7,8-Dihydro-8-oxoguanine (oh8Gua) endonuclease is a DNA repair enzyme in Escherichia coli to remove oh8Gua, a promutagenic DNA adduct. Due to the unique mode of enzyme action and substrate specificity, this DNA repair enzyme has been suggested to be identical to 2,6-diamino-4-hydroxyformamidopyrimidine (Fapy)-DNA glycosylase (Fpg). However, oh8Gua endonuclease had not been definitely identified because it had not been homogeneously purified. In this study, we attempted to purify and identify the enzyme. Through several purification procedures, we obtained two proteins (32 kD and 29 kD). The larger protein co-migrated with Fpg in 12% SDS-PAGE gel. Sequences of N-terminal amino acids of these two proteins were identical to that of Fpg; the smaller one is a degraded product of oh8Gua endonuclease during purification steps. These results indicate that oh8Gua endonuclease is identical to Fpg, implying that oh8Gua in oxidatively damaged DNA rather than Fapy is more physiologically relevant substrate for Fpg.
Chromatography, Affinity ; DNA Damage ; DNA Repair ; Escherichia coli/enzymology* ; Nucleosidases/isolation & purification* ; Sequence Analysis, Protein

The beta-glucosidase encoding gene bglA was cloned from Bacillus polymyxa 1.794. The bglA gene was inserted in expression vector pET28a(+) and transformed into Escherichia coli BL21 (DE3), finally the recombinant strain BL1979 was obtained. Induced by IPTG, the expression P-glucosidase activity reached to 24.7 IU/mL. The optimum temperature and optimum pH of the recombinant expression P-glucosidase in BL1979 were 37 degrees C and 7.0 respectively,the purity can reach to 92.7%. Analysis of the fusion protein by nondenaturing gradient gel electrophoresis, we found the fusion protein exists in dimmer, tetramer,hexamer and octamer, they all have hydrolase activity.
Bacillus ; enzymology ; Escherichia coli ; genetics ; Plasmids ; Recombinant Proteins ; biosynthesis ; isolation & purification ; beta-Glucosidase ; genetics ; isolation & purification ; metabolism

Considering Alcaligenes faecalis pencillin G acylase(AfPGA), which possesses the attractive characteristics for beta-lactam antibiotics conversions, the gene of PGA was cloned into an expressing vector pKKFPGA. The recombinant plasmid contained multicopy replicon(COLE 1), trc promoter, AfPGA gene, rrnB transcript terminator and ampicillin marker transformed Escherichia coli DH5alpha. As both the recombinant plasmid and the host DH5alpha had no laclq gene, the trc promoter was always active and the AfPGA could be constitutively expressed without IPTG induction in the host DH5alpha. In the shaking flask, the recombinant cell was inoculated into the fermentation medium (tryptone 10g/L, yeast extract 5g/L, MgSO4 x 7 H2O 1g, KH2 PO4 2g/L, K2HPO4 x 3H2O 5g/L, Na2HPO4 x 12H2O 7g/L, (NH4)2SO4 1.2g/L, NH4Cl 0.2 g/L, NaCl 0.1g/L, dextrin 30g/L) and cultured at 28 degrees C for 20h. The production of AfPGA reached 2,590u/L(NIPAB method), with a cell-density-specific activity of more than 300(u/L)/A600, this yield increased 432 fold higher than the native expression of Alcaligenes faecalis . Without ammonium sulphate fractionation and dialysis, the supernatant of crude extract was directly loaded on DEAE-Sepharose CL 6B column equilibrated by phosphate buffer (50mmol/L, pH7.8), and the enzyme fraction was not absorbed on the column but impurities were absorbed. Subsequently the effluent was added ammonium sulphate to 1mol/L and loaded on Butyl-Sepharose CL 4B column equilibrated by 50mmol/L phosphate buffer pH7.8-1mol/L ammonium sulphate. The enzyme was eluted as concentration of ammonium sulphate in phosphate buffer decreased to 0, PGA was eluted. After these two column chromatography, the enzyme was enriched 20 times with a 91% activity recovery. The purified enzyme had a specific activity of 68.6u/mg protein. However, the overproduction of PGA was often limited by translocation and/or periplasmic processing steps, subsequently resulted in intracellular accumulation of various types of PGA precursors and then formed inclusion bodies in the cytoplasm and/or periplasm. In this study, 5% PGA precursors formed as inclusion bodies in the cytoplasm while no inclusion bodies formed in the periplasm. It suggested most PGA precursors were transported to the periplasm and matured to active PGA and also explained why PGA gene was highly expressed in the host DH5alpha. On the other hand, inclusion bodies in the cytoplasm indicated that the maturation of PGA in the host DHSalpha was limited by the translocation step.
Alcaligenes faecalis ; enzymology ; Blotting, Western ; Escherichia coli ; genetics ; Penicillin Amidase ; genetics ; isolation & purification ; Recombinant Proteins ; biosynthesis ; isolation & purification

