Lipase Dosage Research Articles

Construction of Pickering emulsion and its application in enzymatic synthesis of phytosteryl oleate

In this study, Janus nanoparticles were prepared and used to construct Pickering emulsion as an emulsifier in presence of lipase. The results showed that Janus nanoparticles could be used to stable water phase in emulsion and the average particle size of emulsion was 60 μm. Phytosterol ester was successfully synthesized in Pickering emulsion catalytic system by using oleic acid as acyl accepter, and the phytosterol conversion reached 91.65 ± 2.07% under the following conditions: emulsifier amount of 1.0 wt%, oil-water ratio of 6:1, phytosterol concentration of 110 mmol/L, lipase dosage of 20 g/L, reaction temperature of 45 °C. Compared with biphasic catalytic system, Pickering emulsion catalytic system could enhance the catalytic efficiency and shorten the reaction time due to large interface area and short molecule diffusion distance. At the end of reaction, Janus nanoparticles could be recovered by simple centrifugation, the relative activity of lipase remained 72.89 ± 1.83% after repeated use for 6 times. Overall, this paper provides a novel and high efficiency interfacial biocatalysis technique for the synthesis of phytosteryl oleate.

Biodiesel production via simultaneous esterification and transesterification of Periplaneta americana oil with liquid lipase Eversa® transform 2.0

Undeveloped Periplaneta americana oil (acid value 38.32 mg KOH/g) was directly used for one-step production of biodiesel with lipase without acid-pretreatment step for the commercial alkaline process. Biodiesel was produced via simultaneous esterification and transesterification of Periplaneta americana oil in the presence of lipase Eversa® Transform 2.0 (ET2) (12 US$/kg) in solvent-free system. The maximum biodiesel yield of 98.63 % was obtained under the optimized conditions of 32 °C, 8.5 wt.% lipase dosage, 8 h, 6.5/1 methanol/oil molar ratio, and 4 wt.% water. Lipase ET2 was recycled 6 times at > 89.52 % biodiesel yield. Biodiesel yield of 93.94 % was further achieved in a 1 L reactor with 15.08 g/kg lipase/biodiesel. Biodiesel cost was estimated as 589.3 US$/ton. Kinetics study gave activation energy of 24.50 kJ/mol with kinetic Michaelis constant of 1.19 mol/L. The physicochemical properties of biodiesel met both Chinese national and US ASTM standards that could be blended with petro-diesel to be applied in both countries. This study suggests that lipase could directly catalyze waste oils for the production of biodiesel at low temperature.

Electrostatically Mediated In Situ Polymerization for Enzyme Immobilization and Activation.

Enzyme immobilization in nanoparticles is of interest for boosting their catalytic applications, yet rational approaches to designs achieving both high enzyme loading and activation remain a challenge. Herein, we report an electrostatically mediated in situ polymerization strategy that simultaneously realizes enzyme immobilization and activation. This was achieved by copolymerizing cationic monomers with a cross-linker in the presence of the enzyme lipase (anionic) as the template, which produces enzyme-loaded nanogels. The effects of different control factors such as pH, lipase dosage, and cross-linker fraction on nanogel formation are investigated systematically, and optimal conditions for enzyme loading and activation have been determined. A central finding is that the cationic polymer network of the nanogel creates a favorable environment that not only protects the enzyme but also boosts enzymatic activity nearly 2-fold as compared to free lipase. The nanogels improve the stability of the lipase to tolerate a broader working range of pH (5.5-8.5) and temperature (25-70 °C) and allow recycling such that after six cycles of reaction, 70% of the initial activity is conserved. The established fabrication strategy can be applied generally to different cationic monomers, and most of these nanogels exhibit adequate immobilization and activation of lipase. Our study confirms that in situ polymerization based on electrostatic interaction provides a facile and robust strategy for enzyme immobilization and activation. The wide variety of ionic monomers, therefore, features great potential for developing functional platforms toward satisfying enzyme immobilization and demanding applications.

