Lufeng Yan

Depolymerization of Fucosylated Chondroitin Sulfate with a Modified Fenton-System and Anticoagulant Activity of the Resulting Fragments

Fucosylated chondroitin sulfate (fCS) from sea cucumber Isostichopus badionotus (fCS-Ib) with a chondroitin sulfate type E (CSE) backbone and 2,4-O-sulfo fucose branches has shown excellent anticoagulant activity although has also show severe adverse effects. Depolymerization represents an effective method to diminish this polysaccharide’s side effects. The present study reports a modified controlled Fenton system for degradation of fCS-Ib and the anticoagulant activity of the resulting fragments. Monosaccharides and nuclear magnetic resonance (NMR) analysis of the resulting fragments indicate that no significant chemical changes in the backbone of fCS-Ib and no loss of sulfate groups take place during depolymerization. A reduction in the molecular weight of fCS-Ib should result in a dramatic decrease in prolonging activated partial thromboplastin time and thrombin time. A decrease in the inhibition of thrombin (FIIa) by antithromin III (AT III) and heparin cofactor II (HCII), and the slight decrease of the inhibition of factor X activity, results in a significant increase of anti-factor Xa (FXa)/anti-FIIa activity ratio. The modified free-radical depolymerization method enables preparation of glycosaminoglycan (GAG) oligosaccharides suitable for investigation of clinical anticoagulant application.

Ultrasound assisted enzymatic hydrolysis of starch catalyzed by glucoamylase: Investigation on starch properties and degradation kinetics

The present work investigates the synergistic impact of glucoamylase and ultrasound on starch hydrolysis. The extent of starch hydrolysis at different reaction parameters (ultrasonic intensity, temperature, reaction time) was analyzed. The hydrolysis extent increased with the reaction time and reached a maximum value under ultrasonic intensity of 7.20W/mL at 10min. Ultrasound did not alter the optimum enzymatic temperature but speeded up the thermal inactivation of glucoamylase. The evaluation of enzymatic kinetics and starch degradation kinetics indicated a promotion of the reaction rate and enzyme-substrate affinity. According to the thermodynamic results, sonoenzymolysis reactions require less energy than enzymolysis reactions. The measurement of molecular weight, solubility, thermal properties, and structures of the substrates revealed that sonoenzymolysis reaction generated greater impacts on starch properties. The molecular weight and radii of gyration decreased by 80.19% and 90.05% respectively while the starch solubility improved by 136.50%.

Structural elucidation of fucosylated chondroitin sulfates from sea cucumber using FTICR-MS/MS:

Fucosylated chondroitin sulfates are complex polysaccharides extracted from sea cucumber. They have been extensively studied for their anticoagulant properties and have been implicated in other biological activities. While nuclear magnetic resonance spectroscopy has been used to extensively characterize fucosylated chondroitin sulfate oligomers, we herein report the first detailed mass characterization of fucosylated chondroitin sulfate using high-resolution Fourier transform ion cyclotron resonance mass spectrometry. The two species of fucosylated chondroitin sulfates considered for this work include Pearsonothuria graeffei (FCS-Pg) and Isostichopus badionotus (FCS-Ib). Fucosylated chondroitin sulfate oligosaccharides were prepared by N-deacetylation–deaminative cleavage of the two fucosylated chondroitin sulfates and purified by repeated gel filtration. Accurate mass measurements obtained from electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry measurements confirmed the oligomeric nature of these two fucosylated chondroitin sulfate oligosaccharides with each trisaccharide repeating unit averaging four sulfates per trisaccharide. Collision-induced dissociation of efficiently deprotonated molecular ions through Na/H+ exchange proved useful in providing structurally relevant glycosidic and cross-ring product ions, capable of assigning the sulfate modifications on the fucosylated chondroitin sulfate oligomers. Careful examination of the tandem mass spectrometry of both species deferring in the positions of sulfate groups on the fucose residue (FCS-Pg-3,4-OS) and (FCS-Ib-2,4-OS) revealed cross-ring products 0,2Aαf and 2,4X2αf which were diagnostic for (FCS-Pg-3,4-OS) and 0,2X2αf diagnostic for (FCS-Ib-2,4-OS). Mass spectrometry and tandem mass spectrometry data acquired for both species varying in oligomer length (dp3–dp15) are presented.

Molecular size is important for the safety and selective inhibition of intrinsic factor Xase for fucosylated chondroitin sulfate

Fucosylated chondroitin sulfate from sea cucumber Isostichopus badionotus (FCS-Ib) showed potent anticoagulant activities without selectivity. The present study focused on developing safe FCS-Ib oligomers showing selective inhibition of intrinsic factor Xase (anti-FXase) prepared through partial N-deacetylation-deaminative cleavage. The N-deacetylation degree was regulated by reaction time, controlling the resulting oligomer distribution. Structure analysis confirmed the selectivity of degradation, and 12 high purity fractions with trisaccharide-repeating units were separated. In vitro anticoagulant assays indicated a decrease in molecular weight (Mw) dramatically reduced activated partial thromboplastin time (APTT), thrombin time (TT), AT-dependent anti-FIIa and anti-FXa activities, while the oligomers retained potent anti-FXase activity until they fell below 3kDa. Meanwhile, human FXII activation and platelet aggregation were markedly reduced with decreasing Mw and were moderate when under 12.0kDa. Thus, fragments of 3-12.0kDa should be safe and effective as selective inhibitors of intrinsic tenase complex for application as clinical anticoagulants.

Ultrasound promotes enzymatic reactions by acting on different targets: Enzymes, substrates and enzymatic reaction systems

With the extensive application of enzyme-catalyzed reactions in numerous fields, improving enzymatic efficiency has attracted wide attention for reducing operating costs and increasing output. There are three targets throughout enzymatic reactions: the enzyme, substrate, and mixed reaction system. Ultrasound has been known to accelerate enzymatic reactions by acting on different targets. It can modify both enzyme and substrate macromolecules, which is helpful for enhancing enzyme activity and product yields. The synergistic effect of ultrasound and enzymes is widely reported to increase catalytic rates. The present review discusses the positive effect induced by ultrasound throughout the enzymatic process, including ultrasonic modification of enzymes, ultrasound assisted immobilization, ultrasonic pretreatment of substrates, and ultrasound assisted enzymatic reactions.