Cyanide-Free Chemoenzymatic Strategies For The Synthesis Of Nitriles

Nitrile play an important role in many fields,but most of the traditional methods for synthesizing nitriles have shortcomings,such as the presence of highly toxic metal cyanide or HCN during the reaction process,resulting in serious safety hazards.Therefore,this study has developed a safe,efficient,and mild chemical enzymatic synthesis of nitriles.Based on the catalytic properties of aldoxime dehydratase,nitriles were synthesized using aldehyde and amine as the substrates,and aldoxime as the reaction intermediate.To improve the catalytic performance of aldoxime dehydratase,some key residues in the binding pocket and substrate channel were mutated in oreder to screen the best mutants using a semi-rational design strategy.Meanwhile,a specific tag peptide segment was designed to covalently immobilize the aldoxime dehydratase polymer onto the resin through a biological orthogonal method mediated by transglutaminase（MTG）,achieving a"one-step"polymerization,purification,and fixation of the target enzyme protein,thus improving its industrial properties.Firstly,the aldoxime dehydratase Oxd Asp derived from the genome of Aspergillus ibericus CBS121593 was obtained through genome mining strategy screening.Select The vector p ET28a was selected to construct a recombinant plasmid and achieved functional expression in E.coli BL21（DE3）.Oxd Asp was characterized using cinnamaldehyde oxime（Cin Ox）as the substrate.The optimum reaction temperature of Oxd Asp was 30℃,and the optimum reaction p H was 6.0.The half-lives of recombinant Oxd Asp were calculated as 3.5 h and 1.6 h at 30 and 35℃,respectively.The K_mand V_maxof Oxd Asp toward Cin Ox were determined as 3.55 m M and 1.54μmol min^-1mg^-1protein,respectively.Through multiple sequence alignment,Oxd Asp has reported that the sequence homology of aldoxime dehydratase is very low,about 20-30%,and does not have a traditional catalytic triplet.Meanwhile,a homologous structure model of Oxd Asp was constructed,and Cin Ox pairs were inserted into catalytic pockets to investigate substrate binding.The catalytic mechanism of Oxd Asp was further predicted based on alanine scanning of key residues.The results demonstrates that after the substrate enters the catalytic pocket,the hydroxyl group is fixed by Glu192,and the adjacent Arg141 polarizes the phenol protons in Tyr138.Then,the protonated Tyr138 donates a proton to remove the water molecules in the aldoxime to produce nitrile.Secondly,a semi-rational protein design was carried out for Oxd F1 in Pseudomonas putida F1 to construct a chemically enzymatic sequential catalytic synthesis of aromatic nitrile chemicals from benzylamine.The crystal structure-based CAVER analysis indicates that Met29,Ala147,Phe306,and Leu318 are located at the entrance of the substrate access tunnel.And the hydrophobic side chain of Leu145might affect the binding direction of the substrate in the catalytic pocket.After two rounds of mutation,the catalytic activity of the mutants L318F and L318F/F306Y towards benzaldoxime was significantly increased to about 2.86μmol min^-1mg^-1.Meanwhile,CAL-B from Candida antarctica lipase type B was functionally expressed in E.coli,using UHP as the oxidant in the reaction system to selectively oxidize benzylamine to aldoxime,and the selective oxidation reaction was optimized.The optimum conditions were obtained with Et OAc as the reaction medium,6 mg/m L lyophilized recombinant E.coli cells as the catalyst,and 2 equiv.UHP as the oxidant,with a conversion rate of approximately 91%within 7 h.The synthesis of aromatic nitrile chemicals from benzylamine was catalyzed by a reduction method using an aqueous solution containing 50 m M of anhydrous Na₂SO₃（1/5 of the oxidation reaction volume）,combining oxidation and dehydration.For almost all substrates,a yield of about 60%can be obtained without separating the intermediate.Finally,using MTG-mediated biorthogonal methods,Oxd F1 was polymerized and covalently fixed to the resin to enhance the industrial application properties of enzyme proteins.Recombinant Escherichia coli K-Oxd F1-L318F-Q preferentially undergoes intramolecular cross-linking between the two residues under the catalysis of MTG,and the enzyme activity has not been improved.Recombinant Escherichia coli K-Oxd F1-L318F-K and Q-Oxd F1-L318F-Q aggregate under the catalysis of MTG,resulting in a significant increase in enzyme activity of about 6.89 U/mg.In conclusion,this thesis has effectively created a chemical enzymatic synthesis technique for the production of nitriles from amines and aldehydes based on the catalytic characteristics of aldoxime dehydratase.This study provides suggestions for nitrile synthesis that is stable and effective,and continuously promots the advancement of green manufacturing techniques for nitrile chemicals with high added value.Aldoxime dehydratase’s catalytic activity has also been enhanced through semi-rational protein design and covalent immobilization,serving as a model for directed enzyme modification,hastening the industrialization of enzymes,and has a wide range of potential applications.