Ultrasound pretreatment for lipase-catalyzed synthesis of stigmasteryl oleate and evaluation of its physicochemical properties

Stigmasterol is an important natural compound with the ability to lower cholesterol and prevent cardiovascular disease. It is often esterified with fatty acids to enhance its range of applications and improve its bioaccessibility. In this paper, stigmasteryl oleate was efficiently synthesized via lipase catalysis under optimized ultrasound pretreatment conditions, and the physicochemical properties of stigmasteryl oleate were investigated. The results showed that the stigmasterol conversion reached 91.37% with an ultrasound power of 200 W, ultrasound pretreatment time of 1 h, lipase dosage of 50 g/L, and ultrasound pretreatment temperature of 50 °C. The chemical structure of the final stigmasteryl oleate product was identified using Fourier transform infrared spectroscopy, mass spectrometry and nuclear magnetic resonance spectroscopy. The melting and crystallization temperatures of stigmasteryl oleate were significantly decreased to 22.8 and 12.9 °C, respectively, while its solubility in edible oil was more than 20 times higher than that of stigmasterol. The peroxide value of stigmasteryl oleate was much lower than that of oleic acid (27.53 and 41.39 mmol/kg). The product of stigmasteryl oleate with more favorable physicochemical properties greatly facilitates the widespread use of stigmasterol in oil-based foods.

Deficiência de Lipase Ácida Lisossômica (LAL): Análise Enzimática em Papel-filtro como Ferramenta Diagnóstica em Paciente com Diagnóstico Prévio de Doença de Niemann-Pick tipo C

Lysosomal acid lipase deficiency (LAL-D) is a lysosomal storage disorder involved in cholesterol ester metabolism. It is a poorly understood genetic cause of cirrhosis, dyslipidemia and premature atherosclerotic disease in children and adults. As the manifestations of LAL-D may resemble those observed in other more common diseases, delayed diagnosis is not uncommon. Wolman disease is an early-onset LAL-D phenotype that is usually fatal in the first year of life, but the most common form of the disease may occur at all ages. Recently, enzyme replacement therapy (ERT) for LAL-D has become available. Case report: A 12- year-old male patient was referred for hepatomegaly. Initially diagnosed as having Niemann-Pick type C disease (NPC), it did not present confirmatory mutations of this disease. Later, with enzymatic dosage of lysosomal acid lipase on filter paper, the diagnosis was redirected to LAL-D (with confirmation in enzymatic dosage in leukocytes and fibroblasts). Discussion: Although LAL-D is considered a rare and uncommon cause of liver disease, recent studies point to a higher prevalence of this disease. Early diagnosis is essential, since specific treatment for this disease is already available. About half of patients with LAL-D die before age 21 in the absence of adequate treatment

Deficiência de Lipase Ácida Lisossômica (LAL): análise enzimática em papel-filtro como ferramenta diagnóstica em paciente com diagnóstico prévio de doença de Niemann-Pick tipo C

INTRODUCTION: Lysosomal acid lipase deficiency (LAL-D) is a lysosomal storage disorder involved in cholesterol ester metabolism. It is a poorly understood genetic cause of cirrhosis, dyslipidemia and premature atherosclerotic disease in children and adults. As the manifestations of LAL-D may resemble those observed in other more common diseases, delayed diagnosis is not uncommon. Wolman disease is an early-onset LAL-D phenotype that is usually fatal in the first year of life, but the most common form of the disease may occur at all ages. Recently, enzyme replacement therapy (ERT) for LAL-D has become available. CASE REPORT: A 12-year-old male patient was referred for hepatomegaly. Initially diagnosed as having Niemann-Pick type C disease (NPC), it did not present confirmatory mutations of this disease. Later, with enzymatic dosage of lysosomal acid lipase on filter paper, the diagnosis was redirected to LAL-D (with confirmation in enzymatic dosage in leukocytes and fibroblasts). DISCUSSION: Although LAL-D is considered a rare and uncommon cause of liver disease, recent studies point to a higher prevalence of this disease. Early diagnosis is essential, since specific treatment for this disease is already available. About half of patients with LAL-D die before age 21 in the absence of adequate treatment.

