Enzymatic production of wax esters by esterification using lipase immobilized via physical adsorption on functionalized rice husk silica as biocatalyst.

The present study consists of developing an enzymatic process for the production of wax esters (lauryl stearate and cetyl stearate) by esterification in a heptane medium. Lipase from Thermomyces lanuginosus (TLL) immobilized via interfacial activation on silica particles from rice husks functionalized with triethoxy(octyl)silane (TLL-Octyl-SiO2 ) was used as biocatalyst. Maximum immobilized protein loading of around 22 mg g-1 (that corresponds to an immobilization yield of ≈55%) of support was observed using an initial protein loading of 40 mg g-1 of Octyl-SiO2 . Its hydrolytic activity (olive oil emulsion hydrolysis) was of 620 U g-1 of biocatalyst. The effect of certain factors on the cetyl estearate production was evaluated using a central composite rotatable design (CCDR). Under optimal conditions (64°C, 21% of mass of biocatalyst per volume of reaction mixture, 170 rpm, and stoichiometric acid:alcohol molar ratio 1 mol L-1 of each reactant), maximum acid conversion percentage of 91% was observed after 60 min of reaction. Lauryl stearate was also produced under such conditions, and an acid conversion of 93% after 60 min of reaction was also achieved. Free lipase exhibited acid conversion of only 15%-20% for both reaction mixtures. After nine successive esterification batches, TLL-Octyl-SiO2 retained 85%-90% of its original activity. These results show the promising use of the prepared biocatalyst in wax esters production due to its high catalytic activity and reusability.

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.

Lipase immobilization via cross-linked enzyme aggregates: Problems and prospects - A review.

In this review we have focused on the preparation of cross-linked enzyme aggregates (CLEAs) from lipases, as these are among the most used enzyme in bioprocesses. This immobilization method is considered very attractive due to preparation simplicity, non-use of supports and the possibility of using crude enzyme extracts. CLEAs provide lipase stabilization under extreme temperature or pH conditions or in the presence of organic solvents, in addition to preventing enzyme leaching in aqueous medium. However, it presents some problems in the preparation and limitations in their use. The problems in preparation refer mainly to the crosslinking step, and may be solved using an aminated feeder. The problems in handling have been tackled designing magnetic-CLEAs or trapping the CLEAs in particles with better mechanical properties, the substrate diffusion problems has been reduced by producing more porous-CLEAs, etc. The enzyme co-immobilization using combi-CLEAs is also a new tendency. Therefore, this review explores the CLEAs methodology aimed at lipase immobilization and its applications.

Production of new lipase from Preussia africana and partial characterization.

The present work aims to study the production of a new extracellular lipase from the endophytic fungus Preussia africana isolated from red lapacho tree (Handroanthus impetiginosus). Tests were carried out in order to evaluate the influence of different inducing oils (sunflower, cotton, corn, palm kernel, canola and linseed) and carbon/nitrogen ratio (C/N of 11.73, 8.63 and 7.05) on submerged fermentation aiming at optimal production of a single extracellular lipase. Results show that an optimal lipase production of 14.5 kDa has been attained after 48 h of fermentation when sunflower oil was used in the C/N ratio of 8.63 in the fermentation medium. The produced lipase showed greater activity for oils that had higher percentage of unsaturated fatty acids in their composition. Characterization was performed using a two-factor central composite rotatable design (CCRD), and the pH 6 was found to be optimal (around 28 U/mL). The temperature range studied (from 20 to 54 °C) showed no difference for the lipase catalytic activity. This is an advantage, especially when aiming at its application in reactions of industrial interest.

Optimization of free fatty acid production by enzymatic hydrolysis of vegetable oils using a non-commercial lipase from Geotrichum candidum.

This study aimed to optimize free fatty acid production by enzymatic hydrolysis of cottonseed, olive and palm kernel oils in stirred-tank reactors using a lipase from Geotrichum candidum (GCL-I). The effect of pH, temperature and substrate concentration on the hydrolytic activity of GCL-I using these vegetable oils was investigated. Thermal stability tests and thermodynamic studies were also performed. A complete hydrolysis of cottonseed oil was obtained after 120 min of reaction, while for olive and palm kernel maximum hydrolysis percentage was 96.4% and 60.1%, respectively. GCL-I exhibited the highest activity in the hydrolysis of vegetable oils that are rich in unsaturated-fatty acids (cottonseed and olive oils). Under optimal conditions (46.8% m/m of oil, 6.6 U/g of the reaction mixture at 40 °C), complete cottonseed oil hydrolysis was observed at 60 min of reaction performed in an emulsifier-free system with no buffer.

