Molecular Modification And Targeted Exploration Of D-Mannose Isomerase

D-Mannose is a functional sugar and performance in regulating the immune system,anti-inflammatory,anti-tumor,antibacterial,etc.It has been widely used in medicine,food and daily chemical industry.At present,the production of D-mannose mainly relies on plant extraction and chemical synthesis.However,the plant extraction is limited by external factors such as environment,season,and region,while the chemical synthesis is not adequate for health demand of consumers due to its harsh reaction conditions and various by-products.Thus,enzymatic catalysis has attracted public attention due to its advantages of green,mild,and high specificity.D-mannose isomerase（MIase）is one of the key enzymes in D-mannose enzymatic production.However,the low enzyme activity and poor thermal stability of MIase severely limit its industrial production.Improving the enzymatic properties of known MIases by molecular modification and mining the gene fragments encoding MIase with high activity and good stability have become the research emphases of this enzyme.In order to provide a new insight into the molecular modification of MIase and a candidate enzyme for industrial production of D-mannose,one the one hand,this paper conducted site-specific saturation mutations on the MIase encoded by the yih S gene with known structure and catalytic mechanism.Mutants with high catalytic level and stability were screened out by a new and effective high throughput screening method.After purification,the enzyme properties of the mutants were further determined,and the relationship between enzyme function,structure and catalytic mechanism was studied.On the other hand,a novel D-mannose isomerase with good enzymatic properties was targeted by gene mining technology.Its structure,catalytic center and key amino acids were studied by homology modeling and molecular simulation.That provided a candidate for industrial enzymatic production of D-mannose.The main research contents and results are as follows:1.The recombinant plasmid p ET-22b（+）-yihs was used as a vector to carry out site-saturation mutation based on R335,E363 and G377 loci selected through molecular dynamics simulation.Through the resorcinol hydrochloric acid method for one round of high-throughput screening and the high-performance liquid phase method for two rounds of screening of crude enzyme solutions,the mutants E363R,R335T,E363V and R335F were preliminarily obtained.The relative enzyme activity of E363R and R335T were higher than 300%.R335T and E363V had good thermal stability and R335F had good acid resistance.After being purified,the enzymatic properties and structural changes of the mutants were determined.The results indicated that the specific enzyme activities of R335T and E363R were 128%and 85%higher than that of wild-type（WT）,respectively.The thermal stability of R335T,E363R and E363V increased by 141%,20%and 28%at 40℃,respectively.The protein structures of R335F,R335T,and E363V have more tyrosine（Tyr）and tryptophan（Trp）residues exposed compared to the wild-type.Theβ-sheet andβ-turn contents of four mutants decreased compared to WT,and the content ofβ-turn of R335T was 7.31%less than that of WT,the content of random coil was 41.63%that was much higher than that of WT,and the content ofα-helix was 2.17%higher than that of WT.2.R335T showed the best thermal stability,the highest enzyme catalytic activity and good acid-base stability among the four mutants,so in-depth study of the structure was of theoretical guiding significance for the molecular modification of R335T.The degree of extension freedom of threonine（Thr335）residue in R335T was smaller than that of arginine（Arg335）residue in WT,and electrostatic redistribution around Thr335 reduced the positive charge.The root mean squared error（RMSD）value of R335T was lower than that of WT,indicating carbon skeleton structure tended to be stable.R335T had higherα-helix content than WT,which could maintain the carbon backbone structure and stability better than WT.The flexibility of loop129～136 above the active center of R335T was better than that of WT,which was more conducive to the substrate entering the active center through the catalytic channel,which was closely related to the higher enzyme activity of R335T.3.The affinity of 19 amino acid sequences of D-mannose isomerase with mannose mined from database was evaluated by Lib Dock Score,and the enzyme activity was used as screening index,it was found that the new D-mannose isomerase from Bifidobacterium（Bifi-MIase）had higher optimal reaction temperature and better thermal stability.Bifi-MIase exhibited the highest activity at55℃,p H 7.5,with enzyme activity of 407 U mg^-1.Its half-lives(t_1/2)at 45,55,60,65 and 75℃were 762.63,483.70,70.8,54.54 and 19.91 min,respectively.Bifi-MIase could catalyze the conversion of fructose to mannose,with a conversion rate of about 29%.The kinetic parameters K_m,V_max and k_cat/K_m of Bifi-MIase were538.7±62.5 m M、11.7±0.9μmol mg^-1 s^-1 and 1.02±0.3 m M^-1 s^-1,respectively.The overall structure of Bifi-MIase,the residues forming the binding pockets and deep fissure,were highly similar to Salmonella MIase（Se Yih S）,which was speculated that His387 may be the main amino acid residue of Bifi-MIase to open the pyranose ring,and the His253 residue was the main acid base catalyst.