Expression of a novel thermostable Cu,Zn-superoxide dismutase from <Emphasis Type="Italic">Chaetomium thermophilum</Emphasis> in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and its antioxidant properties

A new superoxide dismutase (SOD) gene from the thermophilic fungus Chaetomium thermophilum (Ctsod) was cloned and expressed in Pichia pastoris and its gene product was characterized. The specific activity of the purified CtSOD was 2,170 U/mg protein. The enzyme was inactivated by KCN and H₂O₂ but not by NaN₃, confirming that it belonged to the type of Cu, ZnSOD. The amino acid residues involved in coordinating copper and zinc were conserved. The recombinant CtSOD exhibited optimum activity at pH 6.5 and 60°C. The enzyme retained 65% of the maximum activity at 70°C for 60 min and the half-life was 22 and 7 min at 80 and 90°C, respectively. The recombinant yeast exhibited higher stress resistance than the control yeast cells to salt and superoxide-generating agents, such as paraquat and menadione.     

Cloning,expression, and characterization of thermostable Manganese superoxide dismutase from <Emphasis Type="Italic">Thermoascus aurantiacus</Emphasis> var. <Emphasis Type="Italic">levisporus</Emphasis>

A superoxide dismutase (SOD) gene of Thermoascus aurantiacus var. levisporus, a thermophilic fungus, was cloned, sequenced, and expressed in Pichia pastoris and its gene product was characterized. The coding sequence predicted a 231 residues protein with a unique 35 amino acids extension at the N-terminus indicating a mitochondrial-targeting sequence. The content of Mn was 2.46 μg/mg of protein and Fe was not detected in the purified enzyme. The enzyme was found to be inhibited by NaN₃, but not by KCN or H₂O₂. These results suggested that the SOD in Thermoascus aurantiacus var. levisporus was the manganese superoxide dismutase type. In comparison with other MnSODs, all manganese-binding sites were also conserved in the sequence (H88, H136, D222, H226). The molecular mass of a single band of the enzyme was estimated to be 21.7 kDa. The protein was expressed in tetramer form with molecular weight of 68.0 kDa. The activity of purified protein was 2,324 U/mg. The optimum temperature of the enzyme was 55°C and it exhibited maximal activity at pH 7.5. The enzyme was thermostable at 50 and 60°C and the half-life at 80°C was approximately 40 min.     

A highly stable Cu/Zn superoxide dismutase from <Emphasis Type="Italic">Withania somnifera</Emphasis> plant: gene cloning,expression and characterization of the recombinant protein

A gene from Withania somnifera (winter cherry), encoding a highly stable chloroplastic Cu/Zn superoxide dismutase (SOD), was cloned and expressed in Escherichia coli. The recombinant enzyme (specific activity of ~4,200 U mg⁻¹) was purified and characterized. It retained ~90 and ~70% residual activities after 1 h at 80 and 95°C, respectively. At 95°C, thermal inactivation rate constant (K _d) of the enzyme was 2.46 × 10⁻³ min⁻¹ and half-life of heat inactivation was 4.68 h. The enzyme was stable against a broad pH range (2.5–11.0). It also showed a high degree of resistance to detergent, ethanol and protease digestion. This recombinant Cu/Zn SOD could therefore have useful applications.     

Purification and biochemical characterization of a superoxide dismutase from the soluble fraction of the cyanobacterium, <Emphasis Type="Italic">Spirulina platensis</Emphasis>

A superoxide dismutase (SOD) was purified from Spirulina platensis sonicate. The SOD was purified to homogeneity (48-fold and 0.24% yield) through ammonium sulphate precipitation and DEAE-52 anion exchange chromatography. The SOD from S. platensis appeared to be a homodimer with a molecular weight of 30 kDa and a subunit MW of 15 kDa as determined by both native polyacrylamide gel electrophoresis and mass spectrometry. The enzyme activity was stable at pH 6.5–10.0 and 50 °C. Using group-specific chemical modifying reagents, the amino acids arginine, histidine, tryptophan, tyrosine and aspartic acid were identified to be essential for S. platensis SOD activity. The amino acid composition was found to lack methionine and cysteine. The inhibition of activity by H₂O₂ suggests that the enzyme may be an iron containing SOD.     

