Unusual stability of manganese superoxide dismutase from a new species, Tatumella ptyseos ct: Its gene structure, expression, and enzyme properties

Chuian Fu Ken, Chuing Chi Lee, Kow Jen Duan, Chi Tsai Lin

Research output: Contribution to journalArticle

32 Citations (Scopus)

Abstract

A genomic DNA of 1416 bp containing an open reading frame encoding a manganese superoxide dismutase (Mn-SOD) from Tatumella ptyseos ct was cloned. Sequence analysis of this new gene revealed that it translates 205 amino acid residues. The deduced amino acid sequence showed variable identities (41-91%) with sequences of Mn-SODs from other species. The residues required to coordinate the single trivalent manganese ion and the 11 residues putatively involved in the active center are conserved as they are in other reported Mn-SODs. In addition, the gene was introduced into the expression vector, pET-20b(+), and transformed in Escherichia coli BL21(DE3). The Mn-SOD was purified by a His-tag technique. The yield was 0.9 mg from 0.5 L of culture. The specific activity was 6540 U/mg. A dimer is the major form of the enzyme in equilibrium. The half-life of dimer is approximately 50 min and its thermal inactivation rate constant kd was 0.015 min-1 at 80°C. The dimerization of the enzyme was inhibited under an acidic pH (below 4.0), or in the presence of SDS (above 1%) or imidazole (above 0.5 M), whereas it was not affected under an alkaline pH (above 9.0). Furthermore, the dimeric enzyme was much more resistant to proteolytic attack after 3 h of incubation at 37°C with trypsin and chymotrypsin. This unusually stable enzyme can be used as cosmetic to the protection of skin against the unaesthetic effects caused by free radicals.

Original languageEnglish
Pages (from-to)42-50
Number of pages9
JournalProtein Expression and Purification
Volume40
Issue number1
DOIs
Publication statusPublished - 2005 Mar

All Science Journal Classification (ASJC) codes

  • Biotechnology

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