Purification and some properties of a carboxypeptidase B from dogfish Scyliorhinus canicula

A carboxypeptidase B (CPB) has been purified from dogfish (Scyliorhinus canicula) pancreas and partially characterized. The purification procedure included acetone precipitation, ion-exchange chromatography on a CM-cellulose column and gel filtration on Sephadex G-75. The purified enzyme migrates as a single band both on PAGE and SDS-PAGE. Its molecular mass is estimated to be about 32 kDa. The optimum of activity is obtained at pH 7.5–8.2. The enzyme is inhibited by typical metal-chelating agents (EDTA and o-phenanthroline) and by Hg²⁺. It is activated by Co²⁺, l-cysteine and by heat treatment at 40° and 50°C. Kinetic parameters, K_m and k_cat, of native enzyme, Co²⁺-activated CPB and heat-treated CPB have been determined     

NADP-malic enzyme from maize leaf: regulatory properties.

The regulatory properties of purified maize leaf NADP-malic enzyme (EC 1.1.1.40) were studied at three different pHs and the following results were obtained. (a) At pH 7.5 enzyme activity reaches a maximum at 0.4–0.8 mm malate depending on the Mg²⁺ concentration, and higher levels of malate result in marked substrate inhibition; with increasing pH the degree of substrate inhibition is reduced to where at pH 8.4 little or no inhibition is observed. (b) The inhibitory effect of malate is more pronounced at 1 mm Mg²⁺ than at 5–10 mm Mg²⁺ in the pH range of 7.5 to 8.4; a plot of enzyme activity vs Mg²⁺ concentration at 3 mm malate follows Michaelis-Menten kinetics at both pH 7.5 and 8.4; the apparent affinity of the enzyme for Mg²⁺ at pH 8.4 was threefold greater than that at pH 7.5. (c) The activity of NADP-malic enzyme decreases as the ratio of $\text{NADPH}\text{NADP}$ increases, and this effect is enhanced at lower pH. (d) Various α-keto acids including glyoxylate, oxaloacetate, and α-ketoglutarate inhibit NADP-malic enzyme activity, whereas HCO₃^?, pyruvate, and other organic acids, sugar phosphates, and amino acids have little or no effect on the activity of the enzyme. Based on these experimental findings, the regulatory properties of maize leaf NADP-malic enzyme are discussed with respect to its key role in net CO₂ fixation in maize bundle sheath chloroplasts during C₄ photosynthesis.     

Purification of lipase from Cunninghamella verticillata and optimization of enzyme activity using response surface methodology

The fungus Cunninghamella verticillata was selected from isolates of oil-mill waste as a potent lipase producer as determined by the Rhodamine-B plate method. The lipase was purified from C. verticillata by ammonium sulphate fractionation, ion exchange chromatography and gel filtration. The purified enzyme was formed from a monomeric protein with molecular masses of 49 and 42 kDa by SDS–PAGE and gel filtration, respectively. The optimum pH at 40 °C was 7.5 and the optimum temperature at pH 7.5 was 40 °C. The enzyme was stable between a pH range of 7.5 and 9.0 at 30 °C for 24 h. The enzyme activity was strongly inhibited by AgNO₃, NiCl₂, HgCl₂, CdCl₂ and EDTA. However, the presence of Ca²⁺, Mn²⁺ and Ba²⁺ ions enhanced the activity of the enzyme. The activity of purified lipase with respect to pH, temperature and salt concentration was optimized using a Box–Behnken design experiment. A polynomial regression model used in analysing this data, showed a significant lack of fitness. Therefore, quadratic terms were incorporated in the regression model through variables. Maximum lipase activity (100%) was observed with 2 mM CaCl₂, (pH 7.5) at a temperature of 40 °C. Regression co-efficient correlation was calculated as 0.9956.     

