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Purification and characterization of an extracellular lipase from the fungusRhizopus delemar

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Lipids

Abstract

The complete purification and characterization of an extracellular lipase (acylglycerol acylhydrolase, EC 3.1.1.3) fromR. delemar is described. The final product was homogeneous as judged by electrophoresis in denaturing polyacrylamide gels and by isoelectric focusing, and was shown by means of an activity stain to be lipolytic. The purified enzyme had a monomer molecular weight of 30,300, an isoelectric point of 8.6, and approximately one monosaccharide moiety per molecule.N-Terminal sequence data (28 residues) and the amino acid composition of the lipase indicated that it corresponds to the product of a lipase-encoding cDNA previously isolated fromR. delemar. Optimal activity occurred between pH 8.0 and 8.5. The activity and stability of the enzyme were maximum at 30°C. Divalent cations were required for activity, with barium, calcium and manganese conferring maximum activity. Activation by calcium was maximal at and above 10 mM. The lipase was not inactivated by reducing agents, sodium fluoride or phenylmethlsufonyl fluoride. It was resistant toN-ethylmaleimide, and inactivated byp-chloromercuribenzoic acid in a manner which was not reversed by cysteine.

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Abbreviations

BME:

β-mercaptoethanol

DTT:

dithiothreitol

EDTA:

ethylenediaminetetraacetic acid disodium dihydrate

IEF:

isoelectric focusing

kDa:

kilodaltons

NEM:

N-ethylmaleimide

PCMB:

p-chloromercuribenzoic acid

pI:

isoelectric point

PMSF:

phenylmethylsulfonyl fluoride

PVP:

polyvinylpyrrolidone

SDSPAGE:

polyacrylamide gel electrophoresis in sodium dodecyl sulfate

U:

units of lipase activity

UV:

ultraviolet

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Authors and Affiliations

  1. Eastern Regional Research Center, U.S. Department of Agriculture, 600 East Mermaid Lane, 19118, Philadelphia, Pennsylvania

    Michael J. Haas & David G. Bailey

  2. Department of Chemistry, LaSalle University, 19141, Philadelphia, Pennsylvania

    David J. Cichowicz

Authors
  1. Michael J. Haas
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  2. David J. Cichowicz
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  3. David G. Bailey
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Mention of brand or firm names does not constitute an endorsement by the U.S. Department of Agriculture over others of a similar nature not mentioned.

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Haas, M.J., Cichowicz, D.J. & Bailey, D.G. Purification and characterization of an extracellular lipase from the fungusRhizopus delemar . Lipids 27, 571–576 (1992). https://doi.org/10.1007/BF02536112

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  • DOI: https://doi.org/10.1007/BF02536112

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