Significant Hydrolytic Activity Research Articles

Isolation of Thermophilic Bacteria from Extreme Environments in Northern Chile.

The northern region of Chile boasts unique geographical features that support the emergence of geothermal effluents, salt lagoons, and coastal creeks. These extreme climate conditions create polyextreme habitats for microorganisms, particularly adapted to survive these harsh environments. These extremophilic microorganisms hold immense potential as a source of hydrolytic enzymes, among other biotechnological applications. In this study, we isolated 15 strains of aerobic thermophilic bacteria (45-70 °C) from sediment samples collected at five different ecological sites, including hot springs, geothermal fields, and lagoons in the Atacama Desert and Andes high planes. Analyses of the 16S rRNA gene sequences of the isolates showed a close genetic similarity (98-100%) with microorganisms of the genera Parageobacillus, Geobacillus, Anoxybacillus, and Aeribacillus. Notably, these thermophiles exhibited significant hydrolytic enzyme activity, particularly amylases, lipases, and proteases. These findings underscore the potential of using these thermophilic bacterial strains as an invaluable source of thermozymes with wide-ranging applications in diverse industries, such as detergent formulations, pharmaceutical processing, and food technology. This research highlights the ecological significance of these extreme environments in the Atacama Desert and Andes high plains, which serve as vital ecological niches housing extremophilic bacteria as a genetic source of relevant thermozymes, promising great potential for innovation in the biotechnology industry.

Bioengineering Comamonas testosteroni CNB-1: a robust whole-cell biocatalyst for efficient PET microplastic degradation

The escalating crisis of polyethylene terephthalate (PET) microplastic contamination in biological wastewater treatment systems is a pressing environmental concern. These microplastics inevitably accumulate in sewage sludge due to the absence of effective removal technologies. Addressing this urgent issue, this study introduces a novel approach using DuraPETase, a potent enzyme with enhanced PET hydrolytic activity at ambient temperatures. Remarkably, this enzyme was successfully secreted from Comamonas testosteroni CNB-1, a dominant species in the active sludge. The secreted DuraPETase showed significant hydrolytic activity toward p-NPB and PET nanoplastics. Furthermore, the CNB-1 derived whole-cell biocatalyst was able to depolymerize PET microplastics under ambient temperature, achieving a degradation efficiency of 9% within 7 days. The CNB-1-based whole biocatalysts were also capable of utilizing PET degradation intermediates, such as terephthalic acid (TPA) and ethylene glycol (EG), and bis(2-hydroxyethyl)-TPA (BHET), for growth. This indicates that it can completely mineralize PET, as opposed to merely breaking it down into smaller molecules. This research highlights the potential of activated sludge as a potent source for insitu microplastic removal.Graphical

Structural insights into hydrolytic defluorination of difluoroacetate by microbial fluoroacetate dehalogenases.

Fluorine forms the strongest single bond to carbon with the highest bond dissociation energy among natural products. However, fluoroacetate dehalogenases (FADs) have been shown to hydrolyze this bond in fluoroacetate under mild reaction conditions. Furthermore, two recent studies demonstrated that the FAD RPA1163 from Rhodopseudomonas palustris can also accept bulkier substrates. In this study, we explored the substrate promiscuity of microbial FADs and their ability to defluorinate polyfluorinated organic acids. Enzymatic screening of eight purified dehalogenases with reported fluoroacetate defluorination activity revealed significant hydrolytic activity against difluoroacetate in three proteins. Product analysis using liquid chromatography-mass spectrometry identified glyoxylic acid as the final product of enzymatic DFA defluorination. The crystal structures of DAR3835 from Dechloromonas aromatica and NOS0089 from Nostoc sp. were determined in the apo-state along with the DAR3835 H274N glycolyl intermediate. Structure-based site-directed mutagenesis of DAR3835 demonstrated a key role for the catalytic triad and other active site residues in the defluorination of both fluoroacetate and difluoroacetate. Computational analysis of the dimer structures of DAR3835, NOS0089, and RPA1163 indicated the presence of one substrate access tunnel in each protomer. Moreover, protein-ligand docking simulations suggested similar catalytic mechanisms for the defluorination of both fluoroacetate and difluoroacetate, with difluoroacetate being defluorinated via two consecutive defluorination reactions producing glyoxylate as the final product. Thus, our findings provide molecular insights into substrate promiscuity and catalytic mechanism of FADs, which are promising biocatalysts for applications in synthetic chemistry and bioremediation of fluorochemicals.

