Characterization of a spore surface lipase from the biocontrol agent Metarhizium anisopliae

A Metarhizium anisopliae spore surface lipase (MASSL) strongly bound to the fungal spore surface has been purified by ion exchange chromatography on DEAE sepharose followed by ultrafiltration and hydrophobic interaction chromatography on phenyl sepharose. Electrophoretic analyses showed that the molecular weight of this lipase is ～66 kDa and pI is 5.6. Protein sequencing revealed that identified peptides in MASSL shared identity with several lipases or lipase-related sequences. The enzyme was able to hydrolyze triolein, the animal lipid cholesteryl stearate and all ρNP ester substrates tested with some preference for esters with a short acyl chain. The values of K_m and V_max for the substrates ρNP palmitate and ρNP laurate were respectively 0.474 mM and 1.093 mMol min^?1 mg^?1 and 0.712 mM and 5.696 mMol min^?1 mg^?1. The optimum temperature of the purified lipase was 30 °C and the enzyme was most stable within the most acid pH range (pH 3–6). Triton X-100 increased and SDS reduced enzyme lipolytic activity. MASSL activity was stimulated by Ca²⁺, Mg²⁺ and Co²⁺ and inhibited by Mn²⁺. The inhibitory effect on activity exerted by EDTA and EGTA was limited, while the lipase inhibitor Ebelactone B completely inhibited MASSL activity as well as PMSF. Methanol 0.5% apparently did not affect MASSL activity while β-mercaptoethanol activated the enzyme.     

Lipolytic enzyme production by Thermus thermophilus HB27 in a stirred tank bioreactor

In this study, lipolytic enzyme production by Thermus thermophilus HB27 at bioreactor scale has been investigated. Cultivation was performed in a 5-L stirred tank bioreactor in discontinuous mode, at an agitation speed of 200 rpm. Different variables affecting intra- and extra-cellular lipolytic enzyme production such as culture temperature and aeration rate have been analysed. The bacterium was able to grow within the temperature range tested (from 60 to 70 °C) with an optimum value of 70 °C for intra- and extra-cellular lipolytic enzyme production.On the other hand, various aeration levels (from 0 to 2.5 L/min) were employed. A continuous supply of air was necessary, but no significant improvement in biomass or enzyme production was detected when air flow rates were increased above 1 L/min. Total lipolytic enzyme production reached a maximum of 167 U/L after 3 days, and a relatively high concentration of extra-cellular activity was detected (40% of the total amount). Enzyme yield was around 158 U/g cells. Moreover, it is noteworthy that the lipolytic activity obtained operating at optimal conditions (70 °C and air flow of 1 L/min) was about five-fold higher than that attained in shake flask cultures     

A novel organic solvent-stable alkaline protease from organic solvent–tolerant Bacillus licheniformis YP1A

An organic solvent-stable alkaline protease producing bacterium was isolated from the crude oil contaminant soil and identified as Bacillus licheniformis. The enzyme retained more than 95% of its initial activity after pre-incubation at 40 °C for 1 h in the presence of 50% (v/v) organic solvents such as DMSO, DMF, and cyclohexane. The protease was active in a broad range of pH from 8.0 to 12.0 with the optimum pH 9.5. The optimum temperature for this protease activity was 60 °C, and the enzyme remained active after incubation at 50–60 °C for 1 h. This organic solvent-stable protease could be used as a biocatalyst for organic solvent-based enzymatic synthesis.     

Purification and characterization of an extracellular cholesterol oxidase from a Bordetella species

A soil-isolated bacterium (strain B4) was identified as a species of Bordetella and deposited with the China General Microbiological Culture Collection (code, CGMCC 2229). The bacterium grew in a mineral medium, on cholesterol as a sole source of carbon and energy. Only one metabolite of cholesterol was accumulated in detectable amounts during the strain growth. It was identified as 4-cholesten-3-one. Cholesterol oxidase (COD) (EC 1.1.3.6), which catalyzes cholesterol into this metabolite, was evidenced from the strain. The conditions of the bacterium growth were optimized for extracellular enzyme production, which then reached around 1700 UL⁻¹ within 24 h culturing. The enzyme was purified from the spent medium of the strain to homogeneity on SDS-PAGE, and characterized. Its molecular mass, as estimated by this technique, was 55 kDa. COD showed an optimum activity at pH 7.0. It was completely stable at pH 5.0 and 4 °C for 48 h, and retained 80% at least of its initial activity at pH 4.0 or at a pH of 6.0–10.0. The optimum temperature for its reaction was 37 °C. The thermal stability of COD was appreciable, as 90% or 80% of its initial activity was recovered after 1 h or 2 h incubation at 50 °C. Ag⁺ or Hg⁺ at 1 mM, was inhibitor of COD activity, while Cu²⁺, at the same concentration, was activator. The COD K_m, determined at 37 °C and pH 7.0, was 0.556 mM. The enzyme was stable at pH 7.0 and 37 °C during 24 h mechanical shaking in the presence of 33% (v/v) of either of the solvents, dimethylsulfoxide, ethyl acetate, butanol, chloroform, benzene, xylene or cyclohexane.     

