Effect of Surfactants on Mechanical,Thermal, and Photostability of a Monoclonal Antibody

The purpose of this work was to evaluate the effect of commonly used surfactants (at 0.01% w/v concentration) on mechanical, thermal, and photostability of a monoclonal antibody (MAb1) of IgG1 sub-class and to evaluate the minimum concentration of surfactant (Polysorbate 80) required in protecting MAb1 from mechanical stress. Surfactants evaluated were non-ionic surfactants, Polysorbate 80, Polysorbate 20, Pluronic F-68 (polyoxyethylene-polyoxypropylene block polymer), Brij 35 (polyoxyethylene lauryl ether), Triton X-100, and an anionic surfactant, Caprylic acid (1-Heptanecarboxylic acid). After evaluating effect of surfactants and determining stabilizing effect of Polysorbate 80 against mechanical stress without compromising thermal and photostability of MAb1, the minimum concentration of Polysorbate 80 required for mechanical stability was further examined. Polysorbate 80 concentration was varied from 0 to 0.02%. Mechanical stability was evaluated by agitation of MAb1 at 300 rotations per minute at room temperature for 72 h. Samples were analyzed for purity by SEC-HPLC, turbidity by absorbance at 350 nm, visible particles by visual inspection, and sub-visible particles by light obscuration technique on a particle analyzer. All non-ionic surfactants tested showed a similar effect in protecting against mechanical stress and did not exhibit any significant negative effect on thermal and photostability. However, Caprylic acid had a slightly negative effect on mechanical and photostability when compared to the non-ionic surfactants or sample without surfactant. This work demonstrated that polysorbate 80 is better than other surfactants tested and that a concentration of at least 0.005% (w/v) Polysorbate 80 is needed to protect MAb1 against mechanical stress.     

Effect of Polysorbate 80 Quality on Photostability of a Monoclonal Antibody

Polysorbate 80 is one of the key components of protein formulations. It primarily inhibits interfacial damage of the protein molecule due to mechanical stress during shipping and handling. However, polysorbate 80 also affects the formulation photostability. Exposure to light of polysorbate 80 aqueous solution results in peroxide generation, which in turn may result in oxidation of the susceptible amino acid residues in the protein molecule. The purpose of this study was to determine if the photostability of our proprietary IgG(1) monoclonal antibody formulation containing polysorbate 80 is affected by the quality (grade/vendor) of polysorbate 80. Following four types of polysorbate 80 were tested: (1) Polysorbate 80 Super-Refined, Mallinckrodt Baker, (2) Polysorbate 80 NF, Mallinckrodt Baker, (3) Polysorbate 80 NF, EMD Chemicals, and (4) Ultra-pure Polysorbate 80 (HX), NOF Corporation. The samples were exposed to light as per ICH guidelines Q1B. The results of the study show that photostability of the antibody formulation is indeed affected by the quality of polysorbate 80. This study underscores the importance of carefully choosing the quality of polysorbate 80 to ensure the robustness of formulation.     

Cold-Set Gelation of Commercial Soy Protein Isolate: Effects of the Incorporation of Locust Bean Gum and Solid Lipid Microparticles on the Properties of Gels

