Preparation of lyophilized pyrroloquinoline quinone glucose dehydrogenase using trehalose as an additive

Of 8 compounds tested as additives, trehalose at 100 mM was the most effective for the preparation of lyophilized pyrroloquinoline quinone glucose dehydrogenase (PQQGDH). Lyophilized PQQGDH retained about 80% of initial activity after 1 h at 80°C and can be stored at 28°C for more than a month without any significant decrease in enzymatic activity. It is thus suitable for use as a biosensor component.     

Increased thermal stability of glucose dehydrogenase by cross-linking chemical modification

A hetero-oligomeric glucose dehydrogenase (GDH) from a moderate thermophilic bacterium, SM4 was cross-linked with glutaraldehyde (GA) and it now showed only one optimum temperature for reaction at around 65°C, which approximately follows the Arrhenius equation. The native enzyme had shown optima at both 45°C and 75°C. In addition to the alteration of the optimum temperature for reaction, GA cross-linked GDH retained more than 90% of its initial activity even after 30 min of incubation at 65°C.     

Surface charge engineering of PQQ glucose dehydrogenase for downstream processing

The ion-exchange chromatography behavior of recombinant glucose dehydrogenase harboring pyrroloquinoline quinone (PQQGDH) was modified to greatly simplify its purification. The surface charge of PQQGDH was engineered by either fusing a three-arginine tail to the C-terminus of PQQGDH (PQQGDH+Arg3) or by substituting three residues exposed on the surface of the enzyme to Arg by site-directed mutagenesis (3RPQQGDH). During cation exchange chromatography, both surface charge-engineered enzymes eluted at much higher salt concentrations than the wild-type enzyme. After the chromatography purification step, both PQQGDH+Arg3 and 3RPQQGDH appeared as single bands on SDS-PAGE, while extra bands appeared with the wild-type protein sample. Although all tested kinetic parameters of both engineered enzymes are similar to those of wild type, both modifications resulted in enzymes with increased thermal stability. Our achievements have resulted in the greater production of an improved quality PQQGDH by a simplified process.     

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.     

Prevention of mortality induced by perinatal asphyxia: Hypothermia or glutamate antagonism?

Summary Perinatal asphyxia was induced by keeping pups-containing uterus horns, removed by hysterectomy, in a 37°C or a 30°C water bath. Asphyxia for a period of 21–22 min at 37°C led to a 97% mortality within the first 20 min period following delivery. When the asphyctic period was extended to more than 22 min all the pups died following delivery. When the asphyxia was induced at 30°C, 100% of the delivered pups survived and were accepted by surrogate mothers. The protective effect of hypothermia could be observed even when the pups-containing uterus horns were exposed to a 45–46 min asphyctic period. Pretreatment with dizocilpine (0.2 mg/kg s.c.), or 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX) (3–30 mg/kg s.c.), administered to the mothers one hour before hysterectomy, reduced slightly the mortality induced by a 21–22 min asphyctic period at 37°C. An increase in survival following a 22–23 min asphyctic period could only be observed after the highest dose of NBQX.     

Some factors contributing to the survival of rapidly cooled plant cells

The stability of innocuous intracellularly frozen cells was found to be influenced remarkably by the surrounding solutions and the degree of prefreezing. In the cells suspended in isotonic or slightly hypertonic glucose solution, innocuous intracellular freezing readily occurred and these rapidly frozen cells remained stable even at −30 °C for 20 min, while cells prefrozen to −10 °C after suspending in water survived standing at −30 °C only for 5 sec at most. These facts suggest that when the innocuous intracellularly frozen cells are surrounded with concentrated sugar solutions, these cells remain far more stable than those surrounded with ice.     

