Development of a process for large-scale chromatographic purification of an alginate lyase from Klebsiella pneumoniae

Summary A process for purification of an alginate lyase, produced extracellularly by fermentation of Klebsiella pneumoniae, has been developed. The process includes two chromatographic steps and is well suited to large-scale operation. By hydrophobic interaction chromatography on Phenyl-Sepharose FF, followed by anion exchange chromatography on Q-Sepharose FF in a negative mode, the specific activity was increased from 0.09 units (U) mg ^–1 to more than 50 U mg^–1. Due to an extremely low product concentration in the fermentation broth, and large amounts of contaminating proteins, the chromatographic adsorbents had low capacities with respect to alginate lyase. By adsorption on the cation exchanger S-Sepharose FF, the capacity was so low that the enzyme could not be concentrated. The binding capacity of Phenyl-Sepharose FF was approximately 20-fold higher, and a three to tenfold concentration was obtained. The first stage of the process, hydrophobic interaction chromatography, has been applied to the isolation of alginate lyase from fermentation batches of 180 l. Several runs have resulted in a purified product with an average quantity of 30 000–35 000 U per fermentation, and an average specific activity of 4.5 U mg^–1. Although the raw material employed in this work has been particularly unfavourable, the process developed will also be applicable to raw materials with higher product concentrations. Offprint requests to: I. M. Aasen     

Purification and Characterization of an Alginate Lyase from Marine Bacterium Vibrio sp. Mutant Strain 510-64

Marine Vibrio sp. 510 was chosen as a parent strain for screening high producers of alginate lyase using the complex mutagenesis of Ethyl Methanesulphonate and UV radiation treatments. The mutant strain Vibrio sp. 510-64 was selected and its alginate lyase activity was increased by 3.87-fold (reaching 46.12 EU/mg) over that of the parent strain. An extracellular alginate lyase was purified from Vibrio sp. 510-64 cultural supernatant by successive fractionation on DEAE Sepharose FF and two steps of Superdex 75. The purified enzyme yielded a single band on SDS-PAGE with the molecular weight of 34.6 kDa. Data of the N-terminal amino acid sequence indicated that this protein might be a novel alginate lyase. The substrate specificity results demonstrated that the alginate lyase had the specificity for poly G block.     

Isolation and characterization of an alginate lyase from Klebsiella aerogenes

The bacterium Klebsiella aerogenes (type 25) produced an inducible alginate lyase, whose major activity was located intracellularly during all growth phases. The enzyme was purified from the soluble fraction of sonicated cells by ammonium sulfate precipitation, anion- and cation-exchange chromatography and gel filtration. The apparent molecular weight of purified alginate lyase of 28,000 determined by gel filtration and of 31,600 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the active enzyme was composed of a single polypeptide. The alginate lyase displayed a pH optimum around 7.0 and a temperature optimum around 37°C. The purified enzyme depolymerized alginate by a lyase reaction in an endo manner releasing products which reacted in the thiobarbituric acid assay and absorbed strongly in the ultraviolet region at 235 nm. The alginate lyase was specific for guluronic acidrich alginate preparations. Propylene glycol esters of alginate and O-acetylated bacterial alginates were poorly degraded by the lyase compared with unmodified polysaccharide. The guluronate-specific lyase activity was applied in an enzymatic method to detect mannuronan C-5 epimerase in three different mucoid (alginate-synthesizing) strains of Pseudomonas aeruginosa. This enzyme which converts polymannuronate to alginate could not be demonstrated either extracellularly or intracellularly in all strains suggesting the absence of a polymannuronate-modifying enzyme in P. aeruginosa.Abbreviations poly(ManA) (1–4)--D-mannuronan - poly(GulA) (1–4)--L-guluronan - TBA 2-thiobarbituric acid     

Cloning and Characterization of a Novel Oligoalginate Lyase from a Newly Isolated Bacterium Sphingomonas sp. MJ-3

A bacterium possessing alginate-degrading activity was isolated from marine brown seaweed soup liquefied by salted and fermented anchovy. The isolated strain was designated as Sphingomonas sp. MJ-3 based on the analyses of 16S ribosomal DNA sequences, 16S-23S internal transcribed spacer region sequences, biochemical characteristics, and cellular fatty acid composition. A novel alginate lyase gene was cloned from genomic DNA library and then expressed in Escherichia coli. When the deduced amino acid sequence was compared with the sequences on the databases, interestingly, the cloned gene product was predicted to consist of AlgL (alginate lyase L)-like and heparinase-like protein domain. The MJ-3 alginate lyase gene shared below 27.0% sequence identity with exolytic alginate lyase of Sphingomonas sp. A1. The optimal pH and temperature for the recombinant MJ-3 alginate lyase were 6.5 and 50°C, respectively. The final degradation products of alginate oligosaccharides were analyzed by electrospray ionization mass spectrometry and proved to be alginate monosaccharides. Based on the results, the recombinant alginate lyase from Sphingomonas sp. MJ-3 is regarded as an oligoalginate lyase that can degrade oligoalginate and alginate into alginate monosaccharides.     