Highly Efficient Regioselective Acylation of Dihydromyricetin Catalyzed by Lipase in Nonaqueous Solvents

This study aimed to explore the enzymatic acylation of dihydromyricetin (DHM) to synthesized DHM derivatives with a different substituted carbon chain to improve its liposolubility. In the presence of Lipozyme TL IM, DHM was butyrylated in a 96.28% conversion in methyl tert-butyl ether under the optimized conditions (molar ratio of DHM to vinyl butyrate, 1:20; lipase dosage, 0.4 U/mg DHM; temperature, 50 °C; stirrer speed, 200 rpm; reaction time, 72 h). Liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) spectroscopy revealed that two acylation products were formed; these were 7-O-acyl-DHM and 3-O-acyl-DHM. In addition, the liposolubility of the DHM derivatives increased with the increase in the substituted carbon chain length; their antioxidant activities were higher than that of DHM in the lecithin peroxidation system, and C8-DHM had a better effect. Therefore, enzymatic acylation broadens the application of DHM in a lipid system in the food field.

Response Surface Optimization of Lipase‐Catalyzed Surface Esterification for Modification of Cassava Starch with Rosin Acid

The rosin acid starch (RAS) is prepared under the catalysis of lipase, by esterification on the starch particles' surface in a two-phase reaction system, using rosin acid and native cassava starch as starting materials. The effects of reaction time, solvent types, substrate ratio, reaction temperature, and lipase dosage on the degree of substitution (DS) of RAS are investigated. Based on the single factor experiments, the reaction conditions are optimized according to the principle of the combination Box-behnken centered design and response surface methodology analysis. The mathematical model of the influence of reaction temperature, material ratio, and enzyme addition amount on DS is established. The optimum synthesis conditions are determined as follows: solvent, trichloromethane; reaction time, 10 h; reaction temperature, 59 °C; substrate ratio (mRosin acid:mstarch), 4.1; and lipase dosage, 264 µL. Under these conditions, it is found that the DS is 0.0608. The analysis of variance and verification experiments show that the mathematical model has good fit degree and high credibility. Compared with the native cassava starch, RAS has improved contact angle, emulsion capacity, and emulsion stability.

Enhancing biodiesel production via liquid Yarrowia lipolytica lipase 2 in deep eutectic solvents

With the rapid development of effective overexpression of enzymes, the process of liquid lipase catalysis of biodiesel can be concerned in a commercial viewpoint. The main bottleneck is to achieve high yield of biodiesel under a high water content. In this research, firstly, liquid Yarrowia lipolytica Lipase 2 was used to catalyze synthesis of biodiesel and 91.8% yield was obtained in the optimized reaction conditions after 24 h of reaction at 40 °C, water content 30 wt% (water/oil, w/w) and 1500 U/g oil of lipase dosage. To further improve the biodiesel yield, a deep eutectic solvent based on choline chloride and glucose was synthesized and optimized in terms of addition amount and water content, and the highest biodiesel yield of 98.5% was then achieved. The dynamics of each component during the reaction process were totally investigated, and found it was the addition of deep eutectic solvent that led to a deeper completion of the transesterification reaction. It also improved the reusability of the enzyme with a maximum biodiesel yield of 99.6% at the second reuse batch. This study demonstrates that deep eutectic solvent addition strategy has great potential for the commercialization of liquid lipase-catalyzed biodiesel production.

Synthesis of partial glycerides rich in α-linolenic acid efficiently from silkworm pupa oil with immobilized lipase MAS1-H108A

In this paper the ability of immobilized lipase MAS1-H108A to prepare α-linolenic acid-rich partial glycerides was studied. The effects of different reaction conditions on the yield of Diacylglycerol (DAG) and Monoglyceride (MAG) were investigated, including substrate molar ratio, substrate concentration, temperature and lipase dosage. The highest yield (63.92% of MAG and 30.61% of DAG) was obtained when silkworm pupa oil/glycerol ratio of 1:3, lipase dosage was 50 U/g, substrate concentration was 40% (w/v), at 40 °C. Under the optimal conditions, the scale-up (50 times) and single-stage molecular distillation were carried out to separate DAG and MAG, the yield of MAG in the light phase was 99.18%, and the concentration of DAG in the heavy phase was 89.47%, α-linolenic acid in MAG and DAG accounted for 28.96% and 28.23%, respectively. The results can be shown that the immobilized lipase MAS1-H108A has excellent potential for enzymatic glycerolysis to prepare functional partial glycerides.

