Improvement of bacterial endo-1,4-β,-D-glucanase (CMCase) secretion in yeast by mutagenesis of glucoamylase signal sequence = Glucoamylase 분비신호서열의 돌연변이에 의한 효모에서 세균의 Endo-1,4-β-D-glucanase의 분비능 증진

Abstract

Glucoamylase of Saccharomyces diastanticus is produced as a large precursor composed of signal peptide (21 amino acid residues), Thr and Ser-rich region and functional glucoamylase. To evaluate the utility of the glucoamylase signal peptide (GSP) for the secretion of foreign proteins, four types of GSP mutants (m1: Pro-18→Leu-18, m2: Tyr-13→Leu-13, m3: Ser-9→Leu-9, m4: Asn-5→Pro-5) were constructed and secretion efficiency of each mutant was compared with that of native GSP by the expression and secretion of Bacillus subtilis CMCase under the control of GAP promoter and GAL7 terminator in S. diastaticus. Mutants 1, 2 and 3 had a higher hydrophobicity than wild type GSP in N-terminal domain and hydrophobic domain. In mutant 4, a polar amino acid was replaced by a helix - Breaking Pro residue. CMCase activity assay and Western blot analysis revealed that CMCase secretion by GSP mutants replaced by Leu were increased compared with native GSP. In the case of m2 and m3, the substitution of Leu for Tyr-13 and Set-9 in the hydrophobic region resulted in a twofold increase in the extracellular CMCase activity.