A new cold-adapted β-D-galactosidase from the Antarctic Arthrobacter sp. 32c – gene cloning, overexpression, purification and properties

<p>Abstract</p> <p>Background</p> <p>The development of a new cold-active β-D-galactosidases and microorganisms that efficiently ferment lactose is of high biotechnological interest, particularly for lactose removal in milk and dairy products at low temperatures and for cheese whey bioremediation processes with simultaneous bio-ethanol production.</p> <p>Results</p> <p>In this article, we present a new β-D-galactosidase as a candidate to be applied in the above mentioned biotechnological processes. The gene encoding this β-D-galactosidase has been isolated from the genomic DNA library of Antarctic bacterium <it>Arthrobacter </it>sp. 32c, sequenced, cloned, expressed in <it>Escherichia coli </it>and <it>Pichia pastoris</it>, purified and characterized. 27 mg of β-D-galactosidase was purified from 1 L of culture with the use of an intracellular <it>E. coli </it>expression system. The protein was also produced extracellularly by <it>P. pastoris </it>in high amounts giving approximately 137 mg and 97 mg of purified enzyme from 1 L of <it>P. pastoris </it>culture for the AOX1 and a constitutive system, respectively. The enzyme was purified to electrophoretic homogeneity by using either one step- or a fast two step- procedure including protein precipitation and affinity chromatography. The enzyme was found to be active as a homotrimeric protein consisting of 695 amino acid residues in each monomer. Although, the maximum activity of the enzyme was determined at pH 6.5 and 50°C, 60% of the maximum activity of the enzyme was determined at 25°C and 15% of the maximum activity was detected at 0°C.</p> <p>Conclusion</p> <p>The properties of <it>Arthrobacter </it>sp. 32cβ-D-galactosidase suggest that this enzyme could be useful for low-cost, industrial conversion of lactose into galactose and glucose in milk products and could be an interesting alternative for the production of ethanol from lactose-based feedstock.</p

Characterization of exceptionally thermostable single-stranded DNA-binding proteins from Thermotoga maritima and Thermotoga neapolitana

<p>Abstract</p> <p>Background</p> <p>In recent years, there has been an increasing interest in SSBs because they find numerous applications in diverse molecular biology and analytical methods.</p> <p>Results</p> <p>We report the characterization of single-stranded DNA binding proteins (SSBs) from the thermophilic bacteria <it>Thermotoga maritima </it>(<it>Tma</it>SSB) and <it>Thermotoga neapolitana </it>(<it>Tne</it>SSB). They are the smallest known bacterial SSB proteins, consisting of 141 and 142 amino acid residues with a calculated molecular mass of 16.30 and 16.58 kDa, respectively. The similarity between amino acid sequences of these proteins is very high: 90% identity and 95% similarity. Surprisingly, both <it>Tma</it>SSB and <it>Tne</it>SSB possess a quite low sequence similarity to <it>Escherichia coli </it>SSB (36 and 35% identity, 55 and 56% similarity, respectively). They are functional as homotetramers containing one single-stranded DNA binding domain (OB-fold) in each monomer. Agarose mobility assays indicated that the ssDNA-binding site for both proteins is salt independent, and fluorescence spectroscopy resulted in a size of 68 ± 2 nucleotides. The half-lives of <it>Tma</it>SSB and <it>Tne</it>SSB were 10 h and 12 h at 100°C, respectively. When analysed by differential scanning microcalorimetry (DSC) the melting temperature (<it>T</it>_m) was 109.3°C and 112.5°C for <it>Tma</it>SSB and <it>Tne</it>SSB, respectively.</p> <p>Conclusion</p> <p>The results showed that <it>Tma</it>SSB and <it>Tne</it>SSB are the most thermostable SSB proteins identified to date, offering an attractive alternative to <it>Taq</it>SSB and <it>Tth</it>SSB in molecular biology applications, especially with using high temperature e. g. polymerase chain reaction (PCR).</p

A novel cold-active β-D-galactosidase from the Paracoccus sp. 32d - gene cloning, purification and characterization

