Interesterification Activity Research Articles

Enzymatic synthesis of structured lipids rich in α‐linolenic acid: impacts of triacylglycerol profile on physicochemical characteristics

SummaryMedium‐ and long‐chain triacylglycerols rich in α‐linolenic acid (ALA‐MLCT) are a kind of functional structural lipids that provide ω‐3 fatty acid and faster energy supply to human body. To improve the functional and nutritional properties of vegetable oils, we proposed an effective strategy for the synthesis of ALA‐MLCT by enzymatic interesterification of coconut oil and flaxseed oil. Lipase UM1 exhibited the highest interesterification activity compared with commercial Lipozyme RM IM, Lipozyme TL IM and Novozym 435. MLCT (76.59%) and ALA‐MLCT (45.03%) were obtained under the optimum reaction conditions: substrate molar ratio (coconut oil/flaxseed oil) 1:1, temperature 40 °C, Lipase UM1 loading 25 U g‑1. Furthermore, we identified 14 characteristic triacylglycerols molecules between enzymatic interesterification and physical blend by orthogonal partial least squares‐discrimination analysis (OPLS‐DA) model. The content of triunsaturated type triacylglycerols in enzymatic interesterification product (11.29%) was lower than that of physical blend (36.25%). Enzymatic interesterification product had lower solid fat content and 2.44 times oxidative stability than physical blend. These findings provide an effective strategy for the synthesis of functional ALA‐MLCT and advance the understanding of the relationship between triacylglycerols composition and physicochemical characteristics.

Structural and functional insights into the enzymatic activities of lipases from Burkholderia stagnalis and Burkholderia plantarii.

Lipases with high interesterification activity are important enzymes for industrial use. The lipase from Burkholderia stagnalis (BsL) exhibits higher interesterification activity than that from Burkholderia plantarii (BpL) despite their significant sequence similarity. In this study, we determined the crystal structure of BsL at 1.40 Å resolution. Utilizing structural insights, we have successfully augmented the interesterification activity of BpL by over twofold. This enhancement was achieved by substituting threonine with serine at position 289 through forming an expansive space in the substrate-binding site. Additionally, we discuss the activity mechanism based on the kinetic parameters. Our study sheds light on the structural determinants of the interesterification activity of lipase.

Comparison of enzymatic activities of lipases from Burkholderia plantarii and Burkholderia cepacia.

Lipases (EC 3.1.1.3) are enzymes used in the oils and fats industries to modify the physicochemical properties of triacylglycerol (TAG). Lipase-catalyzed interesterification at high temperatures is an effective method for modifying the physicochemical properties of TAG. The lipase from Burkholderia plantarii (BpL) exhibits excellent catalytic activity for non-regiospecific interesterification at high temperatures, with depressed lipase hydrolytic activity. The detailed catalytic mechanism for reactions involving catalytic residues has not been elucidated because of the lack of a conventional method for estimating interesterification activity. We used our original water-in-oil emulsion system to estimate the interesterification activity of lipases. BpL showed 10% hydrolytic and 140% interesterification activities compared to the lipase from Burkholderia cepacia, which has a high sequence homology with BpL. By comparing the sequence and crystal structure data of the lipases, we clarified that two amino acids near the active center are one of the factors controlling the hydrolytic and interesterification activities of the enzyme.

Micro-engineering based Structuring and Valorization of Lipid Foods

Microengineering-based structuring and valorization of lipid foods were investigated. First, to control the fatty acid composition of triglycerides, modified lipases were prepared using emulsifiers and fatty acids, and applied to interesterification of fats and oils. Stearic acid was suitable for lipase activation, showing high interesterification activity. The structure of stearic acid-modified lipase was first analyzed by small-angle X-ray scattering. Next, the membrane separation performance for crude oil was investigated using hydrophobic non-porous membranes. Degumming and decolorization tests for crude soybean oil were conducted, revealing that the rejection of phospholipids and the decolorization ratio were more than 95%, representing higher quality compared to conventional methods. Membrane application to used edible oil also showed better performance than the conventional adsorption method. For practical use, improvement of the permeate flux is needed. Finally, a technology was developed for preparing monodisperse emulsions using fabricated microchannels (MCs). Efficient emulsification was achieved using asymmetrically structured MCs. The slit structure caused large distortion of the oil-water interface, and the interfacial tension induced spontaneous droplet formation. Laboratory-scale MC emulsification equipment was industrialized and used for various applications.

