重组丙酮酸磷酸双激酶与荧光素酶偶联催化ATP-AMP循环反应

丙酮酸磷酸双激酶（pyruvate phosphate dikinase, PPDK; EC 2.7.9.1）能够可逆催化磷酸烯醇式丙酮酸（phosphoenolpyruvate, PEP）、单磷酸腺苷（adenosine monophosphate, AMP）和焦磷酸盐（pyrophosphate, PPi）生成三磷酸腺苷（adenosine triphosphate, ATP）、无机磷酸盐（orthophosphate, Pi）和丙酮酸（pyruvate）.以热玫瑰小双孢菌基因组DNA为模板,PCR扩增得到了编码PPDK的基因,将此基因片段插入表达载体pET24a (+),在大肠杆菌中表达C端融合His-Tag的重组PPDK.与我们先前表达的N端融合His-Tag的PPDK相比,酶的活性提高了20倍,提示该酶的N端对活性十分重要.重组PPDK单体分子量为98 kD.经过镍亲和层析和超滤后,重组PPDK基本达到电泳纯.重组PPDK与荧光素酶偶联能够形成1个ATP-AMP循环反应,在该循环反应中,荧光素酶催化ATP生成的AMP和PPi能够被PPDK重新转化成ATP,产生一个持续稳定的信号.     

Homomultimeric protease in the hyperthermophilic bacterium Thermotoga maritima has structural and amino acid sequence homology to bacteriocins in mesophilic bacteria

A novel homomultimeric protease (>669 kDa), based on 31 kDa subunits, was purified from cell extracts of the hyperthermophilic bacterium Thermotoga maritima. This protease exhibits activity toward chymotrypsin and trypsin substrates, optimally at 90°C and pH 7.1, and has a half-life of 36 min at 95°C. Transmission electron microscopy established that the protease consists of a large globular assembly which appears circular from the front view. The function of this protease in T. maritima remains unclear, although putative homologs include a 29 kDa antigen from Mycobacterium tuberculosis and a 31 kDa monomer of a high molecular weight bacteriocin produced by Brevibacterium linens [Valdes-Stauber, N. and Scherer, S. (1996) Appl. Environ. Microbiol. 62, 1283–1286]. The relationship of these mesophilic proteins to the T. maritima protease suggests that their antibacterial activity may involve elements of proteolysis, and raises the prospect for anti-microbial ecological strategies in hyperthermophilic niches.     

Permeability and reactivity of Thermotoga maritima in latex bimodal blend coatings at 80°C: a model high temperature biocatalytic coating

Thermostable polymers cast as thin, porous coatings or membranes may be useful for concentrating and stabilizing hyperthermophilic microorganisms as biocatalysts. Hydrogel matricies can be unstable above 65°C. Therefore a 55-m thick, two layer (cell coat + polymer top coat) bimodal, adhesive latex coating of partially coalesced polystyrene particles was investigated at 80°C using Thermotoga maritima as a model hyperthermophile. Coating permeability (pore structure) was critical for maintaining T. maritima viability. The permeability of bimodal coatings generated from 0.8 v/v of a suspension of non-film-forming 800 nm polystyrene particles with high glass transition temperature (T_g= 94°C, 26.9% total solids) blended with 0.2 v/v of a suspension of film-forming 158 nm polyacrylate/styrene particles (T_g –5°C, 40.9% total solids) with 0.3 g sucrose/g latex was measured in a KNO₃ diffusion cell. Diffusivity ratio remained above 0.04 (D_eff/D) when incubated at 80°C in artificial seawater (ASW) for 5 days. KNO₃ permeability was corroborated by cryogenic-SEM images of the pore structure. In contrast, the permeability of a mono-dispersed acrylate/vinyl acetate latex Rovace SF091 (T_g~10°C) rapidly decreased and became impermeable after 2 days incubation in ASW at 80°C. Thermotoga maritima were entrapped in these coatings at a cell density of 49 g cell wet weight/liter of coating volume, 25-fold higher than the density in liquid culture. Viable T. maritima were released from single-layer coatings at 80°C but accurate measurement of the percentage of viable entrapped cells by plate counting was not successful. Metabolic activity could be measured in bilayer coatings by utilization of glucose and maltose, which was identical for latex-entrapped and suspended cells. Starch was hydrolyzed for 200 h by latex-entrapped cells due to the slow diffusion of starch through the polymer top coat compared to only 24 h by suspended T. maritima. The observed reactivity and stability of these coatings was surprising since cryo-SEM images suggested that the smaller low T_g polyacrylate/styrene particles preferentially bound to the T. maritima toga-sheath during coat formation. This model system may be useful for concentrating, entrapment and stabilization of metabolically active hyperthermophiles at 80°C.     

