Esterification Activity Of Lipase Research Articles

Effect of ethanol concentrations on rice bran protease activity and ester synthesis during enzymatic synthesis of oleic acid ethyl ester in a fed-batch system using crude rice bran (Oryza sativa) lipase

Crude rice bran lipase was used for the enzymatic synthesis of Oleic Acid Ethyl Ester (OAEE) in a fed-batch system. It was found that rice bran contains protease (4.19 U/g) and lipase (324.03 U/g). The results show that protease inhibits lipase at a low concentration of ethanol (5 μmol/ml), in which lipase esterification activity was only 45.02 U/g. However, protease activity decreased about 11 times at a higher concentration of ethanol (5–50 μmol/ml). It resulted in an increase in the esterification activity of lipase (324.03 U/g). This finding could be used for explaining the effects of ethanol on the enzymatic synthesis of OAEE using crude rice bran lipase in a fed-batch system. When the ethanol concentration in the reaction system was low (≤ 4 μmol/ml), the final OAEE conversion was only 30%. It was caused both by the inhibition of lipase by rice bran protease and by the effect of low ethanol concentration on the equilibrium position of the lipase-catalysed esterification reaction. However, the final OAEE conversion reached 76–92% when higher ethanol concentrations (12.5–50 μmol/ml) were added at initial reaction. It is suggested that the addition of higher ethanol concentrations (12.5–50 μmol/ml) into the reaction system may reduce protease activity and also increase ethanol concentration in the reaction system to be converted into OAEE.

Optimization of esterification activity of lipase from Candida rugosa immobilized using microwave irradiation

Lipases are very efficient biocatalysts with wide application in synthesis of important ingredients of food, cosmetics and pharmaceutical products, due to their capacity to catalyze both, ester synthesis and ester hydrolysis. The preparation of stable and active immobilized derivatives of lipases is necessity for their application in industrial enzymatic processes. In this work, the optimization of lipase from C. rugosa immobilization by microwave irradiation was performed, since it was previously reported that immobilization process can be drastically accelerated by means of microwave irradiation, even resulting with slight increase of lipase activity. Eupergit?, commercial support with active epoxy groups, was used as immobilization support. In first stage of our study, the immobilization time and ionic strength of immobilization buffer were optimized. It was found out that the highest immobilized activity can be achieved at high ionic strengths (1 M buffer) after 3 min, while further increase of immobilization time led to decrease of lipase activity. Then, the immobilized derivative obtained at optimum conditions was applied in synthesis of amyl isobutyrate in organic solvent. Key reaction factors (temperature, water concentration, immobilized lipase concentration, and substrate molar ratio) were optimized using response surface methodology. The substrate conversion higher above 85% was achieved in our study. The statistical analysis revealed that each of analyzed factors had significant effect on yield of ester, with initial enzyme concentration and substrate molar ratio being the most prominent factors. The second-order regression model that describes the effect of all four factors on substrate conversion was established. The optimum values of factors were: temperature 50?C, initial immobilized enzyme concentration 220 mg ml-1, added water concentration 0.1% (v/v), and molar ratio acid/alcohol 2.5.

The Catalytic Activity and Stereoselectivity of Lipase Immobilized in Cetyl Trimethyl Ammonium Bromide/Sorbitan Monostearate Microemulsion-based Gel

Cetyl trimethyl ammonium bromide/sorbitan monostearate(Span-60) microemulsion-based gel(CTAB/Span-60 MBG) having good stability in aqueous solution was successfully prepared.The mass percentage of Span-60 in emulsifier mixture(EM,the mixture of n-butanol and Span-60) was determined.The catalytic activity and stereoselectivity of lipase immobilized in CTAB/Span-60 MBG were tested using the esterification of hexanoic acid and hexanol,the hydrolysis of α-glyceryl monostearate and the stereoselective esterification of racemic ibuprofen and octanol as probes,respectively.The results showed that CTAB/Span-60 MBG with good mechanical strength could be achieved when the mass percentage of Span-60 in EM was less than 57%.In organic solvent,the catalytic esterification activity of lipase in this MBG increased gradually with the increase of the content of Span-60 in EM at the first stage and reached maximum at 35% and then decreased slightly with further increase of the content,and was higher than that in CTAB MBG,within the investigated content of Span-60.This immobilized lipase could catalyze the hydrolysis of α-glyceryl monostearate in aqueous solution and the reaction conversion rate didn′t change any more after 24 h.The catalytic hydrolytic activity decreased by 13.68% after 9 re-usage times.This suggests that lipase in CTAB/Span-60 MBG could be separated and reused.Lipase in this MBG also selectively catalyzed S-enantiomer ibuprofen to S-enantiomer ester and the excessive enantiomeric product(eee) of the latter gradually decreased with the proceeding of the reaction,but showing better stereoselectivity than CTAB MBG.As a promising carrier for immobilizing lipase,CTAB/Span-60 MBG could be used in biosynthesis and biotransformation not only in organic solvent but also in aqueous solution.

