Lipozyme IM Research Articles

Lipase‐catalysed acidolysis of mango kernel fat with capric acid to obtain medium‐ and long‐chain triacylglycerols

SummaryMango kernel fat (MKF) was blended with capric acid (1:5 mole ratio) and subjected to acidolysis using 1,3 specific lipase Lipozyme IM (Mucor miehei) for different time intervals (30 min to 24 h). The capric acid content increased gradually up to 43.5 mol% with corresponding decrease in stearic acid content (45.7–17.4 mol%). DSC crystallisation thermograms showed a new exothermic peak at lower temperature, and HPLC profile showed new peaks both indicating the formation of new lower melting triglycerides. A decrease in higher melting triglycerides and gradual increase in new triglycerides was observed. Two prominent newly formed triglycerides were characterised by ESI/MS studies and identified as 2‐oleo‐dicaprin (10, 18′, 10) and stearo‐oleo‐caprin (18, 18′, 10). Solid fat content decreased at all temperatures and samples interesterified for 5 h remained liquid at ambient conditions. The modified MKF can increase the food application from MKF in addition to contributing to health benefits of medium‐ and long‐chain triglycerides.

Acidolysis of terebinth fruit oil with palmitic and caprylic acids in a recirculating packed bed reactor: optimization using response surface methodology

The acidolysis reaction of terebinth fruit oil with caprylic and palmitic acid has been investigated. The reaction was catalyzed by lipase (Lipozyme IM from Rhizomucormiehei ) and carried out in recirculating packed bed reactor. The effects of reaction parameters have been analyzed using response surface methodology. Reaction time (3.5–6.5 h), enzyme load (10–20%), substrate flow rate (4–8 mL·min -1 ) and substrate mole ratios (Terebinth oil : Palmitic acid : Caprylic acid, 1:1.83:1.22–1:3.07:2.05) were evaluated. The optimum reaction conditions were 5.9 h reaction time, 10% enzyme load, 4 mL·min -1 substrate flow rate and 1:3.10:2.07 substrate mole ratio. The structured lipid obtained at these optimum conditions had 52.23% desired triacylglycerols and a lower caloric value than that of terebinth fruit oil. The melting characteristics and microstructure of the structured lipid were similar to those of commercial margarine fat extracts. The results showed that the structured lipid had the highest oxidative stability among the studied fats.

ChemInform Abstract: Chemoenzymatic Synthesis of Novel 1,4‐Disubstituted 1,2,3‐Triazole Derivatives from 2‐Heteroaryl Substituted Homopropargyl Alcohols.

AbstractThe reaction of aldehydes (I) with propargyl bromide and a subsequent enantiomeric resolution of resulting racemic alcohols (III) using vinyl acetate and Lipozyme IM give rise to enantioenriched benzofuranyl and benzothiophenyl homopropargyl alcohols (III), which are readily converted into chiral triazoles (VII) and (VIII).

Lipase-catalyzed synthesis of palmitanilide: Kinetic model and antimicrobial activity study

Enzymatic syntheses of fatty acid anilides are important owing to their wide range of industrial applications in detergents, shampoo, cosmetics, and surfactant formulations. The amidation reaction of Mucor miehei lipase Lipozyme IM20 was investigated for direct amidation of triacylglycerol in organic solvents. The process parameters (reaction temperature, substrate molar ratio, enzyme amount) were optimized to achieve the highest yield of anilide. The maximum yield of palmitanilide (88.9%) was achieved after 24h of reaction at 40°C at an enzyme concentration of 1.4% (70mg). Kinetics of lipase-catalyzed amidation of aniline with tripalmitin has been investigated. The reaction rate could be described in terms of the Michaelis–Menten equation with a Ping–Pong Bi–Bi mechanism and competitive inhibition by both the substrates. The kinetic constants were estimated by using non-linear regression method using enzyme kinetic modules. The enzyme operational stability study showed that Lipozyme IM20 retained 38.1% of the initial activity for the synthesis of palmitanilide (even after repeated use for 48h). Palmitanilide, a fatty acid amide, exhibited potent antimicrobial activity toward Bacillus cereus.

