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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.108, #1-3)
Adsorption of components of enzymatic synthesis of ampicillin on different hydrophobic resins by Marcelo F. Vieira; Marlei Barboza; Raquel de Lima C. Giordano (pp. 705-713).
This work compared the performance of three hydrophobic resins for the adsorption of ampicillin (AMP), d-phenylglycine (PG), d-phenylglycine methyl ester (PGME), and 6-aminopenicillanic acid (6-APA). The influence of pH on adsorption efficiencies was assessed in the range of 4.5–8.5, at 4 and 25°C. The values at 4°C were slightly higher than those at 25°C. The adsorption efficiency of AMP and 6-APA decreased at higher pHs, for the three resins. An opposite behavior was found for PGME, and the pH did not affect PG adsorption efficiency. Isotherm models were fitted to experimental equilibrium data and the best models were discriminated.
Keywords: Ampicillin; purification; adsorption; β-lactamics
Xylanase production by Trichoderma reesei rut C-30 on rice straw by Alejandro Colina; Betzabé Sulbarán-de-Ferrer; Cateryna Aiello; Alexis Ferrer (pp. 715-724).
Xylanase production of Trichoderma reesei Rut C-30 was examined at different initial pH values (4.8, 5.9, and 7.0) on rice straw in shake flasks, and in a fermentor, for the best pH condition. Enzyme performance was tested on ammonia-treated dwarf elephant grass. The maximum xylanase activities, 92 and 122 IU/mL, were obtained at pH 4.8 in the shake flasks and fermentor, respectively, in which good growth of the fungus was observed during the first 24 h and consumption of proteins dissolved from the rice straw caused the pH to rise later to values between 6.4 and 6.7 (optimal for xylanase production). The xylanases from T. reesei were as effective as Multifect XL, a commercial enzyme preparation, in hydrolyzing ammonia-treated elephant grass.
Keywords: Xylanase; Trichoderma reesei ; rice straw
A different method of measuring and detecting mono-and dioxygenase activities by Roberto Zazueta-Sandoval; Vanesa Zazueta Novoa; Hortencia Silva Jiménez; Roberto Cabrera Ortiz (pp. 725-736).
A spectrophotometric method of measuring oxygenase activity in cell extracts or in zymograms was developed. It is an easy and cheap method that allows spectrophotometric measurement of activity by a colored reaction and reveals activity bands in a polyacrylamide gel electrophoresis (PAGE) gel as brown bands. To prove its usefulness, we report on a study with the oxygenase present in strain YR-1, isolated from petroleum-contaminated soils, that uses hydrocarbons as its sole carbon source. Soluble oxygenase activity was detected (under our conditions of cellular homogenization) in the mycelium of a filamentous fungus strain named YR-1. Oxygenase activity from aerobically grown mycelium was detected in growth medium containing the hydrocarbons decane or hexadecane; the enzyme activity exhibited similar optimum pH for the hydroxylation of different aliphatic or aromatic substrates (decane, hexadecane, benzene, and naphthalene) to the corresponding alcohols. Zymogram analysis conducted with partially purified fractions from cell extracts from the aerobic mycelium of the YR-1 strain indicated the existence of only one oxygenase enzyme. Partially purified samples of enzyme, analyzed by sodium dodecyl sulfate PAGE, indicated the presence of one major protein band with a mol wt of 56 kDa that can be a constituent of the native enzyme. In samples of the enzyme, the 56-kDa protein gave a positive reaction in immuno-detection experiments with antibodies directed against oxygenase from soybean. The partially purified enzyme oxidized different substrates, although higher activity was displayed with benzene. K m values obtained for benzene and decane indicated a higher affinity for the latter
Keywords: Oxygenases; filamentous fungi; hydrocarbon biodegradation; petroleum contamination
Xylanase production by Penicillium canescens 10–10c in solid-state fermentation by Yasser Bakri; Philippe Jacques; Philippe Thonart (pp. 737-748).