This study aimed to evaluate the ability of commercial soy protein isolate (SPI) to form cold-set gels under different pHs (5–11), pre-heating temperatures (60 °C, 80 °C), CaCl₂ (0–15 mM) and SPI (5–15%, w/v) concentrations, and also select a formulation for the investigation of the effects of incorporating locust bean gum (LBG) (0–0.3%, w/v) and solid lipid microparticles (SLM) on gels rheological and microstructural properties. Gels were evaluated in terms of visual aspect, water-holding capacity, microstructure (using confocal laser scanning microscopy and cryo-scanning electronic microscopy) and rheological properties. SPI showed higher solubilities at pHs 7 (32.0%), 9 (51.6%) and 11 (100%). Self-supported gels were obtained under several conditions at alkaline pHs. At pH 7, only systems pre-heated to 80 °C with 15% (w/v) SPI and 10 or 15 mM CaCl₂ gave self-supported gels. At neutral pH, samples showed relative structural instability, which was minimized with LBG incorporation. Formulations G_SPI (pH 7, preheated to 80 °C, 15% (w/v) SPI, 10 mM CaCl₂) and G_MIX (pH 7, preheated to 80 °C, 15% (w/v) SPI, 0.2% (w/v) LBG, 15 mM CaCl₂) were selected for emulsion-filled gels (EFG) production. Power law parameters (K′, K″), calculated from frequency sweep results, revealed that non-filled G_MIX (K′: 472.1; K″: 77.6) was stronger than G_SPI (K′: 170.4; K″: 33.6). Besides, G_MIX showed microphase separation. SLM stabilized with Tween 80-Span 80 were active fillers in EFG, altering microstructures and increasing G’, G” and the Young’s modulus (1.8 to 2.1 kPa for G_SPI and 1.4 to 2.2 kPa for G_MIX).     

Extraordinary denaturant tolerance of keratinolytic protease complex assemblies produced by Meiothermus ruber H328

A moderately thermophilic bacterial strain, Meiothermus ruber H328, can efficiently solubilize intact chicken feathers by aerobic cultivation at 55 °C for 6 days. The keratinolytic proteases extracellularly secreted by the strain were partially purified by an ultrafiltration system and a size-exclusion column chromatography, and thus were found to be two different sizes of macromolecules with an extremely high molecular mass like the sizes of virus and DNA (peak 1 fraction) and with a molecular mass of larger than 500 kDa (peak 2 fraction). They formed protein complex assemblies that were composed of multiple but different proteins. The peak 1 fraction showed more thermophilic characteristics than did the peak 2 fraction in temperature dependence and thermal stability. By contrast, they comparably showed extraordinary resistance to powerful denaturants, SDS at 30 % (w/v) and organic solvents (methanol, ethanol, acetonitrile, acetone, and chloroform) at 40 % (v/v) at 60 °C for 30 min. The extraordinary denaturant tolerance and the large molecular size of the keratinolytic protease complex assemblies suggest the possibility that those may be lipophilic and have the structure of partial membrane fractions, or membrane vesicles, which are exfoliated from the outer membrane of the cells.     

A novel surfactant-, NaCl-, and protease-tolerant β-mannanase from <Emphasis Type="Italic">Bacillus</Emphasis> sp. HJ14

A glycoside hydrolase family 5 β-mannanase-encoding gene was cloned from Bacillus sp. HJ14 isolated from saline soil in Heijing town. Coding sequence of mature protein (without the predicted signal peptide from M1 to A30) was successfully expressed in Escherichia coli BL21 (DE3). Purified recombinant mannanase (rMan5HJ14) exhibited optimal activity at pH 6.5 and 65 °C. The enzyme showed good salt tolerance, retaining more than 56 % β-mannanase activity at 3.0–30.0 % (w/v) NaCl and more than 94 % of the initial activity after incubation with 3.0–30.0 % (w/v) NaCl at 37 °C for 60 min. Almost no mannanase activity was lost after incubation of rMan5HJ14 with trypsin, proteinase K, and Alcalase at 37 °C for 60 min. Surfactants and chelating agents, namely SDS, CTAB, Tween 80, Triton X-100, EDTA, and sodium tripolyphosphate, showed little or no effect (retaining >82.4 % activity) on enzymatic activity. Liquid detergents, namely Tupperware, Walch, Bluemoon, Tide, and OMO, also showed little or no effect (retaining >72.4 % activity) on enzymatic activity at 0.5–2.0 % (v/v). The enzyme further presents a high proportion (11.97 %) of acidic amino acid residues (D and E), which may affect the SDS and NaCl tolerance of the enzyme. Together, the mannanase may be an alternative for potential use in liquid detergent industry.     

