Use of enzymatic cell wall degradation for improvement of protein extraction from Chondrus crispus,Gracilaria verrucosa and Palmaria palmata

The effect of polysaccharidases (κ-carrageenase, β-agarase, xylanase, cellulase) on the protein extraction from three rhodophytes has been studied. The kinetic parameters (apparent V _m, apparent K _m) and the optimum activity conditions (pH, temperature) of each enzyme were determined by using pure substrates. All the tested enzymes possess Michaelis Menten mechanism with estimated substrate saturating concentrations of 8 000 mg l⁻¹(carrageenan) for κ-carrageenase, 8 000 mg l⁻¹ (agar) for β-agarase, 5000 mg l⁻¹ (xylane) for β-xylanase and 6 000 mg l⁻¹ (carboxymethylcellulose) for cellulase. The optimum activity conditions are pH 6.5–6.8 at 45°C for carrageenase, pH 6–6.5 at 55°C for agarase, pH 5 at 55°C for xylanase and pH 3.8 at 50°C for cellulose. Different alga/enzymes couples (κ-carrageenase/Chondrus crispus, β-agarase/Gracilaria verrucosa, β-xylanase/Palmaria palmata) were tested under the optimum activity conditions. Alga/cellulase + specific enzyme (e.g. Chondrus crispus/carrageenase + cellulase) systems were also studied at the optimum activity conditions of a specific enzyme (e.g. carageenase). The use of the only cellulose was also tested on each alga. Except for Palmaria palmata, the highest protein yields were observed with the procedures using cellulase coupled with carrageenase or agarase for an incubation period limited to 2 h. The Chondrus crispus/carrageenase + cellulose and Gracilaria verrucosa/agarase + cellulase systems gave ten-fold and three-fold improvements, respectively, in protein extraction yield as compared to the enzyme-free blank procedure. The combined action of xylanase and cellulose on protein extraction from Palmaria palmata does not significantly improve protein yield. The best overall protein yield for P. palmata is for P. palmata/xylanase with a 14-h incubation time. This study shows the interest in the use of a polysaccharidase mixture for improving protein extractibility from certain rhodophytes. This biotechnology approach, adapted from procedures for protoplast production or enzymatic liquefaction of higher plants, could be tested as an alternative method to obtain proteins from seaweeds of nutritional interest.     

Medium optimization for chitinase production from Trichoderma virens using central composite design

Medium development for chitinase production by Trichoderma virens was first carried out using conventional method of one-factor-at-a-time. The medium was further optimized using Central Composite Design in which response surface was generated later from the derived model. An experimental design of four variables including various initial pH values, chitin, ammonium sulphate, and methanol concentrations were created using Design Expert® Software, Version 6.0. The design consists of 30 experiments, which include 6 replicates at center points. The optimal value for each variable are 3.0 g/L, chitin; 0.1 g/L, ammonium sulphate; 0.4% (v/v), methanol; and initial pH, 4.0 with predicted chitinase activity of 0.1495 U/mL. These predicted parameters were tested in the laboratory and the final chitinase activity obtained was 0.1471 U/mL, which is almost reaching the predicted value. The optimal medium design showed an improvement of chitinase activity of 80.9% compared to activity obtained from the original Absidia medium composition.     

Isolation and characterisation of a fourth hemagglutinin from the red alga, Gracilaria verrucosa, from Japan

