New approach for petroleum hydrocarbon degradation using bacterial spores entrapped in chitosan beads.

Spores of Bacillus subtilis LAMI008 were entrapped in 3-mm chitosan beads and cross-linked with 0.3% glutaraldehyde for n-hexadecane biodegradation and biosurfactant recovery. When exposed to nutrients, the spores generated vegetative cells without morphological alterations as revealed by atomic force microscopy. The entrapped cells degraded almost 100% of 1% of n-hexadecane in medium supplemented with 1% glucose and produce biosurfactant within 48 h, as well as free cells. The number of viable cells inside the beads was maintained throughout the n-hexadecane degradation process and the released biosurfactant was not used as a carbon source. Entrapment of bacterial spores in chitosan beads overcomes problems with stability, storage, and long term cell viability encountered with vegetative cells. This approach can potentially be utilized for biodegradation of complex compounds by entrapping spores of different species of bacteria.

In vivo lymphocyte activation and apoptosis by lectins of the Diocleinae subtribe.

This paper reports the overall effects of three lectins, extracted from Canavalia brasiliensis, Dioclea violacea, and D. grandiflora, on BALB/c mice popliteal draining lymph nodes. These lectins have presented high stimulatory capacity on lymph node T cells. Additionally, they were able to induce apoptosis and inflammation (frequently associated with high endothelial venule necrosis). The data presented here suggest that the Diocleinae lectins studied can stimulate in vivo T cell activation and apoptosis, as well as present important side effects.

The amino-acid sequence of the glucose/mannose-specific lectin isolated from Parkia platycephala seeds reveals three tandemly arranged jacalin-related domains.

A mannose/glucose-specific lectin was isolated from seeds of Parkia platycephala, the most primitive subfamily of Leguminosae plants. The molecular mass of the purified lectin determined by mass spectrometry was 47 946 +/- 6 Da (by electrospray ionization) and 47 951 +/- 9 Da (by matrix-assisted laser-desoption ionization). The apparent molecular mass of the lectin in solutions of pH in the range 4.5-8.5 determined by analytical ultracentrifugation equilibrium sedimentation was 94 +/- 3 kDa, showing that the protein behaved as a non-pH-dependent dimer. The amino-acid sequence of the Parkia lectin was determined by Edman degradation of overlapping peptides. This is the first report of the primary structure of a Mimosoideae lectin. The protein contained a blocked N-terminus and a single, nonglycosylated polypeptide chain composed of three tandemly arranged homologous domains. Each of these domains shares sequence similarity with jacalin-related lectin monomers from Asteraceae, Convolvulaceae, Moraceae, Musaceae, Gramineae, and Fagaceae plant families. Based on this homology, we predict that each Parkia lectin repeat may display a beta prism fold similar to that observed in the crystal structure of the lectin from Helianthus tuberosus. The P. platycephala lectin also shows sequence similarity with stress- and pathogen-upregulated defence genes of a number of different plants, suggesting a common ancestry for jacalin-related lectins and inducible defence proteins.

Purification, chemical, and immunochemical properties of a new lectin from Mimosoideae (Parkia discolor).

A glucose/mannose-binding lectin was isolated from seeds of Parkia discolor (Mimosoideae) using affinity chromatography on Sephadex G-100 gel. The protein presented a unique component in SDS-PAGE corresponding to a molecular mass of 58,000 Da, which is very similar to that of a closely related lectin from Parkia platycephala. Among the simple sugars tested, mannose was the best inhibitor, but biantennary glycans, containing the trimannoside core, present in N-glycoproteins, also seem to be powerful inhibitors of the haemagglutinating activity induced by the purified lectin. The protein was characterised by high content of glycine and proline and absence of cysteine. Rabbit antibodies, anti-P. platycephala seed lectin, recognised the P. discolor lectin. However, no cross-reaction was observed when a set of other legume lectins from sub-family Papilionoideae and others from families Moraceae and Euphorbiaceae were assayed with the Parkia lectins. This suggests that Parkia lectins comprise a new group of legume lectins exhibiting distinct characteristics.