The effect of enzymatic interesterification on the physico-chemical properties and thermo-oxidative stabilities of beef tallow stearin and rapeseed oil blends

Beef tallow stearin (BTS) was blended with rapeseed oil (RSO) and the blends: 40 % BTS + 60 % RSO, 30 % BTS + 70 % RSO, 20 % BTS + 80 % RSO were interesterified using immobilized lipases from Rhizomucor miehei (Lipozyme IM) and Candida antarctica (Novozym 435) as the catalysts. The starting blends and the products of interesterifications were quantitatively separated into the triacylglycerol and non-triacylglycerol fractions containing free fatty acids (FFA) and of mono- and di-acylglycerols. It has been found that after interesterification the contents of the FFA and of MAG and DAG increased. The slip melting temperatures and solid fat contents of triacylglycerols separated from interesterified samples decreased if compared with non-esterified blends. The DSC melting profiles of the blends before and after interesterification were substantially different. The total fatty acid compositions of fats before and after interesterifications remained unchanged. Their distributions into sn-2 and sn-1,3 positions were close to random when Novozym 435 was used. When Lipozyme IM was used, the fatty acid composition at sn-2 position remained practically unchanged compared with the starting blend. Using the DSC and PDSC techniques, the oxidative stabilities of the fats before and after interesterification were also characterized. The interesterified blends were less resistant on thermo-oxidative degradation than the non-esterified ones.

Acidolysis Reaction of Terebinth Fruit Oil with Palmitic and Caprylic Acids to Produce Low Caloric Spreadable Structured Lipid

AbstractThe lipase (Lipozyme IM from Rhizomucor miehei) catalyzed acidolysis reaction of terebinth (Pistacia terebinthus L.) fruit oil with caprylic and palmitic acid in hexane was investigated in a batch system. The effect of reaction conditions and relationship among them were analyzed and optimized by response surface methodology with a four‐factor five‐level central composite rotatable experimental design. The four major factors chosen were enzyme load (10–20 wt%), reaction time (12–20 h), reaction temperature (45–60 °C) and substrate mol ratio (TO:PA:CA, 1:2.3–4.1:1.15–2.05). Optimum reaction conditions for reaction time, temperature, enzyme load and substrate mole ratio were 12 h, 45 °C, 10 wt% and 1:4.1:2.05, respectively. The maximum yield of desired triacylglycerols (TAG) obtained at these optimum conditions was 50.87 %. Produced structured lipid had a caloric value which was 1.5 % lower than that of terebinth fruit oil. Its solid fat content was found comparable with commercially available margarines. The relative activity of lipase was well maintained in up to 10 repeated cycles.

A Simple Enzymatic Approach for Selective Acetylation of Phosphatidylethanolamine

AbstractAcetylation of lecithin improves the fluid properties, water dispersibility and emulsifying properties of a variety of food formulations. Acetylation of lecithin is usually carried out chemically using acetic anhydride in the presence of tertiary amine. In the present study acetylation of soybean lecithin and egg yolk lecithin was carried out using lipozyme IM 20 having 1,3‐specificity from Mucor miehei and vinyl acetate at 70–75 °C. Vinyl acetate played a dual role as acetylating agent and also as the solvent medium. This method selectively acetylated phosphatidylethanolamine without effecting phosphatidylinositol since the enzyme employed was 1,3‐specific.

Enzyme–Catalysed Synthesis of Palm-Based Wax Esters-A Kinetic Study

Wax esters are long chain esters that are derived from fatty acids and alcohols with chain lengths of 12 carbons or more. Wax esters havea wide range of application in industrial especially in cosmetics. The present work focuses on the synthesis of wax esters by alcoholysisreaction from palm oil and oleyl alcohol in hexane by lipase from Rhizomucor meihei (Lipozyme IM). Effect of various concentrations ofpalm oil and oleyl alcohol were studied to deduce the kinetics and mechanism of the reaction. The alcoholysis reaction followed Michaelis-Menten kinetics. The reaction follows a Ping-pong Bi-Bi mechanism. The maximum rate was estimated to be 6 x 10 -3 mmol/h. mg catalystand the Michaelis-Menten constant for palm oil and oleyl alcohol were 4.145 M and 6.120 M, respectively.