Enhancing enzymatic properties by the immobilization method

Effects of some immobilized carriers on enzymatic properties have been studied. The following results were obtained: (1) When cholinesterase was immobilized on the hydrophobic carrier with either α-naphthylamine, benzylamine, orp-methylbenzylamine groups, the affinities of immobilized cholinesterase for toxic organophosphors, GB (Isopnopy 1-methylphophonofluoridate) and Vx [o-ethyl-S-(2-diisopnoylomino-thyl) methyl phosphonothiolate], were enhanced 60–90 times and 700–1200 times, respectively, whereas the thermal stability of the immobilized cholinesterase increased to 110%. Approximately 82–88% activity of the immobilized cholinesterase remained after continuously operating for 8 h; and (2) Lipase was immobilized on the carrier that was made up of 6% polyethylenimine, 1% alginate gel, and 1% glutaraldehyde. The initial reaction rate of the esterification of lauric acid with lauric alcohol catalyzed by this kind of immobilized lipase was increased 21 times, as compared to lipase powder. About 72% esterification activity of lipase remained after continuous operating for 10 d.

Lipase-catalysed interfacial reactions in reverse micellar systems. Role of water and microenvironment in determining enzyme activity or dormancy

Studies of the effect of water content on the equilibrium position of a lipase-catalysed hydrolysis/esterification reaction in an AOT reverse micellar system show that the extent of reaction is apparently independent of ωo, the mole ratio of water to surfactant, over the studied range (2–40). Near-IR spectroscopy is shown to be a useful technique for the in situ monitoring of hydrolysis and synthesis reactions in reverse micelle–water-in-oil (w/o) microemulsion systems, and the methods applied are applicable to other low water systems. The hydrolysis and esterification activity of lipases tends to zero at low ωo where the thermodynamic water activity 1. It is shown that a state of reversible enzyme dormancy can be induced by dehydrating the enzyme-containing w/o microemulsion. Molecular sieve beads were considered unsuitable for this purpose because in addition to water, AOT and lipase were also adsorbed from the microemulsion. Molecular sieve powder was more effective, rapidly dehydrating w/o microemulsions whilst leaving AOT and most of the lipase in solution. Drying through vapour-phase equilibration of the microemulsion with a saturated salt solution in a closed system allowed the water activity to be precisely controlled. Although relatively slow, vapour phase drying is ‘clean’ in that it is specific only to water. AOT reverse micellar media containing dormant Chromobacterium viscosum lipase can be stored for at least a month with the lipase activity immediately regenerated on rehydration, with good recovery of catalytic activity. This phenomenon may have application in the food industry for the instant biogeneration of aroma and flavour compounds.

Sol-gel entrapment of lipase using a mixture of tetramethoxysilane and methyltrimethoxysilane as the alkoxide precursor: Esterification activity in organic media

The sol-gel method, using a mixture of tetramethoxysilane (TMOS) and methyltrimethoxysilane (MTrMOS) as the silicon alkoxide precursor, has been applied to entrapment of lipase catalyzing esterification reactions in organic media. Increasing molar ratio of MTrMOS to TMOS led to formation of opaque and non-shrinkable gels and resulted in an increase in the esterification activity of lipase. The hybrid gel-entrapped lipase based on [MTrMOS]/[TMOS] ratio of 4 exhibited almost equal activity with those of the deposited lipase on different supports at 35°C, and retained ca. ten times higher activity than the deposited counterparts at 65 °C.