Isolation and characterisation of marine algal hemagglutinins or lectins are essential for their potential industrial application as specific carbohydrate affinity ligands. The phosphate buffer extract of the red alga, Gracilaria verrucosa (Huds.) Papenfuss (Gigartinales, Rhodophyta) from Japan is known to contain three different hemagglutinins. The extract of the alga collected in March 1993 from Kagawa Prefecture, Japan, was purified by ammonium sulphate fractionation, ion exchange and gel filtration chromatography. Using gel filtration, two peaks were obtained (hereafter Peak 1 and Peak 2) which differed in molecular size and hemagglutinating activity against horse erythrocytes. Peak 1 corresponded to the known high molecular weight hemagglutinin, H-GVH. Peak 2 contained large amounts of hexose and sulphate along with a small amount of protein. It had a low molecular weight (gel filtration) similar to that of two of the previously reported G.verrucosa hemagglutinins but differed in its electrophoretic behaviour. Peak 2 is therefore a fourth hemagglutinin. Its activity was not inhibited by any of the monosaccharides tested but by the complex glycoproteins such as asialofetuin and fetuin. It had no divalent cation requirement for hemagglutination. The properties of this novel hemagglutinin could prove useful in industrial applications. This revised version was published online in June 2006 with corrections to the Cover Date.     

Modeling of polyphenols extraction from pomegranate by-product using rotatable central composite design of experiments

There is a growing request to find an effective method of polyphenols extraction from agro-industry by-product as pomegranate. In this study, response surface methodology (RSM) was used to explore the effect of three factors on ultrasonic assisted extraction (UAE) of total polyphenols (TP), total flavonoids (TF) and condensed tannins (CT) from pomegranate peels. The optimal conditions were determined for each phenolic compound using regression model equations and 3-D plots. The high TP, TF and CT content were obtained with, respectively, liquid/solid ratio of 20.00, 9.77, 9.77, extraction time of 36.38, 41.82, 30.39 min and 36.00, 83.64, 59.26% of ethanol percentage. In fact, liquid/solid ratio of 20, extraction time of 30.94 min and 59.26% of ethanol gives the highest contents of TP, TF and CT simultaneously. The experimental values using optimal conditions agreed with the predicted values. The pomegranate peels extract obtained under optimum conditions have an effective antioxidant activity as determined by ABTS and DPPH assays.     

Bioconversion of palm oil mill effluent for citric acid production: statistical optimization of fermentation media and time by central composite design

A laboratory-scale study was conducted to evaluate the feasibility of using palm oil mill effluent (POME) as a major substrate and other nutrients for maximum production of citric acid using the potential fungal strain Aspergillus niger (A103). Statistical optimization of medium composition (substrate–POME, co-substrates–wheat flour and glucose, and nitrogen source–ammonium nitrate) and fermentation time was carried out by central composite design (CCD) to develop a polynomial regression model through the effects of linear, quadratic, and interaction of the factors. The statistical analysis of the results showed that, in the range studied, ammonium nitrate had no significant effect whereas substrate, co-substrates and fermentation time had significant effects on citric acid production. The optimized medium containing 2% (w/w) of substrate concentration (POME), 4% (w/w) of wheat flour concentration, 4% (w/w) of glucose concentration, 0% (w/v) of ammonium nitrate and 5 days fermentation time gave the maximum predicted citric acid of 5.37 g/l which was found to be 1.5 g/l in the experimental run. The determination of coefficient (R ²) from the analysis observed was 0.964, indicating a satisfactory adjustment of the model with the response. The analysis showed that the major substrate POME (P < 0.05), glucose (P < 0.01), nutrient (P < 0.05), and fermentation time (P < 0.01) was more significant for citric acid production. The bioconversion of POME for citric acid production using optimal conditions showed the higher removal of chemical oxygen demand (82%) with the production of citric acid (5.2 g/l) on the final day of fermentation process (7 days). The pH and biosolids accumulation were observed during the bioconversion process.     

Development and optimization of solid lipid nanoparticles of amikacin by central composite design

