Propiedades y posible aplicación en la industria alimenticia de las a-Sa-L-arabinofuranosidasas del hongo penicillium purpurogenum

Tesis (Doctor en Biotecnología)La lignocelulosa es la biomasa renovable más abundante en nuestro planeta. Está compuesta por lignina, pectina, celulosa y hemicelulosas. El xilano es el principal componente de las hemicelulosas. Está formado por una cadena principal de residuos P- 1,4-D-xilopiranósidos sustituida por diferentes tipos de residuos: metil glucuronato, Larabinofuranosa y acetato. La biodegradación del xilano es un proceso complejo que necesita la participación de una serie de esterasas y glicanasas. Entre estas Últimas se encuentran las a-L-arabinofuranosidasas (E.C. 3.2.1.55) que hidrolizan residuos de arabinosa unidos a la posición 2 y/ó 3 de la xilosa. El hongo filamentoso Penicillium purpurogenum, utilizado en este trabajo como modelo de estudio, crece en diferentes fuentes de carbono natural, secretando al medio en que se desarrolla una gran variedad de enzimas xilanolíticas, incluyendo tres a-Arabinofuranosidasas, denominadas ABF 1,2 y 3. Este trabajo está enfocado en completar la caracterización de ABF2 y principalmente en el estudio de ABF3. Las dos enzimas han sido purificadas a homogeneidad. El peso molecular determinado por electroforesis en condiciones desnaturantes es de 70.000 para ABF2 y 50.700 para ABF3. Al analizar sus propiedades cinéticas, utilizando el sustrato sintético p-nitrofenil-a- arabinofuranósido (pNPAra), se observó que ambas siguen una cinética de Michaelis- Menten, con valores de KM de 0,098 mM para ABF2 y 0,65 mM para ABF3. La temperatura óptima es de 60°C para ABF2 y de 50°C para ABF3. El pH óptimo es de 5,O para las dos ABFs. ABF3 muestra también actividad frente a p-nitrofeni1-B-Dxilopiranósido (pNPXyl) con una KM de 12 mM; es por lo tanto una enzima bifuncional. La acción sobre sustratos naturales mostró que las tres ABFs (ABF1, 2 y 3) tienen diferente especificidad de sustrato; ABF 1 actúa sobre polisacáridos desramificandolos, ABF2 no actúa sobre polisacáridos y ABF3 muestra mayor preferencia por arabinoxilano y xilooligosacáridos. Estos resultados confirman la hipótesis que las ABFs cumplen distintas funciones en la biodegradación de la lignocelulosa. El gen abf 3 y su cDNA fueron secuenciados y no se encontraron intrones. La proteína madura tiene 433 aminoácidos, con un peso molecular calculado de 47.305. El análisis de la secuencia de ABF3 mostró que es una proteína modular; posee un dominio catalítico y módulo de unión a carbohidratos. De esta forma, ABF3 es la primera enzima bifuncional modular de eucariontes descrita dentro de la Familia 43 de las Glicosil Hidrolasas. Una posible aplicación de las a-L-arabinofuranosidasas ha sido estudiada en relación al aumento del aroma de los vinos. Se utilizó mosto de Moscatel de Alejandría para aislar los glicósidos a través de columnas C18 RP. La cantidad de monosacáridos (glucosa, arabinosa y xilosa) presentes en los glicósidos, fue determinada después de la hidrólisis ácida y fue comparada con la obtenida por hidrólisis enzimática. Para la hidrólisis enzimática se utilizaron las ABFs (1 y 3) y P-glucosidasa de P. purpurogenum. Las dos ABFs fueron activas frente a monoterpenil a-L-arabinofuranosilglucósidos con liberación de arabinosa. Si además se adicionaba P-glucosidasa se generaba glucosa y monoterpenos. Estos resultados son comparables a los obtenidos en experimentos similares utilizando preparaciones comerciales, lo que sugiere que las enzimas de P. purpurogenum pueden ser utilizadas en esta aplicación en particular

Heterologous expression, purification and characterization of a highly thermolabile endoxylanase from the Antarctic fungus Cladosporium sp.

© 2018 British Mycological SocietyNumerous endoxylanases from mesophilic fungi have been purified and characterized. However, endoxylanases from cold-adapted fungi, especially those from Antarctica, have been less studied. In this work, a cDNA from the Antarctic fungus Cladosporium sp. with similarity to endoxylanases from glycosyl hydrolase family 10, was cloned and expressed in Pichia pastoris. The pure recombinant enzyme (named XynA) showed optimal activity on xylan at 50 °C and pH 6–7. The enzyme releases xylooligosaccharides but not xylose, indicating that XynA is a classical endoxylanase. The enzyme was most active on xylans with high content of arabinose (rye arabinoylan and wheat arabinoxylan) than on xylans with low content of arabinose (oat spelts xylan, birchwood xylan and beechwood xylan). Finally, XynA showed a very low thermostability. After 20–30 min of incubation at 40 °C, the enzyme was completely inactivated, suggesting that XynA would be the most thermolabile endoxy

Heterologous expression and biochemical characterization of a novel cold-active α-amylase from the Antarctic bacteria Pseudoalteromonas sp. 2-3