양송이 배지에서 유래한 Lipase 생산균을 이용한 바이오디젤 생산

A lipase producing bacterium was isolated from button mushroom bed, which showing high clear zone on agar media containing Tributyrin as the substrate. The strain was identified as Burkholderia cepacia by analysis of 16S rDNA gene sequence. Crude lipase (CL) was partially purified from 70% ammonium sulfate precipitation using the culture filtrate of B. cepacia. Immobilized lipases were prepared by cross-linking method with CL from B. cepacia and Novozyme lipase (NL) onto silanized Silica-gel as support. Residual activitiy of the immobilized CL (ICL) and immobilized NL (INL) was maintained upto 61% and 72%, respectively. Biodiesel (Fatty acid methyl ester, FAME) was recovered by transesterification and methanolysis of Canola oil using NaOH, CL and ICL as the catalysts to compare the composition of fatty acids and the yield of FAME. Total FAME content was NaOH 781 mg L-1, CL 681 mg L-1 and ICL 596 mg L-1, in which the highest levels of FAME was observed to 50% oleic acid (C18:1) and 22% stearic acid (C18:0). In addition, the unsaturated FAME (C18:1, C18:2) decreased, while saturated FAME (C16:0, C18:0) increased according to increasing the reaction times with both CL and ICL, supporting CL possess both transesterification and interesterification activity. When reusability of ICL and INL was estimated by using the continuous reaction of 4 cycles, the activity of ICL and INL was respectively maintained 66% and 79% until the fourth reaction.

Amberlite에 고정화된 Lipase 제조 및 효소적 Interesterification을 이용한 반응 특성 연구

본 연구는 미생물로부터 유래된 5종(AH, AK, AY, PS와 R)의 lipase들을 Amberlite XAD 7에 흡착법으로 고정화 시킨 후 각 immobilized lipase들의 특성을 알아보았다. 고정화 전과 후의 단백질 함량 및 각 free lipase들과 immobilized lipase들을 이용한 interesterification 반응물의 지방산과 TAG 조성을 분석하였다. 또한, immobilized lipase에 있어 중요한 요인인 reusability를 확인하였다. Free lipase의 단백질 함량은 2.22-11.41%로 AH가 가장 높았던 반면, immobilized lipase에서는 AH, PS와 AK가 mg protein/g support이 높았다. 한편, 반응 특성을 알아보기 위해 카놀라유, PEE와 StEE를 기질로 하여 batch type interesterification을 진행하였을 때, free lipase의 경우 free lipase R을 제외한 다른 free lipase들은 반응시간이 1시간에서 72시간으로 증가함에 따라 총 포화지방산 함량이 증가하였으며 그 중 free lipase AH가 반응성이 가장 높았다. 또한, RP-HPLC를 통해 free lipase AK 반응물을 분석한 결과, 반응시간이 길어질수록 카놀라유(0시간)에서 볼 수 있었던 57.49 area%의 <TEX>${\bigcirc}{\bigcirc}{\bigcirc}$</TEX>가 6.53 area%로 감소하였다. 이는 각 free lipase들이 PEE와 StEE를 효소적 반응에 이용했기 때문이라고 판단된다. 한편, immobilized lipase AY와 R의 경우 반응시간이 1시간에서 48시간으로 증가하여도 카놀라유(0시간)의 총 포화지방산 함량과 큰 차이가 없었으나 immobilized AK의 경우 48시간에서 38.3 area%의 포화지방산 함량으로 가장 높은 활성을 보였다. 또한, 이를 사용하여 continuous type으로 반응하였을 때 유속이 느릴수록 효소와 기질 사이의 접촉 시간이 길어져 반응물의 총 포화지방산 함량이 증가함을 알 수 있었다. Reusability는 immobilized AK, AH와 PS 모두에서 두 번째 반복 반응을 하였을 때, 첫 번째 반응보다 총 포화지방산이 120-196.5% 증가하였다. 그러나 bounding protein 함량이 가장 높았던 immobilized AK는 support에 흡착되었던 free lipase AK의 탈착이 일어나 2번째 반응 후부터 활성이 감소한 반면, immobilized AH와 PS는 활성이 비교적 유지되었다. Immobilized lipases were prepared by physical adsorption using lipase AK, AY, AH, PS and R on Amberlite<TEX>$^{(R)}$</TEX>XAD<TEX>$^{(R)}$</TEX>7 HP resin. With the immobilized lipases (10%), structured lipid was synthesized by enzymatic interesterification of canola oil, palmitic ethyl ester, and stearic ethyl ester in order to study the reaction characteristics. Among the lipase, the highest protein content was obtained from lipase AH (11.41%) before immobilization, while the highest levels of bound protein was observed from immobilized lipase AK (63.91%). Immobilized lipase AK had the highest interesterification activity (38.3% of total saturated fatty acid). Lipase AK was also used for a continuous reaction in which the slow flow of reactant resulted in increased reaction rate. Reusability of immobilized AK, AH and PS increased at the second reaction (120-196.5%). However, the activity of immobilized AK, which had the highest bound protein content (63.91%) decreased after the third reaction, while the activity of immobilized AH and PS was maintained until the sixth reaction.