Formulation Screening and Freeze-Drying Process Optimization of Ginkgolide B Lyophilized Powder for Injection

The purpose of this study was to prepare ginkgolide B (GB) lyophilized powder for injection with excellent appearance and stable quality through a formulation screening and by optimizing the freeze-drying process. Cremophor EL as a solubilizer, PEG 400 as a latent solvent, and mannitol as an excipient were mixed to increase the solubility of GB in water to more than 18 times (about from 2.5 × 10^?4 mol/L (0.106 mg/mL) to 1.914 mg/mL). Formulation screening was conducted by orthogonal design where the content of GB in the solution before lyophilization (using external standard method of HPLC) and reconstitution time after lyophilization were the two evaluation indexes. The optimized formulations were GB in an amount of 2 mg/mL, Cremophor EL in an amount of 16% (v/v), PEG 400 in an amount of 9% (v/v), mannitol in an amount of 8% (w/v), and the solution pH of 6.5. Through four single-factor experiments (GB adding order, preparation temperature of GB solution, adding amount, and adsorption time of activated carbon), the preparation process of GB solution was confirmed. The glass transition temperature of maximally GB freeze-concentrated solution was ? 17.6°C through the electric resistance method. GB lyophilized powder began to collapse at ? 14.0°C, and the fully collapsed temperature was ? 13.0°C, which were determined by freeze-drying microscope. When the collapse temperature was determined, the primary drying temperature was obtained. Thereby, the freeze-drying curve of GB lyophilized powder was initially identified. The freeze-drying process was optimized by orthogonal design, the qualified product appearance and residual moisture content were the two evaluation indexes. The optimized process parameters and process were (1) shelf temperature, decreased from room temperature to ? 45.0°C, at 0.5°C/min in 2 h; (2) shelf temperature increased from ? 45.0 to ? 25.0°C, at 0.1°C/min, maintained for 3 h, and the chamber pressure was held at 10 Pa; (3) shelf temperature was increased from ? 25.0 to ? 15.0°C at 0.1 °C/min, maintained for 4 h, and the chamber pressure was held at 10 Pa; and (4) shelf temperature was increased from ? 15.0 to 20.0°C at 1.0 °C/min, maintained for 4 h, and the chamber pressure was raised up to 80 Pa. In these lyophilization process conditions, the products complied with relevant provisions of the lyophilized powders for injection. Meanwhile, the reproducibility was satisfactory. Post-freezing annealing had no significantly beneficial effects on shortening the freeze-drying cycle and improving the quality of GB lyophilized powder.     

Chitosan/Polyethylene Glycol Beads Crosslinked with Tripolyphosphate and Glutaraldehyde for Gastrointestinal Drug Delivery

This study reports on the preparation of chitosan (CS)/polyethylene glycol (PEG) hydrogel beads using sodium diclofenac (DFNa) as a model drug. Following the optimization of the polymer to drug ratio, the chitosan beads were modified by ionic crosslinking with sodium tripolyphosphate (TPP). The CS/PEG/DFNa beads obtained from a (w/w/w) ratio of 1/0.5/0.5 with crosslinking in 10% (w/v) TPP at pH 6.0 for 30 min yielded excellent DFNa encapsulation levels with over 90% loading efficiency. The dissolution profile of DFNa from CS/PEG/DFNa beads demonstrated that this formulation was able to maintain a prolonged drug release for approximately 8 h. Among the formulations tested, the CS/PEG/DFNa (1/0.5/1 (w/w/w)) beads crosslinked with a combination of TPP (10% (w/v) for 30 min) and glutaraldehyde (GD) (5% (w/v)) were able to provide minimal DFNa release in the gastric and duodenal simulated fluids (pH 1.2 and 6.8, respectively) allowing for a principally gradual drug release over 24 h in the intestinal (jejunum and ileum) simulated fluid (pH 7.4). Thus, overall the CS/PEG beads crosslinked with TPP and GD look to be a promising and novel alternative gastrointestinal drug release system.     