The xylanolytic enzymes found in Thermotoga maritima showed extremely high thermostability and considerable potential in industrial application. Yet expression level of the genes encoding these enzymes was very low. The alpha-glucuronidase gene aguA from T. maritima ATCC 43589 was cloned and expressed in several E. coli strains with different vector. The alpha-glucuronidase was overexpressed in E. coli BL21-CodonPlus(DE3)-RIL with plasmid pET-28a(+), and made up about 20% of the total proteins present in the intracellular soluble fraction. The results proved the assumption that rare codons for arginine (AGA/AGG) and isoleucine (AUA) affect the expression of aguA gene from hyperthermophilic bacterium T. maritima in E. coli. Purification procedure included two steps, heat treatment and immobilized metal affinity chromatography, and over 13.5mg of pure enzyme was obrained from 1L of induced culture. The purified enzyme showed a single band on SDS polyacrylamide gel electrophoresis with a purification of 5.1 fold, and a yield of 55.1%. The optimum activity of recombinant alpha-glucuronidase was found at pH 6.0 and 85 degrees C, the enzyme retained 70% of its activity after 1 h of incubation at 85 degrees C. The induction conditions for expression of recombinant strain BL21-CodonPlus(DE3)-RIL/pET-28a-aguA were studied on induction time and duration by IPTG. The results showed that the activity of thermostable alpha-glucuronidase reach the maximum in 5-hour after inducted at the exponential phase (OD600 of 0.7 - 0.8).
Escherichia coli ; genetics ; Glycoside Hydrolases ; genetics ; isolation & purification ; metabolism ; Plasmids ; Recombinant Proteins ; biosynthesis ; isolation & purification ; Thermotoga maritima ; enzymology

BACKGROUND: This study was performed to investigate the prevalence of qnr genes in clinical isolates of Escherichia coli from Korea that produce extended-spectrum beta-lactamases (ESBLs). METHODS: During the period of May to June 2005, we collected clinical isolates of E. coli that were intermediate or resistant to ceftazidime and/or cefotaxime from 11 Korean hospitals. Antimicrobial susceptibility was determined by the disk diffusion and agar dilution methods. ESBL production was confirmed phenotypically by the double-disk synergy test. ESBL and qnr genes were searched for by PCR amplification, and the PCR products were then subjected to direct sequencing. RESULTS: Double-disk synergy tests were positive in 84.3% (118/140) of ceftazidime- and/or cefotaxime-nonsusceptible E. coli isolates. The most prevalent types of ESBL in E. coli isolates were CTX-M-14 (N=41) and CTX-M-15 (N=58). Other ESBLs were also identified, including CTX-M-3 (N=7), CTX-M-9 (N=8), CTX-M-12 (N=1), CTX-M-57 (N=1), SHV-2a (N=2), SHV-12 (N=17) and TEM-52 (N=4). The qnrA1 and qnrB4 genes were identified in 4 and 7 ESBL-producing isolates, respectively. CONCLUSIONS: CTX-M-type enzymes were the most common type of ESBL in E. coli isolates from Korea, and the qnr genes were not uncommon in ESBL-producing E. coli isolates. Dissemination of E. coli containing both ESBL and qnr genes could compromise the future usefulness of the expanded-spectrum antibiotics for the treatment of infections.
Disk Diffusion Antimicrobial Tests ; Escherichia coli/*enzymology/genetics/isolation & purification ; Escherichia coli Proteins/classification/*genetics ; Humans ; Inhibitory Concentration 50 ; Polymerase Chain Reaction ; beta-Lactamases/biosynthesis/genetics/*metabolism

OBJECTIVETo study the genotype distribution of extended-spectrum beta-lactamases (ESBLs) in ESBLs-producing Escherichia coli (E. coli) isolates from posthepatitic cirrhosis' patients with bloodstream infection.

