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PubMed1991

L-guluronan-specific alginate lyase from a marine bacterium associated with Sargassum.

The major extracellular alginate lyase activities secreted by a Gram-negative, facultative bacterium associated with actively growing Sargassum fluitans have been resolved an examined for substrate specificity. A fraction excluded from Sephadex G-75 was equally active toward (1----4)-beta-D-mannuronan, (1----4)-alpha-L-guluronan, and alginate with the formation of di- and tri-saccharides as apparent limit products and oligo-saccharides indicative of an endolytic mechanism. A second fraction which was included during G-75 filtration was inactive toward D-mannuronan and 4 times more active toward L-guluronan than native alginate. Proton magnetic resonance spectrometry identified the primary product of this enzyme as O-(4-deoxy-alpha-L-erythro-hex-4-enopyranosyluronic acid)-(1----4)-O-(alpha-L-gulopyranosyluronic acid)-(1----4)-O-alpha-L-gulopyranuronic acid. The L-guluronan-specific enzyme requires 0.5 M NaCl for maximal activity and has been purified as a monomeric protein having an apparent molecular mass of 38 kD and an approximate pI of 4.5. The predominant formation of trisaccharide over the course of a reaction showed a primarily exolytic mechanism, indicating an enzyme activity unique from any previously reported.

Carbohydrate Sequence↗
PubMed2000

Force measurements on cell repellant and cell adhesive alginic acid coated surfaces.

The atomic force microscope (AFM) was used to acquire force versus distance curves between the cantilever tip and samples bearing a surface overlayer of covalently linked alginic acid. The alginic acid coating resists cell-adhesion in in vitro experiments involving a normal and a tumor cell line. However, the surface becomes cell adhesive when alginic acid coated samples are subjected to glow discharge treatment. Force curves show in both cases the typical features resulting from the interaction between the cantilever tip and a hydrophilic, compressible polymer overlayer, suggesting that in both cases a diffuse interface with water exists. Following some recent findings on oligoethyleneglycol-terminated self-assembled-monolayers, it is suggested that conformational and molecular aspects of hydrophilic surface layers, rather than steric repulsion effects, could play a significant role in the mechanism that controls resistance to bio-adhesion.

Journal Article↗
PubMed2006

Facile synthesis of polyaniline-sodium alginate nanofibers.

This work demonstrates a facile route to the synthesis of large quantities of uniform polyaniline-sodium alginate (PANI-SA) nanofibers template-guided by SA. This approach is an easy, inexpensive, environmentally friendly, and scalable one-step method to produce uniform nanofibers with controllable average diameters in bulk quantities. We started with biopolymer-monomer complexes formed between the carboxylic groups of SA and the amino group of an organic monomer (aniline). When ammonium persulfate was added, such polymer-monomer complexes could be polymerized. Then, polyaniline-sodium alginate nanofibers with uniform diameters from 40 to 100 nm were successfully obtained in a high yield. The resultant PANI-SA nanofibers were characterized by means of different techniques, such as ultraviolet-visible spectroscopy, thermogravimetric analysis, wide-angle X-ray diffraction, Fourier transform infrared spectroscopy, and scanning and transmission electron microscopy methods. The mechanism governing the formation of the polyaniline-sodium alginate nanofibers is discussed.

Journal Article↗
PubMed2002

Primary structure and chemical modification of some amino acid residues of bifunctional alginate lyase from a marine bacterium Pseudoalteromonas sp. strain no. 272.

The entire amino acid sequence of bifunctional alginate lyase from Pseudoalteromonas sp. strain No. 272 were determined by two approaches, Edman degradation of the peptides obtained from protease digestion of the enzyme protein and analysis of PCR products of the structural gene. The former resulted in incomplete amino acid sequence in the entire sequence, due to lacking of the proper peptides from the protease digestion. To compensate for this lack of sequences we applied the method of PCR of the structural gene that was initially elucidated from the primers designed from N- and C-terminal amino acid sequences of the enzyme. The results of the amino acid sequences from these two approaches showed good agreement. The enzyme consisted of 233 amino acid residues with a molecular mass of 25,549.5, including the sole W and cystine residue. The sequence homology search among the other alginate lyases from different origins indicated that they were very weakly homologous, with the exception of the sequence homology (80.3%) of Pseudoalteromonas elyakovii alginate lyase. The consensus sequence, YFKhG + Y-Q (Wong, T. Y., Preston, L. A., and Schiller, N. L. 2000. Annu. Rev. MicrobioL 54: 289-340) in the C-terminal regions was conserved. The kinetic analyses of chemical modification of some amino acid residues of the enzyme showed that W, K, and Y appeared to be important in the enzyme function.

