Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan1
Department of Plant Protection, National Institute of Fruit Tree Science, 2-1 Fujimoto, Tsukuba, Ibaraki 305-8605, Japan2
Author for correspondence: Takuya Nihira. Tel: +81 6 6879 7433. Fax: +81 6 6879 7432. e-mail: nihira{at}biochem.bio.eng.osaka-u.ac.jp
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ABSTRACT |
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Keywords: Pseudomonas, lactonizing lipase, lipase activation factor, lipase modulator protein
Abbreviations: LAF, lipase activation factor
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INTRODUCTION |
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The gene (lipL) encoding the lactonizing lipase has already been cloned in our laboratory from strain 109, and the amino acid sequence was deduced from the nucleotide sequence (Ihara et al., 1991 ). However, the lipL gene alone was not sufficient to produce active LipL. The co-existence of a downstream limL gene, either on the same plasmid or on a separate plasmid, was found to be essential for the functional expression of LipL (Ihara et al., 1992
). A similar requirement of a secondary gene has been observed for several lipases (Frenken et al., 1993a
; Iizumi et al., 1991; Jørgensen et al., 1991
; Kok et al., 1995
; Ogierman et al., 1997
; Siomi et al., 1992
; Wohlfarth & Winkler, 1992
), and studies of Burkholderia cepacia (LimA; Hobson et al., 1993
), Pseudomonas glumae (LipB; Frenken et al., 1993b
), Pseudomonas aeruginosa (LipB; Hirayama et al., 1993
) and Pseudomonas sp. strain KWI-56 (Act; Iizumi & Fukase, 1994) suggested that the secondary gene product is required for correct lipase folding.
When recombinant LipL (rLipL) and recombinant LimL (rLimL) were overexpressed in Escherichia coli, rLimL was formed as a soluble protein but rLipL was obtained as inactive inclusion bodies. In vitro renaturation experiments with rLipL inclusion bodies in the presence of urea revealed that rLimL was essential to obtain soluble and active rLipL (Ihara et al., 1995 ). However, the renatured rLipL showed only 6% of the activity observed in native and fully active LipL, due to the formation of a tight complex between rLipL and rLimL as detected by immunoprecipitation (Ihara et al., 1995
). This suggests that bound rLimL inhibits hydrolytic activity of rLipL and that some additional mechanism exists that facilitates LimL dissociation. To obtain fully active LipL in large amounts, it is essential to devise an efficient reactivation procedure for the inclusion bodies of rLipL, because the amount of native LipL that can be obtained in Pseudomonas sp. strain 109 is low.
In this study, we found a novel lipase-activating factor (LAF) in Pseudomonas sp. strain 109. This factor synergistically acted with Ca2+ on the rLipLrLimL complex to increase lipase activity 1·8-fold, and was found to be a novel low-Mr compound (Mr 330±30). The increase of lipase activity was confirmed to be associated with the release of free rLipL from the rLipLrLimL complex.
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METHODS |
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Preparation of crude cell-free lysate of Pseudomonas sp. strain 109 and fractionation by ultrafiltration.
Pseudomonas sp. strain 109 was grown in 250 ml LB medium in a 500 ml Sakaguchi flask at 30 °C and 120 r.p.m. for about 12 h to an OD600 of 2·5. Cells were harvested by centrifugation (3000 g, 10 min, 4 °C), suspended in 5 ml 50 mM potassium phosphate buffer (pH 6·5) and sonicated (4x30 s, 4 °C). Cell debris was removed by centrifugation (10000 g, 20 min, 4 °C), and the supematant was used as crude cell-free lysate and stored at -80 °C until use.
To fractionate the crude cell-free lysate by Mr, the lysate was either dialysed with a cellulose dialysis membrane (Wako Pure Chemical) against a 100-fold volume of 50 mM potassium phosphate buffer (pH 6·5) for 12 h at 4 °C, or filtered through a Centriprep 3 (Mr cut 3000) or Centriprep 30 (Mr cut
30000, Amicon). The filtrates from the ultrafiltrations were referred to as <3000 lysate and <30000 lysate, respectively.
Renaturation of recombinant LipL (rLipL).
rLipL and recombinant LimL (rLimL) were overexpressed from plasmids pETY402 and pLIM413, respectively, in E. coli, using an LB medium containing 1% (w/v) glucose, 10 µg ampicillin ml-1 and 2 µg chloramphenicol ml-1, purified as described previously (Ihara et al., 1995 ), and stored at -80 °C until use. For obtaining the rLipLrLimL complex, purified inclusion bodies of rLipL (0·5 mg) were solubilized in 1 ml 6 M urea. After 1 h incubation at room temperature, rLimL (1·15 mg in 50100 µl, two equivalents of rLipL) was added to the solubilized rLipL, and the solution was made up to 5 ml with 6 M urea. The solubilized mixture of rLipL and rLimL was renatured by decreasing the urea concentration in a stepwise manner from 4 to 0 M by successive dialyses against 500 ml 50 mM potassium phosphate buffer (pH 6·5) containing 2 M urea for 3 h at 4 °C and two changes of 500 ml 50 mM potassium phosphate buffer (pH 6·5) for 3 h.
Gel-filtration HPLC.
HPLC was performed with a JASCO model Tri Rotor-V equipped with a UV detector (JASCO UVIDEC-100-V). The <3000 lysate (250 µl) was applied at room temperature onto a Superdex Peptide HR 10/30 column (1·0x30 cm, Pharmacia Blotech) pre-equilibrated with 50 mM potassium phosphate buffer (pH 6·5) containing 0·2 M NaCl. The <3000 lysate was then fractionated from the column with the same buffer at a flow rate of 0·5 ml min-1 and collected in 200 µl fractions.
