Purification, characterization, and cloning of trimethylamine dehydrogenase fromMethylophaga sp. strain SK1

Trimethylamine dehydrogenase (TMADH, EC 1.5.99.7), an iron-sulfur flavoprotein that catalyzes the oxidative demethylation of trimethylamine to form dimethylamine and formaldehyde, was purified fromMethylophaga sp. strain SK1. The active TMADH was purified 12.3-fold through three purification steps. The optimal pH and temperature for enzyme activity was determined to be 8.5 and 55°C, respectively. TheV _max andK _m values were 7.9 nmol/min/mg protein and 1.5 mM. A genomic DNA of 2,983 bp fromMethylophaga sp. strain SK1 was cloned, and DNA sequencing revealed the open reading frame (ORF) of the gene coding for TMADH. The ORF contained 728 amino acids with extensive identity (82%) to that ofMethylophilus methylotrophus W₃A₁.     

Photoaffinity labelling with the cytokinin agonist azido-CPPU of a 34 kDa peptide of the intracellular pathogenesis-related protein family in the moss Physcomitrella patens

As in higher plants, the development of the moss Physcomitrella patens is regulated by environmental signals and phytohormones. At the protonema level transition from chloronema to caulonema cells is under auxin control. The formation on second sub-apical caulonema cells of buds that will give rise to the leafy gametophore requires cytokinins. Using [³H]azidoCPPU (1-(2-azido-6-chloropyrid-4-yl)-3-(4-[³H])phenylurea), a photoactivatable cytokinin agonist, we have specifically photolabelled a soluble 34 kDa protein of P. patens. Urea derivatives were very efficient competitors of photolabelling while purine-type cytokinins were poor competitors. The protein UBP34 was purified by affinity chromatography and the sequences of six internal peptides obtained. A cDNA encoding UBP34 was cloned by screening a P. patens protonema cDNA library with a probe amplified by PCR using degenerate primers designed from the peptide sequences. The UBP34 amino acid sequence shows an average sequence identity of 42% with both intracellular PR proteins and the BetV1-related family of plant allergens. Recombinant UBP34 expressed in Escherichia coli was confirmed to bind azidoCPPU.     

Expression Optimization and Biochemical Characterization of a Recombinant γ-Glutamyltranspeptidase from Escherichia coli Novablue

A truncated Escherichia coli Novablue γ-glutamyltranspeptidase (EcGGT) gene lacking the first 48-bp coding sequence for part of the signal sequence was amplified by polymerase chain reaction and cloned into expression vector pQE-30 to generate pQE-EcGGT. The maximum production of His₆-tagged enzyme by E. coli M15 (pQE-EcGGT) was achieved with 0.1 mM IPTG induction for 12 h at 20 °C. The overexpressed enzyme was purified to homogeneity by nickel-chelate chromatography to a specific transpeptidase activity of 4.25 U/mg protein and a final yield of 83%. The molecular masses of the subunits of the purified enzyme were estimated to be 41 and 21 kDa respectively by SDS-PAGE, indicating EcGGT still undergoes the post-translational cleavage even in the truncation of signal sequence. The optimum temperature and pH for the recombinant enzyme were 40 °C and 9, respectively. The apparent K _m and V _max values for γ-glutamyl-p-nitroanilide as γ-glutamyl donor in the transpeptidation reaction were 37.9 μM and 53.7 × 10⁻³ mM min⁻¹, respectively. The synthesis of L-theanine was performed in a reaction mixture containing 10 mM L-Gln, 40 mM ethylamine, and 1.04 U His₆-tagged EcGGT/ml, pH 10, and a conversion rate of 45% was obtained.     

Expression of truncated phosphoproteins of Nipah virus and Hendra virus in <Emphasis Type="Italic">Escherichia coli</Emphasis> for the differentiation of henipavirus infections

The genus Henipavirus in the family Paramyxoviridae compromises two newly identified dangerous pathogens, Nipah virus and Hendra virus. Phosphoprotein of the two viruses is one of the major immunodominant antigens and the most divergent protein in the viral genomes. We have now expressed two pairs of truncated phosphoproteins of the two viruses in Escherichia coli in a soluble form using a vector tailored from pET32a. The truncated recombinant phosphoproteins were purified with His-Tag affinity chromatography and their antigenicity was determined by western blotting and ELISA. The longer pair of truncated recombinant phosphoproteins, covering amino acid residues 4–550, was more antigenic than the shorter one and of potential utility in the serological differentiation of henipavirus infections.     

