Showing papers in "Process Biochemistry in 2008"

Enzyme stability and stabilization—Aqueous and non-aqueous environment

Abstract: Enzyme stabilization has notable importance due to increasing number of enzyme applications. Stabilization of enzymes in order to realize their full potential as catalysts is discussed in the present review. An overview of the denaturation mechanisms in aqueous and non-aqueous environment is given. Further various methods of enzyme stabilization with respect to their use in aqueous and non-aqueous environment have been given. Using thermophilic enzymes as the reference point, a review of stabilization using various approaches like protein engineering, chemical modifications of enzymes and immobilization has been attempted. Finally, it has been stressed that, for selection of a suitable stabilization approach the intended use and possible interactions between the stabilizer-enzyme have to be taken into consideration.

Electricity production during the treatment of real electroplating wastewater containing Cr6+ using microbial fuel cell

Abstract: Microbial fuel cell (MFC) was employed to dispose Cr 6+ in real electroplating wastewater and generate electricity simultaneously. The experiments were carried out in a dual-chamber MFC. Under the condition of pH 2 and using graphite paper as the cathode electrode chromium removal and power density were highest. Moreover, increasing initial concentration of Cr 6+ could enhance the power density. The results indicated that when treating a real electroplating wastewater containing Cr 6+ with the initial concentration of 204 ppm in the MFC, the maximum power density of 1600 mW/m 2 was generated at a columbic efficiency of 12%. In addition, 99.5% Cr 6+ and 66.2% total Cr were removed through reduction of Cr 2 O 7 2− to Cr 2 O 3 precipitating on the surface of cathode electrode. MFC was proved to be a promising technology for removing Cr 6+ from electroplating wastewater.

Decolorization of synthetic dyes by laccase immobilized on epoxy-activated carriers

Abstract: The laccase from the ascomycete Myceliophthora thermophila was covalently immobilized on polymethacrylate-based polymers (Sepabeads EC-EP3 and Dilbeads NK) activated with epoxy groups. The enzyme immobilized on Sepabeads EC-EP3 exhibited notable activity (203 U/g) along with remarkably improved stability towards pH, temperature and storage time, but no increased resistance to organic solvents. In addition, the biocatalyst also showed good operational stability, maintaining 84% of its initial activity after 17 cycles of oxidation of ABTS. The immobilized laccase was applied to the decolorization of six synthetic dyes (Reactive Black 5, Acid Blue 25, Methyl Orange, Remazol Brilliant Blue B, Methyl Green and Acid Green 27) with or without the redox mediator 1-hydroxybenzotriazole. The laccase immobilized in Sepabeads EC-EP3 retained 41% activity in the decolorization of Methyl Green in a fixed-bed reactor after five cycles. The features of these biocatalysts are very attractive for their application on the decolorization of dyes in the textile industry in batch and continuous fixed-bed bioreactors.

Influence of culture conditions on the productivity and lutein content of the new strain Scenedesmus almeriensis

Abstract: This paper presents the first characterization data of the lutein-rich microalgae Scenedesmus almeriensis, a new strain isolated within a farmer's greenhouse. The main objective is to determine the appropriate conditions for the culture of this microalgae and any factors that might enhance its lutein content. The maximum growth rate was determined first in batch cultures, resulting an initial estimate of 0.63 1/day. Then, the influence of environmental culture conditions such as temperature, pH, culture medium, external irradiance, and salinity were assessed operating in continuous mode at a dilution rate half the maximum previously determined. Finally, the possible interactions between irradiance and temperature were studied by means of a surface response analysis and the optimal conditions of irradiance and temperature were tested in a separate experiment. The results show that the medium proposed by [Mann JE, Myers J. On pigments, growth and photosynthesis of Phaeodactylum tricornutum. J Phycol 1968;4:349–55] was adequate for the growth of this strain and that increasing the initial nutrient concentration did not improve the performance of the cultures. Measurements of chlorophylls fluorescence showed that there was no photoinhibition even under the highest external irradiances tested (1700 μE/m2 s). The optimal pH was found to be 8.0. With regard to the temperature, this strain grew well in the range of 30–40 °C, being clearly distressed in the experiments carried out at 48 °C. Low to medium salinities, between 0 and 5 gNaCl/L, were appropriate to promote growth rate. A response surface analysis predicted a maximum biomass productivity of 0.7 g/L day at 33 °C and 1700 μE/m2 s, and a maximum lutein content of 0.54% d. wt. at 44 °C and 1233 μE/m2 s. The experimental confirmation of the optimal conditions for lutein resulted in a biomass productivity of 0.73 g/L day with a lutein content of 0.53% d. wt., and therefore a lutein productivity of 3.8 mg/L day. The biomass of S. almeriensis is a rich source of lutein, about ten fold richer than the commercial source of this compound, Marigold flowers. The high lutein content and growth rate and the capacity to endure harsh environmental conditions make S. almeriensis a promising source of lutein.