Gel-filtration on a SMART system.
Gel-filtration of rLipLrLimL complexes was performed using a SMART system (Pharmacia Biotech). The rLipLrLlmL complex, either without treatment or activated by LAF, was injected into a Superose12 PC 3·2/30 (0·32x30 cm, Pharmacia Biotech) gel-filtration column equilibrated with 50 mM potassium phosphate buffer (pH 6·5) containing 0·2 M NaCl and 0.1% (w/v) NoigenHC at a flow rate of 25 µl min-1. Protein elution was monitored by absorbance at 280 nm and the eluate was fractionated at room temperature into portions of 25 µl.
Western blot analysis.
The fractions (5 µl samples) from gel-filtration on the SMART system were directly applied to SDS-PAGE after boiling in the presence of 1% SDS, and Western blot analysis was carried out by using the ECL Western blotting detection kit (Amersham Life Science) under conditions recommended by the manufacturer. Polyclonal anti-rLipL or anti-rLimL antibodies have been described previously (Ihara et al., 1995 ). Immunoreactive proteins on Western blots were visualized by the use of horseradish-peroxidase-conjugated anti-rabbit IgG (Amersham Life Science).
Lipase activation assay for LAF.
A typical activation mixture (100 µl) contained 0·05 M potassium phosphate (pH 6·5), 3 µg of the solubilized rLipLrLimL complex, Pseudomonas cell-free lysate, and/or other factors as indicated. Reactions were initiated by the addition of cell-free lysate containing LAF. Samples (10 µl) were withdrawn at appropriate times, mixed with 90 µl potassium phosphate buffer (pH 6·5), and assayed for lipase activity. Each experiment was repeated at least three times, and data are expressed as means of triplicate experiments with all samples. Deviation in the activation by LAF was within ±10%.
Enzyme and protein assay.
Lipase activity was routinely assayed by measuring the amount of p-nitrophenol formed from p-nitrophenyl hexanoate. The substrate (5 mM p-nitrophenyl hexanoate) was emulsified completely by sonication in the presence of 0·5% (w/v) Triton X-100 in 50 mM potassium phosphate buffer (pH 6·5) containing 1 mM MgCl2. After preincubation of the enzyme solution (1·5 ml) in the same buffer at 37 °C for 1 min, reactions were initiated by the addition of an equal volume of the substrate solution, followed by further incubation at 37 °C for 10 min. Reactions were terminated by the addition of ethanol (3 ml), and the absorbance at 400 nm was measured. One unit of lipase activity was defined as the amount of enzyme which liberates 1 µmol p-nitrophenol min-1. Protein concentration was determined by a dye-binding assay (Protein Assay Kit; Bio-Rad) with bovine serum albumin as the standard.
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RESULTS AND DISCUSSION |
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To investigate the possibility that another factor, of either high or low Mr, was required for the activation of the rLipLrLimL complex, we tested several compounds for their ability to enhance lipase activity from the solubilized rLipLrLimL complex. Immediately after the addition of 1 mM CaCl2, lipase activity was enhanced 1·7-fold, while 1 mM ATP or other divalent metal ions (1 mM MgCl2, 1 mM ZnCl2, 1 mM FeCl3) were either inhibitory or had no effect. Because Ca(NO3)2 was similarly effective in enhancing lipase activity (data not shown), Ca2+ was concluded to be an effective component. Addition of Pseudomonas crude cell-free extract resulted in a small increase in activity at 0 h, but after incubation at 4 °C for 24 h, lipase activity increased about 1·8-fold, suggesting that some novel component(s) were present in the lysate. A similar level of activation (1·4-fold) was observed when native LipL was treated similarly after denaturation with 6 M urea. To discriminate between the activation by Ca2+ and that by the lysate, the time-course of activation was compared in the presence of Ca2+ or the lysate (Fig. 1). The effect of Ca2+ was instantaneous and concentration dependent, but the enhanced activity gradually decreased after a 24 h incubation, reaching 4783% of that at 0 h (Fig. 1a
). The effect of the lysate was also concentration dependent, but in this case the activation proceeded gradually with time until 24 h (Fig. 1b
), indicating clearly that the action of the LAF in the lysate was different from that of Ca2+. Atomic absorption spectroscopy indicated that the crude cell-free lysate contained only 70 µM Ca2+. Because at least 0·5 mM Ca2+ was necessary to see the activation, 70 µM Ca2+ could not account for the activation by the lysate; this suggested that the LAF is not Ca2+. Activation by the lysate was faster at higher temperatures (1·8-fold increase after 2 h at 30 °C or after 24 h at 4 °C), although longer incubation at higher temperatures caused gradual loss of lipase activity due to thermal inactivation. To facilitate the reaction, we chose to perform activation by LAF at 30 °C in the following studies.
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To determine its Mr more precisely, the <3000 lysate was separated by gel-filtration HPLC using a Superdex Peptide column (Fig. 2). Only factors eluting from 32·8 min to 33·2 min corresponding to Mr 330±30 from a log(Mr) versus retention time plot showed lipase-activation activity. Therefore, it can be concluded that the LAF of Pseudomonas sp. strain 109 is a negatively charged, hydrophilic compound(s) of Mr 330±30.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 29 January 1999;
revised 19 April 1999;
accepted 7 June 1999.
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