Amino Acid Sequence↗
PubMed1997

Identification of the histidine protein kinase KinB in Pseudomonas aeruginosa and its phosphorylation of the alginate regulator algB.

The exopolysaccharide alginate is an important virulence factor in chronic lung infections caused by the bacterium Pseudomonas aeruginosa. Two positive activators for alginate synthesis, algB and algR, are members of a superfamily of response regulators of the two-component regulatory system. AlgB belongs to the NtrC subfamily of response regulators and is required for high-level production of alginate. In this study, an open reading frame encoding a polypeptide of 66 kDa, designated kinB, was identified immediately downstream of algB. The sequence of KinB is homologous to the histidine protein kinase members of two-component regulatory systems. Western blot analysis of a P. aeruginosa strain carrying a kinB-lacZ protein fusion and studies of kinB-phoA fusions indicate that KinB localizes to the inner membrane and has a NH2-terminal periplasmic domain. A KinB derivative containing the COOH terminus of KinB was generated and purified. In the presence of [gamma-32P]ATP, the purified COOH-terminal KinB protein was observed to undergo progressive autophosphorylation in vitro. Moreover, the phosphoryl label of KinB could be rapidly transferred to purified AlgB. Substitutions of the residues conserved among histidine protein kinases abolished KinB autophosphorylation. These results provide evidence that kinB encodes the AlgB cognate histidine protein kinase.

Amino Acid Sequence↗
PubMed1999

The recombinant Azotobacter vinelandii mannuronan C-5-epimerase AlgE4 epimerizes alginate by a nonrandom attack mechanism.

The Ca2+-dependent mannuronan C-5-epimerase AlgE4 is a representative of a family of Azotobacter vinelandii enzymes catalyzing the polymer level epimerization of beta-D-mannuronic acid (M) to alpha-L-guluronic acid (G) in the commercially important polysaccharide alginate. The reaction product of recombinantly produced AlgE4 is predominantly characterized by an alternating sequence distribution of the M and G residues (MG blocks). AlgE4 was purified after intracellular overexpression in Escherichia coli, and the activity was shown to be optimal at pH values between 6.5 and 7.0, in the presence of 1-3 mM Ca2+, and at temperatures near 37 degrees C. Sr2+ was found to substitute reasonably well for Ca2+ in activation, whereas Zn2+ strongly inhibited the activity. During epimerization of alginate, the fraction of GMG blocks increased linearly as a function of the total fraction of G residues and comparably much faster than that of MMG blocks. These experimental data could not be accounted for by a random attack mechanism, suggesting that the enzyme either slides along the alginate chain during catalysis or recognizes a pre-existing G residue as a preferred substrate in its consecutive attacks.

Azotobacter vinelandii↗
PubMed2001

The catalytic activities of the bifunctional Azotobacter vinelandii mannuronan C-5-epimerase and alginate lyase AlgE7 probably originate from the same active site in the enzyme.