Endoglucanase effects on energy consumption in the mechanical fibrillation of cellulose fibers into nanocelluloses

Enzymatic processing is considered a promising approach for advancing environmentally friendly industrial processes, such as the use of endoglucanase (EG) enzyme in the production of nanocellulose. However, there is ongoing debate regarding the specific properties that make EG pretreatment effective in isolating fibrillated cellulose. To address this issue, we investigated EGs from four glycosyl hydrolase (GH) families (5, 6, 7, and 12) and examined the roles of the three-dimensional structure and catalytic features, with a focus on the presence of a carbohydrate binding module (CBM). Using eucalyptus Kraft wood fibers, we produced cellulose nanofibrils (CNFs) through mild enzymatic pretreatment, followed by disc ultra-refining. Comparing the results with the control (without pretreatment), we observed that GH5 and GH12 enzymes (without CBM) reduced fibrillation energy by approximately 15 %. The most significant energy reduction, 25 and 32 %, was achieved with GH5 and GH6 linked to CBM, respectively. Notably, these CBM-linked EGs improved the rheological properties of CNF suspensions without releasing soluble products. In contrast, GH7-CBM exhibited significant hydrolytic activity, resulting in the release of soluble products, but did not contribute to a reduction in fibrillation energy. This discrepancy can be attributed to the large molecular weight and wide cleft of GH7-CBM, which led to the release of soluble sugars but had little impact on fibrillation. Our findings suggest that the improved fibrillation observed with EG pretreatment is primarily driven by efficient enzyme adsorption on the substrate and modification of the surface viscoelasticity (amorphogenesis), rather than hydrolytic activity or release of products.

Carboxylesterase 2a deletion provokes hepatic steatosis and insulin resistance in mice involving impaired diacylglycerol and lysophosphatidylcholine catabolism

ObjectiveHepatic triacylglycerol accumulation and insulin resistance are key features of NAFLD. However, NAFLD development and progression are rather triggered by the aberrant generation of lipid metabolites and signaling molecules including diacylglycerol (DAG) and lysophosphatidylcholine (lysoPC). Recent studies showed decreased expression of carboxylesterase 2 (CES2) in the liver of NASH patients and hepatic DAG accumulation was linked to low CES2 activity in obese individuals. The mouse genome encodes several Ces2 genes with Ces2a showing highest expression in the liver. Herein we investigated the role of mouse Ces2a and human CES2 in lipid metabolism in vivo and in vitro. MethodsLipid metabolism and insulin signaling were investigated in mice lacking Ces2a and in a human liver cell line upon pharmacological CES2 inhibition. Lipid hydrolytic activities were determined in vivo and from recombinant proteins. ResultsCes2a deficient mice (Ces2a-ko) are obese and feeding a high-fat diet (HFD) provokes severe hepatic steatosis and insulin resistance together with elevated inflammatory and fibrotic gene expression. Lipidomic analysis revealed a marked rise in DAG and lysoPC levels in the liver of Ces2a-ko mice fed HFD. Hepatic lipid accumulation in Ces2a deficiency is linked to lower DAG and lysoPC hydrolytic activities in liver microsomal preparations. Moreover, Ces2a deficiency significantly increases hepatic expression and activity of MGAT1, a PPAR gamma target gene, suggesting aberrant lipid signaling upon Ces2a deficiency. Mechanistically, we found that recombinant Ces2a and CES2 show significant hydrolytic activity towards lysoPC (and DAG) and pharmacological inhibition of CES2 in human HepG2 cells largely phenocopies the lipid metabolic changes present in Ces2a-ko mice including reduced lysoPC and DAG hydrolysis, DAG accumulation and impaired insulin signaling. ConclusionsCes2a and CES2 are critical players in hepatic lipid signaling likely via the hydrolysis of DAG and lysoPC at the ER.

