Keeping an enzyme in its native form with high catalytic activity is of great significance. In the present study, thermal stabilizers of horseradish peroxidase (HRP) were screened. The results indicated that thermal stability of HRP was enhanced by magnesium sulphate and gelatin. A synergic effect of magnesium sulphate and gelatin was observed. In the presence of the stabilizer, the enzymatic activity of HRP remained 89% after kept for 80 h at 50 degrees C and 57% for 90 days at room temperature. Thermal alterations of HRP structure in the absence and presence of the stabilizers were explored by using UV absorption spectra at 402 nm (Soret band), intrinsic fluorescence and 8-anilinonaphthalene-1-sulfonic acid (ANS) fluorescence. The results suggested that magnesium sulphate and gelatin attenuated the extent of unfolding of HRP and therefore the native enzyme structure was stabilized.
Drug Synergism ; Enzyme Stability ; drug effects ; Gelatin ; pharmacology ; Horseradish Peroxidase ; metabolism ; Hot Temperature ; Magnesium Sulfate ; pharmacology

We immobilized Candida sp. lipase onto seven kinds of industrial adsorption and ion exchange resins. By determining the activity of each immobilized enzyme, the weakly basic anionic exchange resin of D301 showed the best results for the immobilization of Candida sp. lipase. Comparing the scanning electron micrographs of D301 with Novozym 435 (immobilized Candida antarctica lipase B from Novo Nordisk Corp.), we selected D301 as a carrier for the immobilization of Candida sp. lipase. And we pretreated the resin D301 with the bifunctional agent glutaraldehyde and crosslinked it with Candida sp. lipase. The optimal conditions for the immobilization of Candida sp. lipase were as follows: 8 mL of the amount of 5% glutaraldehyde solution, five hours of the time pretreated D301 with glutaraldehyde, 1.0 g/L the concentration of Candida sp. lipase used, pH of the phosphate buffered, 6.0 and 10 hours of time for immobilization, respectively. The activity of immobilized enzyme was over 35 U/mg and the efficiency of immobilization was around 3.5 Ul(mg x h).
Candida ; enzymology ; Enzyme Stability ; Enzymes, Immobilized ; chemistry ; drug effects ; metabolism ; Ion Exchange Resins ; pharmacology ; Lipase ; chemistry ; metabolism

Cystatin, which widely distributed in both tissues and body fluids of animal and plant, was a superfamily of cysteine proteinase inhibitors. It could form activity-inhibitor complexes with cysteine proteinases to inhibit the hydrolytic activity of proteinases. Cystatin played important roles not only in the inhibition of the proteolytic degradation of fish muscle, but also in biological defense systems against invaders. To explore the functions of fish cystatin and the potential values in fish disease prevention and cure, as well as seafood processing, the recombinant yeast strains which could express Chinese sturgeon cystatin were constructed. First, the cystatin cDNA of Chinese sturgeon, which had been PCR modified, was subcloned into yeast integrated vector pPICZaA. After extracted and purified, the recombinant plasmids were linearized by Sac I. The yeast Pichia pastoris GS115 strain was transformed by use of the Lithium Chloride transformation method, and the recombinant cystatin yeast strains got. After 0.5% methanol induction, SDS-PAGE analysis of the culture supernatant indicated that the yield of recombinant cystatin was about 215mg x L(-1) with the percentage about 73.6%. The recombinant cystatin was purified through Q-Sepharose anion-exchange chromatography, and the purity reached about 94.2%. The inhibitory activity of recombinant cystatin was measured by inhibiting the proteinase activity of papain. The results showed that about 1 microg recombinant cystatin could inhibit the activity of 15 microg papain. Heat stability assay results showed that there was a decrease in inhibitory activity of cystatin with the increasing of temperature. When solution of recombinant cystatin was kept at 70 degrees C for 5min, the inhibitory activity reduced fast. While the recombinant cystatin was heated to 90 degrees C for 5min, the inhibitory activity of recombinant cystatin was undetected. The inhibitory activity for recombinant Chinese sturgeon cystatin was higher than that of CPI (cysteine proteinase inhibitor) from seeds of corn, that about 1 microg purified CIP could inhibited the activity of 0.278 microg papain. But the heat stability of recombinant cystatin is lower than that of the corn CPI. The expression level and the activity of recombinant cystatin from yeast Pichia pastoris were higher than those from E. coli. Moreover, recombinant cystatin from Pichia pastoris was easier to separate and purify. This paper reported that recombinant fish cystatin was produced in a highly efficient expression system based on the methylotrophic yeast, further work will focus on the function of recombinant Chinese sturgeon cystatin to resist fish disease and explore the value of cystatin as a food additive to inhibit cysteine proteinases during surimi processing.
Animals ; Cystatins ; genetics ; metabolism ; pharmacology ; Cysteine Proteinase Inhibitors ; genetics ; metabolism ; pharmacology ; Enzyme Activation ; drug effects ; Fish Proteins ; genetics ; metabolism ; Pichia ; genetics ; metabolism ; Polymerase Chain Reaction ; Protein Stability ; Temperature

