SOLUBLE EXPRESSION OF BACILLUS LICHENIFORMIS ATCC 27811 α-AMYLASE AND CHARACHTERIZATION OF PURIFIED RECOMBINANT ENZYME
A. Muazzam1,2*, B. Malik2, N. Rashid1, S. Irshad2 and M. Fatima2
1School of Biological Sciences, 2Institute of Biochemistry and Biotechnology, University of the Punjab,Quaid-e-Azam Campus, Lahore-54590, Pakistan.
*Corresponding Author’s Email:ammara.phd.sbs@pu.edu.pk
ABSTRACT
Alpha amylase (α-amylase) from Bacillus licheniformis shows great role in starch hydrolysis for industrial application due to its thermostability, long half-life and specificity. However, it is difficult to produce this enzyme in large quantities because of formation of inclusion bodies, but lowering the temperature to 18 °C leads to a soluble expression of protein. This also highlighted the efficacy of Escherichia coli as one of the best host for secretion of recombinant proteins. A further study to find an approach for a soluble expression of α-amylase was conducted here, using pET-22b(+) as an expression vector and temperature optimization. Purified monomeric enzyme displayed a specific activity of 584.61 U/mg at 37 °C and a molecular mass of 52 kDa. Purification fold of recombinant α-amylase was 2.04 times in comparison to crude amylase extract. Relative activity of enzymewas remarkably enhanced by Ca+2 but strongly impeded by Cu2+ and Na+1, highlighted the significance of Ca+2 as a cofactor for optimal amylase activity.
Key words: Hydrolysis, Soluble, Thermostable, Industrial. |
