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1.
Yamasaki M Moriwaki S Miyake O Hashimoto W Murata K Mikami B 《The Journal of biological chemistry》2004,279(30):31863-31872
Structural and functional analyses of alginate lyases are important in the clarification of the biofilm-dependent ecosystem in Pseudomonas aeruginosa and in the development of therapeutic agents for bacterial disease. Most alginate lyases are classified into polysaccharide lyase (PL) family-5 and -7 based on their primary structures. Family PL-7 enzymes are still poorly characterized especially in structural properties. Among family PL-7, a gene coding for a hypothetical protein (PA1167) homologous to Sphingomonas alginate lyase A1-II was found to be present in the P. aeruginosa genome. PA1167 overexpressed in Escherichia coli cleaved glycosidic bonds in alginate and released unsaturated saccharides, indicating that PA1167 is an alginate lyase catalyzing a beta-elimination reaction. The enzyme acted preferably on heteropolymeric regions endolytically and worked most efficiently at pH 8.5 and 40 degrees C. The specific activity of PA1167, however, was much weaker than that of the known alginate lyase AlgL, suggesting that AlgL plays a main role in alginate depolymerization in P. aeruginosa. In addition to this specific activity, differences were found between PA1167 and AlgL in enzyme properties such as molecular mass, optimum pH, salt effect, and substrate specificity. The first crystal structure of the family PL-7 alginate lyase was determined at 2.0 A resolution. PA1167 was found to form a glove-like beta-sandwich composed of 15 beta-strands and 3 alpha-helices. The structural difference between the beta-sandwich PA1167 of family PL-7 and alpha/alpha-barrel AlgL of family PL-5 may be responsible for the enzyme characteristics. Crystal structures of polysaccharide lyases determined so far indicate that they can be assigned to three folding groups having parallel beta-helix, alpha/alpha-barrel, and alpha/alpha-barrel + antiparallel beta-sheet structures as basic frames. PA1167 is the fourth novel folding structure found among polysaccharide lyases. 相似文献
2.
Fei Xu Xiu-Lan Chen Xiao-Hui Sun Fang Dong Chun-Yang Li Ping-Yi Li Haitao Ding Yin Chen Yu-Zhong Zhang Peng Wang 《The Journal of biological chemistry》2020,295(48):16380
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. 相似文献
3.
Origin and diversity of alginate lyases of families PL-5 and -7 in Sphingomonas sp. strain A1
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Sphingomonas sp. strain A1 has three endotype alginate lyases (A1-I, A1-II [family PL-7], and A1-III [family PL-5]), each of which is encoded by a single gene. In addition to those of these lyases, a gene (the A1-II' gene) showing significant identity with the A1-II gene was present in the bacterial genome and coded for an alginate lyase with broad substrate specificity. Since no expression of A1-II' was observed even in bacterial cells grown on alginate, the A1-II' gene was thought to be a silent gene derived from the A1-II gene, presumably through duplication, modification, and translocation. 相似文献
4.
《Journal of molecular biology》2019,431(24):4897-4909
Alginate lyases, which are important in both basic and applied sciences, fall into ten polysaccharide lyase (PL) families. PL36 is a newly established family that includes 39 bacterial sequences and one eukaryotic sequence. Till now, the structures or catalytic mechanisms of PL36 alginate lyases have yet to be revealed. Here, we characterized a novel PL36 alginate lyase, Aly36B, from Chitinophaga sp. MD30. Aly36B is a polymannuronate specific endolytic alginate lyase. To probe the catalytic mechanism of Aly36B, the structures of wild-type Aly36B and its mutants (K143A/Y185A in complex with alginate tetrasaccharide and K143A/M171A with trisaccharide) were solved. The overall structure of Aly36B belongs to the β-jelly roll scaffold, adopting a typical β-sandwich fold. Aly36B contains a Ca2+, which is far away from the active center and plays an important role in stabilizing the structure of Aly36B. Based on structural and mutational analyses, the catalytic mechanism of Aly36B for alginate degradation was explained. During catalysis, Arg169, Tyr185, and Tyr187 are responsible for neutralizing the negative charge of the substrate, and Lys143 acts as both the catalytic base and the catalytic acid, which represents a new kind of catalytic mechanism of alginate lyases. Sequence alignment shows that these four residues involved in catalysis are highly conserved in all PL36 sequences, suggesting that PL36 alginate lyases may adopt a similar catalytic mechanism. Taken together, this study reveals the molecular structure and catalytic mechanism of a PL36 alginate lyase, broadening our knowledge on alginate lyases and facilitating future biotechnological applications of PL36 alginate lyases. 相似文献
5.
