Expression of Sulfolobus solfataricus alpha-glucosidase in Lactococcus lactis.
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.
Giuliano
M
M
Department of Experimental Medicine, Second University of Naples, Via Luigi De Crecchio 7, Biotechnology and Molecular Biology Section, Medical School, 80138 Naples, Italy.
Schiraldi
C
C
Marotta
M R
MR
Hugenholtz
J
J
De Rosa
M
M
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
D017077:Culture Media, Conditioned; D011994:Recombinant Proteins; D000520:alpha-Glucosidases