Molecular Mechanism of the Hydration of Candida antarctica Lipase B in the Gas Phase: Water Adsorption Isotherms and Molecular Dynamics Simulations

Hydration is a major determinant of activity and selectivity of enzymes in organic solvents or in gas phase. The molecular mechanism of the hydration of Candida antarctica lipase B (CALB) and its dependence on the thermodynamic activity of water (a_w) was studied by molecular dynamics simulations and compared to experimentally determined water sorption isotherms. Hydration occurred in two phases. At low water activity, single water molecules bound to specific water binding sites at the protein surface. As the water activity increased, water networks gradually developed. The number of protein‐bound water molecules increased linearly with a_w, until at a_w=0.5 a spanning water network was formed consisting of 311 water molecules, which covered the hydrophilic surface of CALB, with the exception of the hydrophobic substrate‐binding site. At higher water activity, the thickness of the hydration shell increased up to 10 Å close to a_w=1. Above a limit of 1600 protein‐bound water molecules the hydration shell becomes unstable and the formation of pure water droplets occurs in these oversaturated simulation conditions. While the structure and the overall flexibility of CALB was independent of the hydration state, the flexibility of individual loops was sensitive to hydration: some loops, such as those part of the substrate‐binding site, became more flexible, while other parts of the protein became more rigid upon hydration. However, the molecular mechanism of how flexibility is related to activity and selectivity is still elusive.     

纤维素载体固定化β-葡萄糖苷酶及其在大豆异黄酮水解中的应用

建立纤维素载体固定化β-葡萄糖苷酶系统,用于黑豆浆中大豆异黄酮去糖基化反应,并对该催化系统进行了可行性评价。结果表明,在40 cm3纤维素颗粒上可结合超过40 mgβ-葡萄糖苷酶,并且能有效将4-硝基苯-β-D-葡糖苷酸水解成对硝基苯酚,其反应最适温度为50℃。经动力学测试后可得其米氏常数Km值为(1.38±0.20)mmol/L。将该固定化酶系统用于黑豆浆中大豆异黄酮去糖基化,通过高效液相色谱检测其转化效率,表明该系统可以在30 min内利用40 cm~3含酶载体将50 mL黑豆浆中的异黄酮全部转化为去糖基化形式。该固定化酶催化系统连续化催化稳定性实验表明,在经过15次反应以及15 d之后,该固定化酶系统仍然可维持其60%的催化活性。     

Tropomyosin Contains IgE‐Binding Epitopes Sensitive to Periodate but Not to Enzymatic Deglycosylation

Glycosylation has been reported to affect the epitopes of food allergens, however, there are few reports on its role in crab allergen. In the present study, the effect of glycosylation on the IgE‐binding activity of tropomyosin (TM), a major allergen in Scylla paramamosain, was investigated. The results showed that TM was a glycoprotein with a 0.2% carbohydrate moiety and contained O‐glycan. Moreover, enzymatic deglycosylation of TM by glycosidase had no effect on the IgE‐binding activity of TM. In contrast, treatment with periodate resulted in a significant reduction in its IgE‐binding activity.     

Probing the Role of Mucin‐Bound Glycans in Bacterial Repulsion by Mucin Coatings

Microbial colonization of implanted medical devices in humans can lead to device failure and life‐threatening infections. One strategy to prevent this unwanted colonization is to coat devices with polymers that reduce bacterial attachment. This study investigates how mucins, a class of biopolymers found in mucus, can be used as surface coatings to prevent attachment of selected respiratory pathogens to polystyrene surfaces. Our data show that coatings of porcine gastric mucins or bovine submaxillary mucins reduce surface attachment by Streptococcus pneumoniae and Staphylococcus aureus, but not Pseudomonas aeruginosa. To elucidate how mucin coatings repel S. pneumoniae and S. aureus, the molecular components of mucins are examined. Our data suggest that mucin‐bound glycans are key structural contributors of mucin coatings and are necessary for the repulsive effects toward S. pneumoniae and S. aureus.     

A modified spectrophotometric assay to estimate deglycosylation of steroidal saponin to sapogenin by mixed ruminal microbes

BACKGROUND: The lack of a method for measuring deglycosylation of saponins in ruminal fluid has limited the ability to investigate the impact of these compounds on rumen microorganisms. A simple spectrophotometric assay was adapted and a protocol developed to enable measurement of steroidal saponin and sapogenin in ruminal fluid. The procedure was used for in vitro determination of deglycosylation activity of rumen bacteria obtained from cattle fed or not fed Yucca schidigera saponin, and to determine the relative deglycosylase activities of extracellular and cell‐associated enzymes from ruminal content. RESULTS: Modifications to the spectrophotometric assay (i.e. heating time shortened to 10 min and 0.5 mL dH₂O added to the reaction mixture) improved the stability of the optical density (425 nm) of the chromophore for up to 24 h post‐reaction. Centrifugation (12 000 × g, 20 min) enabled differential estimations of steroidal saponin and sapogenin in ruminal fluid. Steroidal saponin added to defaunated ruminal fluid (dRF) or clarified ruminal fluid (cRF) was recovered completely from the mixture as saponin + sapogenin (99.1% and 100.6%, respectively), whereas saponin recovery from the supernatant of dRF was greatly reduced (P < 0.001) compared to that from supernatant of cRF (58.5 vs. 98.7%). Saponin recoveries from the supernatants of dRF and cRF did not differ between donor cattle fed or not fed Yucca schidigera saponin (59.2 vs. 57.3% and 98.4 vs. 99.3%, respectively). The majority (89–90%) of saponin added to a ruminal extracellular enzyme preparation was recoverable in supernatant after 24 h, compared with only 26–32% remaining in supernatant from incubation with a cell‐associated enzymes fraction. CONCLUSION: Mixed rumen bacteria deglycosylate steroidal saponin to sapogenin, at activity levels unaffected by prior exposure to saponin, but they were unable to degrade the sapogenin core structure. Deglycosylation activity occurred primarily in the cell‐associated enzyme fraction of ruminal content. Copyright © 2008 Crown in the right of Canada. Published by John Wiley & Sons, Ltd     

Structure characterization and IgE-binding of soybean 7S globulin after enzymatic deglycosylation

This study investigated the effects of enzymatic deglycosylation following ultrasound pretreatment on structure and immunoreactivity of soybean 7S globulin. Soybean 7S globulin was pretreated by ultrasound (40 kHz, 300 W) and enzymatically deglycosylated by peptide-N-glycosidase F (PNGase F). Changes in structure of processed soybean 7S globulin were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, reversed-phase high-performance liquid chromatography, ultraviolet absorption spectrum, circular dichroism spectrum, and surface hydrophobicity analysis. Enzyme-linked immunoabsorbent assay was used to evaluate IgE-binding ability. The results showed that the glycan moieties of soybean 7S globulin were effectively removed by PNGase F, which significantly modified protein structures including the secondary and tertiary structures of 7S globulin. Individual enzymatic deglycosylation could reduce IgE-binding capacity of 7S globulin, whereas enzymatic deglycosylation following ultrasound pretreatment enhanced its IgE-binding capacity. In conclusion, soybean 7S globulin treated by single enzymatic deglycosylation can reduce potential allergenicity and may be employed in hypoallergenic food preparation.