Effects of some metal ions on rainbow trout erythrocytes glutathione S-transferase enzyme: an in vitro study

Glutathione S-transferase enzyme (GST) (EC 2.5.1.18) was purified from rainbow trout erythrocytes, and some characteristics of the enzyme and effects of some metal ions on enzyme activity were investigated. For this purpose, erythrocyte glutathione S-transferase enzyme which has 16.54 EU/mg protein specific activities was purified 11,026-fold by glutathione-agarose affinity chromatography with a yield of 59%. Temperature was kept under control (+4°C) during purification. Enzyme purification was checked by performing SDS-PAGE. Optimal pH, stable pH, optimal temperature, and K_M and V_max values for GSH and 1-chloro-2, 4-dinitrobenzene (CDNB) were also determined for the enzyme. In addition, IC₅₀ values, K_i constants and the type of inhibition were determined by means of Line-Weaver-Burk graphs obtained for such inhibitors as Ag⁺; Cd²⁺, Cr²⁺ and Mg²⁺.     

PURIFICATION AND CHARACTERIZATION OF A HOMODIMERIC ENOLASE FROM SYNECHOCOCCUS PCC 6301 (CYANOPHYCEAE)1

Enolase from Synechococcus PCC 6301 was purified 1450‐fold to electrophoretic homogeneity and a final specific activity of 68 μmol of phosphoenolpyruvate produced·min^?1·mg protein^?1. Analytical gel filtration and nondenaturing and SDS‐gel electrophoresis demonstrated that this enolase exists as a 118‐kDa homodimer composed of 56‐kDa subunits. The purified enzyme displayed 1) a broad pH‐activity profile with maximal activity occurring at pH 8.0 and 7.5 for the forward and reverse reactions, respectively, 2) a forward‐to‐reverse maximal activity ratio of about 1.6, 3) a K_m (2‐phosphoglycerate) of 0.28 mM, and 4) an absolute requirement for a divalent metal cation cofactor that was best satisfied by Mg²⁺ (K_m=0.62 mM). Enolase activity increased by about 200% after the first purification step (60° C heat treatment), whereas addition of increasing amounts of a clarified extract led to a progressive 70% inhibition in the activity of the purified enzyme. This was reflected by a reduction in enolase's V_max from 73 to 22 U·mg^?1 and forward‐to‐reverse activity ratio from 1.6 to 1.3. This inhibition was negated when the clarified extract was either preincubated with trypsin or warmed to approximately 40° for 5 min. Results are indicative of a heat‐labile enolase inhibitor protein in Synechococcus PCC 6301. By contrast, the purified enolase lost no activity when incubated at 70° C for up to 5 min. This study represents the first purification of enolase from the Cyanophyceae. Characterization of the purified enzyme's physical and kinetic features has provided insights into the structural and functional properties of cyanobacterial enolase.     

Purification and characterization of a thermostable uricase from Microbacterium sp. strain ZZJ4-1

In order to study the properties of a thermostable uricase produced by Microbacterium sp. strain ZZJ4-1, the enzyme was purified by ammonium sulfate precipitation and DEAE-cellulose ion exchange, hydrophobic and molecular sieve chromatography. The molecular mass of the purified enzyme was estimated to be 34 kDa by SDS-PAGE. The enzyme was stable between pH 7.0 and 10.00. The optimal reaction temperature of the enzyme was 30 °C at pH 8.5. The K _m and K _cat of the enzyme were 0.31 mM and 3.01 s⁻¹, respectively. Fe³⁺ could enhance the enzyme activity, whereas Ag⁺, Hg²⁺, o-phenanthroline and SDS inhibited the activity of the enzyme considerably. After purification, the enzyme was purified 19.7-fold with 31% yield. As compared with uricases from other microbial sources, the purified enzyme showed excellent thermostability and other unique characteristics. The results of this work showed that strains of Microbacterium could be candidates for the production of a thermostable uricase, which has the potential clinical application in measurement of uric acid.     

