Rapid Identification of Genes Encoding DNA Polymerases by Function-Based Screening of Metagenomic Libraries Derived from Glacial Ice

Small-insert and large-insert metagenomic libraries were constructed from glacial ice of the Northern Schneeferner, which is located on the Zugspitzplatt in Germany. Subsequently, these libraries were screened for the presence of DNA polymerase-encoding genes by complementation of an Escherichia coli polA mutant. Nine novel genes encoding complete DNA polymerase I proteins or domains typical of these proteins were recovered.DNA polymerases are essential for DNA replication and DNA repair. Based on sequence similarities and phylogenetic relationships, DNA polymerases are grouped into six different families (A, B, C, D, X, and Y) (17). In this study, we used a DNA polymerase I (polA) mutant of Escherichia coli as a host for the screening of metagenomic libraries. PolA belongs to family A and contains three different domains: a 5′-3′ exonuclease domain at the N terminus, a central proofreading 3′-5′ exonuclease domain, and a polymerase domain at the C terminus of the enzyme (11). These polymerases are employed as tools in molecular biology, including probe labeling, DNA sequencing, and mutagenic PCR (13). To improve their suitability for such applications, various family A DNA polymerases have been modified; e.g., the Klenow fragment of E. coli DNA polymerase I has been redesigned by the removal of the 5′-3′ exonuclease domain (12). Nevertheless, expanding the known DNA polymerase sequence space and discovery of polymerases with novel properties are required for the development of novel or improved molecular methods and tools (13, 20).Metagenomics based on direct isolation of DNA from environmental samples, generation of metagenomic libraries from the isolated DNA, and function-based screening of the constructed libraries has led to identification and characterization of a variety of novel biocatalysts, such as lipases, amylases, amidases, nitrilases, and oxidoreductases (for reviews, see references 6, 7, and 10). In particular, the use of host strains or mutants of host strains that require heterologous complementation for growth under selective conditions has proven to be an efficient strategy to screen complex metagenomic libraries. This approach has been applied to, e.g., the isolation of genes encoding Na⁺/H⁺ antiporters (14), antibiotic resistance (18), or enzymes involved in poly-3-hydroxybutyrate metabolism (21).In this study, we employed the last-named strategy to recover functional genes encoding DNA polymerases. To our knowledge, this is the first report of identification of polymerases or other DNA-modifying enzymes by function-driven screening of metagenomes. For this purpose, we constructed small-insert and large-insert metagenomic libraries from DNA isolated from glacial ice. The employment of glacial ice samples for metagenomic library construction has not been reported by other researchers. The screening for the targeted genes was based on complementation of a cold-sensitive lethal mutation in the polA gene of E. coli (16).     

Identification of a Novel Alkaliphilic Esterase Active at Low Temperatures by Screening a Metagenomic Library from Antarctic Desert Soil

A novel esterase was identified through functional screening of a metagenomic library in Escherichia coli obtained from Antarctic desert soil. The 297-amino-acid sequence had only low (<29%) similarity to a putative esterase from Burkholderia xenovorans. The enzyme was active over a temperature range of 7 to 54°C and at alkaline pH levels and is a potential candidate for industrial application.The cold deserts of the McMurdo Dry Valleys, South Victoria Land, Eastern Antarctica, are widely acknowledged as having the harshest soil environments on Earth (6, 8, 26). Despite the apparent hostility of the environment, we and others have reported both unexpectedly high biomass (9) and phylogenetic diversity (1, 19, 24, 29) in Antarctic soils. The presence of numerous novel taxa suggests that these soils might prove to be valuable sources of genetic material for mining novel industrial enzymes active at low temperatures (9, 23).Esterases (EC 3.1.1.1) and lipases (EC 3.1.1.3) catalyze the hydrolysis and synthesis of ester compounds. Their applications in industry cover a broad spectrum, including as detergent additives, in food processing, in environmental bioremediation, and in biomass and plant waste degradation for the production of useful organocompounds (3, 16).In this study, a novel esterase was isolated from a metagenomic fosmid library obtained from Antarctic soil. The enzyme displays activity over a wide temperature range and has very low similarity (<29% amino acid identity) to esterases in the GenBank database. The study demonstrates the value of Antarctic metagenomes as a source of novel industrial products.     

