Relative efficiency of succinylcholine, xylazine, and carfentanil/xylazine mixtures to immobilize free-ranging moose

We compared the efficiency of succinylcholine chloride, xylazine hydrochloride and carfentanil/xylazine mixtures in immobilizing 364 free-ranging moose (Alces alces) between 1987 and 1997 in Québec (Canada). With succinylcholine chloride (0.070, 0.062, 0.051 mg/kg of estimated body weight for calves, juveniles and adults), 63% of the 252 immobilization attempts led to complete immobilization and marking, whereas 7% of the darted animals died of respiratory paralysis during handling. The moose took an average of 13 min to lay down after darting (down time). Injection of xylazine (3.67-4.22 mg/kg) permitted sedation (the animal laid down but got up again when approached) or complete immobilization in 78% of the 40 darted adult moose, the mean down time being 8.7 min. No mortality was noted with this drug but 58% of the marked animals were only sedated. The use of RX821002A (0.058 mg/kg) as an antagonist, permitted a mean recovery time of 2.8 min after intravenous injection. With the carfentanil/xylazine mixtures (0.0071 and 0.181 mg/kg), 96% of the immobilization trials (n = 72) led to complete (88%) or partial (8%) immobilization, but 6% of the moose died several days after capture. The mean down time was 6.6 min, and injection of naltrexone (0.709 mg/kg) antagonized the effect of the immobilizing agent within 3.7 min. The respiratory rate was higher (P < 0.05) among moose immobilized with xylazine (35/min) than among those immobilized with carfentanil/xylazine mixtures (19/min) but this variation could be related to a longer pursuit time (z = 3.60; P < 0.01) and higher stress levels during handling. Rectal temperature also was higher with xylazine but the difference was small (39.7 vs. 39.3, P = 0.03) and did not differ significantly between the sexes (P > 0.05). Considering loss of materials and helicopter flight time due to non-successful marking trials, carfentanil/xylazine mixtures were the least expensive ($333 Cdn/animal).     

Determination and evaluation of an optimal dosage of carfentanil and xylazine for the immobilization of white-tailed deer (Odocoileus virginianus)

Using an iteration method, optimal hand-injected immobilization dosages of carfentanil/xylazine (CAR/XYL) were determined for 13 adult white-tailed deer (Odocoileus virginianus). Deer were temporarily restrained in a squeeze chute and were repeatedly immobilized one to four times at 2-5-wk intervals from December 2002 to March 2003. A fixed ratio of 1 mg CAR:10 mg XYL intramuscularly was used, increasing or decreasing the dosage until the optimal dosage (defined by an induction time < 3 min and PaCO(2)< 60 mmHg) was reached for each animal. Inductions were video-recorded and reviewed by observers blinded to drugs and dosages, who rated qualitative aspects of each induction. There were significant (P < 0.05) dosage-dependent decreases in induction time, time to first effect, PaO(2), SaO(2), and arterial pH, and significant dosage-dependent increases in PaCO(2) and quality ratings. The median optimal dosage (mOD) was 0.03 (range, 0.015-0.06) mg/kg CAR+0.3 (range, 0.15-0.6) mg/kg XYL. Induction times using the mOD were rapid (median 3.0 min [range, 1.8-10.0]), but quality ratings were considered undesirable for nine of 13 deer. Increased rectal body temperatures of 40.6+/-0.5 C (mean +/- SD) were noted in all deer and hyperthermia (T > 41 C) was noted in three. There was a positive correlation between body temperature and induction time (r=0.44). Heart rates significantly decreased from 5 to 15 min postinduction and remained decreased at the 20-min reading; there was occasional bradycardia. There was a significant increase in pH from 10 to 20 min postinduction, but metabolic acidemia (pH<7.3) persisted throughout the immobilization periods for all deer. Possible hypoxemia (SaO(2) and SpO(2)<90 mmHg but PaO(2)>60 mmHg) was present after induction, while hypercapnea (PaCO(2) > 60 mmHg) did not occur. Reversal times with naltrexone and yohimbine were rapid (mean 3.7+/-1.5 min) and uneventful, with no evidence of renarcotization. Although the median optimal dosage produced rapid inductions, no respiratory depression, complete reversal after antagonist administration, and no renarcotization, negative attributes included elevated body temperatures, acidemia, and undesirable induction qualities.     

