Biodiversity And Histamine Oxidase Of Halophilic Archaea Isolated From Pickling Salt

Histamine is widely present in high-salt foods.Excessive histamine has a negative influence on food quality and safety.Enzymatic degradation is a very promising method for histamine degradation.Seven pickling salt samples from the coast of China were investigated in this study.Culture-independent and culture-dependent approaches were used to analyze the diversity of halophilic archaea.The isolated haloarchaeal strains were screened for their abilities to degrade histamine.The histamine oxidase gene was identified based on genomic data.The histamine oxidase was then heterologously expressed,purified and characterized.Seven pickling salt samples produced from Bohai Sea(Liaoning Dalian,BDL;Tianjin,BTJ;Shandong Shouguang,BSG),Yellow Sea(Jiangsu Yancheng,YYC),East China Sea(Zhejiang Hangzhou,EHZ)and South China Sea(Guangdong Guangzhou,SGZ;Hainan Dongfang,SDF)were investigated.q PCR was used to study the number of microorganisms.The results showed that except for Shouguang and Hangzhou salt samples,all samples had the largest number of archaea.Through high-throughput sequencing technology,about 200 OTUs were obtained from each of the seven samples.The diversity and richness of the samples were not significantly different.However,the community composition was quite different,possibly due to different orign and moisture content.A total of 386 haloarchaeal strains were isolated from 7 samples based on the culture-dependent approach.They belonged to 49 species of 21 genus.A total of 95haloarchaeal strains were isolated from Dalian salt,belonging to 21 species of 13 genus.A total of 64 haloarchaeal strains were isolated from Tianjin salt,belonging to 20species of 9 genus.A total of 26 haloarchaeal strains were isolated from Shouguang salt,belonging to 6 species of 4 genus.30 haloarchaeal strains isolated from Yancheng salt all belonged to Halobacterium hubeiense.A total of 14 haloarchaeal strains were isolated from Hangzhou salt,belonging to 10 species of 5 genus.A total of 65haloarchaeal strains were isolated from Guangzhou salt,belonging to 35 species of 17genus.A total of 92 haloarchaeal strains were isolated from Dongfang salt,belonging to 26 species of 12 genus.Guangzhou salt had the highest Shannon diversity index(H')and Margalef richness index(D_Mg),Hangzhou salt had the highest Shannon uniformity index(E),and Shouguang salt had the highest dominance index(d).42 haloarchaeal strains displayed histamine degrading ability.Genome-wide bioinformatic analysis was conducted on the strain Halovivax sp.BDL50,which displayed a strong ability to degrade histamine.Two potential histamine oxidase genes of BDL50?1662 and BDL50?3264 were present in the genome.BDL50?3264 was determined to have a strong ability to degrade histamine through heterologous expression,purification,and enzyme activity determination.BDL50?3264 was named Hod _Hvv and subject to biochemical characterization.The enzyme displayed optimum activity at 60-65?,2 M Na Cl,and p H 6.5.It was stable at 30?,and the enzyme activity increased with time within 3 h of incubation at 50?.This enzyme had good p H and Na Cl tolerance.Sr²⁺and K⁺inhibited the enzyme activity.Hod _Hvv was able to degrade a variety of biogenic amines,with histamine displaying the highest enzyme activity.The V_max of Hod _Hvv with histamine as the substrate was 30.83?g/min/mg,and the kinetic constant K_m was 0.5321 m M.This study shows that histamine oxidase Hod _Hvv from halophilic archaea has excellent enzymatic properties,and thus has promising applications in high-salt food industries.