Comparative genome analysis among Variovorax species and genome guided aromatic compound degradation analysis emphasizing 4-hydroxybenzoate degradation in Variovorax sp. PAMC26660.

BACKGROUND: While the genus Variovorax is known for its aromatic compound metabolism, no detailed study of the peripheral and central pathways of aromatic compound degradation has yet been reported. Variovorax sp. PAMC26660 is a lichen-associated bacterium isolated from Antarctica. The work presents the genome-based elucidation of peripheral and central catabolic pathways of aromatic compound degradation genes in Variovorax sp. PAMC26660. Additionally, the accessory, core and unique genes were identified among Variovorax species using the pan genome analysis tool. A detailed analysis of the genes related to xenobiotic metabolism revealed the potential roles of Variovorax sp. PAMC26660 and other species in bioremediation. RESULTS: TYGS analysis, dDDH, phylogenetic placement and average nucleotide identity (ANI) analysis identified the strain as Variovorax sp. Cell morphology was assessed using scanning electron microscopy (SEM). On analysis of the core, accessory, and unique genes, xenobiotic metabolism accounted only for the accessory and unique genes. On detailed analysis of the aromatic compound catabolic genes, peripheral pathway related to 4-hydroxybenzoate (4-HB) degradation was found among all species while phenylacetate and tyrosine degradation pathways were present in most of the species including PAMC26660. Likewise, central catabolic pathways, like protocatechuate, gentisate, homogentisate, and phenylacetyl-CoA, were also present. The peripheral pathway for 4-HB degradation was functionally tested using PAMC26660, which resulted in the growth using it as a sole source of carbon. CONCLUSIONS: Computational tools for genome and pan genome analysis are important to understand the behavior of an organism. Xenobiotic metabolism-related genes, that only account for the accessory and unique genes infer evolution through events like lateral gene transfer, mutation and gene rearrangement. 4-HB, an aromatic compound present among lichen species is utilized by lichen-associated Variovorax sp. PAMC26660 as the sole source of carbon. The strain holds genes and pathways for its utilization. Overall, this study outlines the importance of Variovorax in bioremediation and presents the genomic information of the species.

Comparative analysis of genome-based CAZyme cassette in Antarctic Microbacterium sp. PAMC28756 with 31 other Microbacterium species.

BACKGROUND: The genus Microbacterium belongs to the family Microbacteriaceae and phylum Actinobacteria. A detailed study on the complete genome and systematic comparative analysis of carbohydrate-active enzyme (CAZyme) among the Microbacterium species would add knowledge on metabolic and environmental adaptation. Here we present the comparative genomic analysis of CAZyme using the complete genome of Antarctic Microbacterium sp. PAMC28756 with other complete genomes of 31 Microbacterium species available. OBJECTIVE: The genomic and CAZyme comparison of Microbacterium species and to rule out the specific features of CAZyme for the environmental and metabolic adaptation. METHODS: Bacterial source were collected from NCBI database, CAZyme annotation of Microbacterium species was analyzed using dbCAN2 Meta server. Cluster of orthologous groups (COGs) analysis was performed using the eggNOG4.5 database. Whereas, KEGG database was used to compare and obtained the functional genome annotation information in carbohydrate metabolism and glyoxylate cycle. RESULTS: Out of 32 complete genomes of Microbacterium species, strain No. 7 isolated from Activated Sludge showed the largest genomic size at 4.83 Mb. The genomic size of PAMC28756 isolated from Antarctic lichen species Stereocaulons was 3.54 Mb, the G + C content was 70.4% with 3,407 predicted genes, of which 3.36% were predicted CAZyme. In addition, while comparing the Glyoxylate cycle among 32 bacteria, except 10 strains, all other, including our strain have Glyoxylate pathway. PAMC28756 contained the genes that degrade cellulose, hemicellulose, amylase, pectinase, chitins and other exo-and endo glycosidases. Utilizing these polysaccharides can provides source of energy in an extreme environment. In addition, PAMC28756 assigned the (10.15%) genes in the carbohydrate transport and metabolism functional group closely related to the CAZyme for polysaccharides degradation. CONCLUSIONS: The genomic content and CAZymes distribution was varied in Microbacterium species. There was the presence of more than 10% genes in the carbohydrate transport and metabolism functional group closely related to the CAZyme for polysaccharides degradation. In addition, occurrence of glyoxylate cycle for alternative utilization of carbon sources suggest the adaptation of PAMC28756 in the harsh microenvironment.