Cloning and Characterization of Alginate Lyase from a Marine Bacterium Streptomyces sp. ALG-5

A marine bacterium was isolated from seaweeds for its ability to degrade alginate. Analysis of 16S ribosomal DNA sequence and chemotaxonomic characterizations revealed that the strain belongs to Streptomyces. The alginate lyase gene of Streptomyces sp. ALG-5 was cloned by using PCR with the specific primer designed from homologous nucleotide sequences. The consensus sequences of N-terminal YXRSELREM and C-terminal YFKAGXYXQ were conserved in the ALG-5 alginate lyase gene. The recombinant alginate lyase was purified by using Ni-Sepharose affinity chromatography. The alginate lyase appears to be poly-guluronate lyase degrading poly-G block preferentially than poly-M block. The degraded products were determined to be di-, tri-, tetra- and pentasaccharides by using BioGel P-2 gel filtration chromatography and ionization mass spectroscopy method.     

The Release of Alginate Lyase from Growing Pseudomonas syringae pathovar phaseolicola

Pseudomonas syringae pathovar phaseolicola, which produces alginate during stationary growth phase, displayed elevated extracellular alginate lyase activity during both mid-exponential and late-stationary growth phases of batch growth. Intracellular activity remained below 22% of the total activity during exponential growth, suggesting that alginate lyase has an extracellular function for this organism. Extracellular enzyme activity in continuous cultures, grown in either nutrient broth or glucose–simple salts medium, peaked at 60% of the washout rate, although nutrient broth-grown cultures displayed more than twice the activity per gram of cell mass. These results imply that growth rate, nutritional composition, or both initiate a release of alginate lyase from viable P. syringae pv. phaseolicola, which could modify its entrapping biofilm. Received: 14 April 2000 / Accepted: 11 August 2000     

Characterization of alginate lyase (ALYII) from Pseudomonas sp. OS-ALG-9 expressed in recombinant Escherichia coli

An alginate lyase named ALYII was purified to homogeneity from Escherichia coli JM109 carrying a recombinant plasmid, pJK26 harbouring the alyII gene from Pseudomonas sp. OS-ALG-9 by column chromatography with DEAE-cellulose, CM-Sephadex C-50, butyl-Toyopearl 650 M and isoelectric focusing. The molecular size of the purified ALYII was estimated to be 79 kDa by SDS-PAGE and its pI was 8.3. The enzyme was most active at pH 7.0 and 30 °C. Its activity was completely inhibited by Hg²⁺. The enzyme was poly -D-1, 4-mannuronate-specific rather than -D-1, 4-guluronate-specific and it showed a promotion effect in alginate degradation by combination with ALY, an another poly -D-1, 4-mannuronate-specific alginate lyase from the same strain.     

Overexpression of alginate lyase of <Emphasis Type="Italic">Pseudoalteromonas elyakovii</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>, purification,and characterization of the recombinant alginate lyase

The alyPEEC gene encoding alginate lyase from marine bacterium Pseudoalteromonas elyakovii IAM 14594 was subcloned into pBAD24 with arabinose promoter and sequenced, and overexpressed in TOP10 strain of E. coli after arabinose induction. Expression levels of alyPEEC gene in E. coli cells were over 39.6-fold higher than those in P. elyakovii IAM 14594 cells. The molecular mass of purified alginate lyase from the engineered E. coli cells was estimated to be 32.0 kDa. Optimum pH and temperature of the alginate lyase activity were 7.0 and 30 °C, respectively. The enzyme was unstable on heating and in acidic and alkaline solution. The enzyme activity was stimulated by the MgCl₂, NaCl, KCl, CaCl₂, BaCl₂ and MnCl₂, but was inhibited by the addition of 1.0 mM of EGTA, EDTA, SDS, ZnSO₄, AgNO₃, and CoCl₂. All the alginate, polyM and polyG could be converted into oligosaccharides with more than tetrasaccharides by the purified recombinant alginate lyase, suggesting that the recombinant alginate lyase produced by the engineered E. coli has highly potential application in seaweed genetics, food and pharmaceutical industries.     

