Cytoplasmic expression of a thermostable invertase from Thermotoga maritima in Lactococcus lactis.
OBJECTIVE
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.
Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), 20 Biopolis Way, #06-01 Centros, 138668, Singapore, Singapore.
Han Bin
HB
Pek
Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), 20 Biopolis Way, #06-01 Centros, 138668, Singapore, Singapore.
Pei Yu
PY
Lim
Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), 20 Biopolis Way, #06-01 Centros, 138668, Singapore, Singapore.
Chengcheng
C
Liu
Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), 20 Biopolis Way, #06-01 Centros, 138668, Singapore, Singapore.|Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, 117585, Singapore, Singapore.
Dong-Yup
DY
Lee
Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), 20 Biopolis Way, #06-01 Centros, 138668, Singapore, Singapore.
Xuezhi
X
Bi
Molecular Engineering Laboratory, Biomedical Sciences Institutes, A*STAR (Agency for Science, Technology and Research), Proteos, Singapore, Singapore.
Fong Tian
FT
Wong
Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), 20 Biopolis Way, #06-01 Centros, 138668, Singapore, Singapore. dave_ow@bti.a-star.edu.sg.
Dave Siak-Wei
http://orcid.org/0000-0002-0436-1896
DS
Ow
D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004795:Enzyme Stability; D013294:Lactococcus lactis / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics; D043324:beta-Fructofuranosidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D011994:Recombinant Proteins; D043324:beta-Fructofuranosidase
BfrA; Invertase; Lactococcus lactis; Secretary peptide (USP45); Thermogata maritima; Thermostable enzyme