Sheng Dong Tian-Di Wei Xiu-Lan Chen Chun-Yang Li Peng Wang Bin-Bin Xie Qi-Long Qin Xi-Ying Zhang Xiu-Hua Pang Bai-Cheng Zhou Yu-Zhong Zhang 《The Journal of biological chemistry》2014,289(43):29558-29569
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. 相似文献
6.
Anne T?ndervik Geir Klinkenberg Olav A. Aarstad Finn Drabl?s Helga Ertesv?g Trond E. Ellingsen Gudmund Skj?k-Br?k Svein Valla H?vard Sletta 《The Journal of biological chemistry》2010,285(46):35284-35292
Alginates are commercially valuable and complex polysaccharides composed of varying amounts and distribution patterns of 1–4-linked β-d-mannuronic acid (M) and α-l-guluronic acid (G). This structural variability strongly affects polymer physicochemical properties and thereby both commercial applications and biological functions. One promising approach to alginate fine structure elucidation involves the use of alginate lyases, which degrade the polysaccharide by cleaving the glycosidic linkages through a β-elimination reaction. For such studies one would ideally like to have different lyases, each of which cleaves only one of the four possible linkages in alginates: G-G, G-M, M-G, and M-M. So far no lyase specific for only G-G linkages has been described, and here we report the construction of such an enzyme by mutating the gene encoding Klebsiella pneumoniae lyase AlyA (a polysaccharide lyase family 7 lyase), which cleaves both G-G and G-M linkages. After error-prone PCR mutagenesis and high throughput screening of ∼7000 lyase mutants, enzyme variants with a strongly improved G-G specificity were identified. Furthermore, in the absence of Ca2+, one of these lyases (AlyA5) was found to display no detectable activity against G-M linkages. G-G linkages were cleaved with ∼10% of the optimal activity under the same conditions. The substitutions conferring altered specificity to the mutant enzymes are located in conserved regions in the polysaccharide lyase family 7 alginate lyases. Structure-function analyses by comparison with the known three-dimensional structure of Sphingomonas sp. A1 lyase A1-II′ suggests that the improved G-G specificity might be caused by increased affinity for nonproductive binding of the alternating G-M structure. 相似文献
7.
Shangyong Li Linna Wang Xuehong Chen Wenwen Zhao Mi Sun Yantao Han 《Marine biotechnology (New York, N.Y.)》2018,20(1):75-86
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. 相似文献
8.
Wenjun Han Jingyan Gu Yuanyuan Cheng Huihui Liu Yuezhong Li Fuchuan Li 《Applied and environmental microbiology》2016,82(1):364-374
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. 相似文献
9.