Solid lipid nanoparticles (SLNs) have been studied as a drug-delivery system for the controlling of drug release. These colloidal systems have many important advantages, such as biocompatibility, good tolerability, and ease of scale-up. In the preparation of SLNs, many factors are involved in the characteristics of the particles, such as particle size, drug loading, and zeta potential. In this study, fractional factorial design was applied to examine which variables affect the physicochemical properties of amikacin SLNs. Study was continued by a statistical central composite design (CCD) to minimize particle size and maximize drug-loading efficiency of particles. The results showed that three quantitative factors, including the amount of lipid phase, ratio of drug to lipid, and volume of aqueous phase, were the most important variables on studied responses. The best predicted model for particle size was the quadratic model, and for drug-loading efficiency, was the linear model without any significant lack of fit. Optimum condition was achieved when the ratio of drug to lipid was set at 0.5, the amount of lipid phase at 314?mg, and the volume of aqueous phase at 229?mL. The optimized particle size was 149?±?4?nm and the drug-loading efficiency 88?±?5%. Polydispersity index was less than 0.3. The prepared particles had spherical shape, and the drug release from nanoparticles continued for 144 hours (6 days) without significant burst effect.     

Modeling and optimization of lipase-catalyzed production of succinic acid ester using central composite design analysis

Esterification of succinic acid with oleyl alcohol catalyzed by immobilized Candida antarctica lipase B (Novozym 435) was investigated in this study. Response surface methodology (RSM) based on a five-level, four-variable central composite design (CCD) was used to model and analyze the reaction. A total of 21 experiments representing different combinations of the four parameters including temperature (35–65°C), time (30–450 min), enzyme amount (20-400 mg), and alcohol:acid molar ratio (1:1-8:1) were generated. A partial cubic equation could accurately model the response surface with a R² of 0.9853. The effect and interactions of the variables on the ester synthesis were also studied. Temperature was found to be the most significant parameter that influenced the succinate ester synthesis. At the optimal conditions of 41.1°C, 272.8 min, 20 mg enzyme amount and 7.8:1 alcohol:acid molar ratio, the esterification percentage was 85.0%. The model can present a rapid means for estimating the conversion yield of succinate ester within the selected ranges.     

Process design and optimization of bioethanol production from cassava bagasse using statistical design and genetic algorithm

Abstract

Bioethanol production from agro-industrial residues is gaining attention because of the limited production of starch grains and sugarcane, and food–fuel conflict. The aim of the present study is to maximize the bioethanol production using cassava bagasse as a feedstock. Enzymatic liquefaction, by α-amylase, followed by simultaneous saccharification and fermentation (SSF), using glucoamylase and Zymomonas mobilis MTCC 2427, was investigated for bioethanol production from cassava bagasse. The factors influencing ethanol production process were identified and screened for significant factors using Plackett–Burman design. The significant factors (cassava bagasse concentration (10–50?g/L), concentration of α-amylase (5–25% (v/v), and temperature of fermentation (27–37?°C)) were optimized by employing Box–Behnken design and genetic algorithm. The maximum ethanol concentrations of 25.594?g/L and 25.910?g/L were obtained from Box–Behnken design and genetic algorithm, respectively, under optimum conditions. Thus, the study provides valuable insights in utilizing the cost-effective industrial residue, cassava bagasse, for the bioethanol production.     

The origin of red algae and cryptomonad nucleomorphs: A comparative phylogeny based on small and large subunit rRNA sequences of Palmaria palmata, Gracilaria verrucosa, and the Guillardia theta nucleomorph

The complete large subunit rRNA sequences from the red algae Palmaria palmata and Gracilaria verrucosa, and from the nucleomorph of the cryptomonad Guillardia theta, were determined in order to assess their phylogenetic relationships relative to each other and to other eukaryotes. Neighbor-joining, maximum-parsimony, and maximum-likelihood trees were constructed on the basis of small subunit rRNA, large subunit rRNA, and a combination of both molecules. Our results support the hypothesis that the cryptomonad plastid is derived from a primitive red alga, in that an ancient common ancestor of rhodophytes and cryptomonad nucleomorphs is indicated. This cluster shows some affinity with chlorobionts, which could point to a monophyletic origin of green and red plastids. However, the exact branching order of the crown eukaryotes remains uncertain and further research is required.     

