An investigation on acarbose inhibition and the number of active sites in an amylopullulanase (L14-APU) from an Iranian Bacillus sp.

Abstract

An amylopullulanase (L14-APU) from an Iranian thermophilic bacterium was purified and the effect of acarbose, as a general inhibitor of α-amylases, on pullulan and starch hydrolysis catalyzed by L14-APU was investigated. The inhibition is a competitive type whereas inhibition constants for pullulan and starch are 99 µM and 72 µM, respectively. Investigation of the reaction rate in a system contains competitive substrates and the inhibition type of acarbose in presence of different substrates suggests that L14-APU possesses only one active site for two activities. The analysis of metal ions and other reagents effects has shown that Ca 2+ , Mg 2+ , Mn 2+ and Co 2+ enhanced both activities of the enzyme while N-bromosuccinimide treatment leads to the complete inactivation of the enzyme. The enzyme activity increased in the presence of low concentration of SDS as a surfactant.

Cohnella amylopullulanases: Biochemical characterization of two recombinant thermophilic enzymes

“…hydrothermalis did not affect by Na + and Mg 2+ while Mn 2+ increase its activity. Metal cations inhibitory effect such as Ni 2+ and Cu 2+ has been seen for almost all amylopullulanases [ 6 , 18 , 28 , 31 , 36 , 37 ].…”

“…Furthermore starch bioprocessing at higher temperature improves starch solubility, restricts microbial contamination, reduces its viscosity, decreases reaction times and more economical [ 5 ]. Therefore it seems necessity to discover suitable thermoactive and thermostable enzyme which improves saccharification rate and the other process yield [ 6 ]. Amylopullulanases (EC 3.2.1.41) are classified into two groups: Type I that cleaves α-(1→6) linkages in pullulan and branched oligosaccharides such as amylopectin, producing maltotriose and unbranched oligosaccharides, and Type II pullulanase or amylopullulanase that has both amylase and pullulanase activity [ 7 , 8 ].…”