Effect of aqueous ethanol on the triple helical structure of collagen

Collagen, the most abundant protein in mammals, is widely used for making biomaterials. Recently, organic solvents have been used to fabricate collagen-based biomaterials for biological applications. It is therefore necessary to understand the behavior of collagen in the presence of organic solvents at low (≤50 %, v/v) and high (≥90 %, v/v) concentrations. This study was conducted to examine how collagen reacts when exposed to low and high concentrations of ethanol, one of the solvents used to make collagen-based biomaterials. Solubility testing indicated that collagen remains in solution at low concentrations (≤50 %, v/v) of ethanol but precipitates (gel-like) thereafter, irrespective of the method of addition of ethanol (single shot or gradual addition); this behavior is different from that observed recently with acetonitrile. Collagen retains its triple helix in the presence of ethanol but becomes thermodynamically unstable, with substantially reduced melting temperature, with increasing concentration of ethanol. It was also found that the CD ellipticity at 222 nm, characteristic of the triple-helical structure, does not correlate with the thermal stability of collagen. Time-dependent experiments reveal that the collagen triple helix is kinetically stable in the presence of 0–40 % (v/v) ethanol at low temperature (5 °C) but highly unstable in the presence of ethanol at elevated temperature (~34 °C). These results indicate that when ethanol is used to process collagen-based biomaterials, such factors as temperature and duration should be done taking into account, to prevent extensive damage to the triple-helical structure of collagen .     

Protoplast isolation for species in the Chamelaucium group and the effect of antioxidant enzymes (superoxide dismutase and catalase) on protoplast viability

An effective protocol for protoplast isolation from young leaves and somatic embryogenic cells of species in the Chamelaucium group and the use of superoxide dismutase (SOD) and catalase (CAT) to enhance protoplast viability are described. Mesophyll protoplasts were isolated from young leaves of a white Geraldton waxflower (Chamelaucium uncinatum) line 583, using a mixture of 1% (w/v) cellulase R10, 0.5% (w/v) macerozyme R10, and 0.1% (w/v) pectolyase. Viability of isolated mesophyll protoplasts increased dramatically when SOD and CAT were added. The highest increase of 7.61-fold in viability and 4.34-fold of viable protoplast yield were achieved when a combination of SOD at 500 units mL^?1 and CAT at 2,000 units mL^?1 was added to the enzyme mixture. Somatic embryogenic cell-derived protoplasts were isolated from embryogenic suspension cells of C. uncinatum line 583 when 1% (w/v) hemicellulase was added to a combination of 2% (w/v) cellulase R10, and 1% (w/v) macerozyme R10. Addition of SOD at 500 units mL^?1 and CAT at 2,000 units mL^?1 to the enzyme mixture improved viability only slightly, to above 90%, but improved yield significantly (6.6-fold). This combination of enzymes was also used to isolate protoplasts from embryogenic suspension cells of Chamelaucium repens and from young leaves of C. uncinatum, Actinodium calocephalum, Verticordia etheliana, Verticordia grandis, Verticordia hughanii, and Verticordia mitchelliana successfully with viability >80% and viable yield >7?×?10⁵ cells g^?1 fresh weight (or per milliliter packed cell volume in the case of suspension cells).     

Production of a novel compound, 10,12-dihydroxystearic acid from ricinoleic acid by an oleate hydratase from Lysinibacillus fusiformis

A recombinant oleate hydratase from Lysinibacillus fusiformis converted ricinoleic acid to a product, whose chemical structure was identified as the novel compound 10,12-dihydroxystearic acid by gas chromatograph/mass spectrometry, Fourier transform infrared, and nuclear magnetic resonance analysis. The reaction conditions for the production of 10,12-dihydroxystearic acid were optimized as follows: pH?6.5, 30 °C, 15 g?l^?1 ricinoleic acid, 9 mg?ml^?1 of enzyme, and 4 % (v/v) methanol. Under the optimized conditions, the enzyme produced 13.5 g?l^?1 10,12-dihydroxystearic acid without detectable byproducts in 3 h, with a conversion of substrate to product of 90 % (w/w) and a productivity of 4.5 g?l^?1?h^?1. The emulsifying activity of 10,12-dihydroxystearic acid was higher than that of oleic acid, ricinoleic acid, stearic acid, and 10-hydroxystearic acid, indicating that 10,12-dihydroxystearic acid can be used as a biosurfactant.     