A highly thermostable trehalase from the thermophilic bacterium <Emphasis Type="Italic">Rhodothermus marinus</Emphasis>

Trehalases play a central role in the metabolism of trehalose and can be found in a wide variety of organisms. A periplasmic trehalase (α,α-trehalose glucohydrolase, EC 3.2.1.28) from the thermophilic bacterium Rhodothermus marinus was purified and the respective encoding gene was identified, cloned and overexpressed in Escherichia coli. The recombinant trehalase is a monomeric protein with a molecular mass of 59 kDa. Maximum activity was observed at 88°C and pH 6.5. The recombinant trehalase exhibited a K _m of 0.16 mM and a V _max of 81 μmol of trehalose (min)⁻¹ (mg of protein)⁻¹ at the optimal temperature for growth of R. marinus (65°C) and pH 6.5. The enzyme was highly specific for trehalose and was inhibited by glucose with a K _i of 7 mM. This is the most thermostable trehalase ever characterized. Moreover, this is the first report on the identification and characterization of a trehalase from a thermophilic bacterium.     

Characterization of Active Lentinula edodes Glucoamylase Expressed and Secreted by Saccharomyces cerevisiae

The gene encoding Lentinula edodes glucoamylase (GLA) was cloned into Saccharomyces cerevisiae, expressed constitutively and secreted in an active form. The enzyme was purified to homogeneity by (NH₄)₂SO₄ fractionation, anion exchange and affinity chromatography. The protein had a correct N-terminal sequence of WAQSSVIDAYVAS, indicating that the signal peptide was efficiently cleaved. The recombinant enzyme was glycosylated with a 2.4% carbohydrate content. It had a pH optimum of 4.6 and a pH 3.4–6.4 stability range. The temperature optimum was 50°C with stability ≤50°C. The enzyme showed considerable loss of activity when incubated with glucose (44%), glucosamine (68%), galactose (22%), and xylose (64%). The addition of Mn⁺⁺ activated the enzyme by 45%, while Li⁺, Zn⁺⁺, Mg⁺⁺, Cu⁺, Ca⁺⁺, and EDTA had no effect. The enzyme hydrolyzed amylopectin at rates 1.5 and 8.0 times that of soluble starch and amylose, respectively. Soluble starch was hydrolyzed 16 and 29 times faster than wheat and corn starch granules, respectively, with the hydrolysis of starch granules using 10× the amount of GLA. Apparent K_m and V_max for soluble starch were estimated to be 3.0 mg/ml and 0.13 mg/ml/min (40°C, pH 5.3), with an apparent k_cat of 2.9×10⁵ min⁻¹.     

粘虫颗粒体病毒增效蛋白在苏云金芽胞杆菌中的表达及增效活性

【目的】在苏云金芽胞杆菌(Bacillus thuringiensis, Bt)中表达截短后的转宿主粘虫颗粒体病毒(Pseudaletia unipuncta granulovirus-Ps, PuGV-Ps)增效蛋白,为构建增效Bt工程菌提供理论基础。【方法】通过对截短后增效蛋白的密码子进行优化,构建增效蛋白及其融合蛋白表达载体,分析不同启动子指导下增效蛋白表达量的变化,明确增效蛋白对Bt的增效活性。【结果】本研究构建了表达载体pHTP_cry1AcCoEn81、 pHTRHCoEn81和pHTNCCoEn81, SDS-PAGE结果显示pHTP_cry1AcCoEn81和pHTNCCoEn81分别可以产生81 kDa和134 kDa的重组蛋白。启动子Pcry1Ac和P_cry8E指导下的增效蛋白表达量和重组增效蛋白产量均无显著性差异。生物测定结果表明,重组增效蛋白可以显著增加Bt对小菜蛾的杀虫活性。【结论】研究结果表明,密码子优化的PuGV-Ps增效蛋白可以在Bt中表达并具有显著增效活性,为高效苏云金芽胞杆菌工程菌的构建及...     