Purification,biochemical characterisation,and mass spectrometry analysis of phenylalanine aminopeptidase from the shoots of pea plants

Phenylalanine aminopeptidase (Phe-AP) was isolated from the shoots of 3-week-old pea plants and purified to molecular homogeneity using a four-step purification procedure (ammonium sulphate precipitation, chromatography on DEAE-cellulose, phenyl-sepharose HP, and Protein-Pak Q 8HR HPLC columns). The enzyme was purified 513-fold with a recovery of 8%. The molecular weight of the purified enzyme as determined by SDS-PAGE and gel filtration was approximately 60 kDa, and the enzyme appeared to be a monomer. Its pH and temperature optimum were pH 7.5 and 37°C, respectively. The enzyme prefers substrates with Phe at the N-terminus, although a high activity for substrates with N-terminal Tyr, Trp, Leu, and Met was also observed. The activity with Leu-β-naphthylamide was at least two times lower than that with Phe-β-naphthylamide (Phe-β-NA). The K _m value for activity with Phe-β-NA was the lowest amongst the substrates tested, and it was 7.5 × 10⁻⁵ M. The activity of Phe-AP was not inhibited by EDTA, 1,10-phenanthroline and pepstatin A. The most effective inhibitors were pHMB and E-64, which modify sulphydryl groups; however, a significant inhibition in the presence of DFP and PMSF, both of which are serine protease inhibitors, was also observed. By applying mass spectrometry analysis, the peptides derived from the purified Phe-AP were assigned to amino acid sequences of the leucine aminopeptidases of N-type (LAPs-N).     

Purification of ctp: cholinephosphate cytidylyl-transferase from pea stems

CTP:cholinephosphate cytidylyltransferase (EC 2.7.7.15) was purified from pea (Pisum sativum) stems. The purification involved ammonium sulphate fractionation, ion exchange chromatography, removal of proteases with α₂-macroglobulin and gel filtration. The purified enzyme had K_m values for phosphorylcholine and CTP of 2.1 mM and 0.55 mM respectively. It was found to have a pH optimum of 7.5, a requirement for Mg²⁺ and an M_r of 56000. It could not utilize phosphorylethanolamine and its activity was not stimulated by added phospholipids.     

Purification and properties of methylmalonyl coenzyme A mutase from human liver

Methylmalonyl coenzyme A (CoA) mutase has been purified to apparent homogeneity from human liver by a procedure involving column chromatography on DEAE-cellulose, Matrex-Gel Blue A, hydroxylapatite, and Sephadex G-150. The overall purification achieved is 500- to 600-fold, yield 3–5%. Electrophoresis of the native purified protein on nondenaturing polyacrylamide gels shows a single diffuse band coincident with the enzyme activity; dodecyl sulfate/polyacrylamide gels show a single protein band with an apparent molecular weight of 77,500. The native protein has a molecular weight of approximately 150,000 by Sephadex G-150 chromatography, suggesting that it is composed of two identical subunits. The activity of the purified enzyme is stimulated only slightly (10–20%) by the addition of its cofactor, adenosylcobalamin, indicating that the purified enzyme is largely saturated with coenzyme. The spectrum of the enzyme is consistent with the presence of about 1 mole of adenosylcobalamin per mole of subunit. The enzyme displays complex kinetics with respect to dl-methylmalonyl CoA; substrate inhibition by l-methylmalonyl CoA appears to occur. The enzyme activity is stimulated by polyvalent anions (PO₄^3? > SO₄^2? > Cl^?); monovalent cations are without effect, but high concentrations of divalent cations are inhibitory. The enzyme activity is insensitive to N-ethylmaleimide, is rapidly destroyed at temperatures > 50 °C, and shows a broad pH optimum around pH 7.5.     

Purification and properties of milk-clotting enzyme from Bacillus subtilis K-26

A milk-clotting enzyme from Bacillus subtilis K-26 was purified by gel filtration and ion-exchange chromatography resulting in a 24-fold increase in specific activity with an 80% yield. Polyacrylamide gel electrophoresis and ultracentrifugel analysis revealed that the purified enzyme was homogeneous and had a molecular weight of 27,000 and a K_m of 2.77mg/ml for κ-casein. The enzyme was most stable at pH 7.5 and showed increasing clotting activity with decrease in milk pH up to 5.0. The maximum milk-clotting activity was obtained at 60°C, but the enzyme was inactivated by heating for 30 min at 60°C. The enzyme was irreversibly inhibited by EDTA and unaffected by DFP. Heavy-metal ions (Hg²⁺, Pb²⁺) inactivated the enzyme.     