Interfacially activated lipases against hydrophobic supports: Effect of the support nature on the biocatalytic properties

Abstract: Different lipases (lipase B from Candida Antarctica , CAL-B, lipase from Thermomyces lanuginose , TLL and lipase from Bacillus thermocatenulatus , BTL) and a phospholipase (Lecitase ® Ultra) were immobilized by interfacial activation on four different hydrophobic supports (hexyl- and butyl-toyopearl and butyl- and octyl-agarose) and their properties were compared. The results suggested that selection of different supports yielded very different results in terms of recovered activity (ranging from a sevenfold hyperactivation to almost fully inactive biocatalysts), stability, specificity and adsorption strength. Even more interestingly, the enantioselectivity of the enzymes in the hydrolysis of (±)-2- O -butyryl-2-phenylacetic acid was strongly dependent on the support utilized. For example, BTL immobilized on octyl-agarose was fully enantiospecific towards the hydrolysis of ( S )-2- O -butyryl-2-phenylacetic acid ( E > 100), whereas when immobilized on hexyl-toyopearl, the enantiomeric value of the immobilized lipase was only E = 8. However, there is not an optimal support; it depends on the lipase and on the studied parameter. In the asymmetric hydrolysis of phenylglutaric acid diethyl diester, BTL immobilized on hexyl-toyopearl was the most enantioselective catalyst with ee > 99% (A factor >100) in the production of S -monoester product, whereas the enzyme immobilized on butyl-toyopearl only exhibited an A factor of 3. Finally, butyl-agarose was chosen as the most specific support on the lipase adsorption – compared to other proteins – at low ionic strength yielding the best purification of BTL from crude preparations.

Production, purification and characterization of mid-redox potential laccase from a newly isolated Trichoderma harzianum WL1

Abstract: A new strain of Trichoderma harzianum WL1 was isolated from the Western Ghats region of Tamilnadu, India and assayed for laccase activity by ABTS oxidation. Supplementation of CuSO4 (1 mM) in liquid medium yielded high amounts of laccase (4.36 U ml−1) at an incubation period of 4 days. Laccase enzyme produced by T. harzianum in cultures supplemented with copper sulphate has been purified by ultrafiltration, Sephadex G-100 column chromatography and Concanavalin-A affinity chromatography with a final purification fold of 151.7 and a yield of 0.39%. The purified enzyme was identified as a glycoprotein with a molecular mass of 79 kDa by SDS gel electrophoresis. The UV–vis spectrum of the purified laccase had a peak at 608 and 325 nm suggesting the presence of types I and III Cu centers. The redox potential of the enzyme was found to be 692 mV for the type 1 Cu (T1) site. The optimum pH and temperature for enzyme activity were 4.5 and 35 °C, respectively. Laccase was stable for 24 h at 35 °C and had half-life of 60 min at 65 °C. Purified laccase showed Km values of 180 and 60 μM, respectively, and Vmax values of 3.95 and 1.42 U mg−1 protein, respectively, for the substrates ABTS and guaiacol. The susceptibility of laccase towards several putative inhibitors and metal cations was also assessed. The enzyme activity was completely inhibited by sodium azide (NaN3) at the concentration of 20 μM. Laccase activity was also inhibited by several metal cations, especially Hg2+.

Sugar-based growth, astaxanthin accumulation and carotenogenic transcription of heterotrophic Chlorella zofingiensis (Chlorophyta)

Abstract: The green microalga Chlorella zofingiensis can grow and produce the ketocarotenoid astaxanthin in the dark with glucose as sole carbon and energy source. In the present study, we reported that glucose, mannose, fructose, sucrose, galactose and lactose could differentially support the cell growth and astaxanthin biosynthesis. Of the sugars surveyed, glucose and mannose were the best carbon sources for the algal growth in the dark, as indicated by the relatively high specific growth rates (ca. 0.03 h(-1)) and high cell dry densities (ca. 10 g l(-1)). Furthermore, the algal cells cultured with glucose and mannose accumulated the highest amounts of astaxanthin (ca. 1 mg g(-1)), indicating a correlation between cell growth and astaxanthin formation. In addition, various sugars differentially regulated the transcription of three carotenogenic genes encoding phytoene desaturase (PDS), beta-carotene ketolase (BKT), and beta-carotene hydroxylase (CHYb) respectively. By using glucose fed-batch fermentation, high cell dry weight concentration (ca. 53 g l(-1)) and high astaxanthin production (ca. 32 mg l(-1)) were obtained, which are much higher than those ever reported in the alga. The present study suggests that C. zofingiensis is suitable for the production of natural astaxanthin on a massive scale. (C) 2008 Elsevier Ltd. All rights reserved.