Production and stabilization of amylase preparations from rice bran solid medium

A low-cost amylase preparation of dried fermented bran was developed from rice bran solid cultures of Aspergillus oryzae supplemented with soya bean flour (SBF) and cassava starch (3:1) and dried at 50 °C for 4 h. Storage stability of preparations at 4 °C or 30 °C was significantly enhanced (P 0.05) by adding SBF or partially hydrolyzed starch (PHS). While amylase preparations without stabilizer retained 59 and 48% of their activity after 12 weeks storage at 4 and 30 °C respectively, the same preparations fortified with SBF (5% w/v) retained 95 and 94% stability respectively, during the same period. PHS at 5% (w/v) also gave a maximum stability of 94 and 91.8% at 4 and 30 °C, respectively. The unstabilized preparation retained only 42% of its activity compared to the stabilized forms, which retained 82–90% activity after 15 min incubation at 100 °C.     

Purification and properties of urease fromSporobolomyces roseus

Urease (EC 3.5.1.5) catalyses the hydrolysis of urea to ammonia and carbon dioxide. The enzyme fromSporobolomyces roseus was enriched 780-fold and purified to apparent homogeneity using heat treatment, ion exchange chromatography on Q-Sepharose fast flow, hydrophobic interaction chromatography on Phenyl-Sepharose, size exclusion chromatography on Sephacryl S 300 HR, and ion exchange chromatography on MonoQ. Analysis of the purified enzyme by SDS-PAGE demonstrated the presence of subunits with a molecular weight of 90 (± 4) kDa. The M _r of the native enzyme was estimated by size exclusion chromatography to be 340 (± 30) kDa, suggesting a tetrameric structure different from other ureases isolated so far from both prokaryotes and eukaryotes. The enzyme was heat-stable, showing no loss of activity after incubation at 70 °C for 15 min. The highest urease activities were observed after growth on media containing urea as the sole source of nitrogen.     

Characterization of proteases produced by newly isolated and identified proteolytic microorganisms from fermented fish (Budu)

Eight different strains ofBacillus were isolated from fermented fish (Budu) and their proteolytic enzyme activities were determined after 18 h cultivation at room temperature (35° C). Four isolates possessed high protease activities. Optimum pH for these enzymes was between 7.0 and 8.0 and the optimal temperature was 55° C. The proteases retained 40% of their original activity after 20 min at 55° C but lost all activity at 65° C. Three of the four isolates were identified asBacillus subtilis, the fourth asBacillus licheniformis.     

Pyruvate phosphate dikinase and pyrophosphate metabolism in the glycosome of Trypanosoma cruzi epimastigotes

Pyruvate phosphate dikinase (PPDK) was recently reported in trypanosomatids, but its metabolic function is not yet known. The present work deals with the cellular localization and the function of the Trypanosoma cruzi enzyme. First, we show by digitonin titration and cell fractionation that the enzyme was essentially present in the glycosome matrix of the epimastigote form. Second, we address the issue of the direction of the reaction inside the glycosome for one part, our bibliographic survey evidenced a quite exergonic ΔG°′ (at least −5.2 kcal/mol at neutral pH and physiologic ionic strength); for another part, no pyrophosphatase (PPase) could be detected in fractions corresponding to the glycosomes; therefore, glycosomal PPDK likely works in the direction of pyruvate production. Third, we address the issue of the origin of the glycosomal pyrophosphate (PPi): several synthetic pathways known to produce PPi are already considered to be glycosomal. This work also indicates the presence of an NADP⁺-dependent β-oxidation of palmitoyl-CoA in the glycosome. Several pyruvate-consuming activities, in particular alanine dehydrogenase (ADH) and pyruvate carboxylase (PC), were detected in the glycosomal fraction. PPDK appears therefore as a central enzyme in the metabolism of the glycosome of T. cruzi by providing a link between glycolysis, fatty acid oxidation and biosynthetic PPi-producing pathways. Indeed, PPDK seems to replace pyrophosphatase in its classical thermodynamic role of displacing the equilibrium of PPi-producing reactions, as well as in its role of eliminating the toxic PPi.     