A thermo-halo-tolerant and proteinase-resistant endoxylanase from Bacillus sp. HJ14

A glycosyl hydrolase family 10 endoxylanase from Bacillus sp. HJ14 was grouped in a separated cluster with another six Bacillus endoxylanases which have not been characterized. These Bacillus endoxylanases showed less than 52 % amino acid sequence identity with other endoxylanases and far distance with endoxylanases from most microorganisms. Signal peptide was not detected in the endoxylanase. The endoxylanase was expressed in Escherichia coli BL21 (DE3), and the purified recombinant enzyme (rXynAHJ14) was characterized. rXynAHJ14 was apparent optimal at 62.5 °C and pH 6.5 and retained more than 55 % of the maximum activity when assayed at 40–75 °C, 23 % at 20 °C, 16 % at 85 °C, and even 8 % at 0 °C. Half-lives of the enzyme were more than 60 min, approximately 25 and 4 min at 70, 75, and 80 °C, respectively. The enzyme exhibited more than 62 % xylanase activity and stability at the concentration of 3–30 % (w/v) NaCl. No xylanase activity was lost after incubation of the purified rXynAHJ14 with trypsin and proteinase K at 37 °C for 60 min. Different components of oligosaccharides were detected in the time-course hydrolysis of beechwood xylan by the enzyme. During the simulated intestinal digestion phase in vitro, 11.5–19.0, 15.3–19.0, 21.9–27.7, and 28.2–31.2 μmol/mL reducing sugar were released by the purified rXynAHJ14 from soybean meal, wheat bran, beechwood xylan, and rapeseed meal, respectively. The endoxylanase might be an alternative for potential applications in the processing of sea food and saline food and in aquaculture as agastric fish feed additive.     

Calorimetric Study of Cowpea Protein Isolates. Effect of Calcium and High Hydrostatic Pressure

The thermal properties of cowpea protein isolates (CPI) were studied by differential scanning calorimetry under the influence of various conditions. An increase in the pH of protein extraction, from 8.0 to 10.0, during CPI preparation promoted a partial denaturation of cowpea proteins. Increases in enthalpy change of denaturation (ΔH) and temperature of denaturation (Td) were detected with increasing protein concentration from 7.5 to 10.5% (w/w). This behavior suggests that denaturation involves a first step of dissociation of protein aggregates. Calcium induced thermal stabilization in cowpea proteins, the increase in Td was ca. 0.3 °C/mM for protein dispersions of 7.5% (w/w) for 0 to 40 mM CaCl₂. High hydrostatic pressure (HHP) induced denaturation in CPI in a pressure level dependent manner. The presence of calcium protected cowpea proteins towards HHP-induced denaturation when pressure level was 400 MPa, but not when it was 600 MPa. Thermal properties of cowpea protein isolates were very sensitive to processing conditions, these behaviors would have implications in processing of CPI-containing foodstuff.     

Ethanol Production from High-Solid SSCF of Alkaline-Pretreated Corncob Using Recombinant Zymomonas mobilis CP4

In this work, Zymomonas mobilis was genetically improved for pentose utilization to increase the final ethanol concentration. It showed good fermentation ability on both soluble sugar mixture and lignocellulose. Nearly all the glucose and xylose in sugar mixture can be consumed, corresponding to 86 % of theoretic ethanol yield. Simultaneous saccharification and co-fermentation (SSCF) of NaOH-pretreated corncob was then carried out in a high dry matter (DM) loading of 15–25?w/v%. At the DM loading of 15 %, the suitable operating conditions were determined, i.e., Z. mobilis loading of 0.30 g dry weight/L at 30 °C (pH?5.5), under which the ethanol concentration reached 49.2 g/L. Higher final ethanol concentrations were obtained when SSCF was operated at the fed-batch mode. Several amounts of substrate (1 % to 10 %) were added, and the highest final ethanol concentration (60.5 g/L) was obtained at 10 % DM addition.     