Biochemical and biophysical characterization of a peroxidase isolated from Euphorbia tirucalli with antifungal activity

A novel peroxidase from the latex of medicinal plant Euphorbia tirucalli (Pencil tree) belonging to the Euphorbiaceae family is purified to homogeneity using cation exchange chromatography. The enzyme, named Euphorbia tirucalli peroxidase (ETP) has a molecular mass of 38.8?kDa. The isoelectric point of the enzyme is pH 7.5 with optimum pH and temperature of pH 6.0 and 50?°C respectively. The extinction coefficient (?_280 nm^1%) of the enzyme is 20.52 and the molecular structure consists of 13 tryptophan, nine tyrosine, and eight cysteine residues forming four disulfide bridges. Three peptide sequences, ALVHKECGPVVSCSDIVAIAARDSVVLTGGPKYDV, YYVDLMNRQGLFTSDQDLYT DKR, and MGQLEVVTGNQGEIR are obtained by MS/MS analysis which confirms the novelty of the enzyme. ETP belongs to α/β class of proteins with secondary structural features of approximately 10% α-helix, 29% β-sheet, and 61% random coil. ETP exhibits antifungal activity against Aspergillus niger and Candida albicans which shows its role in defense mechanism of plants. The enzyme is stable and retains its activity over a broad range of pH and temperature or prolonged storage at 4?°C. Simple purification, high yield, and stability enable exploration of the peroxidase for structure–function relationship studies as well as other biotechnological applications.     

Cloning, purification, and characterization of chitosanase from Bacillus sp. DAU101

A chitosanase-producing Bacillus sp. DAU101 was isolated from Korean traditional food. This strain was identified on the basis of phylogenetic analysis of the 16S rDNA sequence, gyrA gene, and phenotypic analysis. The gene encoding chitosanase (csn) was cloned and sequenced. The csn gene consisted of an open reading frame of 837 nucleotides and encodes 279 amino acids with a deduced molecular weight of 31,420 Da. The deduced amino acid sequence of the chitosanase from Bacillus sp. DAU101 exhibits 88 and 30 % similarity to those from Bacillus subtilis and Pseudomonas sp., respectively. The chitosanase was purified by glutathione S-transferase fusion purification system. The molecular weight of purified enzyme was about 27 kDa, which suggests the deletion of a signal peptide by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The pH and temperature optima of the enzyme were 7.5 and 50 °C, respectively. The enzyme activity was increased by about 1.6-fold by the addition of 5 or 10 mM Ca²⁺. However, Hg²⁺ and Ni⁺ ions strongly inhibited the enzyme. The enzyme produced, GlcN_2–4, were the major products from a soluble chitosan.     

Purification of calcium-activated neutral proteinase (CANP) from purified myelin of bovine brain white matter

A calcium-activated neutral proteinase was purified from myelin of bovine brain white matter. Myelin purified in the presence of EDTA (2 mM) was homogenized in 50 mM Trisacetate buffer at pH 7.5, containing 4 mM EDTA, 1 mM NaN₃, 5 mM -mercaptoethanol and 0.1% Triton X-100 for two hours. After centrifugation at 87,000g for 1 hour, the supernatant was subjected to purification through successive column chromatography as follows: i) DEAE-cellulose, ii) Ultrogel (AC-34) filtration, iii) Phenyl-Sepharose, iv) a second DEAE-cellulose. The enzyme activity was assayed using azocasein as substrate. The myelin enzyme was purified 2072-fold and SDS-PAGE analysis of the purified enzyme revealed a major subunit of 72–76 K. The enzyme was inhibited by iodoacetate (1 mM), leupeptin (1 mM), E-64C (1.6 mM), EGTA (1 mM), antipain (2 mM) and endogenous inhibitor calpastatin (2 g). It required 0.8 mM Ca²⁺ for half-maximal activation and 5 mM Ca²⁺ for optimal activation. Mg²⁺ (5 mM) was ineffective while Zn²⁺ and Hg²⁺ were inhibitory. The pH optimum was ranged from 7.5–8.5. Treatment of myelin with Triton X-100 increased the enzyme activity by 10-fold suggesting it is membrane bound whereas the purufied enzyme was not activated by Triton X-100 treatment. The presence of CANP in myelin may mediate the turnover of myelin proteins and myelin breakdown in degenerative brain diseases.     