The acute physiological response of polar bears to helicopter capture

Many wildlife species are live captured, sampled, and released; for polar bears (Ursus maritimus) capture often requires chemical immobilization via helicopter darting. Polar bears reduce their activity for approximately 4 days after capture, likely reflecting stress recovery. To better understand this stress, we quantified polar bear activity (via collar-mounted accelerometers) and body temperature (via loggers in the body core [T_abd] and periphery [T_per]) during 2–6 months of natural behavior, and during helicopter recapture and immobilization. Recapture induced bouts of peak activity higher than those that occurred during natural behavior for 2 of 5 bears, greater peak T_per for 3 of 6 bears, and greater peak T_abd for 1 of 6 bears. High body temperature (>39.0°C) occurred in T_per for 3 of 6 individuals during recapture and 6 of 6 individuals during natural behavior, and in T_abd for 2 of 6 individuals during recapture and 3 of 6 individuals during natural behavior. Measurements of T_abd and T_per correlated with rectal temperatures measured after immobilization, supporting the use of rectal temperatures for monitoring bear response to capture. Using a larger dataset (n = 66 captures), modeling of blood biochemistry revealed that maximum ambient temperature during recapture was associated with a stress leukogram (7–26% decline in percent lymphocytes, 12–21% increase in percent neutrophils) and maximum duration of helicopter operations had a similar but smaller effect. We conclude that polar bear activity and body temperature during helicopter capture are similar to that which occurs during the most intense events of natural behavior; high body temperature, especially in warm capture conditions, is a key concern; additional study of stress leukograms in polar bears is needed; and additional data collection regarding capture operations would be useful.     

Redefining physiological responses of moose (Alces alces) to warm environmental conditions

We tested the concept that moose (Alces alces) begin to show signs of thermal stress at ambient air temperatures as low as 14 °C. We determined the response of Alaskan female moose to environmental conditions from May through September by measuring core body temperature, heart rate, respiration rate, rate of heat loss from exhaled air, skin temperature, and fecal and salivary glucocorticoids. Seasonal and daily patterns in moose body temperature did not passively follow the same patterns as environmental variables. We used large changes in body temperature (≥1.25 °C in 24hr) to indicate days of physiological tolerance to thermal stressors. Thermal tolerance correlated with high ambient air temperatures from the prior day and with seasonal peaks in solar radiation (June), ambient air temperature and vapor pressure (July). At midday (12:00hr), moose exhibited daily minima of body temperature, heart rate and skin temperature (difference between the ear artery and pinna) that coincided with daily maxima in respiration rate and the rate of heat lost through respiration. Salivary cortisol measured in moose during the morning was positively related to the change in air temperature during the hour prior to sample collection, while fecal glucocorticoid levels increased with increasing solar radiation during the prior day. Our results suggest that free-ranging moose do not have a static threshold of ambient air temperature at which they become heat stressed during the warm season. In early summer, body temperature of moose is influenced by the interaction of ambient temperature during the prior day with the seasonal peak of solar radiation. In late summer, moose body temperature is influenced by the interaction between ambient temperature and vapor pressure. Thermal tolerance of moose depends on the intensity and duration of daily weather parameters and the ability of the animal to use physiological and behavioral responses to dissipate heat loads.     

A yeast lysis mutant: potential biotechnological applications

The conditional yeast lysis mutant cly8 was studied for potential biotechnological applications. The strain stops to grow immediately after a shift to elevated temperatures ( > 30°C). Cell viability (colony forming capacity) decreases at 37°C at a rate depending on the composition of the medium. However, at the elevated temperature cells still consume glucose and incorporate [¹⁴C]leucine into cell material. With decreasing viability the mutant cells become leaky for small, predominantly cytoplasmic components such as leucine or uridine but not for vacuolar storage products like arginine. No trichloroacetic acid-precipitable material could be detected in the medium after the shift to the elevated temperature indicating that leakiness was restricted to low molecular weight compounds. On acetate medium mutant cells became permeable only after prolonged incubation at 37°C but could be used for the oxidation of exogenous NADH. In comparison to the wild type the mutant also produced more glycerol. When the mutant cells were immobilized, glycerol production was in the same range at room temperature and at 28°C and could be maintained for several days.     

