RESUMEN
In this study, the antibacterial activities of colloidal chitosan, chitosan solution, and chitooligosaccharide solution were evaluated against Xanthomonas axonopodis pv. glycines grown in peptone sucrose broth (PSB) medium. Treatment with colloidal chitosan (0.01, 0.025, and 0.05%) inhibited X. axonopodis pv. glycines growth only until 36 h. Thin-layer chromatography analysis detected some metabolites, consistently with the cell growth pattern. Two chitooligosaccharides (1-3 kDa and 5-10 kDa) dissolved in distilled water and acetic acid did not exhibit antibacterial activity against X. axonopodis pv. glycines at all tested concentrations (0, 0.001, 0.005, 0.01, 0.015, 0.02, 0.025, and 0.05%). Compared to the control, the chitosan solution decreased X. axonopodis pv. glycines cell growth by 58.7% and 99.0% at concentrations of 0.015% and 0.02%, respectively, after 3 d of incubation. The chitosan solution exhibited the highest antibacterial activity at pH 6.5. However, the antibacterial activity of chitosan decreased in the presence of NaCl and MgCl2.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Quitosano/química , Quitosano/farmacología , Xanthomonas axonopodis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Concentración de Iones de Hidrógeno , Soluciones , Xanthomonas axonopodis/citologíaRESUMEN
The present study investigated the expression pattern of chitinase in Xuehuali (Pyrus bretschneiderilia) pollen, as well as its subsequent degradation. The chitinase was purified and collected using chitin affinity column chromatography with regenerated chitin. After purification, four additional chitinase isozymes (chiA, chiB, chiC, and chiD) and chitinase (Chi II) were clearly expressed on SDS-PAGE gels that contained 0.01% glycol chitin. The chitinase reaction products were examined using GlcNAc, (GlcNAc)2, (GlcNAc)3, (GlcNAc)4, (GlcNAc)5, and (GlcNAc)6 as substrates at 2 and 24h after reaction via TLC and HPLC. The (GlcNAc)4 oligosaccharide was slightly degraded to (GlcNAc)2 after 24h of reaction with Xuehuali pollen chitinase on TLC. Meanwhile, (GlcNAc)5 was degraded to (GlcNAc)2-4, and 2300ppm (GlcNAc)6 was degraded to 246ppm (GlcNAc)2, 208ppm (GlcNAc)3, 572ppm (GlcNAc)4, and 336ppm (GlcNAc)5 on HPLC. With regard to temperature, the strongest Xuehuali pollen chitinase activity (0.69 unit/mL) was observed at 37°C after 3h of incubation, and with regard to pH, the strongest activity (0.72unit/mL) was observed at pH 3 after 3h of incubation. The main chitin oligomers degraded from (GlcNAc)6 were (GlcNAc)2 and (GlcNAc)4.
Asunto(s)
Quitinasas/genética , Isoenzimas/genética , Polen/enzimología , Pyrus/enzimología , Secuencia de Aminoácidos/genética , Quitina/análogos & derivados , Quitina/química , Quitinasas/química , Quitinasas/aislamiento & purificación , Clonación Molecular , Regulación Enzimológica de la Expresión Génica , Hidrólisis , Isoenzimas/química , Isoenzimas/aislamiento & purificación , Polen/química , Polen/genética , Especificidad por SustratoRESUMEN
Serratia marcescens PRNK-1, which has strong chitinolytic activity, was isolated from cockroaches (Periplaneta americana L.). The chitinase from S. marcescens PRNK-1 was characterized after incubation in a 0.5% colloidal chitin medium at 30 °C for 3 days. The molecular weights of three bands after staining for chitinase activity were approximately 34, 41, and 48 kDa on an SDS-PAGE gel. S. marcescens PRNK-1 strain strongly inhibited hyphal growth of Rhizoctonia solani and Fusarium oxysporum. Thin-layer chromatography (TLC) and high performance liquid chromatograph (HPLC) analyses were conducted to investigate the degradation patterns of N-acetyl-chitooligosaccharides by PRNK-1 chitinase. The N-acetyl-chitooligosaccharides: N-acetyl-chitin dimer (GlcNAc)2, N-acetyl-chitin trimer (GlcNAc)3, and N-acetyl-chitin tetramer (GlcNAc)4 were degraded to (GlcNAc)1-3 on a TLC plate. In an additional experiment, (GlcNAc)6 was degraded to (GlcNAc)1-4 on a TLC plate. The optimal temperature for chitinase activity of the PRNK-1 was 50 °C, producing 32.8 units/mL. As seen via TLC, the highest degradation of (GlcNAc)4 by PRNK-1 chitinase occurred with 50 °C incubation. The optimal pH for chitinase activity of PRNK-1 was pH 5.5, producing 24.6 units/mL. As seen via TLC, the highest degradation of (GlcNAc)4 by PRNK-1 chitinase occurred at pH 5.0-6.0. These results indicate that chitinase produced from S. marcescens PRNK-1 strain showed strong antifungal activity and potential of production of N-acetyl-chitooligosaccharides.