Gene Cloning And Functional Characteristics Of A L-Histidine Decarboxylase HDC1from Enterobacter Aerogenes SP. GXE3

Biogenic amine is a kind of biological factor which closely related to food safety and human health. Generally, histamine existing in meat, dairy and fermented products, which produced by microorganism, is toxic to human race. So far, the research of histamine-producing bacteria around the world focus on detection method, identification of bacterial and analysis of feature. But research in related synthesis enzyme is less. The author got some strains which produced L-histidine decarboxylase from environment base on functional separation screening strategy, those strains to be proved as histamine-producing bacteria after studying by microbiology, molecular biology and chemical methods. Among those strains, four of them were Enterobacter aerogenes and one of them was Klebsiella after analyzing the morphological characteristics, physiological and biochemical features and16S rDNA sequence.As the object of E. aerogenes sp. GXE3in this study, primers were designed according to the E. aerogenes HDC gene sequence reported in Genbank, with the DNA of genome was extracted, then amplified by PCR and cloned the HDC gene into pET-30a（+）, after that transferred it into Escherichis coli DH5a to construct recombinant clone. Bio-informatics analysis showed that the recombinant plasmid comprising a ORF of1137base pairs, which encoded a protein contained a378amino acid with molecular mass of42.4kDa, the gene was named hdcl. At the amino acid level, The Hdcl protein shared the highest identical100%with L-histidine decarboxylase from E. aerogenes KCTC2190（GenBank accession number:YP₀04590726）. However, there are no related reports on the functional study of L-histidine decarboxylase from it.The recombinant plasmid was transferred in E.coli BL21（DE3） and induced by0.05%L-histidine and IPTG. The recombinant protein Hdcl was purified successfully by Ni-NTA chromatography technology. The biochemical characterization of recombinant protein Hdcl was found that the optimal pH value and optimal temperature for Hdcl activity was6.5and25℃, respectively. The specific activity of Hdcl was2.38U/mg; Its activity was stable at pH7.0-8.0and stable below30℃with its relative residual activity more than85%; The activity of Hdcl could be strong inhibited by Mg2+and Fe3+; The kinetics parameters of recombinant protein Hdcl were found that Km was2.047mM, Vmax was0.639μg/min and kcat was13.155/s. On the other hand, the HDC protein of crude enzyme of E. aerogenes sp. GXE3was found that the optimal pH value and optimal temperature for HDC activity was6.0and30℃, respectively. The specific activity of HDC was3.33U/mg; Its activity was stable at pH5.0-7.0and stable below30℃. The activity of HDC could be weeky prompted by Zn2+and Ca2+; The kinetics parameters of HDC protein were found that Km was1.461mM, Vmax was0.428μg/min.The biochemical characterization study of L-histidine decarboxylase of E. aerogenes will contribute to understand the influence factors of the produced and accumulated of histamine in fermented food and aquatic products, and also provides an important reference on food safety and sanitation.