The Azotobacter vinelandii genome encodes a family of seven secreted Ca(2+)-dependent epimerases (AlgE1--7) catalyzing the polymer level epimerization of beta-D-mannuronic acid (M) to alpha-L-guluronic acid (G) in the commercially important polysaccharide alginate. AlgE1--7 are composed of two types of protein modules, A and R, and the A-modules have previously been found to be sufficient for epimerization. AlgE7 is both an epimerase and an alginase, and here we show that the lyase activity is Ca(2+)-dependent and also responds similarly to the epimerases in the presence of other divalent cations. The AlgE7 lyase degraded M-rich alginates and a relatively G-rich alginate from the brown algae Macrocystis pyrifera most effectively, producing oligomers of 4 (mannuronan) to 7 units. The sequences cleaved were mainly G/MM and/or G/GM. Since G-moieties dominated at the reducing ends even when mannuronan was used as substrate, the AlgE7 epimerase probably stimulates the lyase pathway, indicating a complex interplay between the two activities. A truncated form of AlgE1 (AlgE1-1) was converted to a combined epimerase and lyase by replacing the 5'-798 base pairs in the algE1-1 gene with the corresponding A-module-encoding DNA sequence from algE7. Furthermore, substitution of an aspartic acid residue at position 152 with glycine in AlgE7A eliminated almost all of both the lyase and epimerase activities. Epimerization and lyase activity are believed to be mechanistically related, and the results reported here strongly support this hypothesis by suggesting that the same enzymatic site can catalyze both reactions.

Amino Acid Sequence↗
PubMed2003

Crystallization and preliminary X-ray analysis of alginate lyase, a member of family PL-7, from Pseudomonas aeruginosa.

Alginate lyase depolymerizes alginate, a heteropolysaccharide consisting of alpha-L-guluronate and beta-D-mannuronate, through a beta-elimination reaction. A protein PA1167 with a molecular mass of 25 kDa produced by Pseudomonas aeruginosa is an alginate lyase classified into polysaccharide lyase family PL-7. The enzyme was crystallized at 293 K in a drop solution comprising 1.4 M sodium chloride, 0.1 M potassium sodium phosphate and 0.1 M 2-morpholinoethanesulfonate-sodium hydroxide pH 6.5 by means of the vapor-diffusion method. The crystals were monoclinic and belonged to space group P2(1), with unit-cell parameters a = 43.4, b = 70.3, c = 67.4 A, beta = 94.5 degrees. Diffraction data were collected to 2.0 A from a single crystal.

Carbohydrate Sequence↗
PubMed1996

Nucleoside diphosphate kinase from Pseudomonas aeruginosa: characterization of the gene and its role in cellular growth and exopolysaccharide alginate synthesis.

We report the cloning and determination of the nucleotide sequence of the gene encoding nucleoside diphosphate kinase (Ndk) from Pseudomonas aeruginosa. The amino acid sequence of Ndk was highly homologous with other known bacterial and eukaryotic Ndks (39.9 to 58.3% amino acid identity). We have previously reported that P. aeruginosa strains with mutations in the genes algR2 and algR2 algH produce extremely low levels of Ndk and, as a consequence, are defective in their ability to grow in the presence of Tween 20, a detergent that inhibits a kinase which can substitute for Ndk. Hyperexpression of ndk from the clone pGWS95 in trans in the P. aeruginosa algR2 and algR2 algH double mutant restored Ndk production to levels which equalled or exceeded wild-type levels and enabled these strains to grow in the presence of Tween 20. Hyperexpression of ndk from pGWS95 in the P. aeruginosa algR2 mutant also restored alginate production to levels that were approximately 60% of wild type. Nucleoside diphosphate kinase activity was present in both the cytosolic and membrane-associated fractions of P. aeruginosa. The cytosolic Ndk was non-specific in its transfer activity of the terminal phosphate from ATP to other nucleoside diphosphates. However, the membrane form of Ndk was more active in the transfer of the terminal phosphate from ATP to GDP resulting in the predominant formation of GTP. We report in this work that pyruvate kinase and Ndk form a complex which alters the specificity of Ndk substantially to GTP. The significance of GTP in signal transduction events within the cell and in the production of GDP-mannose, an essential alginate precursor, clearly indicates the importance of Ndk in cellular processes as well as in alginate synthesis.

Amino Acid Sequence↗
PubMed1995

Overproduction and properties of the mannuronate alginate lyase AlxMB.

