Concentrating Autographa californica nuclear polyhedrosis virus and recombinant alkaline phosphatase from insect cells using a temperature-sensitive hydrogel

Recombinant alkaline phosphatase expressed in insect cells was concentrated by a factor of one and half times at a separation efficiency of 54.2% using hydrogel ultrafiltration. Enzyme concentration was confirmed by SDS-PAGE as well as by spectrophotometric measurement. Wild and recombinant Autographa californica nuclear polyhedrosis viruses (AcNPV) were concentrated 1.4 and 1.6 times of the feed solution at 48.5 and 60.0% separation efficiency, respectively. Hydrogel ultrafiltration appears to be an attractive alternative for the concentration of AcNPV and recombinant proteins from insect cells. This revised version was published online in June 2006 with corrections to the Cover Date.     

Hydrogel ultrafiltration of recombinant alkaline phosphatase from baculovirus-infected Spodoptera frugiperda

Recombinant alkaline phosphatase expressed in insect cells was concentrated 1.5 times by hydrogel ultrafiltration by swelling at 20°C and collapsing at 35°C at 53-65% separation efficiency and 78-83% enzyme recovery. Enzyme entrapment between gel particles and attachment of enzymes to the gel surface was the main reason for the poor separation efficiency and low enzyme recovery. Exposure of the enzyme at the collapse temperature during repeated swelling-collapsing cycles also decreased separation efficiency to 37% due to thermal deactivation of the enzyme.     

Concentrating rotavirus and recombinant enhanced green fluorescent protein fromE. coli using a temperature-sensitive hydrogel

Recombinant enhanced green fluorescent protein (EGFP) fromE. coli was concentrated 1.9 times by ultrafiltration using a temperature-sensitive hydrogel. Protein recovery and separation efficiency were 64% and 45%, respectively. Increased concentration of recombinant EGFP was confirmed by SDS-PAGE. Rotavirus was concentrated 3.2 times by ultrafiltration using a temperature-sensitive hydrogel, at 95% of virus recovery and 93% of separation efficiency. Hydrogel ultrafiltration appears to be an attractive alternative for the concentration of rotavirus and recombinant proteins fromE. coli.     

Effect of temperature on the survival and infectivity of <Emphasis Type="Italic">Pseudotheraptus devastans</Emphasis> vector

The aim of this study was to investigate under a controlled environment, the effect of temperature on the survival and infectivity of Pseudotheraptus devastans Distant, a cassava anthracnose disease vector. The insect P. devastans was collected from young cassava (Manihot esculenta Crantz) field plots, at the International Institute of Tropical Agriculture, (IITA), Ibadan, Nigeria. A mixture of the different developmental stages of eggs, first to fifth instar nymphs, and adults, were incubated in controlled environment chambers, under various constant temperatures of: 15, 17, 22, 25, 27, 30, and 35°C. Relative humidity at different temperature conditions were recorded and maintained at 90%, 85%, 80%, 75%, 70%, 65%, and 60%, respectively. A significant increase in insect survival was observed between 22 and 27°C temperature conditions while a significant decrease in survival was observed at 15°C and above 30°C. Lesion number, lesion diameter and infectivity among the insect stages varied as a function of temperature and relative humidity. Infectivity was highest at 22–25°C maintained at 75–80% RH and lowest at 15°C and above 30°C maintained respectively, at 65% RH and 90% RH. There was considerable low vector infectivity due to low survival of the insects at extreme temperatures.     

The ecological energetics of Amoeba proteus (Protozoa)

The consumption, production and respiration of Amoeba proteus were measured in the laboratory for cells cultured over a range of Tetrahymena pyriformis concentrations (125–4 000 cells/0.5 ml) at 10, 15 and 20 °C. Differences were attributed to both temperature and prey availability. A series of generation energy budgets were constructed for amoebae grown under the above conditions. The biological efficiencies linking the parameters of the budget equation were calculated. Assimilation efficiencies ranged from 22–59% regardless of temperature. Net production efficiencies were high at 15 and 20 °C (65–82%) but low at 10 °C (11–49%). Gross production efficiencies were also higher at 15 and 20 °C (16–47%) than at 10 °C (4–29%). The ecological implications of this investigation are discussed.     

