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.

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 Vmaxand 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.

The objective of the present study was the isolation of a yeast strain, from citrus fruit peels, able to produce a polygalacturonase by submerged fermentation with maceration activity of raw cassava roots. Among 160 yeast strains isolated from citrus peels, one strain exhibited the strongest pectinolytic activity. This yeast was identified as Wickerhamomyces anomalus by 5.8S-ITS RFLP analysis and confirmed by amplification of the nucleotide sequence. The yeast produced a polygalacturonase (PG) in Erlenmeyer shake flasks containing YNB, glucose, and citrus pectin. PG synthesis occurred during exponential growth phase, reaching 51 UE.mL-1 after 8 hours of fermentation. A growth yield (Yx/s) of 0.43 gram of cell dry weight per gram of glucose consumed was obtained, and a maximal specific growth rate (µm) of 0.346 h-1 was calculated. The microorganism was unable to assimilate sucrose, galacturonic acid, polygalacturonic acid, or citrus pectin, but it required glucose as carbon and energy source and polygalacturonic acid or citrus pectin as inducers of enzyme synthesis. The crude enzymatic extract of Wickerhamomyces anomalus showed macerating activity of raw cassava. This property is very important in the production of dehydrated mashed cassava, a product of regional interest in the province of Misiones, Argentina.