Laccase Redox Potentials: pH Dependence and Mutants, a QM/MM Study.

We have studied the T. versicolor laccase T1 site redox potential (RP) at the M06/6-311++G**/SDD(Cu) level of theory, employing QM/MM-optimized geometries (RI-BP86/def2-SVP/def2-TZVP(Cu):CHARMM) of the whole protein system with electronic embedding. The oxidation state of the trinuclear cluster was found to affect the T1 site RP by about 0.2-0.3 V, depending on the protein protonation state. The computed laccase RP can be drastically lowered upon introduction of a protonation state corresponding to a neutral environment, by up to -1.37 V, which is likely an overestimation of the effect in vivo. The gradual change of the protonation state by single points without optimization or equilibration results in a change that is even larger, namely up to about -2.6 V. Thus, the preferred protein conformation supports a high redox potential, compensating for the RP-lowering effect of surface charges. The predicted change in RP on going to the F463M mutant, ca. -0.1 V, is consistent with observations for a related laccase. Based on our results, we also propose and test a D206N mutant but find it to be locked in a conformation with slightly lower RP.

Extracellular phospholipase activity of Malassezia strains isolated from individuals with and without dermatological disease.

BACKGROUND: The Malassezia genus includes mainly lipophilic yeasts belonging to the cutaneous microbiota of man and other mammals. Some Malassezia species have been associated with various dermatological diseases. The factors permitting the transformation of yeasts of the Malassezia genus from a commensal organism to a pathogenic agent are still little known but the production of various enzymes such as lipase, phospholipase and lipoxygenase could contribute to the pathogenic activity of these yeasts. AIMS: Here we have determined and compared the extracellular phospholipase activity of sixty human isolates of Malassezia so as to relate this feature to the species of Malassezia and to the origin (from dermatological diseases or not) of the strains examined. METHODS: Phospholipase production was determined using the semi-quantitative egg-yolk plate method. RESULTS AND CONCLUSIONS: Malassezia obtusa, Malassezia slooffiae, Malassezia globosa, Malassezia restricta had difficulty developing in the chosen culture medium so that it was not possible to measure phospholipasic activity. Malassezia pachydermatis showed the highest phospholipase activity. Twenty-nine Malassezia sympodialis strains produced phospholipase; the isolates from patients with pityriasis versicolor had significantly higher phospholipasic activity than those isolated from healthy individuals. This observation suggests that the phospholipasic activity of Malassezia may play a role in the onset of skin lesions, especially in the case of pityriasis versicolor.

Comparison of 3 methods for the determination of sinapic acid ester content in enzymatically treated canola meals.

The enzymatic reduction of sinapic acid ester content in canola meal using polyphenol oxidase from the fungus T. versicolor was investigated. To determine the effectiveness of this new process, the results obtained using two spectrophotometric methods and an HPLC analytical method for assaying sinapic acid ester content in the treated and untreated meals were compared. It was found that all the methods gave practically the same results when the samples from untreated canola meals were analysed. However, both of the spectrophotometric methods overestimated the sinapic acid ester content in the enzymatically treated meal by 7%-20%, as compared to the results obtained using HPLC. It was found that the sensitivity limits for the spectrophotometric methods used for the determination of sinapic acid ester content in enzymatically treated canola meals were 2.67 g and 1.47 g phenolics/kg meal for the direct and chemical spectrophotometric methods respectively. A correlation between the results obtained using the spectrophotometric and HPLC methods is given. The enzymatic treatment resulted in a negligible amount of phenolics in the treated meal.