Immobilization of whole Escherichia coli containing penicillin amidase using cross-linking agents and fillers

To obtain a catalyst with good mechanical stress stability and other operational characteristics, cross-linked aggregates of whole Escherichia coli containing penicillin amidase were reinforced with surface modified precipitated silica and chitosan. The immobilized cells plus mixed fillers possess better performance characteristics i.e. higher stability at 4°C and 30°C, least protein leaching capacity and good settling characteristics. Most suitable conditions to prepare catalyst with mixed fillers were: chitosan, 0.3: silica, 0.2 g/g d.wt cell; and minimum moisture content in catalyst, 30% (w/w).     

Survival kinetics of Salmonella enterica serotype senftenberg (S. senftenberg) after heat and acid stress

The survival kinetics of two clinical isolates of Salmonella senftenberg were studied after heat and acid stress. The strains survived better at 53 and 55 °C after heat shock of 30 min at 50 °C or overnight heat adaptation at 45 °C. An increase in the decimal reduction time, D, of heat-shocked [10.2 min (53 °C) and 9.37 min (55 °C)] and heat-adapted [8.12 min (53 °C) and 7.8 min (55 °C)] cells was observed compared with the non-stressed cells [6.87 min (53 °C), 6.56 min 55 °C)]. A significant difference was also observed in the survival of acid-adapted to acid non-adapted S. senftenberg bacteria.     

Tolerance of brown bear spermatozoa to conditions of pre-freezing cooling rate and equilibration time

Specific protocols for the cryopreservation of endangered Cantabrian brown bear spermatozoa are critical to create a genetic resource bank. The aim of this study was to assess the effect of cooling rates and equilibration time before freezing on post-thawed brown bear spermatozoa quality. Electroejaculates from 11 mature bears were extended to 100 × 10⁶ spermatozoa/mL in a TES–Tris–Fructose–based extender, cryopreserved following performance of the respective cooling/equilibration protocol each sample was assigned to, and stored at −196 °C for further assessment. Before freezing, after thawing, and after 1 hour's incubation post-thawing at 37 °C (thermal stress test), the quality of the samples was assessed for motility by computer-assisted semen analysis, and for viability (SYBR-14/propidium iodide), acrosomal status (peanut agglutinin–fluorescein isothiocyanate /propidium iodide), and sperm chromatin stability (SCSA) by flow cytometry. In experiment 1, three cooling rates (0.25 °C/min, 1 °C/min, and 4 °C/min) to 5 °C were assessed. After thawing, total motility (%TM) was higher and percentage of damaged acrosomes (%dACR) was lower (P < 0.05) for 0.25 °C/min than for 4 °C/min. The thermal stress test data indicated equally poor quality (P < 0.05) for the 4 °C/min cooled samples in viability (%VIAB), %dACR, %TM, and progressive motility (%PM). In experiment 2, the effect of a pre-freezing equilibration period at 5 °C for 1 hour (cooling at 0.25 °C/min) was evaluated. Samples kept at 5 °C for 1 hour showed higher (P < 0.05) values than the nonequilibrated ones for both thawing (%dACR) and thermal stress test (%VIAB, %TM, and %PM). In experiment 3, samples stored without cooling and equilibration (direct freezing) were compared with the samples cooled at 0.25 °C/min and equilibrated for 1 hour (control freezing). Using thermal stress test, we observed that direct freezing causes damage in viability, acrosomal status, and motility of spermatozoa compared with the control group (P < 0.05). In conclusion, our results suggest that slow cooling rates to 5 °C and at least 1 hour equilibration time are necessary for the effective cryopreservation of brown bear sperm.     

The effect of hypothermic treatment on the repair or expression of X-ray damage measured in split dose experiments on L5178Y-S cells

