Biotransformation Of L-tagatose By Using Recombinant Bacteria And The Process Optimization

L-tagatose is an important rare sugar,a functional sweetener with many important physiological functions,and also the precursor of many high value-added composite materials.At present,the production method of L-tagatose is mainly by biotransformation,but the previous biotransformation method has the disadvantages of low conversion rate and high production cost,which has caused the limit of the industrial production of L-tagatose and cannot meet the gr owing demand.In this study,the bioconversion method used the high conversion rate Galactitol Dehydrogenase(GatDH)derived from Rhodobacter sphaeroides D as the catalytic enzyme,and the relatively cheap galactitol was used as the substrate to successfully produce L-tagatose.This study cloned the galactitol dehydrogenase(GatDH)(Genbank:ACM89307.1)from Rhodobacter sphaero ides D.Then the recombinant plasmid pET-Duet-1-gdh was constructed and successfully transformed into E.coli Rosetta(DE3).Then successfully expressed the galactitol dehydrogenase protein(GatDH),and the relative molecular mass of the enzyme was about 28 kDa.The conditions for inducing expression of recombinant bacteria were optimized,and the results showed that the optimal IPTG concentration was 0.5 mM,the optimal induction temperature was 30℃,and the induction was overnight.Large-scale applications require the supply of oxidative cofactors.We have constructed Escherichia coli Rosetta(DE3)(pETDuet-1-gdh-smnox)that simultaneously express galactitol dehydrogenase(GatDH)from Rhodobacter sphaeroides D and highly active S.mutant water-producing NADH oxidase(SmNox),using the recombinant bacteria as a biocatalyst to catalyze galactitol to L-tagatose,and its catalytic conditions were optimized to achieve efficient NAD+regeneration and L-tagatose production,try to establish a clean double-enzyme method for producing L-tagatose.The experimental results showthat SmNox has no effect on the production of L-tagatose.One is that the expression of enzyme protein is too low,and the other is that E.coli itself contains sufficient NAD+to meet the reaction requirements.Then,a series of optimizations on the fermentation medium and transformation reaction conditions have been made to obtain higher yield of L-tagatose,the best medium was:NH4Cl(1.5 g/L),Na2HPO4 12H2O(27 g/L),MgSO4(0.1 g/L),NaCl(0.5 g/L),KH2PO4(2.7 g/L),glycerin(25 g/L),corn steep liquor(15 g/L).The optimal reaction conditions are:pH8.0,40℃,and optimal cell dosage OD600=75.On the basis of the optimization of shake flask fermentation culture,further expand the culture.Optimized at the fermenter level,the constant-rate fed culture has achieved a conversion rate of 98%.The 40 g/L galactitol is almost completely converted into the product L-tagatose,which is an increase of 33%compared to the shake flask level,which provides a good foundation for future industrial production.At last,the sodium alginate embedding method was used to immobilize GatDH to produce L-tagatose.The best immobilization conditions were:sodium alginate concentration 3%,The cell embedding OD600 is 70,the CaCl2 concentration is 2%,the immobilization time is 4 h,the optimal concentration range of glutaraldehyde is 0%to 0.05%,and the cross-linking time is 4 h.The optimal reaction conditions are:45℃,pH 8.5.The stability and pH tolerance of immobilized cells continue to improve,and the temperature stability has increased by 20%;the immobilized enzyme activity is still high after being placed under alkaline conditions for 4 hours At 75%.Compared with current chemical and biocatalytic methods,the developed strategy avoids the formation of by-products and achieves the highest yield of L-tagatose at low cost.