Impact of antioxidants on oxidative stability of structured lipids

There is a growing interest among lipid technologists, lipid chemists, and nutritionistsconcerning structured lipids (SL) due to the physiological benefits these lipids offer.Compared to a mixture of medium- and long-chain TAG, SL provide improved absorptionof fatty acids and an increased delivery of total fat and essential fatty acids to the peripheraltissues [1], enhanced absorption of lipid-soluble vitamins and lipophilic antioxidants [2], andthey help to reduce muscle catabolism and improve nitrogen utilization during metabolicstress [3].Several authors have reported that SL are characterized by a lower oxidative stabilitycomparedtotheinitialoil/fatsources[4].SuchfindingshavebeenobservedforSLsynthesizedfromoliveoilandn-3PUFAorcaprylicacid,ricebranandcaprylicacid,saffloweroil,coconutoil, soybean oil, and fish oil [5–7]. According to Martin et al. [4], themethod employed in theproduction of SL is the main determinant of lipid oxidation. Yet, the results in the literatureare inconsistent. Sometimes the SL product from random chemical synthesis generates fasterandmoreextensivelipidoxidationthanlipase-mediatedsynthesisoftheSL.Atothertimestheoxidativestability ofSLrecoveredfromlipase preparationsisworsecomparedtotherandom-ized method [8].During the processing of SL, natural antioxidants present in the starting oils/fats aredegraded. The most serious concern is the loss oftocopherols and tocotrienols. This has beenobservedduringtheproductionofSLfromamixtureoflowerucicacidrapeseedoilandtallow[9], olive oil and caprylic acid [10], soybean and caprylic acid [11], as well as borage andevening primrose oils containing n-3 fatty acids [12]. According to Hamam and Shahidi [13],during the production of SL tocopherols react with carboxylic acids, giving rise to tocopherolesters. These reaction products can be removed from the reaction mixture during short-timedistillation.Byung and Akoh [14] reported that the oxidative stability of purified SL prepared fromroasted sesame oil and caprylic acid was lower than that of sesame oil itself. There were nodifferences in the contents of unsaponifiables, including tocopherols and phytosterols.However, total sesame lignans content was decreased in the SL due to the loss of sesamolwhen compared to sesame oil. A dramatic decrease of phenolic compounds in SL preparedfrom extra virgin olive oil and CLA or caprylic acid was described by Lee et al. [10].Among the food-grade antioxidants available to protect SL against oxidation,a-tocopherol is employed most often; it has been added at a level between 50 and1000 mg/kg to substrates for SL synthesis such as fish oil and caprylic acid, canola oil andcaprylicacid,soybeanoilandcaprylicacid,sunfloweroilandcaprylicacid,milkfatandlinseedoil, as well as olive oil and n-3 PUFA [11, 15, 16]. In the study of Turan et al. [11] on theoxidative stability of SL synthesized by a specific sn-1,3-lipase catalyzed interesterificationof soybean oil with caprylic acid, no significant difference was observed between theinduction time of SL and SL plus different a-tocopherol concentrations (ranging from50 to 1000 mg/kg). Antioxidant mixtures of a-tocopherol (50 mg/kg) and d-tocopherol(500 mg/kg) with ascorbyl palmitate and lecithin have been reported to effectively decreasethe peroxide value, conjugated diene content, and p-anisidine value during oxidation of SLat 60

Assessment of two immobilized lipases activity and stability to low temperatures in organic solvents under ultrasound-assisted irradiation

Both stability and catalytic activity of two commercial immobilized lipases were investigated in the presence of different organic solvents in ultrasound-assisted system. In a general way, for Novozym 435, the use of ethanol as solvent led to a loss of activity of 35% after 10h of contact. The use of iso-octane conducted to a gradual increase in lipase activity in relation to the contact time, reaching a maximum value of relative activity of 126%. For Lipozyme RM IM, after 5h of exposure, the enzyme presented no residual activity when ethanol was used as solvent. The solvents tert-butanol and iso-octane showed an enhancement of about 20 and 17% in the enzyme activity in 6h of exposure, respectively. Novozym 435 and Lipozyme IM presented high stability to storage after treatment under ultrasound-assisted system using n-hexane and tert-butanol as solvents.

