The enzymatic and molecular characteristics of Saccharomycopsis alpha-amylase secreted from Saccharomyces cerevisiae.
The Saccharomycopsis fibuligera alpha-amylase (Sfamy) gene was expressed in Saccharomyces cerevisiae. The highest productivity of Sfamy was 70 mg per liter of culture broth. We purified Sfamy from the culture broth and identified the NH2 terminal primary sequence. This sequence suggests that the Sfamy gene product is synthesized as a pre-pro-precursor, and the pro-sequence is cleaved after a Lys-Arg sequence with the calpain-like endopeptidase encode by the KEX2 gene, resulting in mature Sfamy protein composed of 468 amino acids. Furthermore, the enzyme Sfamy is a glycoprotein in which one N-linked sugar chain containing mannose residues is attached to the Asn residue at the 198 position. The Km and kcat values were 1.1 x 10(-4) M and 1.4 x 10(2) sec-1, respectively, using amylose (the degree of polymerization n = 18) as a substrate. Moreover, the secondary structure, the location of the secondary elements including alpha-helix, beta-sheet, and loop, and tertiary structure were predicted theoretically on the basis of the molecular structure of Aspergillus oryzae alpha-amylase. Taka-amylase A (TAA). These results indicate that Sfamy protein is composed of main (M) and C-terminal (C) domains. The molecular structure of M domain closely resembles that of TAA, but the C domain appears to be more compact than that of TAA because of deletions at three regions forming turns and one region forming alpha-helix.
Matsui
I
I
National Chemical Laboratory for Industry, Ibaraki, Japan.
Matsui
E
E
Ishikawa
K
K
Miyairi
S
S
Honda
K
K
D000595:Amino Acid Sequence; D002461:Cell Line, Transformed; D003001:Cloning, Molecular; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D012442:Saccharomycopsis / Q000201:enzymology* / Q000235:genetics; D000516:alpha-Amylases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*
D011994:Recombinant Proteins; D000516:alpha-Amylases