Filamentous fungi have been widely used to produce hydrolytic enzymes for industrial applications, including xylanases, whose levels in fungi are generally much higher than those in yeast and bacteria. We evaluated the influence of carbon sources, nitrogen sources, and moisture content on xylanase production by Penicillium canescens 10–10c in solid-state fermentation. Among agricultural wastes tested (wheat bran, untreated wheat straw, treated wheat straw, beet pulp, and soja meal), untreated wheat straw gave the highest production of xylanase. Optimal initial moisture content for xylanase production was 83%. The addition of 0.4 g of xylan or easily metabolizable sugar, such as glucose and xylose, at a concentration of 2% to wheat straw enhanced xylanase production. In solid-state fermentation, even at high concentrations of glucose or xylose (10%), catabolic repression was minimized compared to the effect observed in liquid culture. Yeast extract was the best nitrogen source among the nitrogen sources investigated: peptone, ammonium nitrate, sodium nitrate, ammonium chloride, and ammonium sulfate. A combination of yeast extract and peptone as nitrogen sources led to the best xylanase production.
Keywords: Penicillium canescens ; xylanase; solid-state fermentation
Protease production by Streptomyces sp. isolated from brazilian cerrado soil by Luciana A. I. de Azeredo; Leda R. Castilho; Selma G. F. Leite; Rosalie R. R. Coelho; Denise M. G. Freire (pp. 749-755).
Streptomyces are important microorganisms because of their capacity to produce numerous bioactive molecules. In the present work protease production, by Streptomyces sp. 594 isolated from a Brazilian Cerrado soil, was maximized by optimizing a low-cost culture medium composition (casitone and sugarcane molasses) using statistical experimental design. The final protease activity (56 U/mL) was 2.8-fold and 58-fold higher than that obtained in the beginning of this study, and in a previous work, using an actinomycete selection medium, respectively. Protease production, not growth associated, appeared to be modulated by an inducer system, whereby the C/N ratio seemed to play a significant role.
Keywords: Streptomyces ; sugarcane molasses; protease production; experimental design
Synthesis of monocaprin catalyzed by lipase by Marco A. M. Da Silva; Vitória C. Medeiros; Marta A. P. Langone; Denise M. G. Freire (pp. 757-768).
The production of monoglyceride emulsifiers commonly employed in the food, cosmetic, and pharmaceutical industries can be catalyzed by lipases, biocatalysts that are becoming increasingly attractive in the enzyme market. The aim of this study was to produce monocaprin utilizing a commercial immobilized lipase (Lipozyme IM 20) through the direct esterification of capric acid and glycerol. Experiments were performed for 6 h in an open reactor and the products were analyzed by gas chromatography. The parameters investigated were the amount of enzyme, temperature, and molar ratio between the reagents (capric acid/glycerol). The experimental runs followed an experimental design generated using Statistica® software. The results showed that all the parameters were significant and that monocaprin production was enhanced at the lower ranges of the tested variables. The best conditions established were 55°C, 3% (w/w) enzyme concentration, and molar ratio of 1. The final product, obtained after 6 h of reaction, was 61.3% monocaprin, 19.9% dicaprin, and 18.8% capric acid. This composition satisfies the directives of the World Health Organization food emulsifiers, which requires that these mixtures have at least 70% mono- plus diglyceride, and a minimum of 30% monoacylglycerol.
Keywords: Lipase; monocaprin; capric acid; glycerol; monoglycerides; molar ratio; n-hexane; t-butanol
Effect of dose of xylanase on bleachability of sugarcane bagasse ethanol/water pulps by Denise S. Ruzene; Adilson R. Gonçalves (pp. 769-774).
Pulps obtained from the ethanol/water cooking of sugarcane bagasse were bleached with the xylanase enzyme obtained from the fungus Thermomyces lanuginosus IOC-4145 and with the commercial enzyme Cartazyme HS from Sandoz. By changing the enzyme dose from 4.3 to 36 IU/g of pulp, kappa number and viscosity were maintained when the xylanase from T. lanuginosus was used. On the other hand, by using Cartazyme HS, kappa number decreased by 17%, reaching 35.5. This pulp was further extracted with NaOH without a decrease in viscosity (10 cP), and pulp with a kappa number of 13 was obtained. Xylanases had no significant effect on the ethanol/water pulps.
Keywords: Organosolv pulping; sugarcane bagasse; ethanol/water pulp; xylanase bleaching
CelF of Orpinomyces PC-2 has an intron and encodes a cellulase (CelF) containing a carbohydrate-binding module by Huizhong Chen; Xin-Liang Li; David L. Blum; Eduardo A. Ximenes; Lars G. Ljungdahl (pp. 775-785).
