Abstract

Background: Propeptides of lipases have been demonstrated to influence many properties of their mature proteins as intramolecular chaperones. However, the working mechanism of propeptides may be different.

Objectives: The main objective of this study was to determine the role of pentapeptide prosequence of Thermomyces lanuginosus lipase (TLL) through its effect on the recombinant expression of TLL in P. pastoris, and explore the possible function mechanism with its hydrophobicity.

Methods: We have synthesized a codon-optimized TLL gene coTLL with a Kex2 cleavage site “- KREAEA-” directly after the propeptide “SPIRR-”, and obtained expression vector pP-kTL through cloning it into pPIC9K. TL gene without the propeptide and pTL gene with a propeptide directed linked to mature TLL lipase, were also amplified and cloned into pPIC9K to obtain the expression vector pP-TL and pP-pTL. pTL-P and pTL-VP gene variants with mutation in pentapeptide prosequence of TLL were obtained used the site-directed mutation with the pP-pTL plasmid as the template. The recombinant proteins were expressed in P. pastoris GS115. Lipase activity was determined by a spectrophotometric method previously reported using para-nitrophenyl palmitate (pNPP) as the substrate and the thermostability of lipases of TLL was analyzed by incubating at different temperatures (70°C and 80°C) for 5h and molecular dynamics simulation.

Results: The average lipase activity of recombinant strains GS-pTL reached 434.32 U/mL, higher than that of GS-TL 377.71 U/mL. The fermentation result of the recombinant strains with modified propeptide showed that the extracellular lipase activity of GS-pTL-VP variant reached 483.29 U/mL, increasing by 11.27% compared with that of GS-pTL (434.32 U/mL). Further analysis performed on the lipase stabilities with propeptide variants by molecular dynamics simulation showed that the RMSD of variant pTL-VP was similar to that of pTL.

Conclusion: This study revealed that the propeptide “SPIRR-” sequence is beneficial for enhancing TLL expression. In addition, the function of TLL propeptide was identified to be related to its hydrophobicity, implying that propeptide might play a role in assisting the formation of the hydrophobic protein core and accelerate the protein folding process. This work inspired us to attach more emphasis on the propeptide of other lipases for improving their recombinant expression, structure folding and enzymatic properties.

Keywords: Thermomyces lanuginosus lipase, propeptide, prosequence, recombinant expression, protein folding, molecular imprint, intramolecular chaperone.

Graphical Abstract