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Biodiscovery of Marine Microbial Enzymes in Indonesia
Published in Se-Kwon Kim, Marine Biochemistry, 2023
Ekowati Chasanah, Pujo Yuwono, Dewi Seswita Zilda, Siswa Setyahadi
CDA (E.C. 3.5.1.4.1) is a key enzyme in replacing the unfriendly chemical process of chitosan production by hydrolyzing the acetamido group of chitin polymers. CDAs have been isolated from several microorganisms, first from Mucor rouxii (Araki & Ito, 1975); however, CDA research is not reported on as much as chitinase and chitosanase are, which might be due to a problem in the CDA assay. Besides CDA, chitin oligosaccharide deacetylase (COD; EC 3.5.1.105) is also able to produce β-N-acetyl-D-glucosaminyl-(1,4)-D-glucosamine (GlcNAc-GlcN) from (GlcNAc)2 (Hirano et al., 2015). Enzyme assays for deacetylation activity of CDAs and COD are based on monitoring acetate release by ultraviolet (UV) absorbance changes, radiolabeled substrates and coupled enzymatic assays or formation of free amino groups with chromogenic or fluorogenic reagents, such as fluorescamine, o-phthalaldehyde, or ninhydrin (Pascual & Planas, 2018). COD and chitinase (glycosyl hydrolase [GH] or GH family 18) had been reported to be produced by marine bacteria, Vibrio parahaemolyticus KN1699 isolated from a Yatsu dry beach (Narashino, Chiba Prefecture, Japan) (Hirano et al., 2015). Vibrio parahaemolyticus is a member of Vibrionacea, a family of gram-negative and facultative anaerobes bacteria under the phylum Proteobacteria, inhabitants of fresh- or saltwater/marine environments. This result gives insight into marine environments, including Indonesia’s marine environments, being a rich source of CDA or COD enzymes.
Liquid Chromatography
Published in Ernő Pungor, A Practical Guide to Instrumental Analysis, 2020
The naturally occurring polyamines (putrescence, cadaverine, spermidine, and spermine) are present in all living cells. Elevated levels of intracellular polyamines are correlated with increased rates of cellular proliferation and might be associated with several different pathological conditions. Polyamines have low UV absorbance, no fluorescence activity, cannot be detected by EC detection (amperometric), and the concentration is not sufficient to detect with RI in tissues and body fluids or in any other matrices. There are several reagents for pre-column derivatization. With o-phthalaldehyde-2-mercaptoethanol or fluorescamine or other reagents have been published in.58 Both have disadvantages, even the stability of derivatized compound or the lengthy and laborious derivatization procedure and so on. Derivatization of polyamines with benzoyl chloride has been accepted to get stable and UV active compounds.
Bioconjugation of Biodegradable Poly (lactic/glycolic acid) to Protein, Peptide, and Anti-Cancer Drug: An Alternative Pathway for Achieving Controlled Release from Micro- and Nanoparticles
Published in Raphael M. Ottenbrite, Sung Wan Kim, Polymeric Drugs & Drug Delivery Systems, 2019
Lysozyme (Mw 14,300) was chosen for PLGA conjugation because lysozyme can be molecularly dissolved in DMSO, a polar organic solvent, in which PLGA could be also dissolved [17]. By using dicyclohexyl carbodiimide (DCC) as the coupling agent, PLGA-lysozyme conjugate was synthesized as shown in Figure 2 [18]. Since the carbodiimide conjugation process was known to readily occur in basic conditions, lysozyme was lyophilized at pH 9 prior to the dissolution in DMSO to preserve charge states of various amino acid residues present in the protein in the organic solvent similar to those in the aqueous solution prior to the lyophilization (pH-memory effect) [19]. The degree of PLGA–lysozyme conjugation, determined by a fluorescamine assay, indicated that out of seven lysozyme primary amine groups an average of one amine group was conjugated to PLGA (Mw 8,600) molecule [20]. The PLGA–lysozyme conjugate was soluble only in DMSO and insoluble in other solvents such as water, methanol, dimethylformamide, and methyelne chloride. The microspheres encapsulated with the PLGA-conjugated lysozyme were prepared by a single oil-in-water (O/W) emulsion method using a mixture of DMSO and methylene chloride as an oil phase to dissolve the PLGA-conjugated lysozyme. The above solution was directly emulsified in poly(vinyl alcohol) (PVA)/PBS (phosphate buffered saline, 0.1 M NaCl) solution. Conjugation chemistry of PLGA to lysozyme.
Altering the release of tobramycin by incorporating poly(ethylene glycol) into model silicone hydrogel contact lens materials
Published in Journal of Biomaterials Science, Polymer Edition, 2019
Ivana Postic, Heather Sheardown
For quantification of the released drug, an adapted literature procedure involving conjugation of fluorescamine through the free amine and subsequent fluorimetry was performed [46–48]. Within each well, 150 µL of release solution from the study was incubated with 50 µL of 5 mg/mL fluorescamine dissolved in DMSO. The fluorescent compound was measured at excitation/emission wavelengths set to 380/480 nm. The assay was performed in black 96-well plates in order to reduce background fluorescence and prevent cross-talk between wells.