Some new species and new combinations of marine red algae from the central Pacific

Six new species in five genera of Rhodophyta are described. A new combination for Polyopes hakalauensis is also proposed. These taxa were encountered while preparing a manual of marine red algae of the Hawaiian Islands. Among the Halymeniales, two blade-like species of Halymenia, H. cromwellii sp. nov. and H. stipi-tata sp. nov., are distinguished from other species by habit and anatomical differences, including the possession of a stipe by the second. Halymenia hakalauensis Tilden 1902 is transferred to Polyopes as P. hakalauensis (Tilden) Abbott, replacing P. hawaiiensis Kajimura which is considered a synonym. A new species of Prionitis, P. corymbifera sp. nov., shows a corymbose branching pattern different from the mostly pinnate to subdichotomous branching of Japanese and Californian species. In the Gigartinales, Chondracanthus okamurae Abbott is named for a specimen which Okamura included in his understanding of Gigartina (now Chondracanthus tenellus), but which has terete axes, differing from C. tenellus (Harvey) Hommersand which is com-planate throughout. Chondracanthus intermedius (Sur-ingar) Hommersand is placed in synonymy with C. tenellus. The occurrence of C. tenellus in Hawai'i is a new distribution record from Asia. Three new species are added to the Ceramiales, Ceramium tranquillum Meneses (Ceramiaceae), Dasya kristeniae sp. nov. and Dasya murrayana Abbott & Millar (Dasyaceae). Distinctive features of Ceramium tranquillum are internodes that are three to five times longer than the height of nodes, and nodes that never project beyond the diameter of the internodes, giving straight margins except when fertile. Dasya kristeniae is a diminutive epiphyte which is nearly ecorticate, with tetrasporangial stichidia that are rectangular having apices that are curved or nodding, and cystocarps that have flaring ostioles above a constricted pericarp. Dasya murrayana was earlier misidentified with Dasya iyengarii Børgesen, and re-examination of those plants show continuous cortication throughout, where cortication is almost completely lacking in D. iyengarii except near the base. Dasya murrayana has a tufted habit, with indeterminate branches about the same sizes as the main axes, the plants growing on rock, whereas plants of D. iyengarii are not tufted, with indeterminate branches attenuate, and an epiphytic habit.     

Optimisation of a microwave-assisted method for extracting withaferin A from Withania somnifera Dunal. using central composite design

Introduction – Recently, there have been growing attention on the modification and optimisation of new extraction and quantification methods, caused by the lack of environmentally friendly methodologies for the extraction of phytochemicals from complex matrices. In the case of pharmaceutical compounds, not only the extraction procedure but also the analysis method should be efficient, precise, fast and easy. Objectives – The essential pharmaceutical characteristics and trace concentration of withanolides led us to modify and optimise the previously reported extraction and quantification procedure for withaferin A (WA) as a candidate for withanolides. Matrial and methods – The WA from the air‐dried aerial part of Withania somnifera Dunal. was extracted using a microwave‐assisted extraction (MAE) technique. Four variables affecting the extraction procedure were optimised using the central composite design approach. The method of high‐performance thin‐layer chromatography assay was validated and applied for the quantification of each experiment. Results – The optimum values of factors were: extraction time (150 s), extraction temperature (68°C) and 17 mL of methanol : water in the ratio 25 : 75 as extracting solvent. The solvent system consisted of ethyl acetate : toluene : formic acid : 2‐propanol (7.0 : 2.0 : 0.5 : 0.5, v/v/v/v), and densitometric scanning at 220 nm was applied for the analysis. The dynamic linear range, LOD, LOQ and recovery with the inter‐day, and intra‐day RSDs of the developed method indicated the validity of the method. Conclusion – A pressurised MAE method for extracting WA from the plant's aerial part was optimised using factorial‐based design. The net effect of time, temperature, solvent volume and its ratio suggests that the yield of WA increases until each factor reaches its optimum value, and decreases with further increase in temperature or solvent ratio. Copyright © 2010 John Wiley & Sons, Ltd.     