A cDNA, designated celF, encoding a cellulase (CelF) was isolated from the anaerobic fungus Orpinomyces PC-2. The open reading frame contains regions coding for a signal peptide, a carbohydrate-binding module (CBM), a linker, and a catalytic domain. The catalytic domain was homologous to those of CelA and CelC of the same fungus and to that of the Neocallimastix patriciarum CELA, but CelF lacks a docking domain, characteristic for enzymes of cellulosomes. It was also homologous to the cellobiohydrolase IIs and endoglucanases of aerobic organisms. The gene has a 111-bp intron, located within the CBM-coding region. Some biochemical properties of the purified recombinant enzyme are described.
Keywords: Cellulase; anaerobic fungi; Orpinomyces ; cellulose-binding module; carbohydrate-binding module
Partition behavior and partial purification of hexokinase in aqueous two-phase polyethylene glycol/citrate systems by George G. G. Oliveira; Daniel P. Silva; Ines Conceição Roberto; Michele Vitolo; Adalberto Pessoa Jr. (pp. 787-797).
This study dealt with the partition behavior and partial purification of hexokinase (HK) from baker’s yeast by liquid-liquid extraction using aqueous two-phase polyethylene glycol (PEG)/citrate systems. First, we investigated the effect of agitation type (vortex and 8 rpm rotation) on the stability of the system, and then the effects of sodium citrate concentration, PEG concentration, and molar mass of PEG on the partition coefficient of this enzyme by using a 25 factorial experimental design. The results of this factorial experiment showed the possibility of a partial purification of HK by using two extraction steps, since the enzyme preferentially migrated to the top phase and the total proteins (mainly contaminants) remained in the bottom phase. The purification factor (Pur TOP) of the enzyme in the top phase was 1.87, and the partition coefficient of the total proteins (K Prot ) was 0.47.
Keywords: Hexokinase; liquid-liquid extraction; aqueous two-phase systems; Saccharomyces cerevisiae
Effect of aeration on lignin peroxidase production by Streptomyces viridosporus T7A by Leda M. F. Gottschalk; Ronaldo Nobrega; Elba P. S. Bon (pp. 799-807).
The effect of aeration on lignin peroxidase production by Streptomyces viridosporus T7A was studied in a bench-scale bioreactor using a previously optimized growth medium (0.65% yeast extract and 0.1% corn oil, pH7.0) at 37°C and natural pH. Airflow rates of 0.3, 1.0, and 1.5 vvm and a fixed agitation of 200 rpm were initially studied followed by 1.0 vvm and 200, 300, 400, and 500 rpm. The use of 1.0 vvm and 400 rpm increased enzyme concentration 1.8-fold (100–180 U/L) and process productivity 4.8-fold (1.4–6.7 U/[L·h]) in comparison with the use of 200 rpm and 0.3 vvm. The inexpensive corn oil, used as carbon source, besides its antifoam properties, proved to be nonrepressive for enzyme production.
Keywords: Streptomyces viridosporus ; lignin peroxidase production; aeration; productivity; corn oil
Evaluation of supports and methods for immobilization of enzyme cyclodextringlycosyltransferase by Keli A. Sobral; Regina O. Rodrigues; Rogério D. Oliveira; José E. Olivo; Flávio F. de Moraes; Gisella M. Zanin (pp. 809-819).
An experimental design with factorial planning was used for the immobilization of the enzyme cyclodextringlycosyltransferase (CGTase) from Bacillus firmus (strain no.37) to select the best combination of support, method of immobilization, and conditions that gives primarily higher average values for the specific immobilized enzyme activity, and secondarily, higher average values for the percentage of protein fixation. The experimental design factors were as follows: supports—controlled-pore silica, chitosan, and alumina; immobilization methods—adsorption, and two covalent bonding methods, either with γ-aminopropyltriethoxysilane or hexamethylenediamine (HEMDA); conditions—7°C without agitation and 26°C with stirring. The best combination of factors that lead to higher average values of the response variables was obtained with immobilization of CGTase in silica with HEMDA at 7°C. However, immobilization in chitosan at 7°C gave the highest immobilized CGTase specific activity, 0.25 µmole of β-CD/(min·mg protein). Physical adsorption gave low specific enzyme activities, and, in general, a high load of enzyme leads to lower specific enzyme activity.
Keywords: Cyclodextringlycosyltransferase; immobilized enzyme; controlled-pore silica; alumina; chitosan
Active nuclear shuffling system using a swollen conidium of Trichoderma reesei by Hideo Toyama; Makiko Yano; Akane Gisushi; Takeshi Hotta; Nobuo Toyama (pp. 821-824).