Esterification and transesterification reactions catalysed by addition of fermented solids to organic reaction media

We report for the first time both the production of the lipase of Burkholderia cepacia in solid-state fermentation and the biocatalysis of esterification and transesterification reactions through the direct addition of the lyophilised fermented solids to organic reaction media. B. cepacia produced a lipolytic activity equivalent to 108 U of pNPP-hydrolysing activity per gram of dry solids after 72 h growth on corn bran with 5% (v/w) commercial corn oil as the inducer. The fermented solid material was lyophilised and added directly to the reaction medium in esterification and transesterification reactions. A factorial design was used to study the effects on esterification of temperature, alcohol-to-acid molar ratio and amount of lipolytic activity added. All three variables affected the ester yield significantly, with the amount of enzyme being most important. A 94% ester yield was obtained at 18 h at 37 °C, with an alcohol-to-acid molar ratio of 5:1 and 60 U of added lipolytic activity. For the transesterification reaction, a factorial design was undertaken with the variables being the alcohol-to-acid molar ratio and the added lipolytic activity. Ester yields of over 95% were obtained after 120 h. Our results suggest that biocatalysis using direct addition of fermented solids to organic reaction medium should be further explored.     

Immobilization of phytase on epoxy-activated Sepabead EC-EP for the hydrolysis of soymilk phytate

In this work, an active phytase concentrated extract from soybean sprout was immobilized on a polymethacrylate-based polymer Sepabead EC-EP which is activated with epoxy groups. The immobilized enzyme exhibited an activity of 0.1 U/g of carrier and activity yield of 64.7%. The optimum temperature and pH for the activity of both free and immobilized enzymes were found as 60 °C and pH 5.0, respectively. The immobilized enzyme was more stable than free enzyme in the range of pH 3.0–8.0 and more than 70% of the original activity was recovered. Both the enzymes completely retained nearly about 84% of their original activity at 65 °C. The K_m and V_max values were measured as 5 mM and 0.63 U/mg for free enzyme and 12.5 mM and 0.71 U/mg for immobilized enzyme, respectively. Free and immobilized soybean sprout phytase enzymes were also used in the biodegradation of soymilk phytate. The immobilized enzyme hydrolysed 92.5% of soymilk phytate in 7 h at 60 °C, as compared with 98% hydrolysis observed for the native enzyme over the same period of time. The immobilization procedure on Sepabead EC-EP is very cheap and also easy to carry out, and the features of the immobilized enzyme are very attractive that the potential for practical application is considerable.     

Directed immobilization of CGTase: The effect of the enzyme orientation on the enzyme activity and its use in packed-bed reactor for continuous production of cyclodextrins

In this study, two different approaches were assessed in order to direct the immobilization of a cyclodextrin glycosyltransferase on functionalized silica support, one by amino groups using glutaraldehyde activation (Si-NH-G-CGTase) and other by disulfide bond through the Cys on the enzyme surface (Si-SH-CGTase). The efficiency of the immobilization of the enzyme by the Cys in Si-SH was four times higher than with the amino group linkage in Si-NH-G (2.86% and 11.91%, respectively). After immobilization, the optimum pH remained at 5.5 for the two derivatives and the optimum temperature was 70 °C for the free enzyme, 80 °C for Si-SH-CGTase and 90 °C for Si-NH-G-CGTase. Both preparations were used for continuous production of cyclodextrins, and Si-NH-G-CGTase presented higher total productivity, retaining 100% of its initial activity for at least 200 h, while the Si-SH-CGTase presented only 40% at the same time. The Si-SH-CGTase could be reloaded with new enzymes linked by disulfide bonds and was able to be used for more than 200 h.     

