Purification and biochemical characterization of ionically unbound polyphenol oxidase from Musa paradisiaca leaf

An ionically unbound and thermostable polyphenol oxidase (PPO) was extracted from the leaf of Musa paradisiaca. The enzyme was purified 2.54-fold with a total yield of 9.5% by ammonium sulfate precipitation followed by Sephadex G-100 gel filtration chromatography. The purified enzyme exhibited a clear single band on native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS) PAGE. It was found to be monomeric protein with molecular mass of about 40 kD. The zymographic study using crude extract as enzyme source showed a very clear band around 40 kD and a faint band at around 15 kD, which might be isozymes. The enzyme was optimally active at pH 7.0 and 50°C temperature. The enzyme was active in wide range of pH (4.0-9.0) and temperature (30-90°C). From the thermal inactivation studies in the range 60-75°C, the half-life (t(1/2)) values of the enzyme ranged from 17 to 77?min. The inactivation energy (Ea) value of PPO was estimated to be 91.3?kJ mol(-1). It showed higher specificity with catechol (K(m)?=?8?mM) as compared to 4-methylcatechol (K(m)?=?10?mM). Among metal ions and reagents tested, Cu(2+), Fe(2+), Hg(2+), Mn(2+), Ni(2+), protocatechuic acid, and ferrulic acid enhanced the enzyme activity, while K(+), Na(+), Co(2+), kojic acid, ascorbic acid, ethylenediamine tetraacetic acid (EDTA), sodium azide, β-mercaptoethanol, and L-cysteine inhibited the activity of the enzyme.     

Expression and purification of organic solvent stable lipase from soil metagenomic library

Gene for organic solvent stable lipase was overexpressed from soil metagenomic library. The clone with maximum activity was selected, and enzyme was purified by gel-permeation chromatography with a molecular mass of approx. 40 kDa. The deduced aminoacid sequence indicated that the protein belongs to the lipase family I.3 and containing a C-terminal secretion signal for ABC dependent transport together with possible motifs for Ca(2+) binding sites. The enzyme expressed maximum activity at 30 °C and pH 7.0 and found to be stable in pH and temperature ranging from 6.0-9.0 and 20-60 °C, respectively. Furthermore, the enzyme was found highly resistant to many organic solvents, especially isopropanol, DMSO, methanol, xylene and hexane. The enzyme showed enhanced activity in the presence of divalent cations (Mg(2+), Mn(2+), Ca(2+), Hg(2+), Cu(2+)), whereas the presence of trivalent cation (Fe(3+)) inhibited the activity.     

Characterization of a Glucose-, Xylose-, Sucrose-, and d-Galactose-Stimulated ��-Glucosidase from the Alkalophilic Bacterium Bacillus halodurans C-125

The gene (Bhbgl) encoding a β-glucosidase from the alkalophilic bacterium Bacillus halodurans C-125 was synthesized chemically via the PCR-based two-step DNA synthesis (PTDS) method and expressed in Escherichia coli. Bhbgl contained an open reading frame (ORF) of 1359 bp encoding a 453-amino acid protein belonging to glycoside hydrolase family 1 (GHF1), and the deduced molecular mass of recombinant Bhbgl (52,488 Da) was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme exhibited a high specific activity with o-nitrophenyl-β-D-glucopyranoside (oNPGlu) and an apparent K (m) value of 0.32 mM. With oNPGlu as the substrate, Bhbgl displayed pH and temperature optima of ~7.0 and 50°C, respectively. The enzyme was relatively stable under alkaline conditions and >50% activity was retained after incubation at pH 9.5 for 24 h at 4°C. Recombinant Bhbgl activity was inhibited by 5 mM Zn(2+), Fe(3+), or Cd(2+), but was enhanced by 1 mM Mg(2+) and other metal ions. Enzyme activity was also stimulated by at least four sugars (sucrose, D-galactose, xylose, glucose) at concentrations ranging from 50 to 800 mM.     