Purification and characterization of a hyperthermostable Mn-superoxide dismutase from <Emphasis Type="Italic">Thermus thermophilus</Emphasis> HB27

Thermostable Mn-dependent catalases are promising enzymes in biotechnological applications. In the present study, a Mn-containing superoxide dismutase of the hyperthermophilic Thermus thermophilus HB27 had been purified and characterized by a two-stage ultrafiltration process after being expressed in E. coli. The enzyme was highly stable at 90°C and retained 57% activity after heat treatment at 100°C for 1 h. The native form of the enzyme was determined as a homotetramer by analytical size exclusion chromatography and sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The final purified enzyme had an isoelectric point of 6.2 and a high α-helical content of 70%, consistent with the theoretical values. This showed that the purified SOD folded with a reasonable secondary structure.     

A thermostable manganese-containing superoxide dismutase from the thermophilic fungus Thermomyces lanuginosus

A thermostable superoxide dismutase (SOD) from a Thermomyces lanuginosus strain (P134) was purified to homogeneity by fractional ammonium sulfate precipitation, ion-exchange chromatography on DEAE-Sepharose, Phenyl-Sepharose hydrophobic interaction chromatography, and gel filtration on Sephacryl S-100. The molecular mass of a single band of the enzyme was estimated to be 22.4 kDa, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using gel filtration on Sephacryl S-100, the molecular mass was estimated to be 89.1 kDa, indicating that this enzyme was composed of four identical subunits of 22.4 kDa each. The SOD was found to be inhibited by NaN₃, but not by KCN or H₂O₂, suggesting that the SOD in T. lanuginosus was of the manganese superoxide dismutase type. The SOD exhibited maximal activity at pH 7.5. The optimum temperature for the activity was 55°C. It was thermostable at 50 and 60°C and retained 55% activity after 60 min at 70°C. The half-life of the SOD at 80°C was approximately 28 min and even retained 20% activity after 20 min at 90°C.     

Purification,N-Terminal Amino Acid Sequence and Partial Characterization of A Cu,Zn Superoxide Dismutase From the Pathogenic Fungus Aspergillusfumiga Tus

A superoxide dismutase (SOD) has been purified to homogeneity from the fungal pathogen Aspergillus fumigatus using a combination of cell homogenization, isoelectric focusing and gel filtration FPLC. The N-terminal amino acid sequence of the purified enzyme demonstrated substantial homology to known Cu, Zn superoxide dismutases for a range of organisms, including Neurospora crassa and Saccharomyces cerevisiae. The enzyme subunit has a pl of 5.9, a relative molecular mass of 19 kDa and a spectral absorbance maximum of 550nm. The non reduced enzyme has a relative molecular mass of 95 kDa. The enzyme remained active after prolonged incubation at 70°C and was pH insensitive in the range 7-11. Potassium cyanide and diethyldithiocarbamate, known Cu, Zn SOD inhibitors, caused inhibition of the purified enzyme at working concentrations of 0.25 mM, whilst sodium azide and o-phenanthroline demonstrated inhibition at higher concentrations (10-30 mM). SOD activity was also detectable in culture filtrate of A. fumigatus. This enzyme may have a potential role as a virulence factor in the avoidance of neutrophil and phagocyte oxidative burst killing mechanisms.     

The in vitro germination and storage characteristics of Keteleeria fortunei var. cyclolepis pollen provide a reference for cross breeding

