Study On Phosphoglucomutase And Its Regulation Of Cordyceps Militaris

The polysaccharide of Cordyceps militaris is the main active ingredient of Cordyceps militaris and has a variety of biological activities.It plays an important role in human health and has become a very commol/Lon functional polysaccharide.The preliminary work of our laboratory found that phosphoglucomutase was a key enzyme in the synthesis of polysaccharides from Cordyceps militaris.In order to improve the polysaccharide yield of Cordyceps militaris,this paper was aimed at the extraction,separation,enzymatic properties and regulation of phosphoglucomutase.Firstly,phosphoglucomutase was extracted by homogenate extraction method.Then,separated and purified by ultrafiltration,salting out,ion exchange chromatography and molecular sieve chromatography.The purified phosphoglucomutase was obtained with a fold purification of 4.23 times and a recovery of 20.01%.The specific activity was 356.30±2.45 U/mg protein,the total activity was 8593.34±10.41 U.SDS-PAGE analysis showed that the molecular weight was about 50.01 kDa.The study of enzymatic properties showed that the optimal reaction temperature,pH and reaction substrate concentration of phosphoglucomutase was 30?,7.5,and 0.3 mmol/L,respectively.It was a kind of enzyme that was not resistant to high temperature and weak acid and weak base.The Michaelis constant was 11.62 mmol/L,and its maximum velocity was 416.67 nmol NAD(P)H(mg protein)-1 min-1.The effects of organics and metal ions on the activity of phosphoglucomutase was evaluated,the results showed that glycerol had a weak activation.Mercaptoethanol,SDS and DTT had certain inhibiting effects,and the inhibiting effects of DTT was higher than that of mercaptoethanol and SDS;Li+,Cu2+,Na+,Mn2+and Al3+ had different degrees of inhibiting effects.Li+and Cu2+had strong inhibiting effects.Zn2+,Mg2+,Ca2+,Fe2+and Fe3+had different degrees of activation,in which the activation of Zn2+was strongest.By adding Zn2+ into the culture medium,the influence of its type,concentration and additive time on specific activity of phosphoglucomutase was explored.The study shown that the best condition for improving the specific activity of phosphoglucomutase and the yield of polysaccharides in Cordyceps militaris was the addition of 0.5 mmol/L ZnSO4 into the liquid medium at 0 h.By screening of liquid culture condition,the total activity of phosphoglucomutas-e was regulated.The results showed that the optimal fermentation condition for the polysaccharides from Cordyceps militaris and the total activity of phosphoglucomutas-e were that carbon source,nitrogen source and inorganic salts were 6%sucrose,4%beef extract and 0.05%CaCO3,respectively.The optimal pH,inoculum size and shaking speed were 7,9%and 160 r/min,respectively.Through verification,the yield of polysaccharide was 1.32 times higher than that of the control group,the total enzyme activity was increased by 1.27 times,and the gene expression amount was increased by 2.60 times.