Abstract
In previous work, a new lipase and its cognate foldase were identified and isolated from a metagenomic library constructed from soil samples contaminated with fat. This new lipase, called LipG9, is a true lipase that shows specific activities that are comparable to those of well-known industrially-used lipases with high activity against long-chain triglycerides. In the present work, LipG9 was co-expressed and co-immobilized with its foldase, on an inert hydrophobic support (Accurel MP1000). We studied the performance of this immobilized LipG9 (Im-LipG9) in organic media, in order to evaluate its potential for use in biocatalysis. Im-LipG9 showed good stability, maintaining a residual activity of more than 70% at 50°C after incubation in n-heptane (log P 4.0) for 8 h. It was also stable in polar organic solvents such as ethanol (log P -0.23) and acetone (log P -0.31), maintaining more than 80% of its original activity after 8 h incubation at 30°C. The synthesis of ethyl esters was tested with fatty acids of different chain lengths in n-heptane at 30 °C. The best conversions (90% in 3 h) were obtained for medium and long chain saturated fatty acids (C8, C14 and C16), with the maximum specific activity, 29 U per gram of immobilized preparation, being obtained with palmitic acid (C16). Im-LipG9 was sn-1,3-specific. In the transesterification of the alcohol (R,S)-1-phenylethanol with vinyl acetate and the hydrolysis of the analogous ester, (R,S)-1-phenylethyl acetate, Im-LipG9 showed excellent enantioselectivity for the R-isomer of both substrates (E> 200), giving an enantiomeric excess (ee) of higher than 95% for the products at 49% conversion. The results obtained in this work provide the basis for the development of applications of LipG9 in biocatalysis.
Highlights
IntroductionLipases (glycerol ester hydrolases EC 3.1.1.3) are carboxylesterases that normally catalyze the hydrolysis of long and short-chain acyl esters in aqueous media [1]
Lipases are carboxylesterases that normally catalyze the hydrolysis of long and short-chain acyl esters in aqueous media [1]
We identified a new lipase, LipG9, in a metagenomic library constructed from soil samples contaminated with animal fat [18]
Summary
Lipases (glycerol ester hydrolases EC 3.1.1.3) are carboxylesterases that normally catalyze the hydrolysis of long and short-chain acyl esters in aqueous media [1]. When used in water-restricted media, they can catalyze esterification and transesterification reactions, with high regio- and enantioselectivity [1,2]. Due to these properties, lipases have been used in organic media for the synthesis of biodiesel [3,4] and of chiral compounds that have applications in the chemical, pharmaceutical and cosmetic industries [5,6]. A major barrier to the industrialization of lipase-catalyzed processes is their low productivity when compared to chemical routes of synthesis. It is necessary to obtain new lipases that have a high and stable activity in organic solvents, including short-chain alcohols
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