Characterization of two forms of glucoamylase from aspergillus niger

Abstract

Aspergillus niger glucoamylases GI and GII (E.C. 3.2.1.3) were isolated from a commercial enzyme preparation by ammonium sulfate precipitation followed by DEAE-cellulose ion exchange chromatography. Both enzymes consist of a single glycosylated polypeptide chain. The molecular weights of GI and GII were determined by sedimentation equilibrium ultracentrifugation to 52,000 and 46,000, respectively, and by molecular sieving to 65,000 and 55,000. The amino acid compositions of GI and GII were very similar. Furthermore, the N-terminal amino acid sequence of the intact GI and GII as well as of their cyanogen fragments were identical, suggesting great homology in the primary structure of the two forms. In addition the digests of GI and GII produced respectively by Armillaria mellea protease, Staphylococcus aureus V8 protease, and submaxilla~ protease were analyzed by high pressure gel permeation chromatography. The elution profiles were also consistent with GI and GII having similar polypeptide chains. However, digestion with carboxypeptidase Y showed different C-terminal residues of the two forms. 1. INTRODUCTION Glucoamylase (l,4-aD -glucan glucohydrolase, E.C. 3.2.1.3) catalyzes the release of Dglucose from the non-reducing end of starch, glycogen and gluco-oligosaccharides. Although the ct-i,6-glucosidic linkages are cleaved less readily than the a-l,4-glucosidic linkages (17, 28, 38), the debranching capacity of glucoamylases is sufficient to make them important in the industrial production of glucose from starch, a Abbreviations: GI and GII denote two forms of glucoamylase.

Side chain reactivities of glucoamylase G2 from aspergillus niger evaluated by group-specific chemical modifications

“…5) was computed with an electrostatic interaction factor, to, of 0.025 calculated with the assumption that the charges were uniformly distributed on a spherical molecule of 36 A diameter (22). The reference point of zero net charge was set at the calculated isoionic point, pH 4. l, which agrees well with the value ofpH 4.2 determined experimentally by isoelectric focusing including known reference proteins (47). G2 was stable at pH 3 for at least 2 h, but had lost 25% activity after 2 h at pH l 1.…”

“…It comprises the 512 N-terminal residues of the 616 amino acid residues long G1 form and contains the N-glycosylated Asn~7~ and Asn395 as well as the highly O-glycosylated sequence Ser~j-Thr~H (5,43,45). The two forms are equally active towards soluble substrates, but only G 1 is able to degrade raw starch (47). A number of tryptophanyl residues and carboxyl groups involved in substrate binding and catalysis have been identified by chemical modification and sequencing (8,9,42,46).…”

“…Abbreviations: DEP = diethyl pyrocarbonate; EDTA = ethylene diaminotetraacetic acid; G 1, G2 = glucoamylase G I and G2, respectively (5,43,45,47); RP-HPLC = reverse phase high pressure liquid chromatography; Tris = 2-amino-2(hydroxymethyl)-1,3-propandiol.…”

The complete amino acid sequence of the glycoprotein, glucoamylase G1, from Aspergillus niger

“…The glucoamylases are produced by a wide variety of microorganisms (15,17,28,32,45,48). They frequently exist in multiple forms of which only the larger, referred to as G1, has the capacity to adsorb to and digest raw starch (33,44,49). Partial amino acid sequence analysis of GI and the smaller form G2 from A. niger suggested that the two forms have highly homologous structures, although G1 is extended with a C-terminal fragment that is lacking in G2 (44).…”

Mutational analysis of Aspergillus awamori glucoamylase selectivity to improve glucose yield

“…For oligosaccharides of two to seven glucosyl units, the interactions at subsite +1 provide the largest negative free energy of binding between substrate and enzyme. Svensson et al (1982) found that GA I from^. niger hydrolyzes raw starch granules from various sources about 10-to 30-fold faster than does GA H. The difference in the rates of degradation of raw starch stenns from a significant difference in the binding affinity of GA I and GA 11 to raw starch.…”