中间芽孢杆菌3-19中谷氨酰内肽酶2的分离与鉴定

Isolation and characterization of glutamyl endopeptidase 2 from Bacillus intermedius 3-19.

作者信息

Balaban N P, Mardanova A M, Sharipova M R, Gabdrakhmanova L A, Sokolova E A, Garusov A V, Milgotina E I, Rudenskaya G N, Leshchinskaya I B

机构信息

Faculty of Biology, Kazan State University, Kazan, 420008, Russia.

出版信息

Biochemistry (Mosc). 2003 Nov;68(11):1217-24. doi: 10.1023/b:biry.0000009136.09167.b6.

DOI:10.1023/b:biry.0000009136.09167.b6

PMID:14640964

Abstract

The culture filtrate of Bacillus intermedius 3-19 was used for isolation by chromatography on CM-cellulose and Mono S columns of a proteinase that is secreted during the late stages of growth. The enzyme is irreversibly inhibited by the inhibitor of serine proteinases diisopropyl fluorophosphate, has two pH optima (7.2 and 9.5) for casein hydrolysis and one at pH 8.5 for Z-Glu-pNA hydrolysis. The molecular weight of the enzyme is 26.5 kD. The K(m) for Z-Glu-pNA hydrolysis is 0.5 mM. The temperature and pH dependences of the stability of the proteinase were studied. The enzyme was identified as glutamyl endopeptidase 2. The N-terminal sequence (10 residues) and amino acid composition of the enzyme were determined. The enzyme hydrolyzes Glu4-Gln5, Glu17-Asp18, and Cys11-Ser12 bonds in the oxidized A-chain of insulin and Glu13-Ala14, Glu21-Arg22, Cys7-Gly8, and Cys19-Gly20 bonds in the oxidized B-chain of insulin.

摘要

中间芽孢杆菌3-19的培养滤液用于通过在CM-纤维素柱和Mono S柱上进行色谱分离来分离一种在生长后期分泌的蛋白酶。该酶被丝氨酸蛋白酶抑制剂二异丙基氟磷酸不可逆地抑制，对酪蛋白水解有两个最适pH值（7.2和9.5），对Z-Glu-pNA水解有一个最适pH值为8.5。该酶的分子量为26.5 kD。Z-Glu-pNA水解的K(m)为0.5 mM。研究了该蛋白酶稳定性的温度和pH依赖性。该酶被鉴定为谷氨酰胺内肽酶2。测定了该酶的N端序列（10个残基）和氨基酸组成。该酶水解胰岛素氧化A链中的Glu4-Gln5、Glu17-Asp18和Cys11-Ser12键以及胰岛素氧化B链中的Glu13-Ala14、Glu21-Arg22、Cys7-Gly8和Cys19-Gly20键。