A simple room temperature-static bioreactor for effective synthesis of hexyl acetate

For green synthesis in organic phases, the catalysis of lipases needs to be further improved. Here a strategy is to simulate the activation of lipases at water/oil interface by immobilizing lipase on an available carrier material, which can replace the water at the interface to stabilize enzyme against denaturation from organic phase. The carrier is selected as cotton fiber on which Pseudomonas cepacia lipase is immobilized by physical adsorption in a column glass bottle to form a simple bioreactor together with the bottle. In synthesis of fragrance ester hexyl acetate via transesterification of hexanol with vinyl acetate, the bioreactor exhibits a 3-fold increase in the ability to transform substrate, relative to native lipase in terms of the initial period of reaction at 37°C and 160 rpm. And also the bioreactor is very stable in catalysis for that it has an extra long half life t1/2 = 636 h, calculated from the decrease degree of molar conversions in six times of 6-hour transesterifications. More interestingly, the bioreactor behaves excellent activity at room temperature and in a static state, and can transform nearly 100% hexanol after 48 h. All these indicate that the bioreactor has great potential for industrial application.

Kinetics of Enantiomerically Enriched Synthesis of Solketal Esters Using Native and SBA-15 supported P. Fluorescens Lipase

The studies showed that alkaline lipase from Pseudomonas fluorescens enables an irreversible transesterification of vinyl esters to give enantiomeric excess (eeR) of about 80% using vinyl butyrate as acyl donor and diisopropyl ether as a solvent, at partially optimized conditions. For the native lipase the process was adequately described by a five-parameter Ping-Pong Bi Bi model for both enantiomers plus expression accounting for the formation of enzyme-acyl donor complex, but for the same lipase supported on mesoporous materials of SBA-15-Oc type, R-product inhibition also had to be taken into account. The use of hydrophobic support increased by more than two-fold the rate of the S-solketal conversion but even more that of R-solketal. Thus the immobilization of lipase had very positive effect on the process kinetics but decreased its enantioselectivity.

Activated Carbon as Support for Lipase Immobilization

Lipase from<em> Candida rugosa</em> was immobilized onto four different types of activated carbon; KI/2030, KI/3040, KI/5060 and KI/6070. The immobilized lipase was used in the esterification of oleic acid and 1-butanol in hexane. The effects of difference pore sizes, surface area, reaction temperature, thermostability of the immobilized lipases, storage stability in organic solvent and leaching studies were investigated. Among the four samples, KI/6070 gave the highest activities and stability in all the parameters investigated. Immobilized lipases generally exhibit activities higher than the native lipase for the parameters studied, with optimum temperature of 40°C. Immobilized lipases are more stable than native lipase in hexane at room temperature up to 12 days. Leaching study proved that the immobilization of lipase using physical adsorption is cheap and easy. This method was found to be suitable for the attachment of enzyme on the support.

Determination of the extracellular lipases of Pseudomonas fluorescens spp. in skim milk with the β-naphthyl caprylate assay

SUMMARYA method based on the hydrolysis of β-naphthyl caprylate (β-NC) has been developed for quantitating extracellular lipase from Pseudomonas fluorescens. The assay was extremely sensitive to skim milk (SM); as little as 0·02 ml raw SM in a 2·0 ml reaction mixture resulted in an apparent loss of 50% of the lipase activity. Activity improved 3-fold when trypsin (50 μg/ml) was included in the reaction mixture. When super-simplex optimization was used to determine the optimum levels of β-NC, Na taurocholate (NaTC), SM/lipase mixture and trypsin for maximum activity, NaTC was found to be unnecessary for activity. Subsequent addition of 15 mM-NaTC resulted in 80% loss of activity. On the other hand, NaTC was required for native lipase activity in the presence of SM. Native lipase was completely inhibited by heating at 70 °C for 2 min, while B52 lipase retained 75% of its activity under the same conditions. The assay was able to detect lipase produced by Ps. fluorescens B52 in SM at 5 °C when the cell density exceeded 108 colony forming units/ml. The presence of butterfat (3°5%) in the SM assay inhibited B52 lipase by 97%. The β-NC assay gave results comparable to the tributyrin agar diffusion assay using cell-free extracts of ten strains of common dairy psychrotrophs. The results suggest that the β-NC assay may be useful for determining lipase activity in raw SM.