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.     

海藻酸盐裂解酶研究进展

海藻酸盐裂解酶是一类降解褐藻中海藻酸盐的酶。此酶已经在多种有机体中得到分离。对海藻酸盐裂解酶的生物特性、研究方法及其生物学功能进行了介绍。在酶学特性研究的基础上 ,通过酶解构建新型海藻酸盐多聚物 ,可增强和扩展海藻酸盐裂解酶在工业、农业、医药领域中的应用 ,使其在海藻多糖的高值化应用中发挥重要的作用。概述了海藻酸盐和海藻酸盐裂解酶过去和现在的研究状况 ,展望了海藻酸盐和海藻酸盐裂解酶将来的应用前景。     

Polysaccharide lyases

Abstract: Polysaccharide lyases are the products of various microorganisms, bacteriophage and some eukaryotes. All such enzymes cleave a hexose-1,4-α- or β-uronic acid sequence by β-elimination. They are in some examples, the only known type of enzymes degrading their polyanionic substrates. Although only a small number of these enzymes have been exhaustively studied, the pectin lyases of bacterial origin have proved to be of interesting crystal structure containing a parallel β-helix domain. Alginate and heparin lyases may yield products with biotechnological potential.     

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.     

A group of alpha-1,4-glucan lyases and their genes from the red alga Gracilariopsis lemaneiformis: purification, cloning, and heterologous expression

We present here the first report of a group of alpha-1,4-glucan lyases (EC 4.2.2.13) and their genes. The lyases produce 1, 5-anhydro-D-fructose from starch and related oligomers and polymers. The enzymes were isolated from the red alga Gracilariopsis lemaneiformis from the Pacific coasts of China and USA, and the Atlantic Coast of Venezuela. Three lyase isozymes (GLq1, GLq2 and GLq3) from the Chinese subspecies, two lyase isozymes (GLs1 and GLs2) from the USA subspecies and one lyase (GLa1) from the Venezuelan subspecies were identified and investigated. GLq1, GLq3, GLs1 and GLa1 were purified and partially sequenced. Based on the amino acid sequences obtained, three lyase genes or their cDNAs (GLq1, GLq2 and GLs1) were cloned and completely sequenced and two other genes (GLq3 and GLs2) were partially sequenced. The coding sequences of the lyase genes GLq1, GLq2 and GLs1 are 3267, 3276 and 3279 bp, encoding lyases of 1088, 1091 and 1092 amino acids, respectively. The deduced molecular masses of the mature lyases from the coding sequences are 117030, 117667 and 117790 Da, respectively, close to those determined by mass spectrometry using purified lyases. The amino acid sequence identity is more than 70% among the six algal lyase isozymes. The algal GLq1 gene was expressed in Pichia pastoris and Aspergillus niger, and the expression product was identical to the wild-type enzyme.     

Cloning,Expression, and Biochemical Characterization of Two New Oligoalginate Lyases with Synergistic Degradation Capability

Alginate, the most abundant carbohydrate presents in brown macroalgae, has recently gained increasing attention as an alternative biomass for the production of biofuel. Oligoalginate lyases catalyze the degradation of alginate oligomers into monomers, a prerequisite for bioethanol production. In this study, two new oligoalginate lyase genes, oalC6 and oalC17, were cloned from Cellulophaga sp. SY116, and expressed them in Escherichia coli. The deduced oligoalginate lyases, OalC6 and OalC17, belonged to the polysaccharide lyase (PL) family 6 and 17, respectively. Both showed less than 50% amino acid identity with all of the characterized oligoalginate lyases. Moreover, OalC6 and OalC17 could degrade both alginate polymers and oligomers into monomers in an exolytic mode. Substrate specificity studies demonstrated that OalC6 preferred α-L-guluronate (polyG) blocks, while OalC17 preferred poly β-D-mannuronate (polyM) blocks. The combination of OalC6 and OalC17 showed synergistic degradation ability toward both alginate polymers and oligomers. Finally, an efficient process for the production of alginate monomers was established by combining the new-isolated exotype alginate lyases (i.e., OalC6 and OalC17) and the endotype alginate lyase AlySY08. Overall, our work provides new insights for the development of novel biotechnologies for biofuel production from seaweed.     

