Commercial Lipase Research Articles

In situ immobilization of commercial lipase in rigid polyurethane foam and application in the esterification of geraniol and oleic acid

The aim of this work was to evaluate the immobilization of Candida antarcticalipase B (CalB) using polyurethane (PU) foam. The stability of the enzymatic derivative against temperature (40, 60 and 80 °C) together with the recycle capacity and storage stability (4 and 25°C) were evaluated. The application of the enzymatic derivative of CalB in esterification reactions was accomplished. Results indicated that the molar ratio between polyol and isocyanate monomers of 5:3 (v/v) was the best one for the immobilization. The immobilization process resulted in a 1363% increase in activity for the enzymatic derivative. The enzymatic derivative obtained kept its initial activity even after 21 h of exposure to high temperatures. The enzymatic activity of the complex was maintained during 30 days at low temperatures, and showed stability during 4 consecutive reuse cycles. The synthesis of geranyl oleate catalyzed by the immobilized derivative presented conversion of 87%. Taking into account the promising results obtained, the low cost of the immobilization support employed and the whole technique developed, this work comprise an innovative contribution.

Preparation of structured lipids enriched with medium- and long-chain triacylglycerols by enzymatic interesterification for infant formula

Single cell oils (SCOs) are considered as important sources of the essential fatty acids (EFAs) such as arachidonic acid (ARA), which is of great importance for infant’s growth and development. Medium-chain triacylglycerols (MCTs) induce the residual glyceride lipolysis, which improve fat absorption, and thus reduce its deposition in infants. Medium- and long-chain triacylglycerols (MLCTs)-rich structured lipids (SLs) combine these benefits, and they can be delivered to infants in an easy way. In this study, MLCTs-rich SLs were synthesized by lipase-catalyzed interesterification of ARASCO with MCTs in a solvent-free system. Four commercial immobilized lipases from different sources were compared for their efficiency in the production of MLCTs yield. The results indicated that the highest yield of MLCTs was achieved by Lipozyme 435 from Candida antarctica as a biocatalyst. The best reaction conditions were as follows; enzyme load 8% (w/w), reaction temperature 90°C, reaction time of 3h, and substrates mole ratio 1:1. Under these conditions, MLCTs were obtained in a yield of 53.75%. Thermoprofile displayed that the MLCTs-rich SLs melted below body temperature of 37°C. The obtained MLCTs-rich SLs can be suggested as a nutritional and functional fat analogue with potential applications in infant formula industry.

Improved production of biolubricants from soybean oil and different polyols via esterification reaction catalyzed by immobilized lipase from Candida rugosa

Three commercial lipases, Lipomod 34MDP (free lipase from Candida rugosa), Lipozyme RM IM (immobilized lipase from Rhizomucor miehei) and Novozym 435 (immobilized lipase B from Candida antarctica) were evaluated as catalysts in the production of biolubricant base oils. This was accomplished via the esterification of free fatty acids obtained from soybean-oil hydrolysis and different polyols (neopentyl glycol, NPG; trimethylolpropane, TMP; and pentaerythritol, PE). Reactions carried out with free C. rugosa lipase showed the highest conversions for the three polyols (97% for NPG, 100% for TMP and 55% for PE) after 24 h, while Novozym 435 and Lipozyme RM IM resulted, respectively, in 65% and 92% for NPG, 15% and 39% for TMP and 1% and 0% for PE. Then, Accurel MP1000 (a microporous polypropylene support, PP) was used to immobilize the C. rugosa lipase; enzyme loading used was 0.3 mg/g. The zymography analysis showed that the protein band of 60 KDa present in the C. rugosa lipase extract was the main protein responsible for the extract activity in the biolubricant synthesis and it was successfully immobilized onto the support. The immobilized lipase (34MDP-PP) showed conversions of 99% for NPG and 92% for TMP, after 24 h of reaction, maintaining the results obtained with the free enzyme. The 34MDP-PP could be reused for six consecutive reaction cycles without a reduction in the final conversion, using NPG and TMP as substrates. Using Lipozyme RM IM, the conversion decreased from 92 to 56% after six consecutive reactions. The soybean esters of NPG and TMP showed good viscosity indexes, higher than 200.