Genome-enabled discovery of anthraquinone biosynthesis in Senna tora.

Senna tora is a widely used medicinal plant. Its health benefits have been attributed to the large quantity of anthraquinones, but how they are made in plants remains a mystery. To identify the genes responsible for plant anthraquinone biosynthesis, we reveal the genome sequence of S. tora at the chromosome level with 526 Mb (96%) assembled into 13 chromosomes. Comparison among related plant species shows that a chalcone synthase-like (CHS-L) gene family has lineage-specifically and rapidly expanded in S. tora. Combining genomics, transcriptomics, metabolomics, and biochemistry, we identify a CHS-L gene contributing to the biosynthesis of anthraquinones. The S. tora reference genome will accelerate the discovery of biologically active anthraquinone biosynthesis pathways in medicinal plants.

De novo transcriptome sequence of Senna tora provides insights into anthraquinone biosynthesis.

Senna tora is an annual herb with rich source of anthraquinones that have tremendous pharmacological properties. However, there is little mention of genetic information for this species, especially regarding the biosynthetic pathways of anthraquinones. To understand the key genes and regulatory mechanism of anthraquinone biosynthesis pathways, we performed spatial and temporal transcriptome sequencing of S. tora using short RNA sequencing (RNA-Seq) and long-read isoform sequencing (Iso-Seq) technologies, and generated two unigene sets composed of 118,635 and 39,364, respectively. A comprehensive functional annotation and classification with multiple public databases identified array of genes involved in major secondary metabolite biosynthesis pathways and important transcription factor (TF) families (MYB, MYB-related, AP2/ERF, C2C2-YABBY, and bHLH). Differential expression analysis indicated that the expression level of genes involved in anthraquinone biosynthetic pathway regulates differently depending on the degree of tissues and seeds development. Furthermore, we identified that the amount of anthraquinone compounds were greater in late seeds than early ones. In conclusion, these results provide a rich resource for understanding the anthraquinone metabolism in S. tora.

Characterization of a novel antibacterial N-acyl amino acid synthase from soil metagenome.

In an effort to isolate novel natural antibiotics, we searched for antibacterial long-chain N-acyl amino acid synthase (NAS) genes from 70,000 soil metagenome clones by Bacillus subtilis-overlaying screening. In an antibacterial cosmid clone, YS92B, a single gene nasYPL was responsible for the production of the Nas. nasYPL was 903 bp long, and the deduced amino acid sequence showed the highest 71% identity with a hypothetical protein from Massilia niastensis. Phylogenetic analysis demonstrated that NasYPL belongs to Group 1 Nas. Heterologous expression of the same nasYPL gene in Escherichia coli and two Pseudomonas strains (P. putida and P. koreensis) conferred antibacterial activities against Listeria monocytogenes, Staphylococcus epidermidis, and Bacillus subtilis. Mass spectral analysis of the antibacterial fractions identified 7 peaks corresponding to long-chain N-acyl tyrosine, 5 peaks to N-acyl phenylalanine, and 3 peaks to N-acyl leucine (or isoleucine) derivatives linked with 7 fatty acids, indicating enzymatic products derived by NasYPL. Therefore, NasYPL expression by host-specific manner may provide applicable antibacterial characteristics to biotechnologically important Pseudomonas strains.

