Candida Antarctica Lipase A Research Articles

An sn-2 regioselective lipase with cis-fatty acid preference from Cordyceps militaris: Biochemical characterization and insights into its regioselective mechanism

Lipase with unique regioselectivity is an attractive biocatalyst for elaborate lipid modification. However, the excavation of novel sn-2 regioselective lipases is difficult due to their scarcity in nature, with Candida antarctica lipase A (CALA) being the pronouncedly reported one. Here, we identified a novel CALA-like lipase from Cordyceps militaris (CACML7) via in silico mining. Through chiral-phase high-performance liquid chromatography, we determined that CACML7 displays sn-2 regioselectivity (>68 %) as does CALA, but exhibits distinctive chain length selectivity and bias against unsaturated fats. Notably, the curvature of the acyl-binding tunnel was expected to contribute to the 2.2-fold higher preference for cis-fatty acid (C18:1, cis-Δ9) over trans-fatty acid (C18:1, trans-Δ9) unlike trans-active CALA. Random pose docking of trioleoylglycerol (TOG) into the active site of a lid-truncated mutant of CACML7 revealed that TOG accepts a tuning fork conformation, of which the precise positioning of the reactive ester group towards the catalytic center was only favorable via sn-2 binding mode. The unique active site morphology, which we refer to as an “acyl-binding tunnel with a narrow entrance,” may contribute to the sn-2 regioselectivity of CACML7. Our data provide an attractive model to better understand the mechanism underlying sn-2 regioselectivity.

L-cysteine-coated magnetite nanoparticles as a platform for enzymes immobilization: Amplifying biocatalytic activity of Candida antarctica Lipase A

This study presents the synthesis of magnetite nanoparticles coated with L-cysteine (Fe3O4@LC) and their subsequent utilization as a support matrix for the immobilization of Candida antarctica Lipase A (CALA). The immobilization process involved physical interactions and covalent bonding mediated by glutaraldehyde. Comprehensive characterization was conducted using techniques including X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), and vibrating sample magnetometry (VSM), providing compelling evidence of a successful synthesis. Under optimized conditions, employing 1 mg of protein per gram of support, pH 7 in a 25 mM phosphate buffer, and a 5 h reaction at 25 °C, immobilization on glutaraldehyde-activated support achieved an impressive yield of 85.0 % ± 2.6, accompanied by a specific activity of 212.5 ± 1.3 U/g, outperforming the physical adsorption approach. Remarkably, the immobilized enzyme exhibited higher activity than the free enzyme at alkaline and acidic pH levels. Furthermore, thermal and pH inactivation studies revealed that the biocatalyst's half-life exceeded that of free CALA by more than 8 times at pH 10. These results underscore the potential of the Fe3O4@LC-GLU-CALA system as a robust biocatalytic matrix with promising applications in biodiesel production, ester synthesis, and pharmaceutical manufacturing.

DIMENSIONING OF VINYLSULFONIC SUPPORTS FROM CASHEW APPLE BAGASSE BIOMASS IN THE IMMOBILIZATION OF LIPASES

In this work, the support, cashew apple bagasse (CAB), was chemically modified with divinyl sulfone (DVS), and it was evaluated to immobilize Candida antarctica lipase A (CAL-A). The best activation conditions of CAB support were defined by an advanced experimental design using the Taguchi method, assessing five factors at four levels (concentration of DVS, ionic strength, pH, temperature, and time). The support and biocatalyst (CAB-DVS-CAL-A) were characterized by Fourier transform infrared spectroscopy (FTIR), elemental analysis, thermogravimetry (TGA), scanning microscopy (SEM), fluorescence spectroscopy (XRF) and electrophoresis. The optimal conditions to activate the support were DVS concentration of 1.4 mol L-1 (3 mL of DVS in 20 mL of reaction volume), a concentration of sodium bicarbonate buffer at 5 mmol L-1, pH 3.0 at 30 °C for 12 h. The immobilization on CAB-DVS promoted increased thermal stability at 70 °C and different pHs of CAL-A. Therefore, the selected conditions allowed for a catalyst with a catalytic activity of 6.8 U g-1 and more stable than the free enzyme (CAL-A). This demonstrates that pretreated and DVS-activated CAB is a promising support for enzyme immobilization.

A Chemo-Enzymatic Cascade Strategy for the Synthesis of Phosphatidylcholine Incorporated with Structurally Diverse FAHFAs.

