STUDIES ON MASIKINOSIN,A NOVEL TYPE OF PROTEINASE

以琥珀酰基-L-丙氨基-L-丙氨基-L-丙氨酸对硝基苯胺为底物，对本实验室的菌种进行筛选。在筛选的2800多株菌株中，我们发现能够在灰色链霉菌SN-22的培养滤液中释放对硝基苯胺。培养滤液用硫酸铵分级，然后用DEAE-Cellulose、丁基-丰多酚650S和Sephadex G-75柱层析。通过这些步骤，SN-22蛋白酶(命名为Masikinosin)从培养滤液中纯化了约92.7倍，活力回收率为10.3%。经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法和TSK-Gel G2000SW凝胶过滤法测得其相对分子质量为26,000。等电点为6.4。最适pH为9.0。该酶在8.0~11.0之间保持了80%以上的原酶活性。该酶的最适温度为45ﾟC，在40ﾟC、30min、pH 9.0条件下孵育30min，酶活力可达80%以上。DFP和PMSF对该酶有明显的抑制作用，而SSI、MAPI、弹性体、TLCK、TPCK、止痛剂和EDTA不影响该酶的活性。从胰岛素B链和溶菌酶的裂解部位看，Masikinosin专一性地降解丙氨酸和缬氨酸残基的羧基。该酶还可以分解弹性蛋白、弹性蛋白-地衣蛋白和酪蛋白。综上所述，masikinosin是一种独特的丝氨酸水解酶，能专一性地切割丙氨酸和缬氨酸残基的羧基。

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.