Construction Of Genetic Engineering Strain Using ?-galactosidase And L-arabinose Isomerase And Biosynthesis Of D-tagatose

D-tagatose is a natural low-calorie sweetener.It has been widely used in the food field and medicine because of its potential functions in improving intestinal flora,lowering blood sugar,preventing colon cancer and anti-caries.However,due to the current low production of D-tagatose,it is difficult to satisfy the growing demand.The chemical method of producing D-tagatose has a series of drawbacks including intense pollution,high cost and complex purification process.Recently,biosynthesis of D-tagatose has been the focus of researchers.The theory of D-tagatose biosynthesis is that the D-galactose is isomerized by L-arabinose isomerase(L-AI)to produce D-tagatose,and the D-galactose can be produced by hydrolysis of lactose with?-galactosidase(?-GAL).The whey is a by-product of dairy industry which contains70%-80%lactose.Though research has been done on the production of D-tagatose using enzyme method,it is still difficult to realize the commercial value because of limitations such as inadequacy of enzymatic property and high cost of enzyme purification.Therefore,this study focused on the development of new sources of?-GAL and L-AI,construction of the corresponding engineering strain,investigation of their enzymatic property and production of resting cell to achieve efficient conversion from whey to D-tagatose.The main research contents and results are as follows:(1)A 2830 bp of lac LM from Lactobacillus reuteri ATCC 53608 encoding?-GAL(hereafter,LRGAL)was amplified and introduced into E.coli BL21 to construct recombinant strain,E.coli BL21/p ANY1-lac LM.The LRGAL was expressed by induction with IPTG and analyzed in SDS-PAGE that indicated two bands for L and M subunits at 73 k Da and 35 k Da,respectively.The specific activity of LRGAL was found to be 25.63 U/mg.The LRGAL showed its highest activity at 50?and p H 6.5 in the presence of Ca²⁺and Mn²⁺.The K_m,V_max and K_cat/K_m of LRGAL were found to be 2.19m M,21.68 U/mg and 9.3 m M^-1min^-1for o NPG(o-nitrophenol-?-galactoside).Lactose hydrolysis from of whey powder was done using the resting cells of BL21/p ANY1-lac LM with singal factor technique under various conditions.The optimal temperature and p H for lactose hydrolysis were found similar to those of enzyme activity(50?and6.5,respectively),while the optimal cell concentration was 30 g/L(wet cell weight,WCW)at a reaction time of 40 h.No apparent substrate or product inhibition was observed.(2)A 1425 bp of ara A from Lactobacillus parabuchneri CICC 6004 that encodes L-AI(hereafter,LPAI)was amplified and introduced into E.coli BL21 to construct E.coli BL21/p ANY1-ara A strain.The LPAI was expressed by induction with IPTG,which showed a molecular weight of 55 k Da during SDS-PAGE analysis.The maximum specific activity of LPAI was found to be 15.52 U/mg at the optimal temperature 45?,p H 6.7 and 3 m M of Ca²⁺.The K_m,V_max and K_cat/K_m of LPAI were found to be 4.3 m M,63.1 U/mg and 5.9 m M^-1min^-1 for L-arabinose,respectively,while the respective values for D-galactose were 5.4 m M,35.5 U/mg and 2.8 m M^-1min^-1.The optimal condition for D-tagatose biosynthesis from D-galactose using resting cell was done using singal factor technique under various conditions.The results indicate that the optimal temperature for D-tagatose production was 45?,p H 6.7,cell concentration30 g/L WCW,substrate concentration 140 g/L,and reaction time 50 h.(3)D-tagatose biosynthesis was done from lactose in milk whey powder using coculture of two strains to get the optimal conditions using singal factor technique.The optimal conditions for this resting cell biotransformation consisted of temperature 45?,p H 6.7,a mass ratio of E.coli BL21/p ANY1-lac LM to E.coli BL21/p ANY1-ara A strains 1,cell concentration 50 g/L WCW,substrate concentration 120 g/L and reaction time 50 h.Under the optimum conditions,25.3 g/L of D-tagatose was produced with a conversion efficiency of 21.1%.Subsequently,two-stage biotransformation was done to maximize the conversion of lactose and production of D-tagatose.The concentration of D-tagatose after the first step was 25.3 g/L,which increased to 37.7 g/L after the second stage.The conversion efficiency increased from 21.1%at the first step to 31.5%at the second stage.