Use of Agro-Industrial Wastes for the Production of a Wild Yeast Enzyme with Disintegration Activity on Plant Tissues

Abstract The objective of the present study was to develop a cost-effective medium, using agro-industrial wastes for the production of a polygalacturonase by Wickerhanomyces anomalus of interest in cassava starch industries. The effect of several raw agro-industrial wastes and others nutrients on polygalacturonase production by W. anomalus, were evaluated, in a reference fermentation medium, using statistical designs, by batch culture. The ability of the cell-free supernatant to extract cassava starch was evaluated. Lemon peel was the best inducer for the production of PGase. Statistical analysis of the data showed that lemon peel, Mg+2 and PO4HK2 had significant effect on PGase production, and the others variables (yeast extract, Ca+2, Fe+2, amino acid and trace element solution) were no significant. PGase synthesis reached ~31 EUmL-1, in the OFM (glucose, lemon peel, urea, vitamins, KH2PO4 and MgSO4), after 12 h of culture, at a lab scale bioreactor. PGase of W. anomalus, was able to disintegrate cassava tuber tissue, and the starch granules contained within the cells were released into the reaction medium. Lemon peel can be used as inducer for PGase production by W. anomalus, in a low cost culture medium, appropriate for the production of the enzyme at large scale.

Cultivo por lote de Wickerhamomyces anomaìus en un biorreactor a escala laboratorio para la producción de una poligalacturonasa / Batch culture of Wickerhamomyces anomaìus in a lab scale bioreactor for poligalacturonase production

Wickerhamomyces anomalus, una levadura aislada de frutas cítricas en la provincia de Misiones, Argentina, produce una poligalacturonasa (endo-PG) con capacidad macerante de tejidos vegetales. El objetivo del presente trabajo fue determinai los parámetros cinéticos y estequiométricos del crecimiento de W. anomalus y la producción de la enzima poligalacturonasa en un medio de cultivo sintético, operado en sistema por lote, en un biorreactor a escala laboratorio. Los cultivos se realizaron en un biorreactor de 4 l que contenía 3 l de un medio sintético compuesto por glucosa, pectina de citrus, vitaminas, aminoácidos, sulfato de amonio y sales, y se incubaron con agitación y aireación, a 30 °C durante 12 h. El transcurso de proceso fermentativo se siguió por medidas de biomasa, glucosa residual, actividad poligalacturonasa y contenido de O2 y CO2 de los gases a la salida del reactor. La velocidad específica de crecimiento máxima (-im) de W. anomalus fue de 0,337 h-1 y el rendimiento de biomasa producida (Yx/s) de 0,401 gx/gs. Al finalizar el cultivo, la actividad PG en el sobrenadante fue de PG de ~ 83,7 UE/ml. La actividad específica y la productividad obtenidas fueron de ~ 1,91. 10(4) UE/gx y ~ 9.301 UE/l.h, respectivamente. El cociente respiratorio fue cercano a 1 durante el proceso fermentativo. No se formó ningún otro producto, además de biomasa y CO 2 . El cultivo por lote resultó ser una buena alternativa para la producción de PG a partir de W. anomalus, obteniéndose un extracto con elevada actividad enzimática, en un medio de cultivo sintético y de bajo costo.

Wickerhamomyces anomalus, a yeast isolated from citrus fruit peels in the province of Misiones, Argentina, produces a polygalacturonase (endo-PG) with maceration activity of vegetable tissues. The objective of the present work was to determine kinetic and stoichiometric parameters of W. anomalus growth and polygalacturonase production in a synthetic culture medium, operating in a batch-type bioreactor at laboratory scale. Cultures were performed in a bioreactor of 4 l, containing 3 l of a synthetic medium composed of glucose, citrus pectin, vitamins, amino acids, ammonium sulfate and salts, and were incubated with agitation (450 rpm) and aeration at 30 °C, during 12 h. The course of the fermentation process was followed by measuring biomass, residual glucose, polygalacturonase activity and O2 and CO2 content of outlet gases from the reactor. The maximum specific growth rate (Um) of W. anomalus was 0.337 h-1 and the biomass yield (Yx/s) was 0.40 gx/gs. At the end of the culture, PG activity in the supernatant was ~84 UE/ml. The specific activity and the productivity obtained were ~1.91 104 UE/gx and ~9,301 UE/l.h, respectively. Respiratory quotient was approximately 1.0 throughout the fermentation process. No other product different from biomass and CO2 was detected. Batch culture could be an adequate alternative for the production of polygalacturonase from W. anomalus and an extract with high enzymatic activity using a synthetic and economic culture medium could be obtained.

Purification and Characterization of a Polygalacturonase Produced by Wickerhamomyces anomalus

The aim of this work was to study the purification and physicochemical properties of an endo-polygalacturonase (PG) produced by Wickerhamomyces anomalus isolated from the citrus fruit peels. The enzyme was purified to homogeneity from the culture filtrate of W. anomalus grown on the yeast nitrogen base medium with glucose as carbon and energy source and citrus pectin as inductor. After anion-exchange chromatography and gel filtration chromatography, PG activity was eluted as a single peak, yielding 21% of the original activity. After dialysis and cation-exchange chromatography, only one fraction with PG activity was obtained, recovering 56% of initial enzyme activity and 1.3-fold increase in specific activity. The molecular weight of the enzyme was estimated as 43 kDa by the SDS-PAGE. The enzyme exhibited maximal activity at pH 4.2 and was stable over a pH range from 3.5 to 6.0 and up to 49ºC for 10 h. The Vmax and Km values with polygalacturonic acid as substrate were 0.26 mmol/L.min and 0.173 mg/mL, respectively. Cations such as Cu+2, Fe+3, Mg+2, Mn+2 and Zn+2 did not show any significant effect on PG activity but K+ and Ca+2 reduced it. The purified PG was able to macerate cassava tissues.