Microchip transponder thermometry for monitoring core body temperature of antelope during capture

Hyperthermia is described as the major cause of morbidity and mortality associated with capture, immobilization and restraint of wild animals. Therefore, accurately determining the core body temperature of wild animals during capture is crucial for monitoring hyperthermia and the efficacy of cooling procedures. We investigated if microchip thermometry can accurately reflect core body temperature changes during capture and cooling interventions in the springbok (Antidorcas marsupialis), a medium-sized antelope. Subcutaneous temperature measured with a temperature-sensitive microchip was a weak predictor of core body temperature measured by temperature-sensitive data loggers in the abdominal cavity (R²=0.32, bias >2 °C). Temperature-sensitive microchips in the gluteus muscle, however, provided an accurate estimate of core body temperature (R²=0.76, bias=0.012 °C). Microchips inserted into muscle therefore provide a convenient and accurate method to measure body temperature continuously in captured antelope, allowing detection of hyperthermia and the efficacy of cooling procedures.     

Immobilization and characterization of lipase from an indigenous Bacillus aryabhattai SE3-PB isolated from lipid-rich wastewater

Abstract

Extracellular lipase from an indigenous Bacillus aryabhattai SE3-PB was immobilized in alginate beads by entrapment method. After optimization of immobilization conditions, maximum immobilization efficiencies of 77%?±?1.53% and 75.99%?±?3.49% were recorded at optimum concentrations of 2% (w/v) sodium alginate and 0.2?M calcium chloride, respectively, for the entrapped enzyme. Biochemical properties of both free and immobilized lipase revealed no change in the optimum temperature and pH of both enzyme preparations, with maximum activity attained at 60?°C and 9.5, respectively. In comparison to free lipase, the immobilized enzyme exhibited improved stability over the studied pH range (8.5–9.5) and temperature (55–65?°C) when incubated for 3?h. Furthermore, the immobilized lipase showed enhanced enzyme-substrate affinity and higher catalytic efficiency when compared to soluble enzyme. The entrapped enzyme was also found to be more stable, retaining 61.51% and 49.44% of its original activity after being stored for 30 days at 4?°C and 25?°C, respectively. In addition, the insolubilized enzyme exhibited good reusability with 18.46% relative activity after being repeatedly used for six times. These findings suggest the efficient and sustainable use of the developed immobilized lipase for various biotechnological applications.     

Thermal biology of the Central Asian blunt-nosed viper (Macrovipera lebetina turanica) from Nuratau Crest and the Chernov blunt-nosed viper (Macrovipera lebetina černovi) from Western Kysylkum

The ecology of the Central Asian blunt-nosed viper (Macrovipera lebetina turanica) inhabiting the Nuratau Crest of Uzbekistan is described. The temperature conditions of the environment and the spatialtemporal structure of the viper activity are represented, which made it possible to find some of thermobiological characteristics. The temperature diapason of full activity of this viper constitutes approximately 17–34°C. The temperature of thermostabilization is in the range of 26–31°C, nocturnal temperatures are elevated from 9–15°C to 18–23°C in spring and summer, respectively, and diurnal body temperatures are decreased from 18–22°C to 10–15°C. Feeding and digestion are normal in blunt-nosed vipers at 25°C, which is below the diurnal temperature variation. In spring, the light phase duration is rapidly increased from 3–6 h to 14 h or more. This length of time and the large diurnal variation in the body temperature from 18°C to 20–22°C facilitate active and efficient coupling. Later, the diurnal body temperature is decreased to 13–18°C, which facilitates successful recovery in males and pregnancy in females. Plots for the average viper body temperature in different seasons are represented. The thermobiological characteristics of the bluntnosed viper of Macrovipera lebetina ěrnovi are similar to M. l. turanica.     