Partial purification and characterization of almond seed lipase

A lipase was partially purified from the almond (Amygdalus communis L.) seed by ammonium sulfate fractionation and dialysis. Kinetics of the enzyme activity versus substrate concentration showed typical lipase behavior, with K(m) and V(max) values of 25 mM and 113.63 micromol min(-1) mg(-1) for tributyrin as substrate. All triglycerides were efficiently hydrolyzed by the enzyme. The partially purified almond seed lipase (ASL) was stable in the pH range of 6-9.5, with an optimum pH of 8.5. The enzyme was stable between 20 and 90 degrees C, beyond which it lost activity progressively, and exhibited an optimum temperature for the hydrolysis of soy bean oil at 65 degrees C. Based on the temperature activity data, the activation energy for the hydrolysis of soy bean oil was calculated as -5473.6 cal/mol. Soy bean oil served as good substrate for the enzyme and hydrolytic activity was enhanced by Ca(2+), Fe(2+), Mn(2+), Co(2+), and Ba(2+), but strongly inhibited by Mg(2+), Cu(2+), and Ni(2+). The detergents, sodiumdeoxicholate and Triton X-100 strongly stimulated enzyme activity while CTAB, DTAB, and SDS were inhibitors. Triton X-405 had no effect on lipase activity. The partially purified enzyme retained its activity for more than 6 months at -20 degrees C, beyond which it lost activity progressively.     

An alkaline β-glucosidase isolated from an olive brine strain of Wickerhamomyces anomalus

An efficient β-glucosidase (βG)-producing strain, Wickerhamomyces anomalus BS81, was isolated from naturally fermented olive brine and identified based on PCR/restriction fragment length polymorphism of the rDNA internal transcribed spacer and sequence analysis of the D1/D2 region of the 26S rRNA gene. The hydrolytic activity of the βG had an optimum pH of 8.5 and an optimum temperature of 35 °C. The enzyme had high substrate specificity and high catalytic efficiency (K(m) 0.99 mM, V(max) 14 U g(-1) of cells) for p-nitrophenyl-β-d-glucopyranoside. The enzyme was activated by increasing concentrations of NaCl, with maximum activity at 150 g L(-1) NaCl. Although βGs have been purified and characterized from several other sources, the W. anomalusβG is unique among βGs because its relative maximum activity occurs at alkaline pH and 35 °C. Moreover, the yeast strain has esterase activity that acts synergistically with βG to degrade oleuropein to debitter table olives and olive oil.     

A bioprocess for the production of phytase from Schizophyllum commune: studies of its optimization, profile of fermentation parameters, characterization and stability

Schizophyllum commune produces phytase through solid-state fermentation using different agroindustrial residues. After optimization of phytase production, a maximal level of phytase (113.7 Units/gram of dry substrate) was obtained in wheat bran based medium containing 5% sucrose, 50% humidity, 7.5% of biomass at 33 °C pH 7.0 during 72 h and a 285% improvement in enzyme titre was achieved. Analysis of fermentation parameters profile for phytase production showed the highest productivity (1.466 Units/gram of dry substrate/hour) in 66 h of fermentation. Phytase has an optimal pH of 5.0, an optimal temperature of 50 °C and K (m) and V (max) values of 0.16 mM and 1.85 μmol mL(-1) min(-1), respectively. Phytase activity was stimulated essentially in the presence of K(+), Ca(2+), Mg(2+), Mn(2+), Zn(2+), Cu(2+), Fe(2+), Fe(3+), Co(2+), Ni(2+), acetate and citrate at concentrations of 1 mM. Phytase had the best shelf life when stored at a cooling temperature, maintaining 38% of its initial activity after 112 days of storage, and still presenting enzymatic activity after 125 days of storage. Stability studies of phytase performed in aqueous enzyme extracts showed satisfactory results using polyethyleneglycol 3350, carboxymethylcellulose, methylparaben, mannitol and benzoic acid in concentrations of 0.25, 0.025, 0.025, 0.25, and 0.0025%, respectively. PEG 3350 was shown to be the best stabilizing agent, resulting in 109% of phytase activity from the initial crude extract remaining activity in after 90 days.     

