Purification and characterization of a polysialic acid-specific sialidase from Pseudomonas fluorescens JK-0412

Park, Jae Kweon; Choi, Doo Jin; Kim, Sung Min; Choi, Ha Na; Park, Joo Woong; Jang, Sung Jae; Choo, Young‐Kug; Lee, Choul‐Gyun; Park, Yong Il

doi:10.1007/s12257-011-0495-7

Cited by 3 publications

(2 citation statements)

References 74 publications

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“…Observation of optimal enzyme activity was conducted by incubating the sialidase enzymes in pH buffers, including 50 mM sodium acetate (pH 3-6), 50 mM potassium phosphate (pH 7), and 50 mM Tris-HCl (pH 8-10) at 37°C, and observing its activity [20].…”

Section: Optimal Ph and Enzyme Stabilitymentioning

confidence: 99%

Potency of bacterial sialidase Clostridium perfringens as antiviral of Newcastle disease infections using embryonated chicken egg in ovo model

et al. 2022

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Background and Aim: Clostridium toxins are widely used as medicinal agents. Many active metabolic enzymes, including sialidase (neuraminidase), hyaluronidase, and collagenase, contribute to the mechanism of action of these toxins. Sialidase from Clostridium perfringens recognizes and degrades sialic acid receptors in the host cell glycoprotein, glycolipid, and polysaccharide complexes. Sialic acid promotes the adhesion of various pathogens, including viruses, under pathological conditions. This study aimed to investigate the potential of C. perfringens sialidase protein to inhibit Newcastle disease virus (NDV) infection in ovo model. Materials and Methods: C. perfringens was characterized by molecular identification through polymerase chain reaction (PCR) and is cultured in a broth medium to produce sialidase. In addition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis was conducted to characterize the sialidase protein. In contrast, enzymatic activity and protein concentration were carried out using a neuraminidase assay kit and Bradford to obtain suitable active substances. Furthermore, embryonated chicken egg models were used to observe the toxicity of several sialidase doses. Then, the hemagglutination (HA) titer was obtained, and absolute quantitative reverse transcription–PCR assay was performed to measure the viral replication inhibitory activity of sialidase against NDV. Results: Each isolate had a specific sialidase gene and its product. The sialidase derived from C. perfringens could hydrolyze the sialic acid receptor Neu5Ac (2,6)-Gal higher than Neu5Ac (2,3)Gal in chicken erythrocytes, as observed by enzyme-linked lectin assay. A significant difference (p = 0.05) in the HA titer in the pre-challenge administration group at dosages of 375 mU, 187.5 mU, and 93.75 mU in the competitive inhibition experiment suggests that sialidase inhibits NDV reproduction. Quantification of infective viral copy confirmed the interference of viral replication in the pre-challenge administration group, with a significant difference (p = 0.05) at the treatment doses of 750 mU, 375 mU, and 46.87 mU. Conclusion: The potency of sialidase obtained from C. perfringens was shown in this study, given its ability to reduce the viral titer and copy number in allantoic fluids without adversely impacting the toxicity of the chicken embryo at different concentrations.

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Section: Optimal Ph and Enzyme Stabilitymentioning

confidence: 99%

Potency of bacterial sialidase Clostridium perfringens as antiviral of Newcastle disease infections using embryonated chicken egg in ovo model

et al. 2022

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“…BfGH33C released sialic acid from ␣2,3-, ␣2,6-, and ␣2,8-linked substrates and showed 1.2-fold higher activity for ␣2,8 linkage than for ␣2,3 or ␣2,6 linkages, indicating that the specificity is biased toward the ␣2,8 linkage. The preference for ␣2,8-linked substrates was also found in several other sialidases, such as the enzymes from Pseudomonas fluorescens JK-0412 and bacteriophage K1F (36,37). In contrast, the sialidases CpGH33A, CpGH33B, and CpGH33C from C. perfringens ATCC 13124 and SiaBb2 from B. bifidum JCM1254 showed preference for the ␣2,3 linkage, which was consistent with the hydrolysis specificity of the sialidases from Pasteurella multocida and Salmonella Typhimurium (38,39).…”

