Optimization Of D-mannose Isomerase Expression System Using Short-chain Antisense Rna And Its Aplication Research

With the gradual deepening of scientific research,it has been found that functional D-mannose has broad application prospects in immune function regulation,cancer prognosis markers,tumor suppression and urinary tract infection treatment,and food additives.D-mannose isomerase can convert fructose into mannose by enzyme catalysis.Fructose as a raw material is low in cost and easy to obtain,and bio-enzymatic method is a very efficient,green and economical production method,so the research on the expression of D-mannose isomerase in genetically engineered bacteria is of great value.The recombinant D-mannose isomerase gene is regulated by the lactose operon,which is an inducible expression system and is not suitable for industrial production.Although lactose is usually used as an inducer in industrial production,the amount is still large.A large part of the cost,the subject decided to use antisense RNA technology to block the expression of the repressor gene lacl in the lactose operon,thereby releasing the repressor and removing the inducer to make it a constitutive expression.The subject first solved the problem of the unstable passage of the plasmid in the E.coli host.The stable expression vector was constructed by introducing the parDE gene(Post-segregational killing system,PSK)into the pET vector,and the vector was stably passaged without antibiotics.The results indicated that the expression vector BL21(DE3)/pET-parDE-Fmi retained 100%plasmid stability after being transferred 20 times.Then the antisense RNA sequence targeting the lacl gene was designed.The first generation antisense expression vector BL21(DE3)/pET-SRFmi was constructed,and D-mannose isomerase could be expressed without the inducer,the enzyme activity of D-mannose isomerase was 34.5 U/mL.Subsequently,by optimizing the recognition region,the length of the sequence,the promoter in the transcriptional element,and the terminator of the antisense RNA,it was found that the optimal structure of the antisense RNA was that the recognition region was 60 bp from the start codon of the mRNA,and the T7 promoter was used.The final enzyme activity was 42.3 U/mL.The study on the enzymatic properties of D-mannose isomerase showed that the optimum catalytic temperature of D-mannose isomerase was 60?,and the stability was better in the temperature range of 30?40?.The optimum catalytic pH was 7.0,good stability in the range of pH 7.0?9.0.The Michaelis constant Km of D-mannose isomerase is 292.4 mM/L,and the maximum reaction rate Vmax is 0.139 mM/(L·s-1).Metal ions Mn2+,K+,Na+,Mg2+,Ca2+,Cu2+,Zn2+,Fe2+promoted the activity of D-mannose isomerase,and K+and Mg2+had the strongest improvement effect,and the enzyme activity increased by 47%and 45%,while Mg2+,Ca2+,Cu2+,Zn2+and Fe2+increased the enzyme activity by about 33%,and Na+increased the enzyme activity by only 29%.The subject explored the separation process conditions of fructose and mannose.The results showed that the best separation effect can be obtained when the separation condition was CRS-1Ca ion exchange resin with a loading of 2 mL,an elution flow rate of 1.6 mL/min,and a column length of 2 m.The resolution reached 0.731 and the recovery of mannose reached 95%.According to these data,an 8-column simulated moving bed with a processing capacity of 1 kg/h was designed.The final calculation result showed a circulating flow rate of 23.96 L/h,and the extraction liquid flow rate(fructose-containing)was 14.63 L/h.The flow rate(mannose-containing)was 14.28 L/h,the length of each column was 1.642 m,the inner diameter of the column was 0.066 m,and the switching period of the inlet and outlet was ?T of 12.3 min.