Comparison study of the effects of temperature and free ammonia concentration on nitrification and nitrite accumulation

Abstract: The nitrification and nitrite accumulation were studied in various free ammonia (FA) concentration and temperature combinations. Both ammonia oxidation rate and nitrite oxidation rate increased significantly with the increase in temperature from 10 to 30 °C. The increase in the substrate oxidation rate with temperature was relatively faster for ammonia than nitrite. Nitrite accumulation started to occur at 20 °C when the specific oxidation rate of ammonia showed a higher value than that of nitrite. Subsequently, the nitrite accumulation continuously increased with the increasing temperature from 20 to 30 °C. The broad range in FA concentration exhibited negligible effect on the specific substrate utilization rate as well as the relative nitrite accumulation. On the basis of Arrhenius equation, the activation energies of ammonia oxidation were 87.1 and 38.6 kJ mol−1 in the temperature ranges 10–20 °C and 20–30 °C, respectively. However for the nitrite oxidation, the activation energy was obtained as a constant value of 34.2 kJ mol−1 in the temperature range of 10–30 °C.

Flux and performance improvement in a submerged anaerobic membrane bioreactor (SAMBR) using powdered activated carbon (PAC)

Abstract: A three litre submerged anaerobic membrane bioreactor (SAMBR) was used to examine the effect of powdered activated carbon (PAC) on COD removal and flux. 98% COD removal efficiency was achieved at a high loading rate (16 gCOD l−1 d−1) and a low hydraulic retention time (HRT) of 6 h in the presence of 3.4 g l−1 of PAC. PAC addition improved start-up and performance during a hydraulic shock by buffering the volatile fatty acids (VFAs), however, a large amount of biomass was important in accommodating the higher loading rate in the SAMBR. Batch assays showed that PAC addition improved methane potential both in terms of amount and inoculation time. COD retention inside the SAMBR for three experiments was proportional to the soluble microbial products (SMPs) contribution to COD, as (VFAs) passed through the membrane at high sparging rates (5 litres per minute—LPM). Size exclusion chromatography showed that the membrane acted as an ultra-filter (<30 kDa) as the cake or gel layer significantly reduced the amount of organics passing through the membrane. PAC adsorbed slowly biodegradable low and high MW residual COD from the bulk liquid, and hence improved COD removal and flux. Particle size distributions demonstrated that PAC also adsorbs fine colloids, and flux data showed that only a thin biofilm was formed due to the high backtransport velocity of PAC. The combined effects of adsorption of fine colloids and dissolved organics, and the formation of a thin cake layer resulted in significant flux improvement from 2 to 9 litres per square metre per hour (LMH) in the presence of 1.67 g l−1 PAC. However, addition of 3.4 g l−1 PAC reduced the flux to 5 LMH by the combined effect of viscosity and its inability to completely adsorb the dissolved organics and fine colloids that resulted in high internal fouling. Under the given operating conditions, addition of 1.67 g l−1 PAC appears to be the optimum quantity in terms of cost, flux and soluble COD removal.

Production of 3-hydroxypropionic acid from glycerol by a novel recombinant Escherichia coli BL21 strain

Abstract: 3-Hydroxypropionic acid (3-HP) is an important platform chemical from which several commodity and specialty chemicals can be generated. The present investigation focuses on the construction and evaluation of a recombinant strain Escherichia coli SH254 that produces 3-HP from glycerol. The strain was developed by cloning two genes, dhaB of Klebsiella pneumoniae DSM 2026 encoding glycerol dehydratase and aldH of E. coli K-12 MG1655 encoding aldehyde dehydrogenase, respectively. In vitro assays of crude enzyme extract of glycerol dehydratase (DhaB) showed 37.0 U mg −1 protein on glycerol with coenzyme B 12 , and partially purified aldehyde dehydrogenase (AldH) exhibited 22.8 U mg −1 protein on 3-hydroxypropionaldehyde (3-HPA) with NAD + as a cofactor. When cultivated aerobically on a glycerol medium containing yeast extract, the recombinant E. coli SH254 produced 3-HP at a maximum of 6.5 mmol l −1 (0.58 g l −1 ). The highest specific rate and yield of 3-HP production were estimated as 6.6 mmol g −1 cdw h −1 and 0.48 mol mol −1 glycerol, respectively. Although not optimized extensively, this study is encouraging for further development of a bioprocess to produce 3-HP from glycerol.