The production conditions of extracellular laccase from Armilliria mellea and the characteristics of the enzyme were studied. The experiment proved that initial pH5.5 of the culture medium and temperature at 25 degrees C were favorable for laccase synthesis. As carbon resources, cellobiose and raffinose were better in terms of productivity than maltose, sorbose and galactose. Organic nitrogen source was more suitable for Armilliria mellea to synthesize laccase than inorganic nitrogen source. Peat extract (PE) enhanced notably the yield of laccase; the maximal yield was 7 times as much as that of the control when PE concentration was 50%. Three isozymes were detected in culture supernatant named A, B and C respectively after their mobility on PAGE. After concentrated by (NH4)2SO4 precipitation, laccase A was further purified to homogeneity by preparative native PAGE and anion exchange column chromatography. The native enzyme was a single polypeptide with a molecular mass of approximately 59 kD estimated by SDS-PAGE, while 58 kD by gel filtration chromatography under non-denaturing conditions. Determined by IEF its isoelectric point was 4.0. The optimal pH value and temperature were 5.6 and 60 degrees C respectively in catalytic reaction of oxidizing guaiacol. At 60 degrees C and 65 degrees C, half-lives of laccase A were 45 min and 36.8 min, respectively. Enzyme activity was inhibited with 100 mmol/L Cl-, but was activated with 1 mmol/L SO4(2-). However, if the concentration of NaN3 was only 1 mmol/L, laccase A lost its activity completely. 10 mmol/L EDTA had no effect on laccase A, while 1 mmol/L Cu2+ could enhance its activity. Laccase A showed a good stability when the pH of the buffer varied from 5.2 to 7.2. Using guaiacol as the substrate, the Km was 1.026 mmol/L and the Vmax was 5 mumol/(min.mg); using ABTS instead, the Km was 0.22 mmol/L and Vmax was 69 mumol/(min.mg).
Agaricales ; drug effects ; enzymology ; growth & development ; Carbohydrates ; pharmacology ; Culture Media, Conditioned ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Enzyme Stability ; Hydrogen-Ion Concentration ; Laccase ; Oxidoreductases ; isolation & purification ; metabolism

To enhance the thermostability and storage stability of alpha-cyclodextrin glycosyltransferase (a-CGTase), we added specific chemical additives into the preparation of alpha-CGTase, and studied the effect of additives on the storage stability of alpha-CGTase at different temperatures. Then we measured the protein structure of CGTase in the far UV (200-250 nm) and near UV (250-320 nm) ranges respectively by Circular dichroism (CD) spectra under high temperature and analyzed the relationship between thermostability and protein structure. The results indicated that the addition of selected additives (gelatin, glycerin, CaCl2 and PEG400) enhanced the thermostability of alpha-CGTase dramatically. After 45 days, the preparation of alpha-CGTase still had 100% of the enzyme activity with different additives superimposed at the optimum concentration at 40 degrees C. The CD spectra of alpha-CGTase showed that glycerin could protect the secondary and the tertiary structure of the CGTase under high temperature and therefore the enzyme maintained its high activity. Chemical additives can improve the stability of alpha-CGTase significantly and they preserve the enzyme activity by protecting its secondary structure.
Enzyme Stability ; drug effects ; Escherichia coli ; genetics ; metabolism ; Glucosyltransferases ; biosynthesis ; chemistry ; genetics ; Glycerol ; chemistry ; Paenibacillus ; enzymology ; Recombinant Proteins ; biosynthesis ; chemistry ; genetics