A Novel Alginate Lyase with High Activity on Acetylated Alginate of Pseudomonas aeruginosa FRD1 from Pseudomonas sp. QD03

Summary To exploit alginate lyase which could degrade bacterial alginates, degenerate PCR and long range-inverse PCR (LR-IPCR) were used to isolate alginate lyase genes from soil bacteria. Gene algL, an alginate lyase-encoding gene from Pseudomonas sp. QD03 was cloned, and it was composed of a 1122 bp open reading frame (ORF) encoding 373 amino acid residues with the calculated molecular mass of 42.2 kDa. The deduced protein had a potential N-terminal signal peptide of 20 amino acid residues that was consistent with its proposed periplasmic location. Gene algL was expressed in pET24a (+)/E. coli BL21 (DE3) system. The recombinant AlgL was purified to electrophoretic homogeneity using affinity chromatography. The molecular weight of AlgL was estimated to be 42.8 kDa by SDS-PAGE. AlgL exhibited maximal activity at pH 7.5 and 37 °C. Na⁺, K⁺, Ca²⁺ and Ba²⁺ significantly enhanced the activity of AlgL. AlgL could degrade alginate and mannuronate blocks, but hardly degrade guluronate blocks. In particular, AlgL could degrade acetylated alginate of Pseudomonas aeruginosa FRD1 (approximately 0.54 mol of O-acetyl group per mol of alginate). It might be possible to use alginate lyase AlgL as an adjuvant therapeutic medicine for the treatment of disease associated with P. aeruginosa infection.     

Characterization of alginate lyase gene using a metagenomic library constructed from the gut microflora of abalone

A metagenomic fosmid library was constructed using a genomic DNA mixture extracted from the gut microflora of abalone. The library gave an alginate lyase positive clone (AlyDW) harboring a 31.7-kbp insert. The AlyDW insert consisted of 22 open reading frames (ORFs). The deduced amino acid sequences of ORFs 11–13 were similar to those of known alginate lyase genes, which are found adjacent in the genome of Klebsiella pneumoniae subsp. aerogenes, Vibrio splendidus, and Vibrio sp. belonging to the phylum Gammaproteobacteria. Among the three recombinant proteins expressed from the three ORFs, alginate lyase activity was only observed in the recombinant protein (AlyDW11) coded by ORF 11. The expressed protein (AlyDW11) had the highest alginate lyase activity at pH 7.0 and 45°C in the presence of 1 mM AgNO₃. The alginate lyase activity of ORF 11 was confirmed to be endolytic by thin-layer chromatography. AlyDW11 preferred poly(β-d-mannuronate) as a substrate over poly(α-l-guluronate). AlyDW11 contained three highly conserved regions, RSEL, QIH, and YFKAGVYNQ, which may act to stabilize the three-dimensional conformation and function of the alginate lyase.     

Kinetics and specificity of alginate lyases

A purified preparation of the extracellular alginate lyase has been used to study kinetics and specificity towards purified, homopolymeric fragments of alginate. The enzyme preparation from Bacillus circulans 1351 degraded both block types, although with different efficiency, and thus appears to be nonspecific. Addition of calcium ions markedly enhanced the reaction rate for the polymannuronate block but had little or no effect on the reaction with polyguluronate. Michaelis-Menten kinetics are not obeyed in the absence of calcium ions and only for the polymannuronate in the presence of calciumThe study of progress curves in response to variation in substrate and enzyme concentrations strongly suggests that the abalone lyase is subject to a reversible product inhibition.     

Development of a fermentation process for production of an alginate G-lyase from Klebsiella pneumoniae

A high-density-cell fermentation process for production of an exracellular alginat lyase from Klebseilla pneumoniae on a defined medium has been developed. The process employs a strategy using two carbon sources. One low-molecular-mass, low-viscosity carbon source (sucrose) with high water solubililty is used as the main carbons source for growth, while the high-molecular-mass and viscoous alginate in low concentration is used as an inducer for enzyme synthesis. The repression of algiante lyase production by sucrose and the growth inhibition that we observed at increased levels of ammonia were circumvented by a computer-assisted fed-batch addition of the carbon sources (succrose and alginate) and by supplying nitrogen source as ammonia in the pH control. No enzyme production was observed when dissolved oxygen limited growth at an oxygen uptake rate of 40%–50% of the maximum uptake rate. An optimal composition of the feeding solution (12.5 g alginate and 587.5 g sucrose 1^–1) was found both for the maximum final concentration of enzyme (1330 U 1^–1) and for the maximum volumetric rate of enzyme production (67 U 1^–1 h^–1). The enzyme production dependes of the growth rate in the linear growth phase, giving a maximum enzyme concentration at the highest growth rate tested. The final enzyme concentration shows a fiveflod increase compare with previously reproted daata where alginate was used as a carbon source. In addition, the ratio of alginate lyase by a factor of apporximately 15. A doubling in extracellular specific activity of the enzyme was observed, a property of significant interest, especially for purification of the enzyme. On the othr hand, the final dry cell weight concentration of the bacteria also increased by a factor of 15–20 thus giving a relatively lower specific productivity of 0.4 U (g cell dry weight)^–1 h^–1.     