Relevance of substrates and products on the desorption of lipases physically adsorbed on hydrophobic supports.

Lipase B from Candida antarctica (CALB) has been physically immobilized on octyl-agarose via interfacial activation. The incubation of the enzyme in 80% ethanol at pH 5 and 25°C has not significant effect on enzyme activity. Moreover, the hydrolysis of 100mM tributyrin catalyzed by this biocatalyst exhibited a quite linear reaction course. However, a new cycle of tributyrin hydrolysis showed a drastic drop in the activity. SDS-PAGE gels of the supernatant and the biocatalyst showed a significant enzyme desorption after the reaction. Similar results could be appreciated using triacetin or sunflower oil, while using 300mM methyl phenyl acetate, butyl butyrate or ethyl butyrate most enzyme molecules remained immobilized. The results show that the detergent properties of some reaction products increase the enzyme release from the hydrophobic support, and this problem increased if the concentration of the reactants increased. Using 500mM tributyrin, even in fully aqueous medium, some enzyme desorption from the support may be observed. Thus, the results show a limitation of this kind of biocatalysts that should be considered in the selection of an industrial lipase biocatalyst.

Enzymatic synthesis of isoamyl butyrate catalyzed by immobilized lipase on poly-methacrylate particles: optimization, reusability and mass transfer studies.

Isoamyl butyrate (banana flavor) was synthesized by esterification reaction of isoamyl alcohol and butyric acid in heptane medium. Immobilized Thermomyces lanuginosus lipase (TLL) prepared via physical adsorption on mesoporous poly-methacrylate particles (PMA) was used as biocatalyst. The factors that affect the esterification reaction were optimized by response surface methodology (RSM). Under optimal experimental conditions, maximum ester conversion percentage of 96.1 and 73.6% was reached after 50 and 90 min, respectively, for esterification reaction performed at equimolar ratio alcohol:acid at 500 and 2000 mM of each substrate. Under these experimental conditions, the esterification reaction was not controlled by external and intra-particle mass transfer effects. The product (isoamyl butyrate) was confirmed by proton nuclear magnetic resonance ((1)H NMR) spectroscopy. Reusability tests showed that the biocatalyst retained around 96 and 31% of its initial activity after eight successive esterification cycles performed at 500 and 2000 mM, respectively. The application of the biocatalyst prepared showed to be a promising strategy to catalyze flavor ester synthesis in a non-aqueous medium.

Production of alkyl esters from macaw palm oil by a sequential hydrolysis/esterification process using heterogeneous biocatalysts: optimization by response surface methodology.

The present study deals with the enzymatic synthesis of alkyl esters with emollient properties by a sequential hydrolysis/esterification process (hydroesterification) using unrefined macaw palm oil from pulp seeds (MPPO) as feedstock. Crude enzymatic extract from dormant castor bean seeds was used as biocatalyst in the production of free fatty acids (FFA) by hydrolysis of MPPO. Esterification of purified FFA with several alcohols in heptane medium was catalyzed by immobilized Thermomyces lanuginosus lipase (TLL) on poly-hydroxybutyrate (PHB) particles. Under optimal experimental conditions (mass ratio oil:buffer of 35% m/m, reaction temperature of 35 °C, biocatalyst concentration of 6% m/m, and stirring speed of 1,000 rpm), complete hydrolysis of MPPO was reached after 110 min of reaction. Maximum ester conversion percentage of 92.4 ± 0.4% was reached using hexanol as acyl acceptor at 750 mM of each reactant after 15 min of reaction. The biocatalyst retained full activity after eight successive cycles of esterification reaction. These results show that the proposed process is a promising strategy for the synthesis of alkyl esters of industrial interest from macaw palm oil, an attractive option for the Brazilian oleochemical industry.