Synthesis of Inorganic Pyrophosphatase-Nanodiamond Conjugates Resistant to Calcium and Fluoride.

Pyrophosphate arthropathy is the mineralization defect in humans caused by the deposition of microcrystals of calcium pyrophosphate dihydrate in joint tissues. As a potential therapeutic strategy for the treatment of pyrophosphate arthropathy, delivery of exogenous pyrophosphate-hydrolyzing enzymes, inorganic pyrophosphatases (PPases), to the synovial fluid has been suggested. Previously, we synthesized the conjugates of Escherichia coli PPase (Ec-PPase) with detonation synthesis nanodiamonds (NDs) as a delivery platform, obtaining the hybrid biomaterial retaining high pyrophosphate-hydrolyzing activity in vitro. However, most known PPases including Ec-PPase in the soluble form are strongly inhibited by Ca2+ ions. Because synovial fluid contains up to millimolar concentrations of soluble calcium, this inhibition might limit the in vivo application of Ec-PPase-based material in joint tissues. In this work, we proposed other bacterial PPases from Mycobacterium tuberculosis (Mt-PPase), which are resistant to the inhibition by Ca2+ ions, as an active PPi-hydrolyzing agent. We synthesized conjugates of Mt-PPase with NDs and tested their activity under various conditions. Unexpectedly, conjugates of both Ec-PPase and Mt-PPase with aminated NDs retained significant hydrolytic activity in the presence of well-known mechanism-based PPase inhibitors, fluoride or calcium. The incomplete inhibition of PPases by fluoride or calcium was found for the first time.

A Novel VIII Carboxylesterase with High Hydrolytic Activity Against Ampicillin from a Soil Metagenomic Library.

A novel carboxylesterase gene, named dlfae4, was discovered and sequenced from a soil metagenomic library. The dlfae4 gene was composed of 1017 base pairs encoding 338 amino acid residues with a predicted molecular mass of 37.2kDa. DLFae4 exhibited strong hydrolytic activity towards methyl ferulate under optimum pH and temperature conditions (pH 8.6, 50°C) and displayed remarkable thermostability, with residual activity as high as 50% after incubation for 3h at 60°C. A family VIII esterase DLFae4 was found to contain a typical serine residue within the S-X-X-K motif, which serves as a catalytic nucleophile in class C β-lactamases and family VIII esterases. As a consequence of its high sequence similarity with β-lactamases, DLFae4 exhibited significant hydrolytic activity towards ampicillin. In addition, DLFae4 was found to be the first known member of family VIII carboxylesterases with phthalate-degrading ability. Site-directed mutagenesis studies revealed that Ser11, Lys14, and Tyr121 residues play an essential catalytic role in DLFae4. These new findings, which are of great importance for further in-depth research and engineering development of carboxylesterases, should advance the implementation of biotechnological applications.

ZHO-1, an intrinsic MBL from the environmental Gram-negative species Zhongshania aliphaticivorans.

Our aim was to characterize the putative MBL of the environmental strain Zhongshania aliphaticivorans isolated from a marine environment. The putative MBL was identified in silico using the NCBI database. The β-lactamase gene was cloned into different Escherichia coli backgrounds. Kinetic parameters were determined using the purified enzyme. The enzyme named ZHO-1 shared 51% amino acid identity with the acquired class B carbapenemases IMP-1, KHM-1 and DIM-1. Expression of the blaZHO-1 gene in a susceptible E. coli resulted in a carbapenemase phenotype. Kinetic parameters determined from purified ZHO-1 enzyme showed that it had significant hydrolytic activity against most β-lactams including penicillins, cephalosporins and carbapenems, with the exception of aztreonam and cefepime. This study adds to the knowledge regarding environmental species as a reservoir of possible clinically relevant MBLs.

Implications of dimeric activation of PDE6 for rod phototransduction.

