scholarly journals Biochemical Characterization and Degradation Pattern of a Novel Endo-Type Bifunctional Alginate Lyase AlyA from Marine Bacterium Isoptericola halotolerans

Marine Drugs ◽  
2018 ◽  
Vol 16 (8) ◽  
pp. 258 ◽  
Author(s):  
Benwei Zhu ◽  
Limin Ning ◽  
Yucui Jiang ◽  
Lin Ge
Keyword(s):  
Substrate Specificity ◽  
Biochemical Characterization ◽  
Alginate Lyase ◽  
Ionization Mass ◽  
Broad Substrate Specificity ◽  
Action Mode ◽  
Degradation Pattern ◽  
Km Value ◽  
Alginate Lyases ◽  
Layer Chromatography

Alginate lyases are important tools to prepare oligosaccharides with various physiological activities by degrading alginate. Particularly, the bifunctional alginate lyase can efficiently hydrolyze the polysaccharide into oligosaccharides. Herein, we cloned and identified a novel bifunctional alginate lyase, AlyA, with a high activity and broad substrate specificity from bacterium Isoptericola halotolerans NJ-05 for oligosaccharides preparation. For further applications in industry, the enzyme has been characterized and its action mode has been also elucidated. It exhibited the highest activity (7984.82 U/mg) at pH 7.5 and 55 °C. Additionally, it possessed a broad substrate specificity, showing high activities towards not only polyM (polyβ-d-mannuronate) (7658.63 U/mg), but also polyG (poly α-l-guluronate) (8643.29 U/mg). Furthermore, the Km value of AlyA towards polyG (3.2 mM) was lower than that towards sodium alginate (5.6 mM) and polyM (6.7 mM). TLC (Thin Layer Chromatography) and ESI-MS (Electrospray Ionization Mass Spectrometry) were used to study the action mode of the enzyme, showing that it can hydrolyze the substrates in an endolytic manner to release a series of oligosaccharides such as disaccharide, trisaccharide, and tetrasaccharide. This study provided extended insights into the substrate recognition and degrading pattern of the alginate lyases, with a broad substrate specificity.

scholarly journals Biochemical Characterization and Elucidation of Action Pattern of a Novel Polysaccharide Lyase 6 Family Alginate Lyase from Marine Bacterium Flammeovirga sp. NJ-04

Marine Drugs ◽  
2019 ◽  
Vol 17 (6) ◽  
pp. 323 ◽  
Author(s):  
Qian Li ◽  
Fu Hu ◽  
Benwei Zhu ◽  
Yun Sun ◽  
Zhong Yao
Keyword(s):  
Biochemical Characterization ◽  
Alginate Lyase ◽  
Ionization Mass ◽  
Action Pattern ◽  
Polysaccharide Lyase ◽  
Glycosidic Bonds ◽  
Alginate Oligosaccharides ◽  
Degradation Pattern ◽  
Alginate Lyases ◽  
Layer Chromatography

Alginate lyases have been widely used to prepare alginate oligosaccharides in food, agricultural, and medical industries. Therefore, discovering and characterizing novel alginate lyases with excellent properties has drawn increasing attention. Herein, a novel alginate lyase FsAlyPL6 of Polysaccharide Lyase (PL) 6 family is identified and biochemically characterized from Flammeovirga sp. NJ-04. It shows highest activity at 45 °C and could retain 50% of activity after being incubated at 45 °C for 1 h. The Thin-Layer Chromatography (TLC) and Electrospray Ionization Mass Spectrometry (ESI-MS) analysis indicates that FsAlyPL6 endolytically degrades alginate polysaccharide into oligosaccharides ranging from monosaccharides to pentasaccharides. In addition, the action pattern of the enzyme is also elucidated and the result suggests that FsAlyPL6 could recognize tetrasaccharide as the minimal substrate and cleave the glycosidic bonds between the subsites of −1 and +3. The research provides extended insights into the substrate recognition and degradation pattern of PL6 alginate lyases, which may further expand the application of alginate lyases.