Characterization of an inhibitor-resistant endo-1,4-ß-mannanase from the gut microflora metagenome of Hermetia illucens.

OBJECTIVE: Hermetia illucens is a voracious insect scavenger that efficiently decomposes food waste. To exploit novel hydrolytic enzymes from this insect, we constructed a fosmid metagenome library using unculturable H. illucens intestinal microorganisms. RESULTS: Functional screening of the library on carboxymethyl cellulose plates identified a fosmid clone with a product displaying hydrolytic activity. Fosmid sequence analysis revealed a novel mannan-degrading gene (ManEM17) composed of 1371 base pairs, encoding 456 amino acids with a deduced 54 amino acid N-terminal signal peptide sequence. Conceptual translation and domain analysis revealed that sequence homology was highest (46%) with endo-1,4-ß-mannosidase of Anaerophaga thermohalophila. Phylogenetic and domain analysis indicated that ManEM17 belongs to a novel ß-mannanase containing a glycoside hydrolase family 26 domain. The recombinant protein (rManEM17) was expressed in Escherichia coli, exhibiting the highest activity at 55 °C and pH 6.5. The protein hydrolyzed substrates with ß-1,4-glycosidic mannoses; maximum specific activity (5467 U mg-1) occurred toward locust bean gum galactomannan. However, rManEM17 did not hydrolyze p-Nitrophenyl-ß-pyranosides, demonstrating endo-form mannanase activity. Furthermore, rManEM17 was highly stable under stringent conditions, including polar organic solvents as well as chemical reducing and denaturing reagents. CONCLUSIONS: ManEM17 is an attractive candidate for mannan degradation under the high-organic-solvent and protein-denaturing processes in food and feed industries.

Isolation and characterization of a halotolerant and protease-resistant α-galactosidase from the gut metagenome of Hermetia illucens.

Hermetia illucens is a voracious insect scavenger, decomposing food waste efficiently. To survey novel hydrolytic enzymes, we constructed a fosmid metagenome library using unculturable intestinal microorganisms from H. illucens in our previous study (Lee et al., 2014). Functional screening of the library on carboxymethyl cellulose plates identified a fosmid clone the product of which displayed hydrolytic activity. Sequence analysis of the fosmid revealed a novel α-galactosidase gene, Agas2. The Agas2 gene is composed of 2,007 base pairs encoding 668 amino acids with a deduced 25 amino acid N-terminal signal peptide sequence. The conceptual translation and domain analysis of Agas2 showed the highest sequence identity (84%) with the putative α-galactosidase of Dysgonomonas sp. HGC4, exhibiting well-conserved domain homology with glycosyl hydrolase family 97. Phylogenetic analysis indicated that Agas2 may be a currently uncharacterized α-galactosidase. The recombinant protein, rAgas2, was successfully expressed in E. coli. rAgas2 showed the highest activity at 40 °C and pH 7.0. It displayed great pH stability within a pH range of 5-11 for 15 h at 4 °C. rAgas2 was highly stable under stringent conditions, including polar organic solvents, non-ionic detergents, salt, and proteases. rAgas2 hydrolyzed α-d-galactose substrates, showing the maximum enzymatic activity toward p-nitrophenyl α-d-galactopyranoside (specific activity 128.37 U/mg). However, rAgas2 did not hydrolyze substrates linked with ß-glucose moieties. Overall, Agas2 may be an attractive candidate for the degradation of α-galactose family oligosaccharides in high-salt, protease-rich and high-organic-solvent processes.

Lack of the α1,3-Fucosyltransferase Gene (Osfuct) Affects Anther Development and Pollen Viability in Rice.

