The enzymatic hydrolysis of used frying oil by native lipase

The enzymatic hydrolysis of used frying oil catalyzed by native lipase ofNigelk sativa seed was investigated under different conditions to elucidate the role of different process parameters on the reaction rate. Data fitted the first-order rate equation.     

Enzymatic hydrolysis by Humicola lanuginosa lipase of polycaprolactone monolayers

The enzymatic hydrolysis by Humicola lanuginosa lipase (HLL) of spread insoluble monolayers of polycaprolactone with various molecular weights was studied by measuring the decrease in surface area and in surface potential, in a barostat surface balance. The interfacial hydrolysis under the action of enzymes leads to the progressive fragmentation of the polymer molecules and to the appearance at the interface of charged insoluble and small soluble products. The solubilization of the small soluble fragments was detected by measuring the decrease in surface area during hydrolysis. An independent study showed that, in contrast to poly(lactic-co-glycolic acid) (PLAGA) oligomers, this solubilization is not instantaneous. Taking into account the solubilization rates, one can determine the kinetics of enzymatic hydrolysis. The specific catalytic activity of HLL was estimated in the framework of the random-scission model and compared to those obtained for the hydrolysis of monolayers built up of PLAGA or of simple di- and triglyceride molecules. Received: 25 July 2000 Revised: 28 November 2000 Accepted: 29 November 2000     

Application of xylanase from thermomyces lanuginosus IOC-4145 for enzymatic hydrolysis of corncob and sugarcane bagasse

Xylanases have significant current and potential uses for several industries including paper and pulp, food, and biofuel. For the biofuel industry, xylanases can be used to aid in the conversion of lignocellulose to fermentable sugars (e.g., xylose). We investigated the thermophilic fungus Thermomyces lanuginosus was yielded for xylanase production and found that the highest activity (850 U/mL) was yielded after 96 h of semisolid fermentation. The enzyme was used for hydrolyzing agricultural residues with and without pretreatment. Such residues were characterized in relation to the maximum xylose content by total acid hydrolysis. The highest xylose yields realized by enzymatic hydrolysis were 24 and 52%, achieved by using 3000 U/g (dried material) of sugarcane bagasse and corncob, respectively, which received both alkali and thermal pretreatment.     

Use of corncob for endoxylanase production by thermophilic fungus Thermomyces lanuginosus IOC-4145

The production of cellulase-free end oxylanase by the thermophilic fungus Thermomyces lanuginosus was investigated insemisolid fermentation and liquid fermentation. Different process variables were investigated in semisolid fermentation, employing corncobas the carbon source. The best results were with the following conditions: grain size=4.5 mm, solid:liquid ratio=1:2, and inoculum size=20% (v/v). Corncob, xylan, and xylose were the best inducers for endoxylanase production. Additionally, organic nitrogen sources were necessary for the production of high endoxylanase activities. The crude enzyme had optimum activity at pH 6.0 and 75°C, displaying a high thermostability. The apparent K ₂₅ and V _max were 1.77 mg of xylan/mL and 21.5 U/mg of protein, respectively.     

Isolation of lipase producer and its performance in enantioselective hydrolysis of glycidyl butyrate

A racemic glycidyl butyrate resolving strain, preliminarily identified as a Rhizopus sp., had been isolated from soil. Its extracellular lipase was found to enantioselectively hydrolyze the (S)-enantiomer of the chiral ester, with optimal activities at pH 5.3 and 42°C. Higher en antioselectivity of theenzyme was observed at lower temperatures, while the best anantioselectivity was obtained at pH 5.5–6.0, with an, E value (enantiomeric ratio) of 57.     

Effect of fermentation conditions in the enzymatic activity and stereoselectivity of crude lipase from Candida rugosa

Different fed-batch cultures of Candida rugosa were carried out using oleic acid as the only carbon source. The crude lipases obtained under several operational conditions and downstream processes showed different catalytic activity and isoenzymes ratio. This fact implied that the performance of the lipase produced could be modulated by using different operational fermentation conditions. These powders were compared with commercial lipase from Sigma (St. Louis, MO) in hydrolysis and synthesis reactions. Especially interesting was the fact that the enantioselectivity of a crude lipase was higher than that observed with commercial lipase in the resolution of recemic Ketoprofen. In addition, response of both lipases in the presence of water was different.     