A new generation of flowerlike horseradish peroxides as a nanobiocatalyst for superior enzymatic activity

Although various supports including nanomaterials have been widely utilized as platforms for enzymes immobilization in order to enhance their catalytic activities, most of immobilized enzymes exhibited reduced activities compared to free enzymes. In this study, for the first time, we used iron ions (Fe²⁺) and horseradish peroxidase (HRP) enzyme together to synthesize flowerlike hybrid nanostructures with greatly enhanced activity and stability and reported an explanation of the enhancements in both catalytic activity and stability. We demonstrated that Fe²⁺-HRP hybrid nanoflower (HNF) showed catalytic activity of ∼512% and ∼710%, respectively when stored at +4 °C and room temperature (RT = 20 °C) compared to free HRP. In addition, the HNF stored at +4 °C lost only 2.9% of its original activity within 30 days while the HNF stored at RT lost approximately 10% of its original activity. However, under the same conditions, free HRP enzymes stored at +4 °C and RT lost 68% and 91% of their activities, respectively. We claim that the drastic increases in activities of HNF are associated with to high local HRP concentration in nanoscale dimension, appropriate HRP conformation, less mass transfer limitations, and role of Fe²⁺ ion as an activator for HRP. Further biosensors studies based on enhanced activity and stability of HNF are currently underway.     

Purification and comparative characterization of monophenolase and diphenolase activities from a wild edible mushroom (Macrolepiota gracilenta)

Polyphenol oxidases (PPO) are very important enzymes group in many industrial applications, especially in food, medicine and cosmetics. PPO from Macrolepiota gracilenta, a wild edible mushroom, was purified using a Sepharose 4B-l-tyrosine-p-amino benzoic acid affinity column and characterized in terms of mono- and diphenolase activity. The highest activities for pure enzyme were observed in the presence of PHPPA and DHPPA for monophenolase and diphenolase, respectively. The enzyme showed pH optimum values at 7.0 and 5.0, respectively, for monophenolase and diphenolase activities. K_m values calculated as 0.8 mM for monophenolase and 1 mM for diphenolase activity at the presence of PHPPA and DHPPA as substrate, respectively. V_max values were calculated as 2000 U/mg protein for both activity. Monophenolase and diphenolase activities were conserved approximately 40% and 60%, respectively, in their optimum pH at 4 °C after 5 day incubation. The activities were inhibited most effectively by thiourea. The data obtained from this study showed that this enzyme could be useful for some industrial purposes.     

Extracellular expression of a thermostable phytase (phyA) in Kluyveromyces lactis

Functional expression of a thermostable phytase from A. niger was achieved in Kluyveromyces lactis GG799 cells. Effective secretion of recombinant enzyme (198 U ml⁻¹) in the fermentation broth at 72 h incubation at 22 °C was obtained. Purified enzyme showed a specific activity of 72 U mg⁻¹) and was detected on SDS-PAGE as a heavily glycosylated protein with a molecular weight of ≥140 kDa. Optimum temperature of the enzyme was at 55 °C and it showed a characteristic bi-hump pH profile with two pH optima (at pH 2.5 and 5.5). Enzyme showed considerable pepsin resistance with 60% activity retention after incubation with pepsin at the ratio of 1:1000. Enzyme was thermostable retaining 69 and 37% activity at 90 and 100 °C for 10 min respectively and remained active at these temperatures till 1 h. Deglycosylation studies demonstrated negligible effect of N-linked glycans on thermal properties. Multiple sequence alignment data revealed a conserved Asn at position 345 of this phytase which might contribute to its thermal properties. This thermostable phytase coupled with its noticeable protease resistance could be a better alternative to current commercial phytases.     

