Purification and specificity of recombinant Hormoconis resinae glucoamylase P and endogenous glucoamylase from Trichoderma reesei.
Hormoconis resinae glucoamylase P of high debranching activity was purified from a recombinant Trichoderma reesei strain. Four different purified fractions were obtained. Three had the same amino terminal sequence as the wild-type enzyme and about the same specific activity, and yielded the same single band on SDS-PAGE after deglycosylation. Presumably they resulted from different glycosylation patterns of the recombinant glucoamylase P. One fraction had a much lower specific activity and yielded tryptic peptides that identified it as the host cellobiohydrolase I contaminated with glucoamylase P. The different glycosylation patterns of recombinant glucoamylase P had only minor effects on its thermal inactivation. During purification of the recombinant glucoamylase, a protein with lower debranching activity was found and purified by chromatofocusing to homogeneity as assessed by SDS-PAGE. It had a pI of about 4.0 and a ratio of pullulan- to starch-degrading activity of 15%. Its amino terminal sequence showed 60% identity to the amino terminal sequence of glucoamylases P and S from Hormoconis resinae. Presumably this enzyme is the endogenous glucoamylase of Trichoderma reesei.
Fagerström
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Research Laboratories, Alko Ltd., Helsinki, Finland.
D000595:Amino Acid Sequence; D002852:Chromatography, Ion Exchange / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D003904:Mitosporic Fungi / Q000201:enzymology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014242:Trichoderma / Q000201:enzymology*; D014357:Trypsin
D005656:Fungal Proteins; D007527:Isoenzymes; D010446:Peptide Fragments; D011994:Recombinant Proteins; C086694:GamP protein, Amorphotheca resinae; D005087:Glucan 1,4-alpha-Glucosidase; D014357:Trypsin