N-linked glycosylation is one of the key post-translational modifications. α1,3-Fucosyltransferase (OsFucT) is responsible for transferring α1,3-linked fucose residues to the glycoprotein N-glycan in plants. We characterized an Osfuct mutant that displayed pleiotropic developmental defects, such as impaired anther and pollen development, diminished growth, shorter plant height, fewer tillers, and shorter panicle length and internodes under field conditions. In addition, the anthers were curved, the pollen grains were shriveled, and pollen viability and pollen number per anther decreased dramatically in the mutant. Matrix-assisted laser desorption/ionization time-of-flight analyses of the N-glycans revealed that α1,3-fucose was lacking in the N-glycan structure of the mutant. Mutant complementation revealed that the phenotype was caused by loss of Osfuct function. Transcriptome profiling also showed that several genes essential for plant developmental processes were significantly altered in the mutant, including protein kinases, transcription factors, genes involved in metabolism, genes related to protein synthesis, and hypothetical proteins. Moreover, the mutant exhibited sensitivity to an increased concentration of salt. This study facilitates a further understanding of the function of genes mediating N-glycan modification and anther and pollen development in rice.

UC2 search: using unique connectivity of uncharged compounds for metabolite annotation by database searching in mass spectrometry-based metabolomics.

Summary: For metabolite annotation in metabolomics, variations in the registered states of compounds (charged molecules and multiple components, such as salts) and their redundancy among compound databases could be the cause of misannotations and hamper immediate recognition of the uniqueness of metabolites while searching by mass values measured using mass spectrometry. We developed a search system named UC2 (Unique Connectivity of Uncharged Compounds), where compounds are tentatively neutralized into uncharged states and stored on the basis of their unique connectivity of atoms after removing their stereochemical information using the first block in the hash of the IUPAC International Chemical Identifier, by which false-positive hits are remarkably reduced, both charged and uncharged compounds are properly searched in a single query and records having a unique connectivity are compiled in a single search result. Availability and implementation: The UC2 search tool is available free of charge as a REST web service (http://webs2.kazusa.or.jp/mfsearcher) and a Java-based GUI tool. Contact: sakurai@kazusa.or.jp. Supplementary information: Supplementary data are available at Bioinformatics online.

Differential Metabolic Profiles during the Developmental Stages of Plant-Parasitic Nematode Meloidogyne incognita.

Meloidogyne incognita is a common root-knot nematode with a wide range of plant hosts. We aimed to study the metabolites produced at each stage of the nematode life cycle to understand its development. Metabolites of Meloidogyne incognita were extracted at egg, J2, J3, J4, and female stages and 110 metabolites with available standards were quantified using CE-TOF/MS. Analyses indicated abundance of stage-specific metabolites with the exception of J3 and J4 stages which shared similar metabolic profiles. The egg stage showed increased abundance in glycolysis and energy metabolism related metabolites while the J2 metabolites are associated with tissue formation, motility, and neurotransmission. The J3 and J4 stages indicated amino acid metabolism and urea cycle- related metabolites. The female stage was characterized with polyamine synthesis, antioxidant activity, and synthesis of reproduction related metabolites. Such metabolic profiling helps us understand the dynamic physiological changes related to each developmental stage of the root-knot nematode life cycle.

Stage-Wise Identification and Analysis of miRNA from Root-Knot Nematode Meloidogyne incognita.

In this study, we investigated global changes in miRNAs of Meloidogyne incognita throughout its life cycle. Small RNA sequencing resulted in approximately 62, 38, 38, 35, and 39 Mb reads in the egg, J2, J3, J4, and female stages, respectively. Overall, we identified 2724 known and 383 novel miRNAs (read count > 10) from all stages, of which 169 known and 13 novel miRNA were common to all the five stages. Among the stage-specific miRNAs, miR-286 was highly expressed in eggs, miR-2401 in J2, miR-8 and miR-187 in J3, miR-6736 in J4, and miR-17 in the female stages. These miRNAs are reported to be involved in embryo and neural development, muscular function, and control of apoptosis. Cluster analysis indicated the presence of 91 miRNA clusters, of which 36 clusters were novel and identified in this study. Comparison of miRNA families with other nematodes showed 17 families to be commonly absent in animal parasitic nematodes and M. incognita. Validation of 43 predicted common and stage-specific miRNA by quantitative PCR (qPCR) indicated their expression in the nematode. Stage-wise exploration of M. incognita miRNAs has not been carried out before and this work presents information on common and stage-specific miRNAs of the root-knot nematode.