We examine the implications of a recent report providing evidence that two transducins must bind to the rod phosphodiesterase to elicit significant hydrolytic activity. To predict the rod photoreceptor's electrical response, we use numerical simulation of the two-dimensional diffusional contact of interacting molecules at the surface of the disc membrane, and then we use the simulated PDE activity as the driving function for the downstream reaction cascade. The results account for a number of aspects of rod phototransduction that have previously been puzzling. For example, they explain the existence of a greater initial delay in rods than in cones. Furthermore, our analysis suggests that the ‘continuous’ noise recorded in rods in darkness is likely to arise from spontaneous activation of individual molecules of PDE at a rate of a few tens per second per rod, probably as a consequence of spontaneous activation of transducins at a rate of thousands per second per rod. Hence, the dimeric activation of PDE in rods provides immunity against spontaneous transducin activation, thereby reducing the continuous noise. Our analysis also provides a coherent quantitative explanation of the amplification underlying the single photon response. Overall, numerical analysis of the dimeric activation of PDE places rod phototransduction in a new light.

Characterization of esterase activity from an Acetomicrobium hydrogeniformans enzyme with high structural stability in extreme conditions.

The biotechnological and industrial uses of thermostable and organic solvent-tolerant enzymes are extensive and the investigation of such enzymes from microbiota present in oil reservoirs is a promising approach. Searching sequence databases for esterases from such microbiota, we have identified in silico a potentially secreted esterase from Acetomicrobium hydrogeniformans, named AhEst. The recombinant enzyme was produced in E. coli to be used in biochemical and biophysical characterization studies. AhEst presented hydrolytic activity on short-acyl-chain p-nitrophenyl ester substrates. AhEst activity was high and stable in temperatures up to 75°C. Interestingly, high salt concentration induced a significant increase of catalytic activity. AhEst still retained ~ 50% of its activity in 30% concentration of several organic solvents. Synchrotron radiation circular dichroism and fluorescence spectroscopies confirmed that AhEst displays high structural stability in extreme conditions of temperature, salinity, and organic solvents. The enzyme is a good emulsifier agent and is able to partially reverse the wettability of an oil-wet carbonate substrate, making it of potential interest for use in enhanced oil recovery. All the traits observed in AhEst make it an interesting candidate for many industrial applications, such as those in which a significant hydrolytic activity at high temperatures is required.

Adding Value to Agro-industrial Co-products from Canola and Soybean Oil Extraction Through Lipase Production Using Yarrowia lipolytica in Solid-State Fermentation

The vegetable oil industry generates several materials after oil extraction that can be used as animal feed but that can represent an environmental and public health concern in case of improper disposal. However, the bioconversion of these feedstocks for the production of enzymes of industrial interest, such as lipases, by fermentation is an interesting alternative to obtain a product of high added value from a low cost material. This study aimed to use canola cake and soybean meal as feedstocks for lipase production through solid-state fermentation by Yarrowia lipolytica. The lipase obtained from canola cake, after 28 h fermentation presented the highest activity of 72.6 ± 2.4 U/g when using olive oil as substrate. On the other hand, the activity of the lipase obtained from soybean meal, after 14 h of fermentation reached the highest value of 93.9 ± 2.9 U/g using olive oil as substrate. The lipase produced from canola cake presented optimal performance at alkaline pH and high temperatures, while the lipase extract produced from soybean meal presented better hydrolytic activity under mild conditions. Yarrowia lipolytica demonstrated high biotechnological potential for solid-state fermentation using canola cake and soybean meal, since it was able to produce lipase in both feedstocks in a short period of time, adding value to these agro-industrial co-products. Furthermore, the lipase crude extracts produced are versatile biocatalysts, once they presented significant hydrolytic activity in different conditions of temperature and pH.