Characterization of a cutinase from Myceliophthora thermophila and its application in polyester hydrolysis and deinking process

Abstract A gene encoding cutinase (MtCUT) from Myceliophthora thermophila was expressed in Pichia pastoris. The optimal temperature and pH for MtCUT were 30 °C and 8.5, respectively. MtCUT showed high activities toward a broad range of p-nitrophenyl esters, and the highest specific activity was recorded for p-nitrophenyl butyrate. MtCUT hydrolyzed and broke down apple cutin into C16 and C18 family fatty acids. MtCUT could efficiently degrade polycaprolactone (PCL), the weight loss of which was 78.5% in 36 h. In addition, MtCUT could degrade PVAc, a kind of adhesive that is frequently used in papermaking and in synthetic toner or ink. Its performance in the deinking of waste papers was investigated and compared with that of commercial lipase in this study. The results showed that an ink removal efficiency of 78.4% on laser-printed paper and 81.3% on newspaper was obtained using MtCUT at 30 °C, which was better than that of lipase from Candida rugosa (CrLIP) (77.0% and 76.0%, respectively). The properties of the MtCUT-treated samples were similar to that of the control samples. This revealed that the cutinase MtCUT from M. thermophila has a great potential in PCL hydrolysis and the deinking process.

Application of Enzymatic Extracts from a CALB Standard Strain as Biocatalyst within the Context of Conventional Biodiesel Production Optimization.

The application of biocatalysts in the transesterification process of triglycerides (TG) allows integrating the glycerol in the form of monoglyceride (MG), sharply increasing the yield and the environmental sustainability of the conventional biodiesel production process. This is known as Ecodiesel. To overcome the inconvenient of the high cost of the currently employed highly purified commercial enzymes, the use of scarcely purified extracts obtained from standard strains of the same species of commercial lipases currently applied in this process is being investigated. Thus, Candida antarctica type B (CALB) was chosen to determine the optimal conditions of culture of this yeast. The standard strain was obtained from the Spanish Type Microbial Cultures Collection (CECT) and has been used to carry out several studies to elucidate its optimum growth conditions. Through a process of lyophilization with prior dialysis of the liquid cultures, the enzymatic extracts were obtained. The different obtained cultures have been applied as biocatalysts in the 1,3-selective transesterification reaction of sunflower oil with ethanol to obtain Ecodiesel (FAEE + MG). Selectivity and reaction yields were obtained by gas chromatography. Acceptable yields are obtained during the reaction time as well as in successive reactions, demonstrating the feasibility of using these CALB enzymatic extracts as biocatalysts.

Optimization of enzyme-catalyzed biodiesel production from crude tall oil using Taguchi method

Biodiesel production from crude tall oil (CTO) in presence of a commercial lipase catalyst (Eversa Transform) was optimized using Taguchi method. Process parameters including reaction temperature, molar ratio of CTO to methanol, reaction time, and enzyme dose were examined for their effect on the in-situ esterification of fatty acids (FA) in CTO to fatty acid methyl ester (FAME). Fractional distillation was employed to separate the FAME fraction (biodiesel) from the rest of the CTO components. Using Taguchi (L9) Orthogonal Design, the optimum biodiesel yield was statistically predicted and experimentally verified as 97.02% and 96.57%, respectively. The optimal conversion of CTO to crude biodiesel was obtained at 16 h reaction time, 40 °C reaction temperature, 1:1.5 CTO to methanol molar ratio, and 1.0 wt% enzyme dose. The reaction time and CTO to methanol molar ratio were the two most contributing factors for the optimal conversion of CTO to biodiesel.

Bioremediation of cooking oil waste using lipases from wastes.

