Subsite mapping of <i>Aspergillus niger</i> glucoamylases I and II with malto‐ and isomaltooligosaccharides

Meagher, Michael M.; Nikolov, Z̆ivko L.; Reilly, Peter J.

doi:10.1002/bit.260340512

Cited by 74 publications

(38 citation statements)

References 38 publications

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“…However, the optimal pH for binding is somewhat different: near 5.5 for acarbose and a broader optimum range around 5.5-7.0 for 1 -deoxynojirimycin. Both of these optimal pH values are significantly different from the optimal pH for maltooligodextrin hydrolysis of 4.5 (Meagher & Reilly, 1989;Sierks et al, 1990). The pH optima seem, however, to be related to the PKa values of the nitrogen moiety in each inhibitor: for acarbose pKa z 5.0 (Truscheit et al, 1981), and for 1-deoxynojirimycin pKa = 6.6 (Inouye et al, 1966(Inouye et al, ,1968.…”

Section: Resultsmentioning

confidence: 93%

Thermodynamics of Inhibitor Binding to the Catalytic Site of Glucoamylase from Aspergillus niger Determined by Displacement Titration Calorimetry

Sigurskjold¹,

Berland²,

Svensson³

1994

Biochemistry

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The binding of different inhibitors to glucoamylase G2 from Aspergillus niger and its temperature and pH dependencies have been studied by titration calorimetry. The enzyme binds the inhibitors 1-deoxynojirimycin and the pseudo-tetrasaccharide acarbose with association constants of 3 x 10(4) and 9 x 10(11) M-1, respectively, at 27 degrees C. The binding free energy for both ligands is remarkably temperature-invariant in the interval from 9 to 54 degrees C as the result of large compensating changes in enthalpy and entropy. Acarbose and 1-deoxynojirimycin bound with slightly different free energy-pH profiles, with optima at 5.5 and 5.5-7.0, respectively. Variations in delta H degrees and T delta S degrees as a function of pH were substantially larger than variations in delta G degrees in a partly compensatory manner. Two titratable groups at or near subsite 1 of the catalytic site were found to change their pKa slightly upon binding. The hydrogenated forms of acarbose, D-gluco- and L-ido-dihydroacarbose, bind with greatly reduced association constants of 3 x 10(7) and 2 x 10(5) M-1, respectively, and the pseudo-disaccharide methyl acarviosinide, lacking the two glucose units at the reducing end compared to acarbose, has a binding constant of 8 x 10(6) M-1; these values all result from losses in both enthalpy and entropy compared to acarbose. Three thio analogues of the substrate maltose, methyl alpha- and beta-4-thiomaltoside and methyl alpha-4,5'-dithiomaltoside, bind with affinities from 3 x 10(3) to 6 x 10(4) M-1.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Resultsmentioning

confidence: 93%

Thermodynamics of Inhibitor Binding to the Catalytic Site of Glucoamylase from Aspergillus niger Determined by Displacement Titration Calorimetry

Sigurskjold¹,

Berland²,

Svensson³

1994

Biochemistry

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“…[52,54,55,58] These a-helical segments form the major part of an active-site pocket that is believed to contain seven subsites that can accommodate one glycosyl residue each. [59][60][61] The hydrolytic event occurs between subsites À1 and 1 and requires the substrate to bind in a productive mode in these two terminal sites, thereby exposing the glycosidic linkage to the catalytic residues. The affinities of subsites À1 and 1 estimated from pre-steady-state association constants indicates that the initial binding of substrate is controlled by a very high affinity in subsite À1 with subsite 1 having much lower affinity.…”

Section: Wwwchemeurjorgmentioning

confidence: 99%

Chemoselective Capture of Glycans for Analysis on Gold Nanoparticles: Carbohydrate Oxime Tautomers Provide Functional Recognition by Proteins