Expression and characterization of a new heat-stable endo-type alginate lyase from deep-sea bacterium Flammeovirga sp. NJ-04

Extremophiles - Alginate lyases play an essential role in the production of oligosaccharides by degrading alginate polysaccharide. Although many alginate lyases from various microorganisms have...     

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.     

Structural and molecular basis for the substrate positioning mechanism of a new PL7 subfamily alginate lyase from the arctic

Alginate lyases play important roles in alginate degradation in the ocean. Although a large number of alginate lyases have been characterized, little is yet known about those in extremely cold polar environments, which may have unique mechanisms for environmental adaptation and for alginate degradation. Here, we report the characterization of a novel PL7 alginate lyase AlyC3 from Psychromonas sp. C-3 isolated from the Arctic brown alga Laminaria, including its phylogenetic classification, catalytic properties, and structure. We propose the establishment of a new PM-specific subfamily of PL7 (subfamily 6) represented by AlyC3 based on phylogenetic analysis and enzymatic properties. Structural and biochemical analyses showed that AlyC3 is a dimer, representing the first dimeric endo-alginate lyase structure. AlyC3 is activated by NaCl and adopts a novel salt-activated mechanism; that is, salinity adjusts the enzymatic activity by affecting its aggregation states. We further solved the structure of an inactive mutant H127A/Y244A in complex with a dimannuronate molecule and proposed the catalytic process of AlyC3 based on structural and biochemical analyses. We show that Arg⁸² and Tyr¹⁹⁰ at the two ends of the catalytic canyon help the positioning of the repeated units of the substrate and that His¹²⁷, Tyr²⁴⁴, Arg⁷⁸, and Gln¹²⁵ mediate the catalytic reaction. Our study uncovers, for the first time, the amino acid residues for alginate positioning in an alginate lyase and demonstrates that such residues involved in alginate positioning are conserved in other alginate lyases. This study provides a better understanding of the mechanisms of alginate degradation by alginate lyases.     

Isozymes of Alginate Lyase in the Mid-gut Gland of Turbo cornutus

Alginate lyase, SP2, from Turbo cornutus was separated on an SP-Sephadex C-50 column from SP1, whose properties have already been reported, and purified according to the method employed for SP1, to obtain information on SP2. The profiles of the optimal pH, pH-stability, thermal inactivation and molecular size of SP2 were entirely the same as those of SP1. The isoelectric point of SP1 and SP2 was 7.5 and 7.7, respectively. The action of SP2 on Alginate caused a rapid decrease in solution viscosity. Analysis of digestion products of alginate with SP2 showed that the enzyme had an affinity toward the mannuronate-rich domains of the alginate molecule and released unsaturated oligomers mostly composed of mannuronic acid as final product. Alginate lyases, SP1 and SP2, were shown to be isozymes in the mid-gut gland of Turbo cornutus.     

Optimal production of 4-deoxy-l-erythro-5-hexoseulose uronic acid from alginate for brown macro algae saccharification by combining endo- and exo-type alginate lyases

Algae are considered as third-generation biomass, and alginate is the main component of brown macroalgae. Alginate can be enzymatically depolymerized by alginate lyases into uronate monomers, such as mannuronic acid and guluronic acid, which are further nonenzymatically converted to 4-deoxy-l-erythro-5-hexoseulose uronic acid (DEH). We have optimized an enzymatic saccharification process using two recombinant alginate lyases, endo-type Alg7D and exo-type Alg17C, for the efficient production of DEH from alginate. When comparing the sequential and simultaneous additions of Alg7D and Alg17C, it was found that the final yield of DEH was significantly higher when the enzymes were added sequentially. The progress of saccharification reactions and production of DEH were verified by thin layer chromatography and gas chromatography–mass spectrometry, respectively. Our results showed that the two recombinant enzymes could be exploited for the efficient production of DEH that is the key substrate for producing biofuels from brown macro algal biomass.     