Increased production of γ-lactones from hydroxy fatty acids by whole Waltomyces lipofer cells induced with oleic acid

Among several fatty acids tested, oleic acid was selected as the most efficient inducer for the production of 4-hydroxydodecanoic acid, a metabolite of β-oxidation, by Waltomyces lipofer. Cells were induced by incubation for 12 h in a medium containing 10 g l^?1 yeast extract, 10 g l^?1 peptone, 5 g l^?1 oleic acid, 1 g l^?1 glucose, and 0.05 % (w/v) Tween 80. The optimal reaction conditions for the production of γ-lactones by induced cells were pH 6.5, 35 °C, 200 rpm, 0.71 M Tris, 60 g l^?1 hydroxy fatty acid, and 20 g l^?1 cells. Non-induced cells produced 38 g l^?1 γ-dodecalactone from 60 g l^?1 10-hydroxystearic acid after 30 h, with a conversion yield of 63 % (w/w) and a productivity of 1.3 g l^?1 h^?1 under the optimized conditions, whereas induced cells produced 51 g l^?1 γ-dodecalactone from 60 g l^?1 10-hydroxystearic acid after 30 h, with a conversion yield of 85 % (w/w) and a productivity of 1.7 g l^?1 h^?1. The conversion yield and productivity of induced cells were 22 % and 1.3-fold higher, respectively, than those of non-induced cells. Induced cells also produced 28 g l^?1 γ-decalactone and 12 g l^?1 γ-butyrolactone from 60 g l^?1 12-hydroxystearic acid and 60 g l^?1 10-hydroxydecanoic acid, respectively, after 30 h. The concentration, conversion yield, and productivity of γ-dodecalactone and γ-decalactone are the highest reported thus far. This is the first study on the biotechnological production of γ-butyrolactone.     

Evaluation of the in vitro methods for micropropagation of Chondracanthus acicularis (Roth) Fredericq (Gigartinales, Rhodophyta): tissue culture and production of protoplasts

Tissue was cultured and protoplasts isolated from the carrageenophyte Chondracanthus acicularis with the aim of developing micropropagation as an alternative to harvesting raw material from natural beds. Both adventitious shoots and filamentous calluses were induced by tissue culture on medium solidified with 0.4–1 % (w/v) agar. Adventitious shoots were mainly produced from discoid bases while filamentous calluses were mainly induced from basal zones and sub-apical explants. A gradient of the regeneration ability was observed from the top to the bottom of the thallus. The discoid base was the most reactive explant and produced the highest number of adventitious shoots compared to basal zones and sub-apical explants, irrespective of the concentration of agar. Protoplasts were isolated enzymatically from the whole thallus using a combination of cellulase R-10 Onozuka, macerozyme R-10, and crude extract of the gland gut of algivorous molluscs. The highest mean yield of protoplasts (1.2?×?10⁶ protoplasts g^?1 fresh weight) was obtained after 16 h of digestion with an enzyme mixture containing 2 % (w/v) cellulase R-10, 1 % (w/v) macerozyme R-10 Onozuka, 4 % (v/v) crude extract of gut gland of Haliotis, 0.8 M mannitol, 50 mM sodium citrate, 0.3 % (w/v) bovine serum albumin. Depending on the conditions, mean protoplast yields ranged from 3.14?×?10⁵ to 1.2?×?10⁶ protoplasts g^?1 fresh weight. Different factors (storage duration, mannitol, sodium citrate, crude extract of the gland gut of algivorous molluscs) were tested to improve the yield of protoplasts but none has a significantly effect.     

Temperature-induced changes of malondialdehyde, heat-shock proteins in relation to chlorophyll fluorescence and photosynthesis in Conocarpus lancifolius (Engl.)

