Stability and activity of Dictyoglomus thermophilum GH11 xylanase and its disulphide mutant at high pressure and temperature

The functional properties of extremophilic Dictyoglomus thermophilum xylanase (XYNB) and the N-terminal disulphide-bridge mutant (XYNB-DS) were studied at high pressure and temperature. The enzymes were quite stable even at the pressure of 500 MPa at 80 °C. The half-life of inactivation in these conditions was over 30 h. The inactivation at 80 °C in atmospheric pressure was only 3-times slower. The increase of pressure up to 500 MPa at 80 °C decreased only slightly the enzyme's stability, whereas in 500 MPa the increase of temperature from 22 to 80 °C decreased significantly more the enzyme's stability. While the high temperature (80–100 °C) decreased the enzyme reaction with short xylooligosaccharides (xylotetraose and xylotriose), the high pressure (100–300 MPa) had an opposite effect. The temperature of 100 °C strongly increased the K_m but did not affect the k_cat to the same extent, thus indicating that the interaction of the substrate with the active site suffers before the catalytic reaction begins to decrease as the temperature rises. Circular dichroism spectroscopy showed the high structural stability of XYNB and XYNB-DS at 93 °C.     

Ethyl isovalerate synthesis using Candida rugosa lipase immobilized on silica nanoparticles prepared in nonionic reverse micelles

Uniform and monodispersed silica nanoparticles were synthesized with a mean diameter of 100 ± 20 nm as analyzed by Transmission Electron Microscopy (TEM). Glutaraldehyde was used as a coupling agent for efficient binding of the lipase onto the silica nanoparticles. For the hydrolysis of pNPP at pH 7.2, the activation energy within 25–40 °C for free and immobilized lipase was 7.8 and 1.25 KJ/mol, respectively. The V_max and K_m of immobilized lipase at 25 °C for pNPP hydrolysis were found to be 212 μmol/min/mg and 0.3 mM, whereas those for free lipase were 26.17 μmol/min and 1.427 mM, respectively. The lower activation energy of immobilized lipase in comparison to free lipase suggests a change in conformation of the enzyme leading to a requirement for lower energy on the surface of the nanoparticles. A better yield (7 fold higher) of ethyl isovalerate was observed using lipase immobilized onto silica nanoparticles in comparison to free lipase.     

Copper,zinc superoxide dismutase of Curcuma aromatica is a kinetically stable protein

This study details on cloning and characterization of Cu,Zn superoxide dismutase (Ca–Cu,Zn SOD) from a medicinally important plant species Curcuma aromatica. Ca–Cu,Zn SOD was 692 bp with an open reading frame of 459 bp. Expression of the gene in Escherichia coli cells followed by purification yielded the enzyme with K_m of 0.047 ± 0.008 μM and V_max of 1250 ± 24 units/mg of protein. The enzyme functioned (i) across a temperature range of −10 to +80 °C with temperature optima at 20 °C; and (ii) at pH range of 6–9 with optimum activity at pH 7.8. Ca–Cu,Zn SOD retained 50% of the maximum activity after autoclaving, and was stable at a wide storage pH ranging from 3 to 10. The enzyme tolerated varying concentrations of denaturating agent, reductants, inhibitors, trypsin, was fairly resistant to inactivation at 80 °C for 180 min (k_d, 6.54 ± 0.17 × 10⁻³ min⁻¹; t_1/2, 106.07 ± 2.68 min), and had midpoint of thermal transition (T_m) of 70.45 °C. The results suggested Ca–Cu,Zn SOD to be a kinetically stable protein that could be used for various industrial applications.     

Immobilization of acidic lipase derived from Pseudomonas gessardii onto mesoporous activated carbon for the hydrolysis of olive oil

Mesoporous activated carbon (MAC) derived from rice husk is used for the immobilization of acidic lipase (ALIP) produced from Pseudomonas gessardii. The purified acidic lipase had the specific activity and molecular weight of 1473 U/mg and 94 kDa respectively. To determine the optimum conditions for the immobilization of lipase onto MAC, the experiments were carried out by varying the time (10–180 min), pH (2–8), temperature (10–50 °C) and the initial lipase activity (49 × 10³, 98 × 10³, 147 × 10³ and 196 × 10³ U/l in acetate buffer). The optimum conditions for immobilization of acidic lipase were found to be: time—120 min; pH 3.5; temperature—30 °C, which resulted in achieving a maximum immobilization of 1834 U/g. The thermal stability of the immobilized lipase was comparatively higher than that in its free form. The free and immobilized enzyme kinetic parameters (K_m and V_max) were found using Michaelis–Menten enzyme kinetics. The K_m values for free enzyme and immobilized one were 0.655 and 0.243 mM respectively. The immobilization of acidic lipase onto MAC was confirmed using Fourier Transform-Infrared Spectroscopy, X-ray diffraction analysis and scanning electron microscopy.     