Production of biodiesel by immobilized Candida sp. lipase at high water content

A new process for enzymatic synthesis of biodiesel at high water content (10–20%) with 96% conversion by lipase from Candida sp. 99–125 was studied. The lipase, a no-position-specific lipase, was immobilized by a cheap cotton membrane and the membrane-immobilized lipase could be used at least six times with high conversion. The immobilized lipase could be used for different oil conversion and preferred unsaturated fatty acids such as oleic acid to staturated fatty acids such as palmitic acid. The changes in concentration of fatty acids, diglycerides, and methyl esters in the reaction were studied and a mechanism of synthesis of biodiesel was suggested: the triglycerides are first enzymatically hydrolyzed into fatty acids, and then these fatty acids are further converted into methyl esters.     

Preparation and characterization of pancreatic lipase immobilized in Eudragit-matrix

Pancreatic lipase (EC 3.1.1.3) was immobilized by entrapping in a commercial preparation of acrylic/methacrylic acid ester-based copolymer (Eudragit E 30 D). The activity of the immobilized lipase beads with a diameter of 1.5-2.0 mm was found to be lower than that of the free lipase. The optimum pH was shifted to the alkaline region and the thermal stability increased, whereas the optimum temperature level remained unchanged. The most important reason for the decreased activity was diffusion limitations. The diffusion of the substrate and products became more pronounced, and lipolytic activity increased upon addition of n-hexane into the reaction medium. The storage and operational stabilities of the immobilized lipase were investigated, and both characteristics were found to be increased when compared to the free enzyme. Furthermore, mechanical or magnetic stirring during the operation were found to have no influence on the carrier-matrix as determined by nephelometric measurements.     

Production of glycerides from glycerol and fatty acids by native lipase ofNigella sativa seed

The possible application of native lipase ofNigella sativa seed in the esterification of fatty acids to glycerol was investigated, and the effect of process parameters and the enzyme selectivity on the reaction were determined. For this aim, the esterification of oleic acid, sunflower oil fatty acids, and coco oil fatty acids with glycerol were studied.     

The most favorable condition for lipid hydrolysis by Rhizopus delemar lipase in combination with a sugar–ester and alcohol W/O microemulsion system

The hydrophobic interfacial characterization of sugar–ester alcohol W/O microemulsion was investigated in order to develop a suitable design for reaction media. A W/O microemulsion system was prepared as a combination of sugar–ester, DK-ester-F-110 with alcohol as a cosolvent. The diameter of the sub-micron-sized water pool, the hydrophobicity of the water pool, and the fluidity of the micro-interface were studied to analyze the reactivity of Rhizopus delemar lipase in lipid hydrolysis. The diameter of the sub-micron sized water pool was measured by dynamic laser light scattering. It was proportional to the 0.33 power of W_soln and decayed with increasing alcohol concentration. The initial reaction rate of the hydrolysis of triolein in W/O microemulsion depended on the solubilized water content. The hydrophilic character of the micro-water pool was estimated from the fluorescence wavelength indicated by Coumarin 343. The maximum initial reaction rate appeared at 450–460 nm. The fluidity of the micro-interface was investigated by fluorescence anisotropy using TMA-DPH. A suitable physicochemical reaction condition for high reactivity to occur was found, satisfying both the hydrophilicity and the fluidity of the micro-interface.     

Pretreatment of wood by UCT-solvent for the enzymatic hydrolysis

UCT-solvent pretreatment was carried out on woods (beech and akamatsu (pine)) for the enzymatic hydrolysis, in which pretreatment the ground woods were autoclaved with a mixture of water and cyclo-hexanol (37.5% vol% cyclohexanol) having upper critical temperature (UCT: 184°C) on the mutual solubility curve (named as UCT-solvent). Ninety-five and 92% of Klason lignin were removed from beech and akamatsu, respectively, whereas when the woods were autoclaved with water instead of UCT-solvent, only 43 and 18% of Klason lignin was removed from them, respectively. The excellent ability of UCT-solvent for the removal of Klason lignin is owing to that the solvent disturbs re-coupling between the degradation products. The enzymatic hydrolysis of wood was much improved by UCT-solvent pretreatment: the hydrolytic reactivity of akamatsu was enhanced by 2.8 times comparing with when akamatsu was pretreated with water instead of UCT-solvent.     

