Enzymatic assay with detection by enhanced luminol chemiluminescence in a reversed micellar system: determination of l-amino acids and glucose

A detection system for hydrogen peroxide, i.e., luminol chemiluminescence (CL) in a hexadecyltrimethylammonium bromide (CTAB) reversed micellar system, was coupled to enzyme reactions. The use of CTAB reversed micellar medium allows one to conduct both the oxidase enzymatic and CL detection reactions simultaneously at mild pH (l-amino acid system, pH 8.7; glucose system, pH 8.5) in the absence of any co-oxidant or catalyst. Based on this result, simple and unique determinations of l-amino acids and glucose as substrates were developed. The calibration graph for a representative amino acid, l-phenylalanine, was linear in the concentration range 4.0×10^?6?200×10^?6M with a relative standard deviation of 5.78% (five determinations). The method established for l-phenylalanine was also applicable for the assay of fourteen other l-amino acids. The calibration graph for glucose was linear in the concentration range 5.4×10^??540×10^?6M with a relative standard deviation of 4.27% (eight determinations). This method was compared with a standard spectrophotometric method (hexokinase) and successfully applied to the determination of glucose in human serum.     

Purification and characterization of an extracellular lipolytic enzyme from the fermented fish-originated halotolerant bacterium, <Emphasis Type="Italic">Virgibacillus alimentarius</Emphasis> LBU20907

A halotolerant Virgibacillus alimentarius LBU20907 isolated from fermented fish (Budu) was found to be an efficient producer of extracellular halophilic lipase enzyme. The enzyme was purified 5.99-fold with a 0.15% final yield to homogeneity by ammonium sulfate precipitation, followed by dialysis, Toyopearl DEAE-650 M ion exchange chromatography, Toyopearl butyl-650 M hydrophobic interaction chromatography, and Toyopearl-HW 55 F gel filtration chromatography. SDS-PAGE of purified lipase exhibited a homogenous single band with a very high molecular weight of 100 kDa. The properties of purified lipase revealed maximum activity at pH 7.0 and 40 °C. It was also highly stable in a pH range of 6.0–7.0, retaining more than 90% activity for 24 h. It was stable at the temperature of 30–50 °C and maintained more than 80% activity for 16 h. The purified lipase performing the maximal activity in the presence of 20.0% NaCl indicated halophilic enzyme properties. Its lipolytic activity was highest against p-nitrophenyl palmitate. The lipase activity was found to be enhanced in hexane. The enzyme activity was stimulated in the presence of Zn²⁺, Ca²⁺, Mg²⁺, and Sr²⁺; while, it was completely inhibited by Ba²⁺ and Co²⁺. The enzyme had a K _m and V _max of 108.0 mg and 79.1 U mL^?1, respectively.     

Purification and Characterization of 3-Ketovalidoxylamine A C-N Lyase Produced by Stenotrophomonas maltrophilia

A soluble 3-ketovalidoxylamine A C-N lyase from Stenotrophomonas maltrophilia was purified to 367.5-fold from the crude enzyme, with a yield of 16.4% by column chromatography on High S IEX, Methyl HIC, High Q IEX, and Sephadex G 100. The molecular mass of the enzyme was estimated to be 34 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and the enzyme was a neutral protein having an isoelectric point value at pH?7.0. The optimal pH of 3-ketovalidoxylamine A C-N lyase was around 7.0. The enzyme was stable within a pH range of 7.0–10.5. The optimal temperature was found to be near 40?°C, and the enzyme was sensitive to heat. The enzyme was completely inhibited by ethylenediaminetetraacetic acid, and it was reversed by Ca²⁺. The product, p-nitroaniline, inhibited the enzyme activity significantly at low concentration. The enzyme has C-N lyase activity and C-O lyase activity, and need 3-keto groups. The apparent K _m value for p-nitrophenyl-3-ketovalidamine was 0.14 mM.     

Purification of Peroxidase from Red Cabbage (Brassica oleracea var. capitata f. rubra) by Affinity Chromatography

Peroxidase was purified in a single step using 4-amino benzohydrazide affinity chromatography from red cabbage (Brassica oleracea var. capitata f. rubra), and some important biochemical characteristics of the purified enzyme were determined. The enzyme, with a specific activity of 3,550 EU/mg protein, was purified 120.6-fold with a yield of 2.9 % from the synthesized affinity matrix. The molecular weight of the enzyme was found to be 69.3 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme exhibited maximum activity at pH 7.0 and 30 °C. For guaiacol substrate, the K _m and V _max values were found as 0.048 mM and 1.46 EU/mL/min, respectively. Additionally, the IC₅₀ and K _i values for 4-amino benzohydrazide were calculated to be 1.047 and 0.702?±?0.05 mM, respectively, and 4-amino benzohydrazide showed noncompetitive inhibition.     