Batch and continuous lipase‐catalyzed interesterification of blends containing olive oil for trans‐free margarines

AbstractThe interesterification of natural fats can improve certain physical and nutraceutical properties by modification of their acylglycerol profile. The aim of this study was the production of structured lipids by lipase‐catalyzed interesterification of blends of palm stearin (PS), palm kernel oil (PK), and olive oil (OO), to be incorporated in margarines. Interesterification activity was evaluated by the decrease of the solid fat content at 35°C (SFC35°C) of the blends. The best interesterification conditions, found by response surface methodology (RSM), catalyzed by “Lipozyme® TL IM” or “Lipozyme® RM IM,” were 65°C and a blend of 45% w/w of PS, 30% w/w of PK, and 25% w/w of OO. Under these conditions, after 2 h of batch interesterification, the product had a SFC35°C of 3.0%. The reaction was implemented in a continuous packed‐bed bioreactor, under optimized conditions, for 226 h (“Lipozyme® TL IM”) or 188 h (“Lipozyme® RM IM”), at a residence time of 7 min. The inactivation profile of both biocatalysts followed the first‐order deactivation model: half‐lives of 88 h (“Lipozyme® TL IM”) and 60 h (“Lipozyme® RM IM”) were estimated, respectively. Free fatty acid content of continuous interesterified blends (c.a. 1% w/w) was lower than that of batch‐interesterified blends (c.a. 4–5% w/w).Practical applications: The obtained structured lipids, with low SFC35°C, are adequate for the production of trans‐free table margarines. Using sn‐1,3 specific lipases as catalysts, the original fatty acids at sn‐2 position will be maintained with nutritional benefits. PS and PK are fats used as trans‐free sources of solid fat, for the margarine industry, which are not very sensitive to oxidation. PK, a lauric fat, imparts a fast crystallization rate and confers plasticity to the final products. The incorporation of OO will enrich the blends with natural antioxidants and oleic acid at sn‐2 position in acylglycerols. The development of response surface models, describing both final SFC values of interesterified blends and SFC decrease, will allow predicting results for novel proportions of fats and oils and/or a novel combination composition‐temperature. The implementation of the interesterification in continuous reactors promotes a faster reaction and lower acidity of interesterified blends, with benefits in terms of product separation and purification.

Optimized Production of MLM Triacylglycerols Catalyzed by Immobilized Heterologous Rhizopus oryzae Lipase