In previous studies (Malissard et al., FEMS Microbiol. Lett. (1993) 110, 101-106), the alginate lyase AlxM of the marine bacterium ATCC 433367 was produced in Escherichia coli TC4/pAL-A3 with a yield of 50 micrograms per litre of culture. The polypeptide chain was cleaved between two cysteine residues, C169 and C183, themselves linked by a disulphide bridge. AlxM has now been overproduced in E. coli BL21(DE3)/pAL-Sur/pLysS. Under conditions in which formation of inclusion bodies can be avoided, the enzyme is synthesized as a catalytically active, water-soluble, unnicked polypeptide with a yield of 32 mg per litre of culture. It has been purified to protein homogeneity using a one-step procedure. The nicked AlxMA and unnicked AlxMB alginate lyases have identical alginate-degrading activities at high salt concentrations.

Amino Acid Sequence↗
PubMed2006

Purification and characterization of alginate lyase from marine Vibrio sp. YWA.

Extracellular alginate lyase secreted by marine Vibrio sp. YWA, isolated from decayed Laminaria japonica, was purified by a combination of ammonium sulfate precipitation and diethylaminoethyl-Sephacel column chromatography. The results show that the molecular mass of alginate lyase was approximately 62.5 kDa, with an optimal pH and temperature at pH 7.0 and 25 degrees C, respectively. K(m) was approximately 72.73 g/L. The activity of the enzyme was enhanced by EDTA and Zn(2+), but inhibited by Ba(2+). The substrates specificity analysis shows that it was specific for hydrolyzing poly-beta-D-1,4-mannuronate in alginate.

Enzyme Activation↗
PubMed1982

Isolation of marine bacteria capable of producing specific lyases for alginate degradation.

Alginate lyases (EC 4.2.2.3) were isolated from cultures of several marine bacterial isolates. The lyases were induced by native alginate and had activity toward both the mannuronic acid and the guluronic acid blocks of the alginate polymer. The guluronic acid-specific lyase was recovered from the medium, whereas the mannuronic acid-specific lyase was retained with the bacteria.

Journal Article↗
PubMed1990

Enhancing the Viability of Lactobacillus plantarum Inoculum by Immobilizing the Cells in Calcium-Alginate Beads Incorporating Cryoprotectants.

Many literature reports have cited the importance of the rehydration conditions of lyophilized cultures in determining viability. The rate of rehydration and the volume of fluid used have been identified as two important factors. One possible means of controlling these is by immobilizing the cells before lyophilization within a gel matrix in which the subsequent rehydration rate and fluid volume would be controlled by the properties of the gel. In this study Lactobacillus plantarum was immobilized and lyophilized in Ca-alginate beads in which 1 M glycerol or 0.75 M adonitol with skim milk were incorporated as a cryoprotectant. The properties of these Ca-alginate beads were examined before and after lyophilization and rehydration. The beads incorporating glycerol were smaller and stronger than those with adonitol. After lyophilization, size decreased and strength increased but to a greater extent in the beads with glycerol, indicating that the microenvironment within the two bead types was probably different. The protective effect of the bead microenvironment on immobilized L. plantarum was also examined. Lyophilization and rehydration within the alginate beads with either polyol yielded higher survival rates than that attained with free cell cultures during rehydration in optimal or suboptimal conditions. During rehydration under suboptimal conditions, the immobilized cell survival was greatest when 0.75 M adonitol was the incorporated cryoprotectant.

Journal Article↗
PubMed1991

Cloning of alginate lyase gene (alxM) and expression in Escherichia coli.

The alxM gene encoding a D-mannuronan-specific alginate lyase has been cloned from a marine bacterium isolated as an epiphyte on the brown alga, Sargassum fluitans. Expression of this gene in Escherichia coli provides a source of this enzyme for probing alginate structure and modifying the mannuronan-rich alginate polymers produced by bacterial pathogens.

Blotting, Southern↗
PubMed1993

Involvement of the alginate algT gene and integration host factor in the regulation of the Pseudomonas aeruginosa algB gene.