Purification and Characterization of Lipase from Wheat (Triticum aestivum L.) Germ

A method of isolation and purification of lipase (EC 3.1.1.3) from the germ of wheat (Triticum aestivumL.) is described. An electrophoretically homogeneous preparation of the enzyme (specific activity, 622.5 × 10^–3 mol/min per mg protein) was obtained after 61-fold purification. The molecular weight of the enzyme, determined by gel chromatography, was 143 ± 2 kDa. The optimal conditions for the enzyme were 37°C and pH 8.0. The homogeneous preparation of the lipase exhibited high thermal stability: over 20% of the original activity was retained after incubation of the preparation at high temperatures (60–90°C) for 1 h at pH 8.0.     

Isolation and Characterization of Genes Encoding Thermoactive and Thermostable Dextranases from Two Thermotolerant Soil Bacteria

Thermotolerant Paenibacillus strain Dex70-1B and unidentified strain Dex70-34 produce thermoactive dextran-degrading enzymes. Plasmid-based genomic DNA libraries constructed from mixed bacterial cultures containing Dex70-1B or Dex70-34 were screened for the ability to confer dextranolytic activity at 70°C onto Escherichia coli. One gene, designated dex1, was isolated from each strain. The Dex70-1B and Dex70-34 dex1 gene sequences were non-identical, and encoded proteins containing 597 (M_r 68.6 kDa) and 600 amino acids (M_r 69.2 kDa), respectively. The Dex1 amino acid sequences were most similar to one another, and formed a new clade among the family 66 glycosyl hydrolase sequences. Expression of the Dex1 proteins in E. coli produced dextranolytic activity that converted ethanol-insoluble blue dextran into an ethanol-soluble form, suggestive of endodextranases (EC 3.2.1.11). Both enzymes were most active at about 60°C and pH 5.5, and retained more than 70% maximal activity after incubation at 57°C for 9.5 h in the absence of substrate.     

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.     

Distinct effects of different low temperatures on the induction of NAD-sorbitol dehydrogenase activity in diapause eggs of the silkworm,Bombyx mori

Summary Diapause eggs of the silkworm, Bombyx mori, exposed to 5°C and 0.5°C from 2 or 30 days after oviposition, were examined for changes in contents of glycogen, sorbitol and glycerol. Cold acclimation did not alter the profile of accumulation of sorbitol from that in eggs kept continuously at 25°C. However, acclimation at 5°C resulted in conversion of sorbitol to glycogen, while acclimation at 0.5°C was not accompanied by the utilization of sorbitol. NAD-sorbitol dehydrogenase (NAD-SDH; EC 1.1.1.14) activity was examined in the cold-acclimated eggs. The activity was induced by acclimation at 5°C but not at 0.5°C. Incubation at 0.5°C suppressed any further increase in the activity that had been induced. Temperature-directed changes in NAD-SDH activity paralleled those in sorbitol content. Hatching of the diapause eggs was monitored after cold acclimation for various periods of time and subsequent transfer to 25°C. Incubation at 0.5°C was less effective than 5°C at breaking diapause. The time required for the eggs to hatch in synchrony after acclimation at 5°C coincided with that required for the induction of NAD-SDH activity. These results show that different effects result from acclimation at 5°C and near 0°C with respect to the control of NAD-SDH activity, that utilization of sorbitol is controlled by NAD-SDH activity, and that induction of this activity is temperature-dependent. Furthermore, induction of NAD-SDH activity is involved in the termination of diapause in B. mori.Abbreviations DH diapause hormone - NAD nicotinamide-adenine-dinucleotide - NAD-SDH NAD-sorbitol-dehydrogenase     

Expression of cold-tolerant pyruvate,orthophosphate dikinase cDNA,and heterotetramer formation in transgenic maize plants

Maize is a typical C₄ plant of the NADP-malic enzyme type, and its high productivity is supported by the C₄ photosynthetic cycle, which concentrates atmospheric CO₂ in the leaves. The plant exhibits superior photosynthetic ability under high light and high temperature, but under cold conditions the photosynthetic rate is significantly reduced. Pyruvate orthophosphate dikinase (PPDK), a key enzyme of the C₄ pathway in maize, loses its activity below about 12 °C by dissociation of the tetramer and it is considered as one possible cause of the reduction in the photosynthetic rate of maize at low temperatures. To improve the cold stability of the enzyme, we introduced a cold-tolerant PPDK cDNA isolated from Flaveria brownii into maize by Agrobacterium-mediated transformation. We obtained higher levels of expression by using a double intron cassette and a chimeric cDNA made from F. bidentis and F. brownii with a maximum content of 1mg/g fresh weight. In leaves of transgenic maize, PPDK molecules produced from the transgene were detected in cold-tolerant homotetramers or in heterotetramers of intermediate cold susceptibility formed with the internal PPDK. Simultaneous introduction of an antisense gene for maize PPDK generated plants in which the ratio of heterolologous and endogenous PPDK was greatly improved. Arrhenius plot analysis of the enzyme extracted from one such plant revealed that the break point was shifted about 3 °C lower than that of the wild type.     