Microwave activation of enzymatic catalysts for biodiesel production

The enzymatic microwave assisted biodiesel synthesis from macauba (Acrocomia aculeata) oil and ethanol using Novozyme 435 and Lipozyme IM was studied using statistically designed experiments. The investigated variables were reaction temperature, time and enzyme loading. It was observed a significant effect of the reaction time in reducing the catalytic activity which interpreted in terms enzyme deactivation due microwave exposure. The enzyme loading also played an important role, however the effect of temperature was minor appearing only in the effect of variable interactions. The result comparison between biocatalyst activity in absence and presence of microwave showed that the activity is increased about one order of magnitude due to microwave.

FOUR-FACTOR RESPONSE SURFACE OPTIMIZATION OF THE ENZYMATIC SYNTHESIS OF WAX ESTER FROM PALM KERNEL OIL

The synthesis of wax ester using refined, bleached and deodorized (RBD) palm kernel oil (PKO) and oley alcohol catalyzed by Lipozyme IM was carried out. Response surface methodology (RSM) based on a five-level, four-factor central composite rotatable design (CCRD) was used to evaluate the interactive effects of synthesis, of reaction time (5-20 h), temperature (20-50 oC), amount of enzyme (0.1-0.2 g) and substrate molar ratio (palm kernel oil to oleyl alcohol, 1:1-1:5) on the percentage yield of wax esters. The optimum condition conditions derived via RSM were reaction time 8.46 h, temperature 44.4 oC, amount of enzyme 0.182 g, substrate molar ratio 1 to 3.7. The actual experimental yield was 92.9 % under optimum condition, which good accordance to the maximum predicted value of 92.4 %. Keywords: response surface methodology, central composite rotatable design, palm kernel oil, lipozyme, alcoholysis, wax ester

The role of organic solvent amount in the lipase-catalyzed biodiesel production

This research note addresses the role of organic solvent amount in the production of fatty acid ethyl esters from soybean oil. N-hexane was chosen as solvent and two commercial immobilized lipases as catalysts, Novozym 435 and Lipozyme IM. The reactions were conducted in 6 hours, varying the solvent to oil ratio from zero to 50 (v/wt) and adopting adopting for Novozym 435: 65 ºC, enzyme concentration (E, wt%) = 5, oil to ethanol molar ratio (R) = 1:10, water addition (H, wt%) = 0, and for Lipozyme IM: 35 ºC, E = 5 wt%, R = 1:3, H = 10 wt%. For Lipozyme IM, an increase in solvent amount is shown to lead to an enhancement of reaction conversion, while a negligible effect was found for Novozym 435. When using 30 mL of solvent the reaction conversions were 88% for Lipozyme IM and 15% for Novozym 435.

Structured and Specialty Lipids in Continuous Packed Column Reactors: Comparison of Production Using One and Two Enzyme Beds