A novel family VIII carboxylesterase hydrolysing third- and fourth-generation cephalosporins.

A metagenomic library was constructed from a soil sample of spindle tree-rhizosphere. From this library, one clone with esterase activity was selected. The sequence analysis revealed an open reading frame (EstSTR1) encoded protein of 390 amino acids. EstSTR1 is a family VIII carboxylesterase and retains the S-X-X-K motif conserved in both family VIII carboxylesterases and class C ß-lactamases. The estSTR1 gene was overexpressed in E. coli and the recombinant protein was purified by purified by metal chelating affinity chromatography and size-exclusion chromatography. EstSTR1 hydrolysed p-nitrophenyl esters, exhibited the highest activity toward p-nitrophenyl butyrate. Furthermore, EstSTR1 could hydrolyse third- and fourth-generation cephalosporins (cefotaxime and cefepime) as well as first-generation cephalosporin (cephalothin). Site-directed mutagenesis studies revealed that a catalytic residue, Ser71, of EstSTR1 plays an essential role in hydrolysing both antibiotics and p-nitrophenyl esters. We demonstrate that a metagenome-derived carboxylesterase displays ß-lactam-hydrolysing activities toward third- and fourth-generation cephalosporins.

Identification of a novel alkaline amylopullulanase from a gut metagenome of Hermetia illucens.

A novel pullulanase gene, PulSS4, was identified from the gut microflora of Hermetia illucens by a function-based metagenome screening. The PulSS4 gene had an open reading frame of 4455 base pairs, and encoded a mature protein of 1484 amino acids, with a signal peptide sequence of 44 amino acids. The deduced amino acid sequence of PulSS4 gene showed 51% identity with that of the amylopullulanase of Amphibacillus xylanus, exhibiting no significant sequence homology to already known pullulanases. A conserved domain analysis revealed it to be a pullulanase type II with respective active sites at the N-terminal pullulanase and C-terminal amylase domain. PulSS4 was active in the temperature range of 10-50°C, with an optimum activity at 40°C. It was active in the pH range of 6.5-10.5, with optimum pH at 9.0, and retained more than 80% of its original activity in a broad pH range of 5-11 for 24h at 30°C. Also, PulSS4 was highly stable against many different chemical reagents, including 10% polar organic solvents and 1% non-ionic detergents. Overall, PulSS4 is expected to have the strong potential for application in biotechnological industries that require high activity at moderate temperature and alkaline conditions.

Screening and characterization of a novel cellulase gene from the gut microflora of Hermetia illucens using metagenomic library.

A metagenomic fosmid library was constructed using genomic DNA isolated from the gut microflora of Hermetia illucens, a black soldier fly. A cellulase-positive clone, with the CS10 gene, was identified by extensive Congo-red overlay screenings for cellulase activity from the fosmid library of 92,000 clones. The CS10 gene was composed of a 996 bp DNA sequence encoding the mature protein of 331 amino acids. The deduced amino acids of CS10 showed 72% sequence identity with the glycosyl hydrolase family 5 gene of Dysgonomonas mossii, displaying no significant sequence homology to already known cellulases. The purified CS10 protein presented a single band of cellulase activity with a molecular mass of approximately 40 kDa on the SDS-PAGE gel and zymogram. The purified CS10 protein exhibited optimal activity at 50°C and pH 7.0, and the thermostability and pH stability of CS10 were preserved at the ranges of 20~50°C and pH 4.0~10.0. CS10 exhibited little loss of cellulase activity against various chemical reagents such as 10% polar organic solvents, 1% non-ionic detergents, and 0.5 M denaturing agents. Moreover, the substrate specificity and the product patterns by thinlayer chromatography suggested that CS10 is an endo-ß-1,4-glucanase. From these biochemical properties of CS10, it is expected that the enzyme has the potential for application in industrial processes.