Simultaneous saccharification and fermentation of steam exploded wheat straw pretreated with alkaline peroxide

Abstract: The cellulose content of substrate is one of the most important factors for ethanol production from lignocellulose. To increase the cellulose content of substrate and ethanol yield in Simultaneous saccharification and fermentation (SSF), a pretreatment method coupling steam explosion with alkaline peroxide for wheat straw was studied. After the complex pretreatment, the cellulose content in wheat straw increased from 31.5% to 67.2%. In the hydrolysate of wheat straw pretreated with the complex method and steam explosion, the glucose concentration was 110.9 g/L and 67.8 g/L, respectively. The optimal conditions for SSF were 40 degrees C, 120 h, cellulase loading 40 FPU/(g wheat straw), yeast inoculum 10% (v/v) and substrate concentration 16.7% (w/v). Under the optimal conditions, the total ethanol concentration in SSF of wheat straw pretreated with steam explosion and alkaline peroxide reached 51.5 g/L, and an overall yield of 81.1% was obtained. (c) 2008 Elsevier Ltd. All rights reserved.

Utilization of two agroindustrial by-products for the production of a surfactant by Candida sphaerica UCP0995

Abstract: Medium constituents other than carbon sources affect the production of biosurfactants. In this work we describe a low cost medium for the production of a surfactant by the yeast Candida sphaerica. The medium was formulated only with distilled water supplemented with 5.0% ground-nut oil refinery residue plus 2.5% corn steep liquor as substrates. The isolated biosurfactant was formed with a yield of 4.5 gl−1. The biosurfactant showed high surface tension reducing activity (26 mN m−1), a small CMC value (0.08%), thermal and pH stability with respect to surface tension reducing activity and to emulsification activity against motor oil and a high level of salt tolerance. The biosurfactant was characterized as an anionic glycolipid consisting of 75% lipid and 25% carbohydrate. The potential application of the biosurfactant in oil recovery from sand was demonstrated by the percentile of oil removal (65%). The promising results obtained in this work are noteworthy for possible biosurfactant production from agricultural materials.

Preparation of cross-linked tyrosinase aggregates

Abstract: Tyrosinase from mushroom was immobilized as a cross-linked enzyme aggregate (CLEA) via precipitation with ammonium sulfate and cross-linking with glutaraldehyde. The effects of precipitation and cross-linking on CLEA activity were investigated and the immobilized tyrosinase was characterized. Sixty percent ammonium sulfate saturation and 2% glutaraldehyde were used; a 3-h cross-linking reaction at room temperature, at pH 7.0 was performed; particle sizes of the aggregates were reduced; consequently, 100% activity recovery was achieved in CLEAs with enhanced thermal and storage stabilities. Slight changes in optimum pH and temperature values of the enzyme were recorded after immobilization. Although immobilization did not affect V max , substrate affinity of the enzyme increased. Highly stable CLEAs were also prepared from crude mushroom tyrosinase with 100% activity recovery.

ACE-inhibitory peptides identified from the muscle protein hydrolysate of hard clam (Meretrix lusoria)

Abstract: The meat of hard clam was extracted using hot water. The residual meat was freeze-dried then hydrolyzed at 50 °C for 5 h by Protamex (PX). The inhibitory effects of hot water extract and hydrolysate against angiotensin I converting enzyme (ACE) were investigated. The IC 50 value of hot water extract and hydrolysate on ACE were 1.090 and 0.036 mg/ml, respectively. The PX hydrolysate was separated into five fractions by size exclusion chromatography on a Sephadex G-25 column. The fifth fraction of the hydrolysate having molecular weight ranged 350–300 Da showed the highest inhibitory efficiency ratio (IER) being 5831%/(mg ml). The amino acid sequence of the inhibitory peptide was Tyr-Asn (IC 50 = 51 μM). The hydrolysate showed mixed-type inhibition kinetics while Captopril, the positive control, showed competitive inhibition on ACE. Their K i values were 0.027 mg/ml and 0.0067 μg/ml, respectively.

Changes of microbial population and some components in carrot juice during fermentation with selected Bifidobacterium strains

Abstract: Probiotics have several advantages for human health. Since most of the probiotic foods are dairy products, they cannot be consumed by humans who are allergic to milk proteins or have severe lactose intolerance. While looking for alternative food matrices, the suitability of carrot juice as a raw material for the production of probiotic food with Bifidobacterium strains ( B. lactis Bb-12, B. bifidum B7.1 and B3.2) was investigated. Pasteurization of freshly prepared carrot juice at 80 °C for 20 min decreased the microbial population to below the detection limit (10 colony-forming unit (cfu)/ml). All tested bifidobacteria strains were capable of growing well on pure carrot juice without nutrient supplementation. Moreover, 10 7 cfu/ml initial cell concentrations resulted in 10 8 cfu/ml after 6 h of incubation, and were kept viable up to the end of fermentation (24 h). Volumetric productivities of B. lactis Bb-12, B. bifidum B7.1, and B. bifidum B3.2 were 2.16 × 10 10 cfu/l h, 4.65 × 10 10 cfu/l h, and 3.85 × 10 10 cfu/l h, respectively. Due to intense metabolism of the bacteria strains, carrot juice media were acidified to a pH level of less than 4.5. During the fermentation, the amounts of glucose and sucrose decreased significantly. Meanwhile the fructose concentration did not change. Degradation of carotenoids (α-carotene and β-carotene) was between 15–45% depending on the strain used. Production of lactic and acetic acids was in the range of 14.8–16.7 mg/ml and 3.3–5.3 mg/ml, respectively.

