RESUMEN
The cellulases from Glycoside Hydrolyses family 12 (GH12) play an important role in cellulose degradation and plant cell wall deconstruction being widely used in a number of bioindustrial processes. Aiming to contribute toward better comprehension of these class of the enzymes, here we describe a high-yield secretion of a endoglucanase GH12 from Aspegillus terreus (AtGH12), which was cloned and expressed in Aspergillus nidulans strain A773. The purified protein was used for complete biochemical and functional characterization. The optimal temperature and pH of the enzyme were 55°C and 5.0 respectively, which has high activity against ß-glucan and xyloglucan and also is active toward glucomannan and CMC. The enzyme retained activity up to 60°C. AtGH12 is strongly inhibited by Cu2+, Fe2+, Cd2+, Mn2+, Ca2+, Zn2+ and EDTA, whereas K+, Tween, Cs+, DMSO, Triton X-100 and Mg2+ enhanced the enzyme activity. Furthermore, SAXS data reveal that the enzyme has a globular shape and CD analysis demonstrated a prevalence of a ß-strand structure corroborating with typical ß-sheets fold commonly found for other endoglucanases from GH12 family.
Asunto(s)
Aspergillus , Celulasa , Clonación Molecular , Proteínas Fúngicas , Expresión Génica , Aspergillus/enzimología , Aspergillus/genética , Celulasa/biosíntesis , Celulasa/química , Celulasa/genética , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas RecombinantesRESUMEN
A novel GH1 ß-glucosidase (EaBgl1A) from a bacterium isolated from Antarctica soil samples was recombinantly overexpressed in Escherichia coli cells and characterized. The enzyme showed unusual pH dependence with maximum activity at neutral pH and retention of high catalytic activity in the pH range 6 to 9, indicating a catalytic machinery compatible with alkaline conditions. EaBgl1A is also a cold-adapted enzyme, exhibiting activity in the temperature range from 10 to 40°C with optimal activity at 30°C, which allows its application in industrial processes using low temperatures. Kinetic characterization revealed an enzymatic turnover (Kcat) of 6.92s(-1) (cellobiose) and 32.98s(-1) (pNPG) and a high tolerance for product inhibition, which is an extremely desirable feature for biotechnological purposes. Interestingly, the enzyme was stimulated by up to 200 mM glucose, whereas the commercial cocktails tested were found fully inhibited at this concentration. These properties indicate EaBgl1A as a promising biocatalyst for biotechnological applications where low temperatures are required.
Asunto(s)
Adaptación Biológica , Bacillaceae/enzimología , Bacillaceae/genética , Frío , beta-Glucosidasa/química , beta-Glucosidasa/genética , Carbohidratos/química , Catálisis , Clonación Molecular , Activación Enzimática , Expresión Génica , Concentración de Iones de Hidrógeno , Cinética , Análisis de Secuencia de ADN , Especificidad por Sustrato , beta-Glucosidasa/aislamiento & purificaciónRESUMEN
Efficient, low-cost enzymatic hydrolysis of lignocellulosic residues is essential for cost-effective production of bioethanol. The production of ß-glucosidase, ß-xylosidase and xylanase by Colletotrichum graminicola was optimized using Response Surface Methodology (RSM). Maximal production occurred in wheat bran. Sugarcane trash, peanut hulls and corncob enhanced ß-glucosidase, ß-xylosidase and xylanase production, respectively. Maximal levels after optimization reached 159.3 ± 12.7 U g-1, 128.1 ± 6.4 U g-1 and 378.1 ± 23.3 U g-1, respectively, but the enzymes were produced simultaneously at good levels under culture conditions optimized for each one of them. Optima of pH and temperature were 5.0 and 65 °C for the three enzymes, which maintained full activity for 72 h at 50 °C and for 120 min at 60 °C (ß-glucosidase) or 65 °C (ß-xylosidase and xylanase). Mixed with Trichoderma reesei cellulases, C. graminicola crude extract hydrolyzed raw sugarcane trash with glucose yield of 33.1% after 48 h, demonstrating good potential to compose efficient cocktails for lignocellulosic materials hydrolysis.