Abstract: In order to obtain a multi-copper oxidase with unusual features,a cotA gene from alkalophilic Bacillus clausii KSM-K16 was cloned and expressed in Escherichia coli. According to the CotA sequence from B. clausii KSM-K16 and the codon preference in E. coli,the gene of full-length sequence was designed,synthesized,and cloned into the expression vector of pET28a. Then the pET28a-cotAwas transformed to E. coli BL21（DE3）that was induced to express recombinant protein,which was subsequently purified and biochemically characterized. The purified recombinant CotA was about 62 kD,showed the blue green and had the characteristic absorption peak of Cu²⁺ at 609 nm. Also the recombinant CotA oxidized the substrates such as ABTS,SGZ and bilirubin. When SGZ as substrate,the optimal temperature and pH was 90℃ and 7.5,respectively. CotA maintained more than 70% of its original activity after incubating at 80℃ for 2 h. Furthermore,it was quite stable over pH ranging 4.0-11.0,more than 90% of its original activity after incubating at 37℃ for 1 h still remained. The results showed that the CotA from alkalophilic B. clausii was a typical multi-copper oxidase with the activities of laccase and bilirubin oxidase,and presented strong acidic-,alkali-,and thermo-stability.

Key words: Bacillus clausii, spore coat protein, laccase, multi-copper oxidase