Pretreatment of bagasse by UCT-Solvent for the enzymatic hydrolysis

A thermochemical pretreatment of bagasse for the enzymatic hydrolysis has been carried out, in which pretreatment bagasse was autoclaved with binary solvent, composed of Water and organic solvent having upper critical temperature (UCT) on the mutual solubility curve. The pretreatment was named “UCT-solvent pretreatment.” The hydrophobic decomposition products from lignin and hemicellulose, that dissolved in organic phase at room temperature, could be easily separated from the solid and sugars in the aqueous phase. By using UCT-solvent instead of only water, the sugar recoveries from bagasse through the pretreatment and the enzymatic hydrolysis were much improved. There exists an optimal mixing ratio between organic solvent and water to maximize the effect of the pretreatment for enzymatic hydrolysis. The optimal ratio can be explained by the competitive effect between the ability of water as a reagent for the hydrolysis and the ability of solvent for the extraction of the decomposition product, and furthermore by the competitive effect between affinities of the solvent to hydrophilic hemicellulose and hydrophobic lignin. Decomposition of hemicellulose at lower temperature than 190°C was decreased, and hence the degradation of xylose during the pretreatment decreased. These favorable effects of UCT-solvent pretreatment are significantly attributed to the formation of the homogeneous single phase of organic solvent and water at high temperature and the phase separation at room temperature.     

Eicosapentaenoic acid release from the red alga Pachymeniopsis lanceolata by enzymatic degradation

Forty-eight species of seaweeds from Japanese waters were screened for the valuable polyunsaturated fatty acids eicosapentaenoic acid (EPA). The eight species that contained the highest levels of these compounds were analyzed in detail. Of all species tested the red alga Pachymeniopsis lanceolata contained the highest EPA concentration, and it was present as both the free and bound forms. EPA constituted 38.7% of total fatty acids, and polar lipids were the main constituent of the total lipids in P. lanceolata. EPA was obtained from the marine algae P. lanceolata by enzymatic hydrolysis of the total lipids extract using phospholipase A₂(PLA₂). The release of EPA reached a plateau after 10 min of enzymatic treatment. These results suggest that P. lanceolata is a useful natural source of EPA and that PLA₂ treatment is a convenient method for obtaining EPA from the red alga.     

Influence of the support on the reaction course of tributyrin hydrolysis catalyzed by soluble and immobilized lipases

Lipases from different origins have been immobilized in supports chosen by its different aquaphilicity and used as biocatalysts for the hydrolysis of tributyrin. The changes of the concentration of tri-, di-, monobutyrin, glycerol, and butyric acid during the reactions catalyzed by soluble, as well as immobilized, lipases were evaluated by gas chromatography. The experimental data were fitted to a simple kinetic model for the sequential reaction of tributyrin hydrolysis. The calculated apparent rate constants were different for the biocatalysts used and were apparently related to diffusional effects and aquaphilicity of the supports. Maximal yields of dibutyrin were found with the soluble Candida lipase, whereas the highest yield of monobutyrin (90%) was obtained with the least aquaphylic derivative (Candida-Celite).     

Immobilization of lipase from Candida rugosa on layered double hydroxides for esterification reaction

Synthesis of layered double hydroxides (LDHs) of Zn/Al-NO3- hydrotalcite (HIZAN) and Zn/Al-diocytyl sodium sulfosuccinate (DSS) nanocomposite (NAZAD) with a molar ratio of Zn/Al of 4:1 were carried out by coprecipitation through continuous agitation. Their structures were determined using X-ray diffractometer spectra, which showed that basal spacing for LDH synthesized by both methods was about 8.89 A. An expansion of layered structure of about 27.9 A was observed to accommodate the surfactant anion between the interlayer. This phenomenon showed that the intercalation process took place between the LDH interlayer. Lipase from Candida rugosa was immobilized onto these materials by physical adsorption method. It was found that the protein loading onto NAZAD is higher than HIZAN. The activity of immobilized lipase was investigated through esterification of oleic acid and 1-butanol in hexane. The effects of pore size, surface area, reaction temperature, thermostability of the immobilized lipases, storage stability in organic solvent, and leaching studies were investigated. Stability was found to be the highest in the nanocomposite NAZAD.     

