title: Expression of Sulfolobus solfataricus alpha-glucosidase in Lactococcus lactis.
abstract: The industrial potential to use extreme thermophilic microorganisms and their enzymes lies in applications in which the temperature cannot be adjusted (cooled) at will. The production of enzymes from wild-type thermophiles is very low, therefore, for industrial applications, it is necessary to use recombinant microorganisms. In this paper, the cloning of a heat-stable alpha-glucosidase from Sulfolobus solfataricus using lactic acid bacteria as expression system is reported. The extremophilic alpha-glucosidase was cloned in Lactococcus lactis and correctly folded despite being expressed at a lower temperature. The recombinant cells were assayed for enzyme residual activity at 75 degrees C in order to analyze the direct use of whole cells as biocatalysts. Maximum activity corresponded to 40 U/l in static cultures. The protein yield was further improved by optimizing fermentation and reached 600 U/l in batch mode. Microfiltration led to an even higher enzyme production of 850 U/l as a result of increased biomass. The overall production of alpha-glucosidase using the engineered L. lactis strain in microfiltration fermentation is 1,000-fold higher than obtained using the wild-type.
authors.affiliation: Department of Experimental Medicine, Second University of Naples, Via Luigi De Crecchio 7, Biotechnology and Molecular Biology Section, Medical School, 80138 Naples, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Giuliano
authors.forename: C
authors.initials: C
authors.lastname: Schiraldi
authors.forename: M R
authors.initials: MR
authors.lastname: Marotta
authors.forename: J
authors.initials: J
authors.lastname: Hugenholtz
authors.forename: M
authors.initials: M
authors.lastname: De Rosa
mesh_terms: D003001:Cloning, Molecular; D017077:Culture Media, Conditioned; D005374:Filtration; D006358:Hot Temperature; D007218:Industrial Microbiology; D017045:Lactococcus / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis; D013379:Substrate Specificity; D016944:Sulfolobus / Q000201:enzymology*; D000520:alpha-Glucosidases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D017077:Culture Media, Conditioned; D011994:Recombinant Proteins; D000520:alpha-Glucosidases