BspLS2I--a new site-specific endonuclease from the thermophilic bacteria Bacillus species LS2]

A new restriction endonuclease BspLS2I was isolated from the thermophilic bacterium Bacillus species LS2 and purified by blue sepharose and hydroxyapatite chromatographies. The enzyme is an isoschizomer of SduI from Streptococcus durans. BspLS2I recognizes the sequence 5' G(G/A/T)GC(C/T/A) decreases C 3' on double-stranded DNA and cleaves it is indicated by the arrow to yield sticky-ended DNA fragments. Maximum catalytic activity of endonuclease was found in 10 mM tris-HCl (pH 7.9) in the presence of 15-30 mM MgCl2 at 50 degrees C. The phage T4 glucosylated DNA is not cleaved by the enzyme.     

Constitutive expression, purification and characterization of a phosphoglucomutase from Fusarium oxysporum

The phosphoglucomutase gene from a wild type Fusarium oxysporum strain (F3), was homologously expressed, under the control of the constitutive promoter of gpdA of Aspergillus nidulans. The transformant produced elevated levels of phosphoglucomutase activity compared to the wild type, a fact that facilitated the subsequent purification procedure. The enzyme (FoPGM) was purified to homogeneity applying three anion exchange and one gel filtration chromatography steps. The native enzyme revealed a monomeric structure with a molecular mass of 60 kDa, while the isoelectric point was 3.5. FoPGM was active in pH ranged from 6.0 to 8.0, with an optimum using 3-(N-morpholino)propanesulfonic acid buffer at 7.0, while loss of activity was observed when phosphate buffer was used in the above mentioned pH range. The optimal temperature for activity was 45°C but the enzyme became unstable at temperatures above 40°C. FoPGM requires the presence of a divalent cation for its function with maximum activity being obtained with Co(2+). The apparent K(m) for Co(2+) was found to be 10 μM. The enzyme was also active with other divalent metal ions such as Mn(2+), Mg(2+), Ni(2+) and Ca(2+) but to a lesser extent. The following kinetic constants were determined: v(max), 0.74 μmol mg(protein)(-1)min(-1); k(cat), 44.2 min(-1); K(m)(G1P), 0.10mM; K(m)(G1,6 diP), 1.03 μM; k(cat)/K(m)(G1P), 443 mM(-1)min(-1) and k(cat)/K(m)(G1,6 diP), 42,860 mM(-1)min(-1). The enzyme was considered to follow a Ping Pong substituted enzyme or enzyme isomerization mechanism.     

Purification and characterization of an extracellular, uracil specific ribonuclease from a Bizionia species isolated from the marine environment of the Sundarbans

The first ribonuclease (RNase) from the Cytophaga-Flavobacterium-Bacteroides phylum, dominant in the marine environment, and also from the first Bizionia species isolated from the tropics was purified and characterized. Extracellular RNase production occurred when the culture medium contained 5-7% (w/v) NaCl. The 53.0 kDa enzyme was purified 29 folds with a recovery of 4% and specific activity of 630unit/mg protein. The pH and temperature optima are 6.5 and 35 degrees C, respectively and the enzyme retains more than half of its activity (relative to optimal assay conditions) after 1h pre-incubation separately with 5% (w/v) NaCl or from pH 5.0 to 8.5 or at 50 degrees C. Dithiothreitol and beta-mercaptoethanol do not inhibit whereas human placental RNase inhibitor protein halves the RNase activity. While Mg(2+), Ba(2+) and Ca(2+) enhanced the enzyme activity, Fe(2+), Cu(2+) and Hg(2+) inactivated it. This RNase degrades uracil containing nucleic acids only. Our isolate could be a novel renewable source of deoxyribonuclease (DNase)--free RNase enzyme.     

Isolation and characteristics of new thermostable DNA ligase from archaea of the genus Thermococcus

The DNA ligase gene from thermophilic archaea of the genus Thermococcus (strain 1519) was identified and sequenced in the polymerase chain reaction. The recombinant enzyme LigTh1519 was expressed in Escherichia coli, purified, and characterized. LigTh1519 was capable of ligating the cohesive ends and single-strand breaks in double-stranded DNA (ATP as a cofactor). The optimum conditions for the ligase reaction appeared as follows: 100 mM NaCl, 50 mM MgCl2, pH 7.0-10.5, and temperature 70 degrees C. More than 50% Lig1519 activity were preserved after incubation of the enzyme at 80 degrees C for 30 min. New thermostable DNA ligase LihTh1519 may be used for basic and applied researches in molecular biology and genetic engineering.     

