Optimizing the Culture Medium of Lasiodiplodia sp. to Improve the Yield of Ethyl Acetate Extract as an Antimicrobial Source.

Endophytes often inhabit plant tissues and cause no disease symptoms. Lasiodiplodia is generally considered a pathogenic fungus, but such a genus is capable of producing high-value bioactive molecules, such as enzymes, secondary metabolites including antimicrobials. Therefore, Lasiodiplodia sp. endophyte was cultivated in static mode for 12 days and EtOAc extracts were obtained and evaluated against pathogens afterward. Fermentation parameters (glucose, sucrose and NaNO3) were optimized by the factorial design and response surface methodology, as these are powerful tools to provide reliable information about fungal culture conditions and EtOAc extract yields were considered as response variables. Lasiodiplodia growth curve indicated that optimal production of EtOAc extract mass was achieved after 12 days of fermentation (284 mg 300 mL-1 broth), which is in agreement with values obtained from validation tests. Minimum Inhibitory Concentration (MIC) and Minimum Microbicidal Concentration (MMC) essays suggested that the endophyte produce substances presenting antimicrobial and antifungal activities against ATCC Staphylococcus aureus and Candida albicans strains at optimum point under evaluated conditions. MIC values ranged between 50 and 100 µg mL-1 for both pathogens, while MMC of C. albicans ranged from 100 to 200 µg mL-1, which evidence its fungicidal effect. Furthermore, it was found that the EtOAc extract yield can be increased by optimizing carbon and nitrogen sources in endophyte cultivation, and there was good agreement between predicted and experimental values under optimized conditions. Thus, Lasiodiplodia fungi are promising sources of antimicrobials and changes in carbon and nitrogen sources can improve the yield of secondary metabolites according to the factorial design.

Preparation, functionalization and characterization of rice husk silica for lipase immobilization via adsorption.

Silica has been extracted from rice husks via a simple hydrothermal process and functionalized with triethoxy(octyl)silane -OCTES (Octyl-SiO2) and (3-aminopropyl)triethoxysilane - 3-APTES (Amino-SiO2), with the aim of using it as support to immobilize lipase from Thermomyces lanuginosus (TLL) via adsorption. The supports have been characterized by particle size distribution and elemental analyses, XRD, TGA, SEM, AFM and N2 physisorption so as to confirm their functionalization. Effect of pH, temperature, initial protein loading and contact time on the adsorption process has been systematically evaluated. Maximum immobilized protein loading of 12.3 ± 0.1 mg/g for Amino-SiO2 (5 mM buffer sodium acetate at pH 4.0, 25 °C and initial protein loading of 20 mg/g) and 21.9 ± 0.1 mg/g for Octyl-SiO2 (5 mM buffer sodium acetate at pH 5.0, 25 °C and initial protein loading of 30 mg/g) was observed. However, these biocatalysts presented similar catalytic activity in olive oil emulsion hydrolysis (between 630 and 645 U/g). TLL adsorption was a spontaneous process involving physisorption. Experimental data on Octyl-SiO2 and Amino-SiO2 adsorption were well-fitted to the Langmuir isotherm model. It was also investigated whether these biocatalysts could synthesize cetyl esters via esterification reaction. Thus, it was found that cetyl stearate synthesis required 100-110 min of reaction time to attain maximum conversion percentage (around 94%). Ester productivity of immobilized TLL on Amino-SiO2 was 1.3-3.1 times higher than Octyl-SiO2.

Preparation of a biocatalyst via physical adsorption of lipase from Thermomyces lanuginosus on hydrophobic support to catalyze biolubricant synthesis by esterification reaction in a solvent-free system.

Lipase from Thermomyces lanuginosus (TLL) was immobilized on mesoporous hydrophobic poly-methacrylate (PMA) particles via physical adsorption (interfacial activation of the enzyme on the support). The influence of initial protein loading (5-200mg/g of support) on the catalytic properties of the biocatalysts was determined in the hydrolysis of olive oil emulsion and synthesis of isoamyl oleate (biolubricant) by esterification reaction. Maximum adsorbed protein loading and hydrolytic activity were respectively ≈100mg/g and ≈650 IU/g using protein loading of 150mg/g of support. The adsorption process followed the Langmuir isotherm model (R(2)=0.9743). Maximum ester conversion around 85% was reached after 30min of reaction under continuous agitation (200rpm) using 2500mM of each reactant in a solvent-free system, 45°C, 20%m/v of the biocatalyst prepared using 100mg of protein/g of support. Apparent thermodynamic parameters of the esterification reaction were also determined. Under optimal experimental conditions, reusability tests of the biocatalyst (TLL-PMA) after thirty successive cycles of reaction were performed. TLL-PMA fully retained its initial activity up to twenty two cycles of reaction, followed by a slight decrease around 8.6%. The nature of the product (isoamyl oleate) was confirmed by attenuated total reflection Fourier transform infrared (ATR-FTIR), proton ((1)H NMR) and carbon ((13)C NMR) nuclear magnetic resonance spectroscopy analyses.