RESUMEN

Alginate immobilized microalgae (AIM) was found efficient in algal cells separation and pollutants removal, however, its processing required alginate removal. In present study, polysaccharide-degrading bacterium of Saccharophagus degradans was used to biodegrade alginate and microalgae in AIM and produce polyhydroxybutyrate (PHB). Results showed that AIM cultivated in wastewater contained 34.0% carbohydrate and 45.7% protein. S. degradans effectively degraded and utilized polysaccharide of AIM to maintain five-day continuous growth at 7.1-8.8 log CFU/mL. Compared with glucose, S. degradans metabolism of mixed polysaccharide in AIM maintained the medium pH at 7.1-7.8. Increasing the inoculum concentration did not enhance AIM utilization by S. degradans due to the carbon catabolite repression of glucose which likely inactivated hydrolysis enzymes. PHB production in S. degradans peaked at 64.9 mg/L after 72 h cultivation but was later degraded to provide energy. Conclusively, S. degradans was effective in direct processing of AIM while showing potential in PHB production.

Asunto(s)

Alginatos , Microalgas , Alginatos/metabolismo , Gammaproteobacteria , Glucosa , Microalgas/metabolismo , Polisacáridos/metabolismo

RESUMEN

We investigated the structural and functional properties of SdGA, a glucoamylase (GA) from Saccharophagus degradans, a marine bacterium which degrades different complex polysaccharides at high rate. SdGA is composed mainly by a N-terminal GH15_N domain linked to a C-terminal catalytic domain (CD) found in the GH15 family of glycosylhydrolases with an overall structure similar to other bacterial GAs. The protein was expressed in Escherichia coli cells, purified and its biochemical properties were investigated. Although SdGA has a maximum activity at 39 °C and pH 6.0, it also shows high activity in a wide range, from low to mild temperatures, like cold-adapted enzymes. Furthermore, SdGA has a higher content of flexible residues and a larger CD due to various amino acid insertions compared to other thermostable GAs. We propose that this novel SdGA, is a cold-adapted enzyme that might be suitable for use in different industrial processes that require enzymes which act at low or medium temperatures.

RESUMEN

The cbm6e gene from Saccharophagus degradans 2-40 T was cloned and expressed in Escherichia coli. CBM6E contains a glycoside hydrolase family 128 (GH128) catalytic module and a C-terminal carbohydrate-binding module (CBM) grouped into CBM family 6. The purified recombinant CBM6E displayed high substrate specificity toward curdlan as an endo-ß-1,3-glucanase and had maximal activity at pH 6.0 and 35 ℃. The hydrolytic products against curdlan were predominantly laminaritriose (L3) and laminaritetraose (L4) along with a lower amount of laminaripentaose (L5) and laminarihexaose (L6). The CBM6 module selectively enhanced the enzyme activity against curdlan and displayed strict binding specificity to curdlan, no matter in its powder or high-set gel forms. This study laid a foundation for enzymatic degradation of curdlan to produce high-value ß-1,3-glucooligosaccharides at moderate temperatures and provided a novel CBM tag for enzyme immobilization on curdlan.

Asunto(s)

Celulasa/química , Gammaproteobacteria/enzimología , Glicósido Hidrolasas/química , Polisacáridos Bacterianos/química , beta-Glucanos/química , Biocatálisis , Clonación Molecular/métodos , Escherichia coli/enzimología , Escherichia coli/genética , Gammaproteobacteria/genética , Concentración de Iones de Hidrógeno , Hidrólisis , Oligosacáridos/biosíntesis , Especificidad por Sustrato , Temperatura

RESUMEN

Noncompetitive carbon sources such as algae are unconventional and promising raw material for sustainable biofuel production. The capability of one marine bacterium, Saccharophagus degradans 2-40 to degrade red seaweed Gelidium amansii for production of polyhydroxyalkanoates (PHA) was evaluated in this study. S. degradans can readily attach to algae, degrade algal carbohydrates, and utilize that material as main carbon source. Minimal media containing 8g/L G. amansii were used for the growth of S. degradans. The PHA content obtained was 17-27% of dry cell weight by pure culture of S. degradans and co-culture of S. degradans and Bacillus cereus, a contaminant found with S. degradans cultures. The PHA type was found to be poly(3-hydroxybutyrate) by gas chromatography and Fourier transform-infrared spectroscopy. This work demonstrates PHA production through consolidated bioprocessing of insoluble, untreated red algae by bacterial pure culture and co-culture.