Fatty acid esters of hydroxy fatty acids (FAHFAs), a newly discovered class of human endogenous complex lipids showing great promise for treating diabetes and inflammatory diseases, exist naturally in extremely low concentrations. This work reports a chemo-enzymatic approach for the comprehensive synthesis of phospholipids containing FAHFAs via sequential steps: hydratase-catalyzed hydration of unsaturated fatty acids to generate structurally diverse hydroxy fatty acids (HFAs), followed by the selective esterification of these HFAs with fatty acids mediated by secondary alcohol-specific Candida antarctica lipase A (CALA), resulting in the formation of a series of diverse FAHFA analogs. The final synthesis is completed through carbodiimide-based coupling of FAHFAs with glycerophosphatidylcholine. Optimal reaction conditions are identified for each step, and the substrate affinity of CALA, responsible for the catalytic mechanisms during FAHFA production, is evaluated through molecular docking. Compared to multistep lab-tedious chemical synthesis, this route, relying on natural building blocks and natural biocatalysts, is significantly facile, scalable, and highly selective, affording high yields (74-98 mol %) in each step for the construction of higher FAHFA-PC series (10/12/13-FAHFAs). The developed strategy aims to increase the availability of naturally occurring FAHFA species and provide the tools for the construction of versatile and novel analogs of FAHFA conjugates.

Lipase-catalysed changes in essential oils revealed by comprehensive two-dimensional gas chromatography

Candida antarctica lipase A (CALA) was applied for the chemo-selective enzymatic transesterification of terpene and phenyl alcohols in 35 different essential oil samples. Comprehensive two-dimensional gas chromatography with mass spectrometry (GC×GC‒MS) analysis enabled the separation and tentative identification of a cohort of 125 compounds, allowing the instant visualisation of the reaction process changes, amid the complex chemical background of the samples. The results indicate that 42 out of 79 alcohols so-identified were fully or partially esterified within 48 h of reaction, with primary alcohols being the substrates of preference of the enzyme (90–100% conversion), followed by secondary alcohols (mostly ~ 80–100% conversion). No significant conversion of tertiary alcohols and phenols was observed using the tested conditions. Overall, the enzyme’s performance was consistent for primary alcohol substrates identified in multiple samples of different compositions. The observed selectivity, efficiency, robustness, scalability (enzyme/substrate working concentration ratio > 1:160), potential reusability, mild reaction conditions, and other factors make this process a greener and more sustainable alternative for industry applications, particularly for the manufacture of novel flavours and fragrances.Graphical

Lipase-catalyzed two-step hydrolysis for concentration of acylglycerols rich in ω-3 polyunsaturated fatty acids

In this study, enzymatic two-step hydrolysis of tuna oil was performed to release saturated (SFAs) and monounsaturated fatty acids (MUFAs) from triacylglycerols (TAGs) to obtain acylglycerols rich in ω-3 polyunsaturated fatty acids (PUFAs). At the first step, AY “Amano” 400SD was chosen to mainly hydrolyze MUFAs from tuna oil under the conditions as described previously. At the second step, second hydrolysis by Candida antarctica lipase A (CAL-A) was conducted to mainly remove SFAs in acylglycerols isolated from the hydrolysate obtained at the first step. The second hydrolysis conditions were optimized. Under the optimum conditions, the content of ω-3 PUFAs in acylglycerols increased from initial 34.3% in tuna oil to 57.65% after initial hydrolysis and further to 68.94% after second hydrolysis. The most important finding in this study is that AY “Amano” 400SD and CAL-A selectively hydrolyze MUFAs and SFAs, respectively, which may have the potential application for PUFAs concentration.

Encapsulation of lipases by nucleotide/metal ion coordination polymers: enzymatic properties and their applications in glycerolysis and esterification studies.

