Expression Of Pseudomonas Geniculata AGE Family Epimerase/isomerase In Escherichia Coli And Its Application In D-mannose Preparation

D-mannose is a kind of six-carbon sugar,which is mainly used as a food supplement.It also has physiological functions such as preventing urinary tract infection and anti-inflammation.It has been widely used in medicine,food,feed industry and life sciences.At present,the industrial preparation of D-mannose is mainly carried out by biological enzymatic method.Compared with the plant extraction method and the chemical method,the enzymatic preparation of D-mannose has the advantages of low production cost,high conversion efficiency,simple reaction process and mild reaction conditions.D-mannose isomerase?D-MIase?catalyzes the reversible isomerization reaction between D-fructose and D-mannose and is a key enzyme in D-mannose production.In this study,AGE family epimerase/isomerase?AGEase?derived from P.geniculata was expressed in E.coli BL21?DE3?firstly and characterized as D-MIase by enzymatic properties analysis.Recombinant P.geniculata D-MIase mutant with improved thermal stability was obtained by site-directed mutagenesis,and the process of producing D-MIase using D-fructose as substrate was optimized.The high-efficiency expression of D-MIase was achieved by 3-L fermentor fermentation optimization.In order to explore the reason for the high expression level of recombinant D-MIase,further studies on protein unfolding/refolding were conducted.The main findings are as follows:?1?The gene age of AGEase was obtained from P.geniculata by PCR technique,and the recombinant plasmid pET-24a-age was constructed and transformed into E.coli BL21?DE3?.The recombinant strain E.coli BL21?DE3?/pET-24a-age was cultured for 24 h in shake flasks,the intracellular enzyme activity reached 284790 U·mL^-1,and the protein expression was 5.3mg·mL^-1.Protein electrophoresis showed a target band appeared at 43 kDa,indicating that AGEase derived from Pseudomonas geniculata was successfully expressed in E.coli BL21?DE3?.?2?The recombinant P.geniculata AGEase was purified by ammonium sulfate precipitation and Superdex 200?10/300 GL?gel column,and its enzymatic properties were investied.The results showed that the optimum temperature of recombinant P.geniculata AGEase is 60?,and the optimum pH is 7.5.At pH 7.5,the half-lives at 50?and 60?are 4h and 0.5 h,respectively.The K_m,k_catat and k_cat/K_m of recombinant P.geniculata AGEase for D-mannose were 48.74±8.4 mM,84062±725 s^-1,1724.7±86 s^-1·mM^-1,respectively,and the specific activity was 53208 U·mg^-1.The recombinant enzyme can be classified into the D-MIase of the YihS enzyme in the AGE enzyme family by studying the substrate specificity,enzyme reaction kinetics and three-dimensional structure active center analysis of the recombinant P.geniculata AGEase.?3?The recombinant P.geniculata D-MIase was modified by site-directed mutagenesis to obtain mutants C126A and E402W,the 50?half-life were 50%higher than the wild type.On the basis of this,the above two points were superimposed and mutagenized to obtain mutant C126A/E402W,and its half-life at 50?was increased by 75%compared with wild type.D-mannose was prepared by catalyzing D-fructose using the mutant C126A/E402W,and the process was optimized.The optimal conditions for the preparation of D-mannose by using D-fructose as the substrate using recombinant P.geniculata D-MIase are:The amount of recombinant P.geniculata D-MIase is 450 U·mL^-1,pH 7.5,50?,the substrate D-fructose concentration is 20%,at this time the enzyme conversion efficiency is up to 39.3%,and the D-mannose yield is 78 g·L^-1;when 30%D-fructose was used as the substrate,the yield of D-mannose reached a maximum of 104.4 g·L^-1,and the enzyme conversion efficiency was34.8%.?4?Explored the effect of fermentor induction temperature on the growth and enzyme production of recombinant E.coli BL21?DE3?/pET-24a-age in3 L tank.The results showed that when the induction temperature was 25?,the enzyme activity of recombinant P.geniculata D-MIase was the highest,reached 2535197 U·mL^-1,which was about 9 times of the shake flask level.The protein expression of recombinant P.geniculata D-MIase was the highest,reaching 50 mg·mL^-1,which is about 9.4 times of the shake flask level.In order to investigate the reason for the high expression level of the recombinant E.coli BL21?DE3?/pET-24a-age,further studies on protein unfolding/refolding were conducted.The study found that the slow rate of protein refolding helps its correct folding,and the soluble expression is higher.The protein refolding rate is too fast,which may cause the protein to fold incorrectly during expression and form a large number of inclusion bodies with low expression.