Decay and termite resistance,water absorption and swelling of thermally compressed wood panels

This study evaluated decay and termite resistance of thermally compressed pine wood panels under pressure at either 5 or 7 MPa and either 120 or 150 °C for 1 h. Wood specimens from the panels were exposed to laboratory decay resistance tests by using the wood degrading fungi, Gloeophyllum trabeum and Trametes versicolor. The thermal compression process caused increases in density and decreases in thickness of the panels; however, laboratory decay resistance tests revealed that thermally compressed wood was not resistant against the wood degrading fungi tested. More interesting results were found in laboratory termite resistance tests by using the Eastern subterranean termites, Reticulitermes flavipes. As pressure and temperature applied to the specimens increased to 7 MPa and 120 °C, mass losses in the specimens gradually decreased in comparison with control specimens. However, the specimens compressed at 7 MPa and 150 °C showed higher mass losses when compared to the specimens compressed at 7 MPa and 120 °C. The lowest water absorption and swelling rates were seen in the specimens exposed to a pressure of 7 MPa at 120 °C. The thermal compression process at 7 MPa and 150 °C resulted in the highest water absorption and swelling in the specimens.     

Evaluation of structure and hydrolysis activity of Candida rugosa Lip7 in presence of sub-/super-critical CO2

This work aimed to assess the effect of sub-/super-critical CO₂ on the structure and activity of Candida rugosa Lip7 (CRL7) in its solution form. The structure was examined by SDS-PAGE gel electrophoresis, circular dichroism (CD) and fluorescence spectra photometry. Results revealed that the primary structure remained intact after sub-/super-critical CO₂ treatment, and the secondary structure altered at the pressure of 10 MPa and temperature 40 °C for 30 min incubation, but it was reflex to its native form with increasing incubation time up to 150 min under 10 MPa and 40 °C. Meanwhile, the tertiary structure via fluorescence spectra analysis showed that the intensity of the maximal emission wavelength at 338 nm decreased under the conditions of 10 MPa and 40 °C for 150 min. Furthermore, the residue hydrolysis activity and kinetics constants (V_max and K_m) of CRL7 treated with sub-/super-critical CO₂ were also investigated. In cases of 6 MPa and 35 °C, or 10 MPa and 40 °C for 30 min, activity variance of CRL7 was maybe caused by its secondary structure alteration. But in case of 10 MPa and 40 °C for 150 min, the tertiary structure change was perhaps responsibility for CRL7 activity enhancement.     

Effect of the timing of ice slurry ingestion for precooling on endurance exercise capacity in a warm environment

It has been demonstrated that precooling with ice slurry ingestion enhances endurance exercise capacity in the heat. However, no studies have yet evaluated the optimal timing of ice slurry ingestion for precooling. This study aimed to investigate the effects of varying the timing of ice slurry ingestion for precooling on endurance exercise capacity in a warm environment. Ten active male participants completed 3 experimental cycling trials to exhaustion at 55% peak power output (PPO) after 15 min of warm-up at 30% PPO at 30 °C and 80% relative humidity. Three experimental conditions were set: no ice slurry ingestion (CON), pre-warm-up ice slurry ingestion (−1 °C; 7.5 g kg⁻¹) (PRE), and post-warm-up ice slurry ingestion (POST). Rectal and mean skin temperatures at the beginning of exercise in the POST condition (37.1±0.2 °C, 33.8±0.9 °C, respectively) were lower than those in the CON (37.5±0.3 °C; P<0.001, 34.8±0.8 °C; P<0.01, respectively) and PRE (37.4±0.2 °C; P<0.01, 34.6±0.7 °C; P<0.01, respectively) conditions. These reductions increased heat storage capacity and resulted in improved exercise capacity in the POST condition (60.2±8.7 min) compared to that in the CON (52.0±11.9 min; effect size [ES]=0.78) and PRE (56.9±10.4 min; ES=0.34) conditions. Ice slurry ingestion after warm-up effectively reduced both rectal and skin temperatures and increased cycling time to exhaustion in a warm environment. Timing ice slurry ingestion to occur after warm-up may be effective for precooling in a warm environment.     

