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Isolation and partial characterization of collagenolytic enzyme from the mosquito-parasitizing fungus, Lagenidium giganteum

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Abstract

Lagenidium giganteum Couch produced an extracellular collagenolytic enzyme when grown on peptone-yeast extract-glucose (PYG) broth. The enzyme was purified to apparent homogeneity by chromatography on soybean trypsin inhibitor Sepharose 4B and DEAE cellulose. The purified enzyme hydrolyzed Azocoll, insoluble bovine Achilles tendon collagen, and acid soluble calfskin collagen at neutral pH. No other substrates appeared to be hydrolyzed.

The purified enzyme had an apparent molecular weight of 100,000 and required calcium for optimal activity. The inhibitors PMSF, TPCK, TLCK, D-penicillamine, 8-hydroxyquinoline, cysteine, and 1,10-phenanthroline had little or no effect on enzyme activity.

SDS polyacrylamide gel electrophoresis of collagenase digested collagen indicated that the enzyme cleaved in the helical region of the collagen molecule, although the exact site of cleavage was not determined.

The properties of the Oomycete collagenase, the first ever reported, were found to differ consider considerably from those collagenases reported in higher fungi, with regard to metal-dependence, molecular weight and mode of action.

A possible role for this enzyme was discussed in relation to pathogenesis produced in mosquito larvae by this fungus.

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Abbreviations

BAPA:

N-α-benzoyl-Dl-arginine p-nitroanilide HCl

Bis:

N,N1-methylene-bis-acrylamide

CAPS:

3-(cyclohexylamino)-propane sulfonic acid

EDTA:

disodium ethylenediamine tetraacetate

EGTA:

ethylene glycol-bis-(B-aminoethyl ether)-N,N1 tetraacetic acid

I-RT-63:

α,α′,α″-tris (3-amidinophenoxy) mesitylene-3 HCl

MOPS:

3-(N-morpholino)-propane sulfonic acid

PMSF:

phenylmethylsulfonyl fluoride

PZ-peptide:

4-phenylazobenzyloxycarbonyl-L-Pro-L-Leu-Gly-L-Pro-D-Arg

SDS:

sodium dodecylsulfate

TAME:

p-toluenesulfonyl-L-arginine methylester

TEMED:

N,N,N′,N′-tetramethylethylenediamine

TES:

2-(tris-hydroxymethyl)-aminoethane sulfonic acid

TLCK:

N-tosyl-L-lysine chloromethyl ketone

TPCK:

N-tosyl-L-phenylalanine chloromethyl ketone

Tris:

tris-(hydroxymethyl)-aminomethane

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Dean, D.D., Domnas, A.J. Isolation and partial characterization of collagenolytic enzyme from the mosquito-parasitizing fungus, Lagenidium giganteum . Arch. Microbiol. 136, 212–218 (1983). https://doi.org/10.1007/BF00409847

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  • DOI: https://doi.org/10.1007/BF00409847

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