Cooking oil waste leads to well-known environmental impacts and its bioremediation by lipase-based enzymatic activity can minimize the high cytotoxic potential. In addition, they are among the biocatalysts most commercialized worldwide due to the versatility of reactions and substrates. However, although lipases are able to process cooking oil wastes, the products generated from this process do not necessarily become less toxic. Thus, the aim of the current study is to analyze the bioremediation of lipase-catalyzed cooking oil wastes, as well as their effect on the cytotoxicity of both the oil and its waste before and after enzymatic treatment. Thus, assessed the post-frying modification in soybean oil and in its waste, which was caused by hydrolysis reaction catalyzed by commercial and home-made lipases. The presence of lipases in the extracts obtained from orange wastes was identified by zymography. The profile of the fatty acid esters formed after these reactions was detected and quantified through gas chromatography and fatty acids profile compared through multivariate statistical analyses. Finally, the soybean oil and its waste, with and without enzymatic treatment, were assessed for toxicity in cytotoxicity assays conducted in vitro using fibroblast cell culture. The soybean oil wastes treated with core and frit lipases through transesterification reaction were less toxic than the untreated oils, thus confirming that cooking oil wastes can be bioremediated using orange lipases.

Study of the Influence of Microwave and Conventional Heating on the Lipase-Catalyzed Esterification of Lauric Acid with Different Alcohols

In a systematic study, three commercial lipases were used for the esterification of lauric acid with six different alcohols: methanol, ethanol, propanol, butanol, pentanol, and allyl alcohol, under conventional heating and microwave irradiation. Optimization of reaction conditions included reaction time and effect of temperature under microwave irradiation and conventional heating. The optimum reaction temperature was previously established in enzymatic esterification by conventional heating. Novozyme 435, lipase (Pseudomonas cepacia) immobilized in diatomite (PS-DI) and lipase (P. cepacia) immobilized in ceramic (PS-CI) were used in this study. The latter showed the highest activity, therefore, this enzyme was used to investigate the variation of reaction time under microwave heating. The stability of immobilized lipases under classical heating and under the microwave irradiation was studied by visual analysis of the immobilized matrix by scanning electron microscopy (SEM).

Utilization of Waste Glycerol from Biodiesel Process as a Substrate for Mono-, Di-, and Triacylglycerol Production

Glycerol (or glycerin) is a major by-product in the biodiesel process. Ten percent of crude glycerol are created for every 100 pounds of biodiesel produced. As the utilization of biodiesel increasing, an excess of glycerol is being generated. However, the purify of waste glycerol is expensive, biodiesel producers must search alternative methods for its utilization. This paper demonstrate the feasibility of producing mono-, di- and triacylglycerol from waste glycerol. The acylglycerol was produced through glycerolysis reaction using commercial lipase from Candida sp. Effects of various reaction parameters were evaluated. The optimum conditions for acylglycerol production were a glycerol to fatty acid mole ratio of 6∶1, 100 mg of lipase with the reaction temperature and time at 40°C and 24 h, respectively. Analysis of acylglycerol by Thin Layer Chromatography (TLC) using petroleum ether : diethyl ether :methanol:acetic acid (90:7:2.5:0.5 v/v) as mobile phase found that mono-, di- and triacylglycerol were produced in high purity. Interestingly, the highest production of acylglycerol yeild from commercial lipase and crude lipase from pacific white shrimp’s hepatopancreas were obtained at the same conditions. Attenuated Total Reflectance (ATR)-Fourier Transform Infrared Spectroscopy (FTIR) proved that the final product contains mono-, di- and triacylglycerol.

Application of commercial and non-commercial immobilized lipases for biocatalytic production of ethyl lactate in organic solvents

This study explores the potential for enhancing the production of ethyl lactate (EL), a green solvent, through enzymatic esterification. Different solvents were compared as organic media for conversion of lactate and ethanol into EL, catalyzed by Novozym 435. Chloroform and hexane were the most effective in low acid concentrations (0.01–0.1M) exhibiting maximum EL yields of 88% and 75% respectively. The yield of EL improved as the solvent’s LogP increased up to a value of 2. Non-commercial immobilized biocatalysts consisting heterologous Rhizopous oryzae (rROL) and Candida rugosa (CRL) lipases immobilized on hydrophobic supports were compared to commercial biocatalysts clarifying that Novozym 435 and Lipozyme RM IM could be efficiently applied. Operational stability tests were conducted using Novozym 435, which retained higher activity in chloroform as compared to hexane. Although non-commercial biocatalysts were not competitive in esterification, they exhibited significant activity towards hydrolysis constituting a valuable alternative to higher-cost options.

