RESUMO
Janibacter sp. strain R02 (BNM 560) was isolated in our laboratory from an Antarctic soil sample. A remarkable trait of the strain was its high lipolytic activity, detected in Rhodamine-olive oil supplemented plates. Supernatants of Janibacter sp. R02 displayed superb activity on transesterification of acyl glycerols, thus being a good candidate for lipase prospection. Considering the lack of information concerning lipases of the genus Janibacter, we focused on the identification, cloning, expression and characterization of the extracellular lipases of this strain. By means of sequence alignment and clustering of consensus nucleotide sequences, a DNA fragment of 1272bp was amplified, cloned and expressed in E. coli. The resulting recombinant enzyme, named LipJ2, showed preference for short to medium chain-length substrates, and displayed maximum activity at 80°C and pH 8-9, being strongly activated by a mixture of Na+ and K+. The enzyme presented an outstanding stability regarding both pH and temperature. Bioinformatics analysis of the amino acid sequence of LipJ2 revealed the presence of a consensus catalytic triad and a canonical pentapeptide. However, two additional rare motifs were found in LipJ2: an SXXL ß-lactamase motif and two putative Y-type oxyanion holes (YAP). Although some of the previous features could allow assigning LipJ2 to the bacterial lipase families VIII or X, the phylogenetic analysis showed that LipJ2 clusters apart from other members of known lipase families, indicating that the newly isolated Janibacter esterase LipJ2 would be the first characterized member of a new family of bacterial lipases.
Assuntos
Actinobacteria/enzimologia , Actinobacteria/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Lipase/genética , Lipase/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/classificação , Clonagem Molecular , Sequência Conservada , Estabilidade Enzimática , Escherichia coli/enzimologia , Escherichia coli/genética , Esterases/classificação , Esterases/genética , Esterases/metabolismo , Expressão Gênica , Genes Bacterianos , Temperatura Alta , Concentração de Íons de Hidrogênio , Cinética , Lipase/classificação , Modelos Moleculares , FilogeniaRESUMO
Glycosidases provide a powerful resource for in vitro synthesis of novel anomerically pure glycosides. Generation of new low molecular weight galactosides is of interest since they are potential galectin inhibitors. Galectins are molecular targets for cancer therapy and thus their inhibitors are potential antitumor agents. Here we report the enzymatic synthesis and structural characterization of 2-aminoethyl ß-D-galactopyranoside. Critical parameters for transgalactosylation using either soluble or immobilized enzyme were investigated and optimized for the galactoside synthesis. We found that 0.2 M lactose, and 0.5 M 2-aminoethanol at 50 °C for 30 min were the optimal conditions for synthesis. 2-Aminoethanol proved to be an enzyme inhibitor, fitting a mixed inhibition model with inhibition constants, K(ic)=0.31±0.04 M and K(iu)=0.604±0.035 M.
Assuntos
Aspergillus oryzae/enzimologia , Galactose/biossíntese , beta-Galactosidase/metabolismo , Catálise , Galactosídeos/metabolismo , Glicosídeo Hidrolases/metabolismoRESUMO
The synthesis of novel galactosides is interesting because of their important role in several biological processes. Their properties greatly depend upon the configuration and type of galactoside. Therefore, to study biological activity, it is essential to elucidate the structure of the products. Glycosidases are capable of catalyzing glycosidic linkages with absolute stereoselectivity of the anomeric center. We report the enzymatic synthesis of galactosyl-ethylene glycol, galactosyl-glycerol, and galactosyl-erythritol by immobilized beta-galactosidase from Aspegillus oryzae. The obtained galactosides were isolated and fully characterized by an extensive nuclear magnetic resonance (NMR) study. Complete structure elucidation and full proton and carbon assignments were carried out using 1D ((1)H and (13)C) and 2D (gCOSY, TOCSY, multiplicity-edited gHSQC, and gHMBC) NMR experiments. The beta-galactosidase from A. oryzae showed a strong preference for primary alcohols. For galactosyl-glycerol and galactosyl-erythritol, this preference generated one and two chiral centers, respectively, and a mixture of stereoisomers was obtained as a consequence.
Assuntos
Aspergillus oryzae/enzimologia , Enzimas Imobilizadas/metabolismo , Proteínas Fúngicas/metabolismo , Galactosídeos/química , Lactose/metabolismo , Polímeros/metabolismo , beta-Galactosidase/metabolismo , Aspergillus oryzae/química , Enzimas Imobilizadas/química , Proteínas Fúngicas/química , Galactosídeos/metabolismo , Estereoisomerismo , beta-Galactosidase/químicaRESUMO
Addition of tags [such as His (histidine) tags] is extremely helpful for the affinity purification of recombinant proteins. In several cases, these tags must be removed before performing functional and structural studies. The enzyme most frequently used to cleave tags of recombinant proteins is the TEV-protease (tobacco-etch-virus NIa protease). The continuous production of this enzyme in soluble form is quite an expensive process and not easily accessible to many laboratories. Thus an interesting alternative is the use of TEV-protease in an immobilized form, which may be reutilized several times. The main objective of the present study was to obtain a TEV-protease in an immobilized form, by covalent immobilization on to solid supports through selective use of different amino acid residues, lysine or cysteine. High protein immobilization yields (75-97%) were obtained with both strategies. The TEV-protease immobilized through its exposed cysteine thiol groups maintained its ability for cleaving a 20 kDa substrate. While the activity of the immobilized TEV-protease maintained only 30% of the activity of the enzyme in soluble form, its stability at 4 degrees C was improved three times. Moreover, this enzyme could be reutilized in at least five cycles of cleavage without loss of performance. The present results indicate that the use of a TEV-protease in an immobilized form is a potentially useful tool for the cleavage of His tags of recombinant proteins and may be useful for reducing the cost of the total process of cleavage.
Assuntos
Endopeptidases/metabolismo , Enzimas Imobilizadas/metabolismo , Potyvirus/enzimologia , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Virais/metabolismo , Endopeptidases/química , Estabilidade Enzimática , Enzimas Imobilizadas/química , Glutaral/química , Histidina/metabolismo , Cinética , Oligopeptídeos/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificação , Sefarose/química , Proteínas Virais/químicaRESUMO
beta-Galactosidase (Escherichia coli) was immobilized through its thiol groups on thiolsulfinate-agarose gel. After enzyme immobilization, different nano-environments were generated by reacting the excess of gel-bound thiolsulfinate moieties with 2-mercaptoethanesulfonic acid (S-gel), glutathione (G-gel), cysteamine (C-gel), and mercaptoethanol (M-gel). Concerning thermal stability at 50 degrees C, the G-gel and the M-gel derivatives were the most stable with residual activity values of 67% and 45%, respectively. The stability in several solvent systems was studied: ethyl acetate (1.6% vol/vol), ethylene glycol (50% vol/vol), and 2-propanol (50% vol/vol). In ethyl acetate, both the M-gel and S-gel were highly stabilized; the time required for activity to decay to 80% of the initial activity was increased 29-fold for the M-gel and 20-fold for the S-gel with respect to the soluble enzyme. The G-gel was the least stable of all the derivatives. The different behaviors of the derivatives in thermal and solvent stability studies suggest that each nano-environment contributes differently to the enzyme stability, depending on the denaturing conditions. Therefore, it may be possible to tailor the matrix surface to maximize enzyme stability in particular applications.