A novel oligoalginate lyase from abalone, Haliotis discus hannai, that releases disaccharide from alginate polymer in an exolytic manner

We previously reported the isolation and cDNA cloning of an endolytic alginate lyase, HdAly, from abalone Haliotis discus hannai [Carbohydr. Res.2003, 338, 2841-2852]. Although HdAly preferentially degraded mannuronate-rich substrates, it was incapable of degrading unsaturated oligomannuronates smaller than tetrasaccharide. In the present study, we used conventional chromatographic techniques to isolate a novel unsaturated-trisaccharide-degrading enzyme, named HdAlex, from the digestive fluid of the abalone. The HdAlex showed a molecular weight of 32,000 on SDS-PAGE and could degrade not only unsaturated trisaccharide but also alginate and mannuronate-rich polymers at an optimal pH and temperature of 7.1 and 42 degrees C, respectively. Upon digestion of alginate polymer, HdAlex decreased the viscosity of the alginate at a slower rate than did HdAly, producing only unsaturated disaccharide without any intermediate oligosaccharides. These results indicate that HdAlex degrades the alginate polymer in an exolytic manner. Because HdAlex split saturated trisaccharide producing unsaturated disaccharide, we considered that this enzyme cleaved the alginate at the second glycoside linkage from the reducing terminus. The primary structure of HdAlex was deduced with cDNAs amplified from an abalone hepatopancreas cDNA library by the polymerase chain reaction. The translational region of 822 bp in the total 887-bp sequence of HdAlex cDNA encoded an amino-acid sequence of 273 residues. The N-terminal sequence of 16 residues, excluding the initiation methionine, was regarded as the signal peptide of this enzyme. The amino-acid sequence of the remaining 256 residues shared 62-67% identities with those of the polysaccharide lyase family-14 (PL14) enzymes such as HdAly and turban-shell alginate lyase SP2. To our knowledge, HdAlex is the first exolytic oligoalginate lyase belonging to PL14.     

产双功能褐藻胶裂解酶菌株的筛选与初步研究

目的：双功能褐藻胶裂解酶既能降解聚β-D-甘露糖醛酸,又能降解聚α-L-古罗糖醛酸,可以用一种酶来制备不同结构的褐藻胶寡糖。本文的目的是筛选能产生双功能褐藻胶裂解酶的菌株,对其产酶曲线和降解产物作初步研究。方法：利用唯一碳源培养基筛选产生褐藻胶裂解酶的菌株,通过16SrDNA序列比对进行菌种鉴定,通过在凝胶上检测褐藻胶裂解酶活性来判断发酵上清液中褐藻胶裂解酶的数量及分子量,利用薄层层析确定降解褐藻胶的终产物组成。结果：从褐藻上筛选到一株海洋细菌QY107,鉴定为弧菌属细菌。发酵120h时褐藻胶裂解酶产量为12．32U／mL,其发酵液上清中只含有一种褐藻胶裂解酶,分子量在28kDa左右,并且对聚β—D-甘露糖醛酸和聚α-L-古罗糖醛酸都能降解,降解褐藻胶的终产物主要为三糖。结论：本文筛选到一株弧菌QY107,其发酵液上清中只有一种双功能褐藻胶裂解酶,可用于大量制备褐藻胶三糖。推测该酶具有特殊的催化腔结构,对其结构与功能相互关系的研究可能会发现新的底物结合与催化机制。酶解制备褐藻胶寡糖因其环保高效而越来越受到人们的重视,因此该菌株能促进海洋寡糖类生物制品的开发,在医药、食品、农业、生物燃料等领域具有广阔的应用前景。     

Molecular Insight into the Role of the N-terminal Extension in the Maturation,Substrate Recognition,and Catalysis of a Bacterial Alginate Lyase from Polysaccharide Lyase Family 18