Effect of enzymatic and hydrothermal treatments of rapeseeds on quality of the pressed rapeseed oils: part II. Oil yield and oxidative stability

Response surface methodology was used to evaluate the quantitative effects of three independent variables: rapeseed moisture content (MC), enzymes dosage (ED) and conditioning temperature (T) on rapeseed oil yield (OY), efficiency of pressing (EP), and oxidative stability (OS). The highest OY (16.4%) and EP (42.8%) were obtained from pectolytic enzyme (0.1%) treated seeds (MC = 9%, T = 90 °C). The highest OS (12.6 h) was found for oil pressed from rapeseeds heated at 120 °C (MC = 11%), after the cellulolytic enzyme treatment. Results of OY, EP and OS determinations correlate with the predicted values calculated from the partial cubic models (PCMs) equations (R² = 0.9995, 0.9994, 0.9974 for the cellulolytic enzyme-treated oils and 0.9900, 0.9900, 0.9990 for the pectolytic enzyme-treated oils). The predicted optimum MC = 9.5% and 8.6%, ED = 0.06% and 0.1%, T = 91.2 °C and 90.1 °C resulted in OY = 15.5% and 16.5%, EP = 40.4% and 43.0% for rapeseed oils from seeds treated with cellulolytic and pectolytic enzymes. OS values (12.6 h and 11.8 h) at the optimum conditions of MC = 11.0% and 10.1%, ED = 0.04% and 0.08%, T = 120.0 °C and 119.9 °C for the cellulolytic and pectolytic enzyme-treated oils were also calculated using PCM. Moreover, scanning electron microscopy revealed structural changes in the rapeseed after enzymatic treatment.     

Hen egg white as a feeder protein for lipase immobilization

Enzyme stabilization via immobilization is one of the preferred processes as it provides the advantages of recovery and reusability. In this study, Thermomyces lanuginosus lipase has been immobilized through crosslinking using 2% glutaraldehyde and hen egg white, as an approach towards CLEA preparation. The immobilization efficiency and the properties of the immobilized enzyme in terms of stability to pH, temperature, and denaturants was studied and compared with the free enzyme. Immobilization efficiency of 56% was achieved with hen egg white. The immobilized enzyme displayed a shift in optimum pH towards the acidic side with an optimum at pH 4.0 whereas the pH optimum for free enzyme was at pH 6.0. The immobilized enzyme was stable at higher temperature retaining about 83% of its maximum activity as compared to the free enzyme retaining only 41% activity at 70 °C. The denaturation of lipase in free form was rapid with a half-life of 2 h at 60 °C and 58 min at 70 °C as compared to 12 h at 60 °C and 2 h at 70 °C for the immobilized enzyme. The effect of denaturants, urea and guanidine hydrochloride on the free and immobilized enzyme was studied and the immobilized enzyme was found to be more stable towards denaturants retaining 74% activity in 8 M urea and 98% in 6 M GndHCl as compared to 42% and 33% respectively in the case of free enzyme. The apparent K_m (2.08 mM) and apparent V_max (0.95 μmol/min) of immobilized enzyme was lower as compared to free enzyme; K_m (8.0 mM) and V_max (2.857 μmol/min). The immobilized enzyme was reused several times for the hydrolysis of olive oil.     

Immobilization of porcine pancreatic lipase on poly-hydroxybutyrate particles for the production of ethyl esters from macaw palm oils and pineapple flavor

Poly-hydroxybutyrate particles (PHB) were used as support to immobilize porcine pancreatic lipase (PPL). The biocatalysts prepared were tested in the synthesis of pineapple flavor by esterification of butanol and butyric acid in heptane medium, and in the synthesis of ethyl esters by transesterification of macaw palm pulp (MPPO) and macaw palm kernel (MPKO) oils with ethanol in solvent-free systems. The effect of protein loading on the biocatalyst activity was assessed in olive oil hydrolysis. Maximum hydrolytic activity of 292.8 ± 8.60 IU/g was observed. Langmuir isotherm model was applicable to the adsorption of PPL on PHB particles. Maximum immobilized protein amount was 24.3 ± 1.70 mg/g. The optimal pH and temperature in hydrolysis reaction for the immobilized PPL were at pH 8.5 and 50 °C, while for the crude PPL extract were at pH 8.0 and 45 °C. Immobilized PPL exhibited full hydrolytic activity after 2 h of incubation in non-polar solvents. In esterification reaction, optimal conversion was around 93% after 2 h of reaction. After six esterification cycles, the biocatalyst retained 63% of its initial activity. The biocatalyst prepared attained transesterification yield of 50% after 48 h of reaction for MPKO and 35% after 96 h of reaction for MPPO.     

