Molecular Modification,Targeted Exploration And Food-grade Expression Of Cellobiose 2-epimerase

Food enzymes have a promising industrial application prospect.The enzymes are proteins whose structures are delicate and complex.Currently,the researches on food enzymes primarily focus on applications,but the researches at the theoretical level is little.And the studies of food enzymes at the molecular level are still in the early stage.To solve this problem,the project carried out the study on cellobiose 2-epimerase?CE?.The structure and function of CE are studied theoretically.For the applications of this enzyme in the dairy industry,the molecular modification,targeted exploration,and food-grade expression of CE were carried out by using Molecular Dynamics?MD?simulation and molecular biology techniques.CE is an emerging enzyme in the dairy industry.It can catalyze the isomerization and epimerization of lactose to lactulose and epilactose without using coenzymes or co-substrates.Lactulose and epilactose are functional sweetener with bioactivities which are good for health.However,the currently reported CEs are still of low activities and low thermostabilities,which is hard to satisfy the needs of the industrially high-efficient productions in different working environments.The development of computational techniques and resources provide new ideas for the theoretical research of enzymes.With the help of computational tools,the structural stabilities and mechanisms of enzymes can be studied at the molecular level.In this project,the reported CE was modified by semi-rational method.The activities of the computationally designed mutants were measured.The isomerization activity and thermostability of CsCE-M5/E327D were improved.The lactulose produced by the mutant within 1 h was higher than that produced by the wild-type CE within 4 h under the same condition.The structural flexibilities of the CEs were studied by using MD simulation.The results showed that the computational data?RMSD and hydrogen bonds?of the enzymes have correlations with the experimental data of the enzymes.Compared with the predictors using static information,the methods using dynamic information can predict the thermostability more accurately.The catalytic mechanism of CE was studied by MD simulation.The ring opening process of the substrate was proposed to be catalyzed by another residue.The rate-limiting steps of isomerization and epimerization reaction was determined and lay a foundation for the future protein design.The CE with a high flexibility at low temperature was discovered by searching in the protein sequence database.The plasmid including the gene of the CE from Roseburia intestinalis was transformed into E.coli BL21?DE3?for expression.The enzyme was purified by nickel-affinity chromatography.The relative molecular mass of the purified enzyme was measured to be 43,500.The optimum temperature and optimum pH for the enzyme-catalyzed reaction were 45? and 7.0,respectively.The K_m,k_cat,and k_cat/K_m values of Roin-CE for lactose epimerization were 429.9±57.3 mM,117.0±7.7 s^-1,and 27 mM^-1 s^-1,respectively.The K_m,k_cat,and k_cat/K_m values of Roin-CE for cellobiose epimerization were 131.0±11.6 mM,98.5±2.9 s^-1,0.75 mM^-1 s^-1,respectively.At 8?,the epimerization activity of Roin-CE was 18.5-fold higher than that of Dith-CE,of which the epimerization activity was the highest among all the reported data.Roin-CE is more suitable for epilactose production at low temperature than the other CEs.MD simulation can help the exploration of wild-type CE more efficiently and rationally.The genes of CE together with antibiotic resistance genes were integrated into the chromosomes of B.subtilis 1A751 respectively.The antibiotic resistance genes were knocked out by the Cre/lox system from the recombinant strains.The maximum volumetric activity of the fermented broth of the recombinant B.subtilis based on chromosomal integration was 0.325U/mL;The replicative plasmid containing CE gene and D-alanine racemase gene was designed.These antibiotic resistance gene-free plasmids were constructed in the auxotrophic strains that are deficient in biosynthesis of D-alanine.The maximum volumetric activity of the fermented broth of the recombinant B.subtilis based on the replicative plasmid was more than 7 U/mL.The epilactose production activity of ethanol treated cells was 3.67-fold higher than that of untreated cells.An enzymatic route including degradation of the lactose,yeast fermentation,and cation exchange chromatography was constructed for further epilactose purification.The epilactose with a purity>98%was produced with a yield of 24.0%.