Strains of Pseudomonas aeruginosa causing pulmonary infection in cystic fibrosis patients are often mucoid because of the synthesis of a capsular polysaccharide called alginate. Regulation of alginate biosynthesis includes the algB gene product (AlgB), which belongs to a class of proteins that control gene transcription in response to environmental stimuli. In this study, a homolog of the DNA-binding-and-bending protein integration host factor (IHF) and the positive regulatory gene algT were shown to be involved in algB expression. An algB-cat gene fusion was constructed on a low-copy-number, broad-host-range plasmid. In alginate-producing (Alg+) P. aeruginosa, levels of chloramphenicol acetyltransferase from algB-cat were twofold higher than in spontaneous Alg- or algT::Tn501 mutant strains, indicating that the mucoid status of the cell influences algB transcription. An algB transcription initiation site was identified 286 nucleotides upstream of translation initiation and revealed an Escherichia coli sigma 70-like promoter. Sequences in the algB promoter region were highly similar to the consensus E. coli IHF binding site. In DNA gel band mobility shift assays, a protein present in extracts from IHF+ E. coli strains and IHF purified from E. coli bound specifically to these algB DNA fragments, while extracts prepared from isogenic IHF- E. coli strains failed to alter the mobility of algB DNA fragments containing the consensus IHF binding site. A protein in cell extracts prepared from P. aeruginosa strains also demonstrated binding to algB fragments containing the IHF binding site, and the position of the complex formed with these extracts was identical to that of the complex formed with purified IHF. Moreover, this binding could be inhibited by anti-IHF antibodies. To test the role of the IHF site in algB regulation, site-specific mutations in the algB IHF site, based on changes which severely affect IHF binding in E. coli, were generated. When either purified E. coli IHF or extracts from P. aeruginosa were used in DNA binding studies, the algB mutant DNAs were severely reduced in IHF binding. Mutations affecting IHF binding at the algB promoter were introduced into the algB-cat plasmid, and all resulted in severely impaired transcriptional activity in Alg- and algT mutant strains of P. aeruginosa. However, these mutations resulted in similar or slightly reduced algB-cat transcription in Alg+ and algB::Tn501 mutant strains. Thus, the algT product plays a positive role in the high-level expression of algB in mucoid cells, whereas as protein present in P.aeruginosa extracts which is likely an IHF homolog plays a positive role in maintaining a basal level of algB expression in nonmucoid strains.

Bacterial Proteins↗
PubMed1999

Cloning and expression of the algL gene, encoding the Azotobacter chroococcum alginate lyase: purification and characterization of the enzyme.

The alginate lyase-encoding gene (algL) of Azotobacter chroococcum was localized to a 3.1-kb EcoRI DNA fragment that revealed an open reading frame of 1,116 bp. This open reading frame encodes a protein of 42.98 kDa, in agreement with the value previously reported by us for this protein. The deduced protein has a potential N-terminal signal peptide that is consistent with its proposed periplasmic location. The analysis of the deduced amino acid sequence indicated that the gene sequence has a high homology (90% identity) to the Azotobacter vinelandii gene sequence, which has very recently been deposited in the GenBank database, and that it has 64% identity to the Pseudomonas aeruginosa gene sequence but that it has rather low homology (15 to 22% identity) to the gene sequences encoding alginate lyase in other bacteria. The A. chroococcum AlgL protein was overproduced in Escherichia coli and purified to electrophoretic homogeneity in a two-step chromatography procedure on hydroxyapatite and phenyl-Sepharose. The kinetic and molecular parameters of the recombinant alginate lyase are similar to those found for the native enzyme.

Amino Acid Sequence↗
PubMed2000

Characterization of alginate lyase from Pseudomonas syringae pv. syringae.

The gene encoding alginate lyase (algL) in Pseudomonas syringae pv. syringae was cloned, sequenced, and overexpressed in Escherichia coli. Alginate lyase activity was optimal when the pH was 7.0 and when assays were conducted at 42 degrees C in the presence of 0.2 M NaCl. In substrate specificity studies, AlgL from P. syringae showed a preference for deacetylated polymannuronic acid. Sequence alignment with other alginate lyases revealed conserved regions within AlgL likely to be important for the structure and/or function of the enzyme. Site-directed mutagenesis of histidine and tryptophan residues at positions 204 and 207, respectively, indicated that these amino acids are critical for lyase activity.

Cloning, Molecular↗