Enhanced tolerance against freezing stress in<Emphasis Type="Italic">Escherichia coli</Emphasis> cells expressing an algal cyclophilin gene

Members of the cyclophilin (Cyp) family are known to function as co-chaperones, interacting with chaperones such as heat shock protein 90, and perform important roles in protein folding under high temperature stress. In addition, they have been isolated from a wide range of organisms. However, there have been no reports on the functions of algal Cyps under other stress conditions. To study the functions of the cDNAGjCyp-1 isolated from the red alga (Griffithsia japonica), a recombinant GjCyp-1 containing a hexahistidine tag at the amino-terminus was constructed and expressed inEscherichia coli. Most of the gene product expressed inE. coli was organized as aggregate insoluble particles known as inclusion bodies. Thus, the optimal time, temperature, and concentration ofl(+)-arabinose for expressing the soluble and nonaggregated form of GjCyp-1 inE. coli were examined. The results indicate that the induction of Cyp, at 0.2%l(+)-arabinose for 2 h at 25°C, had a marked effect on the yield of the soluble and active form of the co-chaperone as PPlase. An expressed fusion protein, H₆GjCyp-1, maintained the stability ofE. coli proteins up to-75°C. In a functional bioassay of the recombinant H₆GjCyp-1, the viability ofE. coli cells overexpressing H₆GjCyp-1 was compared to that of cells not expressing H₆GjCyp-1 at −75°C. For all the cycles of a freeze/thaw treatment, a significant increase in viability was observed in theE. coli cells overexpressing H₆GjCyp-1. The results of the GjCyp-1 bioassays, as well asin vitro studies, strongly suggest that the algal Cyp confers freeze tolerance toE. coli.     

Purification and characterization of pectin lyase secreted by <Emphasis Type="Italic">Penicillium citrinum</Emphasis>

The importance of various parameters such as sugarcane juice concentration, pH of the medium, and effects of different solid supports for maximum secretion of pectin lyase from Penicillium citrinum MTCC 8897 has been studied. The enzyme was purified to homogeneity by Sephadex G-100 and DEAE-cellulose chromatography. The molecular mass determined by SDS-PAGE was 31 kDa. The K _m and k _cat values were found to be 1 mg/ml and 76 sec⁻¹, respectively. The optimum pH of the purified pectin lyase was 9.0, though it retains activity in the pH 9.0–12.0 range when exposed for 24 h. The optimum temperature was 50°C, and the pectin lyase was found to be completely stable up to 40°C when exposed for 1 h. The purified pectin lyase was found efficient in retting of Linum usitatissimum, Cannabis sativa, and Crotalaria juncea. Published in Russian in Biokhimiya, 2009, Vol. 74, No. 7, pp. 985–992.     

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.     

Production and harvesting of ionically wall-bound extensin from living cell suspension cultures

Cell-suspension cultures ofSpinacia andRosa accumulated a cell wall protein, extensin, in a form that was amenable to leaching from the surface of the living cells by a brief treatment with non-toxic salts. Cultures ofLycopersicon, Capsicum, Acer andFestuca did not accumulate this class of extensin. InSpinacia andRosa, optimum yields of leachable extensin were achieved from young cultures, in media at relatively low pH, by leaching with 0.1 M CaCl₂. Older cultures, pH values >6.5, and LaCl₃ or higher concentrations of CaCl₂ were less effective.Abbreviation TCA trichloroacetic acid     

Purification and biochemical characterization of a native invertase from the hydrogen-producing Thermotoga neapolitana (DSM 4359)

This is the first report describing the purification and enzymatic properties of a native invertase (β-D-fructosidase) in Thermotogales. The invertase of the hydrogen-producing thermophilic bacterium Thermotoga neapolitana DSM 4359 (hereby named Tni) was a monomer of about 47 kDa having an amino acid sequence quite different from other invertases studied up to now. Its properties and substrates specificity let us classify this protein as a solute-binding protein with invertase activity. Tni was specific for the fructose moiety and the enzyme released fructose from sucrose and raffinose and the fructose polymer inulin was hydrolyzed in an endo-type fashion. Tni had an optimum temperature of 85°C at pH 6.0. At temperatures of 80–85°C, the enzyme retained at least 50% of its initial activity during a 6 h preincubation period. Tni had a K _m and k _cat /K _m values (at 85°C and pH 6.0) of about 14 mM and 5.2 × 10⁸ M⁻¹ s⁻¹, respectively. Dedicated to the memory of Prof. R. A. Nicolaus, founder of the Institute (1968).     