AbstractResponse surface methodology was used to model and optimize the acidolysis of virgin olive oil with caprylic (C8:0) or capric (C10:0) acids, aimed at the production of low caloric triacylglycerols (TAG) of MLM type, in solvent free media, catalyzed by the heterologous Rhizopus oryzae lipase (r‐ROL) immobilized in Eupergit® C. This lipase was produced in the methylotrophic yeast Pichia pastoris Muts phenotype (experiments with C10:0) or a Mut+ phenotype (experiments with C8:0), under different operational conditions. The r‐ROL used in experiments with C10:0 presented a hydrolytic activity about 5 times of that presented by r‐ROL used in acidolysis with C8:0. The experiments were carried out following a central composite rotatable design, as a function of the molar ratio (MR) medium chain fatty acid/TAG (1.6–4.4) and temperature (25–55 °C). Convex surfaces described by second order polynomials as a function of MR and temperature were well fitted to fatty acid incorporation values. After 24‐h reaction, the predicted maximum incorporation of caprylic (15.5 mol%) or capric (33.3 mol%) acids in olive oil occurs at 37 and 35 °C, respectively, and at C8:0/TAG of 2.8:1 or C10:0/TAG of 3:1. These predicted optima were experimentally validated. Fermentation conditions used in r‐ROL production highly affected hydrolytic activity and to a lesser extent interesterification activity.

Production of human milk fat substitutes enriched in omega-3 polyunsaturated fatty acids using immobilized commercial lipases and Candida parapsilosis lipase/acyltransferase

Abstract In human milk fat (HMF), palmitic acid (20–30%), the major saturated fatty acid, is mostly esterified at the sn -2 position of triacylglycerols, while unsaturated fatty acids are at the sn -1,3 positions, conversely to that occurring in vegetable oils. This study aims at the production of HMF substitutes by enzyme-catalyzed interesterification of tripalmitin with (i) oleic acid (system I) or (ii) omega-3 polyunsaturated fatty acids (omega-3 PUFA) (system II) in solvent-free media. Interesterification activity and batch operational stability of commercial immobilized lipases from Rhizomucor miehei (Lipozyme RM IM), Thermomyces lanuginosa (Lipozyme TL IM) and Candida antarctica (Novozym 435) from Novozymes, DK, and Candida parapsilosis lipase/acyltransferase immobilized on Accurel MP 1000 were evaluated. After 24-h reaction at 60 °C, molar incorporation of oleic acid was about 27% for all the commercial lipases tested and 9% with C. parapsilosis enzyme. Concerning omega-3 PUFA, the highest incorporations were observed with Novozym 435 (21.6%) and Lipozyme RM IM (20%), in contrast with C. parapsilosis enzyme (8.5%) and Lipozyme TL IM (8.2%). In system I, Lipozyme RM IM maintained its activity for 10 repeated 23-h batches while for Lipozyme TL IM, Novozym 435 and C. parapsilosis enzyme, linear (half-life time, t 1/2 = 154 h), series-type ( t 1/2 = 253 h) and first-order ( t 1/2 = 34.5 h) deactivations were respectively observed. In system II, Lipozyme RM IM showed linear deactivation ( t 1/2 = 276 h), while Novozym 435 ( t 1/2 = 322 h) and C. parapsilosis enzyme ( t 1/2 = 127 h), presented series-type deactivation. Both activity and stability of the biocatalysts depended on the acyl donor used.

Operational stability of immobilised lipase/acyltransferase during interesterification of fat blends

AbstractThe lipase/acyltransferase from Candida parapsilosis is an unusual enzyme that preferably catalyses alcoholysis over hydrolysis in biphasic aqueous/organic media. The aim of this study was to evaluate the operational stability of an immobilised form of this enzyme during the interesterification of fat blends containing n‐3 polyunsaturated fatty acids, in solvent‐free media, at 60 °C, carried out continuously and batchwise. When the interesterification was performed in a continuous fluidised‐bed reactor, an operational half‐life of 9 h was estimated. The biocatalyst was also reused in consecutive 23‐h batches, in a total of four batches, either using fresh medium with no water addition or adding water to rehydrate the biocatalyst. When no water and extra water was added to the reaction medium, the obtained half‐lives were 10 and 18 h, respectively. Thus, the loss of activity may be explained by a progressive dehydration occurring along the reaction rather than by product or substrate inhibition effects. The interesterification activity was accompanied by changes in the acylglycerol profile. An increase in compounds of low equivalent carbon number (ECN) and in triacylglycerols (TAG) of ECN 42 and 44 was observed. This increase was accompanied by the consumption of TAG of ECN 46, 48 and 50.