Purification and characterization of a novel alginate lyase from the marine bacterium Cobetia sp. NAP1 isolated from brown algae

The application of marine resources, instead of fossil fuels, for biomass production is important for building a sustainable society. Seaweed is valuable as a source of marine biomass for producing biofuels such as ethanol, and can be used in various fields. Alginate is an anionic polysaccharide that forms the main component of brown algae. Various alginate lyases (e.g. exo- and endo-types and oligoalginate lyase) are generally used to degrade alginate. We herein describe a novel alginate lyase, AlgC-PL7, which belongs to the polysaccharide lyase 7 family. AlgC-PL7 was isolated from the halophilic Gram-negative bacterium Cobetia sp. NAP1 collected from the brown algae Padina arborescens Holmes. The optimal temperature and pH for AlgC-PL7 activity were 45 °C and 8, respectively. Additionally, AlgC-PL7 was thermostable and salt-tolerant, exhibited broad substrate specificity, and degraded alginate into monosaccharides. Therefore, AlgC-PL7 is a promising enzyme for the production of biofuels.     

Functional implications of structure-based sequence alignment of proteins in the extracellular pectate lyase superfamily.

Pectate lyases are plant virulence factors that degrade the pectate component of the plant cell wall. The enzymes share considerable sequence homology with plant pollen and style proteins, suggesting a shared structural topology and possibly functional relationships as well. The three-dimensional structures of two Erwinia chrysanthemi pectate lyases, C and E, have been superimposed and the structurally conserved amino acids have been identified. There are 232 amino acids that superimpose with a root-mean-square deviation of 3 A or less. These amino acids have been used to correct the primary sequence alignment derived from evolution-based techniques. Subsequently, multiple alignment techniques have allowed the realignment of other extracellular pectate lyases as well as all sequence homologs, including pectin lyases and the plant pollen and style proteins. The new multiple sequence alignment reveals amino acids likely to participate in the parallel beta helix motif, those involved in binding Ca2+, and those invariant amino acids with potential catalytic properties. The latter amino acids cluster in two well-separated regions on the pectate lyase structures, suggesting two distinct enzymatic functions for extracellular pectate lyases and their sequence homologs.     

Bacterial alginate: physiology, product quality and process aspects

Alginate, a copolymer of beta-D-mannuronic acid and alpha-L-guluronic acid and currently commercially produced from the marine brown algae, can also be biologically produced by bacteria such as Azotobacter vinelandii, A. chroococcum and several species of Pseudomonas. The ever-increasing applications of this polymer in the food and pharmaceutical sectors have led to continuing research interest aimed at better understanding the metabolic pathways, the physiological or biological function of this polymer, the regulation of its formation and composition, and optimising the microbial production process. These aspects are reviewed here, with particular attention to alginate formation in the soil bacterium A. vinelandii. In addition, the biotechnological and industrial applications of alginate are summarised.     

Structural and mutational characterization of the catalytic A-module of the mannuronan C-5-epimerase AlgE4 from Azotobacter vinelandii

Alginate is a family of linear copolymers of (1-->4)-linked beta-d-mannuronic acid and its C-5 epimer alpha-l-guluronic acid. The polymer is first produced as polymannuronic acid and the guluronic acid residues are then introduced at the polymer level by mannuronan C-5-epimerases. The structure of the catalytic A-module of the Azotobacter vinelandii mannuronan C-5-epimerase AlgE4 has been determined by x-ray crystallography at 2.1-A resolution. AlgE4A folds into a right-handed parallel beta-helix structure originally found in pectate lyase C and subsequently in several polysaccharide lyases and hydrolases. The beta-helix is composed of four parallel beta-sheets, comprising 12 complete turns, and has an amphipathic alpha-helix near the N terminus. The catalytic site is positioned in a positively charged cleft formed by loops extending from the surface encompassing Asp(152), an amino acid previously shown to be important for the reaction. Site-directed mutagenesis further implicates Tyr(149), His(154), and Asp(178) as being essential for activity. Tyr(149) probably acts as the proton acceptor, whereas His(154) is the proton donor in the epimerization reaction.     