Molecular identification of a polyM-specific alginate lyase from Pseudomonas sp. strain KS-408 for degradation of glycosidic linkages between two mannuronates or mannuronate and guluronate in alginate

2011 ◽  
Vol 57 (12) ◽  
pp. 1032-1041 ◽  
Author(s):  
Natania Kam ◽  
Yoo Jung Park ◽  
Eun Yeol Lee ◽  
Hee Sook Kim
Keyword(s):  
Signal Peptide ◽  
Alginate Lyase ◽  
Ionization Mass ◽  
Pseudomonas Sp ◽  
H Nmr ◽  
Alginate Oligosaccharides ◽  
Catalytic Active Site ◽  
Isoelectric Points ◽  
Alginate Lyases ◽  
Layer Chromatography

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. 1H 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.

scholarly journals Origin and Diversity of Alginate Lyases of Families PL-5 and -7 in Sphingomonas sp. Strain A1

2004 ◽  
Vol 186 (9) ◽  
pp. 2891-2896 ◽  
Author(s):  
Osamu Miyake ◽  
Akihito Ochiai ◽  
Wataru Hashimoto ◽  
Kousaku Murata
Keyword(s):  
Substrate Specificity ◽  
Single Gene ◽  
Bacterial Genome ◽  
Alginate Lyase ◽  
Bacterial Cells ◽  
Silent Gene ◽  
Broad Substrate Specificity ◽  
Alginate Lyases

ABSTRACT 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.

scholarly journals Biochemical Characteristics and Variable Alginate-Degrading Modes of a Novel Bifunctional Endolytic Alginate Lyase

2017 ◽  
Vol 83 (23) ◽  
Author(s):  
Yuanyuan Cheng ◽  
Dandan Wang ◽  
Jingyan Gu ◽  
Junge Li ◽  
Huihui Liu ◽  
...  
Keyword(s):  
Alginate Lyase ◽  
Fluorescent Labeling ◽  
Structure Identification ◽  
Key Factors ◽  
Content Type ◽  
Biochemical Characteristics ◽  
Action Mode ◽  
Molecular Sizes ◽  
Sugar Chains ◽  
Alginate Lyases

ABSTRACT Bifunctional alginate lyases can efficiently degrade alginate comprised of mannuronate (M) and guluronate (G), but their substrate-degrading modes have not been thoroughly elucidated to date. In this study, we present Aly1 as a novel bifunctional endolytic alginate lyase of the genus Flammeovirga. The recombinant enzyme showed optimal activity at 50°C and pH 6.0. The enzyme produced unsaturated disaccharide (UDP2) and trisaccharide fractions as the final main alginate digests. Primary substrate preference tests and further structure identification of various size-defined final oligosaccharide products demonstrated that Aly1 is a bifunctional alginate lyase and prefers G to M. Tetrasaccharide-size fractions are the smallest substrates, and M, G, and UDP2 fractions are the minimal product types. Remarkably, Aly1 can vary its substrate-degrading modes in accordance with the terminus types, molecular sizes, and M/G contents of alginate substrates, producing a series of small size-defined saturated oligosaccharide products from the nonreducing ends of single or different saturated sugar chains and yielding unsaturated products in distinct but restricted patterns. The action mode changes can be partially inhibited by fluorescent labeling at the reducing ends of oligosaccharide substrates. Deletion of the noncatalytic region (NCR) of Aly1 caused weak changes of biochemical characteristics but increased the degradation proportions of small size-defined saturated M-enriched oligosaccharide substrates and unsaturated tetrasaccharide fractions without any size changes of degradable oligosaccharides, thereby enhancing the M preference and enzyme activity. Therefore, our results provided insight into the variable action mode of a novel bifunctional endolytic alginate lyase to inform accurate enzyme use. IMPORTANCE The elucidated endolytic alginate lyases usually degrade substrates into various size-defined unsaturated oligosaccharide products (≥UDP2), and exolytic enzymes yield primarily unsaturated monosaccharide products. However, it is poorly understood whether endolytic enzymes can produce monosaccharide product types when degrading alginate. In this study, we demonstrated that Aly1, a bifunctional alginate lyase of Flammeovirga sp. strain MY04, is endolytic and monosaccharide producing. Using various sugar chains as testing substrates, we also proved that key factors causing Aly1's action mode changes are the terminus types, molecular sizes, and M/G contents of substrates. Furthermore, the NCR fragment's effects on Aly1's biochemical characteristics and alginate-degrading modes and corresponding mechanisms were discovered by gene truncation and enzyme comparison. In summary, this study provides a novel bifunctional endolytic tool and a variable action mode for accurate use in alginate degradation.

