Halophilicity of Thermolysin.Protein Engineering Studies

嗜热菌蛋白酶的嗜盐性蛋白质工程研究

• 批准号: 09660082
• 负责人: INOUYE Kuniyo
• 金额: $ 1.86万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09660082/
• 关键词: Halophilic Enzyme; Thermolysin; Metalloproteinase; chemical modification; Enzyme Activity; Proteinase; Salt; Protein Engineering; 金属プロテアーゼ; アスパルテーム; プロテアーゼ; 蛋白質分解酵素; 好塩性; 酵素構造; 金属酵素

Thermolysin (TLN) is a thermophilic neutral metalloprpteinase, and we have reported that TLN is highly halophilic and its activity enhances 30-60 times in the presence of saturated concentration of salts in comparison with that in the absence.In this study, the halophilicity was examined from the point of view of the enzyme structure and medium properties.1. The salt activation was shown to be dependent considerably on pH and temperature and the addition of organic solvent, suggesting that the activation might be related with electrostatic interaction on the surface of TLN.2. Temperature stability of TLN was enhanced 2-3 times by the addition of 0.5-1.5 M NaCl. The dependence on the salt concentration was different from that of the activation, suggesting that the stability and activity can be increased by indepenent mechanisms.3. The solubility of TLN is as low as 1 mg/ml in the common buffer. It was found that it increased remarkably by the addition of salts up to 70 mg/ml. TLN was revealed to be cold-soluble.4. Trp 115 is at the S2 suibsite. By the site-directed mutagenesis, an aromatic amino acid was shown to be needed strictly at this position for the activity.5. Nitration and amination of tyrosyl reasidues of TLN showed that introduction of negative charges on the surface of TLN decreased the degree of the salt activation.

热溶酶（TLN）是一种嗜热中性金属蛋白酶，我们报告说，与缺少盐的饱和浓度相比，在存在盐饱和浓度的情况下，TLN具有高度卤素，其活性增强了30-60倍。在这项研究的情况下，卤水从enzyme and of Enzyme and Property和Inform Properties.1中进行了研究。盐的激活显示出很大程度上取决于pH和温度以及加入有机溶剂，这表明激活可能与TLN.2表面上的静电相互作用有关。通过添加0.5-1.5 m NaCl，TLN的温度稳定性增强了2-3倍。对盐浓度的依赖性与激活不同，这表明可以通过独立机制提高稳定性和活性。3。在公共缓冲液中，TLN的溶解度低至1 mg/ml。发现它通过添加高达70 mg/ml的盐来显着增加。 TLN被揭示为冷溶性。4。 TRP 115在S2 Suibsite。通过位置定向的诱变，表明在此位置严格需要芳香氨基酸才能进行活动。5。 TLN的酪酶重生的硝化和胺化表明，TLN表面的负电荷引入降低了盐激活程度。

项目成果

S.OHNAKA: "Deletion of the κ genes from the mouse hybridoma established with NS-l myelomas" Cytotechnology. 24・3. 213-218 (1997)

S.OHNAKA：“从用 NS-1 骨髓瘤建立的小鼠杂交瘤中删除 κ 基因”《细胞技术》24·3（1997）。

T.Sakaki, N.Sawada, K.Inouye他2人: "Enzymatic properties of mouse 25-hydroxy vitamin D3 1α hydroxylase expressed" Eur.J.Biochem.259. 731-738 (1999)

T.Sakaki、N.Sawada、K.Inouye 和其他 2 人：“小鼠 25-羟基维生素 D3 1α 羟化酶表达的酶学特性”Eur.J.Biochem.259 (1999)。

K.Inouye, K.Kuzuya & B.Tonomura: "Effect of salrs on the solubility of thermolysis a remarkable increase in the solubility・・・" J.Biochem. 123・5. 847-852 (1998)

K.Inouye、K.Kuzuya & B.Tonomura：“盐对热解溶解度的影响，溶解度显着增加......”J.Biochem. 123・5（1998）。

K.Inoue, S.-B.Lee, K.Nambu, and B.To no mura: "Effect of pH,temperature, and alcohols on the remarkable activation of thermolysin by salts." J.Biochem.122(2). 358-364 (1997)

K.Inoue、S.-B.Lee、K.Nambu 和 B.To no mura：“pH、温度和醇对盐显着激活嗜热菌蛋白酶的影响。”

T.Sakaki, N.Sawada, K.Takeyama, S.Kato, and K.Inouye: "Enzymatic properties of mouse 25-hydroxyvitamin D3 1alpha-hydroxylase expressed in Escherichia coli." Eur.J.Biochem.259. 731-738 (1999)

T.Sakaki、N.Sawada、K.Takeyama、S.Kato 和 K.Inouye：“在大肠杆菌中表达的小鼠 25-羟基维生素 D3 1α-羟化酶的酶学特性。”