DataSheet_1_An extracellular protease containing a novel C-terminal extension produced by a marine-originated haloarchaeon.pdf

Marine microorganisms have long been acknowledged as a significant reservoir of enzymes required for industrial use. In this study, a novel extracellular protease HslHlyB derived from marine-originated haloarchaeon Halostella pelagica DL-M4T was identified. HslHlyB contained polycystic kidney disease (PKD) domain and pre-peptidase C-terminal (PPC) domain at the C-terminus. Truncation and replacement of the C-terminal extension (CTE) of HslHlyB demonstrated the importance of the CTE in maintaining the protease activity secreted by haloarchaeon. HslHlyB and HslHlyBΔCTE were expressed in Escherichia coli BL21(DE3), and purified by high-affinity column refolding and gel filtration chromatography. The molecular masses of HslHlyB and HslHlyBΔCTE were 42 kDa and 20 kDa, respectively. The optimum catalytic reaction conditions were 50°C, pH 8.5, NaCl 3.5 M and 50°C, pH 7.5, NaCl 3 M, respectively. They showed good stability and hydrolysis capabilities towards a wide range of protein substrates. HslHlyBΔCTE showed higher catalytic reaction rate and better thermal stability than the wild type against azocasein and tetrapeptide substrate. The hydrolysates of soybean protein hydrolyzed by HslHlyBΔCTE had smaller average molecular masses and shorter average peptide chain lengths than those by HslHlyB. These results indicated the diversity of halolysins from marine-originated haloarchaea to harness organic nitrogen in the marine environment and provided promising candidates for application in various industries.</p

DataSheet_1_Halorarius litoreus gen. nov., sp. nov., Halorarius halobius sp. nov., Haloglomus halophilum sp. nov., Haloglomus salinum sp. nov., and Natronomonas marina sp. nov., extremely halophilic archaea isolated from tidal flat and marine solar salt.pdf

Five novel halophilic archaeal strains, named BND22T, ZY10T, ZY41T, ZY58T, and ZY43T, were isolated from the coastal saline sediment of the intertidal zone located in Qingdao and the natural sea salt produced from Huanghua marine solar saltern, PRChina. These five strains demonstrated the typical morphology and growth characteristics of haloarchaea. The comparison of 16S rRNA gene revealed that strain BDN22T was associated with Salinirubellus salinus ZS-35-S2T (95.2% similarity), strain ZY10T was related to Halosegnis rubeus F17-44T (95.1% similarity), both strains ZY41T and ZY58T were closely related to Haloglomus irregulare F16-60T (98.1% and 98.2% similarities, respectively), and strain ZY43T was close to Natronomonas salina YPL13T (98.0% similarity). In addition to the 16S rRNA gene, the rpoB′ gene is undoubtedly another important molecular marker for the identification of halophilic archaea, and concatenated-conserved-protein phylogeny was widely used in archaeal classification in recent years. Phylogenetic and phylogenomic analyses based on rpoB′ genes and 122 concatenated archaeal protein genes showed that these haloarchaea construct three different clades and gathered together with the current members of Haloarculaceae. Strains BND22T and ZY10T formed two distinct clades separated from Salinirubellus salinus ZS-35-S2T and Halosegnis members, strains ZY41T and ZY58T clustered with Haloglomus irregulare F16-60T, and strain ZY43T gathered with the current members of Natronomonas. The ANI, DDH, and AAI values of these five strains against phylogenetic neighbours were no more than 91%, 45%, and 92%, respectively, far below the cut-off values for species delineation, supporting their placements in new taxa. Based on the phenotypic, chemotaxonomic, phylogenetic, and phylogenomic properties, these five strains represent five novel taxa of the family Haloarculaceae, Halorarius litoreus gen. nov., sp. nov. (type strain BND22T = CGMCC 1.18780T = JCM 34966T), Halorarius halobius sp. nov. (type strain ZY10T = CGMCC 1.17475T = JCM 34319T), Haloglomus halophilum sp. nov. (type strain ZY41T = CGMCC 1.17030T = JCM 34161T), Haloglomus salinum sp. nov. (type strain ZY58T = CGMCC 1.17216T = JCM 34163T), and Natronomonas marina sp. nov. (type strain ZY43T = CGMCC 1.17202T = JCM 34162T). This is the first report of description of a novel haloarchaeon isolated from a marine intertidal zone.</p

Novel Archaeal Histamine Oxidase from Natronobeatus ordinarius: Insights into Histamine Degradation for Enhancing Food Safety

Histamine, found abundantly in salt-fermented foods, poses a risk of food poisoning. Natronobeatus ordinarius, a halophilic archaeon isolated from a salt lake, displayed a strong histamine degradation ability. Its histamine oxidase (HOD) gene was identified (hodNbs). This is the first report of an archaeal HOD. The HODNbs protein was determined to be a tetramer with a molecular weight of 307 kDa. HODNbs displayed optimum activity at 60–65 °C, 1.5–2.0 M NaCl, and pH 6.5. Notably, within the broad NaCl range between 0.5 and 2.5 M, HODNbs retained above 50% of its maximum activity. HODNbs exhibited good thermal stability, pH stability, and salinity tolerance. HODNbs was able to degrade various biogenic amines. The Vmax of HODNbs for histamine was 0.29 μmol/min/mg, and the Km was 0.56 mM. HODNbs exhibited high efficiency in histamine removal from fish sauce, namely, 100 μg of HODNbs degraded 5.63 mg of histamine (37.9%) in 10 g of fish sauce within 24 h at 50 °C. This study showed that HODNbs with excellent enzymatic properties has promising application potentials to degrade histamine in high-salt foods

Data_Sheet_1_Mechanism and Structural Insights Into a Novel Esterase, E53, Isolated From Erythrobacter longus.docx

Esterases are a class of enzymes that split esters into an acid and an alcohol in a chemical reaction with water, having high potential in pharmaceutical, food and biofuel industrial applications. To advance the understanding of esterases, we have identified and characterized E53, an alkalophilic esterase from a marine bacterium Erythrobacter longus. The crystal structures of wild type E53 and three variants were solved successfully using the X-ray diffraction method. Phylogenetic analysis classified E53 as a member of the family IV esterase. The enzyme showed highest activity against p-nitrophenyl butyrate substrate at pH 8.5–9.5 and 40°C. Based on the structural feature, the catalytic pocket was defined as R1 (catalytic center), R2 (pocket entrance), and R3 (end area of pocket) regions. Nine variants were generated spanning R1–R3 and thorough functional studies were performed. Detailed structural analysis and the results obtained from the mutagenesis study revealed that mutations in the R1 region could regulate the catalytic reaction in both positive and negative directions; expanding the bottleneck in R2 region has improved the enzymatic activity; and R3 region was associated with the determination of the pH pattern of E53. N166A in R3 region showed reduced activity only under alkaline conditions, and structural analysis indicated the role of N166 in stabilizing the loop by forming a hydrogen bond with L193 and G233. In summary, the systematic studies on E53 performed in this work provide structural and functional insights into alkaliphilic esterases and further our knowledge of these enzymes.</p