Isolation and Characterization of a Novel Lysine Racemase from a Soil Metagenomic Library

A lysine racemase (lyr) gene was isolated from a soil metagenome by functional complementation for the first time by using Escherichia coli BCRC 51734 cells as the host and d-lysine as the selection agent. The lyr gene consisted of a 1,182-bp nucleotide sequence encoding a protein of 393 amino acids with a molecular mass of about 42.7 kDa. The enzyme exhibited higher specific activity toward lysine in the l-lysine-to-d-lysine direction than in the reverse reaction.Amino acids are the building blocks of proteins and play an important role in the regulation of the metabolism of living organisms. Among two enantiomers of naturally occurring amino acids, l-amino acids are predominant in living organisms, while d-amino acids are found in both free and bound states in various organisms like bacteria (36), yeasts (35), plants (47), insects (11), mammals (17), bivalves (39), and fish (28). The d-amino acids are mostly endogenous and produced by racemization from their counterparts by the action of a racemase. Thus, the amino acid racemases are involved in d-amino acid metabolism (29, 46). Since the discovery of alanine racemase in 1951 (42), several racemases toward amino acids, such as those for glutamate, threonine, serine, aspartate, methionine, proline, arginine, and phenylalanine, have been reported in bacteria, archaea, and eukaryotes, including mammals (1, 2, 15, 30, 31, 44). They are classified into two groups: pyridoxal 5′-phosphate (PLP)-dependent and PLP-independent enzymes (9, 36).Lysine racemase (Lyr, EC 5.1.1.5) was first reported in Proteus vulgaris ATCC 4669 (19) and proposed to be involved in the lysine degradation of bacterial cells (5, 19). Catabolism of lysine occurs via two parallel pathways. In one of the pathways, δ-aminovalerate is the key metabolite, whereas in the other l-lysine is racemized to d-lysine, and l-pipecolate and α-aminoadipate (AMA) are the key metabolites (5). d-Lysine catabolism proceeds through a series of cyclized intermediates which are necessary to regenerate an α-amino acid and comprise the following metabolites (AMA pathway): d-lysine→α-keto-ɛ-amino caproate→Δ¹-piperideine-2-carboxylate→pipecolate→Δ¹-piperideine-6-carboxylate→α-amino-δ-formylcaproate→α-AMA→α-ketoadipate (6, 7, 12, 27). The final product is converted to α-ketoglutarate via a series of coenzyme A derivatives and subsequently participates as an intermediate in the Krebs cycle. This pathway suggests that the biological function of d-lysine in the bacteria is that of a carbon or nitrogen source. Racemization of added l-lysine to d-lysine by whole cells of Proteus spp. and Escherichia spp. (19) and by the cell extract of Pseudomonas putida ATCC 15070 (5, 20) has been found. However, the enzyme has not been purified to homogeneity, and thus, its molecular and catalytic characteristics, including its gene structure, have not been elucidated. In this study, we explored a metagenomic library constructed from a garden soil to isolate a novel Lyr enzyme. After expression in Escherichia coli, the purified enzyme was characterized in terms of optimal pH and temperature, thermal stability, and racemization activity.     

Isolation and Characterization of a Novel Endoglucanase from a Bursaphelenchus xylophilus Metagenomic Library

A novel gene (designated as cen219) encoding endoglucanase was isolated from a Bursaphelenchus xylophilus metagenomic library by functional screening. Sequence analysis revealed that cen219 encoded a protein of 367 amino acids. SDS-PAGE analysis of purified endoglucanase suggested that Cen219 was a monomeric enzyme with a molecular mass of 40 kDa. The optimum temperature and pH for endoglucanase activity of Cen219 was separately 50°C and 6.0. It was stable from 30 to 50°C, and from pH 4.0 to 7.0. The activity was significantly enhanced by Mn²⁺ and dramatically reduced by detergent SDS and metals Fe³⁺, Cu²⁺ or Hg²⁺. The enzyme hydrolyzed a wide range of β-1, 3-, and β-1, 4-linked polysaccharides, with varying activities. Activities towards microcrystalline cellulose and filter paper were relatively high, while the highest activity was towards oat gum. The K_m and V_max of Cen219 towards CMC was 17.37 mg/ml and 333.33 U/mg, respectively. The findings have an insight into understanding the molecular basis of host–parasite interactions in B. xylophilus species. The properties also make Cen219 an interesting enzyme for biotechnological application.     