Symbiotic formation of alginate lyase in mixed culture of bacteria isolated from soil

Bacteria having alginate lyase activity were screened by growing cells from various sources in a medium containing alginate as a carbon source. Among the various samples tested, the culture enriched with paddy-field bacteria showed the highest alginate lyase activity, and contained three kinds of bacteria. They were purified and identified to be Flavobacterium, Alcaligenes and Bacillus species. The alginate lyase activity in these strains was low when they were grown separately. The highest alginate lyase activity was obtained when these strains were cultured all together in the same medium.     

The Surface Display of the Alginate Lyase on the Cells of Yarrowia lipolytica for Hydrolysis of Alginate

The alginate lyase structural gene (AlyVI gene) was amplified from plasmid pET24-ALYVI carrying the alginate lyase gene from the marine bacterium Vibrio sp. QY101 which is a pathogen of Laminaria sp. When the gene was cloned into the multiple cloning site of the surface display vector pINA1317-YlCWP110 and expressed in cells of Yarrowia lipolytica, the cells displaying the alginate lyase could form clear zone on the plate containing sodium alginate, indicating that they had high alginate lyase activity. The cells displaying alginate lyase can be used to hydrolyze poly-β-d-mannuronate (M) and poly-α-l-guluronate (G) and sodium alginate to produce different lengths of oligosaccharides (more than pentasaccharides). This is the first report that the yeast cells displaying alginate lyase were used to produce different lengths of oligosaccharides from alginate.     

Heterologous expression of an alginate lyase from Streptomyces sp. ALG-5 in Escherichia coli and its use for preparation of the magnetic nanoparticle-immobilized enzymes

The marine alginate lyase from Streptomyces sp. ALG-5, which specifically degrades poly-G block of alginate, was functionally expressed as a His-tagged form with an Escherichia coli expression system. The recombinant alginate lyase expressed with pColdI at 15 °C exhibited the highest alginate-degrading activity. The recombinant alginate lyase was efficiently immobilized onto two types of magnetic nanoparticles, superparamagnetic iron oxide nanoparticle, and hybrid magnetic silica nanoparticle, based on the affinity between His-tag and Ni²⁺ that displayed on the surfaces of nanoparticles. An alginate oligosaccharide mixture consisting of dimer and trimer was prepared by the immobilized alginate lyase. The immobilized enzymes were re-used repeatedly more than 10 times after magnetic separation.     

Partial purification and characterization of a mannuronan-specific alginate lyase fromPseudomonas aeruginosa

Production of a thick exopolysaccharide coat (alginate) by mucoid strains ofPseudomonas aeruginosa has been shown to contribute to the pathogenicity and persistence of these bacteria in the lungs of patients with cystic fibrosis. Previous studies have shown that some mucoidP. aeruginosa strains produce an enzyme(s) capable of degrading this alginate coat. In this study, an alginate lyase from mucoidP. aeruginosa strain WcM#2 was isolated and characterized. Lyase production was enhanced by the addition of 0.2–0.3m NaCl to the growth media. The lyase was eluted from an alginate-Sepharose affinity column with 0.5m NaCl, which can serve as a simple one-step purification protocol for obtaining semi-pure functional alginate lyase. Fractionation of the enzyme preparation on a Sephadex G-75 sizing column showed that the enzyme has an apparent molecular weight of 40,000, whereas sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) suggested a molecular weight of approximately 43,000. The affinity-purified enzyme had a pH optimum of 9.0, its activity was enhanced in the presence of 0.3m NaCl, and it showed substrate specificity for polymannuronic acid blocks. These results demonstrate the presence of a mannuronan-specific alginate lyase inP. aeruginosa that differs in several respects from previous reports ofP. aeruginosa alginate lyases.     