Agar properties of two species of Gracilariaceae from the Gulf of California,Mexico

Agar properties of two potentially commercial important seaweeds from the Gulf of California were studied. Maximum yield in Gracilaria vermiculophylla (45.7%) occurred during the summer months, coinciding with high water temperatures (31°C) whereas minimum yields (11.6%) were obtained during the coldest months of the year when populations of this species diminish in the bay. Gracilariopsis longissima showed two yield peaks, one in spring and another in fall, before the maximum and minimum seawater temperatures. Gel strength in native agar from the two species was low (<22.5 g cm⁻²) for most of the year. G. vermiculophylla native agar showed a slight increase in gel strength from June to August, which were the hottest months. Maximum value was 85 g cm⁻¹ in August. Maximum gel strength in G. longissima was observed in October (91 g cm⁻¹), and an unusual native agar with no detectable gel strength was observed in March and April samples. Gelling temperatures range from 27.7 to 36.5°C in G. vermiculophyla and from 26.6 to 34.9°C in G. longissima, meanwhile melting points were 73.9 – 53.5°C and 75.5 – 56.6°C, respectively. Sulfate content was high, 6.3–13.9% in G. vermiculophylla and 1.9–11.9% in G. longissima, and on the other hand 3,6 anhydrogalactose content was low 12.1–26.7% and 9.1–23%, respectively compared to other species. Results obtained showed that mean native agar yields of Gracilaria vermiculophylla and Gracilariopsis longissima from the Gulf of California are comparable to other tropical Gracilaria. However, the low gel strength, high sulfate content and low 3,6 anhydrogalactose content observed in the native agar extracted from these species make this an agaroid, thus alternative methods of extraction should be used to evaluate the possibility of commercial utilization of both species.     

Membrane physical state as key parameter for the resistance of the gram-negative <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis> to hyperosmotic treatments

The survival of Bradyrhizobium japonicum under hyperosmotic treatments achieved at various temperatures was investigated. The bacterial viability was measured at a combination of different levels of osmotic pressure (1.4–49.2 MPa) in glycerol solutions and temperature (4–28°C). Viability was dependent on these two variables, with low temperatures (10 and 4°C) exhibiting a protective effect against exposure to high levels of osmotic pressure. To understand these results, the relation between membrane physical state and structure of whole cells and osmotic shock tolerance of B. japonicum was studied. Membrane physical changes were evaluated by using 1,3-diphenyl-1,3,5-hexatriene (DPH) and Laurdan (6-dodecanoil-2-dimethylaminonaphtelene) as probes. The results showed that the membrane of B. japonicum was subjected to a progressive phase transition from the liquid-crystalline to the gel phase during cooling between 28 and 4°C. Accordingly, under isotonic conditions, the Laurdan GP spectra showed that, in the range 12–28°C, membrane lipids were in the liquid-crystalline phase, and in a gel phase at 4°C. The study of the variation in anisotropy of DPH revealed that cooling cells before the hyperosmotic treatment could induce opposite effects to the fluidizing effect of the hyperosmotic shock. Cell resistance was finally related to modifications of the membrane structure depending on combined effects of cooling and dehydration.     

Purification and Characterization of Trypsin-like Enzymes from <Emphasis Type="Italic">Neomysis japonica</Emphasis> Using BApNA as Substrate

Using N-α-benzoyl-l-arginine p-nitroanilide (BApNA) as substrate, trypsin-like enzymes (TLEs) were purified from mysis (Neomysis japonica) following two chromatographic steps, Sephacryl S100 HR gel filtration and Benzamidine-Sepharose 4B affinity. They presented a high stability in the raw material, retaining over 45% of the initial activity after 30 days of storage at pH 8.0, 45 °C. The purified TLEs had relative molecular mass between 32 kDa and 33 kDa. With higher stability and greater activity, they had similar stability and activity profiles (pH 6.0–11.0, 15–65 °C) as bovine trypsin but had a different optimum temperature (35 °C for trypsin and 45 °C for TLEs). Similar to bovine trypsin, the purified TLEs could be activated by Ca²⁺ and Mg²⁺. And the purified TLEs also showed similar inhibitory profiles as bovine trypsin with the exception of chicken egg ovomucoid (CEOM), an effective inhibitor of bovine trypsin but less so for purified TLEs. Having TLEs with physiological efficiency 3.6 times that of bovine trypsin, the use of mysis as a source for commercial production of TLEs is discussed.     

Insecticidal toxin fromXenorhabdus nematophilus, symbiotic bacterium associated with entomopathogenic nematodeSteinernema glaseri

Entomopathogenic nematodes are being used for insect control. We purified a toxin secreted by the insect-pathogenic bacterium,Xenorhabdus nematophilus, which lives in the gut of entomopathogenic nematodes. Culture broth ofX. nematophilus was separated by centrifugation and concentrated by ultrafiltration. The concentrated culture broth was applied to a DEAE Sephadex A-50 column, and proteins were eluted stepwise with increasing concentrations of KCl. Fractions with insect toxicity were further concentrated and then applied to a HPLC with a gel filtration column. The molecular weight of purified toxin was 39 kDa on SDS-PAGE, and Fourier transformed infrared (FTIR) spectroscopy indicated that this toxin could be a new protein exhibiting the characteristics of C=O stretching peak near 1650 cm⁻¹.     

