Production, properties and applications of microbial heparinases
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  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 .
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# 188.8.131.52) 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.
A graphene-printed carbon electrode for real-time measurements of vacant active sites in a cellulase
Analytical Biochemistry, bind 447, 2014, pp. 162-168
Cellulases hydrolyze cellulose into soluble sugars and this process is used in sustainable industries based on lignocellulosic feedstocks. Better analytical tools are needed to understand the basic cellulase mechanisms to enable rational improvements to the industrial process. In this work, we describe a new electrochemical approach to quantify the populations of enzymes, each free in the aqueous bulk, adsorbed to the insoluble substrate with an unoccupied active site, or threaded to the cellulose strand in the active tunnel. A distinction between these three states appears to be essential for identifying the rate-limiting step. The method is based on disposable graphene-modified screen-printed carbon electrodes and we show how the time evolution of the concentrations of the three enzyme forms can be derived from a combination of the electrochemical data and adsorption measurements. The method was tested for the cellobiohydrolase Cel7AHypocrea jecorinaact on microcrystalline cellulose and the threaded enzyme form was found to dominate in this system, while adsorbed enzymes with an unoccupied active site accounted for less than 5 % of the population.
Weak protein-protein interactions are revealed by immiscible surface tension-assisted filtration
Analytical Biochemistry, bind 447, 2014, pp. 133-140
Biological mechanisms are often mediated by transient interactions between multiple proteins. The isolation of intact protein complexes is essential for understanding biochemical processes and an important prerequisite for the identification of new drug targets and biomarkers. However, low-affinity interactions are often difficult to detect. Here we use a recently described method called Immiscible Filtration Assisted by Surface Tension (IFAST) to isolate proteins under defined binding conditions. This method, which allows for near-instantaneous isolation, allows for significantly higher recovery of transient complexes compared to current wash-based protocols that require re-equilibration at each of the multiple wash steps, resulting in protein losses. The method moves proteins or protein complexes captured on a solid phase through one or more immiscible phase barriers that effectively prevent the passage of non-specific material in a single operation. We use a previously described polyol-responsive monoclonal antibody to explore the potential of this new method to study protein binding. In addition, difficult-to-isolate complexes affecting the biologically and clinically important Wnt signaling pathway have been isolated. We anticipate that this simple and rapid method for isolating intact, transient complexes will enable the discovery of new signaling pathways, biomarkers, and drug targets.
Intravital imaging of Bacillus thuringiensis Cry1A toxin binding sites in the silkworm midgut
Analytical Biochemistry, bind 447, 2014, pp. 90-97
Identification of the resistance mechanism of insects toBacillus thuringiensisCry1A toxin is becoming an increasingly challenging task. This fact underscores the need to establish new methods to further study the molecular interactions of the Cry1A toxin with insects and the receptor-binding region of Cry1A toxins for their broader application as biopesticides and gene source for genetically engineered crops. In this paper, a quantum dot-based near-infrared fluorescence imaging method was used to directly and dynamically monitor the specific binding of the Cry1A toxins CrylAa and CrylAc to the silkworm midgut tissue. In vitro fluorescence imaging demonstrated the higher binding specificity of CrylAa-QD probes compared to CrylAc-QD to the brush border membrane vesicles in the silkworm midgut. TheDirectImaging showed that the binding of multiple CrylAa-QDs to the silkworm midgut could be monitored efficiently and clearly in live silkworms. Furthermore, the frozen section analysis clearly showed the broader receptor binding region of Cry1Aa compared to that of Cry1Ac in the midgut part. These observations suggest that the insecticidal activity of Cry toxins may depend on the receptor binding sites, and this sensitive and visual near-infrared fluorescence imaging could provide a new avenue to study the resistance mechanism for maintaining Cry's insecticidal activityB. thuringiensistoxins.
Thermostable single-domain antibody-maltose-binding protein fusion for the detection of the Bacillus anthracis spore protein BclA
Analytical Biochemistry, bind 447, 2014, pp. 64-73
We have engineered a genetic fusion of a single domain antibody (sdAb) with the thermophile's thermally stable maltose-binding proteinPyrococcus gal(PfuMBP). Produced inEscherichia coliHigh yield cytoplasm proved to be a robust and efficient immunoreagent. sdAb-A5 binds BclA, aBacillus anthracisHigh affinity spore protein (KD~5 p.m.). MBPs, including the thermostable onePfuMBP have been shown to be excellent folding chaperones, enhancing the production of many recombinant proteins. A three-step purification ofE coliShake off flask culturesPfuMBP-sdAb yielded approximately 100 mg/L of highly pure product. ThePfuMBP remained stable up to 120 °C, whereas the sdAb-A5 unit unfolded at around 120 °C. 68 to 70 °C but could be refolded to regain activity. This fusion construct was stable upon heating at 1 mg/mL for 1 hour at 70°C and retained nearly 100% of its binding activity; Almost a quarter (24%) of the activity persisted after 1 hour at 90°C. ThePfuThe MBP-sdAb construct also provides a stable and efficient method for coating gold nanoparticles. Most importantly, the design has been proven to allow for improved detectionB. anthracisThe star strain (34F2) tracks sdAb-A5 as both a capture and detection reagent.
Stimulators of translation were identified during a small molecule screening campaign
Analytical Biochemistry, bind 447, 2014, pp. 6-14
By screening a library of natural and synthetic products for eukaryotic translation modulators, we identified two natural products, isohymenialdisine and hymenialdisine, that have translation-stimulating effects. Characterization of these compounds led to the finding that mRNA used to program the translational extracts in the high-throughput assay set-up resulted in the phosphorylation of eIF2α, a potent negative regulatory event mediated by one of four kinases. We identified double-stranded RNA-dependent protein kinase (PKR) as eIF2α kinase activated by exogenously added mRNA template. Characterization of isohymenialdisine's mode of action revealed that it acts directly on PKR by inhibiting autophosphorylation, disrupting the PKR-eIF2α phosphorylation axis and can be modeled into the PKR-ATP binding site. Our results identify a source of "false positives" for high-throughput display campaigns and translation extraction, raising a caveat for these types of display ads.
Revealing the detailed cleavage mode of heparinase I to structurally defined heparin oligosaccharides
International Journal of Biological Macromolecules, verbindlich 141, 2019, s. 756-764
For a more insightful study of the specificity of bacterial heparinase I, a series of structurally well-defined heparin oligosaccharides were synthesized using a highly efficient chemoenzymatic strategy. Apart from the primary cleavage site, five glycosidic bonds of oligosaccharides with different modifications to obtain secondary cleavage sites were degraded by a high concentration of heparinase I. The reactivity of the bonds towards heparinase I was not entirely dependent on 2-.Ö-sulfated iduronic acid is split off or the adjacent 6-Ö-sulphated glucosamine residues, but was dependent on higher degrees of sulphation of the oligosaccharides and dispensableN-substituted glucosamine adjacent to the cleavable bond. In addition, the enzyme showed less preferential cleavage towards glycosidic bonds containing glucuronic acid than those containing iduronic acid of the corresponding oligosaccharides. Biolayer interferometry revealed differences in reactivity that are not fully consistent with different affinities of the substrates for enzymes. Our study provided precise information on the cleavage promiscuity of heparinase I, which is essential for heparin depolymerization.
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