The effect of variable temperatures (10–50 °C) on photosynthesis and chlorophyll fluorescence in Conocarpus lancifolius was evaluated. Additionally, the ability of the species to synthesize heat-shock proteins (HSPs) to protect against high temperatures, and malondialdehyde (MDA) as a by-product of lipid peroxidation was investigated. Plants at 10 °C showed virtually no measurable growth, leaf discoloration and a few brown lesions, while high temperatures (40 and 50 °C) promoted growth and lateral branch development. Chlorophyll content index, photochemical efficiency (F _v/F _m) of PS II, electron transport rate and photosynthetic rate declined with decreasing temperature but increased significantly at higher temperatures. Heat-shock protein (HSP 70 kDa) was produced at temperatures 30–50 °C and an additional 90 kDa protein was also produced at 50 °C. Increase in the efficiency of excitation energy captured by the open PS II reaction centers (F _v/F _m) increased linearly (P ≤ 0.05) with the accumulation of HSP 70 at higher temperatures. However, at low temperatures the concentration of MDA increased significantly, indicating lipid peroxidation due to oxidative stress. The production and accumulation of HSP 70 and 90 kDa coupled with increased electron transport rate and photochemical efficiency can be used to assess survival, growth capacity and to some extent the tolerance of C. lancifolius to elevated temperatures.     

Production,Purification, and Characterization of α-Amylase from Solventogenic Clostridium sp. BOH3

A solventogenic strain of Clostridium sp. BOH3 produces extracellular α-amylase (7.15 U/mg protein) in reinforced clostridial medium supplemented with sugarcane bagasse hydrolysate (1 % w/v) and a small amount of starch (0.1 % w/v), which is essential for the expression of α-amylase. In the presence of α-amylase, BOH3 utilizes starch directly without any pretreatment and produces butanol almost equivalent (～90 %) to the production of butanol from glucose. α-Amylase can be purified from culture supernatant by using one-step weak anion exchange chromatography with a yield of 43 %. In peptide fingerprinting analysis, this enzyme shows homology with α-amylase produced by Clostridium acetobutylicum ATCC824. However, the molecular weight is 54 kDa, which is smaller than α-amylase of ATCC824 (84 kDa). This enzyme has optimum temperature at 45–50 °C and optimum pH at 4.5–5.5. Under this condition, the enzyme activity is 91.32 U/mg protein, and its K _m and V _max values are 1.71?±?0.02 mg/ml and 96.13?±?0.15 μmol/min/mg protein, respectively. Activity of this α-amylase can be enhanced (>1.5 times) by addition of Ca²⁺ and Co²⁺ and its activity can be maintained at an acidic pH (pH 3–5) for about 24 h. These unique characteristics suggest that this enzyme can be used for saccharification of starch for production of biofuel in one pot.     

Acetophenone reductase with extreme stability against a high concentration of organic compounds or an elevated temperature

The gene encoding acetophenone reductase (APRD), a useful biocatalyst for producing optically pure alcohols, was cloned from the cDNA of Geotrichum candidum NBRC 4597. The gene contained an open reading frame that consisted of 1,029 nucleotides corresponding to 342 amino acid residues. The subunit molecular weight was calculated to be 36.7 kDa. The predicted amino acid sequence did not have significant similarity to those of the acetophenone reductase reported previously. The gene was inserted into the pET-21b(+) expression vector and expressed in Escherichia coli Rosetta?(DE3)pLysS by induction with 1 mM of isopropyl-β-d-thiogalactopyranoside. E. coli cell-free extract gave 21.9 U/mg APRD activity, which was 81 times that of the G. candidum cell-free extract. The enzyme was purified with a HisTrap FF crude column. The enzyme exhibited the highest activity at 60 °C, and optimum reducing and oxidizing activity were observed in a pH range around 7.0–8.0 and 8.5, respectively. The enzyme was most stable at 60 °C and pH?6.5–7.5. The Vmax and the apparent Km value of the reductase were 67.6 μmol/min per milligram of protein and 0.146 mM for acetophenone, respectively. From 4 % (v/v) 4-phenyl-2-butanone, (S)-4-phenyl-2-butanol was obtained with a yield >80 % and an enantiomeric excess >99 % in a 20 h reaction recycling NADH with 15 % (v/v) 2-propanol.     

