Optimization of the production of a polygalacturonase from Aspergillus kawachii cloned in Saccharomyces cerevisiae in batch and fed-batch cultures

ROJAS NL; ORTIZ GE; CHESINI M; BARUQUE DJ; CAVALITTO SF

Curitiba

Congreso; 4th International Congress on Bioprocess in Food Industries; 2010

Polygalacturonase (PG; EC 3.2.1.15) catalyses the hydrolysis of pectin and/or pectic acid and is useful for industrial applications such as juice clarification and pectin extraction. Aspergillus kawachii produces an acidic PG, called PG1, which was cloned into S. cerevisiae. Growth of the recombinant yeast and heterologous expression in a synthetic medium was studied in batch and fed batch cultures. Kinetics and stoichiometric parameters were determined for the recombinant yeast and the following results were obtained: Specific growth rate (µmax) 0,28 h-1, yx/s = 0.173 cmol X. cmol S-1, yCO2/s = 0.252 mol CO2. cmol S-1, b = 0.082 mol O2. cmol S-1, yp/s = 0.523 c-mol P. cmol S-1 and a carbon balance of 1.05, which indicates there is no other products but ethanol generated.  In batch cultures, it was determined a maximum of PG1 production between 20 and 24 hours and the total biomass concentration, protein concentration, and enzyme activity 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, the strategies used for galactose feeding were: (a) after a glucose growth phase, the addition of a single pulse of galactose at a final concentration of 10% (w/v); (b) after a glucose growth phase, the introduction of a double pulse of galactose at the same final concentration; and (c) a simultaneous feeding of glucose and galactose, the both at a final concentration of 10% (w/v), as the CES and the inducer, respectively. With strategy (a), the final PG1 concentration and total protein content obtained were 12.2 U/ml and 67 mg/l, respectively, after 63 h of culture to give a productivity of 0.19 U/ml.h. Strategy (b), with the addition of a double pulse of galactose, gave a final PG1 concentration and a total protein content of 16 U/ml and 73.6 mg/l, respectively, after 78 h of culture, corresponding to a productivity of 0.21 U/ml.h. Strategy (c), involving simultaneous growth and induction phases, yielded a final PG1 concentration and a total protein content of 50.0 U/ml and 54 mg/l, respectively, after 38 h of culture to give a productivity of 1.32 U/ml.h. On comparison of the results from these three approaches to the level of PG1 production by the fed-batch mode, we conclude that the simultaneous feeding of glucose and galactose was by far the most suitable strategy for the generation of this enzyme. Based on these results, this last approach would appear to be the most promising one for the recombinant production of PG1 in large-scale experiments. Finally, some biochemical properties of the recombinant enzyme were determined. Since these biochemical characteristics and the stability of PG1 under extreme acid conditions (pH 2.5) are desirable for this enzyme´s utility within an industrial context, the overexpression of the pg1 gene allows obtaining sufficiently large amounts of PG1 for the appropriate industrial applications.