Enhancement of bioethanol production from Gracilaria verrucosa by Saccharomyces cerevisiae through the overexpression of SNR84 and PGM2

Bioprocess and Biosystems Engineering - A total monosaccharide concentration of 47.0&nbsp;g/L from 12% (w/v) Gracilaria verrucosa was obtained by hyper&nbsp;thermal acid hydrolysis with...     

Statistical optimization of culture conditions for 1,3-propanediol by Klebsiella pneumoniae AC 15 via central composite design

A central composite design was used to study the effect of glycerol, rate of stirring, air aeration and pH on the synthesis of 1,3-propanediol (1,3-PD) by Klebsiella pneumoniae AC 15. Among the four variables, glycerol and rate of stirring significantly affected 1,3-PD productivity, whereas air aeration and pH were not effective. A quadratic polynomial equation was obtained for 1,3-PD productivity by multiple regression analysis using response surface methodology. The validation experimental confirmed with the predicted model. The optimum combinations for 1,3-PD productivity was glycerol, rate of stirring, air aeration, and pH of 50 g/l, 318 rpm, 0.6 vvm, 6.48, respectively. The subsequent fed batch experiments produced 1,3-PD of 70 g/l at a fermentation of 30 h.     

A new approach for modeling cellulase-cellulose adsorption and the kinetics of the enzymatic hydrolysis of microcrystalline cellulose

Two fractions of substrate in microcrystalline cellulose which differ in their adsorption capacities for the cellulases and their susceptibility to enzymatic attack have been identified. On the basis of a two-substrate hypothesis, mathematical models to describe enzyme adsorption and the kinetics of hydrolysis have been derived. A new nonequilibrium approach was chosen to predict cellulase-cellulose adsorption. A maximum binding capacity of 76 mg protein per gram substrate and a half-maximum saturation constant of 26 filter paper units (FPU) per gram substrate have been calculated, and a linear relationship of hydrolysis rate vs. adsorbed protein has been found. The fraction of substrate more easily hydrolyzed, as calculated from hydrolysis data, represents 19% of the total effective substrate concentration. This fraction is only slightly different from that of other celluloses and has been estimated to be 27% and 30% for NaOH- and H(3)PO(4)-swollen cellulose, respectively. The effective substrate concentration is equal to the maximum amount of the substrate which can be converted during exhaustive hydrolysis. This in turn is determined by the overall degradability of the substrate by the cellulases (85-90% for microcrystalline cellulose) and by the cellobiose concentration during hydrolysis. The kinetic model is based on a summation of two integrated first-order reactions with respect to the effective substrate concentration. Furthermore, it includes the principal factors influencing the reaction rates: the ratio of filter paper and beta-glucosidase units per gram substrate and the initial substrate concentration. (c) 1993 John Wiley & Sons, Inc.     

Factorial design for the optimization of enzymatic detection of cadmium in aqueous solution using immobilized urease from vegetable waste

Free as well as alginate immobilized urease was utilized for detection and quantitation of cadmium (Cd2+) in aqueous samples. Urease from the seeds of pumpkin (Cucumis melo), being a vegetable waste, was extracted and purified to apparent homogeneity (Sp. Activity 353 U/mg protein; A280/A260=1.12) by heat treatment at 48+/-0.1 degrees C and gel filtration through Sephadex G-200. The homogeneous enzyme preparation was immobilized in 3.5% alginate leading to 86% immobilization and no leaching of the enzyme was found over a period of 15 days at 4 degrees C. Urease catalyzed urea hydrolysis by both soluble and immobilized enzyme revealed a clear dependence on the concentration of Cd2+. The inhibition caused by Cd2+ was non-competitive (Ki=1.41 x 10(-5) M). The time dependent inhibition both in the presence and in absence of Cd2+ ion revealed a biphasic inhibition in the activity. A Response Surface Methodology (RSM) for the parametric optimization of this process was performed using two-level-two-full factorial (2(2)), central composite design (CCD). The regression coefficient, regression equation and analysis of variance (ANOVA) was obtained using MINITAB 15 software. The predicted values thus obtained were closed to the experimental value indicating suitability of the model. In addition to this 3D response surface plot and isoresponse contour plot were helpful to predict the results by performing only limited set of experiments.     