Purification and Characterization of a Zinc-Dependent Cinnamyl Alcohol Dehydrogenase from Leucaena leucocephala,a Tree Legume

A cinnamyl alcohol dehydrogenase (CAD) from the secondary xylem of Leucaena leucocephala has been purified to homogeneity through successive steps of ammonium sulfate fractionation, DEAE cellulose, Sephadex G-75, and Blue Sepharose CL-6B affinity column chromatographies. CAD was purified to 514.2 folds with overall recovery of 13 % and specific activity of 812. 5 nkat/mg. Native and subunit molecular masses of the purified enzyme were found to be ～76 and ～38 kDa, respectively, suggesting it to be a homodimer. The enzyme exhibited highest catalytic efficiency (Kcat/Km 3.75 μM^?1 s^?1) with cinnamyl aldehyde among all the substrates investigated. The pH and temperature optima of the purified CAD were pH 8.8 and 40 °C, respectively. The enzyme activity was enhanced in the presence of 2.0 mM Mg²⁺, while Zn²⁺ at the same concentration exerted an inhibitory effect. The inclusion of 2.0 mM EDTA in the assay system activated the enzyme. The enzyme was inhibited with caffeic acid and ferulic acid in a concentration-dependent manner, while no inhibition was observed with salicylic acid. Peptide mass analysis of the purified CAD by MALDI-TOF showed a significant homology to alcohol dehydrogenases of MDR superfamily.     

Defense-Related Polyphenol Oxidase from Hevea brasiliensis Cell Suspension: Purification and Characterization

Polyphenol oxidase (PPO) was examined from the extract of leaf, seed, and cell suspension of Hevea brasiliensis, a rubber plant. The defense-related isozyme from Hevea cell suspension induced by culture filtrate of Phytophthora palmivora or by agitation stress was isolated through anion exchange and affinity chromatography, respectively. A 104-purification fold, migrated as a single band of 70?kDa on sodium dodecyl sulfate?Cpolyacrylamide gel electrophoresis of PPO, was obtained after further purified by the preparative gel electrophoresis. Based on reaction with catechol and dopamine but not with p-cresol and guaiacol, it is a diphenol-type PPO. The values of V _max /K _m ratio indicated that catechol was the most specific substrate. The optimal activity of the purified PPO was observed at pH?6.0. The PPO activity was retained at pH?4.0?C10.0 and temperature 10?C60?°C. The inhibitors which completely inhibited the activity were ascorbic acid, dithiothreitol, and ??-mercaptoethanol while sodium azide was a poor inhibitor. The PPO obtained from Hevea cell suspension possesses high specific activity and is stable at wide range of pH and temperature. It is therefore suitable for extreme condition uses and may lead to an alternative source of PPO in various industrial applications.     

Expression and Characterization of a GH39 β-Xylosidase II from Caulobacter crescentus

In the present work, the gene xynB2, encoding a ??-xylosidase II of the Glycoside Hydrolase 39 (GH39) family, of Caulobacter crescentus was cloned and successfully overexpressed in Escherichia coli DH10B. The recombinant protein (CcXynB2) was purified using nickel-Sepharose affinity chromatography, with a recovery yield of 75.5?%. CcXynB2 appeared as a single band of 60?kDa on a sodium dodecyl sulfate polyacrylamide gel and was recognized by a specific polyclonal antiserum. The predicted CcXynB2 protein showed a high homology with GH39 ??-xylosidases of the genus Xanthomonas. CcXynB2 exhibited an optimal activity at 55?°C and a pH of 6. CcXynB2 displayed stability at pH values of 4.5?C7.5 for 24?h and thermotolerance up to 50?°C. The K _M and V _Max values were 9.3?±?0.45?mM and 402?±?19???mol?min^?1 for ??-nitrophenyl-??-d-xylopyranoside, respectively. The purified recombinant enzyme efficiently produced reducing sugars from birchwood xylan and sugarcane bagasse fibers pre-treated with a purified xylanase. As few bacterial GH39 family ??-xylosidases have been characterized, this work provides a good contribution to this group of enzymes.     