Herbivorous marine gastropods such as abalone and sea hare ingest brown algae as a major diet and degrade the dietary alginate with alginate lyase (EC 4.2.2.3) in their digestive fluid. To date alginate lyases from Haliotidae species such as abalone have been well characterized and the primary structure analyses have classified abalone enzymes into polysaccharide-lyase-family 14 (PL-14). However, other gastropod enzymes have not been so well investigated and only partial amino-acid sequences are currently available. To improve the knowledge for primary structure and catalytic residues of gastropod alginate lyases, we cloned the cDNA encoding an alginate lyase, AkAly30, from an Aplysiidae species Aplysia kurodai and assessed its catalytically important residues by site-directed mutagenesis. Alginate lyase cDNA fragments were amplified by PCR followed by 5′- and 3′-RACE from A. kurodai hepatopancreas cDNA. The finally cloned cDNA comprised 1313 bp which encoded an amino-acid sequence of 295 residues of AkAly30. The deduced sequence comprised an initiation methionine, a putative signal peptide for secretion (18 residues), a propeptide-like region (9 residues), and a mature AkAly30 domain (267 residues) which showed ∼40% amino-acid identity with abalone alginate lyases. An Escherichia coli BL21(DE3)-pCold I expression system for recombinant AkAly30 (recAkAly30) was constructed and site-directed mutagenesis was performed to assess catalytically important amino-acid residues which had been suggested in abalone and Chlorella virus PL-14 enzymes. Replacements of K99, S126, R128, Y140 and Y142 of recAkAly30 by Ala and/or Phe greatly decreased its activity as in the case of abalone and/or Chlorella virus enzymes. Whereas, H213 that was essential for Chlorella virus enzyme to exhibit the activity at pH 10.0 was originally replaced by N120 in AkAly30. The reverse replacement of N120 by His in recAkAly30 increased the activity at pH 10.0 from 8 U/mg to 93 U/mg; however, the activity level at pH 7.0, i.e., 774.8 U/mg, was still much higher than that at pH 10.0. This indicates that N120 is not directly related to the pH dependence of AkAly30 unlike H213 of vAL-1. 相似文献
10.
A bacterium, Sphingomonas sp. strain A1, can incorporate alginate into cells through a novel ABC (ATP-binding cassette) transporter system specific to the macromolecule. The transported alginate is depolymerized to di- and trisaccharides by three kinds of cytoplasmic alginate lyases (A1-I [66 kDa], A1-II [25 kDa], and A1-III [40 kDa]) generated from a single precursor through posttranslational autoprocessing. The resultant alginate oligosaccharides were degraded to monosaccharides by cytoplasmic oligoalginate lyase. The enzyme and its gene were isolated from the bacterial cells grown in the presence of alginate. The purified enzyme was a monomer with a molecular mass of 85 kDa and cleaved glycosidic bonds not only in oligosaccharides produced from alginate by alginate lyases but also in polysaccharides (alginate, polymannuronate, and polyguluronate) most efficiently at pH 8.0 and 37 degrees C. The reaction catalyzed by the oligoalginate lyase was exolytic and thought to play an important role in the complete depolymerization of alginate in Sphingomonas sp. strain A1. The gene for this novel enzyme consisted of an open reading frame of 2,286 bp encoding a polypeptide with a molecular weight of 86,543 and was located downstream of the genes coding for the precursor of alginate lyases (aly) and the ABC transporter (algS, algM1, and algM2). This result indicates that the genes for proteins required for the transport and complete depolymerization of alginate are assembled to form a cluster. 相似文献
11.
A gene for a polyMG-specific alginate lyase possessing a novel structure was identified and cloned from Stenotrophomas maltophilia KJ-2 by using PCR with homologous nucleotide sequences-based primers. The recombinant alginate lyase consisting of 475 amino acids was purified on Ni-Sepharose column and exhibited the highest activity at pH 8 and 40?°C. Interestingly, the recombinant alginate lyase was expected to have a similar catalytic active site of chondroitin B lyase but did not show chondroitin lyase activity. In the test of substrate specificity, the recombinant alginate lyase preferentially degraded the glycosidic bond of polyMG-block than polyM-block and polyG-block. The chemical structures of the degraded alginate oligosaccharides were elucidated to have mannuronate (M) at the reducing end on the basis of NMR analysis, supporting that KJ-2 polyMG-specific alginate lyase preferably degraded the glycosidic bond in M-G linkage than that in G-M linkage. The KJ-2 polyMG-specific alginate lyase can be used in combination with other alginate lyases for a synergistic saccharification of alginate. 相似文献
12.