Cellulase hyperproducers of Trichoderma reesei can be constructed using autopolyploidization and haploidization techniques. To increase the efficiency of this method, the active nuclear shuffling system in a swollen conidium was effective. A dried mature green conidium of a model strain, T. reesei QM6a (IFO 31326), was swollen to make room for a larger autopolyploid nucleus. After colchicine treatment, a larger autopolyploid nucleus was produced in such a swollen conidium. Benomyl treatment of swollen conidia generated multiple smaller nuclei from one larger autopolyploid nucleus. Those smaller nuclei were transported through conidia to mycelia after germination. This system could contribute to increasing the efficiency of genetic shuffling.
Keywords: Trichoderma reesei ; cellulase; colchicine; cellulose; benomyl
Phenylboronate-chitosan resins for adsorption of β-amylase from soybean extracts by Eduardo J. Arruda; Cesar C. Santana (pp. 829-842).
Isolation and purification of bioproducts from crude extracts can be obtained by affinity methods based on reversible binding of a specific molecule to ligand immobilized in a porous matrix. In the present work, nicrospheres based on chitosan matrix, which incorporated aminophenylboronic acid as a derivative, were prepared and characterized, aimed at developing a β-amylase adsorption process. Kinetic curves and adsorption isotheriru of the crude extracts as well as the breakthrough curves for a frontal chromatographic separation method of a commercial sample of β-amylase from soybean are presented. These results were compared to similar data obtained with a comercial microspheres gel based-on agarose.
Keywords: Purification; β-amylase; soybean; phenylboronate; chitosan
Ester fuels and chemicals from biomass by Edwin S. Olson; Ted R. Aulich; Ramesh K. Sharma; Ronald C. Timpe (pp. 843-851).
Bench-scale research demonstrated that using an efficient esterification step to integrate an ethanol with a carboxylic acid fermentation stream offers potential for producing valuable ester feedstocks and fuels. Polar organic acids from bacterial fermentations are difficult to extract and purify, but formation of the ammonium salts and their conversion to esters facilitates the purifications. An improved esterification procedure gave high yields of esters, and this method will lower the cost of ester production. Fuel characteristics have been determined for a number of ester-gasoline blends with promising results for lowering Reid vapor pressure and raising octane numbers.
Keywords: Biorefinery; esters; esterification; alcohols; ammonium
Purification of glucose-6-phosphate dehydrogenase from baker’s yeast in aqueous two-phase systems with free triazine dyes as affinity ligands by Yan Xu; Michele Vitolo; Cristina Northfleet de Albuquerque; Adalberto Pessoa Jr. (pp. 853-865).
To improve the selectivity of glucose-6-phosphate dehydrogenase (G6PDH) extraction by an aqueous two-phase system, a simple and inexpensive affinity aqueous two-phase system using unbound reactive triazine dyes as ligands was introduced. In a polyethylene glycol (PEG)/hydroxypropyl starch (PES) system, the unbound free triazine dyes, Cibacron Blue F3GA and Procion Red HE3B, partitioned unevenly in the top PEG-rich phase and thus showed an affinity effect on G6PDH, but no influence on hexokinase. The various parameters investigated were pH of the system, buffers, molecular weight of PEG, and ligand type and concentration. A two-step affinity extraction process was established for the purification of G6PDH from baker’s yeast. The total yield of G6PDH was 66.9% and purification factor was 2.35.
Keywords: Aqueous two-phase systems; affinity partitioning; triazine dyes; glucose-6-phosphate dehydrogenase; baker’s yeast
Continuous clavulanic acid adsorption process by Renata M. R. G. Almeida; Marlei Barboza; Carlos O. Hokka (pp. 867-879).
Adsorption kinetics and equilibrium data of clavulanic acid, a β-lactam antibiotic, on ion-exchange resin Amberlite IRA 400 were utilized to carry out the modeling and simulation of a continuous adsorption process. These simulations allowed the estimation of yield, concentration, and purification factors of the process utilizing the product final concentration. Experimental runs of this process were carried out using the conditions pointed out by simulation studies. Comparison of the experimental results and those calculated by the proposed model showed that the model could describe very well the main features of the continuous process.
Keywords: Clavulanic acid; continuous adsorption; purification process
Extraction of nutraceuticals from milk thistle by Jose F. Alvarez Barreto; Sunny N. Wallace; Danielle Julie Carrier; Edgar C. Clausen (pp. 881-889).