Recombinant Expression and Characterization of l-Asparaginase II from a Moderately Thermotolerant Bacterial Isolate

A moderately thermotolerant bacterium belonging to Enterobacteriaceae, which can grow at 44.5?°C, was isolated from cow dung; l-asparaginase II gene was isolated by PCR, cloned, and expressed in pET 20b with pelB leader sequence and 6× Histidine tag at the C-terminal end. The active protein from the soluble sonicated fraction was purified through nickel affinity chromatography. After characterization, the purified protein showed optimum activities at a temperature of 37?°C and in a buffer system of pH?6 to 7. The enzyme exhibited thermostability at 50?°C with a 33% and 28% of activity retention after 45 and 60?min. The kinetic parameters for the enzyme were calculated from Lineweaver?CBurk plot, and K _m and V _max were 0.89?mM and 0.18?U/mg, respectively.     

A Novel α-Amylase from Bacillus mojavensis A21: Purification and Biochemical Characterization

α-Amylase from Bacillus mojavensis A21 (BMA.2) was purified to homogeneity by ultrafiltration, Sephadex G-75 gel filtration and Sepharose mono Q anion exchange chromatography, with a 15.3-fold increase in specific activity and 11% recovery. The molecular weight of the BMA.2 enzyme was estimated to be 58 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration. The optimum temperature and pH were 80?°C and 6.5, respectively. BMA.2 belonged to the EDTA-sensitive α-amylase, but its activity was not stimulated by the presence of Ca²⁺ ions. The major end-products of starch hydrolysis were maltohexaose, maltopentaose and maltotriose. The N-terminal amino acid sequence of the first ten amino acids of the purified α-amylase was ASVNGTLMQY. Compared to sequences of other amylases, the ten amino acid sequence contains Val at position 3, while amylases from Bacillus licheniformis NH1 and Bacillus sp. SG-1 have Leu and Thr at position 3, respectively.     

Purification and Characterization of an Antifungal Chitinase from Citrobacter freundii str. nov. haritD11

The purpose of the research was to study the purification and partial characterization of antifungal alkaline chitinase from a newly isolated Citrobacter freundii haritD11. The enzyme was purified in a three-step procedure involving ammonium sulfate precipitation, dialysis, and Sephadex G-100 gel filtration chromatography. The enzyme was shown to have a relative high molecular weight of 64 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and was purified 7.3-fold with a yield of 18.8 %. It was most active at 35 °C, pH 8.0, with colloid chitin as substrate and was very stable at alkaline pH contradicting the characteristic that most of the bacterial chitinases are active at acidic pH. Further, the purified chitinase exhibited remarkable antifungal activity against pathogenic fungi Aspergillus flavus MTCC 2798 and Aspergillus niger MTCC 9652 showing diametric inhibition zones of 27 mm and 21 mm, respectively.     

Cloning, Expression, and Characterization of a Novel Methylglyoxal Synthase from Thermus sp. Strain GH5

A gene encoding methylglyoxal synthase from Thermus sp. GH5 (TMGS) was cloned, sequenced, overexpressed, and purified by Q-Sepharose. The TMGS gene was composed of 399 bp which encoded a polypeptide of 132 amino acids with a molecular mass of 14.3 kDa. The K _m and k _cat values of TMGS were 0.56 mM and 325 (s^?1), respectively. The enzyme exhibited its optimum activity at pH?6 and 75?°C. Comparing the amino acid sequences and Hill coefficients of Escherichia coli MGS and TMGS revealed that the loss of Arg 150 in TMGS has caused a decrease in the cooperativity between the enzyme subunits in the presence of phosphate as an allosteric inhibitor. Gel filtration experiments showed that TMGS is a hexameric enzyme, and its quaternary structure did not change in the presence of phosphate.     

A Novel Hatching Enzyme from Starfish Asterias amurensis: Purification, Characterization, and Cleavage Specificity

Hatching enzyme (HE) is of importance to degrade egg membrane to let the larvae be free. HE was purified and characterized from starfish blastula. The specific activity and the purification ratio of the purified HE with 110.9 kDa of molecular weight were 449.62 U/mg and 7.42-fold, respectively. Its optimal pH and temperature for activity were pH?8.0 and 30 °C, respectively. This enzyme was relatively stable in the range of pH?4.0–6.0 and 30–40 °C. This enzyme was inhibited by ethylene diamine tetraacetic acid (EDTA) and ethylene glycol tetraacetic acid, and also done moderately by Leupeptin, tosyl-lysine chloromethyl ketone, tosyl-phenylalanine chloromethyl ketone, and phenyl-methanesulfonyl fluoride. Zn²⁺ ion activated HE activity strongly and recovered the EDTA-pretreated activity more than did Ca²⁺, Mg²⁺, and Cu²⁺. Based on the results above, the starfish HE was classified as a zinc metallo- and trypsin-like serine protease. The values of Km, Vmax, and Kcat of the starfish HE on dimethyl casein were 0.31 mg/ml, 0.17 U/ml, and 122.70 s^?1, respectively, whereas 1.09 mg/ml, 0.12 U/ml, and 771.98 s^?1 on type I collagen. Therefore, the starfish HE could be a potential cosmeceutical because of its strong cleavage specificity on type I collagen.     