Asunto(s)

Fermentación , Polihidroxialcanoatos/biosíntesis , Polihidroxialcanoatos/química , Rhodophyta/metabolismo , Carbono/metabolismo , Rhodophyta/crecimiento & desarrollo , Espectroscopía Infrarroja por Transformada de Fourier

RESUMEN

ß-1,6-glucan is a polysaccharide found in brown macroalgae and fungal cell walls. In this study, a ß-1,6-endoglucanase gene from Saccharophagus degradans 2-40T, gly30B, was cloned and overexpressed in Escherichia coli. Gly30B, which belongs to the glycoside hydrolase family 30 (GH30), was found to possess ß-1,6-endoglucanase activity by hydrolyzing ß-1,6-glycosidic linkages of pustulan (ß-1,6-glucan derived from fungal cell walls) and laminarin (ß-1,3-glucan with ß-1,6-branchings, derived from brown macroalgae) to produce gentiobiose and glucose as the final products. The optimal pH and temperature for Gly30B activity were found to be pH 7.0 and 40 °C, respectively. The kinetic constants of Gly30B, V max, K M, and k cat were determined to be 153.8 U/mg protein, 24.2 g/L, and 135.6 s-1 for pustulan and 32.8 U/mg protein, 100.8 g/L, and 28.9 s-1 for laminarin, respectively. To our knowledge, Gly30B is the first ß-1,6-endoglucanase characterized from bacteria. Gly30B can be used to hydrolyze ß-1,6-glucans of brown algae or fungal cell walls for producing gentiobiose as a high-value sugar and glucose as a fermentable sugar.

Asunto(s)

Celulasa/aislamiento & purificación , Celulasa/metabolismo , Gammaproteobacteria/enzimología , Glucanos/metabolismo , Polisacáridos/metabolismo , Celulasa/genética , Clonación Molecular , Disacáridos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Gammaproteobacteria/genética , Expresión Génica , Glucosa/metabolismo , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Phaeophyceae , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Temperatura

RESUMEN

The marine bacteria Saccharophagus degradans (also known as Microbulbifer degradans), are rod-shaped and gram-negative motile γ-proteobacteria, capable of both degrading a variety of complex polysaccharides and fermenting monosaccharides into ethanol. In order to obtain insights into structure-function relationships of the enzymes, involved in these biochemical processes, we characterized a S. degradans ß-glycosidase from glycoside hydrolase family 1 (SdBgl1B). SdBgl1B has the optimum pH of 6.0 and a melting temperature T m of approximately 50 °C. The enzyme has high specificity toward short D-glucose saccharides with ß-linkages with the following preferences ß-1,3 > ß-1,4 â‰« ß-1,6. The enzyme kinetic parameters, obtained using artificial substrates p-ß-NPGlu and p-ß-NPFuc and also the disaccharides cellobiose, gentiobiose and laminaribiose, revealed SdBgl1B preference for p-ß-NPGlu and laminaribiose, which indicates its affinity for glucose and also preference for ß-1,3 linkages. To better understand structural basis of the enzyme activity its 3D model was built and analysed. The 3D model fits well into the experimentally retrieved low-resolution SAXS-based envelope of the enzyme, confirming monomeric state of SdBgl1B in solution.

Asunto(s)

Gammaproteobacteria/enzimología , Glucosidasas/química , Glucosidasas/metabolismo , Sacarosa/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Gammaproteobacteria/química , Gammaproteobacteria/genética , Glucosidasas/genética , Concentración de Iones de Hidrógeno , Modelos Moleculares , Dispersión del Ángulo Pequeño , Especificidad por Sustrato , Temperatura de Transición , Difracción de Rayos X

RESUMEN

Endo-1,4-ß-xylanases are mostly classified into glycoside hydrolase (GH) family 10 or 11. In this study, we examined the catalytic functions of a recombinant endo-1,4-ß-xylanase belonging to GH10 (Xyn10C) from a marine bacterium, Saccharophagus degradans 2-40. Optimal activity of this enzyme was evident at 30 °C and pH 7.0, but activity remained even at low temperatures, indicating its adaptation to cold. With respect to other xylanases known to be active in cold temperatures, Xyn10C is unique in that it showed maximal activity in the presence of 2 M of NaCl. The action patterns of recombinant Xyn10C on xylans from hardwood and softwood differed in part, but the enzyme hydrolyzed polysaccharidic substrates primarily to xylobiose and xylotriose through xylo-oligosaccharides, releasing a small amount of xylose. The K m and V max values on birchwood xylan were 10.4 mg mL(-1) and 253 µmol mg(-1) min(-1), respectively. The efficient catalytic function of Xyn10C on short-length xylo-oligosaccharide chains was similar to the typical function of other known GH10 xylanases.