Summary The nature of the post-irradiation lesions and processes leading to cellular reproductive death or survival were investigated in mouse lymphoblastic leukemia L5178Y-S (LY-S) cells. Post-(x-)irradiation incubation at 25° C protects LY-S cells against the fixation of biologically expressed damage which takes place at 37° C. An optimal condition for the repair of damage, assayed in split-dose experiments as split-dose recovery (SDR), is 1 h at 37° C followed by 4 h holding at 25° C prior to the second half of a split dose, or 5 h holding at 25° C without a 37° C incubation during the interval between doses. Longer incubations at 37° C resulted in progressively decreased survivals. Postirradiation inhibition of DNA synthesis at 37° C was observed only during the first 30 min; thereafter,³H-dThdR incorporation washigher than in unirradiated controls. Theexcess synthesis effect was removed by shifting irradiated cells to 25° C holding. The inhibition observed at 25° C was reversed by shifting to 37° C. Thus the degree of postirradiation DNA synthesis is inversely related to SDR. DNA filter elution shows complete strand break repair by 20 min at 37° C, and by 3 h at 25° C; DNA double-strand break (DSB) repair plateaus at 80% (37° C) and 60% (25° C) after 90 min. An inverse correlation was found between total strand break repair rate, as assayed by filter elution methods, and cell survival. This work was supported by a grant from The Mathers Charitable Foundation.A preliminary report of this work was presented at the 35th Annual Meeting of the Radiation Research Society, Atlanta, GA 1987, USA     

Inactivation of Alicyclobacillus acidoterrestris vegetative cells in model system,apple, orange and tomato juices by high hydrostatic pressure

The objective of this study is to determine the effect of high hydrostatic pressure (HHP) on inactivation of Alicyclobacillus acidoterrestris vegetative cells in a model system (BAM broth) and in orange, apple and tomato juices. The shelf-life stability of pressurized juices is also studied. In general the viability loss was enhanced significantly as the level of pressure and temperature were increased (P < 0.05). 4.70 log cycle reduction was obtained after pressurization at 350 MPa at 50 °C for 20 min in BAM broth whereas thermal treatment at 50 °C for 20 min caused only 1.13 log cycle inactivation showing the effectiveness of HHP treatment on inactivation. The D values for pressure (350 MPa at 50 °C) and temperature (50 °C) treatments were 4.37 and 18.86 min in BAM broth, respectively. All juices were inoculated with A. acidoterrestris cells to 10⁶ c.f.u./ml and were pressurized at 350 MPa at 50 °C for 20 min. More than 4 log cycle reduction was achieved in all juices studied immediately after pressurization. The pressurized juices were also stored up to 3 weeks at 30 °C and the viable cell numbers of A. acidoterrestris in orange, apple and tomato juices were 3.79, 2.59 and 2.27 log cycles, respectively after 3 weeks. This study has indicated that A. acidoterrestris vegetative cells can be killed by HHP at a predictable rate even at temperatures at which the microorganism would normally grow.     

Synthesis and evaluation of physicochemical properties of cross-linked sago starch

Highly substituted sago starch phosphate was synthesized using POCl₃ as cross-linking reagent. Titrimetric and Fourier transform infra red (FT-IR) spectral analysis were used to characterize the substitution. Studying the different factors affecting the reaction parameters showed that the optimal conditions for starch phosphorylation were: 4 h reaction time and reagent concentration 1.5% (w/w). The physicochemical properties of cross-linked sago starch (CLSS) were done using Scanning electron micrograph (SEM), X-ray powder diffractometer (XRD and Thermogravimetric analysis (TGA). The results revealed that crystalline nature of native sago starch was transformed after cross-linking. TGA report exhibited higher thermal stability, which makes it suitable for various industrial applications. Swelling behavior showed high swelling at low temperature (30 and 60 °C) as compared to high temperature (90 °C).     

Fast determination of operational stability of the soluble acetylacetone-cleaving enzyme Dke1 in an enzyme membrane reactor

The main aim of this study was the determination of the operational stability of soluble Dke1 (EC 1.13.11.50) in an enzyme membrane reactor. In order to calculate the half-life of soluble Dke1, the K _M of oxygen must be known. The determination of this constant was done using progress curve analysis (K _M=260 μmol l⁻¹). In a next step, the reactor system was studied by building a mathematical model for calculation of the reactor system, using Berkeley Madonna ver. 8.0.1 software. After that, the determination of the half-life of Dke1 under operational conditions at different temperatures (5, 10, 15, 25, 30, 35°C) was performed. The quantitative criterion for stability was the value of the first-order rate constant of monomolecular inactivation. The experiments showed that soluble Dke1 is poorly stable. The half-life ranged from 308 min at 5°C to 9 min at 35°C. This method for determining the half-life is quite applicable for enzymes which are poorly stable. In addition, both the storage stability and the operational stability can be determined.     