Characterization of Glycosynthase Mutants Derived from Glycoside Hydrolase Family 10 Xylanases

Four xylanases belonging to glycoside hydrolase family 10—Thermotoga maritima XylB (TM), Clostridium stercorarium XynB (CS), Bacillus halodurans XynA (BH), and Cellulomonas fimi Cex (CF)—were converted to glycosynthases by substituting the nucleophilic glutamic acid residues with glycine, alanine, and serine. The glycine mutants exhibited the highest levels of glycosynthase activity with all four enzymes. All the glycine mutants formed polymeric β-1,4-linked xylopyranose as a precipitate during reaction with α-xylobiosyl fluoride. Two glycine mutants (TM and CF) recognized X₂ as an effective acceptor molecule to prohibit the formation of the polymer, while the other two (CS and BH) did not. The difference in acceptor specificity is considered to reflect the difference in substrate affinity at their +2 subsites. The results agreed with the structural predictions of the subsite, where TM and CF exhibit high affinity at subsite 2, suggesting that the glycosynthase technique is useful for investigating the affinity of +subsites.     

Metagenomic Profiling of Antibiotic Resistance Genes and Mobile Genetic Elements in a Tannery Wastewater Treatment Plant

Antibiotics are often used to prevent sickness and improve production in animal agriculture, and the residues in animal bodies may enter tannery wastewater during leather production. This study aimed to use Illumina high-throughput sequencing to investigate the occurrence, diversity and abundance of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in aerobic and anaerobic sludge of a full-scale tannery wastewater treatment plant (WWTP). Metagenomic analysis showed that Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria dominated in the WWTP, but the relative abundance of archaea in anaerobic sludge was higher than in aerobic sludge. Sequencing reads from aerobic and anaerobic sludge revealed differences in the abundance of functional genes between both microbial communities. Genes coding for antibiotic resistance were identified in both communities. BLAST analysis against Antibiotic Resistance Genes Database (ARDB) further revealed that aerobic and anaerobic sludge contained various ARGs with high abundance, among which sulfonamide resistance gene sul1 had the highest abundance, occupying over 20% of the total ARGs reads. Tetracycline resistance genes (tet) were highly rich in the anaerobic sludge, among which tet33 had the highest abundance, but was absent in aerobic sludge. Over 70 types of insertion sequences were detected in each sludge sample, and class 1 integrase genes were prevalent in the WWTP. The results highlighted prevalence of ARGs and MGEs in tannery WWTPs, which may deserve more public health concerns.     

Identification of Mur, an Atypical Peptidoglycan Hydrolase Derived from Leuconostoc citreum

A gene encoding a protein homologous to known bacterial N-acetyl-muramidases has been cloned from Leuconostoc citreum by a PCR-based approach. The encoded protein, Mur, consists of 209 amino acid residues with a calculated molecular mass of 23,821 Da including a 31-amino-acid putative signal peptide. In contrast to most of the other known peptidoglycan hydrolases, L. citreum Mur protein does not contain amino acid repeats involved in cell wall binding. The purified L. citreum Mur protein was shown to exhibit peptidoglycan-hydrolyzing activity by renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An active chimeric protein was constructed by fusion of L. citreum Mur to the C-terminal repeat-containing domain (cA) of AcmA, the major autolysin of Lactococcus lactis. Expression of the Mur-cA fusion protein was able to complement an acmA mutation in L. lactis; normal cell separation after cell division was restored by Mur-cA expression.     

Isolation and Characterization of a Cold-Active Xylanase Enzyme from Flavobacterium sp.

Xylan is the major component of hemicellulose, and xylan should be fully utilized to improve the efficiencies of a biobased economy. There are a variety of industrial reaction conditions in which an active xylanase enzyme would be desired. As a result, xylanase enzymes with different activity profiles are of great interest. We isolated a xylanase gene (xyn10) from a Flavobacterium sp. whose sequence suggests that it is a glycosyl hydrolase family 10 member. The enzyme has a temperature optimum of 30°C, is active at cold temperatures, and is thermolabile. The enzyme has an apparent K_m of 1.8 mg/ml and k_cat of 100 sec⁻¹ for beechwood xylan, attacks highly branched native xylan substrates, and does not have activity against glucans.     