Characterization of Candida rugosa lipase immobilized onto magnetic microspheres with hydrophilicity

Abstract: Magnetic microspheres were prepared by suspension polymerization of glycidyl methacrylate, ethylene glycol dimethacrylate and vinyl acetate in the presence of oleic acid-coated magnetite, and then, alcoholysed to improve their hydrophilicity. Candida rugosa lipase was covalently immobilized on the hydrophilic magnetic microspheres via the active epoxy groups with the activity yield up to 64.2%. The resulting immobilized lipase had better resistance to pH and temperature inactivation in comparison to free lipase, the adaptive pH and temperature ranges of lipase were widened, and it exhibited good thermal stability and reusability. The resistance of immobilized lipase against urea denaturation was also improved, and the immobilized lipase retained 40% of the activity in the presence of 5 M urea whereas free enzyme retained only 8% of activity. Kinetic parameters were also determined for both immobilized and free lipase. The K m value for immobilized (1.03 mg/ml) was higher than that of the free lipase (0.52 mg/ml), whereas the V max value was smaller for the immobilized lipase.

Metal desorption from copper(II)/nickel(II)-spiked kaolin as a soil component using plant-derived saponin biosurfactant

Abstract: This study reports experimental measurements on the washing ability of quillaja saponin biosurfactant in kaolin suspensions, which were presorbed with the binary heavy metals of Cu(II) and Ni(II). A synthetic surfactant (SDS) and chelating agent (EDTA) were also evaluated for comparison studies. The sorption capacity for Cu(II) and Ni(II) onto kaolin were measured through the variation in pH and initial metal ion concentration. Much attention has been paid to understanding the micelle-forming properties of biosurfactant solution and its interaction with metals at the clay–water interface so washing strategies can then seek to achieve the ideal capacities of saponin biosurfactant for desorbing heavy metals from kaolin clays. The desorption of Cu(II) and Ni(II) from kaolin were examined as a function of pH, saponin concentration, temperature, time and mass/volume ratio of kaolin suspension. Because the saponin successfully competes with the kaolin clay for complexing with the metal ions by the Lewis acid–base interaction induced by pH, structure size, or charge of surfactant solution, the highest desorption improvements by 2000 mg/l of saponin were gained at 20 g/l kaolin suspension and pH 5–8, in which a single washing at room temperature could remove ∼83% of the copper and ∼85% of the nickel from kaolin containing 0.45 mg copper/g kaolin and 0.14 mg nickel/g kaolin. These results were interpreted as showing the action of a three-step micellar washing mechanism on the desorption efficiencies for Cu(II) and Ni(II). It is then pointed out quillaja saponin may be applied as a useful biosurfactant for the removal of heavy metals in contaminated clays or soils.

Aqueous two-phase poly(ethylene glycol)–poly(acrylic acid) system for protein partitioning: Influence of molecular weight, pH and temperature

Abstract: The partitioning behavior of two model proteins in poly(ethylene glycol) (PEG)–poly(acrylic acid) (PAA) aqueous two-phase systems has been studied. The effect of polymer molecular weight, tie line length (TLL), pH, temperature and NaCl concentration on the partitioning of proteins (ovalbumin and myoglobin) of different molecular weights and isoelectric points has been studied. Poly(ethylene glycol) is enriched in the top phase while poly(acrylic acid) is found in the bottom phase. An increased partitioning of proteins to the PEG phase has been observed with increased TLL. The protein partitioning decreases on increasing the molecular weight of PEG and temperature, whereas the partitioning increases with increase in pH and NaCl concentration of the system. The protein partitioning is better for both the model proteins using PEG 4000–PAA systems at 20 °C, pH 8.0 with 1 M NaCl. The percentage yield of extraction for myoglobin and ovalbumin is 95.2% and 87.4%, respectively.

Statistical optimization of enzymatic saccharification and ethanol fermentation using food waste.

Abstract: Response surface methodology (RSM) based on the 23 factorial central composite design (CCD) was applied to optimize the conditions of enzymatic saccharification and ethanol fermentation using food waste. Optimum conditions were found to be saccharification pH of 5.20, enzyme reaction temperature of 46.3 °C, enzyme concentration of 0.16% (v/v), fermentation pH of 6.85, fermentation temperature of 35.3 °C, and fermentation time of 14 h. The model predicted that maximum concentration of reducing sugar and ethanol under the above optimum conditions were 117.0 g reducing sugar/L and 57.6 g EtOH/L, respectively. Experimental results were in close agreement with model prediction with 120.1 g reducing sugar/L and 57.5 g EtOH/L, respectively.

