RESUMEN
The gene (1488-bp) encoding a novel GH10 endo-ß-1,4-xylanase (XylM) consisting of an N-terminal catalytic GH10 domain and a C-terminal ricin-type ß-trefoil lectin domain-like (RICIN) domain was identified from Luteimicrobium xylanilyticum HY-24. The GH10 domain of XylM was 72% identical to that of Micromonospora lupini endo-ß-1,4-xylanase and the RICIN domain was 67% identical to that of Actinospica robiniae hypothetical protein. The recombinant enzyme (rXylM: 49kDa) exhibited maximum activity toward beechwood xylan at 65°C and pH 6.0, while the optimum temperature and pH of its C-terminal truncated mutant (rXylMâ³RICIN: 35kDa) were 45°C and 5.0, respectively. After pre-incubation of 1h at 60°C, rXylM retained over 80% of its initial activity, but the thermostability of rXylMâ³RICIN was sharply decreased at temperatures exceeding 40°C. The specific activity (254.1Umg-1) of rXylM toward oat spelts xylan was 3.4-fold higher than that (74.8Umg-1) of rXylMâ³RICIN when the same substrate was used. rXylM displayed superior binding capacities to lignin and insoluble polysaccharides compared to rXylMâ³RICIN. Enzymatic hydrolysis of ß-1,4-d-xylooligosaccharides (X3-X6) and birchwood xylan yielded X3 as the major product. The results suggest that the RICIN domain in XylM might play an important role in substrate-binding and biocatalysis.
Asunto(s)
Actinomycetales/enzimología , Proteínas Bacterianas/química , Endo-1,4-beta Xilanasas/química , Xilanos/química , Actinomycetales/química , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Sitios de Unión , Clonación Molecular , Endo-1,4-beta Xilanasas/genética , Endo-1,4-beta Xilanasas/aislamiento & purificación , Endo-1,4-beta Xilanasas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Cinética , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Termodinámica , Xilanos/metabolismoRESUMEN
OBJECTIVES: To evaluate the biocatalytic characteristics of a new endo-ß-1,4-D-mannan-degrading enzyme (ManP) from Paenibacillus sp. strain HY-8, a gut bacterium of the longicorn beetle Moechotypa diphysis. RESULTS: Purified ManP (32 kDa) with an N-terminal amino acid sequence of APSFAVGADFSYVPG displayed the greatest degree of biocatalytic activity toward locust bean gum (LBG) at 55 °C and pH 7.0. The enzyme degraded LBG, guar gum, ivory nut mannan, and mannooligosaccharides (M2-M5), but did not exhibit any hydrolytic activity against structurally unrelated substrates. The biocatalytic activity of ManP against LBG and guar gum was 695 and 450 U mg-1, respectively. Especially, enzymatic hydrolysis of mannobiose yielded a mixture of mannose (16.6 %) and mannobiose (83.4 %), although the degree of mannobiose degradation by ManP with was relatively limited. CONCLUSION: The present results suggest that ManP is an endo-ß-1,4-mannanase and is distinct from various other characterized endo-ß-1,4-mannanases.
Asunto(s)
Paenibacillus/enzimología , Concentración de Iones de Hidrógeno , Hidrólisis , Manosidasas/genética , Manosidasas/metabolismo , Especificidad por Sustrato , TemperaturaRESUMEN
The gene (1608-bp) encoding a GH6 endo-ß-1,4-glucanase (CelL) from the earthworm-symbiotic bacterium Cellulosimicrobium funkei HY-13 was cloned from its whole genome sequence, expressed recombinantly, and biochemically characterized. CelL (56.0 kDa) is a modular enzyme consisting of an N-terminal catalytic GH6 domain (from Val57 to Pro396), which is 71 % identical to a GH6 protein (accession no.: WP_034662937) from Cellulomonas sp. KRMCY2, together with a C-terminal CBM 2 domain (from Cys429 to Cys532). The highest catalytic activity of CelL toward carboxymethylcellulose (CMC) was observed at 50 °C and pH 5.0, and was relatively stable at a broad pH range of 4.0-10.0. The enzyme was capable of efficiently hydrolyzing the cellulosic polymers in the order of barley ß-1,3-1,4-D-glucan > CMC > lichenan > Avicel > konjac glucomannan. However, cellobiose, cellotriose, p-nitrophenyl derivatives of mono- and disaccharides, or structurally unrelated carbohydrate polymers including ß-1,3-D-glucan, ß-1,4-D-galactomannan, and ß-1,4-D-xylan were not susceptible to CelL. The enzymatic hydrolysis of cellopentaose resulted in the production of a mixture of 68.6 % cellobiose and 31.4 % cellotriose but barley ß-1,3-1,4-D-glucan was 100 % degraded to cellotriose by CelL. The enzyme strongly bound to Avicel, ivory nut mannan, and chitin but showed relatively weak binding affinity to lichenan, lignin, or poly(3-hydroxybutyrate) granules.
