Premium
Facile purification of a C‐terminal extended His‐tagged Vibrio mimicus arylesterase and characterization of the purified enzyme
Author(s) -
Lee YaLin,
Chang ReyChang,
Shaw JeiFu
Publication year - 1997
Publication title -
journal of the american oil chemists' society
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.512
H-Index - 117
eISSN - 1558-9331
pISSN - 0003-021X
DOI - 10.1007/s11746-997-0239-1
Subject(s) - enzyme , arylesterase , biochemistry , esterase , histidine , chemistry , thioesterase , affinity chromatography , trypsin , enzyme assay , chymotrypsin , circular dichroism , microbiology and biotechnology , biology , biosynthesis , pon1 , gene , genotype
Abstract Vibrio mimicus arylesterase, a 20 kDa protein, is a multifunctional enzyme with thioesterase and chymotrypsin‐like activities. Because an affinity His‐tag (six consecutive histidine affinity tag) directly to the protein caused the loss of enzyme activity, a hexadecapeptide with His‐tag, ADPNSSSVDKLAAALE encoded from vector pET‐20b(+) was constructed to extend from the carboxyl terminus of the arylesterase. This Histagged protein retained enzyme functions. Thermal unfolding behavior of both proteins was almost identical, and their T m values were near 54°C as monitored by circular dichroism. Tryptic cleavage of the functional His‐tagged enzyme produced two smaller proteins, which still possessed enzyme activity and which suggested that the additional peptide extended on the protein surface. The spacing peptide between His‐tag and arylesterase successfully prevented the interference of the His‐tag to the enzyme functions. The kinetic studies showed that the esterase and thioesterase activities of the His‐tagged enzyme were similar to those of the wild type. On the other hand, the catalytic efficiency of chymotrypsin‐like activity of the His‐tagged protein was two times higher than that of the wild type.