Keteleeria fortunei var. cyclolepis is an ideal tree species for mountain afforestation, timber forests, and landscaping. Its pollination process can be affected by the rainy season, making it difficult to pollinate the massive female cones, which leads to a high abortion rate and low quality of seeds. Here, we observed the pollen morphology of K. f. cyclolepis using scanning electron and light microscopes, investigated the characteristics of its in vitro germination by the detached method, and explored the effect of different storage temperatures and times on the pollen germination rate and the activity of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). Our results indicated that the pollen of K. f. cyclolepis is a five-cell pollen, comprising one noumenon and two air sacs, both of which were oval in polar view. The optimal condition for pollen germination of K. f. cyclolepis was 240 g/L sucrose + 70 mg/L CaCl₂ + 210 mg/L H₃BO₃ at 24 °C and pH 6.0, resulting in a germination rate of 45.0%. The effects of different storage temperature and time on pollen germination rate varied significantly. The best storage temperature was − 80 °C, at which the germination rate was 20.9% after 365 days of storage, and the activity of three protective enzymes remained relatively high, representing relatively strong antioxidation and antiaging activity. Stepwise regression analysis showed that SOD was the main factor affecting the pollen germination rate of K. f. cyclolepis. The function of the three protective enzymes differed under various temperatures, for example, SOD served as a sensitive protective enzyme at room temperature, − 20 °C and − 80 °C, whereas both SOD and CAT served as sensitive protective enzymes at 4 °C.

     

Purification and Characterization of Cu,Zn Superoxide Dismutase from Ark Shell Scapharca broughtonii

A superoxide dismutase has been purified to apparent homogeneity from the muscular tissue of the ark shell, Scapharca broughtonii, by ammonium sulfate fractionation, and consecutive column chromatographies using DEAE-Sephadex and Sephadex G-100. This enzyme has a molecular weight of 71,700 and is composed of two identical subunits of M _r 35,800, which are joined by noncovalent interactions. The purified enzyme was stable over the range of pH 5.0-10.0 at 4°C for 24 h and at temperatures below 45°C. Cyanide at 0.1 and 1 mM inhibited the activity of the superoxide dismutase 56 and 100%, but 5 mM azide caused 8% inhibition. The optical spectrum of this enzyme had a maximum at 265 nm, and the amino acid composition of the enzyme was similar to that of the other Cu, Zn superoxide dismutases except for the contents of threonine, serine, proline, and leucine. Atomic absorption spectroscopy showed that this enzyme has approximately 2 atoms of Cu²⁺ and Zn²⁺ per mole of enzyme. These results indicate that the purified enzyme from ark shell, Scapharca broughtonii, is a Cu, Zn superoxide dismutase.     

Purification, immobilization and characterization of linoleic acid isomerase on modified palygorskite

Linoleic acid isomerase from Lactobacillus delbrueckii subsp. bulgaricus 1.1480 was purified by DEAE ion-exchange chromatography and gel filtration chromatography. An overall 5.1% yield and purification of 93-fold were obtained. The molecular weight of the purified protein was ~41 kDa which was analyzed by SDS-PAGE. The purified enzyme was immobilized on palygorskite modified with 3-aminopropyltriethoxysilane. The immobilized enzyme showed an activity of 82 U/g. The optimal temperature and pH for the activity of the free enzyme were 30 °C and pH 6.5, respectively; whereas those for the immobilized enzyme were 35 °C and pH 7.0, respectively. The immobilized enzyme was more stable than the free enzyme at 30–60 °C, and the operational stability result showed that more than 85% of its initial activity was retained after incubation for 3 h. The K _m and V _max values of the immobilized enzyme were found to be 0.0619 mmol l⁻¹ and 0.147 mmol h⁻¹ mg⁻¹, respectively. The immobilized enzyme had high operational stability and retained high enzymatic activity after seven cycles of reuse at 37 °C.     

Cloning,expression and characterization of glycoside hydrolase family 11 endoxylanase from <Emphasis Type="Italic">Bacillus pumilus</Emphasis> ARA

An endoxylanase gene, xynA, was cloned from Bacillus pumilus ARA and expressed in Escherichia coli. The open reading frame of the xynA gene was 687 bp encoding a signal peptide and a mature xylanase with a molecular mass of 23 kDa. The enzyme was categorized as a glycosyl hydrolase family 11 member based on the sequence analysis of the putative catalytic domain. The recombinant XynA (Bpu XynA) was purified to homogeneity by Ni–NTA and ion exchange chromatography on DEAE–Sepharose FF. The enzyme exhibited highest activity at pH 6.6 and 50°C. The purified Bpu XynA was stable for at least 2 h at 45°C, and retained over 50% residual activity after being incubated at 60°C for 1 h. The activity of the xylanase was not significantly affected by metal ions and EDTA. The K _m and K _cat /K _m of Bpu XynA for oat-spelt xylan were 5.53 mg/ml and 10.14 ml/mg s at 50°C and pH 6.6. The main product of hydrolysis by Bpu XynA was xylooligosaccharide. The results revealed that the consumption of grass xylan by B. pumilus ARA depended on the synergistic reactions of Bpu XynA and Bpu arabinosidase, and that a typical GH11 xylanase e.g. Tla XynA had capability to remove the side chain of xylan. The properties Bpu XynA make it promising for application in the production of Bifidobacterium growth-promoting factors and in feed industry.     

