상세 정보

underline
Metadata Downloads : dc(xml) or Excel
Cited 0 time in scopus ci

Title 

Substrate specificity and detailed characterization of a bifunctional amylase-pullulanase enzyme from Bacillus circulans F-2 having two different active sites on one polypeptide

Authors 

Chul Ho KimYong Sung Kim

Publisher 

Federation of European Biochemical Societies

Issue Date 

1995

Citation 

European Journal of Biochemistry, vol. 227, no. 0, pp. 687-693

Keywords 

amylase-pullulanase enzymebacillus circulansdifferent active sitekineticssubstrate specificityamylasepullulanaseenzyme active siteenzyme activity

Abstract 

Bacillus circulans F-2 amylase-pullulanase enzyme (APE) displayed dual activity with respect to glycosidic bond cleavage. The enzyme was active on α-1,6 bonds in pullulan, amylopectin, and glycogen, while it showed α-1,4 activity against malto-oligosaccharides, amylose, amylopectin, and soluble starch, but not pullulan. Kinetic analysis of the purified enzyme in a system which contained both pullulan and amylose as two competing substrates was used to distinguish the dual specificity of the enzyme from the single-substrate specificity known for pullulanases and α-amylases. Enzyme activities were inhibited by some metal ions, and by metal-chelating agents with a different mode. The enzyme-inhibitory results of amylase and pullulanase with Hg2+ and Co2+ ions were different, indicating that the activation mechanisms of both enzyme activities are different. Cyclomaltoheptaose inhibited both α-amylase and pullulanase activities with inhibition constants (K(i)) of 0.029 and 0.06 mg/ml, respectively. Modification with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide confirmed a carboxy group at the active sites of both enzymes. The N-terminal sequence of the enzyme was: Ala-Asp-Ala-Lys-Lys-Thr-Pro-Gln-Gln-Gln-Phe-Asp-Ala-Leu-Trp-Ala-Ala-Gl y-ILe-Val-Thr-Gly-Thr-Pro-Asp-Gly-Phe. The purified enzyme displayed Michaelis constant (K(m)) values of 0.55 mg/ml for amylose, and 0.71 mg/ml for pullulan. When both amylose and pullulan were simultaneously present, the observed rate of product formation closely fitted a kinetic model in which the two substrates are hydrolyzed at different active sites. These results suggest that amylopullulanases, which possess both α-1,6 and α-1,4 cleavage activities at the same active site, should be distinguished from APEs, which contain both activities at different active sites on the same polypeptide. Also, it is proposed that the Enzyme Commission use the term 'amylase-pullulanase enzyme' to refer to enzymes which act on starch and cleave both α-1,6-bonds in pullulan and α-1,4 bonds in amylose at different active sites.

ISSN 

0014-2956

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2017-04-19


There are no files associated with this item.
qrcode

FusionCharts.
DSpace Software Coptright(c) 2010 MIT and Hewleft-Packard  /  KRIBB-REPOSITORY ( Email:jakim@kribb.re.kr)