Thermus aquaticus ATCC 33923 amylomaltase gene cloning and expression and enzyme characterization: production of cycloamylose

Abstract

The amylomaltase gene of the thermophilic bacterium Thermus aquaticus ATCC 33923 was cloned and sequenced. The open reading frame of this gene consisted of 1,503 nucleotides and encoded a polypeptide that was 500 amino acids long and had a calculated molecular mass of 57,221 Da. The deduced amino acid sequence of the amylomaltase exhibited a high level of homology with the amino acid sequence of potato disproportionating enzyme (D-enzyme) (41%) but a low level of homology with the amino acid sequence of the Escherichia coli amylomaltase (19%). The amylomaltase gene was overexpressed in E. coli, and the enzyme was purified. This enzyme exhibited maximum activity at 75 degrees C in a 10-min reaction with maltotriose and was stable at temperatures up to 85 degrees C. When the enzyme acted on amylose, it catalyzed an intramolecular transglycosylation (cyclization) reaction which produced cyclic alpha-1,4-glucan (cycloamylose), like potato D-enzyme. The yield of cycloamylose produced from synthetic amylose with an average molecular mass of 110 kDa was 84%. However, the minimum degree of polymerization (DP) of the cycloamylose produced by T. aquaticus enzyme was 22, whereas the minimum DP of the cycloamylose produced by potato D-enzyme was 17. The T. aquaticus enzyme also catalyzed intermolecular transglycosylation of maltooligosaccharides. A detailed analysis of the activity of T. aquaticus ATCC 33923 amylomaltase with maltooligosaccharides indicated that the catalytic properties of this enzyme differ from those of E. coli amylomaltase and the plant D-enzyme.

FIG. 1

SDS-PAGE analysis performed at each purification step. A 10-μg portion of protein from each purification step was analyzed. Lane 1, cell extract; lane 2, cell extract after heat treatment; lane 3, Phenyl-Toyopearl 650 M pool; lane 4, Source 15Q pool; lanes M, marker proteins (Bio-Rad).

FIG. 2

Effects of temperature (A) and pH (B) on activity (●) and stability (○) of the amylomaltase. (A) The enzyme activity was assayed at each temperature in 50 mM CH₃COONa-CH₃COOH (pH 5.5). To determine the thermal stability, the enzyme in 10 mM KH₂PO₄-Na₂HPO₄ (pH 7.0) was incubated at each temperature for 10 min and then immediately transferred to ice. The residual activity was assayed at 70°C in 50 mM CH₃COONa-CH₃COOH (pH 5.5). (B) The enzyme activity was assayed at 70°C in each buffer (50 mM). To determine the pH stability, the enzyme in each buffer (100 mM) was incubated at 70°C for 10 min. After the enzyme solution was diluted with 10 mM KH₂PO₄-Na₂HPO₄ (pH 7.0), the residual activity was assayed at 70°C in 50 mM CH₃COONa-CH₃COOH (pH 5.5). The buffers used were CH₃COONa-HCl (pH 3.0 to 5.0), CH₃COONa-CH₃COOH (pH 4.0 to 6.0), KH₂PO₄-Na₂HPO₄ (pH 5.5 to 8.0), and H₃BO₄ · KCl-NaOH (pH 8.0 to 10.0).

FIG. 3

TLC of reaction products formed from the activity of amylomaltase with maltooligosaccharides. Reaction mixtures (300 μl) containing 1% (wt/vol) substrate in 20 mM sodium acetate buffer (pH 5.5) and 5 mU of enzyme per ml (lanes 1), 10 mU of enzyme per ml (lanes 2), 50 mU of enzyme per ml (lanes 3), or no enzyme (lanes −) were incubated at 70°C for 6 h. Three microliters of each reaction mixture was analyzed by TLC. Lanes M contained standard maltooligosaccharides. G1, glucose; G6, maltohexaose.

FIG. 4

Activity of amylomaltase with amylose AS-110. Reaction were stopped at different times. The absorbance at 660 nm (■) and the reducing power (○) of the reaction mixture were determined. The reducing power when all of the amylose was broken down to glucose was defined as 100%. The amount of glucoamylase-resistant glucans (●) was determined as described in the text.

FIG. 5

HPAEC analysis of products formed from the activity of amylomaltase with amylose AS-110. (A) The glucoamylase-resistant glucans in reaction mixtures (50 μl) prepared as described in the legend to Fig. 4 were precipitated with 10 volumes of ethanol, dried in vacuo, and then redissolved in 50 μl of distilled water. Each 25-μl sample was analyzed by HPAEC by using a sodium acetate gradient. (B) To determine the DP of cycloamylose, the glucoamylase-resistant glucans obtained after 24 h and the cycloamylose standard were analyzed by HPAEC by using a sodium nitrate gradient. The DP of cycloamyloses are indicated above the peaks.