Lipase catalysed synthesis of sugar ester in organic solvents

Abstract

Lipases (EC 3.1.1.3, glycerol ester hydrolase) cat-alyze the hydrolysis of triacylglycerols to glycerol and free fatty acids. Recently, a variety of new applications of lipase have emerged, especially in enantioselective hydrolysis of esters (Nakano et al. in modification of sugars or chiral drugs (Bornemann et al., 1992; Margolin, 1993; Patil et al., 1991). Many different kinds of microbial lipases have been reported, and they have received much attention because of their potential use in industry (Björkling et al., 1991; Har-wood, 1989). However, the known lipases scarcely hy-drolyze esters containing a bulky alcohol moiety, such as tertiary-alcohol esters, although primary-alcohol es-ters (Amaya et al., 1995; Linko et al., 1995; Zaidi et al., 1995) and secondary-alcohol esters (Iwai et al., 1980; Mitsuda et al., 1988) are good substrates for li-pases. This lack of ability to hydrolyze bulky alcohol esters hindered the wide use of lipases for complex compounds. Therefore, we started to obtain a lipase that hydrolyzes t-butyl octanoate (TBO) as a model substrate for bulky esters, and isolated a microorganism that produces a new type lipase from soil samples. In this report, we describe the primary characterization of this lipase and taxonomic identification of the producer. Materials and Methods Preparation of t-butyl esters. The t-butyl alcohol esters used in this work were prepared from t-butyl alcohol and corresponding acid chlorides. The typical protocol for the synthesis of TBO is described below. Octanoyl chloride (48 g, 0.3 M) was added dropwise to a mixture of 2-methyl-2-propanol (80 g, 1.08 M), anhy-drous pyridine (25.6 g, 0.32 M), and 4-dimethyl aminopyridine (1 g, 8.2 mM) in 800 ml of hexane at 0-5°C for 2 h. After 14 h reaction at room temperature, water (400 ml) was added to the mixture and the hexane layer was separated. The hexane solution was washed with water, 1 N HCl solution and sodium carbonate solution. The hexane layer was separated, washed with water, dried over sodium sulfate and concentrated under reduced pressure to yield 39.3 g (65.5%) of oil. The structure was confirmed by 1 H-NMR and IR spectroscopy. Other t-butyl esters were Fatty acid esters composed of sterically hindered alcohol are very poor substrates for known li-pases. In order to obtain a novel lipase, t-butyl octanoate (TBO) was selected as a model substrate to screen for bacteria-producing lipase(s) which can preferentially hydrolyze bulky esters. Of 279 strains isolated from 350 soil samples based on the ability to grow with TBO as a sole carbon source, one strain (YY62) was chosen for its strong TBO-hydrolyzing activity. Strain YY62 is a Gram-negative motile rod and was identified as Burkholderia sp. from the taxonomic characters and phylogenetic analysis of 16S rDNA nucleotide sequences. Using the activity ratio between TBO and p-nitrophenyl acetate as a measure for preference to bulky esters, we confirmed that the lipase of strain YY62 was 100-fold superior to commercial lipases in terms of TBO-hydrolyzing activity.

Quantitative enzymatic production of 1,6‐diacyl sorbitol esters

“…More recently, procedures have been described for obtaining nearly complete conversion of glucose (Ljunger et al, 1994) and sorbitol (Cao et al, 1997) in a reaction medium where most of the precursor sugar is not dissolved. Methods based on formation of phenylboronic complexes with sugars (Schlotterbeck et al, 1993) containing single free primary hydroxyl group may be exploited for selective monoacylation reactions in apolar media such as n-heptane (Oguntimein et al, 1993;Scheckermann et al, 1995). Alternatively, production of sugar monoesters from the isopropylidene sugar and the corresponding fatty acid may be carried out in absence of solvent (Ward et al, 1997).…”

The Use of Biobased Surfactant Obtained by Enzymatic Syntheses for Wax Deposition Inhibition and Drag Reduction in Crude Oil Pipelines

“…A product that is secreted by the metabolism of microorganisms, with amphiphilic molecules and high surface activity, is a biobased surfactant. Compared to classical surfactants, they have some advantages, including low toxicity, biodegradability, a wide variety of structures, synthesis from inexpensive renewable materials and stability in a wide range of pH [26]. Surfactants are traditionally manufactured with chemical processes that cost large quantities of energy, including high acidity and alkalinity and temperatures that exceed 100˝C, and organic solvents may be essential.…”

Effect of salt hydrate pair on lipase-catalyzed regioselective monoacylation of sucrose

“…Because of their different solubilities, however, it is not easy to mix sugar and fatty acids in the reaction medium. To solve this problem a number of different approaches for solubilizing substrates in enzyme reactions have been introduced, for example, by using solvents Riva et al, 1988) such as pyridine and DMF, by using sugar derivatives [alkyl (Mutua and Akoh, 1993) or acetal (Fregapane et al, 1991;Sarney et al, 1994)], or by using solubilizing agents for sugars (Ikeda and Klibanov, 1993;Oguntimein et al, 1993;Schlotterbeck et al, 1993) like organoboronic acids.…”