O. Misset et al., THE STRUCTURE-FUNCTION RELATIONSHIP OF THE LIPASES FROM PSEUDOMONAS-AERUGINOSA AND BACILLUS-SUBTILIS, Protein engineering, 7(4), 1994, pp. 523-529
Within the BRIDGE T-project on lipases we investigate the structure-fu
nction relationships of the lipases from Bacillus subtilis and Pseudom
onas aeruginosa. Construction of an overproducing Bacillus strain allo
wed the purification of > 100 mg lipase from 30 I culture supernatant.
After testing a large variety of crystallization conditions, the Baci
llus lipase gave crystals of reasonable quality in PEG-4000 (38-45%),
Na2SO4 and octyl-beta-glucoside at 22 degrees C, pH 9.0. A 2.5 Angstro
m dataset has been obtained which is complete from 15 to 2.5 Angstrom
resolution. P.aeruginosa wild-type strain PAC1R was fermented using co
nditions of maximum lipase production. More than 90% of the lipase was
cell bound and could be solubilized by treatment of the cells with Tr
iton X-100. This permitted the purification of similar to 50 mg lipase
. So far, no crystals of sufficient quality were obtained. Comparison
of the model we built for the Pseudomonas lipase, on the basis of sequ
ences and structures of various hydrolases which were found to possess
a common folding pattern (alpha/beta hydrolase fold), with the X-ray
structure of the P.glumae lipase revealed that it is possible to corre
ctly build the structure of the core of a protein even in the absence
of obvious sequence homology with a protein of known 3-D structure.