Conversion of levulinic acid into alkyl levulinates: Using lipase immobilized on meso-molding three-dimensional macroporous organosilica as catalyst

For conversion of biomass-derived levulinic acid into alkyl levulinates, a novel kind of lipase-based biocatalyst was prepared through immobilized lipase B from C. antarctica (CALB) on organosilica material with highly ordered 3D macroporous organosilica frameworks and a 2D hexagonal meso-structure (named 3DOM/m-OS) for the first time. The catalytic performance of the immobilized lipase (NER@3DOM/m-OS) was investigated. NER@3DOM/m-OS was used as biocatalyst to catalyze the esterification reaction between levulinic acid (LA) and n-butanol. Under optimized reaction conditions, 74.59% of ester yield was achieved after 12h of reaction. NER@3DOM/m-OS was also used to production of other alkyl levulinates, the ester yields increased to 84.51% (octyl levulinate) and 91.14% (dodecyl levulinate), respectively. When NER@3DOM/m-OS was used repeatedly in batch reactions, the ester yields of n-butyl, octyl, and dodecyl levulinate could retain 46.18%, 82.33% and 81.25% after 9 reaction cycles, respectively, which was better than commercial lipase Novozym 435 under the same condition.

Polyesters from Macrolactones Using Commercial Lipase NS 88011 and Novozym 435 as Biocatalysts.

The demand for environmentally friendly products allied with the depletion of natural resources has increased the search for sustainable materials in chemical and pharmaceutical industries. Polyesters are among the most widely used biodegradable polymers in biomedical applications. In this work, aliphatic polyesters (from globalide and ω-pentadecalactone) were synthesized using a new commercial biocatalyst, the low-cost immobilized NS 88011 lipase (lipase B from Candida antarctica immobilized on a hydrophobic support). Results were compared with those obtained under the same conditions using a traditional, but more expensive, commercial biocatalyst, Novozym 435 (lipase B from C. antarctica immobilized on Lewatit VP OC). When NS 88011 was used in the polymerization of globalide, longer reaction times (240min)-when compared to Novozym 435-were required to obtain high yields (80-90 wt%). However, higher molecular weights were achieved. When poly(ω-pentadecalactone) was synthesized, high yields and molecular weights (130,000gmol-1) were obtained and the enzyme concentration showed strong influence on the polyester properties. This is the first report describing NS 88011 in polymer synthesis. The use of this cheaper enzymatic preparation can provide an alternative for polyester synthesis via enzymatic ring-opening polymerization.

Comparison of cutinases in enzymic deinking of old newsprint

In this study, the impact of cutinases from Thermobifida fusca and Fusarium solani pisi on the deinking of old newsprint were evaluated for the first time. When repulped old newsprint was treated with 8 U/g of T. fusca cutinase at pH 8 and 60 $$^{\circ }$$ C for 30 min, the brightness of the deinked papers reached 42.01%. With 8 U/g F. solani cutinase at pH 8.5 and 35 $$^{\circ }$$ C for 30 min, the brightness reached 41.62%. These brightness values are higher than that achieved using chemical deinking by 5.13 and 4.38%, respectively. These brightness values were also superior to that achieved using commercial lipase in this study and those previously reported using cellulases, hemicellulases, and laccase, which were higher than that achieved using chemical deinking by 2–4%. The mechanical properties of the deinked paper, including tensile index, tear index, and burst index, were also measured. The results showed that the two cutinases had different effects on the paper fibers. In summary, both T. fusca and F. solani pisi cutinases were able to remove ink from old newsprint more efficiently than alkaline chemistry or other enzymes. This study provides a potential strategy to deink efficiently old newsprint using cutinase.

