Study On Heterologous Expression And Enzymatic Properties Of Sialidase And Preparation Of Ganglioside GM1 By Biotransformation

In this work,recombinant Bacillus subtilis WB800N-pHT43-sia for secretory expression of sialidase was constructed,the conditions for sialidase production by recombinant strain were optimized.The sialidase activity reached 401 U/mL under the optimal conditions:concentration of revulsant 1.0 mM,induction time 12 h,temperature 25?,TB medium.Gangliosides(GLS)were converted into monosialoganglioside(GM1)using whole cells,10 g/L of GLS was completely converted to GM1 in a optimized cell system.The concentration of GM1 was increased from 0.12 mg/mL to 2.23 mg/mL.The enzymatic properties of recombinant sialidase were studied.The optimum temperature and pH of the recombinant sialidase were 370? and 6.5 respectively.The enzyme activity was relatively stable in the range of pH 5.5-7.0.The Km and Vmax ofsialidase were 0.15 mmol/L and 0.69 ?ml/min,respectively.The inhibitory type of sialic acid on sialidase was competitive inhibition and inhibitory constant was 0.15 mmol/L.Dialysis,an in situ separation method was applied to produce GM1,and in the result,the inhibition of sialic acid on sialidase was significantly released.The content of GM1 increased from 0.42 mg/mL to 10.88 mg/mL in the dialysis system under the conditions:50 g/L of substrate,10000 U/mL of enzyme,conversion time 12 h.The substrate completely converted to GM1,while the control had residual ganglioside GD1a and the content of GM1 only increased to 6.87 mg/mL.Dialysis effectively removed the inhibitory effect of sialic acid on sialidase and made the reaction go smoothly toward the target product.D301 resin was used to adsorb free sialic acid in the system.The adsorption rate of sialic acid using D301 resin was 89.2%,the desorption rate of formic acid to sialic acid was 83.7%,and the total recovery rate of sialic acid was 74.7%.D301 resin efficiently retrieved free sialic acid from system.