Characterization, kinetics, and subsite mapping of Aspergillus niger glucoamylases I and II, and partial purification and characterization of a Chainia endo-xylanase

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1987
Authors
Meagher, Michael
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Chemical and Biological Engineering
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This dissertation is the account of investigations into the kinetic characterization and subsite mapping of two forms of glucoamylase from Aspergillus niger, and the partial purification and physical and kinetic characterization of an endo-xylanase from the genus Chainia;Glucoamylase I (GAI) and glucoamylase II (GAII) were initially purified from a commercial preparation by ammonium sulfate precipitation and separated from each other by anion-exchange column chromatography with a decreasing linear pH gradient. The two forms were free of transglycosylase, but exhibited microheterogeneity upon disc gel electrophoresis, sodium dodecyl sulfate electrophoresis, and isoelectric focusing;Both forms were subsite-mapped by determining their maximum rates and Michaelis constants with homologous series of malto- and isomaltooligosaccharides. The two forms had almost identical subsite maps, with subsite affinities more positive with maltooligosaccharide substrates than with isomaltooligosaccharides, and intrinsic hydrolysis rate coefficients higher with the former;Maximum rates and Michaelis constants were determined with GAI at several temperatures and pH's for the disaccharides [alpha],[beta]-trehalose, kojibiose, nigerose, maltose, and isomaltose and the trisaccharides panose and isomaltotriose, and with GAII for maltose, maltotriose, and isomaltotriose. The activation energies and standard heats of reaction for binding were less positive and more negative, respectively, for the hydrolysis of [alpha]-1,4-linked substrates than they were for the hydrolyis of substrates linked by other bonds. Variation of kinetic parameters with pH indicated that two carboxyl groups were involved in the binding of substrates;An endo-xylanase from the actinomycete genus Chainia was purified more than 1000-fold from a very crude preparation. Its molecular weight was 4800 and 9000 daltons by Sephadex G-50 and Fractogel HW-40S gel permeation column chromatography, respectively, and 6000 daltons by sodium dodecyl sulfate gel electrophoresis. If these values are sustained on further experimentation, this protein is certainly the smallest endo-xylanase and apparently the smallest enzyme yet described. The enzyme, while yet incompletely purified, has a specific activity higher than that of any other endo-xylanase.

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