Thermophilic lipase from Thermomyces lanuginosus: Gene cloning,expression and characterization

An extracellular lipase gene ln1 from thermophilic fungus Thermomyces lanuginosus HSAUP₀₃80006 was cloned through RT-PCR and RACE amplification. Its coding sequence predicted a 292 residues protein with a 17 amino acids signal peptide. The deduced amino acids showed 78.4% similarity to another lipase lgy from T. lanuginosus while shared low similarity with other fungi lipases. Higher frequencies hydrophobic amino acids related to lipase thermal stability, such as Ala, Val, Leu and Gly were observed in this lipase (named LN). The sequence, -Gly-His-Ser-Leu-Gly-, known as a lipase-specific consensus sequence of mould, was also found in LN. High level expression for recombinant lipase was achieved in Pichia pastoris GS115 under the control of strong AOX1 promoter. It was purified to homogeneity through only one step DEAE-Sepharose anion exchange chromatography and got activity of 1328 U/ml. The molecular mass of one single band of this lipase was estimated to be 33 kDa by SDS-PAGE. The enzyme was stable at 60 °C and kept 65% enzyme activity after 30 min incubation at 70 °C. It kept half-activity after incubated for 40 min at 80 °C. The optimum pH for enzyme activity was 9.0 and the lipase was stable from pH 8.0 to 12.0. Lipase activity was enhanced by Ca²⁺ and inhibited by Fe²⁺, Zn²⁺, K⁺, and Ag⁺. The cell-free enzyme hydrolyzed and synthesized esters efficiently, and the synthetic efficiency even reached 81.5%. The physicochemical and catalytic properties of the lipase are extensively investigated for its potential industrial applications.     

A reduced core to skin temperature gradient,not a critical core temperature,affects aerobic capacity in the heat

The purpose of this study was to determine the impact of the core to skin temperature gradient during incremental running to volitional fatigue across varying environmental conditions. A secondary aim was to determine if a “critical” core temperature would dictate volitional fatigue during running in the heat. 60 participants (n=49 male, n=11 female; 24±5 yrs, 177±11 cm, 75±13 kg) completed the study. Participants were uniformly stratified into a specific exercise temperature group (18 °C, 26 °C, 34 °C, or 42 °C) based on a 3-mile run performance. Participants were equipped with core and chest skin temperature sensors and a heart rate monitor, entered an environmental chamber (18 °C, 26 °C, 34 °C, or 42 °C), and rested in the seated position for 10 min before performing a walk/run to volitional exhaustion. Initial treadmill speed was 3.2 km h⁻¹ with a 0% grade. Every 3 min, starting with speed, speed and grade increased in an alternating pattern (speed increased by 0.805 km h⁻¹, grade increased by 0.5%). Time to volitional fatigue was longer for the 18 °C and 26 °C group compared to the 42 °C group, (58.1±9.3 and 62.6±6.5 min vs. 51.3±8.3 min, respectively, p<0.05). At the half-way point and finish, the core to skin gradient for the 18 °C and 26 °C groups was larger compared to 42 °C group (halfway: 2.6±0.7 and 2.0±0.6 vs. 1.3±0.5 for the 18 °C, 26 °C and 42 °C groups, respectively; finish: 3.3±0.7 and 3.5±1.1 vs. 2.1±0.9 for the 26 °C, 34 °C, and 42 °C groups, respectively, p<0.05). Sweat rate was lower in the 18 °C group compared to the 26 °C, 34 °C, and 42 °C groups, 3.6±1.3 vs. 7.2±3.0, 7.1±2.0, and 7.6±1.7 g m⁻² min⁻¹, respectively, p<0.05. There were no group differences in core temperature and heart rate response during the exercise trials. The current data demonstrate a 13% and 22% longer run time to exhaustion for the 18 °C and 26 °C group, respectively, compared to the 42 °C group despite no differences in beginning and ending core temperatures or baseline 3-mile run time. This capacity difference appears to result from a magnified core to skin gradient via an environmental temperature advantageous to convective heat loss, and in part from an increased sweat rate.     

