Study On The Amino Acid Decarboxylase Gene And Functional Characteristics Of Amine Producing Bacteria From Suan Yu

The formation of biogenic amines is an important safety hazard for fermented fish.Enterobacteria are very important amine producing bacteria,which secrete a variety of amino acid decarboxylases,and the corresponding amino acids are produced by decarboxylation.At present,there are many researches on amine producing microorganisms in fresh fish at home and abroad,but few studies on amine producing microorganisms in fermented fish products.In this study,the diversity of amino acid decarboxylase gene was detected by polymerase chain reaction?PCR?and biogenic amine content was detected by RP-HPLC;the effect of pH on the type and capacity of intestinal bacteria producing amines and the relationship among the amine producing pathways under acid stress were studied;K.aerogenes was also studied objective to study the enzymatic properties of histidine decarboxylase;to study the cloning and bioinformatics analysis of K.aerogenes 10C2 histidine decarboxylase gene.The specific contents and results are as follows:?1?In this study,the biogenic amines formation ability of 97 strains of intestinal bacteria in Suan yu was determined.97 strains were divided into four grades according to their ability to produce amines.Enteric bacteria produced particularly high amounts of putrescine,followed by histamine and cadaverine.About 33.0%?32/97?of the strains produced more than 900 mg/L putrescine.Meanwhile,The content of histamine was between 100 and 500 mg/L,accounting for41.2%?40/97?.However,no strain produced cadaverine content higher than 500 mg/L.The percentage of cadaverine content between 100 and 500 mg/L was 16.5%?16/97?,and 83.5%?81/97?of the strains were below 100 mg/L.Twenty-seven strains of high-yield putrescine,histamine,and cadaverine were selected from 97 strains for follow-up study.The 16S rDNA of 27strains was amplified with the universal primers 27F/1492R,and these sequences were analyzed with the BLAST?NCBI?.Strains were assigned to the different species:2 strains of Enterobacter asburiae,2 strains of Klebsiella aerogenes,3 strains of Klebsiella oxytoca,1 strain of Klebsiella pneumoniae,5 strains of Enterobacter hormaechei,3 strains of Enterobacter ludwigii,7 strains of Morganella morganii,1 strain of Citrobacter youngae,2 strains of Enterobacter cloacae,and 1strain of Enterobacter sp.27 strains carried odc,speA,speB,adiA,and ldc genes.13 carried hdc gene.?2?The effect of different pH on the type and ability of amine production of 27 strains.The results indicated that p H can considerably affect the BA-producing ability of the tested bacteria.odc,adiA,speB,ldc,and hdc were important for enteric bacteria survival under acidic conditions,and these pathways should be collaborative.At pH 6.70 and 6.00,the putrescine-,histamine-production and cadaverine-production dominated in the regulation of extracellular acid environment in most strains of enteric bacteria.At pH 5.00 and 4.50,the the putrescine-and cadaverine-production in the regulation of extracellular acid environment in most strains of enteric bacteria and the putrescine-and histamine-production are the main pathways in a few enteric bacteria.Decarboxylase was strain specific rather than species specific?P<0.01?Acid stress caused the growth delay but can increase the contents of putrescine,histamine,and cadaverine.?3?The enzymatic properties of histidine decarboxylase were studied.The results showed that the optimum temperature was 45?and the optimum pH was 6.00.The temperature stability range of histidine decarboxylase was 4-30?and p H was 5.00-8.00.Mg²⁺could increase the activity of the enzyme,and the relative activity was 106%.Ca²⁺,Mn²⁺,K⁺,Pb²⁺had no effect on the enzyme activity.Fe²⁺and Fe³⁺reduced part of the enzyme activity,but the enzyme activity remained above 89%.However,Zn²⁺and Cu²⁺greatly inhibited the enzyme activity,and the residual relative enzyme activity was 28%and 1%respectively.?4?Gene cloning and bioinformatics analysis.The length of the nucleic acid sequence encoding the enzyme was 1137 bp.The protein size is about 44 kDa.The enzyme was located in the cytoplasm with the molecular formula of C₁₉₀₅H₂₉₁₄N₅₁₀O₅₅₈S₁₇,and the molecular weight was42434.20.The theoretical value of isoelectric point of the protein was 5.86<7.0,so the protein was acidic protein with fat coefficient of 84.68.The total average hydrophilicity was-0.176<0,which indicated that the protein was soluble and had strong hydrophilicity.The enzyme is composed of 378 amino acids,including 20 amino acids.Among them,alanine?Ala?and isoleucine?Ile?were the most commonly used amino acids in the histidine decarboxylase,accounting for 8.7%of the total amino acids,followed by serine?Ser?7.7%,aspartic acid?Asp?7.4%.Pyrrolysine?Pyl?and selenocysteine?Sec?,two special amino acids,were not included.The enzyme has 11 kinase phosphorylation sites,no signal peptide and no transmembrane structure.According to the secondary structure prediction,the protein mainly contains alpha helix?Hh??-helix,extended strand?Ee?extension chain,beta turn?Tt??-turn and random coil?Cc?random coil.?-helix,extended chain,?-turn and random coil structure run through the whole amino acid chain and are evenly distributed.The most common amino acids were?-helix?Hh?and random coil structure?Cc?.There were 151 amino acids?39.95%?in Hh and 150 amino acids?39.68%?in Cc.The second was the extended chain?Ee?,with 59 amino acids,accounting for15.61%.The least is the?-turn?Tt?,only 18 amino acids,accounting for 4.76%.