Biodiesel production by solvent-free ethanolysis of palm oil catalyzed by fermented solids containing lipases of Burkholderia contaminans

Fermented solids containing lipases from Burkholderia contaminans LTEB (denominated BCFS) were obtained by solid-state fermentation using sugarcane bagasse impregnated with soybean oil. These solids were dried and used to catalyze the ethanolysis of palm oil in a solvent-free reaction medium. The reaction was optimized in 25-mL vials, containing 15mmol of palm oil. Molar ratios (MR) of ethanol to palm oil of 3:1 and 4.5:1 were tested, with stepwise addition of ethanol. In these experiments, the maximum conversion of palm oil achieved in 48h was 90%, with a MR of 4.5:1 and ethanol addition in 3 steps. We then used BCFS in a packed-bed reactor with recirculation. Ethanol was added in four steps, with an overall molar ratio of ethanol added to initial oil of 5.5:1, giving a conversion of 89% in 30h. When the same fermented solids were used under the same conditions in successive 30-h batches in this packed-bed bioreactor, with sequential washings with n-hexane and tert-butanol between cycles, the conversion in the 5th cycle was 66%. These results are comparable to those that have been obtained for transesterification of palm oil in solvent-free reaction media with commercial lipases, with our catalyst having the advantage that it is much cheaper to prepare.

Enzymatic esterification of acid oil from soapstocks obtained in vegetable oil refining: Effect of enzyme concentration

Abstract The enzymatic esterification of an acid waste oil was investigated using a commercial lipase ( Thermomyces lanuginosus ) in batch reactors, under the following reaction conditions: temperature of 35 °C, molar ratio of acid:alcohol of 1:1.5, vigorous magnetic stirring and enzyme concentration from 2 to 5 wt.%. The reaction progressed during 24 h. The acid oil obtained from soapstock of vegetable oil refining had an acidity of 65.5 wt.% and a very high sulphur content of 10 400 mg/kg. Thus, a pretreatment to reduce the mineral acidity before enzymatic esterification was necessary. The selected pretreatment consisted of one wash with 1:1 V/V oil:NaOH solution followed by two washings with 1:1 V/V oil:distilled water. The results from the esterification of the pretreated oil showed a clear influence of enzyme concentration in the reduction of the acidity, most of which was achieved in the first 7 h. The amount and type of alcohol had minor influence in the reaction conversion and the fractionated addition of methanol had only expressive effect for lower catalyst concentrations, with final conversions being still unsatisfactory. The best conditions found were 4 wt.% of enzyme, 35 °C, 24 h, and 1:1.5 molar ratio of acid:alcohol, which afforded an 80% reduction of acidity.

Preparation of phytosteryl ester and simultaneous enrichment of stearidonic acid via lipase-catalyzed esterification

Abstract The simultaneous synthesis of a phytosteryl ester and enrichment of stearidonic acid (SDA) were performed via a one-step lipase-catalyzed esterification of fatty acids from Ahiflower™ seed oil with phytosterol. A commercial lipase (Lipase OF) from Candida rugosa was employed as a biocatalyst. Three solvents were screened and cyclohexane was selected as a suitable reaction medium. The effects of enzyme loading, temperature, and solvent amount were investigated. The conversion as well as the SDA content were significantly improved by adding molecular sieves after 1 h of reaction. The optimum conditions were the enzyme loading of 10% (based on the total substrate weight), the temperature of 30 °C, and the solvent amount of 4 mL (based on a substrate weight of 2 g), respectively. The maximum conversion and SDA content in the residual fatty acid were 81 mol% and 58 mol%, respectively, under the optimum conditions. The SDA content increased a 3.4-fold from 17 mol% in Ahiflower™ seed oil to 58 mol%.

Hydration-aggregation pretreatment for drastically improving esterification activity of commercial lipases in non-aqueous media.