Biochemical characterization and degradation pattern analysis of a novel PL-6 alginate lyase from Streptomyces coelicolor A3(2)

2020 ◽  
Vol 323 ◽  
pp. 126852 ◽  
Author(s):  
Danyang Cheng ◽  
Zhen Liu ◽  
Chengcheng Jiang ◽  
Laihao Li ◽  
Changhu Xue ◽  
...  
Keyword(s):  
Pattern Analysis ◽  
Biochemical Characterization ◽  
Streptomyces Coelicolor ◽  
Alginate Lyase ◽  
Degradation Pattern

scholarly journals Biochemical Characterization of the POM-1 Metallo-β-Lactamase from Pseudomonas otitidis

2014 ◽  
Vol 59 (3) ◽  
pp. 1755-1758 ◽  
Author(s):  
Luisa Borgianni ◽  
Filomena De Luca ◽  
Maria Cristina Thaller ◽  
Yunsop Chong ◽  
Gian Maria Rossolini ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Functional Analysis ◽  
Substrate Specificity ◽  
Biochemical Characterization ◽  
Content Type ◽  
Broad Substrate Specificity ◽  
Catalytic Activities

ABSTRACTThe POM-1 metallo-β-lactamase is a subclass B3 resident enzyme produced byPseudomonas otitidis, a pathogen causing otic infections. The enzyme was overproduced inEscherichia coliBL21(DE3), purified by chromatography, and subjected to structural and functional analysis. The purified POM-1 is a tetrameric enzyme of broad substrate specificity with higher catalytic activities with penicillins and carbapenems than with cephalosporins.

scholarly journals Efficient Degradation of Alginate And Preparation of Unsaturated Monosaccharides By A Novel Alginate Lyase And Effects of Domain Truncation On Biochemical Characteristics, Degradation Patterns

2021 ◽  
Author(s):  
Qian Li ◽  
Shengsheng Cao ◽  
Ling Zheng ◽  
Benwei Zhu
Keyword(s):  
Synergistic Effect ◽  
Brown Algae ◽  
Catalytic Efficiency ◽  
Alginate Lyase ◽  
Commercial Application ◽  
Action Mode ◽  
Domain Truncation ◽  
High Conversion Rate ◽  
Modular Domain ◽  
Alginate Lyases

Abstract BackgroundBrown algae are considered promising crops for the production of sustainable biofuels. However, its commercial application has been limited by lack of efficient methods for converting alginate into fermentable sugars. Recently, exo-type alginate lyases have received extensive attention due to their excellent ability of conversion of alginate into 4-deoxy-L-erythro-5-hexoseulose uronate (DEH), a promising material for bioethanol production and biorefinery systems.ResultsHerein, we cloned and characterized a novel alginate lyase AlyPL17 from Pedobacter hainanensis NJ-02. It possessed outstanding catalytic efficiency towards polymannuronic acid (polyM), polyguluronic acid (polyG) and alginate sodium, with kcat of 39.42 + 1.9 s-1, 32.53 + 0.88 s-1, and 38.30 + 2.12 s-1, respectively. In addition, AlyPL17 adopts a unique hybrid action mode to degrade alginate by the synergistic effect of two domains. Furthermore, the combination of AlyPL17 and AlyPL6 exhibited apparently synergistic effect for the preparation of unsaturated monosaccharides. ConclusionOverall, the results show that AlyPL17 is a PL17 exo-type alginate lyase with high activity and a high conversion rate at low/moderate temperatures, which provides a useful enzymatic tool for the conversion of brown algae into biofuels and enhance our understanding of the function of modular domain of alginate lyase.

scholarly journals A Thermostable Monoacylglycerol Lipase from Marine Geobacillus sp. 12AMOR1: Biochemical Characterization and Mutagenesis Study

2019 ◽  
Vol 20 (3) ◽  
pp. 780 ◽  
Author(s):  
Wei Tang ◽  
Dongming Lan ◽  
Zexin Zhao ◽  
Shuang Li ◽  
Xiuting Li ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Biochemical Characterization ◽  
Fermentation Broth ◽  
Catalytic Triad ◽  
Esterification Reaction ◽  
Monoacylglycerol Lipase ◽  
His Tag ◽  
Km Value ◽  
Mutagenesis Study ◽  
Hydrolysis Activity

Lipases with unique substrate specificity are highly desired in biotechnological applications. In this study, a putative marine Geobacillus sp. monoacylglycerol lipase (GMGL) encoded gene was identified by a genomic mining strategy. The gene was expressed in Escherichia coli as a His-tag fusion protein and purified by affinity chromatography with a yield of 264 mg per liter fermentation broth. The recombinant GMGL shows the highest hydrolysis activity at 60 °C and pH 8.0, and the half-life was 60 min at 70 °C. The GMGL is active on monoacylglycerol (MAG) substrate but not diacylglycerol (DAG) or triacylglycerol (TAG), and produces MAG as the single product in the esterification reaction. Modeling structure analysis showed that the catalytic triad is formed by Ser97, Asp196 and His226, and the flexible cap region is constituted by residues from Ala120 to Thr160. A mutagenesis study on Leu142, Ile145 and Ile170 located in the substrate binding tunnel revealed that these residues were related with its substrate specificity. The kcat/Km value toward the pNP-C6 substrate in mutants Leu142Ala, Ile145Ala and Ile170Phe increased to 2.3-, 1.4- and 2.2-fold as compared to that of the wild type, respectively.