Demonstration of a conserved histidine and two water ligands at the Mn2+ site in Diocleinae lectins by pulsed EPR spectroscopy.

Lectins from the Diocleinae subtribe, including Canavalia brasiliensis, Canavalia bonariensis, Canavalia grandiflora, Cratylia floribunda, Dioclea grandiflora, Dioclea guianensis, Dioclea rostrata, Dioclea violacea, and Dioclea virgata, have been recently isolated and characterized in terms of their carbohydrate binding specificities. Although all of the lectins are Man/Glc specific, they possess different biological activities. In the present study, electron paramagnetic resonance (EPR) spectroscopy demonstrates that all nine Diocleinae lectins contain Mn2+. The spectra of C. floribunda and D. rostrata suggest Mn2+ site symmetry different from that of the other seven lectins. However, electron spin-echo envelope modulation (ESEEM) spectroscopy indicates that all nine lectins are coordinated to a histidyl imidazole, with similar electron-nuclear coupling to the Mn2+-bound imidazole nitrogen. ESEEM also demonstrates ligation of two water molecules to Mn2+ in all nine Diocleinae lectins. Thus, the EPR and ESEEM data indicate the presence of a Mn2+ binding site in the above Diocleinae lectins with a conserved histidine residue and two water ligands.

Molecular characterization and crystallization of Diocleinae lectins.

Molecular characterization of seven Diocleinae lectins was assessed by sequence analysis, determination of molecular masses by mass spectrometry, and analytical ultracentrifugation equilibrium sedimentation. The lectins show distinct pH-dependent dimer-tetramer equilibria, which we hypothesize are due to small primary structure differences at key positions. Lectins from Dioclea guianensis, Dioclea virgata, and Cratylia floribunda seeds have been crystallized and preliminary X-ray diffraction analyses are reported.

Purification and characterization of a lectin from seeds of Vatairea macrocarpa Duke.

A lectin from Vatairea macrocarpa Duke seeds (VML) was isolated using affinity chromatography on a guar gum column. The lectin, a glycoprotein without erythrocyte specificity, displays specificity to galactose and some derivatives. On SDS-polyacrylamide gels, V. macrocarpa seed lectin is composed of two major high-Mr bands of 34 and 32 kDa and two minor low-Mr bands of 22 and 13 kDa. N-Terminal sequencing showed that the 34, 32, and 13 kDa products possess identical N-terminal sequence, which display best similarity with the N-terminal portion of Robinia pseudoacacia lectins (RPL). On the other hand, the N-terminal sequence of the 22 kDa band can be aligned with an internal sequence of RPL starting at residue 149 of the cDNA-derived sequence. These data indicate that, like other leguminous lectins, VML is made up of a mixture of one-chain 30-35 kDa glycoforms and of 22 and 13 kDa endogenous C- and N-terminal fragments. Size-exclusion chromatography indicated that, at neutral pH, VML is predominantly a dimeric (70 kDa) protein, although tetramers (115 kDa) and larger aggregates (300 kDa) were also present.

Amino acid sequence, glycan structure, and proteolytic processing of the lectin of Vatairea macrocarpa seeds.