<p>Abstract</p> <p>Background</p> <p>β-<smcaps>D</smcaps>-Galactosidases (EC 3.2.1.23) catalyze the hydrolysis of terminal non-reducing β-<smcaps>D</smcaps>-galactose residues in β-<smcaps>D</smcaps>-galactosides. Cold-active β-<smcaps>D</smcaps>-galactosidases have recently become a focus of attention of researchers and dairy product manufactures owing to theirs ability to: (i) eliminate of lactose from refrigerated milk for people afflicted with lactose intolerance, (ii) convert lactose to glucose and galactose which increase the sweetness of milk and decreases its hydroscopicity, and (iii) eliminate lactose from dairy industry pollutants associated with environmental problems. Moreover, in contrast to commercially available mesophilic β-<smcaps>D</smcaps>-galactosidase from <it>Kluyveromyces lactis </it>the cold-active counterparts could make it possible both to reduce the risk of mesophiles contamination and save energy during the industrial process connected with lactose hydrolysis.</p> <p>Results</p> <p>A genomic DNA library was constructed from soil bacterium <it>Paracoccus </it>sp. 32d. Through screening of the genomic DNA library on LB agar plates supplemented with X-Gal, a novel gene encoding a cold-active β-<smcaps>D</smcaps>-galactosidase was isolated. The <it>in silico </it>analysis of the enzyme amino acid sequence revealed that the β-<smcaps>D</smcaps>-galactosidase <it>Paracoccus </it>sp. 32d is a novel member of Glycoside Hydrolase Family 2. However, owing to the lack of a BGal_small_N domain, the domain characteristic for the LacZ enzymes of the GH2 family, it was decided to call the enzyme under study 'BgaL'. The <it>bgaL </it>gene was cloned and expressed in <it>Escherichia coli </it>using the pBAD Expression System. The purified recombinant BgaL consists of two identical subunits with a combined molecular weight of about 160 kDa. The BgaL was optimally active at 40°C and pH 7.5. Moreover, BgaL was able to hydrolyze both lactose and <it>o</it>-nitrophenyl-β-<smcaps>D</smcaps>-galactopyranoside at 10°C with <it>K</it>_mvalues of 2.94 and 1.17 mM and <it>k</it>_catvalues 43.23 and 71.81 s^-1, respectively. One U of the recombinant BgaL would thus be capable hydrolyzing about 97% of the lactose in 1 ml of milk in 24 h at 10°C.</p> <p>Conclusions</p> <p>A novel <it>bgaL </it>gene was isolated from <it>Paracoccus </it>sp. 32d encoded a novel cold-active β-<smcaps>D</smcaps>-galactosidase. An <it>E. coli </it>expression system has enabled efficient production of soluble form of BgaL <it>Paracoccus </it>sp. 32d. The amino acid sequence analysis of the BgaL enzyme revealed notable differences in comparison to the result of the amino acid sequences analysis of well-characterized cold-active β-<smcaps>D</smcaps>-galactosidases belonging to Glycoside Hydrolase Family 2. Finally, the enzymatic properties of <it>Paracoccus </it>sp. 32d β-<smcaps>D</smcaps>-galactosidase shows its potential for being applied to development of a new industrial biocatalyst for efficient lactose hydrolysis in milk.</p

Physicochemical and bilogical characterization of soils from the vicinity of the Arctowski Polish Antarctic Station

Soil samples collected in 2006 from 4 different sites (Puchalski Hill and a moraine below this hill, an elephant seal colony and a penguin rookery) in the vicinity of Arctowski Polish Antarctic Station at King George Island were characterized in terms of physicochemical properties (pH, humidity, concentration of selected inorganic and organic substances), microbial colonization and the overall activity of selected enzymes (differentglycosidases and esterases). Activity of the latter enzymes was assayed by p-nitrophenyl and 4-methylumbelliferone derivatives of fatty acids (by spectrophotometric and fluorimetric method, respectively). The highest lipolytic activity was found in soils from the elephant seal colony and penguin rookery

Prawidłowe rozpoznanie toksoplazmozy u kobiet ciężarnych - ważność badań diagnostycznych oraz nowe możliwości

Artykuł opisuje problem toksoplazmozy, kliniczne objawy choroby, leczenie, aktualną diagnostykę oraz nowe możliwości testów diagnostycznych. Ponadto, przedstawiono istotne zagadnienia obejmujące aktualnie prowadzone badania naukowe, które dotyczą zastosowania nowych narzędzi diagnostycznych (rekombinantowych antygenów T. gondii) w serodiagnostyce toksoplazmozy. Forum Medycyny Rodzinnej 2010, tom 4, nr 4, 255-26

Analysis of the unique structural and physicochemical properties of the DraD/AfaD invasin in the context of its belonging to the family of chaperone/usher type fimbrial subunits

<p>Abstract</p> <p>Background</p> <p>DraD invasin encoded by the dra operon possesses a classical structure characteristic to fimbrial subunits of the chaperone/usher type. The Ig-fold of the DraD possesses two major characteristics distinguishing it from the family of fimbrial subunits: 1) a distortion of the β-barrel structure in the region of the acceptor cleft, demonstrated by a disturbance of the main-chain hydrogen bonds network, and 2) an unusually located disulfide bond connecting B and F strands - the localization exclusively observed in the subfamily of DraD/AfaD-type subunits.</p> <p>Results</p> <p>To evaluate the influence of the DraD-sc specific structural features on its stability and mechanism of thermal denaturation, a series of DSC and FT-IR denaturation experiments were performed giving following conclusions. 1) The DraD-sc is characterized by a low stability (standard Gibbs free energy and enthalpy of unfolding of 18.4 ±1.4 kJ mol^-1and 131 ±25 kJ mol^-1, respectively) that contrasts strongly with almost infinite stability of the described previously DraE-sc fimbrial protein. 2) The DraD-sc unfolds thermally according to the two state equilibrium model, in contrast to the irreversible kinetically controlled transition of the DraE-sc. 3) The DraD specific disulfide bond is crucial at the folding stage and has little stability effect in the mature protein.</p> <p>Conclusions</p> <p>Data published so far emphasize unique biological properties of the DraD invasin as fimbrial subunit: a chaperone independent folding, an usher independent surface localization and the possibility to exist in two forms: as unbound subunits and as loosely bound at fimbrial tip.</p> <p>Presented calorimetric and FT-IR stability data combined with structural correlations has underlined that the DraD invasin is also characterized by unique physicochemical and structural attributes in the context of its belonging to the family of fimbrial subunits.</p

