A New Method for Microdetermination of Amylase Activity by the Use of Amylose as the Substrate

Fuwa, Hidetsugu

doi:10.1093/oxfordjournals.jbchem.a126476

Cited by 463 publications

(211 citation statements)

References 2 publications

Supporting

Mentioning

200

Contrasting

Unclassified

Order By: Relevance

“…The pH dependence of activity was measured with 0.335% soluble starch at 37°C by the blue-value method of Fuwa [15] in 50mM buffer (pH 3-6, sodium citrate; pH 6.5-8.5, Hepes; pH 9, glycine/NaOH) containing 2 m M CaCl,. The enzyme solution was diluted with 20 mM Tris/ HC1 and 2 mM CaC1, (pH 7.5) containing 1 mg/ml bovine serum albumin (Sigma A7638) and added in small amounts to the starch solution.…”

Section: A-amylase Assays and Kinetic Measurementsmentioning

confidence: 99%

Effect of mutation of an amino acid residue near the catalytic site on the activity of Bacillus stearothermophilusα‐amylase

Takase

1993

European Journal of Biochemistry

View full text Add to dashboard Cite

Site-directed mutagenesis of a thermostable a-amylase from Bacillus stearothermophilus was performed to assess the role of amino acid residues near the catalytic site in catalysis. Asn329 is presumed to be adjacent to the proposed catalytic residue Asp331. Its mutation to Lys, which is found at the corresponding position in pullulanase, resulted in the loss of 99.7% of the activity, while the mutation to Asp or Val did not drastically reduce the activity. The mutation to Val altered the temperature/activity profile so that the activity was reduced to 25% of wild-type a-amylase at 60°C but was over twofold greater at 5°C. This effect could be ascribed to a decrease in the activation enthalpy by 32%. The mutation to Asp or Lys altered the pHactivity profile concomitant with possible changes in the ionization state of the groups introduced. These results show the feasibility of altering and possibly improving the enzyme activity by mutagenesis of residues near the catalytic groups.The present study was aimed to assess the role of residues near the catalytic site in catalysis and their potential use as the targets for site-directed mutagenesis in modifying enzyme function. We have used Bacillus stearothermophilus a-amylase for this study because of its heat stability. a-amylase catalyzes the hydrolysis of a -~-1 , 4 glucosidic linkages of starch and, based on the amino acid sequence similarity of the active site, may be classified in the a-amylase family [l]. This family also includes isoamylase and pullulanase that hydrolyze a-D-1,6 linkages of amylopectin and pullulan, respectively. The differences in specificity and action patterns of the enzymes in this family must reside in their sequences, but no amino acid residues have so far been identified as crucial for such differences. . The three-dimensional structure of B. stearothermophilus a-amylase has not yet been solved; however, the sequence similarity of the active site suggests that the active-site structure closely resembles that of Taka amylase A (Aspergillus oryzae a-amylase) whose three-dimensional coordinates are available [2]. Previous mutagenesis studies on B. stearothermophilus a-amylase [3,4] have shown that the three residues, Asp234, Glu264 and Asp331, are critical for catalysis. This result coincides with those of comparable studies on other aamylases [5 -71 and supports the proposals of these residues as catalytic residues [2, 6, 81. By inspection of the threedimensional structure of Taka amylase A, Am295 (Asn329 in B. stearothennophilus a-amylase) was found to be in close proximity (atomic center distances of 0.4-0.5 nm) to Asp297 (Asp331 in B. stearothermophilus). This Asn residue does not appear to be fully exposed to solvent and, although it is almost conserved among various a-amylases (Ser in some species), no function has been assigned to it. On the other hand, Pseudomonas amyloderamosa isoamylase [9] and Klebsiella aerogenes pullulanase [lo] have Val and Lys at the corresponding position, respectively. Considering the possible roles of the ...

show abstract

Section: A-amylase Assays and Kinetic Measurementsmentioning

confidence: 99%

Effect of mutation of an amino acid residue near the catalytic site on the activity of Bacillus stearothermophilusα‐amylase

Takase

1993

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…The enzyme preparations were incubated with 0.4% soluble starch in 10 mM sodium acetate buffer, pH 5.5 at 80°C and decrease of starch and increase of reducing sugars were measured by the 12/KI method [9] and the 3,5-dinitrosalicylic acid method [lo, 111, respectively. One unit of the enzyme was defined as the amount giving a 10% decrease in the intensity of the blue color of amylose-iodine complexes in 30 min.…”