Manganese superoxide dismutase from a higher plant

A manganese-containing superoxide dismutase (EC 1.15.1.1) was purified to homogeneity from a higher plant for the first time. The enzyme was isolated fromPisum sativum leaf extracts by thermal fractionation, ammonium sulfate salting out, ion-exchange and gel-filtration column chromatography, and preparative polyacrylamide gel electrophoresis. Pure manganese superoxide dismutase had a specific activity of about 3,000 U mg^-1 and was purified 215-fold, with a yield of 1.2 mg enzyme per kg whole leaf. The manganese superoxide dismutase had a molecular weight of 94,000 and contained one g-atom of Mn per mol of enzyme. No iron and copper were detected. Activity reconstitution experiments with the pure enzyme ruled out the possibility of a manganese loss during the purification procedure. The stability of manganese superoxide dismutase at-20°C, 4°C, 25°C, 50°C, and 60°C was studied, and the enzyme was found more labile at high temperatures than bacterial manganese superoxide dismutases and iron superoxide dismutases from an algal and bacterial origin.Abbreviations NBT nitro blue tetrazolium - SOD superoxide dismutase (EC 1.15.1.1)     

Purification and some properties of superoxide dismutase from<Emphasis Type="Italic"> Deinococcus radiophilus</Emphasis>, the UV-resistant bacterium

The superoxide dismutase (SOD, EC 1.15.1.1) of Deinococcus radiophilus, a bacterium extraordinarily resistant to UV, ionizing radiations, and oxidative stress, was purified 1,920-fold with a 58% recovery yield from the cell-free extract of stationary cells by steps of ammonium sulfate fractionation and Superdex G-75 gel-filtration chromatography. A specific activity of the purified enzyme preparation was ca. 31,300 U mg^–1 protein. D. radiophilus SOD is Mn/FeSOD, judging by metal analysis and its insensitivity to cyanide and a partial sensitivity to H₂O₂. The molecular weights of the purified enzyme estimated by gel chromatography and polyacrylamide gel electrophoresis are 51.5±1 and 47.1±5 kDa, respectively. The SOD seems to be a homodimeric protein with a molecular mass of 26±0.5 kDa per monomer. The purified native SOD showed very acidic pI of ca. 3.8. The enzyme was stable at pH 5.0–11.0, but quite unstable below pH 5.0. SOD was thermostable up to 40°C, but a linear reduction in activity above 50°C. Inhibition of the purified SOD activity by -naphthoquinone-4-sulfonic acid, -diazobenzene sulfonic acid, and iodine suggests that lysine, histidine, and tyrosine residues are important for the enzyme activity. The N-terminal peptide sequence of D. radiophilus Mn/FeSOD (MAFELPQLPYAYDALEPHIDA(>D) is strikingly similar to those of D. radiodurans MnSOD and Aerobacter aerogenes FeSOD.Communicated by G. Antranikian     

Superoxide dismutase (SOD) in boar spermatozoa: Purification,biochemical properties and changes in activity during semen storage (16 °C) in different extenders