Improvements in lipase production and recovery from Acinetobacter radioresistens in presence of polypropylene powders filled with carbon sources

Polypropylene powders as the adsorbent for organic solution containing n-hexadecane and olive oil were employed as the carbon source for producing an alkaline lipase from Acinetobacter radioresistens. The best volumetric ratio of n-hexadecane to olive oil around 5 for lipase production was determined from shake-flask and fermentation cultivations. The existence of a maximum time course lipase activity of the aqueous phase was attributed to the compensation effects of olive oil on cell growth and lipase production, repression of lipase synthesis by oleic acid, and lipase adsorption on the supports. A linear relationship between the average cell growth rate in the exponential phase and the ratio of surface areas of the supports was found. The benefits of using the present fermentation process include less foaming and emulsion of the broth, less organic phase used, higher lipase production, and easy recovery of the lipase in the centrifugation step.     

Production of lipase by a newly isolated <Emphasis Type="Italic">Bacillus coagulans</Emphasis> under solid-state fermentation using melon wastes

Summary An extracellular lipase was produced by Bacillus coagulans by solid-state fermentation. Solid waste from melon was used as the basic nutrient source and was supplemented with olive oil. The highest lipase production (78,069 U/g) was achieved after 24h of cultivation with 1% olive oil enrichment. Enzyme had an optimal activity at 37°C and pH 7.0, and sodium dodecyl sulfate increased lipase activity. NH ₄NO₃ increased enzyme production, whereas organic nitrogen had no effect. The effect of the type of carbon sources on lipolytic enzyme production was also studied. The best results were obtained with starch and maltose (148,932 and 141,629 U/g, respectively), whereas a rather low enzyme activity was found in cultures grown on glucose and galactose (approx 118,769 and 123,622 U/g, respectively). Enzyme was inhibited with Mn⁺² and Ni⁺² by 68 and 74%, respectively. By contrast, Ca⁺² enhanced enzyme production by 5%.     

Effect of sulfuric and phosphoric acid pretreatments on enzymatic hydrolysis of corn stover

The pretreatment of corn stover with H₂SO₄ and H₃PO₄ was investigated. Pretreatments were carried out from 30 to 120 min in a batch reactor at 121°C, with acid concentrations ranging from 0 to 2% (w/v) at a solid concentration of 5% (w/v). Pretreated corn stover was washed with distilled water until the filtrate was adjusted to pH 7.0, followed by surfactant swelling of the cellulosic fraction in a 0–10% (w/v) solution of Tween-80 at room temperature for 12 h. The dilute acid treatment proved to be a very effective method in terms of hemicellulose recovery and cellulose digetibility. Hemicellulose recovery was 62–90%, and enzymatic digestibility of the cellulose that remained in the solid was >80% with 2% (w/v) acid. In all cases studied, the performance of H₂SO₄ pretreatment (hemicellulose recovery and cellulose digestibility) was significantly better than obtained with H₃PO₄. Enzymatic hydrolysis was more effective using surfactant than without it, producing 10–20% more sugar. Furthermore, digestibility was investigated as a function of hemicellulose removal. It was found that digestibility was more directly related to hemicellulose removal than to delignification.     

Novel reaction systems for the synthesis of O-glucosides by enzymatic reverse hydrolysis

Our studies are presented to replace alcohols as solvents in reverse hydrolytic reactions catalyzed by immobilized β-glucosidase to synthesize O-substituted β-d-glucopyranosides in preparative-scale. We found that 1,2-diacetoxyethane is a suitable solvent and O-alkyl or aryl β-d-glucosides were synthesized in moderate yields (after isolation 12-19%). In these reactions proportion of glucose and glucosyl acceptor hydroxy compounds was 1:20. We suggest that 1,2-diacetoxyethane can be useful not only for alcohols but for other glucosyl donor compounds unsuitable for the role of solvent (e.g., phenols) in the synthesis of O-β-d-glucosides by reverse hydrolysis.     

Effect of temperature, moisture, and carbon supplementation on lipase production by solid-state fermentation of soy cake by Penicillium simplicissimum

The production of lipases by Penicillium simplicissimum using solid-state fermentation and soy cake as substrate was investigated. The effects of temperature, cake moisture, and carbon supplementation on lipase production were studied using a two-level experimental plan. Moisture, pH, and lipase activity were followed during fermentation. Statistical analysis of the results was performed to evaluate the effect of the studied variables on the maximum lipase activity. Incubation temperature was the variable that most affected enzyme activity, showing a negative effect. Moisture and carbon supplementation presented a positive effect on activity. It was possible to obtain lipase activity as high as 21 U/g of dry cake in the studied range of process variables.