Section: Discussionmentioning

confidence: 67%

Enzymatic Synthesis of 6′-Sialyllactose, a Dominant Sialylated Human Milk Oligosaccharide, by a Novel exo -α-Sialidase from Bacteroides fragilis NCTC9343

Guo

Chen

et al. 2018

Appl Environ Microbiol

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Gut bacteria provide a rich source of glycosidases that can recognize and/or hydrolyze glycans for nutrition. Interestingly, some glycosidases have also been found to catalyze transglycosylation reactions and thus can be used for oligosaccharide synthesis. In this work, six putative and one known-α-sialidase genes-three from NCTC9343, three from ATCC 13124, and one known from JCM1254-were subjected to gene cloning and heterogeneous expression in The recombinant enzymes were purified, characterized for substrate specificity, and screened for transglycosylation activity. A sialidase, named BfGH33C, from NCTC9343 was found to possess excellent transglycosylation activity for the synthesis of sialylated human milk oligosaccharide. The native BfGH33C was a homodimer with a molecular weight of 113.6 kDa. The and values for 4-methylumbelliferyl-acetyl-α-d-neuraminic acid and sialic acid dimer were determined to be 0.06 mM and 283.2 s, and 0.75 mM and 329.6 s, respectively. The enzyme was able to transfer sialyl from sialic acid dimer or oligomer to lactose with high efficiency and strict α2-6 regioselectivity. The influences of the initial substrate concentration, pH, temperature, and reaction time on transglycosylation were investigated in detail. Using 40 mM sialic acid dimer (or 40 mg/ml oligomer) and 1 M lactose (pH 6.5) at 50°C for 10 min, BfGH33C could specifically produce 6'-sialyllactose, a dominant sialylated human milk oligosaccharide, at a maximal conversion ratio above 20%. It provides a promising alternative to the current chemical and enzymatic methods for obtaining sialylated oligosaccharides. Sialylated human milk oligosaccharides are significantly beneficial to the neonate, as they play important roles in supporting resistance to pathogens, gut maturation, immune function, and brain and cognitive development. Therefore, access to the sialylated oligosaccharides has attracted increasing attention both for the study of saccharide functions and for the development of infant formulas that could mimic the nutritional value of human milk. Nevertheless, nine-carbon sialic acids are rather complicated for the traditional chemical modifications, which require multiple protection and deprotection steps to achieve a specific glycosidic bond. Here, the -α-sialidase BfGH33C synthesized 6'-sialyllactose in a simple step with high transglycosylation activity and strict regioselectivity. Additionally, it could utilize oligosialic acid, which was newly prepared in an easy, economical way to reduce the substrate cost, as a glycosyl donor. All the studies laid a foundation for the practical use of BfGH33C in large-scale synthesis of sialylated oligosaccharides in the future.

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Characterization of an alkalophilic extracellular chitosanase from Bacillus cereus GU-02

Goo

Park

2014

Journal of Bioscience and Bioengineering

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Purification and characterization of a polysialic acid-specific sialidase from Pseudomonas fluorescens JK-0412

Cited by 3 publications

References 74 publications

Potency of bacterial sialidase Clostridium perfringens as antiviral of Newcastle disease infections using embryonated chicken egg in ovo model

Potency of bacterial sialidase Clostridium perfringens as antiviral of Newcastle disease infections using embryonated chicken egg in ovo model

Enzymatic Synthesis of 6′-Sialyllactose, a Dominant Sialylated Human Milk Oligosaccharide, by a Novel exo -α-Sialidase from Bacteroides fragilis NCTC9343

Characterization of an alkalophilic extracellular chitosanase from Bacillus cereus GU-02

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