Bacterial alginate lyases, which are members of several polysaccharide lyase (PL) families, have important biological roles and biotechnological applications. The mechanisms for maturation, substrate recognition, and catalysis of PL18 alginate lyases are still largely unknown. A PL18 alginate lyase, aly-SJ02, from Pseudoalteromonas sp. 0524 displays a β-jelly roll scaffold. Structural and biochemical analyses indicated that the N-terminal extension in the aly-SJ02 precursor may act as an intramolecular chaperone to mediate the correct folding of the catalytic domain. Molecular dynamics simulations and mutational assays suggested that the lid loops over the aly-SJ02 active center serve as a gate for substrate entry. Molecular docking and site-directed mutations revealed that certain conserved residues at the active center, especially those at subsites +1 and +2, are crucial for substrate recognition. Tyr³⁵³ may function as both a catalytic base and acid. Based on our results, a model for the catalysis of aly-SJ02 in alginate depolymerization is proposed. Moreover, although bacterial alginate lyases from families PL5, 7, 15, and 18 adopt distinct scaffolds, they share the same conformation of catalytic residues, reflecting their convergent evolution. Our results provide the foremost insight into the mechanisms of maturation, substrate recognition, and catalysis of a PL18 alginate lyase.     

Selective alginate degradation by marine bacteria associated with the algal genusSargassum

Summary Alginase-secreting bacteria associated with actively growing tissues of the marine Phaeophyta speciesSargassum fluitans andS. natans have been isolated and evaluated for their ability to degrade alginate (ALG), carboxymethylcellulose, and agar. Of seven isolates selected for their ability to grow on 2% agar containing 1% sodium alginate, none were able to grow on either 2% agar or 2% agar supplemented with 0.1% carboxymethylcellulose. Two of these with fermentative potential, i.e., ALG-A and ALG-G, showed selective activities with respect to their ability to degrade native alginate and/or take up the products resulting from alginate degradation. The ALG-A isolate was able to rapidly degrade native alginate with the generation of a stable polymer fraction and small oligouronides, most of which were dissimilated for growth. The ALG-G isolate was able to completely degrade native alginate with the accumulation of significant quantities of unsaturated dimeric and trimeric oligouronides. A limit polymer was generated from the action of a polymannuronan-specific extracellular alginate lyase purified from exponential cultures of the ALG-A organism. This product proved to be an effective substrate for the alginate lyase activity obtained from the medium of exponential phase cultures of the ALG-G isolate, and upon incubation with concentrated and dialyzed ALG-G medium was converted to the products that were observed to accumulate in the medium of the ALG-G isolate grown on native alginate. These organisms represent examples of the microflora associated with actively growingSargassum tissues, each with a selective ability to degrade and dissimilate the biomass of the marine brown algae.     

Purification and characterization of a Na/K dependent alginate lyase from turban shell gut Vibrio sp. YKW-34

An alginate lyase with high specific enzyme activity was purified from Vibrio sp. YKW-34, which was newly isolated from turban shell gut. The alginate lyase was purified by in order of ion exchange, hydrophobic and gel filtration chromatographies to homogeneity with a recovery of 7% and a fold of 25. This alginate lyase was composed of a single polypeptide chain with molecular mass of 60 kDa and isoelectric point of 5.5–5.7. The optimal pH and temperature for alginate lyase activity were pH 7.0 and 40 °C, respectively. The alginate lyase was stable over pH 7.0–10.0 and at temperature below 50 °C. The alginate lyase had substrate specificity for both poly-guluronate and poly-mannuronate units. The k_cat/K_m value for alginate (heterotype) was 1.7 × 10⁶ s⁻¹ M⁻¹. The enzyme activity was completely lost by dialysis and restored by addition of Na⁺ or K⁺. The optimal activity exhibited in 0.1 M of Na⁺ or K⁺. This enzyme was resistant to denaturing reagents (SDS and urea), reducing reagents (β-mercaptoethanol and DTT) and chelating reagents (EGTA and EDTA).     

Crystal Structure of Exotype Alginate Lyase Atu3025 from Agrobacterium tumefaciens

