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.     

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.     

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.     

Depolymerization of alginate into a monomeric sugar acid using Alg17C,an exo-oligoalginate lyase cloned from <Emphasis Type="Italic">Saccharophagus degradans</Emphasis> 2-40

Macroalgae are considered to be promising biomass for fuels and chemicals production. To utilize brown macroalgae as biomass, the degradation of alginate, which is the main carbohydrate of brown macroalgae, into monomeric units is a critical prerequisite step. Saccharophagus degradans 2-40 is capable of degrading more than ten different polysaccharides including alginate, and its genome sequence demonstrated that this bacterium contains several putative alginate lyase genes including alg17C. The gene for Alg17C, which is classified into the PL-17 family, was cloned and overexpressed in Escherichia coli. The recombinant Alg17C was found to preferentially act on oligoalginates with degrees of polymerization higher than 2 to produce the alginate monomer, 4-deoxy-l-erythro-5-hexoseulose uronic acid. The optimal pH and temperature for Alg17C were found to be 6 and 40 °C, respectively. The K _M and V _max of Alg17C were 35.2 mg/ml and 41.7 U/mg, respectively. Based on the results of this study, Alg17C could be used as the key enzyme to produce alginate monomers in the process of utilizing alginate for biofuels and chemicals production.     

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.     

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.     

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.     

Cloning and Sequencing Analysis of Alginate Lyase Genes from the Marine Bacterium Vibrio sp. O2

We isolated a new marine bacteria, which displayed alginate-depolymerizing activity in plate assays, from seawater in Mihonoseki Harbor, Japan. Analysis of the 16S ribosomal RNA gene sequence of one of the isolates proved that this alginate-depolymerizing bacterium belonged to the genus Vibrio and it was named Vibrio sp. O2. The alginate lyase genes of Vibrio sp. O2 were cloned and expressed in Escherichia coli. Two alginate lyase-producing clones, pVOA-A4 and pVOA-B5, were obtained. The alginate lyase gene alyVOA from pVOA-A4 was composed of an 858-bp open reading frame (ORF) encoding 285 amino acid residues, while alyVOB from pVOA-B5 was composed of an 828-bp ORF encoding 275 amino acid residues. The degree of identity between the deduced amino acid sequences of AlyVOA or AlyVOB and Photobacterium sp. ATCC43367 alginate poly(ManA)lyase AlxM was 92.3% or 32.6%, respectively. Alginate lyase consensus regions corresponding to the sequences YFKAGXYXQ and RXELR were observed in all three of these sequences. AlyVOA and AlyVOB both degraded polymannuronate in plate assays and were therefore confirmed to be poly(β-D-mannuronate)lyases.     

刺参海带饲料原料褐藻胶降解菌的分离与鉴定

从海带及刺参养殖环境中筛选有效降解褐藻胶,且对刺参无致病性的微生物,对海带饲料原料进行降解处理,以降低海带饲料中刺参难以消化的褐藻胶成分,显著提高饲料利用率,增加海带原料价值。以褐藻胶为唯一碳源选择培养基初筛;DNS法测定褐藻胶裂解酶酶活;16S r DNA测序及生理生化试验对菌种进行鉴定;高浓度腹腔攻毒试验考察筛选所得菌株对刺参的潜在致病性;分子排阻色谱及高效凝胶色谱法对微生物酶解褐藻胶的终产物进行分析。系统发育树分析表明,菌株WB1与Bacillus amyloliquifaciens有最高同源性,对刺参无潜在致病性;其褐藻胶裂解酶酶解褐藻胶的终产物主要为二糖和三糖,相对含量分别为74.1%和25.9%,平均分子量为516 Da。解淀粉芽胞杆菌WB1可作为一种安全的有益微生物用于刺参海带饲料原料中褐藻胶成分的降解。     

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     

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.     

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.     