Aspergillus kawachii produces an inulinase in cultures with yacon (Smallanthus sonchifolius) as substrate

Background: Inulinases have been extracted and characterized from inulin-storing tissues; however, production of microbial inulinases have recently draw much attention as they offer several industrial advantages. Many microorganisms, including filamentous fungi, yeast and bacteria have been claimed as inulinase producers. These hydrolases are usually inducible and their exo-acting forms may hydrolyze fructose polymers (inulin) and oligosaccharides such as sucrose and raffinose. Fungal inulinase extracts are often produced as stable mixture of highly active fructanhydrolases. From a practical prospective, the best known inulinases to date are those produced by species of Penicillium, Aspergillus and Kluyveromyces. Results: The production of extracellular inulinase by A. kawachii in liquid cultures, using either inulin or yacon derived materials as CES as well as inulinase inducers, is reported. In addition, a partial characterization of the enzyme activity is included. Conclusions: Yacon derived products, particularly yacon juice, added to the culture medium proved to be a good CES for fungal growth as well as an inducer of enzyme synthesis. Partial characterization of the enzyme revealed that it is quite stable in a wide range of pH and temperature. In addition, characterization of the reaction products revealed that this enzyme corresponds to an exo-type. These facts are promising considering its potential application in inulin hydrolysis for the production of high fructose syrups.

Biotechnological processes for chitin recovery out of crustacean waste: a mini-review

Background: Chitin is an important natural resource. The annual worldwide production is estimated in approximately 10(10)-10(12) ton. It is produced by arthropods (insects and crustaceans), molluscs and fungi. Its main biological function is structural. Crustacean shells are the most important chitin source for commercial use due to its high content and ready availability. Chitin and its derivatives have great economical value because of their numerous applications: food, cosmetics, pharmaceuticals, textile industries, waste water treatment and agriculture. In nature, chitin is closely associated with proteins, minerals, lipid and pigments, which have to be removed. Results: Several techniques to extract chitin from different sources have been reported. The most common method for recovery of chitin from crustacean shells is the chemical procedure. It involves two mayor steps: elimination of inorganic matter (demineralization) and extraction of protein matter (deproteination) using strong acids and bases. However, these processes may cause depolymerization affecting the polymer properties such as molecular weight, viscosity and degree of acetylation. In addition, the chemical purification of chitin is hazardous, energy consuming and threatening to the environment. As an alternative to the chemical process, different biological processes have been investigated: microbiological fermentation and methodologies using enzymatic crude extracts or isolated enzymes. Conclusions: The results reported are extremely variable; however, they offer new perspectives for the production of chitin with the concomitant reduction of the environmental impact.

Extracellular enzymes of Fusarium graminearum isolates

Fusarium graminearum isolates from three different agroecological regions in Argentina were examined according to the production of different extracellular enzyme activities of potential biotechnological interest: pectinases (PGase: polygalacturonase and PMGase: polymethylgalacturonase), cellulase (CMCase: carboxymethylcellulase) and hemicellulase (xylanase). The isolates were grown in minimum salt medium supplemented with 0.25 percent glucose, 0.125 percent citric pectin and 0.125 percent oat bran as carbon sources and/or enzyme inducers. PGase activity was detected early (after two days of incubation) in all the cultures; it was found to be the highest for all the isolates. PMGase was high only for those isolates of the II region. CMCase and endoxylanase activities were particularly found at late stages (after four and seven days of incubation, respectively) and the maximum values were lower than pectinase activities.

Role of PPase-SE in Geotrichum klebahnii, a yeast-like fungus able to solubilize pectin

Protopectinases (PPases) constitute a heterogeneous group of extracellular enzymes able to release soluble pectin from insoluble protopectin in plant tissues. Geotrichum klebahnii (ATCC 42397) produces PPase-SE with endopolygalacturonase activity. PPase-SE has been used for pectin extraction and maceration of plant tissues. Here, the capacity of G. klebahnii to use different pectins as carbon and energy sources (CES) was studied, in addition to PPase-SE capacity to release pectin from lemon peel. The strain was unable to use pectin from different origins as CES. When G. klebahnii was cultivated with mixtures of different amounts of glucose and citrus pectin as CES, the biomass obtained was proportional to the initial concentration of glucose, which was completely consumed. In addition, it produced PPase-SE in a glucose-containing medium. A culture was used for the extraction of pectin from lemon peels. Pectin was enzymatically extracted simultaneously with tissue maceration, yielding 3.7 g of (dry) pectin per 100 g of (wet) lemon peel. Extracted pectin was not metabolized by the strain. It was concluded that G. klebahnii uses PPase-SE to macerate, invade and colonize plant tissues, thus releasing soluble sugars to be used as CES without metabolizing solubilized pectin.