Cytoplasmic expression of a thermostable invertase from Thermotoga maritima in Lactococcus lactis

• Published in: Biotechnology letters Vol. 39; no. 5; pp. 759 - 765
• Main Authors: Pek, Han Bin, Lim, Pei Yu, Liu, Chengcheng, Lee, Dong-Yup, Bi, Xuezhi, Wong, Fong Tian, Ow, Dave Siak-Wei
• Format: Journal Article
• Language: English
• Published: Dordrecht Springer Netherlands 01.05.2017; Springer Nature B.V
• Subjects: Amino acids; Applied Microbiology; beta-Fructofuranosidase - chemistry; beta-Fructofuranosidase - genetics; beta-Fructofuranosidase - isolation & purification; beta-Fructofuranosidase - metabolism; Biochemistry; Biomedical and Life Sciences; Biotechnology; Chromatography, Affinity; Cloning, Molecular; E coli; Enzyme Stability; Enzymes; Escherichia coli; Invertase; Lactococcus lactis; Lactococcus lactis - genetics; Lactococcus lactis - metabolism; Life Sciences; Microbiology; Models, Molecular; Nickel; Original Research Paper; Peptides; Proteins; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Secretions; Solubility; Temperature; Thermotoga maritima; Thermotoga maritima - enzymology; Thermotoga maritima - genetics; Thermostable enzyme; BfrA; Secretary peptide (USP45); Invertase; Lactococcus lactis; Thermogata maritima
• ISSN: 0141-5492; 1573-6776
• DOI: 10.1007/s10529-017-2295-4

Objectives To evaluate the secretory and cytoplasmic expression of a thermostable Thermogata maritima invertase in Lactococcus lactis . Results 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. Conclusions 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.