Novel Alginate Lyase (Aly5) from a Polysaccharide-Degrading Marine Bacterium,Flammeovirga sp. Strain MY04: Effects of Module Truncation on Biochemical Characteristics,Alginate Degradation Patterns,and Oligosaccharide-Yielding Properties

Alginate lyases are important tools for oligosaccharide preparation, medical treatment, and energy bioconversion. Numerous alginate lyases have been elucidated. However, relatively little is known about their substrate degradation patterns and product-yielding properties, which is a limit to wider enzymatic applications and further enzyme improvements. Herein, we report the characterization and module truncation of Aly5, the first alginate lyase obtained from the polysaccharide-degrading bacterium Flammeovirga. Aly5 is a 566-amino-acid protein and belongs to a novel branch of the polysaccharide lyase 7 (PL7) superfamily. The protein rAly5 is an endolytic enzyme of alginate and associated oligosaccharides. It prefers guluronate (G) to mannuronate (M). Its smallest substrate is an unsaturated pentasaccharide, and its minimum product is an unsaturated disaccharide. The final alginate digests contain unsaturated oligosaccharides that generally range from disaccharides to heptasaccharides, with the tetrasaccharide fraction constituting the highest mass concentration. The disaccharide products are identified as ΔG units. While interestingly, the tri- and tetrasaccharide fractions each contain higher proportions of ΔG to ΔM ends, the larger final products contain only ΔM ends, which constitute a novel oligosaccharide-yielding property of guluronate lyases. The deletion of the noncatalytic region of Aly5 does not alter its M/G preference but significantly decreases the enzymatic activity and enzyme stability. Notably, the truncated protein accumulates large final oligosaccharide products but yields fewer small final products than Aly5, which are codetermined by its M/G preference to and size enlargement of degradable oligosaccharides. This study provides novel enzymatic properties and catalytic mechanisms of a guluronate lyase for potential uses and improvements.     

Intracellular Alginolytic Enzymes of the Marine Bacterium Pseudoalteromonas citrea KMM 3297

The marine bacterium Pseudoalteromonas citrea KMM 3297 is an associate of the holothurian Apostichopus japonicus. When grown in a medium containing glucose, the strain produces two intracellular alginolytic enzymes, AlI and AlII. Fucoidan from the brown alga Fucus evanescens induces synthesis of one more alginolytic enzyme, AlIII. These enzymes were separated using anion-exchange chromatography. The alginate lyase AlI completely retains its activity at 35 degrees C, AlII and AlIII being stable at 45 degrees C. The alginate lyases exhibit maximal activities in the range of pH 7-8. The molecular weights of AlI, AlII, and AlIII determined by gel filtration are 25, 79, and 61 kD, respectively. All the investigated enzymes are endo-type alginate lyases. They catalyze degradation of polyguluronate (poly-G) and polymannuronate (poly-M) yielding oligosaccharides of the polymerization degree of 5 > or = n > or = 3 with the unsaturated bond between the C4 and C5 atoms of the non-reducing terminus. A mixture of these three enzymes exhibits synergism while acting on the polymeric substrate. The Km values of the alginate lyase AlI for poly-G and poly-M are 24 and 34 micro g/ml, respectively. Alginate lyase AlIII exhibits less affinity to poly-M (Km = 130.0 microg/ml) than to poly-G (Km = 40.0 microg/ml). NaCl (0.2 M), MgCl2 and MgSO4 (0.01 M) activate all three enzymes more than twofold. The presence of several alginolytic enzymes of different specificity provides efficient destruction of alginic acids of brown algae by the strain P. citrea KMM 3297.     

Cloning and characterization of pectate lyases expressed in the esophageal gland of the pine wood nematode Bursaphelenchus xylophilus

Two pectate lyase genes (Bx-pel-1 and Bx-pel-2) were cloned from the pine wood nematode, Bursaphelenchus xylophilus. The deduced amino acid sequences of these pectate lyases are most similar to polysaccharide lyase family 3 proteins. Recombinant BxPEL1 showed highest activity on polygalacturonic acid and lower activity on more highly methylated pectin. Recombinant BxPEL1 demonstrated full dependency on Ca2+ for activity and optimal activity at 55 degrees C and pH 8 to 10 like other pectate lyases of polysaccharide lyase family 3. The protein sequences have predicted signal peptides at their N-termini and the genes are expressed solely in the esophageal gland cells of the nematode, indicating that the pectate lyases could be secreted into plant tissues to help feeding and migration in the tree. This study suggests that pectate lyases are widely distributed in plant-parasitic nematodes and play an important role in plant-nematode interactions.     

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.