Antimicrobial Effect of a Lauric Acid on Streptococcus Mutans Biofilm.

Background: The purpose of this study was to compare the antimicrobial activity of a synthetic fatty acid sodium laurate (Lauric acid) comprising dodecanoate fatty acids with chlorhexidine (CHX) or calcium hydroxide (CH) against S. mutans biofilm. Methods: S. mutans was grown on cover glass bottom dishes or human dentin disks for 3 days, and then treated with sodium laurate (20 μg/ml), non-functional fatty acid(sodium decanate, sigma Aldrich, C4151) ( (NP, 20 μg/ml), CH (20 μg/ml), 1% CHX, or saline for 5 days at 37℃. On cover glass, live and dead microbials in the biomass were measured by the Film Tracer™ Biofilm viability assay, and observed by confocal laser scanning microscopy (CLSM). On dentin disk, normal, diminished, or ruptured microbials were observed by field-emission scanning electron microscopy (FE-SEM). The results were subjected to two-tailed t-test, one-way analysis variance and post hoc test at a significance level of P=0.05. Results: Live/Dead Biofilm viability assay and CLSM demonstrated that sodium laurate treated biofilms had a significantly less bio-volume than CH, NP, and saline (P < 0.05), but had no significant difference from the CHX-treated group (P > 0.05). FE-SEM demonstrated that there was a marked decrease in aggregations of microbials and biofilm and wrinkled or ruptured microbials were frequently observed in the CHX and sodium laurate. Conclusion: Synthetic sodium laurate fatty acid exhibited significantly higher antimicrobial activity than CH by inhibiting microbial survival and biofilm growth against S. mutans, but had no significant difference compared to CHX.

Evaluation of Luminescent P450 Analysis for Directed Evolution of Human CYP4A11

Cytochrome P450 4A11 (CYP4A11) is a fatty acid hydroxylase enzyme expressed in human liver. It catalyzes not only the hydroxylation of saturated and unsaturated fatty acids, but the conversion of arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE), a regulator of blood pressure. In this study, we performed a directed evolution analysis of CYP4A11 using the luminogenic assay system. A random mutant library of CYP4A11, in which mutations were made throughout the entire coding region, was screened with luciferase activity to detect the demethylation of luciferin-4A (2-[6-methoxyquinolin-2-yl]-4,5-dihydrothiazole-4-carboxylic acid) of CYP4A11 mutants in Escherichia coli. Consecutive rounds of random mutagenesis and screening yielded three improved CYP4A11 mutants, CP2600 (A24T/T263A), CP2601 (T263A), and CP2616 (A24T/T263A/V430E) with ~3-fold increase in whole cells and >10-fold increase in purified proteins on the luminescence assay. However, the steady state kinetic analysis for lauric acid hydroxylation showed the significant reductions in enzymatic activities in all three mutants. A mutant, CP2600, showed a 51% decrease in catalytic efficiency (k cat/K m) for lauric acid hydroxylation mainly due to an increase in K m. CP2601 and CP2616 showed much greater reductions (>75%) in the catalytic efficiency due to both a decrease in k cat and an increase in K m. These decreased catalytic activities of CP2601 and CP2616 can be partially attributed to the changes in substrate affinities. These results suggest that the enzymatic activities of CYP4A11 mutants selected from directed evolution using a luminogenic P450 substrate may not demonstrate a direct correlation with the hydroxylation activities of lauric acid.

Effect of Bioimprinting by Lauric Acid on Esterification Activity of Lipase / 中国生物工程杂志

Bioimprinting is a new developed technique to improve the characteristics of enzymes.Bioimprinting by lauric acid was conducted to improve the esterification activity of lipase PS in sol-gel immobilization process with methyltrimethoxysila(MTMS) and tetramethoxysila(TMOS) as the precursors.Results generated by checking the esterification activity and scanning electron microscope showed that bioimprinting can enhance the specific activity and thermal stability of lipase PS.The bioimprinting system was optimized by orthogonal experiment,and the optimal condition for lipase bioimprinting is water/silane molar ration(R) 12,polyethylene glycol(PEG) 120?l,and lauric acid 0.15 mmol.Compared with the free enzyme and the non-imprinted enzymes,the specific activity of imprinted enzymes has been improved 44.3 fold and 2.4 fold,respectively.Imprinted lipase show better thermal stability,and the relative activity is 58% after incubated in 80 ℃ for 0.5 h,while no activity was detected for the free enzyme.