Identification and Characterization of Coenzyme B12-Dependent Glycerol Dehydratase- and Diol Dehydratase-Encoding Genes from Metagenomic DNA Libraries Derived from Enrichment Cultures

To isolate genes encoding coenzyme B₁₂-dependent glycerol and diol dehydratases, metagenomic libraries from three different environmental samples were constructed after allowing growth of the dehydratase-containing microorganisms present for 48 h with glycerol under anaerobic conditions. The libraries were searched for the targeted genes by an activity screen, which was based on complementation of a constructed dehydratase-negative Escherichia coli strain. In this way, two positive E. coli clones out of 560,000 tested clones were obtained. In addition, screening was performed by colony hybridization with dehydratase-specific DNA fragments as probes. The screening of 158,000 E. coli clones by this method yielded five positive clones. Two of the plasmids (pAK6 and pAK8) recovered from the seven positive clones contained genes identical to those encoding the glycerol dehydratase of Citrobacter freundii and were not studied further. The remaining five plasmids (pAK2 to -5 and pAK7) contained two complete and three incomplete dehydratase-encoding gene regions, which were similar to the corresponding regions of enteric bacteria. Three (pAK2, -3, and -7) coded for glycerol dehydratases and two (pAK4 and -5) coded for diol dehydratases. We were able to perform high-level production and purification of three of these dehydratases. The glycerol dehydratases purified from E. coli Bl21/pAK2.1 and E. coli Bl21/pAK7.1 and the complemented hybrid diol dehydratase purified from E. coli Bl21/pAK5.1 were subject to suicide inactivation by glycerol and were cross-reactivated by the reactivation factor (DhaFG) for the glycerol dehydratase of C. freundii. The activities of the three environmentally derived dehydratases and that of glycerol dehydratase of C. freundii with glycerol or 1,2-propanediol as the substrate were inhibited in the presence of the glycerol fermentation product 1,3-propanediol. Taking the catalytic efficiency, stability against inactivation by glycerol, and inhibition by 1,3-propanediol into account, the hybrid diol dehydratase produced by E. coli Bl21/pAK5.1 exhibited the best properties of all tested enzymes for application in the biotechnological production of 1,3-propanediol.     

深海宏基因组文库克隆子发酵产物的生物活性筛选

目的:对前期构建的深海宏基因组文库克隆子发酵产物进行生物活性筛选.方法:利用CCK8法及琼脂扩散法和双层琼脂法进行了细胞毒活性及抗菌活性筛选,利用荧光定量PCR法进行了抗乙肝病毒活性筛选.结果:筛选发现3个具有细胞毒活性的混合克隆子.HPLC指纹图谱分析表明这3个混合克隆子具有与大肠杆菌宿主对照不同的指纹图谱,其活性与...     

Purification and Characterization of an Esterase from Micrococcus sp. YGJ1 Hydrolyzing Phthalate Esters

An esterase hydrolyzing phthalate esters has been purified from Micrococcus sp. YGJ1. The enzyme, a monomeric protein (M_r=56 kDa) with a pI of 4.0, hydrolyzes various aliphatic and aromatic carboxylesters. The medium chain (C₃-C₄) esters are the most preferred substrates. The enzyme is inhibited by HgCl₂ and p-chloromercuribenzoate but not by phenylmethyl-sulfonyl fluoride.     

Geochemical Characterization of Soil and Water From a Wastewater Treatment Plant in Gaza

The aim of the study was to determine the interaction between the natural geochemistry and the anthropogenic effects through trace element profiles in one of the environmentally significant areas of the Gaza Strip. Five boreholes were dug in the area of the Gaza wastewater treatment plant. The geology, mineralogy, and geochemistry of the soil profiles were studied, and the geochemistry of wastewater, sludge, soil, and groundwater was identified by several analytical techniques. The study introduced the environmental baselines and the infrastructure needed for further research for the first time: the natural infiltration potential, the artificial recharge, and the agricultural activities of water and wastewater. The results of the geochemical investigations confirmed that the upper 40 cm of soil was found to be the affected zone by wastewater and sludge. Among 26 elements analyzed, only a few metals (As, Cd, Cr, Hg, Zn, and to a lesser extent Pb) showed relevance from the human health point of view. The metal accumulations in the soils were characterized by a large spatial variability, with some “hot spots” of Cu and Zn reaching topsoil concentrations of up to 240 and 2005 mg/kg, respectively. In spite of that, the results of the groundwater revealed that none of them was detected at concentrations that exceeded the WHO (World Health Organization) standards. Moreover, it was shown that both anthropogenic activities as well as seawater intrusion caused the high levels of nitrate and salinity.     