Milk thistle contains compounds that display hepatoxic protection properties. We examined the batch extraction of silymarin compounds from milk thistle seed meal in 50, 70, 85, and 100°C water as a function of time. After 210 min of extraction at 100°C, the yield of taxifolin was 1.2 mg/g of seed, a 6.2-fold increase over the results obtained in a Soxhlet extraction with ethanol on pretreated (defatted) seeds. Similarly, the yield of silychristin was 5.0 mg/g of seed, a 3.8-fold increase. The yields of silybinin A and silybinin B were 1.8 and 3.3 mg/g of seed, respectively, or roughly 30% of the Soxhlet yield. The ratios of the extracted compounds, and particularly the ratios at long extraction times, showed that the more polar compounds (taxifolin and silychristin) were preferentially extracted at 85°C, while the less polar silybinin was favored at 100°C.
Keywords: Milk thistle; extraction; water; silymarin; flavanolignans
Extraction of nutraceuticals from milk thistle by Sunny N. Wallace; Danielle Julie Carrier; Edgar C. Clausen (pp. 891-903).
Seeds from milk thistle (Silybum marianum Gaert L.) contain flavanolignan and dihydroflavanol compounds that have interesting and important therapeutic activities. The recovery of these silymarin compounds generally involves a two-step defatting and extraction process using organic solvents. This study examined the batch, single-stage extraction of whole and defatted seeds using ethanol, methanol, acetonitrile, and acetone as the solvents. In extracting defatted milk thistle seeds with organic solvents, extraction with ethanol resulted in the highest silymarin yield, although some potential degradation was observed. The maximum yields of taxifolin, silychristin, silydianin, silybinin A, and silybinin B in ethanol were 0.6, 4.0, 0.4, 4.0, and 7.0 mg/g of defatted seed, respectively. However, if silybinin A were the diastereoisomer of choice, methanol would be the preferred extraction solvent because it yielded the highest silybinin A to silybinin B ratio. Interestingly, lipid removal is an important extraction step, because defatted material yields twice the silymarin concentration.
Keywords: Milk thistle; extraction; ethanol; methanol; silymarin; acetone; acetonitrile
Effect of lidocaine on ovalbumin and egg albumin foam stability by Vorakan Burapatana; Ernest E. Butler; Gaurav Chauhan; Sean Hartig; Helen Kincaid; Tong Wang; Shayrizal Samsudin; Robert D. Tanner (pp. 905-911).
Foam fractionation is a simple separation process that can remove and concentrate hydrophobic molecules such as proteins, surfactants, and organic wastes from an aqueous solution. Bovine serum albumin and ovalbumin have been widely used as model proteins due to their strong foaming potential and low price. Here, we study the effect of lidocaine on albumin foam, since drugs like lidocaine are known to bind with albumin. We observed that lidocaine not only enhances the amount of foam produced but also the stability of that foam as well. The foam stability was evaluated as the decay rate constant of the foam, determined from a change in height (or volume) of the foam over a given time period.
Keywords: Foam; egg albumin; ovalbumin; lidocaine; foam stability
Estimation of solubility effect on the herbicide controlled-release kinetics from lignin-based formulations by Félix M. Pereira; Adilson R. Gonçalves; André Ferraz; Flávio T. Silva; Samuel C. Oliveira (pp. 913-919).
Understanding the main phenomena involved in the controlled-release kinetics of herbicides in a water bath is a very important requisite for diffusive-parameter estimation, because, some mathematical models based on Fick’s second law for diffusion have been developed to describe the controlled-release kinetic data. However, the validity of these models is restricted to the following assumptions: (1) the formulation is an isothermal slab; (2) the release occurs through the two faces of the slab; (3) the herbicide is dissolved in the water contained in the slab pores at a concentration less than the saturation concentration (c is ); (4) the total sum of the individual volumes of the pores is εAL (ε is the slab porosity, A is the slab area, and L is the slab thickness); and (5) the initial concentration of herbicide in the pores is M 0/εAL (M 0 is the initial amount of herbicide in the matrix). The fourth assumption may be invalid for mathematical description of systems in which the herbicide concentration in the slab may be above the saturation concentration. If this were true, the final assumption would also be invalid, because the initial concentration of herbicide in the pores is c is in this case. This work presents a study of the solubility effect on the controlled-release kinetics of herbicides from lignin matrices.
Keywords: Mathematical modeling; controlled-release; lignin; herbicide; diffusion; solubility