Purification and partial characterization of thermostable serine alkaline protease from a newly isolated<Emphasis Type="Italic">Bacillus subtilis</Emphasis> PE-11

The purpose of the research was to study the purification and partial characterization of thermostable serine alkaline protease from a newly isolatedBacillus subtilis PE-11. The enzyme was purified in a 2-step procedure involving ammonium sulfate precipitation and Sephadex G-200 gel permeation chromatography. The enzyme was shown to have a relative low molecular weight of 15 kd by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and was purified 21-fold with a yield of 7.5%. It was most active at 60°C, pH 10, with casein as substrate. It was stable between pH 8 and 10. This enzyme was almost 100% stable at 60°C even after 350 minutes of incubation. It was strongly activated by metal ions such as Ca²⁺, Mg⁺², and Mn⁺². Enzyme activity was inhibited strongly by phenylmethyl sulphonyl fluoride (PMSF) and diisopropyl fluorophosphates (DFP) but was not inhibited by ethylene diamine tetra acetic acid (EDTA), while a slight inhibition was observed with iodoacetate,p-chloromercuric benzoate (pCMB), and β-mercaptoethanol (β-ME). The compatibility of the enzyme was studied with commercial and local detergents in the presence of 10mM CaCl₂ and 1M glycine. The addition of 10mM CaCl₂ and 1M glycine, individually and in combination, was found to be very effective in improving the enzyme stability where it retained 52% activity even after 3 hours. This enzyme improved the cleansing power of various detergents. It removed blood stains completely when used with detergents in the presence of 10mM CaCl₂ and 1M glycine.     

Purification and characterization of an -amino acid deaminase used to prepare unnatural amino acids

-Amino acid deaminase ( -AAD) from Proteus myxofaciens was cloned and over-expressed in Escherichia coli K12. This enzyme has a broad substrate specificity, working on both natural and unnatural -amino acids. Of the 20 naturally occurring -amino acids, -AAD prefers amino acid substrates that have aliphatic, aromatic or sulfur-containing side chains; those with charged side chains (–CO₂⁻ or –NH₃⁺) are poor or non-substrates. Enzyme activity was monitored using a microtiter-plate-based assay, which measures the formation of phenylpyruvic acid from -phenylalanine. The reaction has an absolute requirement for O₂, releases NH₃ and does not produce H₂O₂. Substrate comparisons were carried out by using an O₂ electrode to measure the O₂ utilization rates. Studies on partially purified enzyme show a pH optimum of 7.5 with a subunit molecular weight of approximately 51 kDa. Additional purification and characterization strategies will be presented. The use of whole cells containing -AAD will be discussed to prepare chiral pharmaceutical intermediates.     

Purification and characterization of a cellulase-free xylanase of a moderate thermophile Bacillus licheniformis A99

Two cellulase-free xylanases were secreted by a thermophile, Bacillus licheniformis A99. Of the two, the predominant one was purified to homogeneity. The enzyme was optimally active at 60 °C, pH 6–7.5, and had a molecular weight of about 45 KDa and isoelectric point of 7.0 ± 0.2. The K _m (for birchwood xylan) and V _max were 3.33 mg/ml and 1.111 mmols mg^–1 protein min^–1 respectively. The half-life of the enzyme was 5 h at 60 °C. All cations except Hg²⁺ and Ag⁺ as well as EDTA were well tolerated and did not adversely affect xylanase activity. However, SDS inhibited the enzyme activity. The release of reducing sugars from unbleached commercial pulp sample on treatment with the enzyme indicated its potential in prebleaching of paper pulp. The enzyme caused saccharification of lignocellulosics such as wheat bran, wheat straw and sawdust. This is the first report on purification and characterization of cellulase-free xylanase from a moderate thermophile Bacillus licheniformis.     