METHODSE. coli were isolated in bloodstream from patients with posthepatitic cirrhosis between January and December in 2011. The strains were identified by VITEK-II. The antibiol susceptibility tests were performed with K-B method. beta-lactamases genes were detected multi-PCR, PCR, sequence and blast.

RESULTSA total of 79 non-duplicate clinical isolates of E coli were consecutively collected from liver cirrhosis' patients with bloodstream infection. There were 20 isolates produced TEM-1 type beta-lactamases and 1 isolate produced SHV-1 typebeta-lactamases. 40 clinical isolates were detected to produce CTX-M type ESBLs, there were 20 CTX-M-1 group and 26 CTX-M-9 group, including 6 stains habouring both CTX-M-1 and CTX-M-9 group. Eight CTX-M genotypes were confirmed by sequencing of the PCR products, including CTX-M-3, CTX-M-14, CTX-M-15, CTX-M-24, CTX-M-28, CTX-M-31, CTX-M-65 and CTX-M-79.

CONCLUSIONCTX-M genotype ESBLs was the most popular extended-spectrum beta-lactamases in E. coli isolated from liver cirrhosis' patients with bloodstream infection. The CTX-M-14 is the dominant epidemic type.

Bacteremia ; microbiology ; Cross Infection ; microbiology ; Drug Resistance, Bacterial ; Escherichia coli ; drug effects ; enzymology ; genetics ; isolation & purification ; Escherichia coli Infections ; microbiology ; Escherichia coli Proteins ; genetics ; Genotype ; Hospitalization ; statistics & numerical data ; Humans ; Liver Cirrhosis ; therapy ; Microbial Sensitivity Tests ; beta-Lactamases ; genetics ; metabolism

OBJECTIVETo investigate the possible differences in antimicrobial resistance of Escherichia coli isolated from different samples in children.

METHODSSix hundred and twenty-nine samples from urine, sputum, blood and secretion were collected from June 2004 to May 2009 for bacterial identification by VITEK-32 automatic system and antimicrobial susceptibility tests by Kirby-Bauer method. The drug resistance rate of Escherichia coli isolated from different samples was compared.

RESULTSTwo hundred and sixty strains of Escherichia coli were isolated , and 108 of which were from urine , 64 from sputum, 54 from secretion and 23 from blood. ESBLs were detected in 96 (36.9%) of the 260 isolates, AmpC enzymes in 32 (12.3%), and ESBLs+AmpC in 8 (3.1%). The ESBLs positive rate of Escherichia coli isolates from sputum was significantly higher than that from other samples (P<0.05). The antimicrobial resistance rate of Escherichia coli strains from different samples to amoxicillin/clavulanic acid, ticarcillin/clavulanic acid, piperacillin, cefotaxime, cefuroxime, cefepime, gentamicin, cotrimoxazole, and nitrofurantoin was different. The resistance rate of the strains from sputum samples was higher than that from the other samples (P<0.05).

CONCLUSIONSEscherichia coli isolated from different samples have different antimicrobial resistance rates in children, so the selection of antibiotics for infections confirmed by bacterial cultures from different samples should based on drug sensitivity results.

Adolescent ; Child ; Child, Preschool ; Drug Resistance, Bacterial ; Escherichia coli ; drug effects ; enzymology ; isolation & purification ; Female ; Humans ; Male ; beta-Lactamases ; analysis

A total of 52 endophytic fungi were isolated from roots and stems of Tibetan medicinal plant Phlomis younghusbandii Mukerjee. These fungal isolates were molecularly identified based on ITS sequnces and 28S sequences distributed to 12 genera, including Phoma, Chaetosphaeronema, Fusarium and Leptosphaeria, etc. Among them, the dominant genus was Phoma. Extracts of all strains were evaluated for anti-HIV-1 integrase activity by using soluable integrase expressed in E. coli BL21 (DE3). The results showed that seven samples from five fungal endophytes PHY-24, PHY-38, PHY-40, PHY-51, PHY-53, which belonged to genus Chaetosphaeronema, inhibited strand transfer reaction catalyzed by HIV-1 integrase with IC50 values, of 6.60, 5.20, 2.86, 7.86, 4.47, 4.56 and 3.23 microg x mL(-1) respectively. In conclusion, the endophytic fungi of Phlomis younghusbandii Mukerjee are valuable for further screening anti-HIV-1 integrase agents.
Ascomycota ; enzymology ; isolation & purification ; Chaetomium ; enzymology ; isolation & purification ; Endophytes ; enzymology ; isolation & purification ; Escherichia coli ; enzymology ; HIV Integrase ; genetics ; metabolism ; HIV Integrase Inhibitors ; pharmacology ; Phlomis ; microbiology ; Phylogeny ; Plant Roots ; microbiology ; Plant Stems ; microbiology ; Plants, Medicinal ; microbiology ; Plasmids ; Recombinant Proteins ; genetics ; metabolism

