Recombinant thermostable cycloinulo-oligosaccharide fructanotransferase produced by Saccharomyces cerevisiae.
A truncated fragment of the cycloinulo-oligosaccharide fructanotransferase (CFTase) gene of Bacillus circulans MCI-2554 was fused to the prepro secretion sequence of the alpha-factor and expressed in Saccharomyces cerevisiae under the control of the 5' upstream region of the isocitrate lyase gene of Candida tropicalis (UPR-ICL). Efficiently secreted recombinant CFTase protein (yeast CFTase) was purified. Yeast CFTase consisted of three protein molecules, each of which had CFTase activity (yeast CFTase 1 [116 kDa], yeast CFTase 2 [117 kDa], and yeast CFTase 3 [116 kDa]). Yeast CFTase 2 was the major product of the expression system employed and was shown to be N glycosylated by endoglycosidase H treatment. Yeast CFTase 1 was N glycosylated but had a short truncation at its N terminus, while yeast CFTase 3 did not contain an N-glycosylated carbohydrate chain(s). Yeast CFTase 2 showed an optimum pH, an optimum temperature, and a pH stability similar to those of CFTase purified from B. circulans but exhibited a significant increase in thermostability. Production of yeast CFTase by the strain which had two copies of the CFTase gene integrated into its chromosomes reached 391 U per liter of culture at 120 h, which corresponded to 8.40 mg of protein per liter, by shake-flask cultivation.
Kanai
T
T
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Japan.
Ueki
N
N
Kawaguchi
T
T
Teranishi
Y
Y
Atomi
H
H
Tomorbaatar
C
C
Ueda
M
M
Tanaka
A
A
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004795:Enzyme Stability; D015870:Gene Expression; D005798:Genes, Bacterial; D006031:Glycosylation; D006602:Hexosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D013696:Temperature
D017931:DNA Primers; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D006602:Hexosyltransferases; C062589:cycloinulo-oligosaccharide fructanotransferase