scholarly journals Expression and Characterization of an Alginate Lyase and Its Thermostable Mutant in Pichia pastoris

Marine Drugs ◽  
2020 ◽  
Vol 18 (6) ◽  
pp. 305
Author(s):  
Suxiao Yang ◽  
Zhemin Liu ◽  
Xiaodan Fu ◽  
Changliang Zhu ◽  
Qing Kong ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Degradation Products ◽  
Alginate Lyase ◽  
Maximal Activity ◽  
Degree Of Polymerization ◽  
Molecular Weights ◽  
Industrial Preparation ◽  
Alginate Oligosaccharides ◽  
Elimination Mechanism ◽  
G Ratio

Alginate is one of the most abundant polysaccharides in algae. Alginate lyase degrades alginate through a β-elimination mechanism to produce alginate oligosaccharides with special bioactivities. Improving enzyme activity and thermal stability can promote the application of alginate lyase in the industrial preparation of alginate oligosaccharides. In this study, the recombinant alginate lyase cAlyM and its thermostable mutant 102C300C were expressed and characterized in Pichia pastoris. The specific activities of cAlyM and 102C300C were 277.1 U/mg and 249.6 U/mg, respectively. Both enzymes showed maximal activity at 50 °C and pH 8.0 and polyG preference. The half-life values of 102C300C at 45 °C and 50 °C were 2.6 times and 11.7 times the values of cAlyM, respectively. The degradation products of 102C300C with a lower degree of polymerization contained more guluronate. The oligosaccharides with a polymerization degree of 2–4 were the final hydrolytic products. Therefore, 102C300C is potentially valuable in the production of alginate oligosaccharides with specific M/G ratio and molecular weights.

scholarly journals Structure Characteristics, Biochemical Properties, and Pharmaceutical Applications of Alginate Lyases

Marine Drugs ◽  
2021 ◽  
Vol 19 (11) ◽  
pp. 628
Author(s):  
Shu-Kun Gao ◽  
Rui Yin ◽  
Xiao-Chen Wang ◽  
Hui-Ning Jiang ◽  
Xiao-Xiao Liu ◽  
...  
Keyword(s):  
Brown Algae ◽  
Catalytic Mechanism ◽  
Degradation Products ◽  
Structural Characteristics ◽  
Catalytic Efficiency ◽  
Alginate Lyase ◽  
Biochemical Properties ◽  
Structure And Property ◽  
Alginate Oligosaccharides ◽  
Alginate Lyases

Alginate, the most abundant polysaccharides of brown algae, consists of various proportions of uronic acid epimers α-L-guluronic acid (G) and β-D-mannuronic acid (M). Alginate oligosaccharides (AOs), the degradation products of alginates, exhibit excellent bioactivities and a great potential for broad applications in pharmaceutical fields. Alginate lyases can degrade alginate to functional AOs with unsaturated bonds or monosaccharides, which can facilitate the biorefinery of brown algae. On account of the increasing applications of AOs and biorefinery of brown algae, there is a scientific need to explore the important aspects of alginate lyase, such as catalytic mechanism, structure, and property. This review covers fundamental aspects and recent developments in basic information, structural characteristics, the structure–substrate specificity or catalytic efficiency relationship, property, molecular modification, and applications. To meet the needs of biorefinery systems of a broad array of biochemical products, alginate lyases with special properties, such as salt-activated, wide pH adaptation range, and cold adaptation are outlined. Withal, various challenges in alginate lyase research are traced out, and future directions, specifically on the molecular biology part of alginate lyases, are delineated to further widen the horizon of these exceptional alginate lyases.

scholarly journals Secretory Expression of an Alkaline Alginate Lyase With Heat Recovery Property in Yarrowia lipolytica

2021 ◽  
Vol 12 ◽  
Author(s):  
Lu Liu ◽  
Zhipeng Wang ◽  
Zhihong Zheng ◽  
Ze Li ◽  
Xiaofeng Ji ◽  
...  
Keyword(s):  
Amino Acids ◽  
Yarrowia Lipolytica ◽  
Heat Recovery ◽  
Degradation Products ◽  
Biological Activities ◽  
Alginate Lyase ◽  
Potential Candidate ◽  
Product Specificity ◽  
Alginate Oligosaccharides ◽  
Recovery Performance