The enzymes of a marine bacterial isolate from the brown alga <Emphasis Type="Italic">Sargassum polycystum</Emphasis> agardh, 1821, that catalyzes the transformation of polyanionic oligo-and polysaccharides

A search for enzymes involved in the degradation of polyanionic polysaccharides (fucoidans and alginic acid) was conducted among bacterial epiphytes of the brown alga Sargassum polycystum that grows in the territorial waters of the Socialist Republic of Vietnam. Two resistant bacterial strains, F10 and F14, have been isolated from the algal microflora that degrade the thallus of the alga under laboratory conditions. These bacterial strains differed in the morphological, physiological, and biochemical characteristics and in the composition of enzymes. The strains were studied for the ability to synthesize intracellular oligo-and polysaccharide hydrolases and alginate lyases. The optimal conditions for the growth of bacterial strain F14 and the biosynthesis of fucoidanase and polymannuronate-specific alginate lyase were determined. The partially purified alginate lyase was stable at a temperature up to 40°C and had an optimal pH 6.0 and an optimal temperature 35°C.     

A method for efficient expression of Pseudomonas aeruginosa alginate lyase in Pichia pastoris

As an eco-friendly biocatalyst for alginate hydrolysis, bacteria-derived alginate lyase (AlgL) has been widely used in research and industries to produce oligosaccharides. However, the cost of AlgL enzyme production remains high due to the low expression and difficulty in purification from bacterial cells. In this study we report an effective method to overexpress the Pseudomonas aeruginosa AlgL (paAlgL) enzyme in Pichia pastoris. Fused with a secretory peptide, the recombinant paAlgL was expressed extracellularly and purified from the culture supernatant through a simple process. The purified recombinant enzyme is highly specific for alginate sodium with a maximal activity of 2,440 U/mg. The enzymatic activity remained stable below 45°C and at pH between 4 and 10. The recombinant paAlgL was inhibited by Zn²⁺, Cu²⁺, and Fe²⁺ and promoted by Co²⁺ and Ca²⁺. Interestingly, we also found that the recombinant paAlgL significantly enhanced the antimicrobial activity of antibiotics ampicillin and kanamycin against Pseudomonas aeruginosa. Our results introduce a method for efficient AlgL production, the characterization, and a new feature of the recombinant paAlgL as an enhancer of antibiotics against Pseudomonas aeruginosa.     

Effect of molecular chaperones on the soluble expression of alginate lyase inE. coli

When the alginate lyase gene (aly) fromPseudoalteromonas elyakovii was expressed inE. coli, most of the gene product was organized as aggregated insoluble particles known as inclusion bodies. To examine the effects of chaperones on soluble and nonaggregated form of alginate lyase inE. coli, we constructed plasmids designed to permit the coexpression ofaly and the DnaK/DnaJ/GrpE or GroEL/ES chaperones. The results indicate that coexpression ofaly with the Dnak/DnaJ/GrpE chaperone together had a marked effect on the yield alginate lyase as a soluble and active form of the enzyme. It is speculated this result occurs through facilitation of the correct folding of the protein. The optimal concentration ofl-arabinose required for the induction of the DnaK/DnaJ/GrpE chaperone was found to be 0.05 mg/mL. An analysis of the protein bands on SDS-PAGE gel indicated that at least 37% of total alginate lyase was produced in the soluble fraction when the DnaK/DnaJ/GrpE chaperone was coexpressed.     

Characterization of AlgMsp,an Alginate Lyase from Microbulbifer sp. 6532A

Alginate is a polysaccharide produced by certain seaweeds and bacteria that consists of mannuronic acid and guluronic acid residues. Seaweed alginate is used in food and industrial chemical processes, while the biosynthesis of bacterial alginate is associated with pathogenic Pseudomonas aeruginosa. Alginate lyases cleave this polysaccharide into short oligo-uronates and thus have the potential to be utilized for both industrial and medicinal applications. An alginate lyase gene, algMsp, from Microbulbifer sp. 6532A, was synthesized as an E.coli codon-optimized clone. The resulting 37 kDa recombinant protein, AlgMsp, was expressed, purified and characterized. The alginate lyase displayed highest activity at pH 8 and 0.2 M NaCl. Activity of the alginate lyase was greatest at 50°C; however the enzyme was not stable over time when incubated at 50°C. The alginate lyase was still highly active at 25°C and displayed little or no loss of activity after 24 hours at 25°C. The activity of AlgMsp was not dependent on the presence of divalent cations. Comparing activity of the lyase against polymannuronic acid and polyguluronic acid substrates showed a higher turnover rate for polymannuronic acid. However, AlgMSP exhibited greater catalytic efficiency with the polyguluronic acid substrate. Prolonged AlgMsp-mediated degradation of alginate produced dimer, trimer, tetramer, and pentamer oligo-uronates.