Expression and characterization of fumarase (FUMR) from Rhizopus oryzae

Fumarase catalyzes the reversible hydration of fumarate to l-malate in Rhizopus oryzae. A recombinant pET22b-fumR harboring a fumarase gene (fumR) from R. oryzae was constructed for high level expression in E. coli BL21 (DE3). The FUMR activity was optimal at 30°C and pH 7.2. The enzyme was stable below 45°C and at pH 3.0-9.0. No effects of Zn(2+), Fe(2+), or EDTA were observed on enzyme activity. A slight inhibition of FUMR activity was seen with Mg(2+), while Ca(2+) had a small stimulatory effect. The K(m) for l-malic acid and fumaric acid were 0.46 mM and 3.07 mM, respectively. The activity of FUMR catalyzing hydration of fumarate to l-malate was completely inhibited by 2mM fumaric acid. The unique enzymatic properties suggested that overexpression of FUMR could enhance fumaric acid accumulation in R. oryzae.     

Purification, properties, and sequence specificity of SslI, a new type II restriction endonuclease from Streptococcus salivarius subsp. thermophilus.

SslI, a type II restriction endonuclease, was purified from Streptococcus salivarius subsp. thermophilus strain BSN 45. SslI is an isoschizomer of BstNI. SslI activity was maximum at pH 8.8, 0 to 50 mM NaCl, 2 to 8 mM Mg2+, and 42 degrees C. Activity against phage DNA in vitro was demonstrated.     

Site-specific endonuclease NspLKI is an isoschizomer of endonuclease HaeIII

Site-specific endonuclease NspLKI has been isolated and purified to functionally pure state from soil bacterium Nocardia species LK by successive chromatography on columns with phosphocellulose, HTP hydroxyapatite, and heparin-Sepharose. The isolated enzyme recognizes the 5'-GG downward arrowCC-3' sequence on DNA and cleaves it as indicated by the arrow, i.e., it is an isoschizomer of HaeIII. The final enzyme yield is 1.105 units per gram of wet biomass. The enzyme is active in the temperature range of 25-60 degrees C with an optimum at 48-55 degrees C; it does not lose activity on storage for three days at room temperature. An optimal buffer is HRB containing 10 mM Tris-HCl, pH 7.4, 200 microgram/ml albumin, 10 mM MgCl2, and 100 mM NaCl.     

Purification, properties, and sequence specificity of SslI, a new type II restriction endonuclease from Streptococcus salivarius subsp. thermophilus.

SslI, a type II restriction endonuclease, was purified from Streptococcus salivarius subsp. thermophilus strain BSN 45. SslI is an isoschizomer of BstNI. SslI activity was maximum at pH 8.8, 0 to 50 mM NaCl, 2 to 8 mM Mg2+, and 42 degrees C. Activity against phage DNA in vitro was demonstrated.     

Purification and properties of a glucuronan lyase from Sinorhizobium meliloti M5N1CS (NCIMB 40472).

A glucuronan lyase extracted from Sinorhizobium meliloti strain M5N1CS was purified to homogeneity by anion-exchange chromatography. The purified enzyme corresponds to a monomer with a molecular mass of 20 kDa and a pI of 4.9. A specific activity was found only for polyglucuronates leading to the production of 4,5-unsaturated oligoglucuronates. The enzyme activity was optimal at pH 6.5 and 50 degrees C. Zn(2+), Cu(2+), and Hg(2+) (1 mM) inhibited the enzyme activity. No homology of the enzyme N-terminal amino acid sequence was found with any of the previously published protein sequences. This enzyme purified from S. meliloti strain M5N1CS corresponding to a new lyase was classified as an endopolyglucuronate lyase.     