Alginate, a major component of the cell wall matrix in brown seaweeds, is degraded by alginate lyases through a β-elimination reaction. Almost all alginate lyases act endolytically on substrate, thereby yielding unsaturated oligouronic acids having 4-deoxy-l-erythro-hex-4-enepyranosyluronic acid at the nonreducing end. In contrast, Agrobacterium tumefaciens alginate lyase Atu3025, a member of polysaccharide lyase family 15, acts on alginate polysaccharides and oligosaccharides exolytically and releases unsaturated monosaccharides from the substrate terminal. The crystal structures of Atu3025 and its inactive mutant in complex with alginate trisaccharide (H531A/ΔGGG) were determined at 2.10- and 2.99-Å resolutions with final R-factors of 18.3 and 19.9%, respectively, by x-ray crystallography. The enzyme is comprised of an α/α-barrel + anti-parallel β-sheet as a basic scaffold, and its structural fold has not been seen in alginate lyases analyzed thus far. The structural analysis of H531A/ΔGGG and subsequent site-directed mutagenesis studies proposed the enzyme reaction mechanism, with His³¹¹ and Tyr³⁶⁵ as the catalytic base and acid, respectively. Two structural determinants, i.e. a short α-helix in the central α/α-barrel domain and a conformational change at the interface between the central and C-terminal domains, are essential for the exolytic mode of action. This is, to our knowledge, the first report on the structure of the family 15 enzyme.     

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.     

Alginate lyase: Structure,property, and application

Alginate is a linear polysaccharide in which β-D-mannuronate (M) and its epimer, α-L-guluronate (G), are covalently (1–4)-linked in different sequences. Alginate is mainly used as a food additive to modify food texture due to its high viscosity and gelling property. Alginate lyase can degrade alginate by cleaving the glycosidic bond through a β-elimination reaction, generating oligomer with 4-deoxy-L-erythro-hex-4-enepyranosyluronate at the nonreducing end. Alginate oligosaccharides have been shown to stimulate the growth of human endothelial cells and the secretion of cytotoxic cytokines from human macrophage. Alginate can be converted into unsaturated monosaccharide by saccharification process using endolytic and exolytic alginate lyases, thus alginate lyases have potential as key biocatalyst for application of alginate as a renewable source for biochemicals and biofuels in near future. In this paper, structures and functions of various alginate lyases are reviewed. Prospects on future applications of alginate lyases are also discussed.     

从海洋中分离的弧菌QY102褐藻胶裂解酶的纯化和性质研究

从马尾藻(Sargassum)表面分离到一株产生高效胞外褐藻胶裂解酶的海洋弧菌（Vibrio sp.） QY102。以褐藻胶为唯一碳源发酵培养后,发酵液上清通过0.22μm滤膜过滤、DEAESepharose离子交换和Superdex75凝胶过滤得到电泳纯的褐藻胶裂解酶。酶的性质研究表明：其分子量约为28.5kD（SDSPAGE）,反应最适温度为40℃,最适pH为7.1,Ca2+、Mg2+对酶活有促进作用,而Ni2+、Al3+、Zn2+、Ba2+对酶活有抑制作用。该酶的活性明显高于已报道的褐藻胶裂解酶,pH稳定范围广（5～10）,并且对聚甘露糖醛酸的活性高于对聚古罗糖醛酸的活性。     

Preparation and structure elucidation of alginate oligosaccharides degraded by alginate lyase from Vibro sp. 510

Alginate that was purified from the fermentation solution of marine bacteria Vibro sp. 510 under specific reaction conditions was hydrolyzed by alginate lyase. Seven oligosaccharides, including di-, tri- and tetrasaccharides, were isolated through low-pressure, gel-permeation chromatography (LP-GPC) and semipreparative strong-anion exchange (SAX) fast-protein liquid chromatography (FPLC). The oligosaccharide structures were elucidated based on ESIMS and 2D NMR spectral analysis. The hydrolytic specificity of this alginate lyase to alginate is discussed.     

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.     

