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STUDIES ON MASIKINOSIN,A NOVEL TYPE OF PROTEINASE

新型蛋白酶马西肌肽的研究

基本信息

批准号：
07660127
负责人：
MURAO Sawao
金额：
$ 1.47万
依托单位：
KUMAMOTO INSTITUTE OF TECHNOLOGY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07660127/
关键词：
Proteinase Streptomyces griseoloalbus Masikinosin Proteinase Streptomyces griseoloalbus Masikinosin Streptomyces

项目摘要

The stock strains in this laboratory were screened using succinyl-L-alanyl-L-alanyl-L-alanine p-nitroanilide as a substrate. Of more than 2,800 strains screened, we found ability to release p-nitroaniline in the culture filtrate of Streptomyces griseoloalbus SN-22. The culture filtrate was fractionated with ammonium sulfate and column chromatographies on DEAE-Cellulose, Butyl-Toyopearl 650S,and Sephadex G-75. By these procedures, SN-22 proteinase (named Masikinosin) was purified about 92.7-fold from culture filtrate with an activity recovery of 10.3%. The molecular weight was estimated to be 26,000 by SDS polyacrylamide gel electrophoresis and by gel filtration on a TSKgel G2000SW column. The isoelectric point was 6.4. The optimum pH was pH 9.0. The enzyme retained more than 80% of the original activity between 8.0 and 11.0. The optimum temperature was 45ﾟC and 80% of the initial activity was observed after incubating at 40ﾟC,for 30 min, and at pH 9.0. The enzyme was markedly inhibited by DFP and PMSF.However, it was not affected by SSI,MAPI,elastatinal, TLCK,TPCK,antipain, and EDTA.From the cleavage sites of the insulin B-chain and lysozyme, Masikinosin specifically hydrolyzed the carboxyl side of alanine and valine residues. The enzyme could also hydrolyze elastin, elastin-orcein, and casein. In conclusion, masikinosin is a unique serine proteinase with specificity for cleavage at the carboxyl side of alanine and valine residues.

以琥珀酰-L-丙氨酰-L-丙氨酰-L-丙氨酸对硝基苯胺为底物，对本实验室的储备菌株进行了筛选。在筛选的2，800多株菌株中，我们发现了在灰白链霉菌SN-22的培养滤液中释放对硝基苯胺的能力。将培养滤液用硫酸铵分级，并在DEAE-Cellulose、Butyl-Toyoprotein 650 S和Sephadex G-75上进行柱层析。经此方法，SN-22蛋白酶（命名为Masikinosin）从培养滤液中纯化了92.7倍，活性回收率为10.3%。通过SDS聚丙烯酰胺凝胶电泳和TSKgel G2000 SW柱上的凝胶过滤估计分子量为26，000。等电点为6.4。最适pH为9.0。该酶在8.0和11.0之间保留了80%以上的原始活性。最适温度为45 ℃，在40 ℃、pH9.0条件下孵育30 min后，可观察到80%的初始活性。该酶被DFP和PMSF显著抑制，而SSI、TPCK、Elastinal、TLCK、TPCK、Antipain和EDTA等对酶活性无影响。该酶也能水解弹性蛋白、弹性蛋白-地衣红和酪蛋白。总之，马司激肽是一种独特的丝氨酸蛋白酶，对丙氨酸和缬氨酸残基的羧基侧具有特异性切割。