Section: Detection Of the Transformants Containing The Amylase Genementioning

confidence: 99%

Cloning and nucleotide sequence of a heat‐stable amylase gene from an anaerobic thermophile, Dictyoglomus thermophilum

Fukusumi

Kamizono

Horinouchi

et al. 1988

European Journal of Biochemistry

View full text Add to dashboard Cite

A highly heat-stable amylase gene from an obligately anaerobic and extremely thermophilic bacterium, Dictyoglomus thermophilum, was cloned and expressed in Escherichia coli. The nucleotide sequence of the amylase gene predicts a 686-amino-acid protein of relative molecular mass 81 200, which is consistent with that determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the purified enzyme. The NH2-terminal sequence determined using the enzyme purified from E. coli cells corresponds precisely to that predicted from the nucleotide sequence, except for the absence of the NH2-terminal methionine in the mature protein. When the amylase gene was expressed in E. coli cells, the enzyme was localized in the cytoplasmic fraction; this is probably explained by the absence of the signal sequence for secretion. By using the amylase purified from the E. coli transformant, some enzymatic properties, such as optimum pH, optimum temperature, pH-stability and heat-stability, were examined. The amylase was found to be a highly liquefying-type.Dictyoglomus thermophilum is a gram-negative, obligately anaerobic and extremely thermophilic bacterium forming a characteristic cell-association structure called 'rotund bodies' and possesses DNA of an extremely low G + C content (29 mol%) [l]. This strain was found to produce more than three different amylases with an extreme heat-stability and acidic optimum pH [2] which will be potentially useful in industrial starch saccharification. We intended to clone the amylase genes and produce the amylases in Escherichia coli which is easy to cultivate. In this paper, we describe the cloning and sequencing of one (tentatively designated as amyA) of the amylase genes along with its expression in E. coli cells. A single species of the amylase purified from the E. coli transformant has enabled us to characterize the enzymatic properties. MATERIALS AND METHODS Bacterial DNA isolationPlasmid DNA from E. coli was prepared by ethidium bromide/CsCl equilibrium centrifugation. For isolation of chromosomal DNA from D. thermophilum, the bacterium was cultured at 73 "C in the anaerobic medium with the gas phase of nitrogen, as previously described [l]. Cells were collected by centifugation and lyzed with lysozyme. DNA was purified by the method of Saito and Miura [4]. Recombinant DNA studiesRestriction endonucleases, T4 DNA ligase and DNAmodifying enzymes were purchased from Takara Shuzo Co. Ltd and New England Biolabs. Transformation of E. coli cells [5], DNA recovery from agarose gel slices [6], and Southern hybridization [7j were performed according to the published procedure. E. coli transformants carrying pBR322-or pYEJOOl -derived plasmids were selected on L agar containing 50 Fg/ml of ampicillin. DNA was sequenced with [IX-~'P]~CTP by the M13-dideoxynucleotide method [8]. Detection of the transformants containing the amylase genePlasmid pYEJOOl was digested with HindIII and the larger fragment was purified by agarose gel electrophoresis. Chromosomal DNA partially digested with Hind...

show abstract

“…1.) in the medium was estimated by the method of Fuwa (12). Activity is expressed as units of amylase, one unit being the amount of amylase in mg of amylose required for 10 reduction of the blue value in 10 minutes at 30 C, as defined by Fuwa.…”

Section: Incubation Of Zymogen Granulesmentioning

confidence: 99%

ROLE OF Ca2+ IN THE SECRETION OF AMYLASE FROM THE PAROTID GLAND

1971

View full text Add to dashboard Cite

A New Method for Microdetermination of Amylase Activity by the Use of Amylose as the Substrate

Cited by 463 publications

References 2 publications

Effect of mutation of an amino acid residue near the catalytic site on the activity of Bacillus stearothermophilusα‐amylase

Effect of mutation of an amino acid residue near the catalytic site on the activity of Bacillus stearothermophilusα‐amylase

Cloning and nucleotide sequence of a heat‐stable amylase gene from an anaerobic thermophile, Dictyoglomus thermophilum

ROLE OF Ca2+ IN THE SECRETION OF AMYLASE FROM THE PAROTID GLAND

Contact Info

Product

Resources

About