Bionic-immobilized recombinant lipase obtained via bio-silicification and its catalytic performance in biodiesel production

In this study, bio-silicification was employed to develop a green and simple bionic-immobilization method for the immobilization of recombinant lipase. The results showed that under the optimized bionic-immobilization conditions, a maximum immobilization efficiency and an activity recovery of 97.71% and 89.62%, respectively, were obtained. Further, bionic-immobilization significantly improved the thermal stability, storage stability, and short-chain alcohol resistance of the bionic-immobilized lipase. Furthermore, molecular dynamics simulation of the bionic-immobilization mechanism revealed that the key amino acid residues that facilitated the binding of the recombinant lipase to silica particles included Arg336, Lys345, His391, His392, His394, and His395, indicating that hydrogen bonds had a special effect in intermolecular recognition, and the active center of the bionic-immobilized lipase favored substrate entry due to the conformational changes of Phe291 and Ile320. Finally, biodiesel was prepared using the bionic-immobilized lipase as catalyst, and after optimizing the enzymatic process, the biodiesel yield reached 91.04% using 20% immobilized lipase dosage within 12 h at 45 °C, and a catalytic activity of 55.02% was observed following the reutilization of the bionic-immobilized lipase seven times. These findings suggest that bionic-immobilization is a novel and rapid method to realize recombinant enzyme immobilization, and can provide economic and ecological support for the biocatalytic preparation of industrial products.

Ultrasound-assisted enzymatic preparation of fatty acid ethyl ester in deep eutectic solvent

In this paper, fatty acid ethyl ester (FAEE) was enzymatically produced from the waste oil and ethanol. It was investigated under three different conditions: ultrasound, deep eutectic solvent (DES) and ultrasound-assisted DES. The effects of lipase dosage, alcohol oil ratio, reaction temperature, ultrasonic power, reaction time and DES dosage on the conversion rate of FAEE were explored. The ultrasonic-assisted DES optimization experiment was carried out through the response surface experiment with five factors and three levels. The experimental results showed that the conversion of FAEE was significantly improved and the reaction time was shortened under the ultrasound-assisted DES system, the ultrasonic power and reaction temperature had synergistic effect on the conversion of FAEE. The conversion rate of FAEE reached 93.61 ± 0.46% on the condition of reaction temperature 59 °C, ultrasonic power 80 W, and reaction time 137 min with addition of 6.4% lipase and 4 mmol alcohol for 1 g oil.

EVALUATION OF THE KINETICS OF A LIPASE CATALYSED BIODIESEL PRODUCTION FROM A MIXTURE OF WASTE COOKING OIL AND SOYBEAN OIL

Commercial lipase Novozym 435 was used to catalyse the transesterification of a mixture of waste cooking oil and soybean oil with methanol. A central composite experimental design was performed to analyze the effects of lipase dosage, waste cooking oil content in the mixture, and reaction time on the methyl ester yield. Lipase dosage and waste cooking oil content were the most significant variables after statistical analysis. The highest yield, 98.04%, was obtained using 12.5% of lipase, a mixture containing 50% of waste cooking oil after 10 h of reaction. The lipase was reutilized and showed a good operational stability since the yield was reduced by 6% even after five batches. Two kinetic models, Power Law and Eley-Rideal, were used to model the production process and correlate the experimental results.

Ultrasonic Pretreatment in Synthesis of Caprylic-Rich Structured Lipids by Lipase-Catalyzed Acidolysis of Corn Oil in Organic System and Its Physicochemical Properties.

The current work was to evaluate the lipase-catalyzed acidolysis of corn oil with caprylic acid (CA) in organic system under bath ultrasonic pretreatment and to analyze the physicochemical properties of structured lipids (SLs). Under optimum conditions (Novozym 40086 lipase, 200 W ultrasound power, 10 min ultrasound pretreatment time, 12% dosage of lipase, Triacylglycerol (TAG)/Free fatty acids (FFA): 1/8, 40 °C for 6 h), a 45.55% CA incorporation was obtained (named SLs-U). The highest CA incorporation was 32.75% for conventional method at reaction time of 10 h (named SLs-N). The predominant TAG types of SLs were MLM (medium-, long- and medium-chain-type TAGs) and MLL (medium-, long- and long-chain-type TAGs). X-ray diffraction analysis revealed that both SLs-U and SLs-N present β form. Differential scanning calorimetry (DSC) analysis showed that both SLs-U and SLs-N show a lower melting and crystallization temperature than corn oil. This study suggested that bath ultrasonic pretreatment can accelerate lipase-catalyzed acidolysis synthesis of MLM structured lipids in an organic system, and two kinds of structured lipids show similar physicochemical properties.

