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Optimization of the Production of Polygalacturonase from Aspergillus kawachii Cloned in Saccharomyces cerevisiae in Batch and Fed-Batch Cultures

Natalia Lorena Rojas1, Gastón Ezequiel Ortiz1, Mariana Chesini1, Diego Jorge Baruque2 and Sebastián Fernando Cavalitto1*

1Research and Development Center for Industrial Fermentation (CINDEFI, UNLP; CCT-La Plata, CONICET), La Plata, Argentina
2Institute of Biology and Experimental Medicine (IByME – CONICET), Ciudad Autónoma de Buenos Aires, Argentina

Article history:
Received: September 15, 2010
Accepted: February 24, 2011

Key words:

Aspergillus kawachii, acid polygalacturonase, heterologous expression, fed-batch culture, co-substrate feeding

Summary:
                                                                                                                                                                                                                                                                                                                                                                                            Polygalacturonases (PG; EC 3.2.1.15) catalyze the hydrolysis of pectin and/or pectic acid and are useful for industrial applications such as juice clarification and pectin extraction. Growth and heterologous expression of recombinant Saccharomyces cerevisiae which expresses an acidic PG from Aspergillus kawachii has been studied in batch and fed-batch cultures. Kinetics and stoichiometric parameters of the recombinant yeast were determined in batch cultures in a synthetic medium. In these cultures, the total biomass concentration, protein concentration, and enzyme activity achieved were 2.2 g/L, 10 mg/L, and 3 U/mL, respectively, to give a productivity of 0.06 U/(mL·h). In fed-batch cultures, various strategies for galactose feeding were used: (i) after a glucose growth phase, the addition of a single pulse of galactose which gave a productivity of 0.19 U/(mL·h); (ii) after a glucose growth phase, a double pulse of galactose at the same final concentration was added, resulting in a productivity of 0.21 U/(mL·h); (iii) a simultaneous feeding of glucose and galactose, yielding a productivity of 1.32 U/(mL·h). Based on these results, the simultaneous feeding of glucose and galactose was by far the most suitable strategy for the production of this enzyme. Moreover, some biochemical characteristics of the recombinant enzyme such as a molecular mass of ~60 kDa, an isoelectric point of 3.7 and its ability to hydrolyze polygalacturonic acid at pH=2.5 were determined.


*Corresponding author:          cavali@biotec.org.ar
                                               ++54 221 483 3794
                                               ++54 221 483 3794

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