Characterization and wash performance analysis of an SDS-stable alkaline protease from a Bacillus sp.

An alkaline, SDS-stable protease optimally active at pH 11 from a Bacillus sp. RGR-14 was produced in a complex medium containing soybean meal, starch and calcium carbonate. The protease was active over a wide temperature range of 20–80 °C with major activity between 45 and 70 °C. The protease was completely stable for 1 h in 0.1% SDS and retained 70% of its activity in the presence of 0.5% SDS after 1 h of incubation. The enzyme was active in presence of surfactants (ionic and non-ionic) with 29% enhancement in activity in Tween-85 and was also stable in various oxidizing agents with 100 and 60% activity in presence of 1% sodium perborate and 1% H₂O₂, respectively. The enzyme was also compatible with commercial detergents (1% w/v) such as Surf, Ariel, Wheel, Fena and Nirma, retaining more than 70% activity in all the detergents after 1 h. Wash performance analysis of grass and blood stains on cotton fabric showed an increase in reflectance (14 and 25% with grass and blood stains, respectively) after enzyme treatment. However, enzyme in conjunction with detergent proved best, with a maximum reflectance change of 46 and 34% for grass and blood stain removal, respectively, at 45 °C. Stain removal was also effective after protease treatment at 25 and 60 °C.     

Purification and properties of a heat-stable and cold-labile NAD-specific glutamate dehydrogenase from Sporosarcina ureae

NAD-specific glutamate dehydrogenase (NAD-GluDH; EC 1.4.1.2) was purified to homogeneity from Sporosarcina ureae DSM 320; the native enzyme (M _r 250,000±25,000) is composed of subunits identical in molecular mass (M _r 42,000±3,000), suggesting a hexameric structure. In cell-free extracts and in its purified form, the enzyme was heat-stable, retaining 50% activity after 15 min incubation at temperatures up to 82°C. When exposed to low temperatures at pH values between 7.0 and 9.0. cell-free extracts and purified preparations lost enzyme activity rapidly and irreversibly. The addition of substrates, glycerol, or sodium chloride improved the stability of the enzyme with respect to cold lability and heat stability.Abbreviation NAD-GluDH nicotinamide-adenine-dinucleotide-specific glutamate dehydrogenase     

Purification and characterization of naringinase from a newly isolated strain of <Emphasis Type="Italic">Aspergillus niger</Emphasis> 1344 for the transformation of flavonoids

Summary An extracellular naringinase (an enzyme complex consisting of α-L-rhamnosidase and β-D-glucosidase activity, EC 3.2.1.40) that hydrolyses naringin (a trihydroxy flavonoid) for the production of rhamnose and glucose was purified from the culture filtrate of Aspergillus niger 1344. The enzyme was purified 38-fold by ammonium sulphate precipitation, ion exchange and gel filtration chromatography with an overall recovery of 19% with a specific activity of 867 units per mg of protein. The molecular mass of the purified enzyme was estimated to be about 168 kDa by gel filtration chromatography on a Sephadex G-200 column and the molecular mass of the subunits was estimated to be 85 kDa by sodium dodecyl sulphate-Polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme had an optimum pH of 4.0 and temperature of 50 °C, respectively. The naringinase was stable at 37 °C for 72 h, whereas at 40 °C the enzyme showed 50% inactivation after 96 h of incubation. Hg²⁺, SDS, p-chloromercuribenzoate, Cu²⁺ and Mn²⁺ completely inhibited the enzyme activity at a concentration of 2.5–10 mM, whereas, Ca²⁺, Co²⁺ and Mg²⁺ showed very little inactivation even at high concentrations (10–100 mM). The enzyme activity was strongly inhibited by rhamnose, the end product of naringin hydrolysis. The enzyme activity was accelerated by Mg²⁺ and remained stable for one year after storage at −20 °C. The purified enzyme preparation successfully hydrolysed naringin and rutin, but not hesperidin.     

Immobilization of endo-polygalacturonase from Aspergillus ustus on silica gel

Endo-polygalacturonase from Aspergillus ustus when immobilized on to modified silica gel retained 28% of its original activity. The immobilized enzyme could be re-used through 10 cycles of reaction with almost 90% retention of its original activity. It had increased thermostability over its soluble form: the half-life of the soluble enzyme at 40 °C was less than 10 h whereas the immobilized enzyme retained 82% of its activity after 10 h at 40 °C. Similarly, at 50 °C the half-life of the soluble enzyme was 30 min whereas that of the immobilized enzyme was 5 h.     

