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Improved isolation, stability and substrate specificity of cucumisin, a plant serine endopeptidase
Author(s) -
Kaneda M.,
Yonezawa H.,
Uchikoba T.
Publication year - 1995
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1111/j.1470-8744.1995.tb00347.x
Subject(s) - chemistry , endopeptidase , hydrolysis , cucumis , enzyme , stereochemistry , chymotrypsin , cleave , trypsin , substrate (aquarium) , urea , biochemistry , biology , ecology
Cucumisin (EC 3.4.21.25), a serine endopeptidase, was isolated by a simple purification procedure from the prince melon (Cucumis melo ssp. melo, cv. ‘Prince Melon’). The enzyme is stable over a wide pH range (4‐11) and to heat, 80% of its initial activity remaining even at pH 11.1 and at 60 degrees C for 20 min. The enzyme was inactive at 72 degrees C and pH 8.0, but 38% of the activity remained in the presence of 10% (w/v) glycerol. Caseinolysis by cucumisin indicated full activity in 8 M urea at pH 9.1 and 50 degrees C. Cucumisin was inactivated by treatment with trypsin at 37 degrees C for 24 h, but was not affected by alpha‐chymotrypsin. The synthetic substrates benzyloxycarbonyltyrosine nitrophenyl ester (Z‐Tyr‐ONp) and benzoyltyrosine ethyl ester (Bz‐Tyr‐OEt) were cleaved, but Z‐Lys‐ONp and tosylarginine methyl ester (Tos‐Arg‐OMe) were not cleaved by cucumisin. Oxidized insulin B‐chain was hydrolysed by cucumisin at 37 degrees C for 24 h, 21 cleavage sites being detected. Cucumisin could not cleave the C‐termini of all the valine residues in the oxidized insulin B‐chain molecule.