Lipases are industrially important enzymes having vast applications in various fields. Cloning and expression of lipase enzyme-encoding genes in suitable host lead to their widespread use in different fields. The present study represents the first attempt towards the expression of the synthetic lipase gene in Pseudomonas aeruginosa. An alkalophilic lipase gene (GenBank accession number: NP_388152) from Bacillus subtilis was synthetically designed and introduced in the pJN105 vector and subsequently cloned in Pseudomonas aeruginosa SDK-6. Agarose gel electrophoresis confirmed the transformation of SDK-6, exhibiting a band difference of ~ 700 bp between native and recombinant pJN105. Further amplification of cloned lipase gene was confirmed using PCR amplification with Lip 1 and Lip 2 primers respectively, followed by restriction analysis. Approximately 15-fold increase in lipase production was observed in recombinant Pseudomonas as compared to the native strain. One factor at a time (OFAT) analysis revealed L-arabinose, inoculum size (0.5%; v/v), and agitation (120 rpm) as significant factors affecting the over-expression of lipase enzyme. Optimization of enzyme induction conditions by central composite design (CCD) led to 1.60-fold increase in the production of lipase at 0.65% (w/v) inducer concentration, OD600-1.075 before induction and 35 °C post induction temperature with overall lipase production of 50.50 IU/mL. Statistical validation of observed value via ANOVA showed an F-value of 138.70 at p < 0.01 with R2 of 0.9921.
- MeSH
- arabinosa metabolismus MeSH
- Bacillus subtilis * genetika enzymologie MeSH
- bakteriální proteiny genetika metabolismus MeSH
- exprese genu MeSH
- genetické vektory genetika MeSH
- klonování DNA MeSH
- lipasa * genetika metabolismus MeSH
- Pseudomonas aeruginosa * genetika enzymologie MeSH
- rekombinantní proteiny * genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
Recent synthetic efforts aimed at reconstructing the beginning of life on our planet point at the plausibility of scenarios fueled by extraterrestrial energy sources. In the current work we show that beyond nucleobases the sugar components of the first informational polymers can be synthesized in this way. We demonstrate that a laser-induced high-energy chemistry combined with TiO2 catalysis readily produces a mixture of pentoses, among them ribose, arabinose and xylose. This chemistry might be highly relevant to the Late Heavy Bombardment period of Earth's history about 4-3.85 billion years ago. In addition, we present an in-depth theoretical analysis of the most challenging step of the reaction pathway, i.e., the TiO2-catalyzed dimerization of formaldehyde leading to glycolaldehyde.
- MeSH
- arabinosa chemická syntéza MeSH
- dimerizace MeSH
- formaldehyd chemie MeSH
- katalýza MeSH
- planetární evoluce MeSH
- původ života MeSH
- ribosa chemická syntéza MeSH
- sacharidy chemická syntéza MeSH
- titan chemie MeSH
- xylosa chemická syntéza MeSH
- Země (planeta) MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A series of lupane-type saponins bearing OSW-1 disaccharide unit as well as its regio- and stereoisomers were prepared and used for the structure-activity relationships (SAR) study. Unexpected preference for 1→4-linked regioisomers and an unusual inversion of the conformation of the sugar rings were noted. Cytotoxic activity of new lupane compounds was evaluated in vitro and revealed that some saponins exhibited an interesting bioactivity profile against human cancer cell lines. Influence of the protecting groups on the cytotoxicity was investigated. These results open the way to the synthesis of various lupane-type triterpene and saponin derivatives as potential anticancer compounds.
- MeSH
- antitumorózní látky chemická syntéza chemie farmakologie MeSH
- arabinosa chemie MeSH
- disacharidy chemická syntéza chemie farmakologie MeSH
- glykosylace MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- saponiny chemie MeSH
- stereoizomerie MeSH
- techniky syntetické chemie MeSH
- triterpeny chemie MeSH
- vodíková vazba MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In Bacillus subtilis, the arabinose repressor AraR negatively controls the expression of genes in the metabolic pathway of arabinose-containing polysaccharides. The protein is composed of two domains of different phylogenetic origin and function: an N-terminal DNA-binding domain belonging to the GntR family and a C-terminal effector-binding domain that shows similarity to members of the GalR/LacI family. The crystal structure of the C-terminal effector-binding domain of AraR in complex with the effector L-arabinose has been determined at 2.2 Å resolution. The L-arabinose binding affinity was characterized by isothermal titration calorimetry and differential scanning fluorimetry; the K(d) value was 8.4 ± 0.4 µM. The effect of L-arabinose on the protein oligomeric state was investigated in solution and detailed analysis of the crystal identified a dimer organization which is distinctive from that of other members of the GalR/LacI family.
- MeSH
- arabinosa metabolismus MeSH
- Bacillus subtilis chemie metabolismus MeSH
- bakteriální proteiny chemie metabolismus MeSH
- krystalografie rentgenová MeSH
- molekulární modely MeSH
- represorové proteiny chemie metabolismus MeSH
- terciární struktura proteinů MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A pBBad22T-derived conditioned arabinose (Ara)-inducible expression system was evaluated in Stenotrophomonas maltophilia (an opportunistic pathogen and has gained increasing attention as a cause of healthcare-associated infection). S. maltophilia cannot grow well when Ara is the sole available carbon source. The induction kinetic study, optimal inducer concentration determination, and depletion experiment were performed by using a xylE gene fusion construct, pBxylE, to monitor the expression of pBBad22T in S. maltophilia. For induction survey, the expression of catechol 2,3-dioxygenase (C23O), encoded by xylE gene, continuously increases during an 8-h induced course and can be modulated by different inducer concentrations. The applied induction condition of pBBad22T in S. maltophilia is the inducer concentration ranging from 0.1% to 0.5% for an induction time of 4 h. For repression evaluation, the C23O expression is rapidly turned off within 30 min after the removal of Ara. Accordingly, the established Ara-inducible system can provide a convenient tool for the study of S. maltophilia.
- MeSH
- arabinosa metabolismus MeSH
- bakteriální proteiny genetika metabolismus MeSH
- katechol-2,3-dioxygenasa genetika metabolismus MeSH
- regulace genové exprese u bakterií MeSH
- Stenotrophomonas maltophilia enzymologie genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- arabinosa chemie izolace a purifikace MeSH
- Chlorella cytologie chemie MeSH
- chromatografie plynová metody využití MeSH
- finanční podpora výzkumu jako téma MeSH
- galaktosa chemie izolace a purifikace MeSH
- kyselina glukarová chemie izolace a purifikace MeSH
- monosacharidy analýza chemie MeSH
- polysacharidy analýza chemie MeSH
- spektrální analýza metody využití MeSH
- MeSH
- arabinosa analogy a deriváty chemie metabolismus MeSH
- Aspergillus niger enzymologie MeSH
- chromatografie na tenké vrstvě MeSH
- finanční podpora výzkumu jako téma MeSH
- glykosidhydrolasy chemie metabolismus MeSH
- karboxylesterhydrolasy chemie izolace a purifikace metabolismus MeSH
- koncentrace vodíkových iontů MeSH
- nitrofenoly analýza chemie MeSH
