RESUMO
OBJECTIVE: To characterize L-rhamnose isomerase (L-RI) from the thermophilic bacterium Clostridium stercorarium and apply it to produce D-allose from D-allulose. RESULTS: A recombinant L-RI from C. stercorarium exhibited the highest specific activity and catalytic efficiency (k cat/K m) for L-rhamnose among the reported L-RIs. The L-RI was applied to the high-level production of D-allose from D-allulose. The isomerization activity for D-allulose was maximal at pH 7, 75 °C, and 1 mM Mn2+ over 10 min reaction time. The half-lives of the L-RI at 65, 70, 75, and 80 °C were 22.8, 9.5, 1.9, and 0.2 h, respectively. To ensure full stability during 2.5 h incubation, the optimal temperature was set at 70 °C. Under the optimized conditions of pH 7, 70 °C, 1 mM Mn2+, 27 U L-RI l-1, and 600 g D-allulose l-1, L-RI from C. stercorarium produced 199 g D-allose l-1 without by-products over 2.5 h, with a conversion yield of 33% and a productivity of 79.6 g l-1 h-1. CONCLUSION: To the best of our knowledge, this is the highest concentration and productivity of D-allose reported thus far.
Assuntos
Aldose-Cetose Isomerases/metabolismo , Proteínas de Bactérias/metabolismo , Clostridium/enzimologia , Frutose/metabolismo , Glucose/metabolismo , Proteínas Recombinantes/metabolismo , Aldose-Cetose Isomerases/química , Aldose-Cetose Isomerases/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Clostridium/genética , Estabilidade Enzimática , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidade por Substrato , TemperaturaRESUMO
Clostidium stercorarium xylanase A (XynA) composed of a family 11 catalytic domain of glycosyl hydrolases and family VI CBDs bound to amorphous cellulose, i.e., acid-swollen cellulose (ASC), but not highly crystalline cellulose, and it was released from the cellulose protein complex by wash with a cellobiose solution. The Ka and [PC]max values of ASC were 0.25 liter/µmol and 26µmol/g.