Numerical simulation and deacidification of nanomagnetic enzyme conjugate in a liquid–solid magnetic fluidized bed

The conventional deacidification method is difficult to achieve a better refining effect due to the high acid value in the rice bran crude oil, and the enzymatic esterification deacidification method can effectively reduce the acid value without generating chemical waste. In this study, the free lipase was immobilized on a magnetic polymer carrier Fe3O4/SiOx-g-P (GMA: glycidyl methacrylate) to obtain a immobilized lipase with a particle size of 105.30 ± 1.1 nm and an enzyme activity of 6580 ± 9.6 PLU/g (PLU: enzyme activity unit). Based on the batch deacidification process parameters, a multi-stage magnetic fluidized bed continuous circulation deacidification system was designed, and then the motion law of nanomagnetic immobilized lipase particles in liquid–solid magnetic fluidized bed was simulated by computer. When the iterative step was 5 × 10−5 s, the open porosity of the porous plate was 35.0%, the rice bran oil flow rate was 3.0 mm/s, and the magnetic field strength was 25.0 mT, which was beneficial to the deacidification reaction of rice bran oil. Under the conditions of magnetic immobilized lipase dosage of 4.0%, the phytosterol dosage of 22.0%, the molecular sieve dosage of 10%, the esterification temperature of 78.0 °C and the FFA (free fatty acid) content in rice bran oil decreased to 1.5%, after 48 h of reaction. The conversion rate is 92.8%, which provides a theoretical basis for the subsequent guidance of magnetic fluidized bed enzymatic continuous deacidification.

Enzymatic esterification of rice bran oil and phytosterol in supercritical CO 2

In this paper, the esterification of phytosterol and free fatty acids (FFA) in rice bran oil (RBO) was studied using Novozym 435 in supercritical CO₂. It was found that with the increase in CO₂ filling pressure, the esterification reaction rate also increased. When the pressure exceeded the critical point, CO₂ fluid had a great effect on phytosterol conversion. Therefore, enzymatic esterification deacidification was studied when the CO₂ filling pressure was 8.0 MPa, the results showed that when the lipase dosage was 3.0%, the phytosterol concentration was 18.0%, the molecular sieve addition was 10.0%, and the esterification temperature was 78°C, after 41 hr the FFA content of RBO decreased to 1.1%, the phytosterol conversion rate reached 93.2%, and the vitamin E (VE) retention in the product was 80.0%. Therefore, the esterification by Novozymes 435 in supercritical CO₂ could have several applications in fats and oils industry. PRACTICAL APPLICATIONS: Much research has been done on the interesterification of vegetable oil in order to produce edible fats and oils. The current study provides a reliable and simple method for the esterification of phytosterol and free fatty acids (FFA) in rice bran oil (RBO). The optimal operating conditions lead to high quality oil. The FFA content of RBO decreased to 1.1%, the phytosterol conversion rate reached 93.2%, and the VE content in the product was 80.0%. The esterification by Novozymes 435 in supercritical CO₂ could be applied in fats and oils industry.

A novel clean process for the combined production of fatty acid ethyl esters (FAEEs) and the ethyl ester of polyunsaturated fatty acids (PUFAs) from microalgae oils

Although the methanolysis of microalgae oil for biodiesel preparation was studied previously, the ethanolysis for the combined production of fatty acid ethyl esters (FAEEs, also can be used as biodiesel) and the ethyl esters of PUFAs (an important health-care product) rarely reported. In this work, a novel two-step process, hydrolysis of microalgae oil followed by lipase-mediated ethanolysis, was developed for the synthesis of FAEEs and the ethyl ester of PUFAs. The hydrolysis was firstly performed and the generated free fatty acids was collected and further used for FAEEs synthesis catalyzed by lipase. For lipase-catalyzed ethanolysis, the adding strategy of methanol, molecular sieve dosage, lipase dosage and temperature had significant influence on the FAEE yield. Under the optimized conditions, a FAEE yield of 97% could be obtained. Novozym435 was found to be equally effective for catalyzing the ethanolysis of conventional free fatty acids and PUFAs. The combined effects of multiple factors on the ethanolysis was further studied using Response Surface Methodology (RSM). Validation statistical analysis suggested that the negative correlation existed between FAEE yield and the water content. This work provided a novel strategy to produce FAEEs and the ethyl esters of PUFA effectively with microalgae oil as the substrate.

