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

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

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

The alpha-amylase (EC 3.2.1.1) from the Gram-positive Alicyclobacillus acidocaldarius was one kind of thermoacidophilic enzyme, with optimal temperature and pH of 75 degrees C and 3, respectively. The nucleotide sequence of the gene amy was cloned by PCR. The gene amy was 3901bp long, comprising one open reading frame encoding a polypeptide of 1301 amino acids. The calculated molecular weight of the alpha-amylase AMY was about 140kD. The gene amy was expressed in E. coli BL21 (DE3) and Pichia pastoris respectively, and both of the cloned proteins had bioactivity. The activity of amylase expressed in P. pastoris was further testified by amylase activity staining. The alpha-amylase expressed in P. pastoris had been purified and characterized. The apparent molecular weight of that was about 160kD according to SDS-PAGE. The optimum of pH for the enzyme was pH 3.2 as the native enzyme was; but the optimum of temperature was 65 degrees C and a little lower than that of the native enzyme. Above 50% of relative activity remained after incubation for 30 minutes in 70 degrees C. So the enzyme expressed by P. pastoris was also thermoacidophilic. Moreover some sequence was cloned by PCR, which ranged from + 1174 bp to + 3288 bp in the gene amy, encoding 705 amino acids with the calculated molecular weight of 79kD. The truncated gene amy' was expressed in E. coli BL21 (DE3) induced by 1 mmol/L IPTG, and the expressed enzyme also retained alpha-amylase activity.
Bacillus ; enzymology ; genetics ; Bacterial Proteins ; genetics ; isolation & purification ; metabolism ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Pichia ; genetics ; metabolism ; alpha-Amylases ; genetics ; isolation & purification ; metabolism

Heparinase III is an enzyme that specifically cleaves certain sequences of heparan sulfate. Previous reports showed that this enzyme expressed in Escherichia coli was highly prone to aggregation in inclusion bodies and lacks detectable biological activity. In this paper, we fused a glutathione-S-transferase (GST) tag to the N-terminus of heparinase III gene and expressed the fusion protein in Escherichia coli to develop an expression system of soluble heparinase III. As a result, approximately 80% of the fusion protein was soluble. The protein was then purified to near homogeneity via one-step affinity chromatography. A 199.4-fold purification was achieved and the purified enzyme had a specific activity of 101.7 IU/mg protein. This represented 32.3% recovery of the total activity of recombinant GST-heparinase III. The maximum enzyme production was achieved when bacteria were induced with 0.5 mmol/L isopropyl-beta-D-thiogalactoside at 15 degrees C for 12 h. The enzyme showed maximum activity at 30 degrees C and pH 7.5. And the enzyme activity was stimulated by 1 mmol/L Ca2+ and 150 mmol/L NaCl.
Escherichia coli ; genetics ; metabolism ; Flavobacterium ; enzymology ; genetics ; growth & development ; Glutathione Transferase ; biosynthesis ; genetics ; Heparin Lyase ; biosynthesis ; genetics ; isolation & purification ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification

The gldA gene coding glycerol dehydrogenase (GDH) was amplified by PCR with the genomic DNA of Klebsiella pneumoniae as the template. The gldA were inserted in pMD-18T to construct the recombinant cloning vector pMD-gldA. After the DNA sequence was determined, the gldA was subcloned into expression vector pET-32a (+) to construct the recombinant expression vector pET-32gldA. Upon lactose induction, soluble GDH was over-produced by E. coli BL21 (DE3) harboring the expression construct. Recombinant GDH purified by Ni-NTA affinity chromatography showed a single band about 54 kD on SDS-PAGE gel, and the specified activity was about 188 u/mg, the purification fold is 3 times and the activity recovery is 67.5%.
Chromatography, Affinity ; Cloning, Molecular ; DNA, Bacterial ; genetics ; Escherichia coli ; genetics ; isolation & purification ; metabolism ; Klebsiella pneumoniae ; enzymology ; genetics ; Sugar Alcohol Dehydrogenases ; biosynthesis ; genetics ; isolation & purification

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

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

The structure gene pelA from Thermotoga maritima MSB8 encoding pectate lyase was amplified and ligated into pHsh, resulting pHsh-pelA. Through structural optimization on pHsh-pelA, the ultimate plasmid, pHsh-pelC, which possessed the most appropriate structure and free energy of mRNA, was obtained. Pectate lyase C (PelC) was obtained after expressing pHsh-pelC in Escherichia coli JM109. The optimum activity of PelC was determined at pH 8.5 at 90 degrees C, with a half-life for almost 2 h at 95 degrees C. PelC was stable at the pH range of 8.2-9.8, and was dependent on Ca2+ for activity and stability. The enzyme kept stable for a long time and possessed a high level of activity at 60 degrees C. The kinetic assay using polygalacturonic acid (PGA) as substrate gave K(m) and V(max) of 0.11 mmol/L and 327 U per mg of protein. SDS-PAGE analysis showed that the molecular mass of the expressed recombinant PelC was about 43 kD, which was exactly the size predicted. The expression vector system of the heat shock plasmid pHsh owned such advantages as high expression level and cheap induction. Moreover, the superior stability of the recombinant enzyme laid the base for large-scale fermentation application.
Enzyme Stability ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Hot Temperature ; Polysaccharide-Lyases ; biosynthesis ; genetics ; isolation & purification ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; Recombination, Genetic ; Thermotoga maritima ; enzymology