This paper deals with the purification of a class III endochitinase from Euphorbia characias latex. Described purification method includes an effective novel separation step using magnetic chitin particles. Application of magnetic affinity adsorbent noticeably simplifies and shortens the purification procedure. This step and the subsequently DEAE-cellulose chromatography enable to obtain the chitinase in homogeneous form. One protein band is present on PAGE in non-denaturing conditions and SDS-PAGE profile reveals a unique protein band of 36.5 ± 2 kDa. The optimal chitinase activity is observed at 50 °C, pH 5.0. E. characias latex chitinase is able to hydrolyze colloidal chitin giving, as reaction products, N-acetyl-D-glucosamine, chitobiose and chitotriose. Moreover, we observed that calcium and magnesium ions enhance chitinase activity. Finally, we cloned the cDNA encoding the E. characias latex chitinase. The partial cDNA nucleotide sequence contains 762 bp, and the deduced amino acid sequence (254 amino acids) is homologous to the sequence of several plant class III endochitinases.
- MeSH
- chitin metabolismus MeSH
- chitinasy chemie izolace a purifikace metabolismus MeSH
- chromatografie DEAE-celulózová MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- Euphorbia chemie enzymologie MeSH
- hydrolýza MeSH
- molekulární sekvence - údaje MeSH
- sekvence aminokyselin MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The biochemical properties of the alkaline phosphatases (AlPs) produced by Rhizopus microsporus are described. High enzymic levels were produced within 1-2 d in agitated cultures with 1 % wheat bran. Intra- and extracellular AlPs were purified 5.0 and 9.3x, respectively, by DEAE-cellulose and ConA-sepharose chromatography. Molar mass of 118 and 120 kDa was estimated by gel filtration for both forms of phosphatases. SDS-PAGE indicated dimeric structures of 57 kDa for both forms. Mn(2+), Na(+) and Mg(2+) stimulated the activity, while Al(3+) and Zn(2+) activated only the extracellular form. Optimum temperature and pH for both phosphatases were 65 degrees C and pH 8.0, respectively. The enzymes were stable at 50 degrees C for at least 15 min. Hydrolysis of 4-nitrophenyl phosphate exhibited a K (m) 0.28 and 0.22 mmol/L, with upsilon (lim) 5.89 and 4.84 U/mg, for intra- and extracellular phosphatases, respectively. The properties of the reported AlPs may be suitable for biotechnological application.
- MeSH
- alkalická fosfatasa izolace a purifikace metabolismus MeSH
- chromatografie agarózová MeSH
- chromatografie DEAE-celulózová MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- fungální proteiny izolace a purifikace metabolismus MeSH
- gelová chromatografie MeSH
- kationty farmakologie MeSH
- koncentrace vodíkových iontů MeSH
- molekulová hmotnost MeSH
- Rhizopus enzymologie MeSH
- vysoká teplota MeSH
- MeSH
- chromatografie DEAE-celulózová MeSH
- fosfopyruváthydratasa analýza MeSH
- izoenzymy analýza MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- mozková hypoxie enzymologie krev MeSH
- mozková kůra enzymologie růst a vývoj MeSH
- novorozenec MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- novorozenec MeSH
- zvířata MeSH
