High-yield production of Saccharomycopsis fibuligera glucoamylase in Escherichia coli, refolding, and comparison of the nonglycosylated and glycosylated enzyme forms.
The truncated GLA1 gene encoding the mature form of glucoamylase from the yeast Saccharomycopsis fibuligera has been over-expressed in Escherichia coli using the IPTG inducible pET system. Over-expression has led to the accumulation of insoluble glucoamylase in inclusion bodies from which an electrophoretically homogeneous active enzyme has been prepared yielding 30 mg per litre medium. This protein represents an N-terminus Met-free, non-glycosylated product which displays the identical specific activity of 45 units/mg and reduced thermal stability when compared to glycosylated enzymes isolated from Saccharomyces cerevisiae carrying the GLA1 gene. These data suggest that S. cerevisiae glycosylation of S. fibuligera glucoamylase does not play a critical role in enzymatic activity but that it does contribute to its thermal stability.
Solovicová
A
A
Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovakia.
Gasperík
J
J
Hostinová
E
E
D001483:Base Sequence; D018360:Crystallography, X-Ray; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012442:Saccharomycopsis / Q000201:enzymology*; D013329:Structure-Activity Relationship
D017931:DNA Primers; D011994:Recombinant Proteins; D005087:Glucan 1,4-alpha-Glucosidase