We investigated a novel, simple method for activating lipases in non-aqueous reaction media. Lipase powders were suspended in n-fatty alcohols and were then hydrated by adding a small amount of water. A paste-like aggregate was recovered from the mixture followed by lyophilization for obtaining activated lipases as dry powders. Lipase activity was evaluated for esterification between myristic acid and methanol in n-hexane. The activated lipases exhibited high esterification activity depending on the experiment conditions during hydration-aggregation pretreatment such as the amount of added water, the temperature, the pH of added buffer solutions, and the carbon chain length of the n-fatty alcohols used as pretreatment solvents. Various commercial lipases from different origins could be activated by this method. Changes in lipase conformation induced by the hydration-aggregation pretreatment were studied based on fluorescence and Fourier-transform infrared spectroscopy.

Screening of commercial enzymes for poly(ethylene terephthalate) (PET) hydrolysis and synergy studies on different substrate sources.

Poly(ethylene terephthalate) (PET) is one of the most consumed plastics in the world. The development of efficient technologies for its depolymerization for monomers reuse is highly encouraged, since current recycling rates are still very low. In this study, 16 commercial lipases and cutinases were evaluated for their abilities to catalyze the hydrolysis of two PET samples. Humicola insolens cutinase showed the best performance and was then used in reactions on other PET sources, solely or in combination with the efficient mono(hydroxyethyl terephthalate)-converting lipase from Candida antarctica. Synergy degrees of the final titers of up to 2.2 (i.e., more than double of the concentration when both enzymes were used, as compared to their use alone) were found, with increased terephthalic acid formation rates, reaching a maximum of 59,989µmol/L (9.36g/L). These findings open up new possibilities for the conversion of post-consumer PET packages into their minimal monomers, which can be used as drop in at existing industrial facilities.

Enzymatic tailoring of oleuropein from Olea europaea leaves and product identification by HRMS/MS spectrometry

Oleuropein, a bioactive compound found in all parts of olive tree, especially in leaves and branches, presents numerous health promoting properties that increase research and market interest the last few years. In addition, oleuropein degradation products, such as hydroxytyrosol, elenolic acid, and the aglycones also exhibit biological activities with different properties compared to the starting compound. Under this view, a commercial lipase preparation Lipolase 100L and a thermophilic β-glucosidase from Myceliophthora thermophila were used for the regioselective hydrolysis of oleuropein towards the production of the corresponding biologically active compounds. The enzymatic degradation products of oleuropein, such as hydroxytyrosol, elenolic acid and its glucoside, and oleuropein aglycones were identified by LC-HRMS/MS and NMR spectroscopy. The latter, was found as a mix of diastereomers of the monoaldehydic form of oleuropein aglycone, identified as (5S, 8R, 9S)-, (5S, 8S, 9S)- and (5S, 8R, 9R). The high substrate specificity exhibited by both lipase and β-glucosidase allows the successful tailoring of oleuropein towards the production of different biologically active compounds with significant potential in the cosmeceutical and food industry.

Lipase-catalyzed synthesis of biodiesel from black soldier fly (Hermetica illucens): Optimization by using response surface methodology

Black soldier fly larvae (BSFL), oleaginous insects, can assimilate several organic wastes for fat accumulation, thus serving as a promising feedstock for biodiesel production. In this study, the transesterification of BSFL fat and methanol was catalyzed using lipases to produce environmentally friendly biodiesel. Different commercial lipases were evaluated for their catalytic activity in the reaction. Among the examined biocatalysts, Novozym 435, an immobilized enzyme, showed the highest activity level. Response surface methodology was subsequently used to optimize the lipase-catalyzed reaction and to develop a reliable empirical model for prediction. A maximum biodiesel yield of 96.18% was obtained at a temperature of 26°C, methanol:fat molar ratio of 6.33, enzyme loading of 20%, and reaction time of 9.48h. Under the optimal reaction conditions and enzyme regeneration process, Novozym 435 could be reused up until 20 times, yielding 92.5% biodiesel. The properties of BSFL biodiesel were also studied; all the properties met the standard EN 14214. This study suggests that the lipase-catalyzed conversion of BSFL fat to biodiesel is a promising method toward producing a green alternative fuel.