Cytoplasmic expression of a thermostable invertase from Thermotoga maritima in Lactococcus lactis
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To evaluate the secretory and cytoplasmic expression of a thermostable Thermogata maritima invertase in Lactococcus lactis.
The thermostable invertase from T. maritima was cloned with and without the USP45 secretory peptide into the pNZ8148 vector for nisin-inducible expression in L. lactis. The introduction of an USP45 secretion peptide at the N-terminal of the enzyme led to a loss of protein solubility. Computational homology modeling and hydrophobicity studies indicated that the USP45 peptide exposes a stretch of hydrophobic amino acids on the protein surface resulting in lower solubility. Removal of the USP45 secretion peptide allowed a soluble and functional invertase to be expressed intracellularly in L. lactis. Immobilized metal affinity chromatography purification of the cell lysate with nickel-NTA gave a single protein band on SDS-PAGE, while E. coli-expressed invertase consistently co-purified with an additional band. The yields of the purified invertase from E. coli and L. lactis were 14.1 and 6.3 mg/l respectively.
Invertase can be expressed in L. lactis and purified in a functional form. L. lactis is a suitable host for the production of food-grade invertase for use in the food and biotechnology industries.
KeywordsBfrA Invertase Lactococcus lactis Secretary peptide (USP45) Thermogata maritima Thermostable enzyme
We would like to thank the Microbial Cell Group with special thanks to Crystal Tan Lee Ling for their technical support. This work was supported by the Biomedical Research Council of A*STAR (Agency for Science, Technology and Research) and A*STAR Joint Council Office Grant Call (No. 1431AFG126).
- Khemariya P, Singh S, Nath G, Gulati AK (2016) A review on industrially important Lactococcus lactis: general information, metabolism and genotypic identification tools. Anchor Academic Publishing, New YorkGoogle Scholar
- Kleerebezem M, Beerthuyzen MM, Vaughan EE, de Vos WM, Kuipers OP (1997) Controlled gene expression systems for lactic acid bacteria: transferable nisin-inducible expression cassettes for Lactococcus, Leuconostoc, and Lactobacillus spp. Appl Environ Microbiol 63:4581–4584PubMedPubMedCentralGoogle Scholar
- Kulshrestha S, Tyagib P, Sindhia V, Yadavillic KS (2013) Invertase and its applications—a brief review. J Pharm Res 7:792–797Google Scholar
- Perez de los Santos AI, Cayetano-Cruz M, Gutierrez-Anton M, Santiago-Hernandez A, Plascencia-Espinosa M, Farres A, Hidalgo-Lara ME (2016) Improvement of catalytical properties of two invertases highly tolerant to sucrose after expression in Pichia pastoris. Effect of glycosylation on enzyme properties. Enz Microb Technol 83:48–56CrossRefGoogle Scholar
- Robichon C, Luo J, Causey TB, Benner JS, Samuelson JC (2011) Engineering Escherichia coli BL21(DE3) derivative strains to minimize E. coli protein contamination after purification by immobilized metal affinity chromatography. Appl Environ Microbiol 77:4634–4646CrossRefPubMedPubMedCentralGoogle Scholar
- Tuite MF, Oliver SG (2013) Saccharomyces. Springer, USGoogle Scholar
- Walsh G (2002) Proteins: biochemistry and biotechnology. Wiley, New YorkGoogle Scholar