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Biotechnology and Applied Biochemistry (2002) 36, (47–55) (Printed in Great Britain)

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Characterization and physicochemical properties of a lipase from Pseudomonas mendocina 3121-1

Birute Surinenaite*¹, Vida Bendikiene*, Benediktas Juodka*, Irena Bachmatova† and Liucija Marcinkevichiene†

*Department of Biochemistry and Biophysics, Vilnius University, M. K. Ciurlionio 21, LT-2009, Vilnius, Lithuania, and †Institute of Biochemistry, Mokslininku 12, LT-2600, Vilnius, Lithuania

Key words: alkaline pH optimum, dimeric enzyme, lipolysis, Tween 80, metal ions.

Abbreviations used: c.m.c., critical micellar concentration; p-NPB, p-nitrophenyl butyrate.

¹To whom correspondence should be addressed (e-mail birute.surinenaite@gf.vu.lt).

The lipase from Pseudomonas mendocina 3121-1 was found to be homogeneous with a molecular mass of 30 kDa by SDS/PAGE. It is composed of two identical subunits. A molecular mass of 62 kDa was determined by gel chromatography on a Toyopearl HW-55F column. Some physicochemical properties of the lipase were investigated using p-nitrophenyl butyrate (p-NPB), Tween 80 solution and Sigma olive-oil emulsion as substrates. The optimum temperature was determined to be 52 °C with p-NPB, in the range 50–60 °C with Tween 80 and in the range 50–65 °C with olive-oil emulsion. The optimum pH was determined to be in the pH range 7.2–7.5, both with Tween and the emulsion, but was unusually alkaline (pH 9.5) with p-NPB. The enzyme was activated for p-NPB hydrolysis by thermal treatment up to 60 min at 60 °C, pH 7.0–8.2, but was rapidly inactivated at 70–80 °C and at pH 7.0. The lipase was shown to be more thermolabile at 60 °C with respect to other two substrates. Using the emulsified substrate, no activity was obtained after preincubating the enzyme for 30 min at 70 °C. The enzyme was found to be pH-tolerant when stored at 20 °C, pH 6.3–10.3 (100 mM Briton–Robson buffer) as the half-life (t_1/2) was more than 240 h when p-NPB was used as the substrate. By contrast, the pH-stability range was more narrow (pH 8.0–10.5) with olive-oil emulsion. The effect of various metal ions and EDTA depended on the nature of the substrate.

Received 21 February 2002/9 April 2002; accepted 18 April 2002

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