Purification and characterization of Lip2 and Lip3 isoenzymes from a Candida rugosa pilot-plant scale fed-batch fermentation

Previous purification of a crude extracellular enzyme preparation from Candida rugosa ATCC 14830 pilot-plant fed-batch fermentations showed the presence of two lipase isoenzymes, Lip2 and Lip3, differing in their molecular masses (58 and 62 kDa, respectively). These enzymes were purified but the lip...

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Bibliographic Details
Published in:Journal of biotechnology 2000-11, Vol.84 (2), p.163-174
Main Authors: Pernas, M.A., López, C., Pastrana, L., Rúa, M.L.
Format: Article
Language:English
Subjects:
Aggregation
Biological and medical sciences
Biotechnology
Candida - chemistry
Candida - enzymology
Candida rugosa
Characterization
Chromatography, Gel
Enzyme Activation - physiology
Enzyme engineering
Fermentation - physiology
Fundamental and applied biological sciences. Psychology
Glycosylation
Hydrogen-Ion Concentration
Improved methods for extraction and purification of enzymes
Industrial Microbiology - methods
Isoenzymes
Isoenzymes - chemistry
Isoenzymes - isolation & purification
Isoenzymes - metabolism
Lip2 protein
Lip3 protein
Lipase - chemistry
Lipase - isolation & purification
Lipase - metabolism
Methods. Procedures. Technologies
Molecular Weight
Pilot Projects
Purification
Triacetin - metabolism
Triglycerides - metabolism
Triolein - metabolism
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Summary:Previous purification of a crude extracellular enzyme preparation from Candida rugosa ATCC 14830 pilot-plant fed-batch fermentations showed the presence of two lipase isoenzymes, Lip2 and Lip3, differing in their molecular masses (58 and 62 kDa, respectively). These enzymes were purified but the lipases were forming active aggregates with a molecular mass higher than 200 kDa. In this work we developed a purification method following three steps: ammonium sulfate precipitation, sodium cholate treatment and ethanol/ether precipitation, and anion exchange chromatography which allowed the sequential disaggregation of the isoenzymes. Pure and monomeric Lip2 and Lip3 were characterized according to p I, glycosylation and activity for p-nitrophenol esters and triacylglycerols of varying acyl chain. Lip3 was the best catalyst for the hydrolysis of the simple esters and triacylglycerols with short and medium acyl chains.
ISSN:0168-1656
1873-4863
DOI:10.1016/S0168-1656(00)00351-5