Study On Stereo-Recognition Mechanisms And Performance Enhancement Of Lipase By Structure Modification

Lipase is an important biocatalyst, so its molecular basis of stereo-recognition of chiral substrates, and performance enhancement in non-conventional reaction media are continuous hotspots in enzyme engineering research. Currently, enantioselectivity of lipase towards chiral secondary alcohols have been well investigated. However, the recognition mechanisms of chiral primary alcohols and chiral acids have been rarely studied. In this work, these mechanisms were studied using residue-specific modification, MALDI-TOF-MS (Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry) characterization and MD (Molecular Dynamics) simulation.The stereo-recognition of chiral primary alcohol by lipase was firstly studied. In the ligand-receptor-coupled research approach,14 pairs of primary alcohols esters with different acyl or alcohol moieties were designed, and the correlation between the structural differences of substrates and enantioselectivity was revealed. PcL (Pseudomonas cepacia lipase) was then modified by 9 specific reagents respectively at the aqua-hexane interface, and the modified lipase was characterized with MALDI-TOF-MS and simulated using MD. According to the experimental and computational results, PcL distinguishes enantiomers of chiral primary alcohol by the{PcL-Tyr29-Oη-H...Oalcy-substrate} hydrogen bond quantitatively, R2= 0.985.The stereo-recognition of chiral acid by lipase was studied afterwards. The acyl-chiral-center-selective lipase, RmL (Rhizomucor miehei lipase), and a popular chiral acid ester, BPP (n-butyl 2-phenoxypropionate), were taken as the model enzyme and model substrate respectively. According to MD simulation, the structural difference between the intermediate complexes of (R)-and (S)-BPP + RmL indicated Loopβ1-β2 and α2 helix of RmL are the key domains correlated to the stereo-recognition. Interaction analysis of these domains suggested the {Loopβ1-β2-Asp61-Arg86-α2} electrostatic interaction significantly decided the enantioselectivity of lipase towards chiral acids.1,2-cyclohexanedione modification of Arg86 weakening this interaction decreased the enantioselectivity, and 1-iodo-2,3-butanedione modification of Asp61 and Arg86 stengthen the interaction increased the enantioselectivity.The performance enhancement of lipase in non-conventional media was studied additionally. New immobilization methods was developed targeted to the high-viscosity heterogeneous reaction system of 1,3-diacylglycerol synthesis. MjL (Mucor javanicus lipase) was immobilized on poly(MMA-co-DVB)-Fe3O4 porous magnetic carrier through physical/hydrophobic adsorption. Catalytic activity of the resulting immobilized lipase increased by 170 fold compared with the native lipase, and the immobilized lipase can be reused for 30 cycles with no activity loss, but the thermalstability remained the same as the native lipase. By cross-linking MjL to a surface-activated nano-sized magnetite, NSM-CLEA (Nano-Sized Magnetic Cross-Linked Enzyme Aggregate) was then prepared, also with high activity, regioselectivity and stability, together with excellent thermalstability. The NSM-CLEA remains 80% residue activity after 36 h of heating at 75 ℃, and the regioselectivity of the lipase increased from re= 75% to 90%.