Characterization,application And Thermostability Engineering Of Recombinant Sucrose Phosphorylase

Sucrose phosphorylase(SPase)catalyzes sucrose phosphorylation to produce α-Dglucose-1-phosphate and D-fructose and their reversible reactions.The SPase has glucosyl transfer ability and can transfer one glucosyl group of sucrose molecule to different receptors.It has a wide range of applications in food,medicine,cosmetics and other fields.In recent years,the use of SPase to produce fine chemicals such as α-arbutin and 2-O-α-D-glucosylglycerol(αGG)has received increasing attention.However,the low expression of SPase,low enzyme activity and poor thermostability have directly affected the industrial application of SPase.In this study,a genetically engineered strain with high yield of SPase was constructed to prepare recombinant SPase.the recombinant SPase was used to produce α-arbutin and αGG.And then we use the semi-rational strategy to increase the thermostability of SPase.The main contents are as follows:1.the SPase gene from L.mesenteroides was synthesised after codon optimization and heterologous expressed in E.coli.The recombinant SPase was purified and the enzyme activity was 214.0 U·mg-1,and the purification ratio was 1.5-fold,and the enzyme activity recovery rate was 87.8%.Enzymatic identification results showed that the optimal temperature of the recombinant SPase was 45℃ and the optimal p H was 6.5.When the temperature was not higher than 40℃,the enzyme activity of the recombinant SPase was relatively stable.It can still maintain more than 95.0% of the activity after incubated at 40℃ for 1h,the tolerability under the optimal temperature 45 ℃ was determined,and the half-life was 3.9 h;It was relatively stable from p H 5.0 to p H 7.5,when the p H was higher than 7.5,the enzyme activity decreases rapidly.When the metal ions concentration of Co2+ and Fe3+ was 1 mmol·L-1,they can activate recombinant SPase,but the same concentration of Ni2+,Mn2+,and Cu2+,they can inhibit it.When the metal ion concentration was 5 mmol·L-1,Ni2+,Mn2+,Cu2+,and Zn2+ had a strong inhibitory effect.The Km of SPase for sucrose was 128.8 mmol·L-1,the Vmax was 2.2 μmol·mL-1·min-1,and the kcat was 39237.9 min-1.2.Recombinant SPase catalyzes the synthesis of α-arbutin from hydroquinone.The optimal conditions were 40.0 g·L-1 hydroquinone,5:1 molar ratio of sucrose and hydroquinone,and 250 U·m L-1 recombinant SPase at p H 7.0 and 30℃ for 24.0 h in the dark,and then 500 U·m L-1 glucoamylase was added at 40℃ for 2.5 h.Under the optimized process,the yield of α-arbutin reached 97.9 g·L-1,and the hydroquinone conversion rate was close to 99.0%.After that,the macroporous adsorption resin H103 was used to purify α-arbutin.The purity of the purified α-arbutin was 99.5%,and the recovery rate was 90.4%.3.Catalytic synthesis of αGG using recombinant SPase.The optimized reaction conditions were: 1.2 mol·L-1 sucrose,3.2 mol·L-1 glycerol,and recombinant SPase 30 U·m L-1 in 50mmol·L-1 MES buffer(p H 6.5)at 37℃,and the Speed 200 r·min-1 for 72.0 h.The yield of αGG reached 191.8 g·L-1.4.Thermostability engineering of SPase by semi-rational strategy and obtained two positive mutants with improved thermostability.Based on homology modeling,a threedimensional simulation structure of wild-type SPase was constructed,and Fire Prot was used to predict the stability of the simulated structure,a small mutation library was constructed,and four mutants were preliminarily selected,and then the combination mutation was conducted combined the forward four mutation points.The final measurement results showed that the melting temperature Tm values of the combination mutant and single point mutant T219 L were increased by about 4℃ compared with the wild-type.The half-life of the combination mutant and single point mutant T219 L at 50℃ were about 50 min,which were about 35 min higher than that of the wild type,and had better temperature stability.