Isolation and properties of β-xylosidase from Aspergillus niger GS1 using corn pericarp upon solid state fermentation

There is growing interest in developing high-yield and low-cost production of xylanolytic enzymes for industrial applications using agroindustrial byproducts. A native strain of Aspergillus niger GS1 was used to produce β-xylosidase (EC 3.2.1.37) on solid state fermentation using corn pericarp (CP) with innovative alkaline electrolyzed water (AEW) pretreatment at room temperature. β-xylosidase was purified by ammonium sulfate fractionation followed by anion exchange and hydrophobic interaction chromatographies. β-Xylosidase showed a molecular weight of 111 kDa, isoelectric point of 5.35 and specific activity of 386.7 U (mg protein)^?1, using p-nitrophenyl-β-d-xylopyranoside as substrate, at pH 5 and 60 °C, and optimal activity at pH 4.5. Optimal temperature was 65 °C, showing full activity after 1 h at 60 °C. Activity was reduced by 1 mM β-mercaptoethanol (55.6 ± 0.1%), and enhanced by 1 mM SDS (11.0 ± 0.03%). K_m and V_max were 6.1 ± 0.9 mM and 1364 ± 105 U (mg protein)^?1, respectively, whereas k_cat was 5.1 s^?1. A predominant α-helix (41%) was determined from circular dichroism on β-xylosidase, while thermal transition profiles produced a T_m of 54.1 ± 5.8 °C, enthalpy change for unfolding of 67.4 ± 6.7 kJ/mol, and onset temperature of 37 °C. Pre-treatment of CP using AEW is an ecologically friendly alternative to chemical and heat treatments for the production of relatively high levels of β-xylosidase.     

The effect of temperature on protein refolding at high pressure: Enhanced green fluorescent protein as a model

The application of high hydrostatic pressure (HHP) impairs electrostatic and hydrophobic intermolecular interactions, promoting the dissociation of recombinant inclusion bodies (IBs) under mild conditions that favor subsequent protein refolding. We demonstrated that IBs of a mutant version of green fluorescent protein (eGFP F64L/S65T), produced at 37 °C, present native-like secondary and tertiary structures that are progressively lost with an increase in bacterial cultivation temperature. The IBs produced at 37 °C are more efficiently dissociated at 2.4 kbar than those produced at 47 °C, yielding 25 times more soluble, functional eGFP after the lower pressure (0.69 kbar) refolding step. The association of a negative temperature (−9 °C) with HHP enhances the efficiency of solubilization of IBs and of eGFP refolding. The rate of refolding of eGFP as temperature increases from 10 °C to 50 °C is proportional to the temperature, and a higher yield was obtained at 20 °C. High level refolding yield (92%) was obtained by adjusting the temperatures of expression of IBs (37 °C), of their dissociation at HHP (−9 °C) and of eGFP refolding (20 °C). Our data highlight new prospects for the refolding of proteins, a process of fundamental interest in modern biotechnology.     

Behavioral thermoregulation and critical thermal limits of giant keyhole limpet Megathura crenulata (Sowerby 1825) (Mollusca; Vetigastropoda)

The thermoregulatory behavior of the giant keyhole limpet Megathura crenulata was determined in a horizontal thermal gradient during the day at 18.9 °C and 18.3 °C for the night. The final preferendum determined for giant keyhole limpets was of 18.6±1.2 °C.Limpets' displacement velocity was 10.0±3.9 cm h⁻¹ during the light phase and 8.4±1.6 cm h⁻¹ during the dark phase. The thermotolerance (measured as CTMax at 50%) was determined in a keyhole limpet in three acclimation temperatures 17, 20, and 23 °C. Limpets were subjected to water increasing temperatures at a rate of 1 °C every 30 min, until they detached from the substrate. The critical thermal maximum at 50% was 27.2, 27.9 and 28.3 °C respectively.     

Improved high thermal stability of pullulanase from a newly isolated thermophilic Bacillus sp. AN-7

A thermophilic Bacillus sp. strain AN-7, isolated from a soil in India, produced an extracellular pullulanase upon growth on starch–peptone medium. The enzyme was purified to homogeneity by ammonium sulfate precipitation, anion exchange and gel filtration chromatography. The optimum temperature and pH for activity was 90 °C and 6.0. With half-life time longer than one day at 80 °C the enzyme proves to be thermostable in the pH range 4.5–7.0. The pullulanase from Bacillus strain lost activity rapidly when incubated at temperature higher than 105 °C or at pH lower than 4.5. Pullulanase was completely inhibited by the Hg²⁺ ions. Ca²⁺, dithiothreitol, and Mn²⁺ stimulated the pullulanase activity. Kinetic experiments at 80 °C and pH 6.0 gave V_max and K_m values of 154 U mg⁻¹ and 1.3 mg ml⁻¹. The products of pullulan were maltotriose and maltose. This proved that the purified pullulanase (pullulan-6-glucanohydrolase, EC 3.2.1.41) from Bacillus sp. AN-7 is classified under pullulanase type I. To our knowledge, this Bacillus pullulanase is the most highly thermostable type I pullulanase known to date.     