Immobilization of pycnoporus coccineus laccase on Eupergit C: Stabilization and treatment of olive oil mill wastewaters

The use of olive oil mill wastewaters (OMW) as an organic fertilizer is limited by their phytotoxic effect, due to the high concentration of phenolic compounds. As an alternative to physico-chemical methods for OMW detoxification, the laccase from Pycnoporus coccineus, a white-rot fungus with the ability to decrease the chemical oxygen demand (COD) and color of the industrial effluent, is being studied. In this work, the P. coccineus laccase was immobilized on two acrylic epoxy-activated resins, Eupergit C and Eupergit C 250L. The highest activity was obtained with the macroporous Eupergit C 250L, reaching 110 U g^?1 biocatalyst. A substantial stabilization effect against pH and temperature was obtained upon immobilization. The soluble enzyme maintained ≥80% of its initial activity after 24 h at pH 7.0–10.0, whereas the immobilized laccase kept the activity in the pH range 3.0–10.0. The free enzyme was quickly inactivated at temperatures >50°C, whereas the immobilized enzyme was very stable up to 70°C. Gel filtration profiles of the OMW treated with the immobilized enzyme (for 8 h at room temperature) showed both degradation and polymerization of the phenolic compounds.     

Immobilization of Candida antarctica lipase B (CALB) on surface-modified rice husk ashes (RHA) via physical adsorption and cross-linking methods

In the present study, the recovery of activity of Candida antarctica lipase B (CALB) immobilized onto surface-modified rice husk ash (RHA) was 90% for both cross-linking and adsorption methods. Both cross-linked and adsorbed immobilized preparations were very stable, retaining more than 48% of their activity over the range of temperatures studied. The optimum temperature and optimum pH values were 37?°C and 7.0, respectively for both immobilized preparations, while the relative activities after storage at 4.0?°C for 60 days were 55% and 65% using cross-linking and adsorption methods, respectively. Also, the activity of the immobilized lipase began to decrease after 10 cycles, more than 58% of the initial activities were still retained after 10 cycles for both immobilization methods. These results indicated that lipase immobilized by cross-linking and adsorption not only effected activity recovery, but also remarkably effected stability, reusability and application adaptability. It can be concluded that, surface-modified RHA can be used as alternative supports for immobilization of CALB for polymerization reactions.     

Immobilization and characterization of cellulase on concanavalin A (Con A)-layered calcium alginate beads

Cellulase has been immobilized on hybrid concanavalin A (Con A)-layered calcium alginate–starch beads. Immobilized cellulase retained about 82% of its activity. Con A was extracted from jack bean and the obtained crude protein was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The immobilized beads showed high mechanical and storage stability; immobilized cellulase retained 100% and 85% activity at 4°C and 30°C, respectively, over one month. The immobilized cellulase retained about 70% of its activity after five cycles of use. The immobilized cellulase retained 70% activity after 120-min exposure to 60°C, whereas the soluble form only retained about 20%, showing that immobilization improved thermal stability. Surface morphology and elemental analysis of immobilized cellulase were examined using scanning electron microscope equipped with energy-dispersive X-ray. Based on the enzyme stability and reuse, this method of immobilization is both convenient and cheap.     

Novel grafted agar disks for the covalent immobilization of β‐D‐galactosidase

Novel grafted agar disks were prepared for the covalent immobilization of β‐D‐galactosidase (β‐gal). The agar disks were activated through reacting with ethylenediamine or different molecular weights of Polyethyleneimine (PEI), followed by glutaraldehyde (GA). The modification of the agar gel and the binding of the enzyme were verified by Fourier Transform Infrared (FTIR) and elemental analysis. Moreover, the agar's activation process was optimized, and the amount of immobilized enzyme increased 3.44 folds, from 38.1 to 131.2 U/g gel, during the course of the optimization process. The immobilization of β‐gal onto the activated agar disks caused its optimum temperature to increase from 45°C to 45–55°C. The optimum pH of the enzyme was also shifted towards the acidic side (3.6–4.6) after its immobilization. Additionally, the Michaelis‐Menten constant (K_m) increased for the immobilized β‐gal as compared to its free counterpart whereas the maximum reaction rate (V_max) decreased. The immobilized enzyme was also shown to retain 92.99% of its initial activity after being used for 15 consecutive times. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 675–684, 2015.     

