Optimiranje proizvodnje poligalakturonaze iz Aspergillus kawachii, klonirane u Saccharomyces cerevisiae, u šaržnom i prihranjivanom šaržnom uzgoju

Abstract

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.Poligalakturonaze (EC 3.2.1.15) kataliziraju hidrolizu pektina i/ili pektinske kiseline i mogu se primijeniti u prehrambenoj industriji, npr. za bistrenje soka ili ekstrakciju pektina. U radu je ispitan rast rekombinantnog kvasca Saccharomyces cerevisiae, te heterologna ekspresija kisele poligalakturonaze iz Aspergillus kawachii u šaržnom i prihranjivanom šaržnom uzgoju. U sintetičkom su mediju šaržnim uzgojem određeni kinetički i stehiometrijski parametri rekombinantnog kvasca. Pritom su dobivene ove vrijednosti: koncentracija ukupne biomase od 2,2 g/L; koncentracija proteina od 10 mg/L; enzimska aktivnost od 3 U/mL i produktivnost od 0,006 U/(mL·h). U prihranjivanom su šaržnom uzgoju primijenjene razne strategije prihranjivanja galaktozom: (i) nakon što kvasac utroši glukozu za rast, jednokratnim dodatkom galaktoze postiže se produktivnost od 0,19 U/(mL·h); (ii) nakon utroška glukoze, dvaput se doda galaktoza u jednakim koncentracijama, pri čemu je produktivnost 0,21 U/(mL·h) i (iii) simultanim prihranjivanjem glukozom i galaktozom dobivena je produktivnost od 1,32 U/(mL·h). Zaključeno je da se najbolji rezultati postižu simultanim prihranjivanjem glukozom i galaktozom. Određene su i biokemijske značajke rekombinantnog enzima: molekularna masa od približno 60 kDa, izoelektrična točka od 3,7 i sposobnost enzima da hidrolizira poligalakturonsku kiselinu pri pH=2,5