BACKGROUND: Accurate and rapid detection of extended-spectrum beta-lactamases (ESBLs) is important in guiding proper antimicrobial therapy for infected patients. We evaluated the performance of MicroScan NegCombo Type 44 panel (Dade Behring, USA), which was developed to confirm ESBL-producing Enterobacteriaceae using ceftazidime/clavulanate and cefotaxime/clavulanate. METHODS: From August 30 to September 20, 2007, 206 non-duplicate clinical isolates, including 106 Escherichia coli, 81 Klebsiella pneumoniae, 11 Klebsiella oxytoca, and 8 Proteus mirabilis were subcultured and tested with Type 32 and Type 44 panels. The results were compared with those of the CLSI phenotypic confirmatory test (CLSI-PCT) and disk approximation test (DAT). Isolates not susceptible to cefotetan or flagged as "Possible ESBL, unable to interpret confirm test (Possible ESBL)" on Type 44 panel were tested with boronic acid disks to confirm AmpC beta-lactamases (AmpC) production. RESULTS: Of the 206 isolates tested, 44 (21.4%) produced ESBL by CLSI-PCT or DAT, including 27 E. coli, 14 K. pneumoniae, 2 K. oxytoca, and 1 P. mirabilis. Thirty-eight isolates flagged as "Confirmed ESBL" on Type 44 panel were all confirmed as ESBL-producers. Of 14 K. pneumoniae flagged as "Possible ESBL", 6 were confirmed as ESBL and AmpC co-producers and 8 as AmpC-producers. CONCLUSIONS: Type 44 panel showed an excellent performance in detecting ESBL-producing E. coli, Klebsiella spp., and P. mirabilis. When flagged as "Confirmed ESBL", no other confirmatory test was necessary to report as ESBL; however, "Possible ESBL" required a differential test for AmpC production.
Bacterial Proteins/*biosynthesis ; Cefotetan/pharmacology ; Disk Diffusion Antimicrobial Tests ; Drug Resistance, Bacterial ; Escherichia coli/*enzymology/isolation & purification ; Humans ; Klebsiella/*enzymology/isolation & purification ; Proteus mirabilis/*enzymology/isolation & purification ; Reagent Kits, Diagnostic ; Sensitivity and Specificity ; beta-Lactamases/*biosynthesis

Dye-decolorizing peroxidase (DyP-type peroxidase) represents a group of heme-containing peroxidases able to decolour various organic dyes, most of which are xenobiotics. To identify and characterize a new DyP-type peroxidase (ZmDyP) from Zymomonas mobilis ZM4 (ATCC 31821), ZmDyP was amplified from the genomic DNA of Z. mobilis by PCR, and cloned into the Escherichia coli expression vector pET-21b(+). Alignment of the amino acid sequence of ZmDyP with other members of the DyP-type peroxidases revealed the presence of the active site conserved residues D149, R239, T254, F256 as well as the typical GXXDG motif, indicating that ZmDyP is a new member of the Dyp-type peroxidase family. pET-21b(+) containing ZmDyP gene was expressed in E. coli by IPTG induction. The expressed enzyme was purified by Ni-Chelating chromatography. SDS-PAGE analysis of the purified enzyme revealed a molecular weight of 36 kDa, whereas activity staining gave a molecular weight of 108 kDa, suggesting that the enzyme could be a trimer. In addition, ZmDyP is a heme-containing enzyme as shown by a typical heme absorption peak of Soret band. Moreover, ZmDyP showed high catalytic efficiency with 2, 2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) as a substrate. These results enrich the pool of DyP-type peroxidases and lay a foundation for further studies.
Amino Acid Sequence ; Catalysis ; Coloring Agents ; metabolism ; Escherichia coli ; genetics ; metabolism ; Molecular Sequence Data ; Peroxidases ; biosynthesis ; genetics ; isolation & purification ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Zymomonas ; enzymology