The antioxidant system in semen is composed of enzymes, low-molecular weight antioxidants and seminal plasma proteins. Loss of enzymatic activity of superoxide dismutase (SOD) during semen preservation may cause insufficient antioxidant defense of boar spermatozoa. The aim of this study was to isolate and characterize SOD molecular forms from spermatozoa and to describe changes in SOD activity in boar sperm during preservation at 16 °C. Sperm extracts were prepared from fresh or diluted semen and used for SOD purification or activity measurement. Ion-exchange chromatography and gel filtration was used to purify SOD molecular forms. BTS, Dilu Cell, M III and Vitasem were used as diluents for 5-day storage of semen at +16 °C. The molecular form of SOD released from spermatozoa after cold shock and homogenization had a molecular weight of approximately 67 kDa. The activity of the SOD form was the highest at pH 10 within the temperature range between 20 and 45 °C. The enzymatic activity of form released after cold shock was inhibited by H₂O₂ and diethyldithiocarbamate (DDC; by 65 and 40%, respectively). The SOD form released by homogenization was inhibited by H₂O₂ and DDC (40%). The molecular form released after urea treatment was a 30 kDa protein with maximum activity at 20 °C and pH 10. Enzymatic activity of this form was inhibited by H₂O₂ by 35%, DDC by 80% and 2-mercaptoethanol by 15%. The antigenic determinants of SOD isolated from boar seminal plasma and spermatozoa were similar to each other. Susceptibility of spermatozoa to cold shock increased during storage, but the differences between extenders were statistically non-significant.     

Expression,purification and functional characterization of a recombinant 2,3-dihydroxybiphenyl-1,2-dioxygenase from <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis>

A 2,3-dihydroxybiphenyl (2,3-DHBP) dioxygenase gene from a Rhodococcus sp. strain, named RrbphCI and involved in the degradation of polychlorinated biphenyls (PCBs), was synthesized. RrbphCI was expressed in Escherichia coli and its encoded enzyme was purified. SDS–PAGE analysis indicated that the size of the protein encoded by RrbphCI was about 32 kDa. The activity of the 2,3-DHBP dioxygenase was 82.8 U/mg when the substrate was 2,3-DHBP, with optimum pH 8.0 at 30°C, and optimum temperature was 40°C at pH 8.0. The RrbphCI gene was transformed into Pseudomonas putida strain EG11, to determine the ability of the enzyme to degrade 2,3-DHBP. The wild type EG11 degraded 61.86% of supplied 2,3-DHBP and the transformed EG11 (hosting the RrbphCI gene) utilized 52.68% after 2 min of treatment at 30°C. The overexpressed and purified enzyme was able to degrade 2,3-DHBP. The 2,3-DHBP dioxygenase is a key enzyme in the PCB degradation pathway. RrbphCI and its encoded 2,3-DHBP dioxygenase may have transgenic applications in bioremediation of PCBs.     

Characterization of a thermostable lipase showing loss of secondary structure at ambient temperature

A gene encoding extracellular lipase was cloned and characterized from metagenomic DNA extracted from hot spring soil. The recombinant gene was expressed in E. coli and expressed protein was purified to homogeneity using hydrophobic interactions chromatography. The mature polypeptide consists of 388 amino acids with apparent molecular weight of 43 kDa. The enzyme displayed maximum activity at 50°C and pH 9.0. It showed thermal stability up to 40°C without any loss of enzyme activity. Nearly 80% enzyme activity was retained at 50°C even after incubation for 75 min. However above 50°C the enzyme displayed thermal instability. The half life of the enzyme was determined to be 5 min at 60°C. Interestingly the CD spectroscopic study carried out in the temperature range of 25–95°C revealed distortion in solution structure above 35°C. However the intrinsic tryptophan fluorescence spectroscopic study revealed that even with the loss of secondary structure at 35°C and above the tertiary structure was retained. With p-nitrophenyl laurate as a substrate, the enzyme exhibited a K _m , V _max and K _cat of 0.73 ± 0.18 μM, 239 ± 16 μmol/ml/min and 569 s⁻¹ respectively. Enzyme activity was strongly inhibited by CuCl₂, HgCl₂ and DEPC but not by PMSF, eserine and SDS. The protein retained significant activity (~70%) with Triton X-100. The enzyme displayed 100% activity in presence of 30% n-Hexane and acetone.     

