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Chitinase and β-N- acetylglucosaminidase from the digestive fluid of the spider, Cupiennius salei

Authors
  • Mommsen, Thomas P.
Type
Published Article
Journal
Biochimica et Biophysica Acta (BBA) - Enzymology
Publisher
Elsevier
Publication Date
Jan 01, 1980
Volume
612
Issue
2
Pages
361–372
Identifiers
DOI: 10.1016/0005-2744(80)90119-9
Source
Elsevier
Keywords
License
Unknown

Abstract

Two different enzymes which cause the breakdown of nutritional chitin in the hunting spider Cupiennius salei were purified to homogeneity and characterized. One chitinase (EC 3.2.1.14) was enriched about 80-fold (molecular weight 48 000). Its action upon solubilized chitin yields N,N′- diacetylchitobiose (88%), trimers (5%) and monomers (7%) of N- acetylglucosamine , respectively. The enzyme has a pH-optimum around pH 7.2 and it liberates about 30 nmol of N,N′- diacetylchitobiose from chitin per mg protein per s. The enthalpy of activation is 9.5 kcal/mol at 30°C. N,N′- Diacylchitobiose , in turn, is cleaved by a specific β-N- acetylglycosaminidase (EC 3.2.1.30), which hydrolyzes N,N′- diacetylchitobiose , the 4-methylumbelliferyl β-derivatives of N- acetylglucosamine and N- acetylgalactosamine , and p- nitrophenyl-β-N- acetylglucosaminide at nearly equal rates (approx. 250 nkat/mg protein). The pH optima range around pH 5.4 for the chromogenic substrates and around pH 6.5 for N,N′- diacetylchitobiose . Heat stability for the enzyme is better at pH 6.5 than at pH 5.4; activity is not inhibited by any of the substrates used. β-N- acetylglucosaminidase was purified about 160-fold (molecular weight 108 000) and is characterized by kinetic and thermodynamic parameters and inhibitors. Through inhibition studies with N- acetylglucosamine-1 → 5 lactone, it could be shown that all activities pertain to only one enzyme and bind to an identical active centre.

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