The unusual substrate specificity of a virulence associated serine hydrolase from the highly toxic bacterium, Francisella tularensis

Francisella tularensis is the causative agent of the highly, infectious disease, tularemia. Amongst the genes identified as essential to the virulence of F. tularensis was the proposed serine hydrolase FTT0941c. Herein, we purified FTT0941c to homogeneity and then characterized the folded stability, enzymatic activity, and substrate specificity of FTT0941c. Based on phylogenetic analysis, FTT0941c was classified within a divergent Francisella subbranch of the bacterial hormone sensitive lipase (HSL) superfamily, but with the conserved sequence motifs of a bacterial serine hydrolase. FTT0941c showed broad hydrolase activity against diverse libraries of ester substrates, including significant hydrolytic activity across alkyl ester substrates from 2 to 8 carbons in length. Among a diverse library of fluorogenic substrates, FTT0941c preferred α-cyclohexyl ester substrates, matching with the substrate specificity of structural homologues and the broad open architecture of its modeled binding pocket. By substitutional analysis, FTT0941c was confirmed to have a classic catalytic triad of Ser115, His278, and Asp248 and to remain thermally stable even after substitution. Its overall substrate specificity profile, divergent phylogenetic homology, and preliminary pathway analysis suggested potential biological functions for FTT0941c in diverse metabolic degradation pathways in F. tularensis.

2-O-α-D-Glucosylglycerol Phosphorylase from Bacillus selenitireducens MLS10 Possessing Hydrolytic Activity on β-D-Glucose 1-Phosphate

The glycoside hydrolase family (GH) 65 is a family of inverting phosphorylases that act on α-glucosides. A GH65 protein (Bsel_2816) from Bacillus selenitireducens MLS10 exhibited inorganic phosphate (Pi)-dependent hydrolysis of kojibiose at the rate of 0.43 s−1. No carbohydrate acted as acceptor for the reverse phosphorolysis using β-d-glucose 1-phosphate (βGlc1P) as donor. During the search for a suitable acceptor, we found that Bsel_2816 possessed hydrolytic activity on βGlc1P with a k cat of 2.8 s−1; moreover, such significant hydrolytic activity on sugar 1-phosphate had not been reported for any inverting phosphorylase. The H2 18O incorporation experiment and the anomeric analysis during the hydrolysis of βGlc1P revealed that the hydrolysis was due to the glucosyl-transferring reaction to a water molecule and not a phosphatase-type reaction. Glycerol was found to be the best acceptor to generate 2-O-α-d-glucosylglycerol (GG) at the rate of 180 s−1. Bsel_2816 phosphorolyzed GG through sequential Bi-Bi mechanism with a k cat of 95 s−1. We propose 2-O-α-d-glucopyranosylglycerol: phosphate β-d-glucosyltransferase as the systematic name and 2-O-α-d-glucosylglycerol phosphorylase as the short name for Bsel_2816. This is the first report describing a phosphorylase that utilizes polyols, and not carbohydrates, as suitable acceptor substrates.

A new mechanism and kinetic model for the enzymatic synthesis of short-chain fructooligosaccharides from sucrose

A kinetic model based on a ping-pong mechanism was developed under the steady-state hypothesis to account for the short-chain fructooligosaccharides (sc-FOS) synthesis using the commercial cellulolytic enzyme preparation, Rohapect CM. This new mechanism takes into account the interactions between the enzyme species and potential substrates (sucrose and sc-FOS) as a single complex reaction, allowing a better understanding of the reaction kinetics.The initial reaction rate laws appropriately describe the kinetic profiles of the examined substrates. Whereas sucrose exhibited Michaelis–Menten behavior with substrate inhibition, 1-kestose and nystose followed Michaelis–Menten and sigmoid enzyme kinetics. In addition, the enzyme was competitively inhibited by glucose and exhibited significant hydrolytic activity in the presence of nystose.The overall model was simultaneously fitted to experimental data from three initial sucrose concentrations (0.5, 1.5 and 2.1M) using a multi-response regression with kinetic parameters that have biochemical relevance and are independent of the enzyme concentration. According to the model, sucrose acts almost exclusively as a fructosyl donor substrate. The mathematical development described herein is expected to be suitable for modeling similar enzymatic reaction systems.

