Cloning and Expression of Laccase Enzyme from B. pumilus strain GAZ23

Introduction: Laccases (benzene diol oxygen oxidoreductase: EC 1.10.3.2 are one of the multicopper oxidase family members that catalyze the oxidation of various phenolic compounds by using molecular oxygen Materials and methods Laccase gene was from strain GAZ23 amplified by cloning primers. PCR product cloned in the expression vector (pET21a) and transferred to BL21 strain of E. coli and sequence analysis were carried out. Biochemical properties were investigated using common laccase substrates, 2,2ˊ-azino-bis(3-ethylbenzothioazolin-6-sulphonicacid )ABTS). Results: 16S rRNA gene of strain GAZ23 isolated from Iran soils, showed high similarity to Bacillus pumilus (100%). The gene of the GAZ23 has an open reading frame composed of 1533 bases, which encode 510 amino acid residues. Discussion and conclusion: The laccase gene from GAZ23 shows 67% similarity with CotA from B. subtilis. The expression was performed under microaerobic condition and decreased temperature in order to obtain high amounts of soluble protein. This protein contains four histidine rich copper-binding domains.