A new thermostable and organic solvent-tolerant lipase from Aneurinibacillus thermoaerophilus strain HZ

A thermostable and organic solvent-tolerant lipase produced by Aneurinibacillus thermoaerophilus strain HZ was purified and characterised. The lipase was purified to apparent homogeneity with two steps: anion exchange chromatography on Q-Sepharose and gel filtration on Sephadex-G75. A final specific activity of 43.5 U/mg was obtained with an overall recovery of 19.7% and 15.6 purification fold. The molecular mass of the HZ lipase was estimated to be 50 kDa. The optimum pH for the activity of the purified HZ lipase was 7.0. The stability showed a broad range of pH values between pH 4.0 and 9.0 at 30 °C. The purified HZ lipase exhibited an optimum temperature of 65 °C with a half-life of 3 h and 10 min at 65 °C. The activity of the purified HZ lipase was stimulated in the presence of Ca²⁺. Organic solvents such as dimethyl sulfoxide (DMSO), methanol, n-tetradecane and n-hexadecane enhanced the lipase activity. Studies on the effect of oil showed that the lipase preferred natural oil, such as sunflower oil, over synthetic substrates.     

Stability and structural changes of horseradish peroxidase: Microwave versus conventional heating treatment

Effects of conventional heating (CH) and microwave (MW) on the structure and activity of horseradish peroxidase (HRP) in buffer solution were studied. CH incubation between 30 and 45 °C increased activity of HRP, reaching 170% of residual activity (RA) after 4–6 h at 45 °C. CH treatment at 50 and 60 °C caused HRP inactivation: RA was 5.7 and 16.7% after 12 h, respectively. Secondary and tertiary HRP structural changes were analyzed by circular dichroism (CD) and intrinsic fluorescence emission, respectively. Under CH, activation of the enzyme was attributed to conformational changes in secondary and tertiary structures. MW treatment had significant effects on the residual activity of HRP. MW treatment at 45 °C/30 W followed by CH treatment 45 °C regenerated the enzyme activity. The greatest loss in activity occurred at 60 °C/60 W/30 min (RA 16.9%); without recovery of the original activity. The inactivation of MW-treated HRP was related to the loss of tertiary structure, indicating changes around the tryptophan environment.     

Utilization of hydrolytic enzymes for the extraction of cycloalliin from garlic (Allium sativum L.)

In the present study, enzyme assisted extraction of organosulfur compounds, especially cycloalliin, from garlic (Allium sativum L.) was examined using various commercial cellulases, and the changes of the cycloalliin contents in garlic extract were investigated after storage at 40 °C and 60 °C for 30 days. Among the commercial enzymes tested, Ultraflo L showed the greatest yield of cycloalliin compared to other cellulases. The conditions were optimized to include 2.5% (v/w) addition of Ultraflo L, 1 h incubation at 40 °C and a pH of 6.0. Under the optimum conditions, the contents of cycloalliin achieved 1.5-folds increase in the enzyme-assisted garlic extract compared with the non-enzymatic extraction. In addition, the cycloalliin content was also significantly increased 3.8-fold after storage at 60 °C for 15 days. The polyphenol content was also significantly increased by 3-fold after at 60 °C for 30 days. Overall, Ultraflo L proved to be an efficient method to extract cycloalliin from garlic.     

Cloning of a GH5 endoglucanase from genus Penicillium and its binding to different lignins

The cel5C gene, coding for an endoglucanase (Cel5C) of Penicillium brasilianum was cloned and heterologously expressed in Aspergillus oryzae. This is only the second GH5 EG from the genus Penicillium reported in the CAZy database. The promoter region of the gene has putative binding sites for both the carbon catabolite repressor CreA and the activator XlnR. The pH optimum of Cel5C was found to be 4.0 and the temperature optimum was 70 °C. At a typical temperature for lignocellulose hydrolysis Cel5C retained full residual activity after 20 h of incubation at pH 5.0 and 6.0. Adsorption to Avicel and steam pretreated spruce, was found to follow the Langmuir isotherm, and the maximum adsorption was similar for both substrates, 40 and 49 mg/g, respectively. The affinity for Avicel was 10 times higher than for steam pretreated spruce, 0.040 and 0.0035 L/mg, respectively. Non-productive binding of cellulolytic enzymes to lignin is an important obstacle to overcome for commercial biomass to ethanol production. Therefore, the adsorption on residual lignin produced from various biomass samples was investigated. Both substrate and pretreatment conditions resulted in different adsorptions of Cel5C to the residual lignin.     