VML is a galactose-binding lectin isolated from Vatairea macrocarpa seeds. By SDS-polyacrylamide gel electrophoresis, VML is a glycoprotein composed of a major 32-34 kDa double band (alpha-chain) and minor 22 kDa and 13 kDa bands. N-terminal sequencing of electroblotted samples showed that the 22 and 13 kDa bands corresponded to C-(beta) and N-(gamma) terminal fragments of the alpha-chain, respectively. The primary structure of VML displays similarity with other leguminous lectins, particularly with Erythrina variegata, Robinia pseudoacacia and Sophora japonica lectins. VML is N-glycosylated at asparagine residues at positions 111 and 183 with one major glycan structure. Tandem mass spectrometry and methylation analysis indicated the presence of Manalpha1-6[(Manalpha1-3)(Xylbeta1-2)]Manbeta1-4 -GlcNAcbeta1-4(Fucalpha1-3)GlcNAc, a typical plant Nglycan. Equilibrium sedimentation analysis by analytical centrifugation showed that VML had a mass of 122-130 kDa, which did not change within the pH range 2.5-8.5. These data indicated that VML is a pH-independent homotetrameric protein and that a small proportion of the alpha-subunits is cleaved into noncovalently associated N- and C-terminal fragments. Mass spectrometric analysis suggested a mechanism for the proteolytic processing of VML. V. macrocarpa lectin contains a mixture of doubly (28,525 Da) and singly (27,354 Da) glycosylated alpha-chains. Deglycosylation of Asn-111 correlates with proteolytic cleavage of the Asn-114-Lys-115 bond yielding glycosylated gamma (residues 1-114, 12,304 Da) and nonglycosylated beta-(residues 115-239, 14,957 Da) chains. Some beta-chain molecules are further deglycosylated and N-terminally processed yielding products of molecular masses of 13,783 Da and 13,670 Da.

Molecular cloning and characterization of ConBr, the lectin of Canavalia brasiliensis seeds.

ConBr, a lectin isolated from Canavalia brasiliensis seeds, shares with other legume plant lectins from the genus Canavalia (Diocleinae subtribe) primary carbohydrate recognition specificity for D-mannose and D-glucose. However, ConBr exerts different biological effects than concanavalin A, the lectin of Canavalia ensiformis seeds, regarding induction of rat paw edema, peritoneal macrophage spreading in mouse, and in vitro human lymphocyte stimulation. The primary structure of ConBr was established by cDNA cloning, amino acid sequencing, and mass spectrometry. The 237-amino-acid sequence of ConBr displays Ser/Thr heterogeneity at position 96, indicating the existence of two isoforms. The mature Canavalia brasiliensis lectin monomer consists of a mixture of predominantly full-length polypeptide (alpha-chain) and a small proportion of fragments 1-118 (beta-chain) and 119-237 (gamma-chain). Although ConBr isolectins and concanavalin A differ only in residues at positions 58, 70, and 96, ConBr monomers associate into dimers and tetramers in a different pH-dependent manner than those of concanavalin A. The occurrence of glycine at position 58 does not allow formation of the hydrogen bond that in the concanavalin A tetramer exists between Asp58 of subunit A and Ser62 of subunit C. The consequence is that the alpha carbons of the corresponding residues in ConBr are 1.5 A closer that in concanavalin A, and ConBr adopts a more open quaternary structure than concanavalin A. Our data support the hypothesis that substitution of amino acids located at the subunit interface of structurally related lectins of the same protein family can lead to different quaternary conformations that may account for their different biological activities.

The crystal structure of Canavalia brasiliensis lectin suggests a correlation between its quaternary conformation and its distinct biological properties from Concanavalin A.

Canavalia brasiliensis lectin was isolated from the seeds of a Brazilian autochthonous Leguminosae plant. Despite extensive amino acid sequence similarity with Concanavalin A, C. brasiliensis lectin exerts in vitro and in vivo cellular effects that are markedly different from those displayed by Concanavalin A. We have solved the crystal structure of the C. brasiliensis lectin at 3.0 A resolution. The three-dimensional structure of the lectin monomer can be superimposed onto that of Concanavalin A with a root-mean-square deviation for all C alpha atoms of 0.65 A. However, this parameter is 0.84 and 1.62 A when the C. brasiliensis lectin dimer and tetramer, respectively, are compared with the same structures of Concanavalin A. We suggest that these differences in quaternary structure may account for the different biological properties of these two highly related Leguminosae lectins.