Extracellular lipase of Pseudomonas sp. strain ATCC 21808: purification, characterization, crystallization, and preliminary X-ray diffraction data.
A procedure for the purification of a very hydrophobic lipase from Pseudomonas sp. strain ATCC 21808 was elaborated by avoiding the use of long-chain detergents in view of subsequent crystallization of the enzyme. The purification procedure included chromatography on Q-Sepharose in the presence of n-octyl-beta-D-glucopyranoside, Ca2+ precipitation of fatty acids, and Octyl-Sepharose chromatography. The enzyme was purified 260-fold to a yield of 35% and a specific activity of 3,300 U/mg. The molecular weight was determined as 35,000; a polyacrylamide gel under nondenaturing conditions revealed a band at 110,000, and the isoelectric point proved to be at 4.5 to 4.6. The lipase crystallized with different salts and ethylene glycol polymers in the presence of n-octyl-beta-D-glucopyranoside and one alkyloligooxyethylene compound (CxEy) in the range from C5E2 to C8E4. The crystals diffract to a resolution of about 0.25 nm. Precession photographs revealed that they belong to space group C2 with lattice constants of a = 9.27 nm, b = 4.74 nm, c = 8.65 nm, and beta = 122.3 degrees, indicating a cell content of one molecule per asymmetric unit of the crystal. In hydrolysis of triglycerides, the lipase showed substrate specificity for saturated fatty acids from C6 to C12 and unsaturated long-chain fatty acids. Monoglycerides were hydrolyzed very slowly. The N-terminal sequence is identical to that of the lipase from Pseudomonas cepacia. Treatment with diethyl-p-nitrophenylphosphate affected the activities toward triolein and p-nitrophenylacetate to the same extent and with the same velocity.
Kordel
M
M
Department of Enzyme Technology, GBF-Gesellschaft für Biotechnologische Forschung m.b.H., Braunschweig, Federal Republic of Germany.
Hofmann
B
B
Schomburg
D
D
Schmid
R D
RD
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D003460:Crystallization; D004795:Enzyme Stability; D008049:Lipase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D011549:Pseudomonas / Q000201:enzymology*; D013379:Substrate Specificity; D014961:X-Ray Diffraction
D001426:Bacterial Proteins; D008049:Lipase