Phase Behaviour and Formation of Fatty Acid Esters Nanoemulsions Containing Piroxicam

Fatty acid esters are long-chain esters, produced from the reaction of fatty acids and alcohols. They possess potential applications in cosmetic and pharmaceutical formulations due to their excellent wetting behaviour at interfaces and a non-greasy feeling when applied on the skin surfaces. This preliminary work was carried out to construct pseudo-ternary phase diagrams for oleyl laurate, oleyl stearate and oleyl oleate with surfactants and piroxicam. Then, the preparation and optimization study via ‘One-At-A-Time Approach’ were carried out to determine the optimum amount of oil, surfactants and stabilizer using low-energy emulsification method. The results revealed that multi-phase region dominated the three pseudo-ternary phase diagrams. A composition was chosen from each multi-phase region for preparing the nanoemulsions systems containing piroxicam by incorporating a hydrocolloid stabilizer. The results showed that the optimum amount (w/w) of oil for oleyl laurate nanoemulsions was 30 and 20 g (w/w) for oleyl stearate nanoemulsions and oleyl oleate nanoemulsions. For each nanoemulsions system, the amount of mixed surfactants and stabilizer needed for the emulsification to take place was found to be 10 and 0.5 g (w/w), respectively. The emulsification process via high-energy emulsification method successfully produced nano-sized range particles. The nanoemulsions systems passed the centrifugation test and freeze–thaw cycle with no phase failures, and stable for 3 months at various storage temperatures (3°C, 25°C and 45°C). The results proved that the prepared nanoemulsions system cannot be formed spontaneously, and thus, energy input was required to produce nano-sized range particles.     

Membrane leakage of solutes after thermal shock or freezing

Washed human erythrocytes were cooled at different rates from +37 °C to 0 °C in hypertonic solutions of either NaCl (1.2 m) or of a mixture of sucrose (40% $\text{w}\text{v}$) with NaCl (2.53% $\text{w}\text{v}$). Thermal shock hemolysis was measured and the surviving cells were examined for their mass and cell water content and also for net movements of sodium, potassium, and ¹⁴C-sucrose. The results were compared with those obtained from cells in sucrose (40% $\text{w}\text{v}$) initially, cooled at different rates to ?196 °C and rapidly thawed.The cells cooled to 0 °C in NaCl (1.2 m) showed maximal hemolysis at the fastest cooling rate studied (39 °C/min). In addition in the surviving cells this cooling rate induced the greatest uptake of ¹⁴C-sucrose and increase in cell water and cell mass and also entry of sodium and loss of cell potassium. A different dependence on cooling rate was seen with the cells cooled from +37 °C to 0 °C in sucrose (40% $\text{w}\text{v}$) with NaCl (2.53% $\text{w}\text{v}$). In this solution, survival decreased both at slow and fast cooling rates correlating with the greatest uptake of cell sucrose and increase in cell water. There was extensive loss of cell potassium and uptake of sodium at all cooling rates, the cation concentrations across the cell membrane approaching unity.The cells frozen to ?196 °C at different cooling rates in sucrose (40% $\text{w}\text{v}$) initially, also showed sucrose and water entry on thawing together with a loss of cell potassium and an uptake of cell sodium. More sucrose entered the cells cooled slowly (1.8 ° C/min) than those cooled rapidly (318 ° C/min).These results show that cooling to 0 °C in hypertonic solutions (thermal shock) and freezing to ?196 °C both induce membrane leaks to sucrose as well as to sodium and potassium. These leaks are not induced by the hypertonic solutions themselves but are due to the effects of the added stress of the temperature reduction on the membranes modified by the hypertonic solutions. The effects of cooling rate are explicable in terms of the different times of exposure to the hypertonic solutions. These results indicate that the damage observed after thermal shock or slow freezing is of a similar nature.