scholarly journals A Haloalkane Dehalogenase from a Marine Microbial Consortium Possessing Exceptionally Broad Substrate Specificity

2017 ◽  
Vol 84 (2) ◽  
Author(s):  
Tomas Buryska ◽  
Petra Babkova ◽  
Ondrej Vavra ◽  
Jiri Damborsky ◽  
Zbynek Prokop
Keyword(s):  
Substrate Specificity ◽  
Active Site ◽  
Biochemical Characterization ◽  
Environmental Pollutants ◽  
Haloalkane Dehalogenase ◽  
Broad Substrate Specificity ◽  
Organic Cosolvents ◽  
Marine Metagenome ◽  
Ph Range

ABSTRACTThe haloalkane dehalogenase enzyme DmmA was identified by marine metagenomic screening. Determination of its crystal structure revealed an unusually large active site compared to those of previously characterized haloalkane dehalogenases. Here we present a biochemical characterization of this interesting enzyme with emphasis on its structure-function relationships. DmmA exhibited an exceptionally broad substrate specificity and degraded several halogenated environmental pollutants that are resistant to other members of this enzyme family. In addition to having this unique substrate specificity, the enzyme was highly tolerant to organic cosolvents such as dimethyl sulfoxide, methanol, and acetone. Its broad substrate specificity, high overexpression yield (200 mg of protein per liter of cultivation medium; 50% of total protein), good tolerance to organic cosolvents, and a broad pH range make DmmA an attractive biocatalyst for various biotechnological applications.IMPORTANCEWe present a thorough biochemical characterization of the haloalkane dehalogenase DmmA from a marine metagenome. This enzyme with an unusually large active site shows remarkably broad substrate specificity, high overexpression, significant tolerance to organic cosolvents, and activity under a broad range of pH conditions. DmmA is an attractive catalyst for sustainable biotechnology applications, e.g., biocatalysis, biosensing, and biodegradation of halogenated pollutants. We also report its ability to convert multiple halogenated compounds to corresponding polyalcohols.

scholarly journals Expression and Characterization of a Cold-Adapted Alginate Lyase with Exo/Endo-Type Activity from a Novel Marine Bacterium Alteromonas portus HB161718T

Marine Drugs ◽  
2021 ◽  
Vol 19 (3) ◽  
pp. 155
Author(s):  
Huiqin Huang ◽  
Shuang Li ◽  
Shixiang Bao ◽  
Kunlian Mo ◽  
Dongmei Sun ◽  
...  
Keyword(s):  
Marine Bacterium ◽  
Reducing Power ◽  
Alginate Lyase ◽  
Maximum Activity ◽  
Ionization Mass ◽  
Cold Adapted ◽  
Guluronic Acid ◽  
Type Activity ◽  
Alginate Oligosaccharides ◽  
Alginate Lyases

The alginate lyases have unique advantages in the preparation of alginate oligosaccharides and processing of brown algae. Herein, a gene alg2951 encoding a PL7 family alginate lyase with exo/endo-type activity was cloned from a novel marine bacterium Alteromonas portus HB161718T and then expressed in Escherichia coli. The recombinant Alg2951 in the culture supernatant reached the activity of 63.6 U/mL, with a molecular weight of approximate 60 kDa. Alg2951 exhibited the maximum activity at 25 °C and pH 8.0, was relatively stable at temperatures lower than 30 °C, and showed a special preference to poly-guluronic acid (polyG) as well. Both NaCl and KCl had the most promotion effect on the enzyme activity of Alg2951 at 0.2 M, increasing by 21.6 and 19.1 times, respectively. The TCL (Thin Layer Chromatography) and ESI-MS (Electrospray Ionization Mass Spectrometry) analyses suggested that Alg2951 could catalyze the hydrolysis of sodium alginate to produce monosaccharides and trisaccharides. Furthermore, the enzymatic hydrolysates displayed good antioxidant activity by assays of the scavenging abilities towards radicals (hydroxyl and ABTS+) and the reducing power. Due to its cold-adapted and dual exo/endo-type properties, Alg2951 can be a potential enzymatic tool for industrial production.

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