Heterologous Expression,Isolation,Purification And Enzymatic Properties Of Biogenic Amine Degrading Enzymes From Lactobacillus Plantarum

Biogenic amine(BA)is a product produced by amino acid decarboxylation during fermentation,especially in fruit wine.Among them,histamine has the highest content and the most toxic.In this work we investigated two novel histamine degrading enzymes,namely glyceraldehyde 3-phosphate dehydrogenase(GAPDH)and enolase from Lactobacillus plantarum.Two different expression vectors were successfully constructed,and the heterologous expression and purification of GAPDH and enolase were achieved The enzymatic properties of the two enzymes were studied1.Constructed two different expression vectors in two modes(with and without histidine tag),cloned GAPDH and enolase and successfully introduced the recombinant plasmid into E.coli host2.The E.coli introduced into the recombinant plasmid was successfully expressed Unlabeled and labeled GAPDH were expressed in E.coli DH5? and BL21(DE3),respectively,and the labeled enolase was successfully expressed in E.coli BL21(DE3)Then using different purification techniques,GAPDH without histidine tag was separated and purified by ammonium sulfate precipitation(optimal saturation 70%?80%)and Superdex 200 gel filtration to obtain pure enzyme.After labeling GAPDH and enolase,the wall was successfully separated and purified by Ni column to obtain pure enzyme Unlabeled GAPDH was purified 2500 times,the molecular weight of the subunit was 36 kDa,and the specific activity was 12.50 U/mg.His-GAPDH was identified with a 39.9 kDa subunit,a specific activity of 1.93 U/mg,and a yield of 45.2%.The enolase was purified 3.89 times,the molecular weight of the subunit was 51.58 kDa,the specific activity of 6.20 U/mg and the yield of 22.7%3.The enzymatic properties of the purified enzyme were studied.The optimal pH and temperature of the two enzymes were 5.5 and 40?,respectively.In addition,GAPDH is sensitive to high temperature and is almost inactivated at 70?.It has high stability at pH 5.5-8.5;they are acid resistant,but moderately sensitive to high temperature.For enolase,the optimal temperature is at 50?,and it can maintain its activity in the temperature range of 20-70?,which is resistant to high temperatures;it has the highest activity at pH 7.5 and has a wide range of pH resistance.(pH 4.5-9.5)4.Ethanol and sulfur dioxide have little effect on the activity of recombinant GAPDH and enolase.These properties indicate that recombinant GAPDH and enolase have potential for application in the brewing industry.