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(MTS)结合，金属硫蛋白是富含半胱氨酸的低分子金属结合蛋白。如果MTS可以被一种特定的酶降解，我们就可以很容易地从扇贝肝胰腺中去除镉，这是一种海洋废物。微生物处理具有通过生态过程清理海洋废物的潜力。此外，由于目前还不知道一种能特异性地降解MTS的蛋白酶，因此该酶有望具有独特的性质。我们的研究发现，从土壤中分离出的烟酒节杆菌23-0-11是一种产生一种蛋白酶的微生物，可以将扇贝肝胰腺中的镉释放到液体介质中。用两种离子交换层析对该酶进行纯化。该酶的最适pH为7.0，最适温度为50℃。该酶在pH 3.0~9.0范围内，在60℃以下的温度下活性稳定。对蛋白水解酶抑制剂的敏感性表明，该酶属于丝氨酸蛋白酶家族，其N-端和内部均有部分氨基酸序列。根据这些序列设计简并寡核苷酸引物，并进行聚合酶链式反应。将扩增产物的核苷酸序列作为辅助手段，获得了该蛋白的全核苷酸序列，该蛋白的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的蛋白酶的氨基酸序列，但它们的同源性并不是很高。虽然该酶基因已被克隆并导入到大肠杆菌中，但其表达形式为不溶于水且无活性。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.