Distinct states of lipid mobility in bovine rod outer segment membranes Resolution of spin label results

Freely diffusable lipid spin labels in bovine rod outer segment disc membranes display an apparent two-component ESR spectrum. One component is markedly more immobilized than that found in fluid lipid bilayers, and is attributed to lipid interacting directly with rhodopsin. For the 14-doxyl stearic acid spin label this more immobilized component has an outer splitting of 59 G at 0°C, with a considerable temperature dependence, the effective outer splitting decreasing to 54 G at 24°C. Spin label lipid chains covalently attached to rhodopsin can also display a two-component spectrum in rod outer segment membranes. In unbleached, non-delipidated membranes the 16-doxyl stearoyl maleimide label shows an immobilized component which has an outer splitting of 59 G at 0°C and a considerable temperature dependence. This component which is not resolved at high temperatures (24–35°C), is attributed to the lipid chains interacting directly with the monomeric protein, as with the diffusable labels. In contrast, in rod outer segment membranes which have been either delipidated or extensively bleached, a strongly immobilized component is observed with the 16-doxyl maleimide label at all temperatures. This immobilized component has an outer splitting of 62–64 G at 0°C, with very little temperature dependence (61–62 G at 35°C), and is attributed to protein aggregation.     

A comparison of carfentanil/xylazine and Telazol/xylazine for immobilization of white-tailed deer

October 2001 to January 2002, captive free-ranging white-tailed deer (Odocoileus virginianus) were immobilized with a combination of carfentanil citrate and xylazine hydrochloride. From this study, we selected a dose of carfentanil/xylazine for the purpose of comparing immobilization parameters and physiologic effects with those of a combination of tiletamine and zolazepam (Telazol) and xylazine. Animals were initially given intramuscular injections of 10 mg xylazine and one of four doses of carfentanil (i.e., 0.5, 1.0, 1.5, and 2.0 mg). A carfentanil dose of 1.2 mg (x +/- SD = 23.5 +/- 3.2 microg/kg) and 10 mg xylazine (0.2 +/- 0.03 mg/kg) were selected, based on induction times and previously published reports, to compare with a combination of 230 mg of Telazol (4.5 +/- 0.6 mg/kg) and 120 mg xylazine (2.3 +/- 0.3 mg/kg). Time to first observable drug effects and to induction were significantly longer for deer treated with carfentanil/xylazine than with Telazol/xylazine (P < 0.01). Hyperthermia was common in deer immobilized with carfentanil/xylazine, but heart rate, respiration rate, and hemoglobin saturation were within acceptable levels. Degree of anesthesia of deer immobilized with Telazol/xylazine was superior to deer immobilized with carfentanil/xylazine. The combination of 120 mg of naltrexone hydrochloride and 6.5 mg of yohimbine hydrochloride provided rapid and complete reversal (1.9 +/- 1.1 min) of carfentanil/xylazine immobilization. Animals immobilized with Telazol/xylazine had long recovery times with occasional resedation after antagonism with 6.5 mg of yohimbine. The combination of carfentanil and xylazine at the doses tested did not provide reliable induction or immobilization of white-tailel (leer even though drug reversal was rapid and safe using naltrexone and yohimbine.     