Asunto(s)
Celulasa/genética , Celulasa/metabolismo , Cellulomonas/enzimología , Oligoquetos/microbiología , Secuencia de Aminoácidos , Animales , Carboximetilcelulosa de Sodio/metabolismo , Celobiosa/metabolismo , Celulasa/química , Celulasa/aislamiento & purificación , Cellulomonas/genética , Quitina/metabolismo , Activación Enzimática , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Lignina/metabolismo , Mananos/metabolismo , Datos de Secuencia Molecular , Proteoglicanos , Xilanos/metabolismo , beta-Glucanos/metabolismoRESUMEN
The gene (1350-bp) encoding a modular ß-1,4-xylanase (XylU), which consists of an N-terminal catalytic GH10 domain and a C-terminal carbohydrate-binding module 2 (CBM 2), from Streptomyces mexicanus HY-14 was cloned and functionally characterized. The purified His-tagged recombinant enzyme (rXylU, 44.0 kDa) was capable of efficiently hydrolyze diverse xylosidic compounds, p-nitrophenyl-cellobioside, and p-nitrophenyl-xylopyranoside when incubated at pH 5.5 and 65°C. Especially, the specific activities (649.8 U/mg and 587.0 U/mg, respectively) of rXylU toward oat spelts xylan and beechwood xylan were relatively higher than those (<500.0 U/mg) of many other GH10 homologs toward the same substrates. The results of enzymatic degradation of birchwood xylan and xylooligosaccharides (xylotriose to xylohexaose) revealed that rXylU preferentially hydrolyzed the substrates to xylobiose (>75%) as the primary degradation product. Moreover, a small amount (4%<) of xylose was detected as the degradation product of the evaluated xylosidic substrates, indicating that rXylU was a peculiar GH10 ß-1,4-xylanase with substrate specificity, which was different from its retaining homologs. A significant reduction of the binding ability of rXylU caused by deletion of the C-terminal CBM 2 to various insoluble substrates strongly suggested that the additional domain might considerably contribute to the enzyme-substrate interaction.
Asunto(s)
Endo-1,4-beta Xilanasas/metabolismo , Streptomyces/enzimología , Xilanos/metabolismo , Secuencia de Aminoácidos , Animales , Clonación Molecular , ADN Bacteriano/química , ADN Bacteriano/genética , Endo-1,4-beta Xilanasas/química , Endo-1,4-beta Xilanasas/genética , Endo-1,4-beta Xilanasas/aislamiento & purificación , Estabilidad de Enzimas , Glucuronatos/metabolismo , Concentración de Iones de Hidrógeno , Insectos/microbiología , Datos de Secuencia Molecular , Peso Molecular , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Oligosacáridos/metabolismo , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Eliminación de Secuencia , Streptomyces/genética , Streptomyces/aislamiento & purificación , Especificidad por Sustrato , Temperatura , Xilosa/metabolismoRESUMEN
The XylH gene (1,167-bp) encoding a novel hemicellulase (41,584 Da) was identified from the genome of Microbacterium trichothecenolyticum HY-17, a gastrointestinal bacterium of Gryllotalpa orientalis. The enzyme consisted of a single catalytic domain, which is 74% identical to that of an endo-ß-1,4-xylanase (GH10) from Isoptericola variabilis 225. Unlike other endo-ß- 1,4-xylanases from invertebrate-symbiotic bacteria, rXylH was an alkali-tolerant multifunctional enzyme possessing endo-ß-1,4-xylanase activity together with ß-1,3/ß-1,4- glucanase activity, which exhibited its highest xylanolytic activity at pH 9.0 and 60°C, and was relatively stable within a broad pH range of 5.0-10.0. The susceptibilities of different xylosebased polysaccharides to the XylH were assessed to be as follows: oat spelts xylan > beechwood xylan > birchwood xylan > wheat arabinoxylan. rXylH was also able to readily cleave p-nitrophenyl (pNP) cellobioside and pNP-xylopyranoside, but did not hydrolyze other pNP-sugar derivatives, xylobiose, or hexose-based materials. Enzymatic hydrolysis of birchwood xylan resulted in the product composition of xylobiose (71.2%) and xylotriose (28.8%) as end products.