AbstractIn this study, speciality lipids based on fish oil (FO) and capric acid (CA) were produced in packed bed bioreactors using immobilized Lipozyme IM from Rhizomucor miehei in a solvent‐free environment. Our goal was to compare the product quality and yield among reactors consisting of one or two enzyme columns. Response surface methodology (RSM) was used to optimize process variables for maximum incorporation (Inc) of CA for each reactor configuration. The studied process variables were substrate molar ratio (1:1–3:1 CA/FO), temperature (35–55 °C) and flow rate (0.5–1.5 mL/min). All experiments were conducted based on a face‐centered cube design. The maximum predicted Inc of CA into FO (31.7 mol%) using one column was obtained when substrate molar ratio, temperature and flow rate of substrates were 2.70:1 (CA/FO), 55 °C and 0.5 mL/min. The corresponding optimal Inc of CA into FO (22.7 mol%) using two columns was predicted at 2.95:1 (CA/FO), 55 °C and 0.86 mL/min. Analysis of variance (ANOVA) showed that Inc of CA into the one‐bed design was significantly influenced by all experimental conditions, with substrate molar ratio having the greatest impact. In the two‐bed design, only temperature and flow rate had an effect; the molar ratio of substrates was not significant. Coefficients of determination were low for both designs, indicating a poor fit of our data to the model. However, the main purpose of this study was to assess the effect of process factors on Inc of CA into FO, rather than generate a model useful for prediction of responses under conditions not examined with this design. Therefore, the significant ANOVA results are much more important, stressing the real relationship between parameters and response, than the low coefficients of determination.

Preparation of (S)-1-Halo-2-octanols Using Ionic Liquids and Biocatalysts

Preparation of (S)-1-chloro-2-octanol and (S)-1-bromo-2-octanol was carried out by the enzymatic hydrolysis of halohydrin palmitates using biocatalysts. Halohydrin palmitates were prepared by various methods from palmitic acid and 1,2-octanediol. A tandem hydrolysis was carried out using lipases from Candida antarctica (Novozym® 435), Rhizomucor miehei (Lipozyme IM), and “resting cells” from a Rhizopus oryzae strain that was not mycotoxigenic. The influence of the enzyme and the reaction medium on the selective hydrolysis of isomeric mixtures of halohydrin esters is described. Novozym® 435 allowed preparation of (S)-1-chloro-2-octanol and (S)-1-bromo-2-octanol after 1–3 h of reaction at 40 °C in [BMIM][PF6].

Production and structural analysis of triacylglycerols containing capric acid and conjugated linoleic acid isomers obtained by enzymatic acidolysis

AbstractBACKGROUND: Structured lipids containing medium‐chain fatty acids have interesting applications as reduced‐calorie fats; moreover, conjugated linoleic acid (CLA) isomers have shown interesting biological properties. The aim of this study was to synthesize triacylglycerols (TAGs) containing capric acid in the sn‐1‐ and sn‐3‐ positions and CLA isomers in the sn‐2‐ position, using different commercial available lipases.RESULTS: The homogeneous CLA‐TAGs (Tri‐CLA) were chemically synthesized starting from glycerol and CLA isomers, 9‐cis,11‐trans and 10‐trans,12‐cis CLA. The acidolysis reactions of Tri‐CLA with capric acid were carried out at 55 °C for different times in hexane; after 96 h the acidolysis average yield was 65%. The best capric acid incorporation in total TAGs was obtained after 96 h with Lipozyme IM (56.6%). The results of structural analysis carried out on the obtained TAGs showed that both Novozyme 435 and anhydrous Lipozyme IM gave the best incorporation of capric acid in sn‐1(3)‐ positions (61.8%). However, anhydrous Lipozyme IM gave also the highest CLA percent content in sn‐2‐ position (73.2%).CONCLUSION: Anhydrous Lipozyme IM appears to be the more effective enzyme in acidolysis reactions to obtain structured TAGs containing CLA isomers in the central position and capric acid at external positions. Copyright © 2009 Society of Chemical Industry

Synthesis of structured lipid with balanced omega-3: Omega-6 ratio by lipase-catalyzed acidolysis reaction: Optimization of reaction using response surface methodology

The present study deals with the production of structured lipid containing omega-3 and omega-6 fatty acids in the ratio of 1:1 by incorporating omega-3 fatty acids (α-linolenic acid) from linseed oil into groundnut oil using lipase (Lipozyme IM from Rhizomucor miehei) catalyzed acidolysis reaction in hexane. The reaction conditions were optimized by response surface methodology with a four-variable five-level central composite rotatable experimental design. The influence of four independent parameters, namely ratio of fatty acid concentrate from linseed to groundnut oil (0.66–1.98, w/w), reaction temperature (30–60 °C), enzyme concentration (1–5%) and reaction time (2–54 h) on omega-3 fatty acids incorporation into groundnut oil were optimized. Optimal conditions for the structured lipid containing omega-3 to omega-6 fatty acids in the ratio of 1:1 were determined to be; enzyme concentration 3.75% (w/w), temperature 37.5 °C, incubation time 30.81 h and ratio of free fatty acid concentrate from linseed oil to groundnut oil 1.16 (w/w).