Purification and characterization of laccase from <Emphasis Type="Italic">Pycnoporus sanguineus</Emphasis> and decolorization of an anthraquinone dye by the enzyme

The white rot fungus Pycnoporus sanguineus produced high amount of laccase in the basal liquid medium without induction. Laccase was purified using ultrafiltration, anion-exchange chromatography, and gel filtration. The molecular weight of the purified laccase was estimated as 61.4 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme oxidized typical substrates of laccases including 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonate), 2,6-dimethoxyphenol, and syringaldazine. The optimum pH and temperature for the purified laccase were 3.0 and 65°C, respectively. The enzyme was stable up to 40°C, and high laccase activity was maintained at pH 2.0–5.0. Sodium azide, l-cysteine, and dithiothreitol strongly inhibited the laccase activity. The purified enzyme efficiently decolorized Remazol Brilliant Blue R in the absence of added redox mediators. The high production of P. sanguineus laccase as well as its decolorization ability demonstrated its potential applications in dye decolorization.     

Concentrating alkaline serine protease,subtilisin, using a temperature-sensitive hydrogel

Summary The alkaline serine protease, subtilisin, produced by Bacillus licheniformis was concentrated using hydrogel ultrafiltration. Separation efficiency at 15° and 20°C was 84 % but decreased above 25°C.     

Production of a <Emphasis Type="Italic">Trichoderma reesei</Emphasis> QM9414 xylanase in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and its application in biobleaching of wheat straw pulp

The purpose of this study was to produce a Trichoderma reesei xylanase (XYN2) in Pichia pastoris and to test its potential application for pulp bleaching. The recombinant xylanase was purified by a two-step process of ultrafiltration and gel filtration chromatography. The molecular mass of the recombinant enzyme was 21 and 25 kDa by SDS–PAGE analysis, due to different glycosylation of the native protein. The optimum pH and temperature of the recombinant XYN2 was 5.0 and 50 °C. Enzyme activity was stable at 50 °C and at pH 5.0–7.0. The bleaching ability of the recombinant xylanase was also studied at 50 °C and pH 6.0, using wheat straw pulp. Biobleaching of the xylanase produced chlorine dioxide savings of up to 60%, while retaining brightness at the control level and led to a lower kappa number and small enhancements in tensile, burst and tear strength of pulp fibers.     

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.     

Purification and characterization of an extracellular acid trehalase fromLentinula edodes

Trehalase from the culture filtrate ofLentinula edodes was purified and characterized. Molecular masses were estimated to be 158 kDa and 79–91 kDa by gel filtration and SDS-PAGE under the reduced condition, respectively. The enzyme was composed of two identical subunits and contained carbohydrate molecules. The optimum temperature and pH were obtained at around 40°C and pH 5.0, respectively. The enzyme was stable up to 40°C and in a range pH of 4–10 at 30°C. It cleaved α-1,1 linkages of trehalose, but not α-1,4, α-1,6 or β-1,4 glycosyl linkages, and was defined as an acid trehalase.     

Identification and characterization of fermentation inhibitors formed during hydrothermal treatment and following SSF of wheat straw

A pilot plant for hydrothermal treatment of wheat straw was compared in reactor systems of two steps (first, 80°C; second, 190–205°C) and of three steps (first, 80°C; second, 170–180°C; third, 195°C). Fermentation (SSF) with Sacharomyces cerevisiae of the pretreated fibers and hydrolysate from the two-step system gave higher ethanol yield (64–75%) than that obtained from the three-step system (61–65%), due to higher enzymatic cellulose convertibility. At the optimal conditions (two steps, 195°C for 6 min), 69% of available C6-sugar could be fermented into ethanol with a high hemicellulose recovery (65%). The concentration of furfural obtained during the pretreatment process increased versus temperature from 50 mg/l at 190°C to 1,200 mg/l at 205°C as a result of xylose degradation. S. cerevisiae detoxified the hydrolysates by degradation of several toxic compounds such as 90–99% furfural and 80–100% phenolic aldehydes, which extended the lag phase to 5 h. Acetic acid concentration increased by 0.2–1 g/l during enzymatic hydrolysis and 0–3.4 g/l during fermentation due to hydrolysis of acetyl groups and minor xylose degradation. Formic acid concentration increased by 0.5–1.5 g/l probably due to degradation of furfural. Phenolic aldehydes were oxidized to the corresponding acids during fermentation reducing the inhibition level.     

