Molecular cloning and characterization of the glucoamylase gene of Aspergillus awamori.

Nunberg, Jack H.; Meade, James H.; Cole, G; Lawyer, F C; McCabe, Peter C.; Schweickart, Vicki L.; Tal, Rony; Wittman, Vaughan; Flatgaard, Jeffrey E.; Innis, Michael A.

doi:10.1128/mcb.4.11.2306

Cited by 160 publications

(63 citation statements)

References 65 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…X I 0 0 has not been cloned and we are unable to make specific mutant forms. However, the corresponding gene from A. awamori [24], which is identical to the gene isolated from A. niger [15], is available for molecular engineering. We are therefore able to compare the NMR spectra of glucoamylases from both of these with mutant forms.…”

mentioning

confidence: 99%

NMR spectroscopy of exchangeable protons of glucoamylase and of complexes with inhibitors in the 9–15‐ppm range

Firsov¹,

Neustroev²,

Aleshin

et al. 1994

European Journal of Biochemistry

View full text Add to dashboard Cite

'H-NMR spectra have been recorded for glucoamylases I and I1 from Aspergillus awamori var. X I 0 0 and from A. niger in the 9-15-ppm region. At least 17 distinct peaks, many of them arising from single protons, are observed. These are designated A-Q, A being the furthest downfield. At least 9 of these are lost rapidly by exchange when the enzyme is placed in D,O. Peaks A, B, E and H undergo distinct shifts with pH change in the pH region 3-7. Several others undergo smaller shifts. Small differences are also seen between the enzymes from the two different sources. Binding of the pseudotetrasaccharide inhibitor acarbose leads to a 0.50-ppm downfield shift of peak B, other smaller changes, and retention of two additional protons in D,O. 8-D-Gluconolactone induces shifts in peaks E, H, and L. The slow substrate maltitol causes peak A to broaden and shift, peaks J and K to shift and a new or greatly shifted resonance to appear at 15.4 ppm. It disappears as the maltitol is hydrolyzed.Treatment with iodoacetamide or diethyl pyrocarbonate leads to disappearance of peak D at 12.3 ppm, When this peak was irradiated strong nuclear Overhauser effects (NOE) were observed at 8.01 ppm and 7.22 ppm, positions expected for the Cel and C82 protons of an uncharged imidazole ring. We identify D as arising from the Ne2 proton of His254 which is uncharged except at the lowest pH values. Other NOE and two-dimensional NOE spectra have provided additional information.Three mutant forms of the A. niger enzyme, in which tryptophan residues have been replaced by phenylalanine, have been examined. Because of shifts induced by changes in ring current and other environmental effects it is hard to make a direct identification of the resonances from the replaced indole NH protons. However, on the basis of a distinct NOE between peaks E and H we have identified these resonances as arising from the indole NH protons of Trp52 and Trpl20. Other possible assignments are considered.The NMR spectra of the glucoamylases I, which have a starch binding domain of about 104 residues at the carboxyl terminus, show four sharp resonances in the 9.7-10.6-ppm range that are not present in the glucoamylases 11, which lack this domain. These resonances no doubt represent the four indole NH ring protons from Trp543, Trp562, Trp590 and Trp615. Three of these are very sharp suggesting a high mobility of this domain.

show abstract

mentioning

confidence: 99%

NMR spectroscopy of exchangeable protons of glucoamylase and of complexes with inhibitors in the 9–15‐ppm range

Firsov¹,

Neustroev²,

Aleshin

et al. 1994

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…N-terminal amino acid sequence of GA I was ATLDSWLSNEATVARTA. This sequence showed complete homology with glucoamylase from A. awamori (Nunberg et al, 1984), A. niger (Boel et al, 1984) and A. shirousamii (Shibuya et al, 1990). However, only 11 residues were homologous with that of glucoamylase from A. oryzae (Hata et al, 1991).…”

Section: Resultsmentioning

confidence: 93%

“…Nunberg reported that raw starch adsorption domain of glucoamylase is located near the C-terminal region, and that the TS domain serves as hinge portion between the N-terminal and the C-terminal regions (Nunberg et al, 1984). α-amylases of Aspergillus sp., including Amyl I, do not have TS domain and they are unable to digest raw starch.…”

Section: Discussionmentioning

confidence: 99%

Molecular Cloning and Determination of the Nucleotide Sequence of Raw Starch Digesting α-Amylase from Aspergillus awamori KT-11

Matsubara¹,

Ammar²,

Anindyawati³

et al. 2004

BMB Reports

View full text Add to dashboard Cite

Complementary DNAs encoding α-amylases (Amyl I, Amyl III) and glucoamylase (GA I) were cloned from Aspergillus awamori KT-11 and their nucleotide sequences were determined. The sequence of Amyl III that was a raw starch digesting α-amylase was found to consist of a 1,902 bp open reading frame encoding 634 amino acids. The signal peptide of the enzyme was composed of 21 amino acids. On the other hand, the sequence of Amyl I, which cannot act on raw starch, consisted of a 1,500 bp ORF encoding 499 amino acids. The signal peptide of the enzyme was composed of 21 amino acids. The sequence of GA I consisted of a 1,920 bp ORF that encoded 639 amino acids. The signal peptide was composed of 24 amino acids. The amino acid sequence of Amyl III from the N-terminus to the amino acid number 499 showed 63.3% homology with Amyl I. However, the amino acid sequence from the amino acid number 501 to C-terminus, including the rawstarch-affinity site and the TS region rich in threonine and serine, showed 66.9% homology with GA I.

show abstract

“…M. pusillus and Rhizopus belong to the same class, the Zygomycetes, but the MPR gene lacks an intron in contrast to the glucoamylase gene of the latter. The pattern of codon usage in the MPR gene is similar to those in several filamentous fungi such as Aspergillus (25,28), Trichoderma (27) and Neurospora (23), but different from that in S. cerevisiae (17) especially with respect to the codons for Arg, Cys, Gln, Leu and Pro. Correlation of the gene structures with phylogenic groups will be a future problem in the taxonomy of filamentous fungi and yeasts.…”

Section: Nucleic Acids Researchmentioning

confidence: 82%

“…although the genes of the extracellular glucoamylase from Aspergillus and Rhizopus fungi (25,26) and the extracellular cellulase from Trichoderma reesei (27) have been found to possess intron sequences. M. pusillus and Rhizopus belong to the same class, the Zygomycetes, but the MPR gene lacks an intron in contrast to the glucoamylase gene of the latter.…”

Section: Nucleic Acids Researchmentioning

confidence: 99%

Cloning and sequencing of a gene for Mucor rennin, an aspartate protease fromMucor pusillus

et al. 1986

View full text Add to dashboard Cite

Molecular cloning and characterization of the glucoamylase gene of Aspergillus awamori.

Cited by 160 publications

References 65 publications

NMR spectroscopy of exchangeable protons of glucoamylase and of complexes with inhibitors in the 9–15‐ppm range

NMR spectroscopy of exchangeable protons of glucoamylase and of complexes with inhibitors in the 9–15‐ppm range

Molecular Cloning and Determination of the Nucleotide Sequence of Raw Starch Digesting α-Amylase from Aspergillus awamori KT-11

Cloning and sequencing of a gene for Mucor rennin, an aspartate protease fromMucor pusillus

Contact Info

Product

Resources

About