Identification of a New Gene Encoding EPSPS with High Glyphosate Resistance from the Metagenomic Library

Glyphosate, a powerful nonselective herbicide, acts as an inhibitor of the activity of the enzyme 5-enoylpyruvylshikimate-3-phosphate synthase (EPSPS) encoded by the aroA gene involved in aromatic amino acid biosynthesis. An Escherichia coli mutant AKM4188 was constructed by insertion a kanamycin cassette within the aroA coding sequence. The mutant strain is an aromatic amino acids auxotroph and fails to grow on M9 minimal media due to the inactive aroA. A DNA metagenomic library was constructed with samples from a glyphosate-polluted area and was screened by using the mutant AKM4188 as recipient. Three plasmid clones, which restored growth to the aroA mutant in M9 minimal media supplemented with chloramphenicol, kanamycin, and 50 mM glyphosate, were obtained from the DNA metagenomic library. One of them, which conferred glyphosate tolerance up to 150 mM, was further characterized. The cloned fragment encoded a polypeptide, designated RD, sharing high similarity with other Class II EPSPS proteins. A His-tagged RD fusion protein was produced into E. coli to characterize the enzymatic properties of the RD EPSP protein.     

Isolation and Characterization of Metalloproteases with a Novel Domain Structure by Construction and Screening of Metagenomic Libraries

Small-insert metagenomic libraries from four samples were constructed by a topoisomerase-based and a T4 DNA ligase-based approach. Direct comparison of both approaches revealed that application of the topoisomerase-based method resulted in a higher number of insert-containing clones per μg of environmental DNA used for cloning and a larger average insert size. Subsequently, the constructed libraries were partially screened for the presence of genes conferring proteolytic activity. The function-driven screen was based on the ability of the library-containing Escherichia coli clones to form halos on skim milk-containing agar plates. The screening of 80,000 E. coli clones yielded four positive clones. Two of the plasmids (pTW2 and pTW3) recovered from positive clones conferred strong proteolytic activity and were studied further. Analysis of the entire insert sequences of pTW2 (28,113 bp) and pTW3 (19,956 bp) suggested that the DNA fragments were derived from members of the genus Xanthomonas. Each of the plasmids harbored one gene (2,589 bp) encoding a metalloprotease (mprA, pTW2; mprB, pTW3). Sequence and biochemical analyses revealed that MprA and MprB are similar extracellular proteases belonging to the M4 family of metallopeptidases (thermolysin-like family). Both enzymes possessed a unique modular structure and consisted of four regions: the signal sequence, the N-terminal proregion, the protease region, and the C-terminal extension. The architecture of the latter region, which was characterized by the presence of two prepeptidase C-terminal domains and one proprotein convertase P domain, is novel for bacterial metalloproteases. Studies with derivatives of MprA and MprB revealed that the C-terminal extension is not essential for protease activity. The optimum pH and temperature of both proteases were 8.0 and 65°C, respectively, when casein was used as substrate.Proteolytic enzymes catalyze the hydrolytic cleavage of peptide bonds. These enzymes are present in all living organisms and are essential for cell growth and differentiation. Microorganisms produce a variety of intracellular and/or extracellular proteases. Intracellular microbial proteases are highly specific and are involved in several cellular and metabolic processes such as activation of inactive precursors, maintenance of the cellular protein pool, and sporulation. Extracellular proteases degrade proteins in cell-free environments. The resulting hydrolytic products (small peptides and amino acids) can be transported into the cells and utilized as carbon or nitrogen sources (13, 39). Especially, extracellular proteases are of industrial importance and are used as cleaning agents and food and feed additives.Proteases can be divided into various groups with respect to the functional group present at the active site and the pH profile of the activity. Microbial alkaline proteases, which are defined to be active in a neutral to alkaline pH range (13), possess either a serine center (serine proteases) or are of metal-type (metalloproteases). Extracellular serine proteases are one of the most important groups of industrial enzymes (13, 23). In addition, many extracellular metalloproteases play an important role in pathogenesis (36). Both types of proteases have been cloned and sequenced from many different individual microorganisms, i.e., Bacillus subtilis (56), Enterobacter sakazakii (22), Listeria monocytogenes (4), and Alteromonas sp. (33, 34).In the present study, we sought to isolate proteases by a metagenomic approach. Functional metagenomics comprising the isolation of DNA from environmental samples without prior enrichment of individual microorganisms, the construction of libraries from the recovered DNA, and function-driven screening of the generated libraries has led to the identification and characterization of a variety of novel enzymes (7, 14, 43), i.e., amylases (44, 58), oxidoreductases (21), lipolytic enzymes (9, 16, 44), monooxygenases (53), and proteins conferring nickel resistance (32). Despite the wealth of different biotechnologically important enzyme activities derived from metagenomes, little information on the characteristics of metagenome-derived proteases is available. One metagenome-derived fibrinolytic metalloprotease has been recovered (26). In addition, one serine protease has been cited in reviews (13, 29). In other metagenome projects, activity-based screening was unsuccessful (19, 44), or the thereby recovered proteolytic clones were not characterized (47).We report here on the identification and characterization of two metagenome-derived metalloproteases. We constructed metagenomic DNA libraries from environmental samples by a T4 DNA ligase-based and a topoisomerase-based cloning method. The suitability of both approaches for the construction of small-insert metagenomic libraries was compared The resulting library-containing Escherichia coli clones were screened for proteolytic activity. The plasmids were recovered from the positive E. coli strains and the insert sequences of two plasmids (pTW2 and pTW3), which conferred strong proteolytic activity, were sequenced. The targeted protease-encoding genes and the corresponding gene products were characterized.     

