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Minerva Biotecnologica 2014 December;26(3):295-300


lingua: Inglese

Cloning, expression and purification of laccase enzyme gene from Bacillus subtilis in Escherichia coli

Ghasemi Y. 1, 2, Yarahmadi E. 2, 3, Ghoshoon M. B. 1, 2, Dabbagh F. 1, 2, Hajighahramani N. 2, 3, Ebrahimi N. 1, 2, Mobasher M. A. 1, 2, Montazeri Najafabady N. 1, 2

1 Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran; 2 Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran; 3 Student research committee, International branch, Shiraz University of Medical Sciences, Shiraz, Iran


AIM: Laccases (benzendiol: oxygen oxidoreductases) being the most numerous members of blue multicopper oxidases are widely distributed in nature among some of plants, insects, fungi and bacteria. Bacterial laccases are very stable at high temperature and high pH values. Laccase biotechnological and industrial applications include: biocatalyst in synthetic chemistry for synthesis of polymer, anticancer drugs, novel penicillins and cephalosporins; biosensor in nanobiotechnology for drug, biomedical, environmental and industrial analysis; biofuel cells, bioremediation, biobleaching; food, petrochemical, textile, paper, wood, dye, cosmetic industries; breast cancer and hepatoma treatment, AIDS treatment and antifungal effect.
METHODS: Bacillus subtilis (B. subtilis) PTCC1023 was grown in LB medium at 37°C and its genomic DNA for amplification and cloning was extracted in logarithmic phase and PCR reaction was carried out using specific primers to amplify the gene of 1542 bp encoding laccase (CotA). The resulting PCR amplicon was transformed in expression host Escherichia coli BL21 (DE3) after ligation into suitable vector. The recombinant protein was purified under denaturing and native conditions and analyzed with SDS-PAGE method and enzyme activity was assayed using specific laccase substrate (syringaldazine).
RESULTS: The DNA sequence alignment resulting from the BLAST search of laccase, showed maximum 99% sequence identity with some strains of B. subtilis, whereas other bacteria identity between 87-99%. Laccase cloned gene is different at least in 3 nucleotides with other recorded sequences, which results in 2 amino acids differences. Being active of produced recombinant enzyme was proved using syringaldazine oxidation.
CONCLUSION: We cloned, expressed, and purified a new laccase from B. subtilis that it would be a significant step for applying the recombinant enzyme in biotechnological and industrial processes.

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