Abstract
The primary structure of glucoamylase G1 (EC 3.2.1.3) from Aspergillus niger has been determined. Fragments of G1 were obtained by cleavage with either cyanogen bromide, hydroxylamine, or S. aureus V8 protease. The resulting peptides were separated using ion exchange chromatography on DEAE-Sephacel, gel filtration, and affinity chromatography on Con A-Sepharose. Secondary fragments were generated by cleavage with either o-iodosobenzoic acid or BNPS-skatole as well as by digestion with S. aureus V8 protease, trypsin, and endoproteinase Lys-C. These peptides were purified by the procedures mentioned above and by reverse phase HPLC. The present fragments were amino acid sequenced and this permitted, in combination with the tryptic peptides (Carlsberg Res. Commun. 48, 517–527 (1983)), identification of 574 of the 614 amino acid residues in G1. Sequencing of glucoamylase G1 cDNA, constructed from A. niger total mRNA, enabled deduction of the sequence of the remaining 40 amino acid residues localized to 6 short stretches. From the alignment of the fragments the complete primary structure of the enzyme was established. The amino acid sequence corresponds to a molecular weight of the polypeptide moiety of 65,424. Including both hexosamine and neutral carbohydrate contents the molecular weight of the present sample of G1 was calculated to be about 82,000.
The majority of the carbohydrate of G1 is found in a highly glycosylated region of 70 amino acid residues which comprises about 35 O-glycosyl serine and threonine residues. This region ends approximately 100 residues from the C-terminus of the enzyme. Two N-glycosylated positions were found in the central part of the polypeptide chain. The molecule contains 9 half-cystine residues. No homology is apparent between the sequence of glycoamylase and various α-amylases.
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Abbreviations
- BNPS-skatole:
-
2-(2-nitrophenylsulfenyl)-3-methyl-3′-bromoindolenine
- ca:
-
citraconyl
- Con A:
-
concanavalin A
- DFP:
-
diisopropylfluorophosphate
- DPCC:
-
diphenylcarbamyl chloride
- EDTA:
-
ethylenediaminetetraacetic acid, disodium salt
- Hepes:
-
N-2-hydroxyethyl piperazine-N′-2-ethanesulfonic acid
- Nemac:
-
N-ethylmorpholine acetate
- HPLC:
-
high pressure liquid chromatography
- PTH:
-
phenythiohydantoin
- SDS-PAGE:
-
polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate
- 2-pe:
-
2-pyridylethyl
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G1 designates the larger and G2 the smaller of the forms of glucoamylase from A. niger (44)
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Svensson, B., Larsen, K., Svendsen, I. et al. The complete amino acid sequence of the glycoprotein, glucoamylase G1, from Aspergillus niger. Carlsberg Res. Commun. 48, 529–544 (1983). https://doi.org/10.1007/BF02907555
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DOI: https://doi.org/10.1007/BF02907555