Purification and characterization of two sugarcane bagasse-absorbable thermophilic xylanases from the mesophilic Cellulomonas flavigena.
We report the purification and characterization of two thermophilic xylanases from the mesophilic bacteria Cellulomonas flavigena grown on sugarcane bagasse (SCB) as the only carbon source. Extracellular xylanase activity produced by C. flavigena was found both free in the culture supernatant and associated with residual SCB. To identify some of the molecules responsible for the xylanase activity in the substrate-bound fraction, residual SCB was treated with 3 M guanidine hydrochloride and then with 6 M urea. Further analysis of the eluted material led to the identification of two xylanases Xyl36 (36 kDa) and Xyl53 (53 kDa). The pI for Xyl36 was 5.0, while the pI for Xyl53 was 4.5. Xyl36 had a Km value of 1.95 mg/ml, while Xyl53 had a Km value of 0.78 mg/ml. In addition to SCB, Xyl36 and Xyl53 were also able to bind to insoluble oat spelt xylan and Avicel, as shown by substrate-binding assays. Xyl36 and Xyl53 showed optimal activity at pH 6.5, and at optimal temperature 65 and 55 degrees C, respectively. Xyl36 and Xyl53 retained 24 and 35%, respectively, of their original activity after 8 h of incubation at their optimal temperature. As far as we know, this is the first study on the thermostability properties of purified xylanases from microorganisms belonging to the genus Cellulomonas.
Santiago-Hernández
Alejandro
A
Departamento de Biotecnología y Bioingeniería, CINVESTAV, Av. Instituto Politécnico Nacional, 2508, Mexico D. F., CP 07360, Mexico.
Vega-Estrada
Jesús
J
del Carmen Montes-Horcasitas
María
M
Hidalgo-Lara
María Eugenia
ME
D040143:Cellulomonas / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism*; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D031786:Saccharum / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
D007527:Isoenzymes; D002482:Cellulose; C027433:bagasse; D043364:Endo-1,4-beta Xylanases