Lipase modulator protein (LimL) of Pseudomonas sp. strain 109

Abstract

Plasmids containing a Pseudomonas sp. strain 109 extracellular lipase gene (lipL) lacking NH 2 -terminal sequence and a lipase modulator gene (limL) lacking the NH 2 -terminal hydrophobic region were constructed and expressed independently in Escherichia coli by using the T7 promoter expression vector system. Recombinant LipL (rLipL) was produced as inclusion bodies, whereas recombinant LimL (rLimL) was present as a soluble protein. During in vitro renaturation of the purified rLipL inclusion bodies after they had been dissolved in 8 M urea, addition of rLimL was essential to solubilize and modulate rLipL. The solubility and activity of rLipL were influenced by the rLimL/rLipL molar ratio; the highest level of solubility was obtained at an rLimL/rLipL ratio of 4:5, whereas the highest activity level was obtained at an rLimL/rLipL ratio of 4:1. After renaturation, rLipL and rLimL were coprecipitated with anti-rLipL antibody, indicating the formation of an rLipL-rLimL complex. Activity of the native lipase purified from Pseudomonas sp. strain 109 was also inhibited by rLimL. By Western blotting (immunoblotting) with anti-rLimL antibody, native LimL was detected in Pseudomonas cells solubilized by sarcosyl treatment. LimL was purified from Pseudomonas sp. strain 109, and the NH 2 -terminal amino acid sequence was determined to be NH 2 -Leu-Glu-Pro-Ser-Pro-AlaPro-. We propose that to prevent membrane degradation, LimL weakens lipase activity inside the cell, especially in the periplasm, in addition to modulating lipase folding.

Lipase and Its Modulator from Pseudomonas sp. Strain KFCC 10818: Proline-to-Glutamine Substitution at Position 112 Induces Formation of Enzymatically Active Lipase in the Absence of the Modulator

“…In addition, the addition of the crude extract of E. coli JM109 harboring pACTM12 to the crude extract of E. coli JM109 harboring pKL11 induced the activation of the inactive lipase (data not shown). These results suggest that, despite its low sequence homology with Pseudomonas Lifs, LimK may interact directly with lipase and be required for its correct folding in the same manner that Lifs interact with lipase (9,13,17,30).…”

“…However, much difficulty has been encountered in expressing the lipases in E. coli, which is due mainly to their requirements for specific Lifs (13,17,35). We expect that the novel finding of this study and further characterization of LipK P112Q will be useful in studying the folding pathway of Pseudomonas lipases and provide an effective method for the expression of industrially important lipases.…”

In Vitro Analysis of Roles of a Disulfide Bridge and a Calcium Binding Site in Activation of Pseudomonas sp. Strain KWI-56 Lipase

“…These genes were reported to direct the synthesis of chaperone-like proteins, named modulator, activator, or chaperone, to assist the correct folding of lipase specifically. The inactive lipases produced by recombinant Escherichia coli cells could be reactivated by an in vitro denaturation-renaturation method with the assistance of such chaperones (10,13,16,33,34).…”

Small-Scale Production of Burkholderia cepacia ATCC21808 Lipase Adapted to High-Throughput Screening

“…The lipase:Lif ratio needed for full activation of newly synthesized lipase in vivo is unknown, 17 but in vitro refolding experiments have shown that a 1:1 to 1:4 ratio was optimal for good folding. 12,18,19 Consequently, to increase the Lif expression, the lip-hp genes were placed under the control of the more powerful T7 and tac promoters in the construction of pET-OmpA-Lip-Hp and pFLAG-ATS-Lip-Hp, respectively. Notably, the pFLAG-ATS vector, known to be efficient for eukaryotic gene expression, had not been assayed before for bacterial lipase expression.…”