Quantification of Heparosan by Heparin Lyase III and Spectrophotometry (2023)

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  • Production, properties and applications of microbial heparinases

    2022, Biochemistry


    Heparosan is depolymerized by heparinase III, producing unsaturated disaccharides that can be measured at 232 nm and used to quantify heparosan. A method for quantification of heparosan based on the complete depolymerization of heparosan using excess heparinase III [28] and with which a minimum concentration of heparosan of 0.12 g/l in buffer and 0.6 g/l in buffer has been reported. l can be detected in the fermentation broth. The method overcomes the safety and specificity limitations of the traditionally used carbazole assay, can be applied directly to fermentation broth without the need for time-consuming purification, and unlike CE- and NMR-based determinations, requires no special equipment.

    Heparinases are enzymes that selectively cleave heparin and heparan sulfate chains by cleaving the glycosidic bond between hexosamines and uronic acids to produce disaccharide and oligosaccharide products. While heparin is known as an anticoagulant, heparin and heparan sulfate are also involved in biological processes such as inflammation, cancer and angiogenesis, as well as viral and bacterial infections, and are of increasing interest due to their therapeutic potential. Recently, potential roles of heparin and heparan sulfate in the context of COVID-19 infection have been highlighted. The ability of heparinases to selectively cleave heparin chains has been exploited industrially to produce low molecular weight heparin, which has replaced heparin in several clinical applications. Other uses of heparinases include analyzing the structure of heparin and heparan sulfate, neutralizing heparin in the blood, and removing the inhibitory effects of heparin on various enzymes. Heparinases are known to inhibit neovascularization and heparinase III is of interest for the treatment of cancer and inhibition of tumor cell growth. Heparinase activity, isolated for the first time fromPedobacter heparinus, has since been reported from several other microorganisms. Significant advances have been made in the production, characterization, and improvement of microbial heparinases to meet application requirements in terms of heparinase yield and purity, which will likely expand their usefulness in various applications. This Review focuses on current developments in the identification, characterization, and improvement of microbial heparinases and their established and emerging industrial, clinical, and therapeutic applications.

  • Increased soluble heterologous expression of rat brain 3-O-sulfotransferase 1 – a key enzyme in heparin biosynthesis

    2018, Protein Expression and Purification


    The results show that the GlcNS3S-H1 peak was observed at 5.52 ppm and the GlcNS3S-H3 peak at 3.82 ppm, indicating that NSNA3SHp was successfully produced. In the last decade, an increasing number of publications reported on the heparin production strategy by chemical and enzymatic modification of heparosan from E. coli K5, including high-density fermentation and metabolic engineering modification of K5 strain and relative enzymes [26-33]. Both heparin and HS biosynthesis require a number of different enzymes, but the final enzymes in the process, the 3-OSTs, are critically involved in many of the critical biological functions of these GAGs [9].

    Heparan sulfate (HS) is a glycosaminoglycan (GAG) involved in various biological processes including blood clotting, wound healing and embryonic development. HS 3-Ö-Sulfotransferases (3-OST), which transfer the sulfo group to the 3-hydroxyl group of certain glucosamine residues, are a key enzyme in the biosynthesis of a number of biologically important HS chains. The 3-OST-1 isoform is one of the 7 known 3-OST isoforms and is important for the biosynthesis of anticoagulant HS chains. In this study, we cloned 3-OST-1 from rat brain by reverse transcription polymerase chain reaction (RT-PCR). After codon optimization and removal of the signal peptide, the recombinant plasmid was transformedEscherichia coliBL21 (DE3) to obtain a His-tagged-3-OST-1 fusion protein. SDS-PAGE analysis showed that the expressed 3-OST-1 was mainly found in inclusion bodies. 3-OST-1 was purified by a Ni affinity column and refolded by dialysis. The activity of 3-OST-1 obtained was 0.04 U/ml with a specific activity of 0.55 U/mg after renaturation. In addition, a co-expressed recombinant plasmid pET-28a-3-OST-1 containing the chaperone expression system (pGro7) was constructed and transferredE coliBL21 (DE3) to co-express the recombinant strainE coliBL21 (DE3)/pET-28a-3-OST-1+pGro7. Soluble expression of 3-OST-1 was markedly increased in the co-expressed recombinant strain, with enzyme activity reaching 0.06 U/ml and specific activity of 0.83 U/mg.N-Sulfo,N-Acetylheparosan (NSNAH) was modified by the recombinantly expressed 3-OST-1 and the product was confirmed by1H NMR shows that the sulfo group has been transferred to NSNAH.

  • Preparation of Low Molecular Weight Heparin Production Using Recombinant Glucuronidase

    2015, Carbohydrate Polymers


    where 2 is the conversion factor from 30 minutes to 1 hour; 10 is the conversion coefficient from 1 μmoL to 0.10 μmoL; 3 is the total volume of the reaction mixture (ml); df is the dilution factor of the enzyme solution; 5.50 is the extinction coefficient of unsaturated uronic acid at 235 nm and at the μmol level; 0.2 is the volume of the enzyme solution (mL). Heparin, HS and Heparosan were used as substrates Hep I, Hep II and Hep III (prepared in our laboratory, Huang et al., 2013, 2014) to test the activity of Δ4,5Δ20-glucuronidase on its various depolymerization products. After lyase digestion, the depolymerization products were treated with A4,5A20 glucuronidase.

    The Δ4,5 unsaturated uronate (4-deoxy-α-l-threo-hex-4-eno-pyranosyluronic acid) residue is formed by depolymerization of heparin, heparosan and heparan sulfate with heparin lyases. The recovery of unsaturated uronate-containing products is required to produce low molecular weight heparin (LMWH) from heparin or heparosan. In this study, the gene Δ4.5 and Δ4.5 was usedD20unsaturated glucuronidase (EC# fromPedobacter heparinus(earlierFlavobacterium heparinum) was cloned into the pMAL-c2x plasmid. Its fusion protein with MBP was expressed inEscherichia coliTB1. After purification, Δ4.5 unsaturated glucuronidase was evaluated. Δ4.5D20Glucuronidase showed excellent activity at the unsaturation of the various depolymerized products of Hep I, Hep II and Hep III to heparin, heparosan and heparan sulfate.

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Copyright © 2013 Elsevier Inc. Published by Elsevier Inc. All rights reserved.


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