Patterns of activity and body temperature of Aldabra giant tortoises in relation to environmental temperature

We studied the temperature relations of wild and zoo Aldabra giant tortoises (Aldabrachelys gigantea) focusing on (1) the relationship between environmental temperature and tortoise activity patterns (n = 8 wild individuals) and (2) on tortoise body temperature fluctuations, including how their core and external body temperatures vary in relation to different environmental temperature ranges (seasons; n = 4 wild and n = 5 zoo individuals). In addition, we surveyed the literature to review the effect of body mass on core body temperature range in relation to environmental temperature in the Testudinidae. Diurnal activity of tortoises was bimodally distributed and influenced by environmental temperature and season. The mean air temperature at which activity is maximized was 27.9°C, with a range of 25.8–31.7°C. Furthermore, air temperature explained changes in the core body temperature better than did mass, and only during the coldest trial, did tortoises with higher mass show more stable temperatures. Our results, together with the overall Testudinidae overview, suggest that, once variation in environmental temperature has been taken into account, there is little effect of mass on the temperature stability of tortoises. Moreover, the presence of thermal inertia in an individual tortoise depends on the environmental temperatures, and we found no evidence for inertial homeothermy. Finally, patterns of core and external body temperatures in comparison with environmental temperatures suggest that Aldabra giant tortoises act as mixed conformer–regulators. Our study provides a baseline to manage the thermal environment of wild and rewilded populations of an important island ecosystem engineer species in an era of climate change.     

Improving of Catalase Stability Properties by Encapsulation in Alginate/Fe3O4 Magnetic Composite Beads for Enzymatic Removal of H2O2

The aim of this study was enhancing of stability properties of catalase enzyme by encapsulation in alginate/nanomagnetic beads. Amounts of carrier (10–100 mg) and enzyme concentrations (0.25–1.5 mg/mL) were analyzed to optimize immobilization conditions. Also, the optimum temperature (25–50°C), optimum pH (3.0–8.0), kinetic parameters, thermal stability (20–70°C), pH stability (4.0–9.0) operational stability (0–390 min), and reusability were investigated for characterization of the immobilized catalase system. The optimum pH levels of both free and immobilized catalase were 7.0. At the thermal stability studies, the magnetic catalase beads protected 90% activity, while free catalase maintained only 10% activity at 70°C. The thermal profile of magnetic catalase beads was spread over a large area. Similarly, this system indicated the improving of the pH stability. The reusability, which is especially important for industrial applications, was also determined. Thus, the activity analysis was done 50 times in succession. Catalase encapsulated magnetic alginate beads protected 83% activity after 50 cycles.     

Simultaneous purification and immobilization of soybean hull peroxidase with a dye attached to chitosan mini-spheres

Soybean hull peroxidase (EC 1.11.1.7, SBP) was simultaneously purified and immobilized by dye affinity chromatography with Reactive Blue 4 attached to chitosan mini-spheres. Under optimized conditions, 96% of SBP was adsorbed to the matrix. Under the most stringent condition, only 49% was desorbed, whereas 2 M NaCl failed to desorb a significant amount of SBP. This behaviour allowed proposing the dye matrix as a support to immobilize SBP from a crude extract. The pH of maximum activity shifted from 7 to 3–5. SBP gained thermostability after immobilization: after 5?h at 85?°C, the remaining activity was 54%, whereas that of the free enzyme was 31%. The optimum temperature for the immobilized SBP was 75?°C, whereas that of the free enzyme was 55?°C. After two months at 4?°C, the activity loss of the immobilized SBP was only 3%. Immobilized SBP removed 80% of 2-bromophenol from wastewater in 180?min and, after five cycles of use, the activity loss was only 12.8%.     

Increasing ocean temperatures reduce activity patterns of a large commercially important coral reef fish

