RESUMEN
The genes of endoglucanases EG2 (36.2 kDa) Penicillium verruculosum and LAM (30.8 kDa) Myceliophthora thermophila were cloned in P. verruculosum recombinant strain. New enzyme preparations with highly stable activity against ß-glucan and laminarin were obtained and investigated, homogeneous enzymes EG2 (EC 3.2.1.4) and LAM (EC 3.2.1.6) being purified and characterized. For ß-glucan, the EG2 Km value was found to be 10 times higher than that for LAM; however, EG2 demonstrated greater processivity due to its higher kcat. The pH and temperature optima of EG2 and LAM activity against barley ß-glucan overlapped and were 4.3-4.9 and 61-67°C, respectively, and EG2 appeared to be more stable than LAM. Oligosaccharides with degree of polymerization 2-10 were formed by hydrolysis of ß-glucan and laminarin by the studied enzymes. The recombinant enzyme preparations were faster and more effective in decreasing the reduced viscosity of wholegrain barley extract than some commercial enzyme preparations. Thus, the new enzyme preparations seem to be rather perspective as feed additives for degradation of non-starch polysaccharides in grain animal feed.
Asunto(s)
Celulasa/metabolismo , Penicillium/enzimología , Sordariales/enzimología , Celulasa/genética , Celulasa/aislamiento & purificación , Hidrólisis , Cinética , Polisacáridos/metabolismo , Especificidad por SustratoRESUMEN
Genes of ß-mannosidase 97 kDa, GH family 2 (bMann9), ß-mannanase 48 kDa, GH family 5 (bMan2), and α-galactosidase 60 kDa, GH family 27 (aGal1) encoding galactomannan-degrading glycoside hydrolases of Myceliophthora thermophila C1 were successfully cloned, and the recombinant enzymes were purified to homogeneity and characterized. bMann9 displays only exo-mannosidase activity, the K(m) and k(cat) values are 0.4 mM and 15 sec(-1) for p-nitrophenyl-ß-D-mannopyranoside, and the optimal pH and temperature are 5.3 and 40°C, respectively. bMann2 is active towards galactomannans (GM) of various structures. The K(m) and k(cat) values are 1.3 mg/ml and 67 sec(-1) for GM carob, and the optimal pH and temperature are 5.2 and 69°C, respectively. aGal1 is active towards p-nitrophenyl-α-D-galactopyranoside (PNPG) as well as GM of various structures. The K(m) and k(cat) values are 0.08 mM and 35 sec(-1) for PNPG, and the optimal pH and temperature are 5.0 and 60°C, respectively.
Asunto(s)
Proteínas Fúngicas/metabolismo , Glicósido Hidrolasas/metabolismo , Sordariales/enzimología , Secuencia de Aminoácidos , Clonación Molecular , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Glicósido Hidrolasas/química , Glicósido Hidrolasas/genética , Concentración de Iones de Hidrógeno , Cinética , Datos de Secuencia Molecular , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Especificidad por Sustrato , TemperaturaRESUMEN
An enzyme preparation has been produced on the basis of Penicillium canescens strains with the activity of cellibiohydrolase I, II; endo-1,4-beta-gluconase of Penicillium verruculosum; and beta-glucosidase of Aspergillus niger. It was shown that for the most effective hydrolysis of aspen wood pulp the optimal ratio of cellobiohydrolase and endo- 1,4-3-gluconase in enzyme preparations was 8 : 2 (by protein). It was also established that the homologous xylanase secreted by the Penicillium canescens fungus is a required component for the enzyme complex for hydrolysis of the hemicellulose matrix of aspen wood.
Asunto(s)
Aspergillus niger/enzimología , Celulosa/metabolismo , Proteínas Fúngicas/metabolismo , Penicillium/enzimología , Populus/química , Madera/química , Aspergillus niger/genética , Celulasa/genética , Celulasa/metabolismo , Celulosa 1,4-beta-Celobiosidasa/genética , Celulosa 1,4-beta-Celobiosidasa/metabolismo , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Endo-1,4-beta Xilanasas/genética , Endo-1,4-beta Xilanasas/metabolismo , Proteínas Fúngicas/genética , Hidrólisis , Cinética , Ingeniería Metabólica , Penicillium/genética , Polisacáridos/metabolismo , beta-Glucosidasa/genética , beta-Glucosidasa/metabolismoRESUMEN
The Bxl5-gene encoding a GH3 glycoside hydrolase of Chrysosporium lucknowense C1 was successfully cloned, the homologous recombinant product was secreted, purified and characterized. Bxl5 (120 ± 5 kDa) was able to hydrolyze low molecular weight substrates and polysaccharides containing ß-glucosidic as well as ß-xylosidic residues. The K(m) and V(max)/E values were found to be 0.3mM and 88 s(-1) on p-nitrophenyl-ß-d-glucopyranoside (PNPG), and 13.5mM and 1.8s(-1) on p-nitrophenyl-ß-d-xylopyranoside (PNPX). Optimal pH and temperature for Bxl5 were 4.6 and 75°C for the PNPG hydrolysis, and 5.0-5.5 and 70°C for PNPX hydrolysis. The enzyme was quite stable when incubated at elevated temperatures up to 65°C. Bxl5 hydrolyzes polymeric ß-glucans by the exo-mechanism allowing their complete conversion to d-glucose and is effective for xylan hydrolysis in combination with endo-acting xylan-degrading enzymes. The enzyme seems to be a very promising for bioconversion purposes.
Asunto(s)
Chrysosporium/enzimología , Glicósido Hidrolasas/metabolismo , Xilanos/metabolismo , beta-Glucanos/metabolismo , Chrysosporium/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Estabilidad de Enzimas/efectos de los fármacos , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/enzimología , Glicósido Hidrolasas/antagonistas & inhibidores , Glicósido Hidrolasas/aislamiento & purificación , Hordeum/efectos de los fármacos , Hordeum/metabolismo , Concentración de Iones de Hidrógeno/efectos de los fármacos , Hidrólisis/efectos de los fármacos , Cinética , Espectrometría de Masas , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Especificidad por Sustrato/efectos de los fármacos , TemperaturaRESUMEN
Statistical modeling was applied for describing structural features of ß-(1â4)-D-galactomannans. According to the model suggested theoretical ratios of limiting degrees of locust bean, tara gum and guar gum galactomannan conversions by two ß-(1â4)-mannanases of different origin (Myceliophthora thermophila and Trichoderma reesei) were calculated. Then the enzymes were tested for enzymatic hydrolysis of three considered galactomannans. Experimentally observed results were compared with theoretically calculated ones. It was shown that T. reesei ß-mannanase attacks sequences of four and more unsubstituted mannopyranosyl residues in a row, while M. thermophila ß-mannanase is a more specific enzyme and attacks sequences of five and more mannopyranosyl residues in a row. Considered statistical model and approach allows to characterize both galactomannan structures and enzyme requirements for regions of unsubstituted mannose residues for substrate hydrolysis.