Enhancement of endothelial cell migration by constitutively active LPA(1)-expressing tumor cells

2,3-Dihydroxybiphenyl-1,2-dioxygenase plays an important role in the degradation of polychlorinated biphenyls. The gene (BsbphCI) encoding a 2,3-DHBP dioxygenase from Bacillus sp. JF8 is 960 bp. We synthesized a 960 bp BsbphCI gene encoding a 2,3-DHBP dioxygenase derived from Bacillus sp. JF8 and expressed it in Escherichiacoli. The recombinant protein was about 36 kDa, confirmed by SDS-PAGE. The concentration of the purified protein was about 1.8 mg/mL. With 2,3-DHBP as a substrate, the optimal temperature for enzyme activity at pH 8.5 was 50 °C. The optimal pH for the 2,3-DHBP dioxygenase was 8.5. The enzyme retained 33% activity after heating at 60 °C for 60 min. We found that Cu(2+), K(+), Zn(2+), Mg(2+), Ni(2+), Co(2+), and Cd(2+) activated the enzyme. However, Ca(2+), Fe(2+), Li(+), and Cr(3+) inhibited it. Enzyme activity was reduced by exposure to H(2)O(2), SDS, and KI. The results of HPLC indicated that the transgenic E. coli strain with the BsbphCI gene degraded 2,3-DHBP more quickly than the wild type strain.     

Characterization of a cysteine protease from wheat Triticum aestivum (cv. Giza 164)

Enzymes, especially proteases, have become an important and indispensable part of the processes used by the modern food and feed industry to produce a large and diversified range of products for human and animal consumption. A cysteine protease, used extensively in the food industry, was purified from germinated wheat Triticum aestivum (cv. Giza 164) grains through a simple reproducible method consisting of extraction, ion exchange chromatography and gel filtration. The molecular weight of the enzyme was estimated to be 61000+/-1200-62000+/-1500 by SDS-PAGE and gel filtration. The cysteine protease had an isoelectric point and pH optimum at 4.4 and 4.0, respectively. The enzyme exhibited more activity toward azocasein than the other examined substrates with K(m) 2.8+/-0.15 mg azocasein/ml. In addition, it had a temperature optimum of 50 degrees C and based on a heat stability study 55% of its initial activity remained after preincubation of the enzyme at 50 degrees C for 30 min prior to substrate addition. All the examined metal cations inhibited the enzyme except Co(2+), Mg(2+), Mn(2+) and Li(+). The proteolytic activity of the enzyme was inhibited by thiol-specific inhibitors, whereas iodoacetate and p-hydroxymercuribenzoate caused a competitive inhibition with Ki values 6+/-0.3 mM and 21+/-1.2 microM, respectively. Soybean trypsin inhibitor had no effect on the enzyme. The enzyme activity remained almost constant for 150 days of storage at -20 degrees C. The properties of this enzyme, temperature and pH optima, substrate specificity, stability and sensitivity to inhibitors or activators, meet the prerequisites needed for food industries.     

Purification and characterization of an endo-exonuclease from adult flies of Drosophila melanogaster.

An endo-exonuclease (designated nuclease III) has been purified to near homogeneity from adult flies of Drosophila melanogaster. The enzyme degrades single- and double-stranded DNA and RNA. It has a sedimentation co-efficient of 3.1S and a strokes radius of 27A The native form of the purified enzyme appears to be a monomer of 33,600 dalton. It has a pH optimum of 7-8.5 and requires Mg2+ or Mn2+ but not Ca2+ or Co2+ for its activity. The enzyme activity on double-stranded DNA was inhibited 50% by 30 mM NaCl, while its activity on single-stranded DNA required 100 mM NaCl for 50% inhibition. Under the latter conditions, its activity on double-stranded DNA was inhibited approximately 98%. The enzyme degrades DNA to complete acid soluble products which are a mixture of mono- and oligonucleotides with 5'-P and 3'-OH termini. Supercoiled DNA was converted by the enzyme to nicked and subsequently to linear forms in a stepwise fashion under the condition in which the enzyme works optimally on single-stranded DNA. The amino acid composition and amino acid sequencing of tryptic peptides from purified nuclease III is also reported.