βIII-Gal is Involved in Galactan Reduction During Phloem Element Differentiation in Chickpea Stems

βIII-Gal, a member of the chickpea β-galactosidase family, is the enzyme responsible for the cell wall autolytic process. This enzyme, whose activity increases during epicotyl growth, displays significant hydrolytic activity against cell wall pectins, and its natural substrate has been determined as an arabinogalactan from the pectic fraction of the cell wall. In the present work, the localization of βIII-Gal in different seedling and plant organs was analyzed by using specific anti-βIII-Gal antibodies. Our results revealed that besides its possible role in cell wall loosening and in early events during primary xylem and phloem fiber differentiation βIII-Gal acts on the development of sieve elements. Localization of the enzyme in this tissue, both in epicotyls and radicles from seedlings and in the different stem internodes, is consistent with the reduction in galactan during the maturation of phloem elements, as can be observed with LM5 antibodies. Thus, βIII-Gal could act on its natural substrate, the neutral side chains of rhamnogalacturonan I, contributing to cell wall reinforcement allowing phloem elements to differentiate, and conferring the necessary strengthening of the cell wall to fulfill its function. This work completes the immunolocation studies of all known chickpea β-galactosidases. Taken together, our results reflect the broad range of developmental processes covered by different members of this protein family, and confirm their crucial role in cell wall remodeling during tissue differentiation.

Self-surface assembly of cellulosomes with two miniscaffoldins on Saccharomyces cerevisiae for cellulosic ethanol production

Yeast to directly convert cellulose and, especially, the microcrystalline cellulose into bioethanol, was engineered through display of minicellulosomes on the cell surface of Saccharomyces cerevisiae. The construction and cell surface attachment of cellulosomes were accomplished with two individual miniscaffoldins to increase the display level. All of the cellulases including a celCCA (endoglucanase), a celCCE (cellobiohydrolase), and a Ccel_2454 (β-glucosidase) were cloned from Clostridium cellulolyticum, ensuring the thermal compatibility between cellulose hydrolysis and yeast fermentation. Cellulases and one of miniscaffoldins were secreted by α-factor; thus, the assembly and attachment to anchoring miniscaffoldin were accomplished extracellularly. Immunofluorescence microscopy, flow cytometric analysis (FACS), and cellulosic ethanol fermentation confirmed the successful display of such complex on the yeast surface. Enzyme-enzyme synergy, enzyme-proximity synergy, and cellulose-enzyme-cell synergy were analyzed, and the length of anchoring miniscaffoldin was optimized. The engineered S. cerevisiae was applied in fermentation of carboxymethyl cellulose (CMC), phosphoric acid-swollen cellulose (PASC), or Avicel. It showed a significant hydrolytic activity toward microcrystalline cellulose, with an ethanol titer of 1,412 mg/L. This indicates that simultaneous saccharification and fermentation of crystalline cellulose to ethanol can be accomplished by the yeast, engineered with minicellulosome.

Characterization of the M32 metallocarboxypeptidase of Trypanosoma brucei: Differences and similarities with its orthologue in Trypanosoma cruzi

Metallocarboxypeptidases (MCP) of the M32 family of peptidases have been identified in a number of prokaryotic organisms but they are absent from eukaryotic genomes with the remarkable exception of those of trypanosomatids. The genome of Trypanosoma brucei, the causative agent of Sleeping Sickness, encodes one such MCP which displays 72% identity to the characterized TcMCP-1 from Trypanosoma cruzi. As its orthologue, TcMCP-1, Trypanosoma brucei MCP is a cytosolic enzyme expressed in both major stages of the parasite. Purified recombinant TbMCP-1 exhibits a significant hydrolytic activity against the carboxypeptidase B substrate FA (furylacryloil)-Ala-Lys at pH 7.0–7.8 resembling the T. cruzi enzyme. Several divalent cations had little effect on TbMCP-1 activity but increasing amounts of Co2+ inhibited the enzyme. Despite having similar tertiary structure, both protozoan MCPs display different substrate specificity with respect to P1 position. Thus, TcMCP-1 enzyme cleaved Abz-FVK-(Dnp)-OH substrate (where Abz: o-aminobenzoic acid and Dnp: 2,4-dinitrophenyl) whereas TbMCP-1 had no activity on this substrate. Comparative homology models and sequence alignments using TcMCP-1 as a template led us to map several residues that could explain this difference. To verify this hypothesis, site-directed mutagenesis was undertaken replacing the TbMCP-1 residues by those present in TcMCP-1. We found that the substitution A414M led TbMCP-1 to gain activity on Abz-FVK-(Dnp)-OH, thus showing that this residue is involved in specificity determination, probably being part of the S1 sub-site. Moreover, the activity of both protozoan MCPs was explored on two vasoactive compounds such as bradykinin and angiotensin I resulting in two different hydrolysis patterns.