Rapid monitoring of autolysis process of proteases by capillary electrophoresis

A protease, MCP-01, produced by a deep-sea psychrotrophic strain of Pseudoaltermonas sp. SM9913 was purified and its autolysis reaction at 20 °C–50 °C was monitored by capillary electrophoresis. Capillary electrophoresis provides a rapid assay because the degree and state of autolysis of protease MCP-01 could be observed within 6 min. The autolysis rate increased as the temperature rose in the tested range. After 30 min incubation at 30 °C, 77% of MCP-01 autolyzed into peptides. However, its activity for the hydrolysis of casein was reduced by only 4%. The rate of loss of activity of MCP-01 was thus slower than that of autolysis of MCP-01 at 30 °C. Similar results were obtained when MCP-01 was incubated at 20 °C, 40 °C and 50 °C. Large peptides produced by autolysis of MCP-01 therefore still have catalytic activity. When these large peptides autolyzed further into smaller peptides, the enzyme conformation that retained its catalytic activity was destroyed and activity was lost.     

Cryopreservation of microencapsulated canine sperm

The objective was to develop a method for cryopreserving microencapsulated canine sperm. Pooled ejaculates from three beagle dogs were extended in egg yolk tris extender and encapsulated using alginate and poly-L-lysine at room temperature. The microcapsules were cooled at 4 °C, immersed in pre-cooled extender (equivalent in volume to the microcapsules) to reach final concentration of 7% (v/v) glycerol and 0.75% (v/v) Equex STM paste, and equilibrated for 5, 30 and 60 min at 4 °C. Thereafter, microcapsules were loaded into 0.5 mL plastic straws and frozen in liquid nitrogen. In Experiment 1, characteristics of microencapsulated canine sperm were evaluated after glycerol addition at 4 °C. Glycerol exposure for 5, 30 and 60 min did not significantly affect progressive motility, viability, or acrosomal integrity of microencapsulated sperm compared with pre-cooled unencapsulated sperm (control). In Experiment 2, characteristics of frozen-thawed canine microencapsulated sperm were evaluated at 0, 3, 6, and 9 h of culture at 38.5 °C. Pre-freeze glycerol exposure for 5, 30, and 60 min at 4 °C did not influence post-thaw quality in unencapsulated sperm. Post-thaw motility and acrosomal integrity of microencapsulated sperm decreased more than those of unencapsulated sperm (P < 0.05) following glycerol exposure for 5 min. However, motility, viability and acrosomal integrity of microencapsulated sperm after 30 and 60 min glycerol exposure were higher than unencapsulated sperm cultured for 6 or 9 h (P < 0.05). In conclusion, since microencapsulated canine sperm were successfully cryopreserved, this could be a viable alternative to convention sperm cryopreservation in this species.     

Comparative studies on physical characteristics and resting metabolism between young male highlanders of Papua New Guinea and young male Japanese

Anthropometric measurements and measurements of resting metabolism were made on 15 young male highlanders in Beha village at altitudes between 1,500 m and 1,800 m in the Eastern Highland of Papua New Guinea in August in 1978 and 10 young male Japanese in Nishinomiya in September. New Guineans showed significantly lower height, considerably lower body weight than Japanese but heavier body weight for height and significantly greater mean values of Rohrer's index and Brugsch's index than Japanese. Skinfold thicknesses for New Guineans were significantly smaller than those for Japanese. Thus, physically, New Guineans were more muscular and athletic when compared with Japanese. The mean value of resting metabolic rate for New Guineans, 46.35 W/m², at 25°C was considerably lower than that for Japanese, 51.01 W/m². New Guineans showed significantly lower mean value of resting metabolism 47.57 W/m² at 30°C than Japanese 55.16 W/m². The mean values of respiratory quotient for New Guineans (RQ = 0.950 at 25°C and 0.971 at 30°C) were significantly greater than those for Japanese (0.81 at 25°C and 0.81 at 30°C). New Guineans showed considerably lower mean value of heart rate at 30°C (71.1 beats/min) than Japanese (79.2 beats/min). The smaller physique of New Guineans might be the result of lower caloric intake and protein intake as well as of living in a tropical climate.     