Alginate lyase possesses wide application prospects for the degradation of brown algae and preparation of alginate oligosaccharides, and its degradation products display a variety of biological activities. Although many enzymes of this type have been reported, alginate lyases with unique properties are still relatively rare. In the present work, an alginate lyase abbreviated as Alyw203 has been cloned from Vibrio sp. W2 and expressed in food-grade Yarrowia lipolytica. The Alyw203 gene consists of an open reading frame (ORF) of 1,566 bp containing 521 amino acids, of which the first 17 amino acids are considered signal peptides, corresponding to secretory features. The peak activity of the current enzyme appears at 45°C with a molecular weight of approximately 57.0 kDa. Interestingly, Alyw203 exhibits unique heat recovery performance, returning above 90% of its initial activity in the subsequent incubation for 20 min at 10°C, which is conducive to the recovery of current enzymes at low-temperature conditions. Meanwhile, the highest activity is obtained under alkaline conditions of pH 10.0, showing outstanding pH stability. Additionally, as an alginate lyase independent of NaCl and resistant to metal ions, Alyw203 is highly active in various ionic environments. Moreover, the hydrolyzates of present enzymes are mainly concentrated in the oligosaccharides of DP1–DP2, displaying perfect product specificity. The alkali suitability, heat recovery performance, and high oligosaccharide yield of Alyw203 make it a potential candidate for industrial production of the monosaccharide and disaccharide.

scholarly journals Cloning and Characterization of a Novel Alginate Lyase from Paenibacillus sp. LJ-23

Marine Drugs ◽  
2022 ◽  
Vol 20 (1) ◽  
pp. 66
Author(s):  
Mingpeng Wang ◽  
Lei Chen ◽  
Zhengyu Lou ◽  
Xueting Yuan ◽  
Guiping Pan ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Water Solubility ◽  
Degradation Products ◽  
Three Dimensional ◽  
Alginate Lyase ◽  
Dimensional Structure ◽  
Amino Acid Residues ◽  
Alginate Oligosaccharides ◽  
Paenibacillus Sp ◽  
Comprehensive Health

As a low molecular weight alginate, alginate oligosaccharides (AOS) exhibit improved water solubility, better bioavailability, and comprehensive health benefits. In addition, their biocompatibility, biodegradability, non-toxicity, non-immunogenicity, and gelling capability make them an excellent biomaterial with a dual curative effect when applied in a drug delivery system. In this paper, a novel alginate lyase, Algpt, was cloned and characterized from a marine bacterium, Paenibacillus sp. LJ-23. The purified enzyme was composed of 387 amino acid residues, and had a molecular weight of 42.8 kDa. The optimal pH of Algpt was 7.0 and the optimal temperature was 45 °C. The analysis of the conserved domain and the prediction of the three-dimensional structure indicated that Algpt was a novel alginate lyase. The dominant degradation products of Algpt on alginate were AOS dimer to octamer, depending on the incubation time, which demonstrated that Algpt degraded alginate in an endolytic manner. In addition, Algpt was a salt-independent and thermo-tolerant alginate lyase. Its high stability and wide adaptability endow Algpt with great application potential for the efficient preparation of AOS with different sizes and AOS-based products.

scholarly journals Imunomodulator Activity of Alginate Oligosaccharides from Alginate Sargassum crassifolium

2017 ◽  
Vol 20 (1) ◽  
pp. 63
Author(s):  
Subaryono Subaryono ◽  
Rosmawati Perangiangin ◽  
Maggy Thenawidjaja Suhartono ◽  
Fransiska Rungkat Zakaria
Keyword(s):  
Biological Activities ◽  
Human Lymphocytes ◽  
Alginate Lyase ◽  
Enzyme Concentration ◽  
Degree Of Polymerization ◽  
Cytotoxic T Cell ◽  
Alginate Oligosaccharides ◽  
Bacterium Bacillus ◽  
Production Conditions

Alginate oligosaccharides (AOS) are oligosaccharides produced from depolimerization of the alginate polymer, and is reported to have various biological activities. The study aims is to determine the effect of AOS<br />production conditions and their effects on products and its activities as an immunomodulatory compound. Production of alginate oligosaccharides (AOS) enzymatically carried out with the help of alginate lyase enzyme produced from the bacterium Bacillus megaterium S245. Variation of incubation time is 2, 4, 6 and 8 hours at concentrations of alginate lyase enzyme addition of 25, 50, 75 and 100U. Treatment of enzyme concentration and the duration of incubation in the production of AOS produces a degree of polymerization (DP) 2-7. In vitro activity test showed AOS is have ability to induce cell proliferation of human lymphocytes.<br />This type of cell lymphocytes proliferation induced by AOS is a CD 8 cells or cytotoxic T cell and non cell CD4 / CD8. AOS production conditions with the addition of alginate lyase enzyme 50 U and incubation period 2 hours has produce AOS with the highest index of lymphocyte proliferation  117.6+3.6% or an increase of 43.24% compared to the native alginat polymer.<br /><br /><br />

scholarly journals Elucidation of a Unique Pattern and the Role of Carbohydrate Binding Module of an Alginate Lyase

