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CRYSTALLIZATION AND X-RAY STUDIES OF LIPASES
Cambillau, Christian
Cambillau, Christian
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Issue Date
1991
Submitted date
2024-03-06
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Abstract
Several structure determinations of lipases are underway in Marseille. Most projects are at a heavy-atom derivative search stage. These projects belong to three classes: i. Cutinases are a group of extracellular fungal hydrolytic enzymes capable of degrading the insoluble lipid polyester matrix, i.e. cutin, which covers the surface ofplants. Their weight is 22,000 Da, signifantly lower than all other lipases. A recombinant cutinase from F. solani pisi is expressed and excreted with very high yields in E.coli cultures. Cutinase was crystallized (PEG 6000 15-20% ‚pH 7.0 to 10.0,20°C) in space group P2, with cell dimensions 35.1A,67.4A,37.05 A, B=94°. They diffract to 1.5 resolution (Rsym=4.41%). Data from native and derivatives have been collected. MIR phasingis in progress. ii, Gastric lipases are pH resistant enzymes with a molecular weight of ca 50,000, including 15% sugars. Every glyco-variant from different species have been purified by ief, and submitted to crystallization, using parameters found by an incomplete factorial analysis method. All attempts were successful, but most crystals were unsuitable to Xray studies due to huge cell dimensions. Dog gastric lipase crystals, the most suitable for X-ray study (pH 6.8, 12% PEG 6000, 20°C; P2,2,2 :182A, 211A, 98 A), contain ca 8 moleculesin the asymetric unit , and therefore diffract to only 4A on lab sources. iii, Crystals of a complex between porcine pancreatic lipase (50000 Da) and its co-factor, colipase (11000 Da), have been obtained. These 1/1 complex crystals are disordered, and diffract weakly. Horse pancreatic lipase has been crystallized in space group P2,2,2,, (89A,97A,145A ; pH 6.0, PEG 8000 10%, 20°C)and a 2.3 A native data set has been collected. One PCMBS derivative was not sufficient to produce a good map, and weare still looking for other derivatives. Two crystal forms of human pancreaticlipase (purified from pancreas juice) have been obtained, different from the one reported in the litterature. They crystallize in the same conditions and space groups are P2, and Py. Native data sets have been collected.
Citation
Lipases : structure, mechanism and genetic engineering, 17 - 26
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Book chapter
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conference paper
Language
en
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Series/Report no.
GBF monographs ; Volume 16
ISSN
0930-4320
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ISBN
156081165X
3527283323
3527283323
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Attribution-NonCommercial-ShareAlike 4.0 International