ATF-2/CREB/IRF-3-targeted anti-inflammatory activity of Korean red ginseng water extract.

ETHNOPHARMACOLOGICAL RELEVANCE: Korean Red Ginseng (KRG) is one of the representative traditional herbal medicines prepared from Panax ginseng Meyer (Araliaceae) in Korea. It has been reported that KRG exhibits a lot of different biological actions such as anti-aging, anti-fatigue, anti-stress, anti-atherosclerosis, anti-diabetic, anti-cancer, and anti-inflammatory activities. Although systematic studies have investigated how KRG is able to ameliorate various inflammatory diseases, its molecular inhibitory mechanisms had not been carried out prior to this study. MATERIALS AND METHODS: In order to investigate these mechanisms, we evaluated the effects of a water extract of Korean Red Ginseng (KRG-WE) on the in vitro inflammatory responses of activated RAW264.7 cells, and on in vivo gastritis and peritonitis models by analyzing the activation events of inflammation-inducing transcription factors and their upstream kinases. RESULTS: KRG-WE reduced the production of nitric oxide (NO), protected cells against NO-induced apoptosis, suppressed mRNA levels of inducible NO synthase (iNOS), cyclooxygenase (COX)-2, and interferon (IFN)-ß, ameliorated EtOH/HCl-induced gastritis, and downregulated peritoneal exudate-derived NO production from lipopolysaccharide (LPS)-injected mice. The inhibition of these inflammatory responses by KRG-WE was regulated through the suppression of p38, c-Jun N-terminal kinase (JNK), and TANK-binding kinase 1 (TBK1) and by subsequent inhibition of activating transcription factor (ATF)-2, cAMP response element-binding protein (CREB), and IRF-3 activation. Of ginsensides included in this extract, interestingly, G-Rc showed the highest inhibitory potency on IRF-3-mediated luciferase activity. CONCLUSION: These results strongly suggest that the anti-inflammatory activities of KRG-WE could be due to its inhibition of the p38/JNK/TBK1 activation pathway.

Characterization of a novel cold-active esterase isolated from swamp sediment metagenome.

A functional screen of a metagenomic library from "Upo" swamp sediment in Korea identified a gene EstL28, the product of which displayed lipolytic properties on a tributyrin-supplemented medium. The EstL28 sequence encodes a 290 amino acid protein (designated as EstL28), with a predicted molecular weight of 31.3 kDa. The encoded EstL28 protein exhibited the highest sequence similarity (45 %) to a hydrolase found in Streptococcus sanguinis. Phylogenetic analysis indicated that EstL28 belongs to a currently uncharacterized family of esterases. Within the conserved α/ß-hydrolase 6 domain, the EstL28 retains the catalytic triad Ser103-Asp248-His268 that is typical of esterases. The Ser103 residue in the catalytic triad is located in the consensus pentapeptide motif GXSXG. The purified EstL28 enzyme worked optimally at 35 °C and pH 8.5 and remained stable at temperatures lower than 20 °C. The catalytic activity of EstL28 was maximal with p-nitrophenyl butyrate, indicating that it was an esterase. This enzyme also exhibited stable activity in the presence of methanol, ethanol, isopropanol, and dimethyl sulfoxide. Therefore, the level of stability in organic solvents and cold temperature suggests that EstL28 has potential for many biotechnological applications.

Structural basis for the ß-lactamase activity of EstU1, a family VIII carboxylesterase.