Marine Drugs ◽  
2019 ◽  
Vol 18 (1) ◽  
pp. 32 ◽  
Author(s):  
Fu Hu ◽  
Benwei Zhu ◽  
Qian Li ◽  
Heng Yin ◽  
Yun Sun ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Degradation Products ◽  
Alginate Lyase ◽  
Product Distribution ◽  
Industrial Applications ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Release Process ◽  
Alginate Oligosaccharides ◽  
Degradation Pattern

Alginate oligosaccharides with different degrees of polymerization (DPs) possess diverse physiological activities. Therefore, in recent years, increasing attention has been drawn to the use of enzymes for the preparation of alginate oligosaccharides for food and industrial applications. Previously, we identified and characterized a novel bifunctional alginate lyase Aly7A, which can specifically release trisaccharide from three different substrate types with a unique degradation pattern. Herein, we investigated its degradation pattern by modular truncation and molecular docking. The results suggested that Aly7A adopted a unique action mode towards different substrates with the substrate chain sliding into the binding pocket of the catalytic domain to position the next trisaccharide for cleavage. Deletion of the Aly7A carbohydrate binding module (CBM) domain resulted in a complex distribution of degradation products and no preference for trisaccharide formation, indicating that the CBM may act as a “controller” during the trisaccharide release process. This study further testifies CBM as a regulator of product distribution and provides new insights into well-defined generation of alginate oligosaccharides with associated CBMs.

scholarly journals Cloning and Characterization of an Alginate Lyase from Marine Vibrio. sp. QD-5

2017 ◽  
Author(s):  
Yaxi Chao ◽  
Shuyan Wang ◽  
Suqi Wu ◽  
Jiaqiang Wei ◽  
Hao Chen
Keyword(s):  
Large Scale ◽  
Ph Value ◽  
Alginate Lyase ◽  
Degree Of Polymerization ◽  
Large Scale Preparation ◽  
Low Degree ◽  
Alginate Oligosaccharides ◽  
Theoretical Molecular Mass ◽  
Scale Preparation

The string of bacteria, Vibrio. sp. QD-5 utilizing alginate, was separated from rotten kelp. The results of genome sequencing showed that the strain QD-5 contained four alginate lyase genes.One of the alginate genes Aly-IV was cloned and linked to the plasmid pET-22b (+). The heterologous expressed alginate lyase aly-IVwas characterized，which possessed the theoretical molecular mass of 62 kDa, and theoretical isoelectric point (pI) of 5.12. - The enzyme aly-IV was purified and the activity reached 1256.78 U/mg, with optimal temperature of 35 oC and pH value of 8.9. Nurtured in the temperature below 25 oC for 30 minutes, the activity was almost stable. The result also suggested that the activity of enzyme was strongly affected by - NaCl whose optimal concentration was 15 mM in a lab environment The TLC and ESI-TOF-MS analysis suggested that the enzyme aly-IV could degrade sodium alginate and polyG in endo-lytic type, producing monomer, dimer and trimmer. So, aly-IV can also be widely applied to make large scale preparation of alginate oligosaccharides with low degree of polymerization (DP).

Permeability Enhancing and Chemotactic Activities of Lower Molecular Weight Degradation Products of Human Fibrinogen

1981 ◽  
Vol 45 (01) ◽  
pp. 090-094 ◽  
Author(s):  
Katsuo Sueishi ◽  
Shigeru Nanno ◽  
Kenzo Tanaka
Keyword(s):  
Molecular Weight ◽  
Degradation Products ◽  
Electron Microscopic Observation ◽  
Chemotactic Activity ◽  
Lower Molecular Weight ◽  
Electron Microscopic ◽  
Human Fibrinogen ◽  
Rabbit Skin ◽  
Molecular Weights ◽  
Active Fragments

SummaryFibrinogen degradation products were investigated for leukocyte chemotactic activity and for enhancement of vascular permeability. Both activities increased progressively with plasmin digestion of fibrinogen. Active fragments were partially purified from 24 hr-plasmin digests. Molecular weights of the permeability increasing and chemotactic activity fractions were 25,000-15,000 and 25,000 respectively. Both fractions had much higher activities than the fragment X, Y, D or E. Electron microscopic observation of the small blood vessels in rabbit skin correlated increased permeability with the formation of characteristic gaps between adjoining endothelial cells and their contraction.These findings suggest that lower molecular weight degradation products of fibrinogen may be influential in contributing to granulocytic infiltration and enhanced permeability in lesions characterized by deposits of fibrin and/or fibrinogen.