LysinB (LysB) in mycobacteriophage D29 was cloned and expressed and its enzymatic properties were analysed. The lysB gene was amplified by PCR from mycobacteriophage D29 genomic DNA and inserted into pET22b vector. The constructed recombinant plasmid was transformed into Escherichia coli BL21(DE3) to express fusion protein, which was purified by Ni-NTA column and enzymatic activity detected. The results showed that expression plasmid pET22b-lysB was constructed successfully. Highly purified recombination protein (His-LysB) was obtained 33.2 mg from 1 L LB culture medium. A screening for His-LysB activity on esterase and lipase substrates confirmed the lipolytic activity. With p-nitrophenyl butyrate as substrate, the thermal stability of the enzyme was poor when the temperature was above 30 degrees C. The enzyme exhibited higher stability at pH 5.0-9.5. The optimum temperature and pH for the lipolytic activity of His-LysB were 23 degrees C and 7.5 respectively. Under the optimum conditions, the specific activity of His-LysB was 1.3 U/mg. Zn2+, CU2+, Mg2+, Mn2+ and phenylmethane sulfonyl fruoride severely inhibited the lipolytic activity of His-LysB. The result provides a new option for tuberculosis drug research and development.
Cloning, Molecular ; Enzyme Stability ; Escherichia coli ; genetics ; metabolism ; Mycobacteriophages ; enzymology ; Mycobacterium tuberculosis ; drug effects ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; metabolism ; Viral Proteins ; biosynthesis ; genetics

The lipase labeled with the fluorescein isothiocyanat (FITC) was immobilized on the derivatives of the polyethylene glycol. The article discussed the effect of factors on the characters of lipase and analyzed the relationships among the activity of lipase, conformation, and fluorescence spectrum while the activity and the fluorescence spectrum of immobilized lipase were determined. The results demonstrated that polyethylene glycol 400-diacrylate could form appropriate network to improve the activity of enzyme. Adding ligand induced the lipase's catalytic conformation to increase the activity twice more than before. The active centre of lipase could be released by the extraction of ligand thus increasing the activity. After immobilization, the stability of labeled lipase improved greatly: immobilized lipases retained more than 70% and 60% of initial activity under conditions of 90 degrees C and strong acid or alkali, respectively. After immersing immobilized lipases into guanidine hydrochloride or urea for 15 days, the lipases retained upwards of 70% activity. The fluorescence spectrum could obviously reflect the changes of the activity and conformation of lipase. The fluorescence intensity was the minimum in the optimal pH and temperature. In the denaturing agent it declined as time passed. These results indicated that the unfolded processes of immobilized lipases are different under different conditions.
Dextrans ; chemistry ; Enzyme Stability ; Enzymes, Immobilized ; chemistry ; metabolism ; Fluorescein-5-isothiocyanate ; analogs & derivatives ; chemistry ; Fluorescent Dyes ; chemistry ; Lipase ; chemistry ; metabolism ; Polyethylene Glycols ; chemistry ; Protein Unfolding ; drug effects

OBJECTIVEInhibition of acetylcholinesterase (AChE) in human brain caused by phoxim or phoxim oxon, their reactivation with oxime and aging of phosphorylated AChE were studied and compared in vitro.

METHODSMicro-colorispectrophotometric assay was used to determine the activity of AChE.

RESULTSThe pI(50) of inhibition of AChE in human brain by phoxim and phoxim oxon were 5.39 and 5.77, respectively, whereas the pI(90) were 4.60 and 5.00, respectively. The reactivation rate of 0.1 mmol/L of pralidoxime (2-PAM), obidoxime (LüH(6)), trimedoxime (TMB-4) and pyramidoxime (HI-6) for phoxim-inhibited AChE in human brain was 65%, 97%, 91% and 56%, respectively, and their reactivation rate for phoxim oxon-inhibited AChE in human brain was 97%, 87%, 99% and 89%, respectively. The optimal reactivator for phoxim and phoxim oxon-inhibited AChEs was LüH(6) and TMB-4, respectively. The half aging time of phoxim and phoxim oxon inhibited phosphorylated AChEs were 39 and 28 hours, respectively, and the 99% aging time were 256 and 186 hours, respectively.

CONCLUSIONSLüH(6) or TMB-4 should be used at the earlier as possible after poisoning with phoxim and phoxim oxon, and the reactivator should be consecutively used for more than seven days, even after their acute symptoms have been well controlled.

