domain, was isolated and partially characterized.     

Characterization and Fungal Inhibition Activity of Siderophore from Wheat Rhizosphere Associated Acinetobacter calcoaceticus Strain HIRFA32

Acinetobacter calcoaceticus HIRFA32 from wheat rhizosphere produced catecholate type of siderophore with optimum siderophore (ca. 92 % siderophore units) in succinic acid medium without FeSO₄ at 28 °C and 24 h of incubation. HPLC purified siderophore appeared as pale yellow crystals with molecular weight [M⁺¹] m/z 347.18 estimated by LCMS. The structure elucidated by ¹H NMR, ¹³C NMR, HMQC, HMBC, NOESY and decoupling studies, revealed that siderophore composed of 2,3-dihydroxybenzoic acid with hydroxyhistamine and threonine as amino acid subunits. In vitro study demonstrated siderophore mediated mycelium growth inhibition (ca. 46.87 ± 0.5 %) of Fusarium oxysporum. This study accounts to first report on biosynthesis of acinetobactin-like siderophore by the rhizospheric strain of A. calcoaceticus and its significance in inhibition of F. oxysporum.     

Expression, Purification and Characterization of the Proline Dehydrogenase Domain of PutA from Pseudomonas putida POS-F84

The objective of the present work was to express a truncated form of Pseudomonas putida PutA that shows proline dehydrogenase (ProDH) activity. The putA gene encoding ProDH enzyme was cloned into pET23a vector and expressed in Escherichia coli strain BL-21 (DE3) plysS. The recombinant P. putida enzyme was biochemically characterized and its three dimensional structure was also predicted. ProDH encoding sequence showed an open reading frame of 1,035-bp encoding a 345 amino acid residues polypeptide chain. Purified His-tagged enzyme gave a single band with a molecular mass of 40 kDa on SDS-PAGE. The molecular mass of the isolated enzyme was found to be about 40 kDa by gel filtration. This suggested that the enzyme of interest consists of one subunit. The K _m and V _max values of recombinant P. putida ProDH were estimated to be 31 mM and 132 μmol/min, respectively. The optimum pH and temperature for the catalytic activity of the enzyme was about pH 8.5 and 30 °C. The modeling analysis of the three dimensional structure elucidated that Ser-165, Lys-195 and Ala-252 were key residues for the ProDH activity. This study provides data on the cloning, sequencing and recombinant expression of PutA ProDH domain from P. putida POS-F84.     

Pyridine nucleotide transhydrogenase from Pseudomonas aeruginosa: Purification by affinity chromatography and physicochemical properties

Pyridine nucleotide transhydrogenase from Pseudomonas aeruginosa was purified 150-fold by affinity chromatography on immobilized 2′-AMP. The binding of the enzyme is pH dependent. Elution was achieved with 2′-AMP, NADP⁺, or NADPH but not with 5′-AMP, NAD⁺, or NADH. The enzyme preparations appeared to be homogeneous in gel chromatography and ultracentrifugation, but only if these procedures were carried out in the presence of 2′-AMP or NADP⁺. With 2′-AMP a sedimentation coefficient of 34 S, a molecular weight of 1.6–1.7 million, and a Stokes' radius of 11.7 nm were determined. In the presence of NADP⁺ the sedimentation coefficient was 42 S and the molecular weight was 6.4 million. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed one kind of subunit with a molecular weight of 54,000. This was consistent with results from amino acid analyses and paper chromatography of peptides. Eight molar urea inactivated the enzyme but did not dissociate it into subunits. Full activity was restored after dialysis against urea-free buffer by mercaptoethanol and flavin-adenine dinucleotide.     

Purification and characterization of trans-3-(4-methoxyphenyl) glycidic acid methyl ester hydrolyzing lipase from Pseudomonas aeruginosa

Enantiospecific lipase was purified from Pseudomonas aeruginosa MTCC 5113 and it was used for the hydrolysis of (±)-methyl trans-3(4-methoxyphenyl) glycidate, a key intermediate in the synthesis of cardiovascular drug, diltiazem. Enzyme from broth supernatant was precipitated with acetone and purified by anion exchange and gel filtration chromatography. The purified lipase was a homogenous protein having a molecular weight of 59.4 kDa as determined by SDS-PAGE. Isoelectric point was found to be approximately 5.5 after 2D electrophoresis. This organic solvent tolerant enzyme was found to be active in presence of EDTA, Tween-80 and β-mercaptoethanol whereas sodium dodecyl sulphate and dithiothreitol inhibited its activity. The K_m and V_max of the enzyme were 50 mM and 27.1 μmol/min mg, respectively using p-nitrophenyl palmitate as a substrate. The activity of lipase was confirmed by (±)-MPGM hydrolysis and zymography.     

Purification, Characterization, and Sequence Analysis of 2-Aminomuconic 6-Semialdehyde Dehydrogenase from Pseudomonas pseudoalcaligenes JS45

2-Aminonumconic 6-semialdehyde is an unstable intermediate in the biodegradation of nitrobenzene and 2-aminophenol by Pseudomonas pseudoalcaligenes JS45. Previous work has shown that enzymes in cell extracts convert 2-aminophenol to 2-aminomuconate in the presence of NAD⁺. In the present work, 2-aminomuconic semialdehyde dehydrogenase was purified and characterized. The purified enzyme migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular mass of 57 kDa. The molecular mass of the native enzyme was estimated to be 160 kDa by gel filtration chromatography. The optimal pH for the enzyme activity was 7.3. The enzyme is able to oxidize several aldehyde analogs, including 2-hydroxymuconic semialdehyde, hexaldehyde, and benzaldehyde. The gene encoding 2-aminomuconic semialdehyde dehydrogenase was identified by matching the deduced N-terminal amino acid sequence of the gene with the first 21 amino acids of the purified protein. Multiple sequence alignment of various semialdehyde dehydrogenase protein sequences indicates that 2-aminomuconic 6-semialdehyde dehydrogenase has a high degree of identity with 2-hydroxymuconic 6-semialdehyde dehydrogenases.