Study On The Discovery And Molecular Engineering Of Monooxygenases And Its Application In Cascade Reactions

Hydroxylation is an important C-H bond activation reaction that can achieve the synthesis of hydroxyl compounds with higher added value from inert C-H activation.The development of highly active and regioselective hydroxylation biocatalysts is of important research significance and application value.Through screening the monooxygenases library,we found that cytochrome P450BM3 from Bacillus megaterium has the activity to hydroxylize cyclohexane and p-xylene.Through semi-rational engineering the hydroxylation activity of2,5-dimethylpyrazine（DMP）was achieved from scratch,and several mutants were obtained with high regioselectivity and hydroxylation ability toward substrate analogues.Different multi enzyme cascade reaction systems were further developed to synthesize products with higher added value.The main research content is as follows:（1）Screening of monooxygenases.Aiming at higher catalytic activity and solubility,a monooxygenases library was screened and six different sources of single oxygenases were obtained.Among them,P450BM3 exhibited certain hydroxylation activity,and wild-type（WT）could hydroxylize cyclohexane（CHX）and p-xylene（PX）with total turnover number（TTN）of 13.8 and 7.3,respectively.Moreover,P450BM3 is a type of solublely expressed self-sufficient monooxygenase.Therefore,P450BM3 was regarded as the starting enzyme for semi-rational engineering to futher increase the catalytic performance.（2）Semi-rational engineering of P450BM3.Based on,molecular docking analysis,nine potential sites L75,V78,F81,A82,F87,L181,I263,A264,A328 within the range of 10（?）near the substrate were selected for site directed mutagenesis to obtain mutants with significantly enhanced hydroxylation activity.Among them,A82F and A328F displayed increased activity toward CHX to 12.5 and 13.4 times compared wiht WT,and 27.7 and 33.4times toward PX,respectively.The double mutant P450BM3_A82F/A328Fshowed a 30.4 and 65.6folds increase in activity toward CHX and PX compared with WT,respectively.Furthermore,based on the differences in substrate structure a new round of semi-rational engineering was conducted for increasing the hydroxylation activity toward DMP.Three single mutant L75E,F82E,and F328E were obtained by performing aspartic acid and glutamic acid scanning on the above nine key sites,with TTNs of 3.3,11.9,and 13.7,respectively.Double mutant L75E/F82E had a TTN of 14.6,and tirple mutants L75E/F82E/F328S and L75E/F82E/F328T had TTNs of 15.5 and 26.1,respectively.A breakthrough in DMP hydroxylation activity from scratch was achieved.（3）Development of a multi enzyme cascade reaction system.To achieve efficient synthesis of various high value-added products,P450BM3_A82F/A328Fand P450BM3_{L75E/F82E/F328T}were coupled with benzyl alcohol dehydrogenase（BADH）and benzaldehyde dehydrogenase（BLDH）from Pseudomonas putida to construct a multi enzyme cascade reaction for separate catalyzing substrates PX（10 m M）and DMP（5 m M）.Within 6 hours of the reaction,the yields of p-methylbenzoic acid and 5-methylpyrazine-2carboxylic acid were 73.5%and37.2%,respectively.Cascade reaction was constructed by coupling P450BM3_A82F/A328Fwith alcohol dehydrogenase Kp ADH and monooxygenase AjBVMO from Acinetobacter johansonii to catalyze substrate CHX（10 m M）.Within 6 hours,the yield ofε-caprolactone reached 68.4%.To further promote the synthesis ofε-caprolactone,two multi enzyme cascade reactions were designed and optimized.Substrate supplied enzyme cascade reaction was constructed using Kp ADH_T215Vand AjBVMO for the synthesis ofε-caprolactone from cyclohexanol.As much as 100 m M cyclohexanol was oxidized to 68.6 m Mε-caprolactone,with a maximum TOF of 17.8 min^-1and TTN of 1273.Product convergent enzyme cascade reaction was developed employing alcohol dehydrogenase Cg KR1 from Candida glablata and AjBVMO for the synthesis ofε-caprolactone from cyclohexanone and 1,6-hexanediol.As much as 115.1 m Mε-caprolactone was obtained from 100 m M cyclohexanone,with a maximum TOF of 9.2 min^-1and TTN of 1231.These two enzyme cascade systems can also be used for synthesisγ-butyrolactone andδ-pentolactone.From the perspectives of product concentration and green chemistry,the product convergent system is more promising than the substrate supply system.