A cold-adapted tyrosinase with an abnormally high monophenolase/diphenolase activity ratio originating from the marine archaeon Candidatus Nitrosopumilus koreensis.

OBJECTIVES: To obtain an acidic and cold-active tyrosinase, which potentially minimizes unwanted self-oxidation of tyrosinase-catalyzed catechols, including 3,4-dihydroxyphenylalanine at elevated pH and high temperature. RESULTS: A putative psychrophilic tyrosinase (named as tyrosinase-CNK) was identified from the genome information of the marine archaeon Candidatus Nitrosopumilus koreensis. This protein contains key tyrosinase domains, such as copper-binding domains and an O2-binding motif, and phylogenetic analysis revealed that it was distinct from other known bacterial tyrosinases. Functional tyrosinase-CNK was produced by applying a co-expression strategy together with chaperone proteins in Escherichia coli with a yield of approx. 30 mg l(-1) and a purity >95 %. The purified enzyme showed optimal activity at pH 6 and 20 °C and still had 50 % activity at 0 °C. Surprisingly, the enzyme exhibited an abnormally high monophenolase/diphenolase activity ratio. CONCLUSIONS: The acidic and cold-adapted tyrosinase-CNK, as a new type of tyrosinase, could expand potential applications of tyrosinases including the production of catechols through minimizing unwanted self-oxidation and the modification of existing materials at low temperature.

Recombinant production of a shell matrix protein in Escherichia coli and its application to the biomimetic synthesis of spherulitic calcite crystals.

OBJECTIVES: To overcome the limited production capability of shell matrix proteins and efficiently conduct in vitro CaCO3 biomineralization studies, a putative recombinant shell matrix protein was prepared and characterized. RESULTS: A glycine-rich protein (GRP_BA) was found in Pinctada fucata as a putative shell matrix protein (NCBI reference sequence; BAA20465). It was genetically redesigned for the production in Escherichia coli. The recombinant protein was obtained in a 400 ml shake-flask culture at approx. 30 mg l(-1) with a purity of >95 %. It efficiently formed a complex with Ca(2+). Ca(2+)-induced agglomeration was like other calcification-related proteins. Spherulitic calcite micro-particles, 20-30 µm diam. with rosette- and sphere-like structures were synthesized in the presence of the recombinant shell protein, which could be formed by stacking and/or aggregation of calcite nanograins and the bound protein. CONCLUSIONS: Recombinant production of a shell matrix protein could overcome potential difficulties associated with the limited amount of protein available for biomineralization studies and provide opportunities to fabricate biominerals in practical aspects.