Carsten Andersen

Structural and mechanistic studies of chloride induced activation of human pancreatic alpha-amylase

The mechanism of allosteric activation of α‐amylase by chloride has been studied through structural and kinetic experiments focusing on the chloride‐dependent N298S variant of human pancreatic α‐amylase (HPA) and a chloride‐independent TAKA‐amylase. Kinetic analysis of the HPA variant clearly demonstrates the pronounced activating effect of chloride ion binding on reaction rates and its effect on the pH‐dependence of catalysis. Structural alterations observed in the N298S variant upon chloride ion binding suggest that the chloride ion plays a variety of roles that serve to promote catalysis. One of these is having a strong influence on the positioning of the acid/base catalyst residue E233. Absence of chloride ion results in multiple conformations for this residue and unexpected enzymatic products. Chloride ion and N298 also appear to stabilize a helical region of polypeptide chain from which projects the flexible substrate binding loop unique to chloride‐dependent α‐amylases. This structural feature also serves to properly orient the catalytically essential residue D300. Comparative analyses show that the chloride‐independent α‐amylases compensate for the absence of bound chloride by substituting a hydrophobic core, altering the manner in which substrate interactions are made and shifting the placement of N298. These evolutionary differences presumably arise in response to alternative operating environments or the advantage gained in a particular product profile. Attempts to engineer chloride‐dependence into the chloride‐independent TAKA‐amylase point out the complexity of this system, and the fact that a multitude of factors play a role in binding chloride ion in the chloride‐dependent α‐amylases.

Development of new α-amylases for raw starch hydrolysis

This paper describes the discovery of a new 4 domain α-amylase from Anoxybacillus contaminans which very efficiently hydrolyses raw starch granules. Compared to traditional starch liquefying α-amylases, this new 4 domain α-amylase contains a starch binding domain. The presence of this starch-binding domain enables the enzyme to efficiently hydrolyse starch at a temperature below the gelatinisation temperature. At a reaction temperature of 60°C and in combination with a glucoamylase from Aspergillusniger it was possible to liquefy 99% of the starch obtaining a DX value of 95%. Furthermore, we describe how the current HFCS process can be turned into a low temperature simultaneous liquefaction and saccharification process by using this new 4 domain α-amylase in combination with a glucoamylase.