EstU1 is a unique family VIII carboxylesterase that displays hydrolytic activity toward the amide bond of clinically used ß-lactam antibiotics as well as the ester bond of p-nitrophenyl esters. EstU1 assumes a ß-lactamase-like modular architecture and contains the residues Ser100, Lys103, and Tyr218, which correspond to the three catalytic residues (Ser64, Lys67, and Tyr150, respectively) of class C ß-lactamases. The structure of the EstU1/cephalothin complex demonstrates that the active site of EstU1 is not ideally tailored to perform an efficient deacylation reaction during the hydrolysis of ß-lactam antibiotics. This result explains the weak ß-lactamase activity of EstU1 compared with class C ß-lactamases. Finally, structural and sequential comparison of EstU1 with other family VIII carboxylesterases elucidates an operative molecular strategy used by family VIII carboxylesterases to extend their substrate spectrum.

Burkholderia denitrificans sp. nov., isolated from the soil of Dokdo Island, Korea.

A novel, Gram-negative, bacterial strain KIS30-44(T) was identified from wet forest soil collected on the Korean island of Dokdo. Growth of the strain was observed at 15-30°C, pH 5-9, 0-3% NaCl, and 950 mM KNO(3). KIS30-44(T) reduced nitrate to nitrogen gas. Analysis of the 16S rRNA gene sequence showed that KIS30-44(T) was phylogenetically related to Burkholderia sacchari, Burkholderia mimosarum, and Burkholderia oxyphila (98.1%, 98.0%, and 98.0% sequence similarity, respectively). The genomic G+C content was 63.5 mol%. KIS30-44(T) exhibited less than 52% DNA-DNA relatedness with the type strains of 9 closely related Burkholderia species. The major isoprenoid quinone was Q-8. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and two unknown aminolipids. The major fatty acids in KIS30-44(T) were C(16:0), C(18:1) ω7c and summed feature 3 (iso-C(15:0) 2-OH and C(16:1) ω7c), and the strain contained half the amount of C(17:0) cyclo found in the 9 closely related Burkholderia species. The results of these phenotypic, 16S rRNA gene sequence, DNA-DNA hybridization, and chemotaxonomic data indicate that KIS30-44(T) represents a novel species within the genus Burkholderia, for which the name Burkholderia denitrificans (Type strain KIS30-44(T) =KACC 12733(T) =DSM 24336(T)) is proposed.

Novel metagenome-derived carboxylesterase that hydrolyzes ß-lactam antibiotics.

It has been proposed that family VIII carboxylesterases and class C ß-lactamases are phylogenetically related; however, none of carboxylesterases has been reported to hydrolyze ß-lactam antibiotics except nitrocefin, a nonclinical chromogenic substrate. Here, we describe the first example of a novel carboxylesterase derived from a metagenome that is able to cleave the amide bond of various ß-lactam substrates and the ester bond of p-nitrophenyl esters. A clone with lipolytic activity was selected by functional screening of a metagenomic library using tributyrin agar plates. The sequence analysis of the clone revealed the presence of an open reading frame (estU1) encoding a polypeptide of 426 amino acids, retaining an S-X-X-K motif that is conserved in class C ß-lactamases and family VIII carboxylesterases. The gene was overexpressed in Escherichia coli, and the purified recombinant protein (EstU1) was further characterized. EstU1 showed esterase activity toward various chromogenic p-nitrophenyl esters. In addition, it exhibited hydrolytic activity toward nitrocefin, leading us to investigate whether EstU1 could hydrolyze ß-lactam antibiotics. EstU1 was able to hydrolyze first-generation ß-lactam antibiotics, such as cephalosporins, cephaloridine, cephalothin, and cefazolin. In a kinetic study, EstU1 showed a similar range of substrate affinities for both p-nitrophenyl butyrate and first-generation cephalosporins while the turnover efficiency for the latter was much lower. Furthermore, site-directed mutagenesis studies revealed that the catalytic triad of EstU1 plays a crucial role in hydrolyzing both ester bonds of p-nitrophenyl esters and amide bonds of the ß-lactam ring of antibiotics, implicating the predicted catalytic triad of EstU1 in both activities.