Amano Lipase PS-catalyzed Hydrolysis of Pine Nut Oil for the Fatty Acids Production Using Deep Eutectic Solvent as Co-solvent.

Free fatty acids (FFAs) are the important material used in food, personal care, emulsifiers, adhesives and surfactants. In order to enhance the preparation of FFAs, the effects of reaction variables, optimization, thermodynamic property for the Amano lipase PS catalyzed hydrolysis of pine nut oil (PNO) using deep eutectic solvents (DESs) as co-solvents were studied. The results showed that FFAs could be successfully prepared from pine nut oil through Amano lipase PS catalyzed hydrolysis using Choline chloride:Urea (ChCl:U, 1:2, mol/mol) as co-solvent. Under the optimal conditions (reaction temperature 46°C, water amount 38%, DES addition 43%, lipase dosage 7.6%, reaction time 13 h), the maximum content of FFAs in the products and degree of hydrolysis (DH) of oil were up to 89.1 ± 1.9% and 92.7 ± 2.2%, respectively. The effects of reaction variables on the hydrolysis increased in the order of DES addition < reaction temperature < reaction time < lipase dosage < water amount. The thermodynamics (Arrhenius equation) for the triglycerides hydrolysis was V = 4289.39·exp(-22942.09/RT) with the activation energy (Ea) of 22.94 kJ/mol. The Gibbs free energy (ΔG), enthalpy (ΔH) and entropy (ΔS) were 81.50 ± 2.64 kJ/mol, 20.18 ± 0.12 kJ/mol and -184.59 ± 0.36 J/mol/K, respectively. The lipase in the aqueous DES could be directly re-used for 3 times.

Lysosomal Acid Lipase Deficiency Leading to Liver Cirrhosis: a Case Report of a Rare Variant Mutation

Lysosomal acid lipase deficiency is a poorly diagnosed genetic disorder, leading to accumulation of cholesterol esters and triglycerides in the liver, with progression to chronic liver disease, dyslipidemia, and cardiovascular complications. Lack of awareness on diagnosis of this condition may hamper specific treatment, which consists on enzymatic replacement. It may prevent the progression of liver disease and its complications. We describe the case of a 53-year-old Brazilian man who was referred to our center due to the diagnosis of liver cirrhosis of unknown etiology. He was asymptomatic and had normal body mass index. He had dyslipidemia, and family history of myocardial infarction and stroke. Abdominal imaging tests showed liver cirrhosis features and the presence of intrahepatic calcifications. Initial investigation of the etiology of the liver disease was not elucidated, but liver biopsy showed microgoticular steatosis and cholesterol esters deposits in Kuppfer cells. The dosage of serum lysosomal acid lipase was undetectable and we found the presence of a rare homozygous mutation in the gene associated with the lysosomal acid lipase deficiency, (allele c.386A &gt; G homozygous p.H129R).

Sugar-Improved Enzymatic Synthesis of Biodiesel with Yarrowia lipolytica Lipase 2

In this research, it was found that in Yarrowia lipolytica Lipase 2 (YLLIP2)-catalyzed biodiesel production, the content of fatty acid methyl esters (FAMEs) was increased by about 10% with the addition of d(+)-glucose. The result indicated that the d(+)-glucose could be used as an effective additive in YLLIP2-catalyzed biodiesel production. In accordance with the results above, the single factor experiments of key parameters in the process were first carried out. On the basis of the single factor experiment results, a five-factor, three-level response surface method was adopted to obtain the optimal reaction conditions: lipase dosage 40 IU/g oil, d(+)-glucose to lipase 1:1.05 (w/w), water content 1.95%, and reaction temperature 39.4 °C. A stoichiometric amount of methanol (11.5%, methanol/waste oil) was added in 6 times (every 4 h for each addition). A subsequent pilot scale production in a 5-ton reactor was carried out to check the performance of this method, and a good biodiesel content of 91.4% was obt...