The multiple soluble malate dehydrogenase of Hoplias malabaricus(Erythrinidae, Characiformes)

To explain the six-banded pattern obtained upon electrophoresis of the soluble form of malate dehydrogenase (sMDH, EC 1.1.1.37) from the characiform Hoplias malabaricus, a recent locus duplication of its A isoform (sMDH-A*), in addition to its sMDH-B* isoform, was proposed. Klebe’s serial dilutions carried out using skeletal muscle, heart and liver extracts showed that the A1 and A2 subunits have the same visual end-points, indicating that these A-duplicated genes have a nondivergent pattern. Since there is no evidence of polyploidy in the Erythrinidae family, the MDH-A* loci have probably evolved from regional gene duplication. While these sMDH-A* loci encode nondivergent thermostable isoforms, the sMDH-B* encodes a thermolabile one. Thermostable sMDHs differ from the thermolabile sMDHs in that they have a higher K_m of oxaloacetate. Liver, muscle and heart unfractionated sMDH levels at three different temperature and two pH regimens were analysed and the results showed that, in the adaptative temperature range of Hoplias, the variation in K_m under conditions of constant pH (imidazole buffer) was less (approximately threefold) than that measured in the presence of temperature-dependent pH imidazole buffer (sevenfold). Estimation of the ratio of both isoforms in these tissues by Klebe’s method showed that, in unfractionated liver – where K_m values were the highest and the minimum K_m was obtained at 30^C (both for temperature-dependent pH and constant-pH imidazole buffer) – the duplicate A (thermostable, A1 and A2) and B (thermolabile) subunits were detected in a ratio of 2:1. On the other hand, in muscle extracts – in which the lowest K_m values were measured, with the minimum K_m at 10–20^C (temperature-dependent pH and constant-pH imidazole buffer, respectively) – a ratio of two thermolabile to one thermostable subunits was observed.     

Screening of chitin deacetylase from Mucoralean strains (Zygomycetes) and its relationship to cell growth rate

Chitin deacetylase (CDA) is an enzyme that catalyzes the hydrolysis of acetamine groups of N-acetyl-d-glucosamine in chitin, converting it to chitosan in fungal cell walls. In the present study, the activity in batch culture of CDA from six Mucoralean strains, two of them wild type, isolated from dung of herbivores of Northeast Brazil, was screened. Among the strains tested, Cunninghamella bertholletiae IFM 46114 showed a high intracellular enzyme activity of 0.075 U/mg protein after 5 days of culture, and a wild-type strain of Mucor circinelloides showed a high intracellular enzyme activity of 0.060 U/mg protein, with only 2 days of culture, using N-acetylchitopentaose as substrate. This enzyme showed optimal activity at pH 4.5 in 25 mM glutamate-sodium buffer at 50°C, and was stable over 1 h preincubation at the same temperature. The kinetic parameters of CDA did not follow Michaelis-Menten kinetics, but rather Hill affinity distribution, showing probable allosteric behavior. The apparent K_HILL and V_max of CDA were 288±34 nmol/l and 0.08±0.01 U mg protein^–1 min^–1, respectively, using N-acetylchitopentaose as substrate at pH 4.5 at 50°C.     

Purification of cytochrome a 1 c 1 from Nitrobacter agilis and characterization of nitrite oxidation system of the bacterium

Cytochrome a ₁ c ₁ was highly purified from Nitrobacter agilis. The cytochrome contained heme a and heme c of equimolar amount, and its reduced form showed absorption peaks at 587, 550, 521, 434 and 416 nm. Molecular weight per heme a of the cytochrome was estimated to be approx. 100,000–130,000 from the amino acid composition. A similar value was obtained by determining the protein content per heme a. The cytochrome molecule was composed of three subunits with molecular weights of 55,000, 29,000 and 19,000, respectively. The 29 kd subunit had heme c.Hemes a and c of cytochrome a ₁ c ₁ were reduced on addition of nitrite, and the reduced cytochrome was hardly autoxidizable. Exogenously added horse heart cytochrome c was reduced by nitrite in the presence of cytochrome a ₁ c ₁; K _m values of cytochrome a ₁ c ₁ for nitrite and N. agilis cytochrome c were 0.5 mM and and 6 M, respectively. V _max was 1.7 mol ferricytochrome c reduced/min·mol of cytochrome a ₁ c ₁ The pH optimum of the reaction was about 8. The nitrite-cytochrome c reduction catalyzed by cytochrome a ₁ c ₁ was 61% and 88% inhibited by 44M azide and cyanide, respectively. In the presence of 4.4 mM nitrate, the reaction was 89% inhibited. The nitrite-cytochrome c reduction catalysed by cytochrome a ₁ c ₁ was 2.5-fold stimulated by 4.5 mM manganous chloride. An activating factor which was present in the crude enzyme preparation stimulated the reaction by 2.8-fold, and presence of both the factor and manganous ion activated the reaction by 7-fold.Cytochrome a ₁ c ₁ showed also cytochrome c-nitrate reductase activity. The pH optimum of the reaction was about 6. The nitrate reductase activity was also stimulated by manganous ions and the activating factor.     