Purification and Characterization of a Cold-active Iron Superoxide Dismutase from a Psychrophilic Bacterium, Marinomonas sp. NJ522

Marinomonas sp. NJ522, isolated from Antarctic sea ice, produces a cold-active iron superoxide dismutase (SOD; EC 1.15.1.1). The purified SOD was dimeric and had an approx. Mr of 48 kDa. Highest activity was detected from pH 8 to 10 and at 40 °C (assayed over 10 min). Activity at 0 °C was nearly 35% of the maximum activity. Received 25 August 2005; Revisions requested 30 August 2005 and 26 September 2005; Revisions received 12 September 2005 and 25 October 2005; Accepted 1 November 2005     

Molecular and biochemical characterization of an extracellular serine-protease from <Emphasis Type="Italic">Vibrio metschnikovii</Emphasis> J1

A protease-producing bacterium was isolated from an alkaline wastewater of the soap industry and identified as Vibrio metschnikovii J1 on the basis of the 16S rRNA gene sequencing and biochemical properties. The strain was found to over-produce proteases when it was grown at 30°C in media containing casein as carbon source (14,000 U ml⁻¹). J1 enzyme, the major protease produced by V. metschnikovii J1, was purified by a three-step procedure, with a 2.1-fold increase in specific activity and 33.3% recovery. The molecular weight of the purified protease was estimated to be 30 kDa by SDS-PAGE and gel filtration. The N-terminal amino acid sequence of the first 20 amino acids of the purified J1 protease was AQQTPYGIRMVQADQLSDVY. The enzyme was highly active over a wide range of pH from 9.0 to 12.0, with an optimum at pH 11.0. The optimum temperature for the purified enzyme was 60°C. The activity of the enzyme was totally lost in the presence of PMSF, suggesting that the purified enzyme is a serine protease. The kinetic constants K _m and K _cat of the purified enzyme using N-succinyl-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide were 0.158 mM and 1.14 × 10⁵ min⁻¹, respectively. The catalytic efficiency (K _cat /K _m) was 7.23 × 10⁸ min⁻¹ M⁻¹. The enzyme showed extreme stability toward non-ionic surfactants and oxidizing agents. In addition, it showed high stability and compatibility with some commercial liquid and solid detergents. The aprJ1 gene, which encodes the alkaline protease from V. metschnikovii J1, was isolated, and its DNA sequence was determined. The deduced amino acid sequence of the preproenzyme differs from that of V. metschnikovii RH530 detergent-stable protease by 12 amino acids, 7 located in the propeptide and 5 in the mature enzyme.     

Molecular characterization of an endochitinase from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> subsp. konkukian

A chitinase gene from Bacillus thuringiensis serovar konkukian S4 was cloned, sequenced, and heterologously expressed in Escherichia coli M15. Recombinant enzyme (Chi74) was purified by Ni-NTA affinity column chromatography. The chi74 gene contains an open reading frame (ORF), with a capacity to encode an endochitinase with a deduced molecular weight 74 kDa and predicted isoelectric point of 5.67. Comparison of Chi74 with other chitinases has shown that it contains a modular structure with an N-terminal family 18 catalytic-domain, a Fibronectin-III like domain and a C-terminal carbohydrate binding module (CBM-II). Turn over rate (K _cat ) of the enzyme was determined using colloidal chitin (28.3 ± 0.70 S⁻¹) as substrate. The Purified enzyme was active at a broad range of pH (pH 3.5–7.5) and temperature (20–70°C) with a peak activity at pH 5.5 and 55°C. However, the enzyme was found to be stable up to 30°C for longer incubation periods. Moreover, the purified enzyme was shown to inhibit fungal spore germination and hyphal growth in the pathogenic fungi Fusarium oxysporum and Aspergillus niger. These studies will lead us to develop broad spectrum resistance in the crop plants via co-expression of the chitinases and the insecticidal proteins.     

Cloning, purification and characterisation of a recombinant purine nucleoside phosphorylase from Bacillus halodurans Alk36

A purine nucleoside phosphorylase from the alkaliphile Bacillus halodurans Alk36 was cloned and overexpressed in Escherichia coli. The enzyme was purified fivefold by membrane filtration and ion exchange. The purified enzyme had a V _max of 2.03 × 10⁻⁹ s ⁻¹ and a K _m of 206 μM on guanosine. The optimal pH range was between 5.7 and 8.4 with a maximum at pH 7.0. The optimal temperature for activity was 70°C and the enzyme had a half life at 60°C of 20.8 h.