Lipase/acyltransferase‐catalysed interesterification of fat blends containing n‐3 polyunsaturated fatty acids

AbstractThe lipase/acyltransferase from Candida parapsilosis is an original biocatalyst that preferentially catalyses alcoholysis over hydrolysis in biphasic aqueous/organic media. In this study, the performance of the immobilised biocatalyst in the interesterification in solvent‐free media of fat blends rich in n‐3 polyunsaturated fatty acids (n‐3 PUFA) was investigated. The interesterification activity of this biocatalyst at a water activity (aw) of 0.97 was similar to that of commercial immobilised lipases at aw values lower than 0.5. Thus, the biocatalyst was further used at an aw of 0.97. Response surface modelling of interesterification was carried out as a function of medium formulation, reaction temperature (55–75 °C) and time (30–120 min). Reaction media were blends of palm stearin (PS), palm kernel oil and triacylglycerols (TAG) rich in n‐3 PUFA (“EPAX 4510TG”; EPAX AS, Norway). The best results in terms of decrease in solid fat content were observed for longer reaction time (&gt;80 min), lower temperature (55–65 °C), higher “EPAX 4510TG” content and lower PS concentration. Reactions at higher temperature led to final interesterified fat blends with lower free fatty acid contents. TAG with high equivalent carbon number (ECN) were consumed while acylglycerols of lower ECN were produced.

Interesterification of fat blends rich in ω-3 polyunsaturated fatty acids catalysed by immobilized Thermomyces lanuginosa lipase under high pressure

The interesterification of natural fats will improve certain physical and nutraceutical properties by modification of their acylglycerol profile. In this study, the effect of high pressure in the interesterification kinetics of fat blends, in solvent-free medium, catalysed by a commercial immobilized lipase from Thermomyces lanuginosa was evaluated. Reaction media were ternary blends of palm stearin, palm kernel oil and a concentrate of triacylglycerols (TAG) rich in ω-3 polyunsaturated fatty acids. Reactions were carried out at 60 °C, at 0.1, 50, 100 and 150 MPa. The interesterification was followed (i) by the decrease in “Solid Fat Content” values of the blend at 35 °C (SFC 35 °C ) and (ii) by the changes in the acylglycerol profile. The biocatalyst presented interesterification activity at least up to 150 MPa. Results obtained at 0.1 MPa, with no agitation of the reaction medium, were found to be similar to those obtained under high-pressure conditions. The observed decrease in SFC 35 °C values was accompanied by important changes in the acylglycerol profile. An increase in compounds of low equivalent carbon number (ECN) and in TAG of ECN 44 and 46 were observed. This increase was accompanied by a consumption of TAG of ECN 48 and 50 for all pressure values, and also of trilaurin (ECN = 36) at normal pressure. High pressures seem to affect lipase selectivity towards lauric acid. Batch operational stability tests showed a linear inactivation profile for each pressure. Half-lives of about 15, 6 and 4 h were estimated for the biocatalyst under 0.1, 50 and 150 MPa, respectively.

Interesterification activity of Rhizopus delemar lipase in phospholipid microemulsions

The interesterification of olive oil with palmitic acid catalyzed by Rhizopus delemar lipase was investigated in phospholipid microemulsion systems. Soybean lecithin was used as the amphiphilic component. The maximal reaction rate was obtained at a buffer pH of 5.5–6.0. The reaction rate was also dependent on the W L (= [H 2O]/[lecithin]) value and attained a maximum at W L = 5. The reaction rate reached a maximum at a palmitic acid concentration of 350 mM. The molar fraction of the interesterified product 1,3-dipalmitoyl-3-oleoyl glycerol (POP) in the olive oil was enhanced from 2.8 to 65.6 mol% after 24 h of the reaction.

Orlistat-induced molecular bio-imprinting of microbial lipase

Some microbial lipases bio-imprinted with the reversible lipase inhibitor, Orlistat. Comparison of interesterification activities indicated that Orlistat bio-imprinting is equally effective as amphiphile imprinting and yields a 3.5–4.0-fold activity enhancement. Solvent-free incubation medium was as effective as hexane medium. Water addition into the incubation medium erased the bio-imprinting effect; hence, lid opening should be the mechanism of activation. This study provides additional proof to the hypothesis that lipase bio-imprinting is an active-site related phenomenon.

