Enhanced secretion of cyclodextrin glucanotransferase in lactococcus lactis using heterologous signal peptide and optimization of induction condition for cultivation

Abstract

Protein secretion is preferable compared to intracellular production due to its easy subsequent purification process. The secretion generally requires a particular N-terminal signal peptide to lead the precursor protein to the secretion machinery. In this study, a strategy to secrete a cyclodextrin glucanotransferase (CGTase) from Bacillus sp G1 into the culture medium of Lactococcus lactis using three different signal peptides was developed. Heterologous signal peptides which are G1 (native signal peptide of CGTase from Bacillus sp G1) and M5 ( mutated form of G1 signal peptide by introduction of helix breaker at H-region signal peptides) were used for inducible and secretory expression of CGTase in L. lactis. The effectiveness of these heterologous signal peptides was compared to the homologous signal peptides which is SPUsp45 signal peptide (derived from Unknown Secreted 45 kDa Protein of L. lactis). Secretion activity of CGTase led by G1 signal peptide was significantly increased by 46.2% and 75.0% compared to CGTase fused to M5 and SPUsp45 signal peptide, respectively after 6 hour post-induction. Sequence analysis showed there is no correlation between signal peptide characteristics (N-terminal signal peptide, hydrophobic signal peptide and C-terminal cleavage site) and secretion level of CGTase. In addition, Response Surface Methodology (RSM) was applied to CGTase led by G1 signal peptide (G1-CGTase) to optimize culture cultivation for post induction temperature, nisin concentration and inducer starting point (OD600). The G1-CGTase activity increased approximately 2.81 fold from 5.79 U/mL to 16.89 U/mL at the optimized post induction temperature, nisin concentration and inducer starting point (OD600) of 20.1°C, 3.086 ng/mL and 0.09, respectively. Hence, G1 signal peptide has a great potential to be incorporated in an expression vector to increase the level of recombinant protein secretion in L. lactis.