Acetylcholinesterase ; metabolism ; Brain ; drug effects ; enzymology ; Cholinesterase Inhibitors ; pharmacology ; Cholinesterase Reactivators ; pharmacology ; Enzyme Stability ; Humans ; In Vitro Techniques ; Obidoxime Chloride ; pharmacology ; Organothiophosphorus Compounds ; pharmacology ; Oximes ; pharmacology ; Paraoxon ; pharmacology ; Pralidoxime Compounds ; pharmacology ; Time Factors ; Trimedoxime ; pharmacology

Angiogenesis is critical and indispensable for tumor progression. Since VEGF is known to play a central role in angiogenesis, the disruption of VEGF-VEGF receptor system is a promising target for anti-cancer therapy. Previously, we reported that a hexapeptide (RRKRRR, RK6) blocked the growth and metastasis of tumor by inhibiting VEGF binding to its receptors. In addition, dRK6, the D-form derivative of RK6, retained its biological activity with improved serum stability. In the present study, we developed a serum-stable branched dimeric peptide (MAP2-dRK6) with enhanced anti-VEGF and anti-tumor activity. MAP2-dRK6 is more effective than dRK6 in many respects: inhibition of VEGF binding to its receptors, VEGF- and tumor conditioned medium-induced proliferation and ERK signaling of endothelial cells, and VEGF-induced migration and tube formation of endothelial cells. Moreover, MAP2-dRK6 blocks in vivo growth of VEGF-secreting colorectal cancer cells by the suppression of angiogenesis and the subsequent induction of tumor cell apoptosis. Our observations suggest that MAP2-dRK6 can be a prospective therapeutic molecule or lead compound for the development of drugs for various VEGF-related angiogenic diseases.
Amino Acid Sequence ; Angiogenesis Inhibitors/*pharmacology ; Animals ; Cell Movement/drug effects ; Cell Proliferation/drug effects ; Colorectal Neoplasms/*pathology/secretion ; Endothelial Cells/cytology/drug effects/enzymology ; Enzyme Activation/drug effects ; Extracellular Signal-Regulated MAP Kinases/metabolism ; Humans ; Mice ; Mice, Nude ; Molecular Sequence Data ; Neovascularization, Pathologic/pathology/prevention & control ; Neovascularization, Physiologic/drug effects ; Peptides/chemistry/*pharmacology ; Protein Multimerization/*drug effects ; Protein Stability/drug effects ; Rats ; Serum ; Vascular Endothelial Growth Factor A/*antagonists & inhibitors/secretion

NDM-1 (New Delhi metallo-beta-lactamase) gene encodes a metallo-beta-lactamase (MBL) with high carbapenemase activity, which makes the host bacterial strain easily dispatch the last-resort antibiotics known as carbapenems and cause global concern. Here we present the bioinformatics data showing an unexpected similarity between NDM-1 and beta-lactamase II from Erythrobacter litoralis, a marine microbial isolate. We have further expressed these two mature proteins in E. coli cells, both of which present as a monomer with a molecular mass of 25 kDa. Antimicrobial susceptibility assay reveals that they share similar substrate specificities and are sensitive to aztreonam and tigecycline. The conformational change accompanied with the zinc binding visualized by nuclear magnetic resonance, Zn(2+)-bound NDM-1, adopts at least some stable tertiary structure in contrast to the metal-free protein. Our work implies a close evolutionary relationship between antibiotic resistance genes in environmental reservoir and in the clinic, challenging the antimicrobial resistance monitoring.
Amino Acid Sequence ; Anti-Bacterial Agents ; pharmacology ; Aztreonam ; pharmacology ; Cephalosporinase ; chemistry ; genetics ; metabolism ; Computational Biology ; methods ; Drug Resistance, Bacterial ; genetics ; Enzyme Stability ; drug effects ; Evolution, Molecular ; Minocycline ; analogs & derivatives ; pharmacology ; Molecular Sequence Data ; Phylogeny ; Protein Structure, Tertiary ; drug effects ; Sequence Homology, Nucleic Acid ; Sphingomonadaceae ; drug effects ; enzymology ; genetics ; Tigecycline ; Zinc ; pharmacology ; beta-Lactamases ; chemistry ; genetics ; metabolism