Mathematical modeling of Microcystis aeruginosa growth and [D-Leu1] microcystin-LR production in culture media at different temperatures

The effect of temperature (26 °C, 28 °C, 30 °C and 35 °C) on the growth of native CAAT-3-2005 Microcystis aeruginosa and the production of Chlorophyll-a (Chl-a) and Microcystin-LR (MC-LR) were examined through laboratory studies. Kinetic parameters such as specific growth rate (μ), lag phase duration (LPD) and maximum population density (MPD) were determined by fitting the modified Gompertz equation to the M. aeruginosa strain cell count (cells mL⁻¹). A 4.8-fold increase in μ values and a 10.8-fold decrease in the LPD values were found for M. aeruginosa growth when the temperature changed from 15 °C to 35 °C. The activation energy of the specific growth rate (Eμ) and of the adaptation rate (E₁/LPD) were significantly correlated (R² = 0.86). The cardinal temperatures estimated by the modified Ratkowsky model were minimum temperature = 8.58 ± 2.34 °C, maximum temperature = 45.04 ± 1.35 °C and optimum temperature = 33.39 ± 0.55 °C.Maximum MC-LR production decreased 9.5-fold when the temperature was increased from 26 °C to 35 °C. The maximum production values were obtained at 26° C and the maximum depletion rate of intracellular MC-LR was observed at 30–35 °C. The MC-LR cell quota was higher at 26 and 28 °C (83 and 80 fg cell⁻¹, respectively) and the MC-LR Chl-a quota was similar at all the different temperatures (0.5–1.5 fg ng⁻¹).The Gompertz equation and dynamic model were found to be the most appropriate approaches to calculate M. aeruginosa growth and production of MC-LR, respectively. Given that toxin production decreased with increasing temperatures but growth increased, this study demonstrates that growth and toxin production processes are uncoupled in M. aeruginosa. These data and models may be useful to predict M. aeruginosa bloom formation in the environment.     

An organic solvent–tolerant lipase from Streptomyces sp. CS133 for enzymatic transesterification of vegetable oils in organic media

The enzymatic route for biodiesel production has been noted to be cost ineffective due to the high cost of biocatalysts. Reusing the biocatalyst for successive transesterification cycles is a potential solution to address such cost inefficiency. However, when organic solvent like methanol is used as acyl-acceptor in the reaction, the biocatalyst (lipase) gets severely inactivated due to the inhibitory effect of undissolved methanol in the reaction medium. Thus, organic solvent–tolerant lipase is highly desirable for enzymatic transesterification. In response to such desirability, a lipase (LS133) possessing aforesaid characteristic was extracted from Streptomyces sp. CS133. Relative molecular mass of the purified LS133 was estimated to be 39.8 kDa by SDS-PAGE. Lipase LS133 was stable in pH range 5.0–9.0 and at temperature lower than 50 °C while its optimum lipolytic activity was achieved at pH 7.5 and 40 °C. It showed the highest hydrolytic activity towards long chain p-nitrophenyl palmitate with K_m and V_max values of 0.152 mM and 270.2 mmol min^?1 mg^?1, respectively. It showed non-position specificity for triolein hydrolysis. The first 15 amino acid residues of its N-terminal sequence, AIPLRQTLNFQAXYQ, were noted to have partial similarity with some of the previously reported microbial lipases. Its catalytic involvement in biodiesel production process was confirmed by performing enzymatic transesterification of vegetable oils with methanol.     