Purification and identification of novel angiotensin-I-converting enzyme inhibitory peptides from shark meat hydrolysate

Abstract: Proteins, especially the proteins of marine origin, are potential resources of natural drugs and food additives. Our previous results showed that shark meat hydrolysate obtained with protease SM98011 digestion showed high angiotensin-I-converting enzyme (ACE) inhibitory activity, with an IC 50 value of 0.4 mg/mL. In this article, ACE inhibitory peptides were separated from shark meat hydrolysate and identified. By ultrafiltration, gel filtration and RP-HPLC, 4 peptides with high ACE inhibitory activity were purified. Their sequences identified by Secondary Ion Mass Spectrometry were Cys-Phe, Glu-Tyr, Met-Phe and Phe-Glu. Cys-Phe, Glu-Tyr and Phe-Glu were conformed to be novel ACE inhibitory peptides, with IC 50 values of 1.96, 2.68 and 1.45 μM, respectively. They may have potential in the treatment of hypertension or in clinical nutrition. This is the first report about novel ACE inhibitory peptides from hydrolysate of shark meat. This research may provide an efficient utilization of shark meat.

Catalytic studies of lipase on FAME production from waste cooking palm oil in a tert-butanol system

Abstract: In this present work, fatty acid methyl ester (FAME) was produced from waste cooking palm oil (WCPO) by lipase-catalyzed reaction. The catalytic activities of several commercial lipases and different organic solvent were screened. Novozyme 435 was found to be more effective in catalyzing the transesterification of WCPO with methanol. tert-Butanol was used as the reaction medium, which eliminated both negative effect caused by excessive methanol and glycerol as the byproduct. Several variables such as effect of methanol/oil molar ratio, effect of lipase quantity and effect of agitation speed (rpm) were examined in a batch system. Transesterification of WCPO can reach up to 88% FAME yield under optimum condition (methanol/oil molar ratio 4:1, 4% Novozyme 435 based on oil weight, 200 rpm and 12 h reaction time). Kinetics of lipase-catalyzed transesterification of WCPO in tert-butanol system was also investigated. A model based on Ping Pong Bi Bi with only inhibition by methanol was found to fit the initial rate data and the kinetics parameters were evaluated by non-linear regression analysis.

Aqueous two-phase extraction (ATPE): An attractive and economically viable technology for downstream processing of Aspergillus oryzae α-galactosidase

Abstract: Aqueous two-phase systems (ATPSs) have many applications for protein purification in biochemistry and biotechnology. One of the major challenges in the biotechnology industry is the large-scale purification of a desired protein from a fermentation broth containing a wide variety of biomolecules. One possible strategy for addressing this challenge is to use an aqueous two-phase system as the initial primary downstream processing (DSP) for partial purification of industrial enzymes. The aim of this work is to extract and purify Aspergillus oryzae α-galactosidase using an aqueous two-phase system. The α-galactosidase was extracted by partitioning in ATPS composed of polyethylene glycol (PEG) and phosphate. The effects of phase composition, molecular weight of the PEG, PEG concentration, phosphate salt concentration, pH, temperature and neutral salt (sodium chloride) concentration on enzyme partition and purification were studied. The optimal system was found at pH 5.0, containing 12% (w/w) PEG 4000 and 11.9% (w/w) phosphate with a Kα-galactosidase of 0.156, purification factor of 3.6 and 87.71% of yield of enzyme activity in the bottom phase. After purification by ATPS, the α-galactosidase optimum pH and temperature were not altered.

Purification and biochemical characterization of a novel α-amylase from Bacillus licheniformis NH1: Cloning, nucleotide sequence and expression of amyN gene in Escherichia coli