Biochemical mapping of human NEIL1 DNA glycosylase and AP lyase activities

Base excision repair of oxidized DNA in human cells is initiated by several DNA glycosylases with overlapping substrate specificity. The human endonuclease VIII homologue NEIL1 removes a broad spectrum of oxidized pyrimidine and purine lesions. In this study of NEIL1 we have identified several key residues, located in three loops lining the DNA binding cavity, important for lesion recognition and DNA glycosylase/AP lyase activity for oxidized bases in double-stranded and single-stranded DNA. Single-turnover kinetics of NEIL1 revealed that removal of 5-hydroxycytosine (5-OHC) and 5-hydroxyuracil (5-OHU) is ～25 and ～10-fold faster in duplex DNA compared to single-stranded DNA, respectively, and also faster than removal of dihydrothymine (DHT) and dihydrouracil (DHU), both in double-stranded and single-stranded DNA. NEIL1 excised 8-oxoguanine (8-oxoG) only from double-stranded DNA and analysis of site-specific mutants revealed that Met81, Arg119 and Phe120 are essential for removal of 8-oxoG. Further, several arginine and histidine residues located in the loop connecting the two β-strands forming the zincless finger motif and projecting into the DNA major groove, were shown to be imperative for lesion processing for both single- and double-stranded substrates. Trapping experiments of active site mutants revealed that the N-terminal Pro2 and Lys54 can alternate to form a Schiff-base complex between the protein and DNA. Hence, both Pro2 and Lys54 are involved in the AP lyase activity. While wildtype NEIL1 activity almost exclusively generated a δ-elimination product when processing single-stranded substrates, substitution of Lys54 changed this in favor of a β-elimination product. These results suggest that Pro2 and Lys54 are both essential for the concerted action of the β,δ-elimination in NEIL1.     

A rapid,sensitive, simple plate assay for detection of microbial alginate lyase activity

Screening of microorganisms capable of producing alginate lyase enzyme is commonly carried out by investigating their abilities to grow on alginate-containing solid media plates and occurrence of a clearance zone after flooding the plates with agents such as 10% (w/v) cetyl pyridinium chloride (CPC), which can form complexes with alginate. Although the CPC method is good, advantageous, and routinely used, the agar in the media interferes with the action of CPC, which makes judgment about clearance zones very difficult. In addition, this method takes a minimum of 30 min to obtain the zone of hydrolysis after flooding and the hydrolyzed area is not sharply discernible. An improved plate assay is reported herein for the detection of extracellular alginate lyase production by microorganisms. In this method, alginate-containing agar plates are flooded with Gram's iodine instead of CPC. Gram's iodine forms a bluish black complex with alginate but not with hydrolyzed alginate, giving sharp, distinct zones around the alginate lyase producing microbial colonies within 2–3 min. Gram's iodine method was found to be more effective than the CPC method in terms of visualization and measurement of zone size. The alginate-lyase-activity area indicated using the Gram's iodine method was found to be larger than that indicated by the CPC method. Both methods (CPC and Gram's iodine) showed the largest alginate lyase activity area for Saccharophagus degradans (ATCC 43961) followed by Microbulbifer mangrovi (KCTC 23483), Bacillus cereus (KF801505) and Paracoccus sp. LL1 (KP288668) grown on minimal sea salt medium. The rate of growth and metabolite production in alginate-containing minimal sea salt liquid medium, followed trends similar to that of the zone activity areas for the four bacteria under study. These results suggested that the assay developed in this study of Gram's iodine could be useful to predict the potential of microorganisms to produce alginate lyase. The method also worked well for screening and identification of alginate lyase producers and non-producers from environmental samples on common laboratory media. They did this by clearly showing the presence or absence of clearance zones around the microbial colonies grown. This new method is rapid, efficient, and could easily be performed for screening a large number of microbial cultures. This is the first report on the use of Gram's iodine for the detection of alginate lyase production by microorganisms using plate assay.     

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     

Effect of substrate mechanics on chondrocyte adhesion to modified alginate surfaces

This study characterized the attachment of chondrocytes to RGD-functionalized alginate by examining the effect of substrate stiffness on cell attachment and morphology. Bovine chondrocytes were added to wells coated with 2% alginate or RGD-alginate. The alginate was crosslinked with divalent cations ranging from 1.25 to 62.5 mmol/g alginate. Attachment to RGD-alginate was 10-20 times higher than attachment to unmodified alginate and was significantly inhibited by antibodies to integrin subunits α₃ and β₁, cytochalasin-D, and soluble RGD peptide. The equilibrium level and rate of attachment increased with crosslink density and substrate stiffness. Substrate stiffness also regulated chondrocyte morphology, which changed from a rounded shape with nebulous actin on weaker substrates to a predominantly flat morphology with actin stress fibers on stiffer substrates. The dependence of attachment on integrins and substrate stiffness suggests that chondrocyte integrins may play a role in sensing the mechanical properties of the matrices to which they are attached.     

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.     