Molecular cloning,sequence analysis,expression and characterization of the endochitinase gene from <Emphasis Type="Italic">Trichoderma</Emphasis> sp. in <Emphasis Type="Italic">Escherichia coli</Emphasis> BL21

The endochitinase DNA and cDNA from Trichoderma sp. were cloned, sequenced and expressed. The cloned DNA and cDNA sequences were 1,476 and 1,275 bp in length, respectively. There were three introns in DNA sequence in comparison with the cDNA sequence. The endochitinase protein contained three regions: the signal peptide, the prepro-region and the mature protein region. The gene fragment encoding the mature endochitinase was ligated into the expression vector pET-28a+, yielding pET-1. The plasmid pET-1 was transformed into the Escherichia coli BL21 (DE3). The clone bearing pET-1 was picked and cultured at 30°C for the expression of endochitinase. SDS-PAGE analysis showed that the endochitinase was expressed in the periplasmic space and the purified protein showed a single band. The activity of 70.2 U/mg was obtained from the cellular extract of the recombinant strain. The activity of endochitinase was 2.5-fold higher at 24 h than at 16 h in the periplasmic space. The optimal pH and temperature of the recombinant endochitinase were determined to be 7.0 and 35°C, respectively. It was relatively stable within the pH range of 5–8. Significant activity stimulation by 1 mM Mg²⁺ and 5 mM Fe²⁺ and inhibition by 5 mM Co²⁺ and 5 mM Hg²⁺ were observed. The kinetic constants K_m, V_max and K_cat for the hydrolysis of the colloidal chitin were 1.5 mM, 1.37 μmol min⁻¹ and 6.23 min⁻¹, respectively.     

Changes in the activity of the multifunctional β-glycosyl hydrolase (Cel44C-Man26A) from Paenibacillus polymyxa by removal of the C-terminal region to minimum size

Paenibacillus polymyxa GS01 secretes Cel44C-Man26A as a multifunctional enzyme with cellulase, xylanase, lichenase, and mannanase activities. Cel44C-Man26A consists of 1,352 amino acids in which present a catalytic domain (CD) of the glycosyl hydrolase family 44 (GH44), fibronectin domain type 3 (Fn3), catalytic domain of glycosyl hydrolase family 26 (GH26), and a cellulose-binding module type 3 (CBM3). A truncated Cel44C-Man26A protein, consisting of 549 amino acid residues, reacted as a multifunctional mature enzyme despite the absence of the 10 amino acids containing GH44, Fn3, GH26, and CBM3. However, the multifunctional activity was not found in the mature Cel44C-Man26A protein truncated to less than 548 amino acids. The truncated Cel44C-Man26A proteins showed the optimum pH for the lichenase activity was pH 7.0, pH 6.0 for the xylanase and mannanase, and pH 5.0 for the cellulase. The truncated Cel44C-Man26A proteins exhibited enzymatic activity 40–120% higher than the full-length Cel44C.     

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.     

Enzymatic characterization of the Plasmodium vivax chitinase, a potential malaria transmission-blocking target

The chitinase (EC 3.2.1.14) of the human malaria parasite Plasmodium falciparum, PfCHT1, has been validated as a malaria transmission-blocking vaccine (TBV). The present study aimed to delineate functional characteristics of the P. vivax chitinase PvCHT1, whose primary structure differs from that of PfCHT1 by having proenzyme and chitin-binding domains. The recombinant protein rPvCHT1 expressed with a wheat germ cell-free system hydrolyzed 4-methylumbelliferone (4MU) derivatives of chitin oligosaccharides (β-1,4-poly-N-acetyl glucosamine (GlcNAc)). An anti-rPvCHT1 polyclonal antiserum reacted with in vitro-obtained P. vivax ookinetes in anterior cytoplasm, showing uneven patchy distribution. Enzymatic activity of rPvCHT1 shared the exclusive endochitinase property with parallelly expressed rPfCHT1 as demonstrated by a marked substrate preference for 4MU-GlcNAc₃ compared to shorter GlcNAc substrates. While rPvCHT1 was found to be sensitive to the general family-18 chitinase inhibitor, allosamidin, its pH (maximal in neutral environment) and temperature (max. at ~ 25 °C) activity profiles and sensitivity to allosamidin (IC₅₀ = 6 µM) were different from rPfCHT1. The results in this first report of functional rPvCHT1 synthesis indicate that the P. vivax chitinase is enzymatically close to long form Plasmodium chitinases represented by P. gallinaceum PgCHT1.