CTX-M-14 and CTX-M-15 enzymes are the dominant type of extended-spectrum β-lactamase in clinical isolates of Escherichia coli from Korea

This study was performed to assess the prevalence and genotypes of plasmid-borne extended-spectrum β-lactamases (ESBLs) and AmpC β-lactamases in Escherichia coli in Korea. A total of 576 isolates of E. coli was collected from 12 Korean hospitals during May and July 2007. A phenotypic confirmatory test detected ESBLs in 82 (14.2 %) of the 576 E. coli isolates. The most common types of ESBLs identified were CTX-M-14 (n=32) and CTX-M-15 (n=27). The prevalence and diversity of the CTX-M mutants, including CTX-M-15, CTX-M-27 and CTX-M-57, with significant hydrolytic activity against ceftazidime were increased. PCR experiments detected genes encoding plasmid-borne AmpC β-lactamases in 15/56 cefoxitin-intermediate or cefoxitin-resistant isolates, and the most common type of AmpC β-lactamase identified was DHA-1 (n=10). These data suggest that the incidence of ESBLs in E. coli has increased as a result of the dissemination of CTX-M enzymes in Korea. In addition, CTX-M-22, CTX-M-27 and CTX-M-57 have appeared in Korea.

Characterization of an Evolutionarily Conserved Metallophosphoesterase That Is Expressed in the Fetal Brain and Associated with the WAGR Syndrome

Among the human diseases that result from chromosomal aberrations, a de novo deletion in chromosome 11p13 is clinically associated with a syndrome characterized by Wilms' tumor, aniridia, genitourinary anomalies, and mental retardation (WAGR). Not all genes in the deleted region have been characterized biochemically or functionally. We have recently identified the first Class III cyclic nucleotide phosphodiesterase, Rv0805, from Mycobacterium tuberculosis, which biochemically and structurally belongs to the superfamily of metallophosphoesterases. We performed a large scale bioinformatic analysis to identify orthologs of the Rv0805 protein and identified many eukaryotic genes that included the human 239FB gene present in the region deleted in the WAGR syndrome. We report here the first detailed biochemical characterization of the rat 239FB protein and show that it possesses metallophosphodiesterase activity. Extensive mutational analysis identified residues that are involved in metal interaction at the binuclear metal center. Generation of a rat 239FB protein with a mutation corresponding to a single nucleotide polymorphism seen in human 239FB led to complete inactivation of the protein. A close ortholog of 239FB is found in adult tissues, and biochemical characterization of the 239AB protein demonstrated significant hydrolytic activity against 2',3'-cAMP, thus representing the first evidence for a Class III cyclic nucleotide phosphodiesterase in mammals. Highly conserved orthologs of the 239FB protein are found in Caenorhabditis elegans and Drosophila and, coupled with available evidence suggesting that 239FB is a tumor suppressor, indicate the important role this protein must play in diverse cellular events.

Purification and N-terminal sequence of a serine proteinase-like protein (BMK-CBP) from the venom of the Chinese scorpion ( Buthus martensii Karsch)

A serine proteinase-like protein was isolated from the venom of Chinese red scorpion ( Buthus martensii Karsch) by combination of gel filtration, ion-exchange and reveres-phase chromatography and named BMK-CBP. The apparent molecular weight of BMK-CBP was identified as 33 kDa by SDS-PAGE under non-reducing condition. The sequence of N-terminal 40 amino acids was obtained by Edman degradation. The sequence shows highest similarity to proteinase from insect source. When tested with commonly used substrates of proteinase, no significant hydrolytic activity was observed for BMK-CBP. The purified BMK-CBP was found to bind to the cancer cell line MCF-7 and the cell binding ability was dose-dependent.