LIPASE MODULATOR PROTEIN (LIML) OF PSEUDOMONAS SP STRAIN-109

Plasmids containing a Pseudomonas sp, strain 109 extracellular lipase gene (lipL) lacking NH2-terminal sequence and a lipase modulator gene (limL) lacking the NH2-terminal hydrophobic region were constructed an d expressed independently in Escherichia coli by using the T7 promoter expression vector system, Recombinant LipL (rLipL) was produced as in clusion bodies, whereas recombinant LimL (rLimL) was present as a solu ble protein. During in vitro renaturation of the purified rLipL inclus ion bodies after they had been dissolved in 8 M urea, addition of rLim L was essential to solubilize and modulate rLipL. The solubility and a ctivity of rLipL were influenced by the rLimL/rLipL molar ratio; the h ighest level of solubility was obtained at an rLimL/rLipL ratio of 4:5 , whereas the highest activity level was obtained at an rLimL/rLipL ra tio of 4:1, After renaturation, rLipL and rLimL were coprecipitated wi th anti-rLipL antibody, indicating the formation of an rLipL-rLimL com plex, Activity of the native lipase purified from Pseudomonas sp. stra in 109 was also inhibited by rLimL. By Western blotting (immunoblottin g) with anti-rLimL antibody, native LimL was detected in Pseudomonas c ells solubilized by sarcosyl treatment. LimL was purified from Pseudom onas sp, strain 109, and the NH2-terminal amino acid sequence was dete rmined to be NH2-Leu-Glu-Pro-Ser-Pro-Ala-Pro-. We propose that to prev ent membrane degradation, LimL weakens lipase activity inside the cell , especially in the periplasm, in addition to modulating lipase foldin g.