Screening and identification of a novel lipase from Burkholderia sp. YY62 which hydrolyzes t-butyl esters effectively.

Abstract

Lipases (EC 3.1.1.3, glycerol ester hydrolase) catalyze 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., 1994;Tsai and Wei, 1994;Waagen et al., 1994;Wu et al., 1990), ester synthesis in organic solvents (Anderson and Callahan, 1960;Armstrong et al., 1988;Bornscheuer, 1995;Klibanov, 1989;Loffet et al., 1989;Makita et al., 1987;Turner and Winterman, 1995) and 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;Harwood, 1989). However, the known lipases scarcely hydrolyze esters containing a bulky alcohol moiety, such as tertiary-alcohol esters, although primary-alcohol esters (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 lipases. 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), anhydrous 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.

Production, purification, characterization, and applications of lipases

“…This feature made the C. parapsilosis enzyme the catalyst of choice for the direct bioconversion of oils in aqueous medium (Vaysse et al, 1997). Yeo et al (1998) reported a novel lipase produced by Burkholderia sp., which could preferentially hydrolyze a bulky ester, t-butyl octanoate (TBO). This lipase was confirmed to be 100-fold superior to commercial lipases in terms of its TBO-hydrolyzing activity.…”

Characterization of the Initial Reactions during the Cometabolic Oxidation of Methyl tert -Butyl Ether by Propane-Grown Mycobacterium vaccae JOB5

“…4). Although tert-butyl esterhydrolyzing esterases are uncommon (54,55), these enzymes are expected to hydrolyze TBF, TBAc, and TBP to TBA and the corresponding acid. We have not quantified formate production from TBF hydrolysis, but we have confirmed that M. vaccae JOB5 does not grow on formate, MTBE, TBA, TBF, or formaldehyde as sole sources of carbon and energy.…”

Microbial lipases and their industrial applications: a comprehensive review

“… Cheese Industry for improvement of flavor [ 709 ] Penicillium roquefortii Cheese Industry for cheese ripening [ 710 ] Staphylococcus warneri, S. xylosus Production of flavour esters [ 711 ] Pseudomonas cepacia Biodiesel fuel production [ 712 ] Pseudomonas sp. Formation of (−)-15-deoxyspergualin 23) in drug industry as antitumor antibiotic and immunosuppressive agent [ 713 ] …”