Hee Sook Kim Choul-Gyun Lee Eun Yeol Lee 《Biotechnology and Bioprocess Engineering》2011,16(5):843-851
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. 相似文献
13.
A structural basis for depolymerization of alginate by polysaccharide lyase family-7 总被引:2,自引:0,他引:2
Alginate lyases depolymerize alginate, a heteropolysaccharide consisting of alpha-L-guluronate and beta-D-mannuronate, through a beta-elimination reaction. Their structure/function relationships are expected to provide information valuable to future industrial alginate processing and drug design for Pseudomonas aeruginosa alginate biofilm-dependent infection, but much remains unknown. Here, we present the crystal structure at 1.0 A resolution and the results of mutational analysis of Sphingomonas sp. A1 alginate lyase A1-II', which is grouped into the polysaccharide lyase (PL) family-7. The overall structure of A1-II' uses a beta-sandwich fold, and it has a large active cleft covered by two short flexible loops. Comparison with other family PL-7 structures indicated that loop opening is necessary for substrate binding when the catalytic reaction is initiated. In contrast to the disorder in many side-chains on the protein surface, the three adjacent beta-strands at the center of the active cleft are well ordered. This results from hydrogen bond networks and stacking-like associations identical with those in other family PL-7 structures. Disruption of these interactions by site-directed mutagenesis (R146A, E148A, R150A, Q189A, and K280A) makes the protein insoluble or greatly decreases its activity. The A1-II' structure includes two sulfate ions in the active cleft. Ammonium sulfate was a potent inhibitor with a Ki of 2.5 mM, indicating that our structure represents a model of the inhibitory state. Results of mutational analysis and continuous hydrogen bond networks suggest that Arg146, Gln189, His191, and Tyr284 form an active center. Tyr284OH appears particularly crucial to the catalytic reaction, which is supported by sulfate ion binding and the proximity to the C5 and O4 atoms of subsite +1 in the model obtained by energy minimization calculations using tri-mannuronate. The structural basis shown by this study is similar in many respects to that of the family PL-5 enzymes. 相似文献
14.
Akihito Ochiai Masayuki Yamasaki Bunzo Mikami Wataru Hashimoto Kousaku Murata 《The Journal of biological chemistry》2010,285(32):24519-24528
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. 相似文献
15.
《Bioscience, biotechnology, and biochemistry》2013,77(11):1853-1857
Pseudomonas sp. OS-ALG-9 produces several kinds of alginate-degrading enzymes both intra- and extracellularly. As a second alginate lyase of this bacterium, the gene encoding alyII has been cloned in Escherichia coli JM109 by shotgun techniques and then sequenced. The alyII gene has an open reading frame of 2141 bp encoding 713 amino acid residues with a calculated molecular mass of 79,803 Da. The deduced amino acid sequence did not show any extensive similarity with those of other known alginate lyases, however, hydrophobic cluster analysis showed that alyII belonged to class 3 of alginate lyases. The alginate lyase from E. coli harboring the alyII gene showed a single active band, which coincided with one of four major alginate lyases from the crude cell extracts of Pseudomonas sp. OS-ALG-9 on a zymogram. 相似文献
16.
Kohsuke Uchimura Masayuki Miyazaki Yuichi Nogi Tohru Kobayashi Koki Horikoshi 《Marine biotechnology (New York, N.Y.)》2010,12(5):526-533
Four alginate lyase genes were cloned and sequenced from the genomic DNAs of deep-sea bacteria, namely members of Vibrio and Agarivorans. Three of them were from Vibrio sp. JAM-A9m, which encoded alginate lyases, A9mT, A9mC, and A9mL. A9mT was composed of 286 amino acids and 57% homologous
to AlxM of Photobacterium sp. A9mC (221 amino acids) and A9mL (522 amino acids) had the highest degree of similarity to two individual alginate lyases
of Vibrio splendidus with 74% and 84% identity, respectively. The other gene for alginate lyase, A1mU, was shotgun cloned from Agarivorans sp. JAM-A1m. A1mU (286 amino acids) showed the highest homology to AlyVOA of Vibrio sp. with 76% identity. All alginate lyases belong to polysaccharide lyase family 7, although, they do not show significant
similarity to one another with 14% to 58% identity. Among the above lyases, the recombinant A9mT was purified to homogeneity
and characterized. The molecular mass of A9mT was around 28 kDa. The enzyme was remarkably salt activated and showed the highest
thermal stability in the presence of NaCl. A9mT favorably degraded mannuronate polymer in alginate. We discussed substrate
specificities of family 7 alginate lyases based on their conserved amino acid sequences. 相似文献
17.
