Research Of Nitrite Reductase In Lactobacillus Plantarum

Nitrite in food and drinking water is very harmful to human health. Lactobacillusis a probiotic bacterium that can produce nitrite reductase. Nitrite reductase hasattracted research interest in recent years because its potential application in foodindustry and dinking water. In this paper, we used Lactobacillus plantarum, whichwas screened and identified in our laboratory, produces nitrite reductase, but enzymeactivity is not enough as we need. By mutagenesis and optimizing the medium, theenzyme production was improved. The enzyme purified from cells, and the enzymaticproperties was studied. In addition, we studied the protein expression and purificationof engineering bacteria which had been structured successfully in our laboratory. Therecombinant protein was able to degrade nitrite. Results as the follows:The original strain Lactobacillus plantarum was mutated by UV, then the newstrain D27was screened. Its nitrite reductase was13.4U/mL, which incerased by22.9%as compared with the starting stain. Another new strain DS31mutated byNaNO2was screened after the strain D27, and its enzyme productivity incerased by6.7%as compared with the strain D27. The nitrite reductase productivity was stablewhen the mutant strain was propagated to the fifth generation. The effects ofnutritional conditions on nitrite reductase production by Lactobacillus plantarumDS31were studied. The optium medium were: Glucose20.97g/L, Beef extract10.l9g/L, Soy peptone10.19g/L, NaAc5.00g/L, Ammonium citrate2.00g/L,K₂HPO₄·3H₂O2.00g/L，MgSO₄·7H₂O0.60g/L, MnSO₄·H₂O0.27g/L, Tween801mL/L.By using the enzymolysis combined with ultrasonic cell-wall breaking method toextract crude enzyme, the liquid enzyme activity reached1347.6U/mL. Through thesteps as ammonium sulfate salting-out, DEAE52ion exchange chromatography andSephadex G150gel filtration, the nitrite reductase was purified. Its subunit molecularwas67.6kDa. The optimal temperature for enzmye was35℃. The enzyme stillremained52.7%activity under the condition of65℃after6h, this indicated that it hasgood thermal stability. The optimal pH for enzmye was6.0, and it was relatively stable with the scope of5.6to7.2. Research found that Ca²⁺、Mn²⁺Cu²⁺and Ba²⁺could improve enzymatic activity and Cu²⁺could significantly improve the enzymeactivity（the relative enzyme activity reached183%）. The nitrite reductase wasinhibited by Zn²⁺+、Cd²⁺、K⁺and strongly inhibited by EDTA(Mg²⁺23、Fe+、Al+hadno evident effects on it). With sodium nitrite as substrate, the enzyme Km and Vmaxwere22.88mmol/L and1.94nmol/min μL.After inducing and wall-breaking the engineering bacteria, the recombinantproteins were divided into supernate and precipitation. Through nickel affinitychromatography and Sephadex G-150gel chromatography, the recombinant proteinyield in supernate reached58%. Inclusion body protein yield reached84%afterwashing. After denaturing in urea，Inclusion body protein yield reached44%and25%by dialysis and nickel affinity chromatography.