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Purification, characterization and cloning of a chitinase from Stenotrophomonas rhizophila G22

Purification, characterization and cloning of a chitinase from Stenotrophomonas rhizophila G22

Urszula Jankiewicz, Bartosz Baranowski, Maria Swiontek Brzezinska, Magdalena Frąk

3 Biotech. 2020 Jan;10(1):16.

PMID: 31879580

Abstract:

In the presented research the extracellular chitinase of Stenotrophomonas rhizophila G22 was biochemically and molecularly characterized. The studied enzyme was purified from a 72-h bacterial culture about 14 times, with a recovery of 63%. The molecular weight of the purified protein was estimated at 50 kDa by SDS-PAGE. The enzyme showed high activity against colloidal chitin. Significantly lower activities were observed with native chitin powder and chitosan. Adsorption of the enzyme to colloidal chitin and to powdered chitin at the level of 75% and 37%, respectively, was observed after 30 min of reaction. Optimum temperature and pH were 37 °C and 5.9, respectively. The enzyme demonstrated higher activity against nitrophenyl-β d N, N', N″-triacetylchitotriose and approx. 5 times lower activity for 4-nitrophenyl-N, N'-diacetyl-β-d-chitobiose. The enzyme is an endochitinase, which is confirmed by the K m and V max values determined in the studies. S. rhizophila G22 endochitinase was inhibited in the presence of cysteine-specific inhibitors, which indicates the role of cysteine moieties in the mechanism of catalysis or in stabilisation of the enzyme molecule. Also Ca2+ and Mn2+ ions may stabilise the protein's spatial structure. SDS and ions: Fe2+, Cu2+, Co2+, Zn2+ inhibited the activity of enzyme. A full-length (2109 bp) gene coding chitinase from S. rhizophila G22 was obtained. Four domains typical for glycoside hydrolase family 18 (GH 18) chitinases were identified: catalytic Gly_18, chitin-binding-ChtBD3, type-III fibronectin-FN3 and polycystic kidney disease domain-PKD domain.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP7699389	4-Nitrophenyl β-D-N,N′,N′′-triacetylchitotriose		7699-38-9	Price

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