Development of cadmium releasing technology from marine waste using the metallothionein proteolytic enzyme.

在生物体中，镉与特定的蛋白质金属硫蛋白（MT）结合，MT是富含半胱氨酸的低分子量金属结合蛋白。如果MT可以被蛋白酶特异性水解，我们可以很容易地从扇贝肝胰脏中去除镉。微生物处理具有通过生态过程清理海洋废物的潜力。另外，由于能够特异性地水解MT的蛋白酶还不为人所知，因此期待该酶具有独特的特性，本研究从土壤中发现了嗜烟节杆菌23-0-11，该菌是一株能够产生蛋白酶的微生物，该蛋白酶能够将扇贝肝胰腺中的镉释放到液体培养基中。用两种类型的离子交换层析纯化蛋白酶。该蛋白酶的分子量为27 kDa，最适pH为7.0，最适温度为50℃。该酶在pH3.0 ~ 9.0范围内稳定，在60℃以下稳定。该蛋白酶对蛋白酶抑制剂敏感，属于丝氨酸蛋白酶家族。根据这些序列设计简并寡核苷酸引物并进行PCR。以PCR产物的核苷酸序列为辅助，获得该蛋白酶的全核苷酸序列，其氨基酸序列具有丝氨酸蛋白酶的保守基序。虽然从数据库中检索到了一些已知蛋白酶的氨基酸序列，但它们的同源性并不高。虽然蛋白酶基因被克隆并导入大肠杆菌，但蛋白酶以不溶性和无活性的形式表达。因此，需要进一步考虑载体、宿主和表达条件。

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.