An alkaline active maltohexaose forming ?-amylase from Bacillus halodurans LBK 34: activity and stability features of Amy 34.

HASHIM S; DELGADO O D; MARTINEZ M A; HATTI-KAUL R; MATTIASSON B

Eslovakia

Simposio; Second Symposium on the Alpha-Amylase Family (Alamy 2); 2004

The gene encoding Amy 34, a maltohexaose forming a-amylase from Bacillus halodurans LBK 34 isolated from Lake Bogoria, Kenya [1], has been cloned, sequenced and expressed in E. coli [2]. Within the amino acid sequence of the mature peptide of Amy 34, the four conserved regions and the three proposed catalytic residues within the a-amylase family could be identified while a carbohydrate binding module, (family 25) is also present at the C-terminal end of the sequence. Occurrence of five isoforms was detected from both the wild type and recombinant enzymes. The 119 kDa recombinant Amy 34 enzyme exhibits optimum activity at 60°C and pH 10.5 - 11.5.  Maltohexaose is the main initial product formed by the hydrolytic action of Amy 34 on starch, while it produces mainly maltotetraose from amylose, amylopectin and maltodextrin. Pullulan, α and β cyclodextrin are not hydrolysed by the enzyme while g-cyclodextrin is hydrolysed to produce glucose, maltose and maltotetraose. Differential Scanning Calorimetry (DSC) studies have revealed that the thermal unfolding of Amy 34 is irreversible with four transitions, as determined by curve fitting using Gaussian curves. A melting temperature, Tm of 70.8°C is obtained at pH 9.0, which increases by 5°C in presence of 100-fold molar excess of CaCl2 and decreases by 10.4°C upon heating in presence of 100-fold molar excess of metal chelator, EDTA. Aggregation is observed when the enzyme is heated in presence of 1000-fold molar excess CaCl2. The effect of calcium on the activity and thermal unfolding of Amy 34 suggests that calcium plays a role in entropic stabilisation rather than having a direct role in the catalytic activity of Amy 34.