Abstract
Acetyl xylan esterase A (AxeA) from Streptomyces lividans belongs to a large family of industrially relevant polysaccharide esterases. AxeA and its truncated form containing only the catalytically competent domain, AxeA tr, catalyze both the deacetylation of xylan and the N-deacetylation of chitosan. This broad substrate specificity lends additional interest to their characterization and production. Here, we report three systems for extracellular production of AxeAtr: secretion from the native host S. lividans with the native signal peptide, extracellular production in Escherichia coli with the native signal peptide, and in E. coli with the OmpA signal peptide. Over five to seven days of a shake flask culture, the native host S. lividans with the native signal peptide secreted AxeAtr into the extracellular medium in high yield (388 mg/L) with specific activity of 19 U/mg corresponding to a total of 7000 U/L. Over one day of shake flask culture, E. coli with the native secretion signal peptide produced 84-fold less in the extracellular medium (4.6 mg/L), but the specific activity was higher (100 U/mg) corresponding to a total of 460 U/L. A similar E. coli culture using the OmpA signal peptide, produced 10 mg/L with a specific activity of 68 U/mg, corresponding to a total of 680 U/L. In 96-well microtiter plates, extracellular production with E. coli gave ∼30 and ∼86 μg/mL in S. lividans. Expression in S. lividans with the native signal peptide is best for high level production, while expression in E. coli using the OmpA secretion signal peptide is best for high-throughput expression and screening of variants in microtiter plate format.
Original language | English (US) |
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Pages (from-to) | 274-284 |
Number of pages | 11 |
Journal | Protein Expression and Purification |
Volume | 46 |
Issue number | 2 |
DOIs | |
State | Published - Apr 2006 |
Bibliographical note
Funding Information:We thank Isabelle Boucher (ISM Biopolymer) for helpful discussions, Roger Dubuc for his technical assistance, and Liette Biron for technical advice and for S. lividans manipulations. This work was supported by NSERC Strategic Grant STPGP 257885-02.
Keywords
- Acetyl xylan esterase
- Deacetylase activity
- Escherichia coli
- OmpA
- Secretion
- Streptomyces lividans