Mannitol Biosynthesis in Pyrenochaeta terrestris II. D-Mannitol-l-phosphatase

Cell-free extracts of mycelial mats of Pyrenochaeta terrestris contained an enzyme which hydrolyzed mannitol-l-phosphate to mannitol and inorganic phosphate. Greatest mannitol-1-phosphatase activity occurred early in the growth period when the mannitol content of the mats was at a maximum. The enzyme was active over a broad pH range with optimum activity between pH 6.5–7.0 in 0.05 M Tris-maleate buffer. Maiinitnl-1-phosphatase was inhibited by reagents known to inhibit enzymes containing -SH groups. A 10-fold purification was attained by a combination of (NII₄)₂ SO₄ fractionation and gel filtration on Sephadex G-100. The partially purified enzyme required Mg^?2 for activity and did not hydrolyze a number of sugar phosphates. Km values for mannitol-l-phosphate and Mg^?2 with the partially purified extract were 3 × 10^?3 M and 1 × 10^?4 M respectively.     

Purification and Properties of a Phytate-degrading Enzyme from <Emphasis Type="Italic">Pantoea agglomerans</Emphasis>

A periplasmatic phytate-degrading enzyme from Pantoea agglomerans isolated from soil was purified about 470-fold to apparent homogeneity with a recovery of 16% referred to the phytate-degrading activity in the crude extract. It behaved as a monomeric protein with a molecular mass of about 42 kDa. The purified enzyme exhibited a single pH optimum at 4.5. Optimum temperature for the degradation of phytate was 60°C. The kinetic parameters for the hydrolysis of sodium phytate were determined to be K_M = 0.34 mmol/l and k_cat = 21 s^-1 at pH 4.5 and 37°C. The enzyme exhibited a narrow substrate selectivity. Only phytate and glucose-1-phosphate were identified as good substrates. Since this Pantoea enzyme has a strong preference for glucose-1-phosphate over phytate, under physiological conditions glucose-1-phosphate is its most likely substrate. The maximum amount of phosphate released from phytate by the purified enzyme suggests myo-inositol pentakisphosphate as the final product of enzymatic phytate degradation.     

Purification and characterization of isocitrate lyase fromEscherichia coli

Isocitrate lyase has been purified to homogeneity, as determined by SDS-polyacrylamide gel electrophoresis and subsequent silver staining, fromEscherichia coli D₅H₃G₇. The enzyme was found to have a subunit molecular weight of 48,000 and a native molecular weight of 188,000 as determined by gel filtration chromatography. Thus, the enzyme appears to have tetrameric structure. The isoelectric point was determined to be 4.6, and the enzyme displayed a pH optimum at 7.3. The K_m of isocitrate lyase forthreo-Ds-isocitrate was determined to be 8 M. The purification procedure is highly reproducible and results in a 39% net yield of purified protein.     

Purification and characterization of arginine decarboxylase from cucumber (Cucumis sativus) seedlings

A simple, reproducible and rapid protocol for the purification of arginine decarboxylase fromCucumis sativus seedlings has been standardised. The purification steps involved ion-exchange chromatography on diethylaminoethyl-cellulose followed by gel filtration on Sephadex G-l 50. The purified enzyme preparation migrated as a single stainable band on Polyacrylamide gels at both basic and acidic pH, but under denaturing and reducing conditions on sodium dodecyl sulphate-polyacrylamide gels resolved into polypeptides of molecular weight 48,000,44,000 and 15,000. However, in the absence of 2-mercaptoethanol on electrophoresis on sodium dodecyl sulphate-polyacrylamide gels, the enzyme moved as single band with a molecular weight of 150,000. Evidence was obtained to indicate that these three polypeptides were probably derived from a single larger molecular weight enzyme. On storage of the purified protein, the 48,000 species was preferentially degraded to smaller polypeptides. The preliminary data suggested that the 48,000 and 44,000 species shared many common tryptic peptides as revealed by finger printing of the [¹²⁵I ]-labelled protein. The purified enzyme was a glycoprotein and had aK _m of 0.5 mM for arginine. Its activity was stimulated by dithiothrietol and pyridoxal phosphate. EDTA did not inhibit the enzyme activity. Mn²⁺ at 1 mM stimulated arginine decarboxylase activity but was inhibitory at higher concentration