Purification and characterization of tubulin from the catfishHeteropneustes fossilis

Tubulin was purified from the brain of the catfishHeteropneustes fossilis by cycles of temperature-dependent assembly and disassembly. Fish tubulin assembles into microtubules in the absence of high molecular weight microtubule associated proteins. Its subunits comigrate with goat brain α andβ tubulin subunits and is composed of 4 major α andβ tubulins each as analyzed by isoelectric focusing and two dimensional gel electrophoresis. Peptide mapping showed it to be very similar to goat brain tubulin. Polymerization of catfish brain tubulin occurs optimally between 18–37°C and the critical protein concentrations of assembly at 18°C and 37°C are the same, as opposed to mammalian brain tubulins.     

Purification and characterization of polygalacturonase produced by thermophilic Thermoascus aurantiacus CBMAI-756 in submerged fermentation

An extracellular polygalacturonase was isolated from 5-day culture filtrates of Thermoascus aurantiacus CBMAI-756 and purified by gel filtration and ion-exchange chromatography. The enzyme was maximally active at pH 5.5 and 60–65°C. The apparent K _m with citrus pectin was 1.46 mg/ml and the V _max was 2433.3 μmol/min/mg. The apparent molecular weight of the enzyme was 30 kDa. The enzyme was 100% stable at 50°C for 1 h and showed a half-life of 10 min at 60°C. Polygalacturonase was stable at pH 5.0–5.5 and maintained 33% of initial activity at pH 9.0. Metal ions, such as Zn⁺², Mn⁺², and Hg⁺², inhibited 50, 75 and 100% of enzyme activity. The purified polygalacturonase was shown to be an endo/exo-enzyme, releasing mono, di and tri-galacturonic acids within 10 min of hydrolysis.     

A case for a free-running circannual rhythm in soldier developmental time of Formosan subterranean termites

The success of all insect societies relies on their ability to maintain optimal levels of different castes. Here we report on an apparent free-running circannual rhythm that optimizes the developmental time of the soldier caste of Coptotermes formosanus Shiraki. Over a 3 year period, bioassays were conducted each month (except June) with groups of 100 termite workers in a 28°C incubator in total darkness. The number of days needed for C. formosanus soldiers to develop varied depending on the time of the year (month). In March, just prior to the major swarming exodus for alates (April to June), 9 days were required before a worker molted to a presoldier. Longer times were required for such a molt in all other months, with an increasing trend from April to December (from 13 to 30 days) and a decreasing trend from January to February (from 25 to 12 days). Colony origin or the length of time that termites were kept in the laboratory under constant conditions (26 – 28°C, 70 – 80% RH) before testing (7 days – 1 year) did not affect this rhythm. This is the first demonstrated evidence of a free-running circannual rhythm in a social insect. Received 23 July 2007; revised 9 and 21 August 2007; accepted 23 August 2007.     

Characteristics of the amylase of Arthrobacter psychrolactophilus

Properties of the extracellular amylase produced by the psychrotrophic bacterium, Arthrobacter psychrolactophilus, were determined for crude preparations and purified enzyme. The hydrolysis of soluble starch by concentrated crude preparations was found to be a nonlinear function of time at 30 and 40 °C. Concentrates of supernatant fractions incubated without substrate exhibited poor stability at 30, 40, or 50 °C, with 87% inactivation after 21 h at 30 °C, 45% inactivation after 40 min at 40 °C and 90% inactivation after 10 min at 50 °C. Proteases known to be present in crude preparations had a temperature optimum of 50 °C, but accounted for a small fraction of thermal instability. Inactivation at 30, 40, or 50 °C was not slowed by adding 20 mg/ml bovine serum albumin or protease inhibitor cocktail to the preparations or the assays to protect against proteases. Purified amylase preparations were almost as thermally sensitive in the absence of substrate as crude preparations. The temperature optimum of the amylase in short incubations with Sigma Infinity Amylase Reagent was about 50 °C, and the amylase required Ca⁺² for activity. The optimal pH for activity was 5.0–9.0 on soluble starch (30 °C), and the amylase exhibited a K _m with 4-nitrophenyl-α-D-maltoheptaoside-4,6-O-ethylidene of 120 μM at 22 °C. The amylase in crude concentrates initially hydrolyzed raw starch at 30 °C at about the same rate as an equal number of units of barley α-amylase, but lost most of its activity after only a few hours.     

Survival of the mosquito predator,Notonecta unifasciata[Hemiptera: Notonectidae] embryos at low thermal gradients

Notonecta unifasciata Guerin eggs maintained at different stages of embryonic development in water at variable temperatures (2.2–25.6 °C) and for periods of 4–12 weeks revealed maximum viability (>80 %) at the highest temperature. However, optimum nondevelopmental viability was at 14.4 °C with eight-day-old embryos (>35 %). Short term (4 weeks) storage at 14.4 °C significantly increased egg viability. Survival was poor (<20 %) at the 2 lowest temperatures. Eggs held at 14.4 °C for 12 weeks and sustainingca. 50 % mortality, may be a practical procedure for biological control.