微泡菌属菌株YPW1和YPW16多糖降解特性分析

【背景】海洋环境中分离到的微泡菌属菌株具有多糖降解能力,在环境中可以作为糖类代谢的重要执行者参与海洋碳循环过程。【目的】测定2株微泡菌属菌株的多糖降解活性,通过与微泡菌属其他菌株基因组比较分析2株菌的多糖降解基因特征。【方法】通过3,5-dinitrosalicylicacid(DNS)定糖法测定多糖降解活性,同时利用高通量测序技术对菌株基因组序列进行测定与组装,并与其他基因组注释结果进行比较分析。【结果】分离得到2株微泡菌属菌株YPW1和YPW16,二者均为潜在新种。结果表明,菌株YPW1能够降解琼胶、褐藻胶、果胶、几丁质、木聚糖、淀粉、普鲁兰等7种多糖,而菌株YPW16仅可降解淀粉和普鲁兰。基因组分析表明,YPW1具有上述7种多糖的降解酶基因,但菌株YPW16只具有淀粉酶与普鲁兰酶降解基因。相较于其他微泡菌属菌株,菌株YPW1多糖降解范围、多糖降解酶基因种类与丰度较高,但菌株YPW16多糖降解范围却较为狭窄。由此可知,多糖降解酶基因在微泡菌属基因组中的分布差异性较大。【结论】本研究为微泡菌属提供了2株潜在的新型菌株资源,为生物多糖降解提供了生化工具,也为研究微泡菌属菌株中多糖降解基...     

An effective method for isolating alginate lyase-producing Bacillus sp. ATB-1015 strain and purification and characterization of the lyase

A new alginate lyase-producing micro-organism, designated as Bacillus sp. strain ATB-1015, was effectively isolated from soil samples pretreated for 3 months with a substrate of the enzyme, sodium alginate. Alginate lyase activity was assayed by the degrading activity of biofilm on Teflon sheet discs, which was formed by a mucoid strain of Pseudomonas aeruginosa PAM3 selected from clinical isolates. The extracellular alginate lyase was precipitated with ammonium sulphate from the culture broth, and purified by gel filtration and anion exchange chromatography. The molecular weight of the lyase was estimated to be 41 kDa by SDS polyacrylamide gel electrophoresis and Sephacryl S-200 HR column chromatography. The optimum pH and temperature for the enzyme activity were around 7·5 and 37 °C, respectively, and the Km value was 0·17% with the substrate, sodium alginate. The lyase activity was completely inhibited by treatment with 1 mmol l⁻¹ of EDTA and the decreased activity was almost completely recovered by the addition of 2 mmol l⁻¹ of CaCl₂. The activity was not affected by treatment with the protein denaturants, 0·01 mol l⁻¹ of SDS or 1 mmol l⁻¹ of urea. The lyase had substrate specificity for both the poly-guluronate and poly-mannuronate units in the alginate molecule.     

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.     

Cloning of a gene for intracellular alginate lyase in a bacterium isolated from a ditch

A DNA fragment with a gene for intracellular alginate lyase in a bacterium A1 isolated from a ditch was cloned using a vector plasmid pKK223-3 and the gene was weakly expressed in Escherichia coli DH1 cells. The alginate lyase produced by E. coli DH1 cells was thought to correspond to A1-I among three kinds of alginate lyases (A1-I, A1-I-1 and A1-I-2) produced by the strain A1. Through this study, CaCl₂ was found to be a useful agent for the screening of microbial alginate lyase-producing colonies on agar plates.     

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.     

Distribution of glyphosate and methylphosphonate catabolism systems in soil bacteria <Emphasis Type="Italic">Ochrobactrum anthropi</Emphasis> and <Emphasis Type="Italic">Achromobacter</Emphasis> sp

Bacterial strains capable of utilizing methylphosphonic acid (MP) or glyphosate (GP) as the sole sources of phosphorus were isolated from soils contaminated with these organophosphonates. The strains isolated from MP-contaminated soils grew on MP and failed to grow on GP. One group of the isolates from GP-contaminated soils grew only on MP, while the other one grew on MP and GP. Strains Achromobacter sp. MPS 12 (VKM B-2694), MP degraders group, and Ochrobactrum anthropi GPK 3 (VKM B-2554D), GP degraders group, demonstrated the best degradative capabilities towards MP and GP, respectively, and were studied for the distribution of their organophosphonate catabolism systems. In Achromobacter sp. MPS 12, degradation of MP was catalyzed by C–P lyase incapable of degrading GP (C–P lyase I). Adaptation to growth on GP yielded the strain Achromobacter sp. MPS 12A, which retained its ability to degrade MP via C–P lyase I and was capable of degrading GP with formation of sarcosine, thus suggesting the involvement of a GP-specific C–P lyase II. O. anthropi GPK 3 also degraded MP via C–P lyase I, but degradation of GP in it was initiated by glyphosate oxidoreductase, which was followed by product transformation via the phosphonatase pathway.     

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.