Galacto-oligosaccharide production by a thermostable recombinant β-galactosidase from <Emphasis Type="Italic">Thermotoga maritima</Emphasis>

Summary A β-galactosidase from Thermotoga maritima produced galacto-oligosaccharides (GOS) from lactose by transgalactosylation when expressed in Escherichia coli. The enzyme activity for GOS production was maximal at pH 6.0 and 90 °C. In thermal stability experiments, the enzyme followed first-order kinetics of pH and thermal inactivation, and half-lives at pH 5.0, pH 8.0, 80 °C, and 95 °C were 27 h, 82 h, 41 h, and 14 min, respectively, suggesting that the enzyme was stable below 80 °C and in the pH range of 5.0–8.0. Mn²⁺ was the most effective divalent cation for GOS production. Cu²⁺ and EDTA inhibited more than 84% of enzyme activity. GOS production increased with increasing lactose concentrations and peaked at 500 g lactose/l. Among tested enzyme concentrations, the highest production of GOS was obtained at 1.5 units enzyme/ml. Under the optimal conditions of pH 6.0, 80 °C, 500 g lactose/l, and 1.5 units enzyme/ml, GOS production was 91 g/l for 300 min, with a GOS productivity of 18.2 g/l · h and a conversion yield of GOS to lactose of 18%.     

Phytase production by the yeast, Pichia anomala

Pichia anomala, isolated from dried flower buds of Woodfordia fruticosa, produced a high activity of an intracellular phytase, at 68 U per g dry biomass, when grown at 20 °C for 24 h in a medium containing glucose (40 g l^–1) and beef extract (10 g l^–1) supplemented with Fe²⁺ (0.15 mM). Partially purified phytase was optimally active at 60 °C and pH 4 with a half life of 7 days at 60 °C. It retained 85% of its activity at 80 °C for 15 min. The enzyme is suitable for supplementing animal feeds to improve the availability of phosphate from phytate.     

Thermostable esterase from Thermoanaerobacter tengcongensis: high-level expression, purification and characterization

The lipA gene encoding a thermostable esterase was cloned from Thermoanaerobacter tengcongensis and overexpressed in Escherichia coli. The recombinant esterase, with a molecular mass of approx. 43 kDa determined by SDS-PAGE, was purified to homogeneity through Sephadex G-100 gel filtration. The purified enzyme actively hydrolyzed tributyrin but not olive oil. Maximum activity was observed on p-nitrophenyl (NP)-propionate (C3) and p-NP-butyrate (C4), with little activity towards p-NP-palmitate (C16). The esterase was optimally active at 70 °C (over 15 min) and at pH 9. It is highly thermostable, with a residual activity greater than 80% after incubation at 50 °C for more than 10 h. The activity was not inhibited by 5 mM EDTA and PMSF, indicating the esterase is not a metalloenzyme and may contain a specific structure around the catalytic serine residue. In addition, it was stable for 1 h at 37 °C in 1% CHAPS and Triton X-100 but not stable in 1% Tween 20 or SDS.     

Reassessment of the cold‐labile nature of phosphofructokinase from a hibernating ground squirrel

This study reassesses the proposal that cellular conditions of low temperature and relative acidosis during hibernation contribute to a suppression of phosphofructokinase (PFK) activity which, in turn, contributes to glycolytic rate suppression during torpor. To test the proposal that a dilution effect during in vitro assay of PFK was the main reason for activity loss (tetramer dissociation) at lower pH values, the influence of the macromolecular crowding agent, polyethylene glycol 8000 (PEG), on purified skeletal muscle PFK from Spermophilus lateralis was evaluated at different pH values (6.5, 7.2 and 7.5) and assay temperatures (5, 25 and 37°C). A 78 ± 2.5% loss of PFK activity during 1 h incubation at 5°C and pH 6.5 was virtually eliminated when 10% PEG was present (only 7.0 ± 1.5% activity lost). The presence of PEG also largely reversed PFK inactivation at pH 6.5 at warmer assay temperatures and reversed inhibitory effects by high urea (50 or 400 mM). Analysis of pH curves at 5°C also indicated that ~ 70% of activity would remain at intracellular pH values in hibernator muscle. The data suggest that under high protein concentrations in intact cells that the conditions of relative acidosis, low temperature or elevated urea during hibernation would not have substantial regulatory effects on PFK.