Purification,Characterization of Amylase from Indigenously Isolated <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> Cau 19 and Its Bioconjugates with Gold Nanoparticles

The amylase from Aureobasidium pullulans Cau 19 was purified by ammonium sulfate precipitation and Sephadex G-100 chromatography with a 9.25-fold increase in specific activity as compared to crude enzyme. Km and turn over values of the enzyme were 6.25 mg/mL and 5.0 × 10²/min, respectively. Effect of different metal ions on the purified enzyme was investigated; 1 mM calcium (Ca) and cobalt (Co) enhanced enzyme activity by twofold; copper (Cu) had no effect on the activity of the enzyme. Mercury (Hg) 1 mM caused 90% inactivation whereas iron (Fe) and manganese (Mn) caused 10 to 16% inhibition. Amylase from A. pullulans Cau 19 was bioconjugated to gold nanoparticles synthesized using the biomass of A. pullulans Cau 19. Fourier transform infrared spectroscopy confirmed the conjugation of the enzyme to the gold nanoparticles. Though, only 20% of the added enzyme was adsorbed/conjugated on gold nanoparticles, 80% of the adsorbed activity could be estimated in the assay. The conjugated enzyme exhibited better tolerance to a broad pH range of 3.0–9.0 and higher temperatures compared with native enzyme.     

In Vitro Inhibition Effect of Some Dihydroxy Coumarin Compounds on Purified Human Serum Paraoxonase 1 (PON1)

Human serum paraoxonase 1 (PON1; EC 3.1.8.1) is a high-density lipoprotein associated, calcium-dependent enzyme that hydrolyses aromatic esters, organophosphates and lactones and can protect the low-density lipoprotein against oxidation. In this study, in vitro inhibition effect of some dihydroxy coumarin compounds namely 6,7-dihydroxy-3-(2-methylphenyl)-2H-chromen-2-one (A), 6,7-dihydroxy-3-(3-methylphenyl)-2H-chromen-2-one (B) and 6,7-dihydroxy-3-(4-methylphenyl)-2H-chromen-2-one (C) on purified PON1 were investigated by using paraoxon as a substrate. PON1 was purified using two-step procedures, namely ammonium sulphate precipitation and Sepharose-4B-l-tyrosine-1-naphthylamine hydrophobic interaction chromatography. The purified enzyme had a specific activity of 11.76?U/mg. The dihydroxy coumarin derivatives of A and B compounds inhibited PON1 enzyme activity in a noncompetitive inhibition manner with K _i of 0.0080?±?0.256 and 0.0003?±?0.018?mM values, respectively. C compound exerted an uncompetitive inhibition of PON1 enzyme activity with K _i of 0.0010?±?0.173?mM. Moreover, dihydroxy coumarin derivatives of A, B and C compounds were effective inhibitors on purified human serum PON1 activity with IC₅₀ of 0.012, 0.022 and 0.003?mM values, respectively. IC₅₀ value of unsubstituted 6,7 dihydroxy coumarin was found as 0.178?mM. The present study has demonstrated that PON1 activity is very highly sensitive to studied coumarin derivatives.     

Partial purification and characterization of protease enzyme from Bacillus subtilis and Bacillus cereus

The aim of this experimental study was to isolate and partially purify protease enzyme from Bacillus cereus and Bacillus subtilis. Protease enzyme is obtained by inducing spore genesis of bacteria from Bacillus species in suitable nutrient plates. The partial purification was realized by applying, respectively, ammonium sulfate precipitation, dialysis, and DEAE-cellulose ion-exchange chromatography to the supernatant that was produced later. Optimum pH, optimum temperature, pH stability, and temperature stability were determined, as well as the effects of pH, temperature, substrate concentration, reaction time, and inhibitors and activators on enzyme activity. In addition, the molecular mass of the obtained enzyme was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The specific activity of partially purified enzyme from B. subtilis was determined to be 84 U/mg. The final enzyme preparation was eight-fold more pure than the crude homogenate. The molecular mass of the partially purified enzyme was found to be 45 kDa by using SDS-PAGE. The protease enzyme that was partially purified from B. cereus was purified 1.2-fold after ammonium sulfate precipitation. The molecular mass of the partially purified enzyme was determined to be 37 kDa by using SDS-PAGE.     