Screening and optimization of nutritional factors for higher dextransucrase production by Leuconostocmesenteroides NRRL B-640 using statistical approach

To improve dextransucrase production from Leuconostocmesenteroides NRRL B-640 culture medium was screened and optimized using the statistical design techniques of Plackett-Burman and response surface methodology (RSM). Plackett-Burman design with six variables viz. sucrose, yeast extract, K2HPO4, peptone, beef extract and Tween 80 was performed to screen the nutrients that were significantly affecting dextransucrase production. The variables sucrose, K2HPO4, yeast extract and beef extract showed above 90% confidence levels for dextransucrase production and were considered as significant factors for optimization using response surface methodology. 2(4)-central composite design was used for RSM optimization. The experimental results were fitted to a second-order polynomial model which gave a coefficient of determination R2=0.95. The optimized composition of 30g/l sucrose, 18.9g/l yeast extract, 19.4g/l K2HPO4 and 15g/l beef extract gave an experimental value of dextransucrase activity of 10.7U/ml which corresponded well with the predicted value of 10.9U/ml by the model.     

A novel approach for the extraction of herbicides and pesticides from water using liquid-core microcapsules

A novel perstraction system using liquid-core microcapsules for pesticide and herbicide removal from aqueous environments is proposed. The microcapsules contain an oil, dibutyl sebacate, surrounded by a hydrogel membrane. The extraction efficiency of the capsules was demonstrated with atrazine, methylparathion, ethylparathion, and 2,4-dichloro-phenoxyacetic acid. The results show that all of the tested compounds could be rapidly extracted, typically 75% extraction within 10 minutes using a capsule: liquid volume ratio of only 3.5% for ethylparathion, and that the rate of extraction increased with increasing hydrophobicity of the compound to be extracted. Higher rates of extraction could be achieved by changing the capsule: liquid volume ratio. The effect of different liquid core solvents, size of capsules, agitation rate, and treatment with complexing agents on the properties of the microcapsules and extraction rate were studied. Capsules of a diameter smaller than 0.800 mm show little external resistance to mass transfer. The main resistance to mass transfer of the pesticides/herbicides was found to reside in the hydrogel membrane composed of cross-linked alginate/polyacrylamide. Removal of divalent cations from the membrane by the addition of citrate, resulted in a 50% increase in the mass transfer coefficient, probably as a result of solubilization and exo-diffusion of alginate.     

Response surface design for the optimization of enzymatic detection of mercury in aqueous solution using immobilized urease from vegetable waste

Soluble and alginate immobilized urease was utilized for detection and quantitation of mercury in aqueous samples. Urease from the seeds of pumpkin, being a vegetable waste, was extracted and purified to apparent homogeneity (sp. activity 353 U/mg protein; A₂₈₀/A₂₆₀ = 1.12) by heat treatment at 48 ± 0.1 °C and gel filtration through Sephadex G-200. Homogeneous enzyme preparation was immobilized in 3.5% alginate leading to 86% immobilization, no leaching of enzyme was found over a period of 15 days at 4 °C. Urease catalyzed urea hydrolysis by soluble and immobilized enzyme revealed a clear dependence on the concentration of Hg²⁺. Inhibition caused by Hg²⁺ was non-competitive (K_i = 1.2 × 10⁻¹ μM for soluble and 1.46 × 10⁻¹ μM for alginate immobilized urease.). Time-dependent inhibition both in presence and in absence of Hg²⁺ ion revealed a biphasic inhibition in activity. For optimization of this process response surface methodology (RSM) was utilized where two-level-two-full factorial (2²) central composite design (CCD) has been employed. The regression equation and analysis of variance (ANOVA) were obtained using MINITAB^® 15 software. Predicted values thus obtained were closed to experimental value indicating suitability of the model. 3D response surface plot, iso-response contour plot and process optimization curve were helpful to predict the results by performing only limited set of experiments.