C-Terminal proline-rich sequence broadens the optimal temperature and pH ranges of recombinant xylanase from Geobacillus thermodenitrificans C5

Efficient utilization of hemicellulose entails high catalytic capacity containing xylanases. In this study, proline rich sequence was fused together with a C-terminal of xylanase gene from Geobacillus thermodenitrificans C5 and designated as GthC5ProXyl. Both GthC5Xyl and GthC5ProXyl were expressed in Escherichia coli BL21 host in order to determine effect of this modification. The C-terminal oligopeptide had noteworthy effects and instantaneously extended the optimal temperature and pH ranges and progressed the specific activity of GthC5Xyl. Compared with GthC5Xyl, GthC5ProXyl revealed improved specific activity, a higher temperature (70 °C versus 60 °C) and pH (8 versus 6) optimum, with broad ranges of temperature and pH (60–80 °C and 6.0–9.0 versus 40–60 °C and 5.0–8.0, respectively). The modified enzyme retained more than 80% activity after incubating in xylan for 3 h at 80 °C as compared to wild −type with only 45% residual activity. Our study demonstrated that proper introduction of proline residues on C-terminal surface of xylanase family might be very effective in improvement of enzyme thermostability. Moreover, this study reveals an engineering strategy to improve the catalytic performance of enzymes.     

Immobilization of α-galactosidase on galactose-containing polymeric beads

Industrial application of α-galactosidase requires efficient methods to immobilize the enzyme, yielding a biocatalyst with high activity and stability compared to free enzyme. An α-galactosidase from tomato fruit was immobilized on galactose-containing polymeric beads. The immobilized enzyme exhibited an activity of 0.62 U/g of support and activity yield of 46%. The optimum pH and temperature for the activity of both free and immobilized enzymes were found as pH 4.0 and 37 °C, respectively. Immobilized α-galactosidase was more stable than free enzyme in the range of pH 4.0–6.0 and more than 85% of the initial activity was recovered. The decrease in reaction rate of the immobilized enzyme at temperatures above 37 °C was much slower than that of the free counterpart. The immobilized enzyme shows 53% activity at 60 °C while free enzyme decreases 33% at the same temperature. The immobilized enzyme retained 50% of its initial activity after 17 cycles of reuse at 37 °C. Under same storage conditions, the free enzyme lost about 71% of its initial activity over a period of 7 months, whereas the immobilized enzyme lost about only 47% of its initial activity over the same period. Operational stability of the immobilized enzyme was also studied and the operational half-life (t_1/2 was determined as 6.72 h for p-nitrophenyl α-d-galactopyranoside (PNPG) as substrate. The kinetic parameters were determined by using PNPG as substrate. The K_m and V_max values were measured as 1.07 mM and 0.01 U/mg for free enzyme and 0.89 mM and 0.1 U/mg for immobilized enzyme, respectively. The synthesis of the galactose-containing polymeric beads and the enzyme immobilization procedure are very simple and also easy to carry out.     

Temperature modulates hepatic carbohydrate metabolic enzyme activity and gene expression in juvenile GIFT tilapia (Oreochromis niloticus) fed a carbohydrate-enriched diet

The effects of rearing temperature on hepatic glucokinase (GK), glucose-6-phosphatase (G6Pase) and Glucose-6-phosphate dehydrogenase (G6PD) activity and gene expression were studied in GIFT (genetically improved farmed tilapia) tilapia fed a high carbohydrate diet containing 28% crude protein, 5% crude lipid and 40% wheat starch. Triplicate groups of fish (11.28 g initial body weight) were fed the diet for 45 days at 22 °C, 28 °C or 34 °C. At the end of the trial, final body weight of juvenile at 28 °C (59.12 g) was higher than that of the fish reared at 22 °C (27.13 g) and 34 °C (43.17 g). Feed intake, feed efficiency and protein efficiency ratio were also better at 28 °C. Liver glycogen levels were higher at 28 °C, while plasma glucose levels were higher in the 22 °C group. Significant (P<0.05) effects of water temperature on enzymes activities and gene expression were observed. Hepatic GK activity and mRNA level were higher at 28 °C than at 34 °C. Higher G6Pase and G6PD activity and gene expression were observed at 22 °C. Overall, the data show that juveniles reared at 28 °C exhibited enhanced liver glycolytic capacity. In contrast, hepatic gluconeogenesis and lipogenesis were increased by low temperature (22 °C).