Purification And Characterization Of An Enzyme Capable Of Histamine Degradation From Lactobacillus Plantarum

Biogenic amines?BA?are toxic metabolites that are formed during the wine-making process.Lactobacillus plantarum has great potential in fruit wine,especially wine because of its effectively degradation of BA.However,there are academic divergences about the nature of the enzyme capable of biogenic amines degradation expressed by Lactobacillus plantarum.Therefore,it is an urgent problem to obtain the enzyme capable of histamine degradation expressed by Lactobacillus plantarum and reveal its chemical nature.Therefore,this study independently screened Lactobacillus plantarum which can degrade biogenic amines,and used it as the initial strain to isolate and purify the enzyme capable of BA degradation and study the properties of the enzyme.The main results are as follows:?1?A multifunctional lactic acid bacterias with biogenic amine degradation and organic acid degradation ability were obtained.Finally,a total of 346 LAB strains were obtained,among those,39 strains have the ability of BA degradation.Then we screened a multifunctional strain with excellent performance through screening for biodegradability and tolerance?low pH,high alcohol concentration and high SO₂ concentration?.Identification was carried out by 16S rDNA and rec A sequences and the strain turned out to Lactobacillus plantarum species.?2?The enzyme capable of histamine degradation of Lactobacillus plantarum SGJ-24 was screened and purified,and the electrophoretic pure protein was obtained.By measuring the activity of total liquid,extracellular fluid and intracellular fluid,it was found that the enzyme existed mainly in the intracellular fluid.At the same time,the best crushing condition was determined–5s/5s 10 min.After ammonium sulfate precipitation?optimum saturation 70%?,DEAE anion exchange chromatography and Superdex 75 gel filtration chromatography,the pure enzyme capable of hitamine degradation was obtained,and its molecular weight was about 36 kDa.?3?In addition,the biochemical characteristics and enzymatic properties of enzyme capable of histamine degradation of Lactobacillus plantarum SGJ-24 were analyzed.The research content includes N-terminal amino acid analysis,the effect of pH and temperature on the stability and activity of hisamine-degrading enzyme,the effect of metal ions and inhibitors on the enzyme activity.The final results are as follows:The N-terminal sequence of the enzyme is Ser,Val,Lys,Ile,Gly,Ile,Asn,Gly,Phe,Gly,Arg,Ile,Gly,Arg,Leu.After BLAST protein comparison,the essence of enzyme isGlyceraldehyde-3-phosphate dehydrogenase.The optimum reaction temperature is40?.The enzyme is stable below 55?and has a wide range of heat resistance.The optimum pH was pH 7.5,which could be stable under the conditions of pH 6.5^pH 8.5.DTT,EDTA,?-mercapto alcohol and Mg²⁺inhibited the activity of the enzyme.SDS,Fe²⁺,Fe³⁺,K⁺,Na⁺,Pb²⁺and Ca²⁺can promote the enzyme activity.