响应面法优化类芽胞杆菌HB172198产褐藻胶裂解酶发酵培养基

为了提高类芽胞杆菌新种HB172198产褐藻胶裂解酶活力,本研究采用响应面法对该菌株液体发酵培养基进行了优化实验。在单因素实验和Plackett-Burman试验筛选出海藻酸钠、胰蛋白胨、NaCl、MgSO4·7H2O等4个显著影响产酶因素的基础上,通过Box-Behnken设计及响应面法进行回归分析,得出产褐藻胶裂解酶最佳发酵培养基,其成分为:海藻酸钠7.50 g/L、胰蛋白胨13.57 g/L、NaCl 29.75 g/L、MgSO4·7H2O 0.08 g/L。优化条件下该菌株最大酶活性达14.60 U/mL,是优化前的1.87倍。本研究为菌株HB172198产褐藻胶裂解酶的大规模生产和工业应用提供了重要的理论依据。     

Purification and characterisation of a bifunctional alginate lyase from novel Isoptericola halotolerans CGMCC 5336

A novel halophilic alginate-degrading microorganism was isolated from rotten seaweed and identified as Isoptericola halotolerans CGMCC5336. The lyase from the strain was purified to homogeneity by combining of ammonium sulfate fractionation and anion-exchange chromatography with a specific activity of 8409.19 U/ml and a recovery of 25.07%. This enzyme was a monomer with a molecular mass of approximately 28 kDa. The optimal temperature and pH were 50 °C and pH 7.0, respectively. The lyase maintained stability at neutral pH (7.0–8.0) and temperatures below 50 °C. Metal ions including Na⁺, Mg²⁺, Mn²⁺, and Ca²⁺ notably increased the activity of the enzyme. With sodium alginate as the substrate, the K_m and V_max were 0.26 mg/ml and 1.31 mg/ml min, respectively. The alginate lyase had substrate specificity for polyguluronate and polymannuronate units in alginate molecules, indicating its bifunctionality. These excellent characteristics demonstrated the potential applications in alginate oligosaccharides production with low polymerisation degrees.     

Substrate recognition by family 7 alginate lyase from Sphingomonas sp. A1

Sphingomonas sp. A1 alginate lyase A1-II′, a member of polysaccharide lyase family 7, shows a broad substrate specificity acting on poly α-L-guluronate (poly(G)), poly β-D-mannuronate (poly(M)) and the heteropolymer (poly(MG)) in alginate molecules. A1-II′ with a glove-like β-sandwich as a basic scaffold forms a cleft covered with two lid loops (L1 and L2). Here, we demonstrate the loop flexibility for substrate binding and structural determinants for broad substrate recognition and catalytic reaction. The two loops associate mutually over the cleft through the formation of a hydrogen bond between their edges (Asn141 and Asn199). A double mutant, A1-II′ N141C/N199C, has a disulfide bond between Cys141 and Cys199, and shows little enzyme activity. Adding dithiothreitol to the enzyme reaction mixture leads to a tenfold increase in its molecular activity, suggesting the significance of flexibility in lid loops for accommodating the substrate into the active cleft. In alginate trisaccharide (GGG or MMG)-bound A1-II′ Y284F, the enzyme interacts appropriately with substrate hydroxyl groups at subsites + 1 and + 2 and accommodates G or M, while substrate carboxyl groups are strictly recognized by specific residues. This mechanism for substrate recognition enables A1-II′ to show the broad substrate specificity. The structure of A1-II′ H191N/Y284F complexed with a tetrasaccharide bound at subsites − 1 to + 3 suggests that Gln189 functions as a neutralizer for the substrate carboxyl group, His191 as a general base, and Tyr284 as a general acid. This is, to our knowledge, the first report on the structure and function relationship in family 7.     

海藻酸裂解酶酶学与基因工程研究进展及应用

海藻中富含海藻酸盐,海藻酸裂解酶降解后产生的寡糖物质具有很强的生物活性及益生作用,酶法降解海藻酸盐的生物降解取代传统的化学降解已日益受到人们的关注,就海藻酸盐降解酶的来源、作用机制、应用效果和影响因素进行了全面综述,阐明了海藻酸盐降解酶的研究具有显著的理论意义和应用价值。