Abstract: A thermostable a-amylase from a newly isolated Bacillus licheniformis NH1 was purified, characterized and the gene was isolated, sequenced and expressed in Escherichia coli BL21. The enzyme (BLA.NH1) was purified to homogeneity by 40‐60% ammonium sulphate precipitation, Sephadex G-100 gel filtration and Sepharose mono Q anion exchange chromatography, with a 3.08-fold increase in specific activity and 15.9% recovery. The molecular weight of the BLA.NH1 was estimated to be 58 kDa by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and gel filtration. The enzyme was highly active over a wide range of pH from 5.0 to 10.0. The relative activities at pH 5.0, 9.0 and 10.0 were about 89, 96.6 and 90%, of that at pH 6.5, respectively. The optimum temperature of the purified enzymewas 90 8C. BLA.NH1 belonged to the EDTA-sensitive a-amylase, but its activity was not stimulated by the presence of Ca 2+ ions. The purified enzyme showed extreme stability towards surfactants (SDS, Tween 20 and Triton X-100) and excellent compatibility with a wide range of commercial solid and liquid detergents at 40 8C, suggesting potential application in the detergent industry. In addition, BLA.NH1 was relatively stable towards oxidizing agents, retaining 57% of its initial activity after 1 h incubation in the presence of 1% (w/v) sodium perborate. The amyN gene, which encodes the a-amylase from B. licheniformis NH1, was isolated and its DNA sequence was determined. It showed 92% homology to the sequence encoding a-amylase from B. licheniformis NCIB 8061. The sequence of the BLA.NH1 differs from that of B. licheniformis NCIB 8061 by 21 amino acids. The region, encoding the mature a-amylase was heterologously expressed in E. coli cells using the pDEST17 expression system. The recombinant (His)6-tag enzymewas purified in a single affinity chromatography step and biochemical properties of the recombinant enzyme were determined and compared to those of the native-type enzyme. Interestingly, the recombinant a-amylase showed improved thermostability compared to the native enzyme. At 85 8C, recombinant BLA.NH1 and native BLA.NH1 showed half-lives of 60 and 8 min, respectively. # 2008 Elsevier Ltd. All rights reserved.

Bisphenol A removal by a membrane bioreactor

Abstract: Endocrine disruptingchemicals(EDCs)arepotentially harmfulchemicalsduringwastewaterreclamation.BisphenolA(BPA)isa typicalEDC, and itsremovalusing asubmergedmembranebioreactor (MBR)wasinvestigated.Forcomparison, aconventionalactivated sludgereactor(CASR) was simultaneously tested using the same BPA sludge loadings as the MBR. The results showed that MBR could remove BPA a little more effectively than CASR, despite changes in sludge loadings ranging from 0.046 to 10.2 g kg 1 d 1 . However, MBR could bear much higher volume loadings than CASR and still achieve the same BPA removal efficiencies. In MBR, HRT did not obviously influence the removal of BPA. The results also showed that the contributions of sludge adsorption to BPA removal were quite low in both reactors. In addition, one metabolite of BPA biodegradation, 4-hydroxy-acetophenone, was detected. These results suggested that biodegradation dominated the BPA removal process. # 2008 Elsevier Ltd. All rights reserved.

Nitrite effect on nitrous oxide emission from denitrifying activated sludge

Abstract: Laboratory-scale experiments were conducted to examine the N 2 O emission during the denitrification process. For each of the 6 runs carried out, synthetic effluent was fed in a 10 l batch mixed liquor to investigate the effect of nitrite on N 2 O emission and Helium was continuously bubbled through the reactor at constant rate (0.12 l/min) to favour N 2 O transfer and detection. An increasing COD/NO 3 − -N influent ratio from 3 to 7 was firstly applied (runs 1–3). Secondly, NO 2 − pulse additions were performed during run 4 and 5 (10 and 20 mg N/l, respectively). Finally, the reactor was fed with influent containing both NO 2 − and NO 3 − . We showed that N 2 O emission was detected shortly after NO 2 − accumulation, few minutes after the substrate feeding. The highest emission occurred at the lower COD/NO 3 − -N ratio (=3) and at the higher NO 2 − addition (20 mg N/l). In addition, the higher nitrogen conversion to N 2 O gas (14.4%) was obtained with an influent containing initially both NO 2 − and NO 3 − . Our results suggest a direct effect of the NO 2 − concentration on the N 2 O emission. We have also confirmed the inhibitory effect of NO 2 − concentration on N 2 O reduction.

Effect of preservatives for food grade C-PC from Spirulina platensis

Abstract: The effect of selected edible preservatives, citric acid, sucrose and calcium chloride on the stability of C-phycocyanin (C-PC) at 0 ± 5 °C and 35 ± 5 °C was studied in aqueous solution. While screening the edible preservatives for a protein like C-phycocyanin, the denaturation of C-PC with urea as a denaturant and thermal unfolding studies through differential scanning calorimetry (DSC) was carried out to select a stabilizing agent having Hofmeister series behaviour acting on hydrophobic interactions. While studying the efficacy of edible preservatives, citric acid (4 mg/ml) was observed to be one of the best preservative for phycocyanin at 35 ± 5 °C in aqueous solution for 45 days with negligible loss which is comparable to the stability of C-PC at 0 ± 5 °C. Calcium chloride and sucrose were also found to be effective in maintaining the stability of C-PC in aqueous phase, but at lower temperature. Citric acid was able to maintain the stability even at higher temperature lasting for more than 1 month in aqueous solution.

