Construction of a fully active truncated alternansucrase partially deleted of its carboxy-terminal domain

• Published in: FEBS letters Vol. 580; no. 3; pp. 763 - 768
• Main Authors: Joucla, Gilles, Pizzut, Sandra, Monsan, Pierre, Remaud-Simeon, Magali
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 06-02-2006
• Subjects: Alternansucrase; Amino Acid Motifs - genetics; Amino acid repeats; Amino Acid Sequence; ASR; Cell wall binding repeats; Domain truncation; Escherichia coli - enzymology; Escherichia coli - genetics; GBD; Gene Expression; glucan binding domain; Glucansucrase; glycoside-hydrolase; Glycosyltransferases - biosynthesis; Glycosyltransferases - genetics; high pressure size exclusion chromatography; HP-SEC; Leuconostoc - enzymology; Leuconostoc - genetics; Protein Engineering; Protein Structure, Tertiary - genetics; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Sequence Deletion; Substrate Specificity - genetics; ASR; Alternansucrase; GH; Glucansucrase; CW; Domain truncation; HP-SEC; GBD; Amino acid repeats; Cell wall binding repeats
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/j.febslet.2006.01.001

Recombinant expression of the large alternansucrase (2057 amino acids) was hindered in E. coli due to poor enzyme solubility and protein degradation. The effects of deletions of the alternansucrase C-terminal CW-like and APY repeated motifs on enzyme solubility and specificity were investigated. A truncated variant deleted of the APY repeats but harboring four C-terminal CW-like repeats displayed a high specific activity and the same specificity of product synthesis as the native enzyme. It is more soluble and suffers less degradation than full length alternansucrase. Hence this truncated variant is a promising tool for the further structural and kinetic study of this interesting enzyme.