Research On Reversible Photocontrol Of Lipase Activity

Photoregulating biological function by non-invasive way greatly promote the development of biological catalysis system with light response characteristics.These systems have the potential application for many fields,including signal transmission,diagnosis and treatment.It is almost no disturbance for the fine control of enzymatic activity especially,which provides a nonparallel advantage.Azobenzene has the extended trans-and the more compact cis-isomer.This photoisomerization could be realized through irradiation with different wavelength of light.When both ends of the azobenzene derivative are cross-linked with two cysteines residues with a certain distance of the target protein,the structure of the azobenzene derivative is changed by irradiation,and the structure of the protein is reversibly changed to realize the reversible regulation of the target protein activity.This article develops a strategy which could achieve to reversibly control activity by the photoresponse change of the lipase lid.A photoswitchable lid was designed by covalent-coupling a water-soluble azobenzene derivative as photosensitive molecule to a-helix of lipase lid structure.Applying UV-and blue-light as the control condition,photocontrol the lipase activity by adjusting conformation of the lip structure.The details were as follows:1?A water soluble and thiol-reactive azobenzene derivative 3,3'-double(sulfoacid)4,4'-(acetamido)azobenzene was synthesized,which were identificated by 1H NMR and 13C NMR.2?Through analyzing the crystal structure of Lip K107,and combining azobenzene derivative's features,10 sets of mutantion was designed,which include 4 sets of i-i+11 cysteine mutantion,4 sets of i-i+7 cysteine mutantion strain as well as 2 sets as control.All mutations were gained by applying seamless clone technology.3?All variants were cross-linked with azobenzene derivatives,and then carryed out enzyme activity by irradiation with UV-and blue-light.The best of photocontrol effects of variant,C127C138,was selected under the precondition of activity was well maintained.Finally,BSBCA-C127C138 was demonstrated by MOLDI-TOF,MS/MS,UV-VIS absorption spectrum and heat relaxation analysis,which further show the success crosslinking between BSBCA and C127C138.4?It shows 3-fold reversible activity change after irradiation with UV-and blue-light,when C127C138A corsslinked with azobenzene derivatives.As well,the activity shows no loss after 5-rounds circulations.In addition,photocontrol the enzymatic activity was caused by conformation change of a-helix on the lid,which can be proved by circular dichroism analysis.