A method for the production of D-tagatose using a recombinant Pichia pastoris strain secreting β-D-galactosidase from Arthrobacter chlorophenolicus and a recombinant L-arabinose isomerase from Arthrobacter sp. 22c

Abstract Background D-Tagatose is a natural monosaccharide which can be used as a low-calorie sugar substitute in food, beverages and pharmaceutical products. It is also currently being tested as an anti-diabetic and obesity control drug. D-Tagatose is a rare sugar, but it can be manufactured by the chemical or enzymatic isomerization of D-galactose obtained by a β-D-galactosidase-catalyzed hydrolysis of milk sugar lactose and the separation of D-glucose and D-galactose. L-Arabinose isomerases catalyze in vitro the conversion of D-galactose to D-tagatose and are the most promising enzymes for the large-scale production of D-tagatose. Results In this study, the araA gene from psychrotolerant Antarctic bacterium Arthrobacter sp. 22c was isolated, cloned and expressed in Escherichia coli. The active form of recombinant Arthrobacter sp. 22c L-arabinose isomerase consists of six subunits with a combined molecular weight of approximately 335 kDa. The maximum activity of this enzyme towards D-galactose was determined as occurring at 52°C; however, it exhibited over 60% of maximum activity at 30°C. The recombinant Arthrobacter sp. 22c L-arabinose isomerase was optimally active at a broad pH range of 5 to 9. This enzyme is not dependent on divalent metal ions, since it was only marginally activated by Mg2+, Mn2+ or Ca2+ and slightly inhibited by Co2+ or Ni2+. The bioconversion yield of D-galactose to D-tagatose by the purified L-arabinose isomerase reached 30% after 36 h at 50°C. In this study, a recombinant Pichia pastoris yeast strain secreting β-D-galactosidase Arthrobacter chlorophenolicus was also constructed. During cultivation of this strain in a whey permeate, lactose was hydrolyzed and D-glucose was metabolized, whereas D-galactose was accumulated in the medium. Moreover, cultivation of the P. pastoris strain secreting β-D-galactosidase in a whey permeate supplemented with Arthrobacter sp. 22c L-arabinose isomerase resulted in a 90% yield of lactose hydrolysis, the complete utilization of D-glucose and a 30% conversion of D-galactose to D-tagatose. Conclusions The method developed for the simultaneous hydrolysis of lactose, utilization of D-glucose and isomerization of D-galactose using a P. pastoris strain secreting β-D-galactosidase and recombinant L-arabinose isomerase seems to offer an interesting alternative for the production of D-tagatose from lactose-containing feedstock.</p

Identification and properties of the Deinococcus grandis and Deinococcus proteolyticus single-stranded DNA binding proteins (SSB)

To study the biochemical properties of SSB's from Deinococcus grandis (DgrSSB) and Deinococcus proteolyticus (DprSSB), we have cloned the ssb genes obtained by PCR and have developed Escherichia coli overexpression systems. The genes consist of an open reading frame of 891 (DgrSSB) and 876 (DprSSB) nucleotides encoding proteins of 296 and 291 amino acids with a calculated molecular mass of 32.29 and 31.33 kDa, respectively. The amino-acid sequence of DgrSSB exhibits 45%, 44% and 82% identity and the amino-acid sequence of DprSSB reveals 43%, 43% and 69% identity with Thermus aquaticus (TaqSSB), Thermus thermophilus (TthSSB) and Deinococcus radiodurans SSBs, respectively. We show that DgrSSB and DprSSB are similar to Thermus/Deinococcus SSBs in their biochemical properties. They are functional as homodimers, with each monomer encoding two single-stranded DNA binding domains (OB-folds). In fluorescence titrations with poly(dT), both proteins bind single-stranded DNA with a binding site size of about 33 nt per homodimer. In a complementation assay in E. coli, DgrSSB and DprSSB took over the in vivo function of EcoSSB. Thermostability with half-lives of about 1 min at 65-67.5°C make DgrSSB and DprSSB similar to the known SSB of Deinococcus radiodurans (DraSSB)