Functional Screening of a Metagenomic Library Reveals Operons Responsible for Enhanced Intestinal Colonization by Gut Commensal Microbes

Evidence suggests that gut microbes colonize the mammalian intestine through propagation as an adhesive microbial community. A bacterial artificial chromosome (BAC) library of murine bowel microbiota DNA in the surrogate host Escherichia coli DH10B was screened for enhanced adherence capability. Two out of 5,472 DH10B clones, 10G6 and 25G1, exhibited enhanced capabilities to adhere to inanimate surfaces in functional screens. DNA segments inserted into the 10G6 and 25G1 clones were 52 and 41 kb and included 47 and 41 protein-coding open reading frames (ORFs), respectively. DNA sequence alignments, tetranucleotide frequency, and codon usage analysis strongly suggest that these two DNA fragments are derived from species belonging to the genus Bacteroides. Consistent with this finding, a large portion of the predicted gene products were highly homologous to those of Bacteroides spp. Transposon mutagenesis and subsequent experiments that involved heterologous expression identified two operons associated with enhanced adherence. E. coli strains transformed with the 10a or 25b operon adhered to the surface of intestinal epithelium and colonized the mouse intestine more vigorously than did the control strain. This study has revealed the genetic determinants of unknown commensals (probably resembling Bacteroides species) that enhance the ability of the bacteria to colonize the murine bowel.     

Identification and Characterization of Hundreds of Potent and Selective Inhibitors of Trypanosoma brucei Growth from a Kinase-Targeted Library Screening Campaign

In the interest of identification of new kinase-targeting chemotypes for target and pathway analysis and drug discovery in Trypanosomal brucei, a high-throughput screen of 42,444 focused inhibitors from the GlaxoSmithKline screening collection was performed against parasite cell cultures and counter-screened against human hepatocarcinoma (HepG2) cells. In this way, we have identified 797 sub-micromolar inhibitors of T. brucei growth that are at least 100-fold selective over HepG2 cells. Importantly, 242 of these hit compounds acted rapidly in inhibiting cellular growth, 137 showed rapid cidality. A variety of in silico and in vitro physicochemical and drug metabolism properties were assessed, and human kinase selectivity data were obtained, and, based on these data, we prioritized three compounds for pharmacokinetic assessment and demonstrated parasitological cure of a murine bloodstream infection of T. brucei rhodesiense with one of these compounds (NEU-1053). This work represents a successful implementation of a unique industrial-academic collaboration model aimed at identification of high quality inhibitors that will provide the parasitology community with chemical matter that can be utilized to develop kinase-targeting tool compounds. Furthermore these results are expected to provide rich starting points for discovery of kinase-targeting tool compounds for T. brucei, and new HAT therapeutics discovery programs.     

Isolation of α-Glucuronidase Enzyme from a Rumen Metagenomic Library

α-Glucuronidase enzymes play an essential role in the full enzymatic hydrolysis of hemicellulose. Up to this point, all genes encoding α-glucuronidase enzymes have been cloned from individual, pure culture strains. Using a high-throughput screening strategy, we have isolated the first α-glucuronidase gene (rum630-AG) from a mixed population of microorganisms. The gene was subcloned into a prokaryotic vector, and the enzyme was overexpressed and biochemically characterized. The RUM630-AG enzyme had optimum activity at pH 6.5 and 40 °C. When birchwood xylan was used as substrate, the RUM630-AG functioned synergistically with an endoxylanase enzyme to hydrolyze the substrate.