| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2005: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 2004: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
In organisms, cadmium is bound to specific protein, metallothioneins (MTs), which are cystein-rich, low-molecular-weight, metal-binding proteins. If MTs can be specifically hydrolyzed by a protease, we can easily remove cadmium from scallop hepatopancreas ; a marine waste. Microbial treatments have the potential to clean up marine waste by ecological processes. In addition, because a protease which can specifically hydrolyze MTs is still unknown, the enzyme is expected to have unique characteristics.
Our study found Arthrobacter nicotnovorans 23-0-11 from soil as a microbe producing a protease which can release cadmium from scallop hepatopancreas into liquid medium. The protease was purified with two types of ion-exchange chromatographies. The molecular mass of the purified protease was estimated to be 27 kDa.
The optimum pH and temperature of the enzyme was pH7.0 and 50℃, respectively. The activity was stable from pH3.0 to 9.0, and at temperature below 60℃. Sensitivity to protease inhibitors indicated that this protease belongs to serine protease family.
Partial amino acid sequences were obtained from the N-terminal and the internal parts of the protease. Degenerate oligo-nucleotide primers were designed from those sequences and PCR was carried out. The nucleotide sequence of the PCR product was used as an aid to obtain whole nucleotide sequence of the protease.
The amino acid sequence of the protease had the conserved motif of the serine proteases. Although amino acid sequences of some known proteases were retrieved from the database, their homologies were not so high.
Mass production of the protease was performed using E.coli as a host. Although the protease gene was cloned and introduced into E.coli, the protease was expressed as the insoluble and inactive form. Therefore, further considerations of the vector, host and conditions for expression are needed.
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