Identification of Crosslinked Fibrin Degradation Products in Plasma of Patients During Fibrinolytic Therapy Using a Heat Extraction/SDS-Polyacrylamide Gradient Gel Electrophoretic Technique

1979 ◽  
Author(s):  
C.W. Francis ◽  
V. J. Marder ◽  
S.E. Martin
Keyword(s):  
Degradation Products ◽  
Fibrinolytic Therapy ◽  
Heat Extraction ◽  
Molecular Weights ◽  
Fibrin Degradation Products ◽  
Normal Plasma ◽  
Electrophoretic Technique ◽  
Gradient Gel Electrophoresis ◽  
Derivatives Of

To quantitate plasmic degradation products of crosslinked fibrin in plasma, a technique has been developed which employs heat precipitation, SDS-polyacrylamide gradient gel electrophoresis of the dissolved, reduced heat precipitate, and quantitation by densitometrie analysis of γ-γ derivatives identified in the stained get. When studied with this sensitive electrophoretic technique, plasmic digests of purified crosslinked fibrin were found to contain a heterogeneous group of γ-γ chain derivatives with molecular weights between 76,000 and 100,000 daltons. In samples of normal plasma to which digests of crosslinked fibrin had been added, this heat extraction/ge1 electrophoretic technigue allowed the detection of γ-γ derivatives with a sensitivity of 20 µg/ml. Derivatives of γ-γ chains with molecular weights of 82,000 and 86,000 daltons have been identified in the plasma of patients with DIC and during fibrinolytic therapy but were not found in normal plasma or in normal plasma treated in vitro with urokinase. This quantitative assay can be performed in 24 hours and appears to be of value in judging the efficacy of thrombolytic therapy.

Extensive autolytic fragmentation of membranous versus cytosolic calpain following myocardial ischemia–reperfusion

2010 ◽  
Vol 88 (5) ◽  
pp. 584-594 ◽  
Author(s):  
James S.C. Gilchrist ◽  
Tom Cook ◽  
Bernard Abrenica ◽  
Babak Rashidkhani ◽  
Grant N. Pierce
Keyword(s):  
Degradation Products ◽  
Ischemia Reperfusion ◽  
Large Subunit ◽  
Western Blots ◽  
Molecular Weights ◽  
Myocardial Ischemia Reperfusion ◽  
Fragmentation Patterns ◽  
Developed Pressure ◽  
Calpain Substrates ◽  
Calpain Inhibitors

We investigated calpain activation in the heart during ischemia–reperfusion (I–R) by immunologically mapping the fragmentation patterns of calpain and selected calpain substrates. Western blots showed the intact 78 kDa large subunit of membrane-associated calpain was autolytically fragmented to 56 and 43 kDa signature immunopeptides following I–R. Under these conditions, the 78 kDa calpain large subunit from crude cytosolic fractions was markedly less fragmented, with only weakly stained autolytic peptides detected at higher molecular weights (70 and 64 kDa). Western blots also showed corresponding calpain-like degradation products (150 and 145 kDa) of membrane-associated α-fodrin (240 kDa) following I–R, but in crude myofibrils α-fodrin degradation occurred in a manner uncharacteristic of calpain. For control hearts perfused in the absence of ischemia, autolytic fragmentation of calpain and calpain-like α-fodrin degradation were completely absent from most subcellular fractions. The exception was sarcolemma-enriched membranes, where significant calpain autolysis and calpain-like α-fodrin degradation were detected. In purified sarcoplasmic reticulum membranes, RyR2 and SERCA2 proteins were also highly degraded, but for RyR2 this did not occur in a manner characteristic of calpain. When I–R-treated hearts were perfused with peptidyl calpain inhibitors (ALLN or ALLM; 25 µmol/L), calpain autolysis and calpain-like degradation of α-fodrin were equally attenuated by each inhibitor. However, only ALLN protected against early loss of developed pressure in hearts following I–R, with no functionally protective effect of ALLM observed. Our studies suggest calpain is preferentially activated at membranes following I–R, possibly contributing to impaired ion channel function implicated by others in I–R injury.

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