© 2018 Elsevier Inc.α-Amylase is an endo-acting enzyme which catalyzes random hydrolysis of starch. These enzymes are used in various biotechnological processes including the textile, paper, food, biofuels, detergents and pharmaceutical industries. The use of active enzymes at low temperatures has a high potential because these enzymes would avoid the demand for heating during the process thereby reducing costs. In this work, the gene of α-amylase from Pseudoalteromonas sp. 2–3 (Antarctic bacteria) has been sequenced and expressed in Escherichia coli BL21(DE3). The ORF of the α-amylase gene cloned into pET22b(+) is 1824 bp long and codes for a protein of 607 amino acid residues including a His6-tag. The mature protein has a calculated molecular mass of 68.8 kDa. Recombinant α-amylase was purified with Ni-NTA affinity chromatography. The purified enzyme is active on potato starch with a Km of 6.94 mg/ml and Vmax of 0.27 mg/ml*min. The pH optimum is 8.0 and the optimal temperature is 2

Cinnamic acid, ethanol and temperature interaction on coumarate decarboxylase activity and the relative expression of the putative cd gene in D. bruxellensis

Dekkera bruxellensis is one of the main contaminating yeasts in wine due to its ability to metabolize cinnamic acids into volatile phenols. This yeast metabolizes p-coumaric acid into 4-vinylphenol through a coumarate decarboxylase (CD) and then transforms it into to 4-ethylphenol (EF) through a vinylphenol reductase. In this work we investigated the influence of the interaction between the concentration of p-coumaric acid, ferulic acid and ethanol as well as growth temperature on the production of CD activity and the expression of a putative gene that codes for this enzymatic activity. For this, a Box Behnken experimental design was used. The concentration of p-coumaric acid (5-26 ppm) and ferulic acid (3-9 ppm) alone did not show any significant effect on any of the studied response variables. However, the interaction between (ethanol concentration * cinnamic acid concentration) and (ethanol concentration * temperature) had a significant statistical effect on the production of CD activity. Additionally, a higher growth temperature negatively affected the expression of the putative cd gene and the production of CD activity. This is the first work that studies the effect of cinnamic acids on the production of CD activity and the relative expression of its putative gene, using natural concentrations of cinnamic acid found in wine

The role of alginate lyases in the enzymatic saccharification of brown macroalgae, Macrocystis pyrifera and Saccharina latissima

In this work, we have compared the carbohydrate content and the enzymatic saccharification of the brown algae Macrocystis pyrifera from Chile and Saccharina latissima from Norway. M. pyrifera contained 40% mannitol, 31% uronic acids and 15% glucose, while S. latissima contained 37% glucose, 30% uronic acids and 25% mannitol. Thus, the ratio between mannitol and glucose was much higher for M. pyrifera. Acid pre-treated and untreated algae were enzymatically saccharified in two steps; first at pH 7.5, 25 degrees C for 12 h with a blend of recombinant alginate and oligoalginate lyases, then the pH was changed to 5.2, a commercial cellulase cocktail was added and saccharification continued at 50 degrees C for 4 h. These experiments showed that the use of recombinant alginate lyases and oligoalginate lyases in combination with cellulases increased the release of glucose from untreated seaweed. However, for saccharification of pretreated algae, only cellulases were needed to achieve high glucose yields. Finally, it was shown that brown algae hydrolysates could be used as a growth medium to produce microbial ingredients, such as Candida utilis yeast.CONICYT REDES150014 Algal Biorefinery Network: Foods of Norway-ICDB of Chile Centre for Biotechnology and Bioengineering (CeBiB) FB-0001 Foods of Norway Centre for Research-based Innovation (Research Council of Norway) 237841/030 Research Council of Norway 23900

Cold-active xylanase produced by fungi associated with Antarctic marine sponges

Despite their potential biotechnological applications, cold-active xylanolytic enzymes have been poorly studied. In this work, 38 fungi isolated from marine sponges collected in King George Island, Antarctica, were screened as new sources of cold-active xylanases. All of them showed xylanase activity at 15 and 23 C in semiquantitative plate assays. One of these isolates, Cladosporium sp.; showed the highest activity and was characterized in detail. Cladosporium sp. showed higher xylanolytic activity when grown on beechwood or birchwood xylan and wheat bran, but wheat straw and oat bran were not so good inducers of this activity. The optimal pH for xylanase activity was 6.0, although pH stability was slightly wider (pH 5-7). On the other hand, Cladosporium sp. showed high xylanase activity at low temperatures and very low thermal stability. Interestingly, thermal stability was even lower after culture media were removed and replaced by buffer, suggesting that low molecular component(s

Pectin: An overview of sources, extraction and applications in food products, biomedical, pharmaceutical and environmental issues

Pectin is a complex versatile heteropolysaccharide of great importance to food, pharmaceutical and cosmetic industries. It is widely used in the food industry due to its thickening, gelling and emulsification properties and in biomedical and biomaterial applications on account of its potential anti-inflammatory and immunomodulatory effects as well as its biodegradability and biocompatibility properties. Pectin is also a soluble dietary fiber with several beneficial gastrointestinal and physiological effects. The multifunctionality of pectin is related to the nature of its molecule that has diverse chemical structures, physicochemical properties and potential functionalities depending on the sources where it is extracted and on the extraction methods. Therefore, this review focuses on the importance of pectin for today's food, pharmaceutical and cosmetic industries, compiling information on its composition and properties as determined by its origins, especially from waste biomass of the fruits and vegetables processing industry, on commercial applications and research needs. The suitability of the different extraction methods was also discussed, considering cost, energy consumption and productivity. Furthermore, the biodegradation of pectin as a complex process performed by a set of enzymes was also reviewed along with application purposes. Finally, future perspectives reveal pectin to be an astounding functional food ingredient requiring continuous research work