Partial characterization ofAspergillus fumigatus polygalacturonases for the degumming of natural fibers

Summary Aspergillus fumigatus strain 4, cultured on citrus pectin as the sole carbon source, produced polygalacturonases whose activity was optimum at 65°C and pH 3.5–4.5. The enzymes presented a bimodal thermostability for 10 min, but not 60 min, of incubation. Polygalacturonases showed pH stability between 3.0 to 9.0. The enzymes were stable when stored at 4–6°C for 90 days, but their activity was reduced by 24% when they were stored at 26–30°C. Orange pulp was the best pectic carbon source tested for the production of pectinases capable of retting ramie fibers. The reutilization of these enzymes was possible, suggesting the viability of industrial use of pectinases for degumming ramie fibers.     

Properties of extracellular proteases from three psychrotolerant Stenotrophomonas maltophilia isolated from Antarctic soil

Three Antarctic psychrotolerant Stenotrophomonas maltophilia were isolated and the characteristics of their extracellular serine proteases were described. The isolates were able to grow at 14 and 34°C, but grew better between 20 and 28°C. The highest protease secretion was reached at 20–24°C. The purified enzyme preparations had maximal activity at 55–60°C and alkaline pH. They showed high pH stability, retaining more than 60% of residual activity after 3 h of incubation at a pH range of 4–12. The thermal stability was slightly lower compared with a commercial mesophilic protease, with 74–79% residual activity after 90 min at 40°C and 50% inactivation at 50°C between 43 and 69 min. These properties suggest that the Antarctic isolates could be adapted to cold by means of synthesising more enzymes with high activity but that the proteases they produce are not truly cold-active, being more similar to mesophilic enzymes.     

Protamine induced intracellular uptake of horseradish peroxidase and vacuolation in mouse skeletal muscle in vitro

Summary The uptake in vitro of horseradish peroxidase (HRP) in mouse skeletal muscle was examined by electron microscopy and chemical determination.In muscles exposed to an HRP solution for 60 min at +37°C, HRP infiltrated the basal lamina of muscle fibres and caused an intense labelling of their sarcolemma. In addition HRP was found within the transverse tubules. Exposure to HRP for 30 min at +37°C followed by HRP together with a polycationic protein (protamine) for 30 min at +37°C caused an intracellular vesicular uptake of HRP. Intracellular HRP was found in numerous vesicles, membrane limited bodies and vacuoles. Protamine also induced focal autophagic vacuolation with progressive muscle fibre degeneration. An intracellular HRP uptake or muscle cell vacuolation could not be detected in the absence of protamine or when the incubation temperature was + 4°C. Chemical determination of HRP uptake was in general agreement with the morphological results. The uptake of HRP in the presence of protamine was stimulated at +31°C and blocked at +4°C.The results suggest that in skeletal muscle in vitro intracellular uptake of macromolecules occurs by endocytosis.     

Operational and storage stability of neutral β-mannanase from Bacillus licheniformis

The stability of neutral -mannanase from Bacillus licheniformis during operation and storage was investigated. The enzyme activity decreased by 70% with a hydrolysate of glucomannan at 20 g l^–1 over 30 min at 25 °C. In an enzymatic membrane reactor operated at 50 °C after 24 h, the loss of enzyme activities were 23% and 9% in the absence/presence of the substrate. The residual activities of the enzyme were 21% and 90%, respectively, when stored in 30% (v/v) glycerol solution and in solid state at 4 °C after one year.