Lishuxin Huang Jungang Zhou Xiao Li Qiang Peng Hong Lu Yuguang Du 《Journal of industrial microbiology & biotechnology》2013,40(1):113-122
Alginate lyase is a promising biocatalyst because of its application in saccharification of alginate for the production of biochemicals and renewable biofuels. This study described the isolation of a new alginate metabolizing bacterium, Flavobacterium sp. S20, from sludge samples and the characterization of its alginate lyase Alg2A. The alginate lyase gene, alg2A, was obtained by constructing and screening the genomic library of the strain S20 and overexpressed in Escherichia coli. Substrate specificity assays indicated Alg2A preferred poly-α-l-guluronate as a substrate over poly-β-d-mannuronate. In the saccharification process of a high content (10 %, w/v) of sodium alginate, the recombinant alginate lyase Alg2A yielded 152 of mM the reducing sugars after 69 h of reaction, and the amounts of oligosaccharides with a different degree of polymerization (DP) generated by Alg2A gradually accumulated without significant variation in the distribution of oligosaccharide compositions. These results indicated that Alg2A possessed high enzymatic capability for saccharifying the alginate, which could be used in saccharifying the alginate biomass prior to the main fermentation process for biofuels. In addition, Alg2A had a different endolytic reaction mode from both the two commercial alginate lyases and other alginate lyases from polysaccharide lyase family 7 owing to high yields of penta-, hex-, and hepta-saccharides in the hydrolysis products of Alg2A. Thus, Alg2A could be a good tool for the large-scale preparation of alginate oligosaccharides with high DP. 相似文献
18.
海藻酸盐裂解酶研究进展 总被引:5,自引:0,他引:5
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。 相似文献
19.
An alginate lyase gene of a newly isolated Pseudomonas sp. strain KS-408 was cloned by using PCR with the specific primers designed from homologous nucleotide sequences. A partial protein sequence of KS-408 alginate lyase was homology-modeled on the basis of the crystal structure of A1-III alginate lyase from Sphingomonas sp. strain A1. The proposed 3-D structure of KS-408 alginate lyase shows that Asn-198, His-199, Arg-246, and Tyr-253 residues are conserved for the catalytic active site. The recombinant KS-408-1F (with signal peptide) and KS-408-2F (without signal peptide) alginate lyases with the (His)(6) tag consist of 393 (44.5 kDa) and 372 (42.4 kDa) amino acids with isoelectric points of 8.64 and 8.46, respectively. The purified recombinant KS-408 alginate lyase was very stable when it was incubated at 40 °C for 30 min. Alginate oligosaccharides produced by the KS-408-2F alginate lyase were purified on a Bio-Gel P2 column and analyzed by thin-layer chromatography, fast-protein liquid chromatography, and electrospray ionization mass spectrometry. (1)H NMR data showed that the KS-408-2F alginate lyase cleaved the glycosidic linkages between two mannuronates (mannuronate-β(1-4)-mannuronate) or mannuronate and guluronate (mannuronate-β(1-4)-guluronate), indicating that the KS-408 alginate lyase is a polyM-specific lyase. 相似文献
20.
Isolation of Marine Bacteria Capable of Producing Specific Lyases for Alginate Degradation 总被引:6,自引:3,他引:3
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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. 相似文献