The Study On N-acetylation Reaction In Water And Immobilization Of Acyltransferase MsAcT

Amides have found significant utility in agriculture,chemistry,and pharmaceutical fields.However,common enzyme-catalyzed acylation reactions typically occur in non-aqueous systems,posing potential risks and limitations for industrial applications.This approach is also not in line with the current trend of environmental friendliness.With the development of green chemistry,there has been continuous exploration of more environmentally friendly reaction systems.Water,as an inexpensive,readily available,safe,and environmentally friendly solvent,has attracted widespread attention in the field of catalysis.Enzymes in aqueous catalytic systems have high solubility and can operate under mild conditions.Compared to non-aqueous systems,this eliminates the need for cumbersome dehydration processes during reactions and minimizes enzyme activity loss.In recent years,the acyltransferase from Mycobacterium smegmatis(MsAcT)has gained increasing attention.It can catalyze acylation reactions using various acyl donors and acceptors in aqueous environments,yielding a variety of amide compounds,and it has gradually become one of the most valuable enzymes in industrial production.Cinnamamides play a vital role in natural product chemistry and medicinal chemistry.Many natural cinnamamides and their derivatives exhibit diverse biological activities.Currently,the synthesis of cinnamamides heavily relies on chemical methods,which entails potential risks,side reactions,and environmental unfriendliness.The acyltransferase MsAcT can catalyze simple N-acetylation reactions in water,however,it has limitations when dealing with larger acyl donors,such as aromatic compounds,and cannot catalyze the synthesis of cinnamamides.In this study,we expanded the diameter and volume of the entrance channel of enzyme’s active site through virtual screening and site-directed mutagenesis.This modification allowed substrates to smoothly access the active site,overcoming the substrate restrictions of MsAcT.As a result,we obtained the target variant MsAcT-L12 A,which could catalyze the synthesis of cinnamamides.Furthermore,MsAcT-L12 A exhibited a clear preference for acyl acceptors with electron-donating groups,such as methoxy.After optimizing reaction conditions,when vinyl cinnamate serveed as acyl donor and 4-methoxyaniline or 3-methoxyaniline as acyl acceptor,MsAcT-L12 A not only catalyzed the reactions,but the yields of the reactions could reach 86.7 ± 2.1%and 71.3 ± 1.1%,respectively.In contrast,the wild-type MsAcT could hardly catalyze the synthesis of cinnamamides under the same reaction conditions.However,free MsAcT often faces various challenges in industrial applications.These challenges include limited reusability,low stability,high sensitivity to environmental factors,and tedious purification processes during production.Addressing these issues,this study utilized metal-organic framework materials(MOFs)MIL-88 A,which contained coordinatively unsaturated metal site(CUS).Through affinity interactions,the histidine-tagged MsAcT was simultaneously immobilized and purified from cell lysates,resulting in the immobilized enzyme MsAcT@MIL-88 A.After optimizing the immobilization conditions,the final MsAcT@MIL-88 A demonstrated an activity recovery of 152.6 ± 4.0% and an immobilization efficiency of 52.9 ± 0.8%.Importantly,in the reaction which utilized ethyl acetate and aniline as acyl donor and acceptor,the initial rate of the catalysis by MsAcT@MIL-88 A was 1.97 times that of MsAcT.This immobilization modification using MIL-88 A not only simplified the purification process but also enhanced the catalytic activity of the enzyme in N-acetylation reactions.The simultaneous purification and immobilization of the target protein using MIL-88 A was a straightforward and practical immobilization method.MsAcT@MIL-88 A exhibited good stability,reusability,and improved enzyme activity.Immobilized enzymes obtained through affinity interaction is a straightforward immobilization method.However,the weak physical bonds between enzymes and carriers can lead to enzyme detachment,and some immobilization carriers may exhibit poor stability in specific environments.To further enhance the stability of immobilized enzymes,this study developed a protective strategy by introducing a silica layer on the surface of immobilized enzymes.Tetraethyl orthosilicate was selected as the precursor material.When negatively charged silicate interacted with positively charged groups on MsAcT@MIL-88 A,the surfactant CTAB readily induced the formation of a silica layer around the surface of MsAcT@MIL-88 A,resulting in MsAcT@MIL-88A@silica.Compared to MsAcT@MIL-88 A,which losed almost all of its activity after five repeated uses,immobilized enzyme MsAcT@MIL-88A@silica exhibited excellent stability and reusability,maintaining69.8 ± 0.2% of its initial activity even after 7th cycle of reuse.Furthermore,MsAcT@MIL-88A@silica demonstrated enhanced catalytic capabilities in N-acetylation reactions in water.Its catalytic activity in the reaction between vinyl acetate and aniline was 3.3 times higher than that of free MsAcT and 2.9 times higher than that of MsAcT@MIL-88 A.This protective strategy enhanced the ability of immobilized enzymes to withstand adverse physical and chemical environments,further boosting their catalytic activity.In order to reduce environmental pollution caused by the carrier,enhance the purification efficiency of the immobilization process for target enzymes,and improve the reusability,the low-cost,widely available,biocompatible,and environmentally friendly aspen powder(AP)was utilized.After undergoing deep eutectic solvent(DES)treatment,oxidation,NTA chelation,and other modifications,Ni-NAP was obtained as the immobilization carrier.Ni-NAP enabled simultaneous purification and immobilization of both MsAcT and MsAcT-L12 A.Compared to free ones,immobilized enzymes showed significantly improved stability and excellent reusability.Specifically,MsAcT-L12A-Ni-NAP,after undergoing 7th cycle of reuse,maintained a relative yield of 83.3 ± 2.2%.Moreover,MsAcT-L12A-Ni-NAP exhibited a preference for substrates containing electron-donating groups in water.In reactions catalyzed by MsAcT-L12A-Ni-NAP involving 3-methoxyaniline and vinyl cinnamate,the yield reached 89.1 ± 0.8%,and the yield for the reaction involving4-methoxyaniline and vinyl cinnamate was 4.6 times higher than that of the reaction involving 4-fluoroaniline and vinyl cinnamate.MsAcT-L12A-Ni-NAP combined excellent stability and reusability with retained catalytic performance,making it a promising candidate for industrial applications.