Purification and biochemical characterization of a novel α-amylase from Bacillus licheniformis NH1

• DOI: 10.1016/j.procbio.2008.01.017
• Publication Date: 2008-02-05T13:43:44Z
• Authors: Noomen Hmidet; Ahmed Bayoudh; Jean Guy Berrin; Safia Kanoun; Nathalie Juge; Moncef Nasri
• Keywords: Bacillus licheniformis; Ammonium sulfate precipitation; Sephadex

A thermostable a-amylase from a newly isolated Bacillus licheniformis NH1 was purified, characterized and the gene was isolated, sequenced and expressed in Escherichia coli BL21. The enzyme (BLA.NH1) was purified to homogeneity by 40‐60% ammonium sulphate precipitation, Sephadex G-100 gel filtration and Sepharose mono Q anion exchange chromatography, with a 3.08-fold increase in specific activity and 15.9% recovery. The molecular weight of the BLA.NH1 was estimated to be 58 kDa by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and gel filtration. The enzyme was highly active over a wide range of pH from 5.0 to 10.0. The relative activities at pH 5.0, 9.0 and 10.0 were about 89, 96.6 and 90%, of that at pH 6.5, respectively. The optimum temperature of the purified enzymewas 90 8C. BLA.NH1 belonged to the EDTA-sensitive a-amylase, but its activity was not stimulated by the presence of Ca 2+ ions. The purified enzyme showed extreme stability towards surfactants (SDS, Tween 20 and Triton X-100) and excellent compatibility with a wide range of commercial solid and liquid detergents at 40 8C, suggesting potential application in the detergent industry. In addition, BLA.NH1 was relatively stable towards oxidizing agents, retaining 57% of its initial activity after 1 h incubation in the presence of 1% (w/v) sodium perborate. The amyN gene, which encodes the a-amylase from B. licheniformis NH1, was isolated and its DNA sequence was determined. It showed 92% homology to the sequence encoding a-amylase from B. licheniformis NCIB 8061. The sequence of the BLA.NH1 differs from that of B. licheniformis NCIB 8061 by 21 amino acids. The region, encoding the mature a-amylase was heterologously expressed in E. coli cells using the pDEST17 expression system. The recombinant (His)6-tag enzymewas purified in a single affinity chromatography step and biochemical properties of the recombinant enzyme were determined and compared to those of the native-type enzyme. Interestingly, the recombinant a-amylase showed improved thermostability compared to the native enzyme. At 85 8C, recombinant BLA.NH1 and native BLA.NH1 showed half-lives of 60 and 8 min, respectively. # 2008 Elsevier Ltd. All rights reserved.