Transesterification of Waste Cooking Oil by an Organic Solvent-Tolerant Alkaline Lipase from Streptomyces sp. CS273

The aim of this present study was to produce a microbial enzyme that can potentially be utilized for the enzymatic transesterification of waste cooking oil. To that end, an extracellular lipase was isolated and purified from the culture broth of Streptomyces sp. CS273. The molecular mass of purified lipase was estimated to be 36.55 kDa by SDS PAGE. The optimum lipolytic activity was obtained at alkaline pH 8.0 to 8.5 and temperature 40 °C, while the enzyme was stable in the pH range 7.0?～?9.0 and at temperature ≤40 °C. The lipase showed highest hydrolytic activity towards p-nitrophenyl myristate (C14). The lipase activity was enhanced by several salts and detergents including NaCl, MnSo₄, and deoxy cholic acid, while phenylmethylsulfonyl fluoride at concentration 10 mM inhibited the activity. The lipase showed tolerance towards different organic solvents including ethanol and methanol which are commonly used in transesterification reactions to displace alcohol from triglycerides (ester) contained in renewable resources to yield fatty acid alkyl esters known as biodiesel. Applicability of the lipase in transesterification of waste cooking oil was confirmed by gas chromatography mass spectrometry analysis.     

Expression and Characterization of a Novel Enantioselective Lipase from Aspergillus fumigatus

A 1,080-bp cDNA (CGMCC 2873) encoding of a cold-active lipase of Aspergillus fumigatus (AFL67) was cloned and expressed in Escherichia coli for the first time. The new lipase, AFL67, was one-step purified by 8.30 folds through Ni?CNTA affinity chromatography with a recovery of 86.8?%. The specific activity of purified AFL67 was 449?U?mg^?1 on p-NP hexanoate. AFL67 preferentially hydrolyzed p-nitrophenyl esters of short- and medium-chain fatty acids, with p-nitrophenyl hexanoate the maximum. The optimum temperature and pH was 15?°C and 7.5, respectively. The purified AFL67 was stable at 10?C25?°C for 30?min, and in the pH range of 6.0?C9.0 for 16?h (at 4?°C). Its activity was increased by 47 and 50?%, in the presence of 10?% (v/v) ethanol and isopropanol, respectively. The new lipase AFL67 highly enantioselectively deacylated (S)-??-acetoxyphenylacetic acid (APA) and o-Cl-APA, m-Cl-APA, and p-Cl-APA to (S)-mandelic acid and its derivates. These features render this cold-active novel lipase AFL67 attractive for biotechnological applications in the field of enantioselective synthesis of chiral mandelic acids, o-acylated mandelic acids, and their derivates and detergent additives.     

Rapid,High-Yield Purification of Rat Liver Glutathione Peroxidase by High Performance Liquid Chromatography

Abstract

Glutathione peroxidase (GSH:H₂O₂ oxidoreductase, EC 1.11.1.9) was purified 3500-fold from rat liver with a yield of 42% using high performance liquid chromatography. The crucial purification step was size-exclusion chromatography on a Spherogel TSK-3000SW column, and the purified enzyme eluted as a single peak. The enzyme stained as a single band following SDS-gel electrophoresis. The molecular weight of the enzyme was estimated to be 105,000, and the subunit molecular weight determined by SDS-gel electrophoresis was 25,000. Polyacrylamide gel electrophoresis indicated five bands of protein with a broad of enzymatic activity. Isoelectric focusing resulted in a peak of enzymatic activity at pH 6.9 with a shoulder at pH 7.3. The specific activity of the purified enzyme was 1,100 μmol of NADPH oxidized per minute per milligram of protein.     

Purification and Properties of a Thermostable Xylanase GH 11 from Penicillium occitanis Pol6

An extracellular, endo-??-1,4-xylanase was purified to homogeneity from the culture filtrate of the filamentous fungus Penicillium occitanis Pol6, grown on oat spelt xylan. The purified enzyme (PoXyn2) showed a single band on SDS?CPAGE with an apparent molecular weight of 30?kDa. The xylanase activity was optimal at pH?3.0 and 65?°C. The specific activity measured for oat spelt xylan was 2,368?U?mg^?1. The apparent K _m and V _max values were 8.33?mg?ml^?1 and 58.82???mol?min^?1?ml^?1, respectively, as measured on oat spelt xylan. Thin-layer chromatography experiments revealed that purified PoXyn2 degrades xylan in an endo-fashion releasing xylobiose as main end product. The genomic DNA and cDNA encoding this protein were cloned and sequenced. This PoXyn2 presents an open reading frame of 962?bp, not interrupted by any introns and encoding for a mature protein of 320 amino acids and 29.88?kDa.