Site-specific Modification Of Lipase Via Catechol-functionalized Polyethylene Glycol

The introduction of novel functionalities into proteins by PEGylation has been extensively studied for better application in industrial production in recent decades. In order to increase the homogeneity of conjugates and thus to better control the fidelity of structure and function of modified biocatalysts, site-specific PEGylation plays a more important role than random conjugation of PEG chains. Recent developments focus on linking proteins more selectively, more rapidly and more quantitatively in the field of site-specific PEGylation despite the large number of publications.This article is intended as a new strategy for protein site-specific modification via functional PEG in combination with bio-inspired catechol group contributing to adhesion. Lipase is investigated as a model enzyme for our study. Thanks to site-directed mutagenesis, which allows the introduction of a cysteine residue, thiol group of the cysteine is used as a covalent binding site for the highly selective catecholic PEG. The influence of the chain length of PEG and the binding site on the structure and bioactivity of modified lipases is fully studied. The details are as follows:First, two routes were used for the synthesis of catecholic PEG with different molecular weights. The products were called DP5k, DTP5k, DTP12k, and DTP20k, which were further identified by1H-NMR.Second, according to previous study, the lipase structure had been solved. Two different mutants were introduced into the lid region (T138C) and the random coil (Q201C) far away from the lid domain, respectively. Each derivative of LipK107possessed a free cysteine. Q201C and T138C were constructed and expressed.Third, the selective bioconjugation procedure was totally explored. The reaction factors such as temperature, time, and pH were further examined. The best reaction condition was at pH7.4and25℃. Our results revealed the bioconjugation of either Q201C or T138C to DTP5k for3h was100%complete.Forth, the specificity of the conjugation was demonstrated by SDS-PAGE, Western-Blotting, SEC-HPLC, and MALDI-TOF, showing site-specific modification.Finally, CD Spectra demonstrated the secondary structure of lipases was not influenced by PEGylation. After the entire modification procedure, catalytic activity of lipases was almost retained, at the same time, the thermal stability and pH stability of the biocatalysts were significantly increased compared to unmodified lipases.In conclusion, the catecholic PEG can be used to modify protein in a controlled way without any influence on the structure and function of modified biocatalysts, which provides a novel, convenient, and flexible approach for wide applications.