Large‐bodied fish are critical for sustaining coral reef fisheries, but little is known about the vulnerability of these fish to global warming. This study examined the effects of elevated temperatures on the movement and activity patterns of the common coral trout Plectropomus leopardus (Serranidae), which is an important fishery species in tropical Australia and throughout the Indo West‐Pacific. Adult fish were collected from two locations on Australia's Great Barrier Reef (23°S and 14°S) and maintained at one of four temperatures (24, 27, 30, 33 °C). Following >4 weeks acclimation, the spontaneous swimming speeds and activity patterns of individuals were recorded over a period of 12 days. At 24–27 °C, spontaneous swimming speeds of common coral trout were 0.43–0.45 body lengths per second (bls^?1), but dropped sharply to 0.29 bls^?1 at 30 °C and 0.25 bls^?1 at 33 °C. Concurrently, individuals spent 9.3–10.6% of their time resting motionless on the bottom at 24–27 °C, but this behaviour increased to 14.0% at 30 °C and 20.0% of the time at 33 °C (mean ± SE). The impact of temperature was greatest for smaller individuals (<45 cm TL), showing significant changes to swimming speeds across every temperature tested, while medium (45–55 cm TL) and large individuals (>55 cm TL) were first affected by 30 °C and 33 °C, respectively. Importantly, there was some indication that populations can adapt to elevated temperature if presented with adequate time, as the high‐latitude population decreased significantly in swimming speeds at both 30 °C and 33 °C, while the low‐latitude population only showed significant reductions at 33 °C. Given that movement and activity patterns of large mobile species are directly related to prey encounter rates, ability to capture prey and avoid predators, any reductions in activity patterns are likely to reduce overall foraging and energy intake, limit the energy available for growth and reproduction, and affect the fitness and survival of individuals and populations.     

Stabilization of multimeric sucrose synthase from Acidithiobacillus caldus via immobilization and post-immobilization techniques for synthesis of UDP-glucose

Sucrose synthases (SuSys) have been attracting great interest in recent years in industrial biocatalysis. They can be used for the cost-effective production of uridine 5′-diphosphate glucose (UDP-glucose) or its in situ recycling if coupled to glycosyltransferases on the production of glycosides in the food, pharmaceutical, nutraceutical, and cosmetic industry. In this study, the homotetrameric SuSy from Acidithiobacillus caldus (SuSyAc) was immobilized-stabilized on agarose beads activated with either (i) glyoxyl groups, (ii) cyanogen bromide groups, or (iii) heterogeneously activated with both glyoxyl and positively charged amino groups. The multipoint covalent immobilization of SuSyAc on glyoxyl agarose at pH 10.0 under optimized conditions provided a significant stabilization factor at reaction conditions (pH 5.0 and 45 °C). However, this strategy did not stabilize the enzyme quaternary structure. Thus, a post-immobilization technique using functionalized polymers, such as polyethyleneimine (PEI) and dextran-aldehyde (dexCHO), was applied to cross-link all enzyme subunits. The coating of the optimal SuSyAc immobilized glyoxyl agarose with a bilayer of 25 kDa PEI and 25 kDa dexCHO completely stabilized the quaternary structure of the enzyme. Accordingly, the combination of immobilization and post-immobilization techniques led to a biocatalyst 340-fold more stable than the non-cross-linked biocatalyst, preserving 60% of its initial activity. This biocatalyst produced 256 mM of UDP-glucose in a single batch, accumulating 1 M after five reaction cycles. Therefore, this immobilized enzyme can be of great interest as a biocatalyst to synthesize UDP-glucose.

     

Covalent Immobilization of Alcohol Dehydrogenase (ADH2) from Haloferax volcanii: How to Maximize Activity and Optimize Performance of Halophilic Enzymes

Alcohol dehydrogenase from halophilic archaeon Haloferax volcanii (HvADH2) was successfully covalently immobilized on metal-derivatized epoxy Sepabeads. The immobilization conditions were optimized by investigating several parameters that affect the halophilic enzyme–support interaction. The highest immobilization efficiency (100 %) and retention activity (60 %) were achieved after 48 h of incubation of the enzyme with Ni-epoxy Sepabeads support in 100 mM Tris–HCl buffer, pH 8, containing 3 M KCl at 5 °C. No significant stabilization was observed after blocking the unreacted epoxy groups with commonly used hydrophilic agents. A significant increase in the stability of the immobilized enzyme was achieved by blocking the unreacted epoxy groups with ethylamine. The immobilization process increased the enzyme stability, thermal activity, and organic solvents tolerance when compared to its soluble counterpart, indicating that the immobilization enhances the structural and conformational stability. One step purification–immobilization of this enzyme has been carried out on metal chelate-epoxy Sepabeads, as an efficient method to obtain immobilized biocatalyst directly from bacterial extracts.