Lipase-catalyzed acidolysis of fish liver oil with dihydroxyphenylacetic acid in organic solvent media

Lipase-catalyzed acidolysis reaction of fish liver oil with dihydroxyphenylacetic acid (DHPA) was investigated in terms of enzyme specificity as well as the effects of enzyme concentration, molar substrate ratio and organic solvent mixture on the bioconversion yield. The highest bioconversion yield of 83% was obtained when Novozym 435 was used as biocatalyst in a hexane:2-butanone mixture of 75:25 (v/v) at a fish liver oil to DHPA substrate molar ratio of 4:1; however, lower bioconversion yield (15%) was obtained when Lipozyme IM 20 was used. The bioconversion yield of phenolic monoacylglycerols (MAGs) increased from 11 to 70% when the ratio of the hexane/2-butanone reaction medium was changed from 85:15 to 75:25 (v/v), whereas that of phenolic diacylglycerols (DAGs) remained relatively unchanged (13–16%). The results also showed that the acidolysis reaction resulted in an increase of C 20:5 ω-3 and C 22:6 ω-3 proportions from 11.5 and 20.2% in the original fish liver oil to 22.6–27.1 and 22.8–23.1% in the phenolic lipids, respectively. The radical scavenging ability of phenolic lipids was determined to be about half-time lower than that of α-tocopherol.

Preparation of isoxazolidinyl nucleoside enantiomers by lipase-catalysed kinetic resolution

An efficient biocatalytic procedure to obtain chiral N,O-nucleoside derivatives consisting of a lipase-catalysed resolution of the corresponding racemates in organic solvent has been developed. Enantioselective esterification of thymine and cytosine derivatives, (±)- 9a and (±)- 9b, has been investigated by comparing the efficiency of different lipases and acyl donors. Since esterification of (±)- 9a and (±)- 9b, occurred with low enantioselectivity ( E ⩽ 14) in the presence of the lipases considered, for preparative purposes we resorted, with success, to a double sequential kinetic resolution, using Lipozyme IM as the best catalyst. This approach enables us to obtain all the enantiomers with ee ⩾ 95%. The absolute configuration of the new chiral N-acetyl cytosine derivative 9b was established by circular dichroism spectroscopy.

Lipase-catalyzed synthesis and antibacterial activity of N-vanillylnonanamide

N-vanillylnonanamide (VAN) was successfully synthesized from vanillylamine hydrochloride by enzymatic catalysis in supercritical carbon dioxide (SC–CO 2). Five commercial lipases, Novozyme 435, Lipozyme IM, Amano PS, Amano G and Sigma Candida cylindracea type VII, as biocatalysts for VAN synthesis were compared. Lipozyme IM exhibited best yields of tested lipases. Various parameters such as time, temperature, pressure and vanillylamine hydrochloride/nonanoic anhydride ratio that influenced the reaction were investigated. Nonanoic anhydride showed the best acyl donor of the employed substrates. An amidation yield of 40% was obtained when nonanoic anhydride and Lipozyme IM were used at 170 bar and 50 °C for 23 h in SC–CO 2. Besides, addition of 2 mM divalent salts (CuCl 2 and ZnCl 2) significantly increased 11–23% yield of the VAN. The enzyme operational stability suggested that Lipozyme IM maintained over 50 °C of the initial activity for the synthesis of VAN after reuse for 69 h. Furthermore, in vitro, VAN behaved as a potential antibacterial against Escherichia coli.