Thygesen¹,

Sauer²,

Jensen³

2009

Chemistry A European J

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Nanoparticles functionalized with glycans are emerging as powerful solid-phase chemical tools for the study of protein-carbohydrate interactions using nanoscale properties for detection of binding events. Methods or reagents that enable the assembly of glyconanoparticles from unprotected glycans in two consecutive chemoselective steps with meaningful display of the glycan are highly desirable. Here, we describe a novel bifunctional reagent that 1) couples to glycans by oxime formation in solution, 2) aids in purification through a lipophilic trityl tag, and 3) after deprotection then couples to gold nanoparticles through a thiol. NMR studies revealed that these oximes exist as both the open-chain and N-glycosyl oxy-amine tautomers. Glycan-linker conjugates were coupled through displacement of ligands from preformed, citrate-stabilized gold nanoparticles. Recognition of these glycans by proteins was studied with a lectin, concanavalin A (ConA), in an aggregation assay and with a processing enzyme and glucoamylase (GA). We demonstrate that the presence of the N-glycosyl oxy-amines clearly enables functional recognition in sharp contrast to the corresponding reduced oxy-amines. This concept is then realized in a novel reagent, which should facilitate nanoglycobiology by enabling the operationally simple capture of glycans and their biologically meaningful display.

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“…Glucoamylases hydrolyze -1,4 and -1,6 linkages of starch and related polymers to produce glucose as the sole end product. Glucoamylases also hydrolyze other starchrelated oligo-and polysaccharides, and show a preference for maltooligosaccharides of at least six residues [22]. One of most important applications of glucoamylases is the production of high glucose syrups from starch, and these enzymes are also used in the production of ethanol and in the baking and brewing industries [35].…”

Section: Introductionmentioning

confidence: 99%

Properties of a purified thermostable glucoamylase from Aspergillus niveus

Silva

Maller

Damásio

et al. 2009

J Ind Microbiol Biotechnol

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A glucoamylase from Aspergillus niveus was produced by submerged fermentation in Khanna medium, initial pH 6.5 for 72 h, at 40 degrees C. The enzyme was purified by DEAE-Fractogel and Concanavalin A-Sepharose chromatography. The enzyme showed 11% carbohydrate content, an isoelectric point of 3.8 and a molecular mass of 77 and 76 kDa estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or Bio-Sil-Sec-400 gel filtration, respectively. The pH optimum was 5.0-5.5, and the enzyme remained stable for at least 2 h in the pH range of 4.0-9.5. The temperature optimum was 65 degrees C and retained 100% activity after 240 min at 60 degrees C. The glucoamylase remained completely active in the presence of 10% methanol and acetone. After 120 min hydrolysis of starch, glucose was the unique product formed, confirming that the enzyme was a glucoamylase (1,4-alpha-D-glucan glucohydrolase). The K(m) was calculated as 0.32 mg ml(-1). Circular dichroism spectroscopy estimated a secondary structure content of 33% alpha-helix, 17% beta-sheet and 50% random structure, which is similar to that observed in the crystal structures of glucoamylases from other Aspergillus species. The tryptic peptide sequence analysis showed similarity with glucoamylases from A. niger, A. kawachi, A. ficcum, A. terreus, A. awamori and A. shirousami. We conclude that the reported properties, such as solvent, pH and temperature stabilities, make A. niveus glucoamylase a potentially attractive enzyme for biotechnological applications.

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Subsite mapping of Aspergillus niger glucoamylases I and II with malto‐ and isomaltooligosaccharides

Cited by 74 publications

References 38 publications

Thermodynamics of Inhibitor Binding to the Catalytic Site of Glucoamylase from Aspergillus niger Determined by Displacement Titration Calorimetry

Thermodynamics of Inhibitor Binding to the Catalytic Site of Glucoamylase from Aspergillus niger Determined by Displacement Titration Calorimetry

Chemoselective Capture of Glycans for Analysis on Gold Nanoparticles: Carbohydrate Oxime Tautomers Provide Functional Recognition by Proteins

Properties of a purified thermostable glucoamylase from Aspergillus niveus

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