Immobilization of Baker's yeast invertase in PVA–alginate matrix using innovative immobilization technique

Abstract: This paper presents an innovative technique of immobilizing Baker's yeast invertase using polyvinyl alcohol (PVA)-alginate matrix. Previous immobilization technique was improved by adding treatment solutions such as boric acid and sodium sulphate. PVA-alginate beads with four different compositions were investigated in terms of enzyme activity within the beads, immobilization yield, diffusion coefficient and also chemical and mechanical stability. The enzyme activity within the beads was also compared with the free enzyme activity. Finally, the microstructure of the beads was analyzed using SEM. Amongst others, the results revealed that the fabricated beads remained insoluble in aqueous solution owing to the innovative technique. In addition, beads produced from 12% (w/v) PVA concentration and 5% (w/v) boric acid possess at least 10% higher enzyme activity while those produced from 12% (w/v) PVA concentration and 7% (w/v) boric acid have at least 28% higher mechanical stability compared to the other formulations.

Evidence for a halotolerant-alkaline laccase in Streptomyces psammoticus: Purification and characterization

Abstract: An unusual halotolerant-alkaline laccase from Streptomyces psammoticus has been purified to homogeneity through anion exchange and gel filtration chromatography steps with an overall purification fold of 12.1. The final recovery of the enzyme was 22.1%. The molecular mass of the purified laccase was about 43 kDa. The enzyme was active in the alkaline pH range with pH optima at 8.5 and 97% activity retention at pH 9.0. The optimum temperature was 45 °C. The enzyme was stable in the pH range 6.5–9.5 and up to 50 °C for 90 min. The enzyme was tolerant to NaCl concentrations up to 1.2 M. It was inhibited by all the putative laccase inhibitors while the enzyme was activated by metal ions like Fe, Zn, Cu, Na and Mg. Fe enhanced the enzyme activity by twofold (204%). The enzyme showed lowest Km value with pyrogallol (0.25 mM) followed by ABTS (0.39 mM). The purified enzyme was a typical blue laccase with an absorption peak at 600 nm.

Hexavalent chromate reductase activity in cytosolic fractions of Pseudomonas sp. G1DM21 isolated from Cr(VI) contaminated industrial landfill

Abstract: Hexavalent chromate reductase activity was localized and characterized in vitro in cytosolic fraction of a newly isolated Pseudomonas sp. G1DM21. The suspended culture of the bacterium reduced 99.7% of 500 μM Cr(VI) and 93.06% of 1000 μM Cr(VI) in 48 h. The suspended culture repeatedly reduced 100 μM Cr(VI) within 6 h up to four consecutive inputs. The permeabilized cells of the bacterium reduced 92% of 100 μM Cr(VI) within 6 h. The cell-free extracts (CFE) reduced 90% of 100 μM Cr(VI) in 120 min. The Km and Vmax determined for chromate reductase activity in the CFE were 175 μM Cr(VI) and 1.6 μmoles/min/mg of protein, respectively, the Km and Vmax determined in the presence of 0.5 mM NADH were 150 μM Cr(VI) and 2.0 μmoles/min/mg of protein, respectively. Hexavalent chromate reductase activity was maximum at 30 °C and pH 7.0. Relative molecular mass (Mr) of the native Cr(VI) reductase in the cytosolic fraction was estimated as 61.7 kDa. The Cr(VI) reductase activity increased in the presence of metal ions like Cu2+, Mg2+, Na+ and electron donors like citrate, succinate, acetate and was significantly inhibited in the presence of metal ions like Hg2+, Ag+, Cd2+, disulfide reducers like 2-mercaptoethanol, while the respiratory inhibitors had a minute effect on the activity. Scanning probe atomic force microscopy (AFM) analysis indicates that exposure of Pseudomonas sp. G1DM21 to 1 mM Cr(VI) for 24 h, leads to an increase in cell length and height.

Development of a continuous process to adapt the textile wastewater treatment by fungi to industrial conditions

Abstract: The scale-up of a 10 L air pulsed bioreactor for the continuous treatment of textile wastewater by pellets of the white rot fungus Trametes versicolor has been carried out, based on the geometric similitude with lab-scale bioreactors (0.5 and 1.5 L). Decolourisation experiments of 150 mg L −1 Grey Lanaset G dye solution carried out in the pilot-scale bioreactor showed that in both discontinuous and continuous treatment with an HRT of 48 h, the decolourisation levels were higher than 90%. Some operational changes were carried out in the continuous decolourisation treatment of the dye solution in order to adapt the process to industrial conditions such as, non-sterilization of the dye solution, use of tap water instead of distilled water plus macronutrients and micronutrients and the use of industrial quality co-substrate instead of reagent grade. The pilot system was working continuously during 3 months and over 70 days without sterilization of the dye feeding solution, achieving good decolourisation levels (78% average during the treatment). Continuous treatment of real industrial textile wastewater under non-sterile conditions was carried out during 15 days in the pilot-scale bioreactor, with colour reduction levels between 40 and 60%. These dye concentrations are regarded as environmentally acceptable to be discharged into a municipal wastewater treatment plant if necessary according to the local regulation.