Extracellular cold-active lipase of Microbacterium luteolum isolated from Gangotri glacier,western Himalaya: Isolation,partial purification and characterization

A psychrophilic bacterium producing cold-active lipase upon growth at low temperature was isolated from the soil samples of Gangotri glacier and identified as Microbacterium luteolum. The bacterial strain produced maximum lipase at 15 °C, at a pH of 8.0. Beef extract served as the best organic nitrogen source and ammonium nitrate as inorganic for maximum lipase production. Castor oil served as an inducer and glucose served as an additional carbon source for production of cold-active lipase. Ferric chloride as additional mineral salt in the medium, highly influenced the lipase production with an activity of 8.01 U ml^?1. The cold-active lipase was purified to 35.64-fold by DEAE-cellulose column chromatography. It showed maximum activity at 5 °C and thermostability up to 35 °C. The purified lipase was stable between pH 5 and 9 and the optimal pH for enzymatic hydrolysis was 8.0. Lipase activity was stimulated in presence of all the solvents (5%) tested except with acetonitrile. Lipase activity was inhibited in presence of Mn²⁺, Cu²⁺, and Hg²⁺; whereas Fe⁺, Na⁺ did not have any inhibitory effect on the enzyme activity. The purified lipase was stable in the presence of SDS; however, EDTA and dithiothreitol inhibited enzyme activity. Presence of Ca²⁺ along with inhibitors stabilized lipase activity. The cold active lipase thus exhibiting activity and stability at a low temperature and alkaline pH appears to be practically useful in industrial applications especially in detergent formulations.     

Formulation of Pseudomonas chlororaphis strains for improved shelf life

Formulations of Pseudomonas strains with long-term shelf life are needed for commercial use in biological disease control and growth promotion in crops. In the present work Pseudomonas chlororaphis (Pc) 63-28 formulated with coconut fiber [moisture content (MC) of 80%], talc (MC 8%) or peat (MC 40%), with or without the addition of carboxymethylcellulose or xanthan gum, and formulations of Pc 63-28 and P. chlororaphis TX-1 in coconut fiber with water contents (v:v) of 75%, 45%, and 25%, were evaluated in terms of shelf life and cell viability. The shelf life of Pc 63-28 was longer when formulated in coconut fibre with a MC was 80% than in the other formulations and longer at 3 ± 1 °C compared to 22 ± 1 °C. Densities of viable Pc 63-28 cells in coconut fiber stored at 3 ± 1 °C did not decline significantly during 224 days when the MC was 80% and within 120 days at 75% MC. Densities of Pc TX-1 in coconut fiber of 75% MC did not decline within 60 days at 3 ± 1 °C. P. chlororaphis 63-28 survived longer in deionized water and buffer than in canola oil. Cells of Pc 63-28 cells formulated in coconut fibre of 80% MC after storage for 140 days at 3 ± 1 °C in coconut fiber improved hydroponic growth of hydroponic lettuce and better than cells freshly recovered from culture. We conclude that coconut fiber is a carrier of superior performance in maintaining shelf life of Pseudomonas strains. The observed shelf life would be sufficient for practical use of Pseudomonas strains as tools for disease control and growth promotion in crops.     

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.     

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.     

Seed storage and germination in Kumara plicatilis,a tree aloe endemic to mountain fynbos in the Boland,south-western Cape,South Africa

Seed storage under appropriate conditions is a relatively inexpensive means of safeguarding plant genetic material for ex situ conservation. Post-storage germination trials are used to determine the viability of stored seeds, and hence the efficacy of the particular storage treatment. Kumara plicatilis (= Aloe plicatilis) is a tree aloe endemic to mountain fynbos in the Boland, south-western Cape. The viability and germination behaviour of K. plicatilis seeds were assessed for seeds stored for four and nine months at − 80 °C, 4 °C, 25 °C and under ambient conditions in a laboratory. Seeds were germinated under controlled conditions and germination rates and percentages determined. Ungerminated seeds were tested for viability using tetrazolium salt. Seed viability was not significantly reduced during storage. Seeds stored at − 80 °C for four and nine months exhibited the fastest germination rate overall (both 5.9 ± 0.3 weeks, mean ± S.E.), and slowest was for seeds stored under ambient conditions for four and nine months (both 7.8 ± 0.4 weeks). All seed lots showed similar percentage germination after four months of storage (78.0–90.4%). The highest percentage germination overall was for seeds stored at − 80 °C for four months (90.4%) and the lowest was for seeds kept at 4 °C and − 80 °C for nine months (39.2 and 39.6%, respectively). Respective percentage viability for ungerminated seeds in these two treatments was 82% and 87%, respectively, indicating the induction of secondary dormancy. Induced dormancy triggered by protracted cold temperatures may be an adaptation that enables seeds to survive prolonged extreme conditions that are unfavourable for germination. Further research on the long-term storage of aloe seeds would be beneficial for developing long-term seed storage and germination testing protocols for ex situ conservation.