Interesterification and hydrolysis catalyzed by fatty acid-modified lipases

Native lipases often exhibit poor interesterification activity. We previously developed a fatty acid modification method to improve the activity of lipases. In this study, we applied this fatty acid modification method to several lipases and evaluated their interesterification and hydrolytic activities. The resulting interesterification activity was strongly dependent on the modifying fatty acid used. Of the saturated fatty acids tested, stearic acid modification substantially improved the interesterification activity of three lipases. Hydrolytic activity was affected slightly by the modifying fatty acid used. Substrate specificity of the modified lipase with triglycerides was also investigated and it was found that fatty acid modification changed the substrate specificity of some lipases.

Combining the bioimprinting technique with lipase immobilization for interesterification

Lipases were bioimprinted, immobilized and bioimprinted–immobilized (coupled technique), and interesterification activities were compared. Bioimprinted–immobilized enzyme preparations gave 4–6.2-fold activity enhancements compared to solid enzyme preparations supplied by manufacturers. This increase was higher than that of bioimprinting alone and immobilization alone. Solvent-free media were equally effective and water addition caused erasure of bioimprinting. Also, new preparations showed higher thermostability and reusability.

Bio-imprinting of microbial lipase at air–water interface

Bio-imprinting has been introduced as a technique of interfacial activation of lipase for anhydrous reaction applications. In this study, air–water (bubble) interfaces were compared to amphiphile and substrate interfaces in microbial lipase bio-imprinting. Results indicated that the bubble interface is equally effective on lipase interesterification activity and produces a 4–4.5-fold increase compared with the enzymes as supplied. Interesterification activity can be explained in terms of effects upon the accessibility of the lipase active site. This technique provides an easier, cheaper and product-friendly way of lipase bio-imprinting.

Improving the application of microbial lipase by bio-imprinting at substrate-interfaces

Lipases have bio-imprinted with common substrate-interfaces and interesterification activities compared with amphiphile bio-imprinted counterparts. Bio-imprinting has yielded a 3.5- to 4.5-fold activity enhancement. Solvent-free medium was equally effective as hexane medium. Water addition erased the bio-imprinting effect. Bio-imprinting caused rate acceleration in the interesterification reaction and increased thermostability of the enzyme.

Effect of Hydrocarbon-Water Interfaces on Synthetic and Hydrolytic Activities of Lipases.

We have developed a new method of lipase activation for interesterification, in which lipase, in contact with a hydrocarbon-water interface, has been found to have high interesterification activity in an anhydrous solvent. We have applied this activation method to various lipase and obtained high synthetic activity in n-hexane, and have investigated the effect of various hydrocarbon-water interfaces on the synthetic and hydrolytic activities of lipases. The esterification and/or interesterification activity of lipases tested was improved by this activation method, using an n-tetradecane-water interface. From the initial group of lipases, three representative lipases (from Rhizopus japonicus, Chromobacterium viscosum and porcine pancreas) were selected for further study. The effect of various hydrocarbon-water interfaces on synthetic (interesterification or esterification) activity was studied. We demonstrated that the resulting synthetic activity was affected by the choice of hydrocarbon-water interface and that there were differences in the effects of interfaces on the synthetic activity of these lipases.

Effect of hydrocarbon-water interfaces on synthetic and hydrolytic activities of lipases

We have developed a new method of lipase activation for interesterification, in which lipase, in contact with a hydrocarbon-water interface, has been found to have high interesterification activity in an anhydrous solvent. We have applied this activation method to various lipase and obtained high synthetic activity in n-hexane, and have investigated the effect of various hydrocarbon-water interfaces on the synthetic and hydrolytic activities of lipases. The esterification and/or interesterification activity of lipases tested was improved by this activation method, using an n-tetradecane-water interface. From the initial group of lipases, three representative lipases (from Rhizopus japonicus, Chromobacterium viscosum and porcine pancreas) were selected for further study. The effect of various hydrocarbon-water interfaces on synthetic (interesterification or esterification) activity was studied. We demonstrated that the resulting synthetic activity was affected by the choice of hydrocarbon-water interface and that there were differences in the effects of interfaces on the synthetic activity of these lipases.