Asunto(s)
Actinomycetales/enzimología , Actinomycetales/genética , Proteínas Bacterianas/metabolismo , Endo-1,4-beta Xilanasas/metabolismo , Tracto Gastrointestinal/microbiología , Gryllidae/microbiología , Actinomycetales/clasificación , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Endo-1,4-beta Xilanasas/química , Endo-1,4-beta Xilanasas/genética , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Especificidad por Sustrato , TemperaturaRESUMEN
The gene (2304-bp) encoding a novel xylanolytic enzyme (XylK2) with a catalytic domain, which is 70% identical to that of Cellulomonas flavigena DSM 20109 GH6 ß-1,4-cellobiohydrolase, was identified from an earthworm (Eisenia fetida)-symbiotic bacterium, Cellulosimicrobium sp. strain HY-13. The enzyme consisted of an N-terminal catalytic GH6-like domain, a fibronectin type 3 (Fn3) domain, and a C-terminal carbohydrate-binding module 2 (CBM 2). XylK2ΔFn3-CBM 2 displayed high transferase activity (788.3 IU mg(-1)) toward p-nitrophenyl (PNP) cellobioside, but did not degrade xylobiose, glucose-based materials, or other PNP-sugar derivatives. Birchwood xylan was degraded by XylK2ΔFn3-CBM 2 to xylobiose (59.2%) and xylotriose (40.8%). The transglycosylation activity of the enzyme, which enabled the formation of xylobiose (33.6%) and xylotriose (66.4%) from the hydrolysis of xylotriose, indicates that it is not an inverting enzyme but a retaining enzyme. The endo-ß-1,4-xylanase activity of XylK2ΔFn3-CBM 2 increased significantly by approximately 2.0-fold in the presence of 50mM xylobiose.
Asunto(s)
Endo-1,4-beta Xilanasas/metabolismo , Bacterias Gramnegativas/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Metabolismo de los Hidratos de Carbono , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Endo-1,4-beta Xilanasas/química , Glicosilación , Bacterias Gramnegativas/clasificación , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
A fibrinolytic enzyme was found in a Gram-negative bacterium, Aeromonas sp. JH1. SDS-PAGE and fibrinzymography showed that it was a 36 kDa, monomeric protein. Of note, the enzyme was highly specific for fibrinogen molecules and the hydrolysis rate of fibrinogen subunits was highest for α, ß, and γ chains in that order. The first 15 amino acids of N-terminal sequence were X-D-A-T-G-P-G-G-N-V-X-T-G-K-Y, which was distinguishable from other fibrinolytic enzymes. The optimum pH and temperature of the enzyme were approximately 8.0 and 40°C, respectively. Therefore, these results provide a fibrinolytic enzyme with potent thrombolytic activity from the Aeromonas genus.
Asunto(s)
Aeromonas/enzimología , Proteínas Bacterianas/metabolismo , Fibrinógeno/metabolismo , Fibrinolíticos/metabolismo , Aeromonas/química , Aeromonas/genética , Aeromonas/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Estabilidad de Enzimas , Fibrinolíticos/química , Datos de Secuencia Molecular , Oligoquetos/microbiología , TemperaturaRESUMEN
The gene (1272-bp) encoding a ß-1,4-mannanase from a gut bacterium of Eisenia fetida, Cellulosimicrobium sp. strain HY-13 was cloned and expressed in Escherichia coli. The recombinant ß-1,4-mannanase (rManH) was approximately 44.0 kDa and has a catalytic GH5 domain that is 65% identical to that of the Micromonospora sp. ß-1,4-mannosidase. The enzyme exhibited the highest catalytic activity toward mannans at 50 °C and pH 6.0. rManH displayed a high specific activity of 14,711 and 8498 IU mg⻹ towards ivory nut mannan and locust bean gum, respectively; however it could not degrade the structurally unrelated polysaccharides, mannobiose, or p-nitrophenyl sugar derivatives. rManH was strongly bound to ivory nut mannan, Avicel, chitosan, and chitin but did not attach to curdlan, insoluble oat spelt xylan, lignin, or poly(3-hydroxybutyrate). The superior biocatalytic properties of rManH suggest that the enzyme can be exploited as an effective additive in the animal feed industry.