Asunto(s)

Organismos Acuáticos/enzimología , Proteínas Bacterianas/química , Glicósido Hidrolasas/química , Bacilos y Cocos Aerobios Gramnegativos/enzimología , Xilanos/química , Organismos Acuáticos/genética , Proteínas Bacterianas/genética , Glicósido Hidrolasas/genética , Bacilos y Cocos Aerobios Gramnegativos/genética , Calor , Concentración de Iones de Hidrógeno , Proteínas Recombinantes/química , Proteínas Recombinantes/genética

RESUMEN

In the Carbohydrate-Active Enzyme (CAZy) database, glycoside hydrolase family 5 (GH5) is a large family with more than 6,000 sequences. Among the 51 described GH5 subfamilies, subfamily GH5_26 contains members that display either endo-ß(1,4)-glucanase or ß(1,3;1,4)-glucanase activities. In this study, we focused on the GH5_26 enzyme fromSaccharophagus degradans(SdGluc5_26A), a marine bacterium known for its capacity to degrade a wide diversity of complex polysaccharides.SdGluc5_26A displays lichenase activity toward ß(1,3;1,4)-glucans with a side cellobiohydrolase activity toward ß(1,4)-glucans. The three-dimensional structure ofSdGluc5_26A adopts a stable trimeric quaternary structure also observable in solution. The N-terminal region ofSdGluc5_26A protrudes into the active site of an adjacent monomer. To understand whether this occupation of the active site could influence its activity, we conducted a comprehensive enzymatic characterization ofSdGluc5_26A and of a mutant truncated at the N terminus. Ligand complex structures and kinetic analyses reveal that the N terminus governs the substrate specificity ofSdGluc5_26A. Its deletion opens the enzyme cleft at the -3 subsite and turns the enzyme into an endo-ß(1,4)-glucanase. This study demonstrates that experimental approaches can reveal structure-function relationships out of reach of current bioinformatic predictions.

Asunto(s)

Proteínas Bacterianas/química , Celulosa 1,4-beta-Celobiosidasa/química , Gammaproteobacteria/química , Glicósido Hidrolasas/química , beta-Glucanos/química , Secuencia de Aminoácidos , Organismos Acuáticos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biocatálisis , Dominio Catalítico , Celulosa 1,4-beta-Celobiosidasa/genética , Celulosa 1,4-beta-Celobiosidasa/metabolismo , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Gammaproteobacteria/enzimología , Expresión Génica , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Hidrólisis , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Especificidad por Sustrato , beta-Glucanos/metabolismo

RESUMEN

Quantitative proteomic analysis was conducted to assess the assimilation processes of Saccharophagus degradans cultured with glucose, pectin, and alginate as carbon sources. A liquid chromatography-tandem mass spectrometry approach was used, employing our unique, long monolithic silica capillary column. In an attempt to select candidate proteins that correlated to alginate assimilation, the production of 23 alginate-specific proteins was identified by statistical analyses of the quantitative proteomic data. Based on the analysis, we propose that S. degradans has an alginate-specific gene cluster for efficient alginate utilization. The alginate-specific proteins of S. degradans were comprised of alginate lyases, enzymes related to carbohydrate metabolism, membrane transporters, and transcription factors. Among them, the short-chain dehydrogenase/reductase Sde_3281 annotated in the alginate-specific cluster showed 4-deoxy-L-erythro-5-hexoseulose uronic acid reductase (DehR) activity. Furthermore, we found two different genes (Sde_3280 and Sde_0939) encoding 2-keto-3-deoxy-D-gluconic acid (KDG) kinases (KdgK) that metabolize the KDG derived from alginate and pectin in S. degradans. S. degradans used Sde_3280 to phosphorylate the KDG derived from alginate and Sde_0939 to phosphorylate the KDG derived from pectin. The distinct selection of KdgKs provides an important clue toward the elucidation of how S. degradans recognizes and processes polysaccharides.

Asunto(s)

Alginatos/metabolismo , Organismos Acuáticos/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Gammaproteobacteria/metabolismo , Proteoma/metabolismo , Ácido Glucurónico/metabolismo , Ácidos Hexurónicos/metabolismo , Transducción de Señal/fisiología