In the present study, lipases of TLL (lipase from Thermomyces lanuginosus), AOL (lipase from Aspergillus oryzae), RML (lipase from Rhizomucor miehei), BCL (lipase from Burkholderia cepacia), CALA (Candida antarctica lipase A) and LU (Lecitase® Ultra) were encapsulated into nucleotide-hybrid metal coordination polymers (CPs). Enzyme concentration was optimized for encapsulation and the enzymatic properties of the obtained lipases were investigated. In addition, their performance in glycerolysis and esterification was evaluated, and glycerolysis conditions (water content, temperature and time) were optimized. Hydrolysis activity over 10 000 U g-1 and activity recovery over 90% were observed from AOL@GMP/Tb, TLL@GMP/Tb and RML@GMP/Tb. GMP/Tb encapsulation (of AOL, TLL, RML and LU) improved their thermostability when incubated in air. The encapsulated lipases exhibited moderate [triacylglycerols (TAG) conversion 30-50%] and considerable glycerolysis activity (TAG conversion over 60%). TAG conversions from 69.37% to 82.35% and diacylglycerols (DAG) contents from 58.62% to 64.88% were obtained from CALA@GMP/metal samples (except for CALA@GMP/Cu). Interestingly, none of the encapsulated lipases initiated the esterification reaction. AOL@GMP/Tb, TLL@GMP/Tb, RML@GMP/Tb and CALA@GMP/Tb showed good reusability in glycerolysis, with 88.80%, 94.67%, 89.85% and 78.16% of their initial glycerolysis activity, respectively, remaining after five cycles of reuse. The relationships between temperature and TAG conversion were LnV0 =6.5364-3.7943/T and LnV0 =13.8820-6.4684/T for AOL@GMP/Tb and CALA@GMP/Tb, respectively; in addition, their activation energies were 31.55 and 53.78 kJ mol-1 , respectively. Most of the present encapsulated lipases exhibited moderate and considerable glycerolysis activity. In addition, AOL@GMP/Tb, TLL@GMP/Tb, RML@GMP/Tb and CALA@GMP/Tb exhibited good reusability in glycerolysis reactions and potential in practical applications. © 2022 Society of Chemical Industry.

Concentration of n-3 polyunsaturated fatty acid glycerides by Candida antarctica lipase A-catalyzed selective methanolysis

n-3 polyunsaturated fatty acids (n-3 PUFA) have biologically important functions in human beings, but their contents in natural oils are usually low. This study investigated the concentration of n-3 PUFA-enriched glycerides by Candida antarctica lipase A (CAL-A)-catalyzed selective methanolysis of algal oil. First, lipases and acyl acceptors were screened. Subsequently, the methanolysis conditions including methanol concentration in aqueous solution, molar ratio of methanol to oil, reaction temperature, lipase loading and reaction time, were optimized. The results indicated that CAL-A could effectively identify n-3 PUFA during methanolysis. Saturated fatty acids and monounsaturated fatty acids were released from algal oil by CAL-A-catalyzed methanolysis, causing the production of n-3 PUFA-enriched glycerides. Under optimal conditions (3:1 of molar ratio of methanol to algal oil, 20% methanol aqueous solution, 6% CAL-A dosage, 25 °C, 18 h), the n-3 PUFA content increased from the initial 45.96% to 73.96%, with an n-3 PUFA yield of 81.76%. The reusability of CAL-A demonstrated that under optimal conditions the lipase could be used for 8 times. Therefore, this enzymatic process is efficient for synthesis of n-3 PUFA-enriched glycerides.

Enhancement of the catalytic efficiency of Candida antarctica lipase A in enantioselective hydrolysis through immobilization onto a hydrophobic MOF support

Immobilization of Candida antarctica lipase A (CAL-A) onto MOF (metal-organic framework) supports were investigated to enhance the efficiency of CAL-A in catalysis of an enantioselective hydrolysis reaction. CAL-A@ZIF-8 prepared through physical adsorption of CAL-A onto ZIF-8 was characterized and further employed as the catalyzer in kinetic resolution of carprofen (CP) enantiomers through enantioselective hydrolysis of (R,S)-CP methyl ester. Results show that CAL-A is immobilized on ZIF-8 with a loading amount of 227 mg/g. Efficiency of CAL-A is significantly improved after immobilization, which is evaluated by the yield (YS) and enantiomeric excess (eep) of (S)-CP in the hydrolysis products, and the enhancement is ascribed for the hydrophobic nature of ZIF-8. The YS of 90.62 % and eep of 97.42 % are achieved with CAL-A@ZIF-8, while only 59.79 % of YS and 92.61 % of eep are obtained with free CAL-A under the identical conditions. Moreover, CAL-A@ZIF-8 possesses superior thermal stability and pH-tolerance than free CAL-A. Immobilization of the lipase onto a hydrophobic MOF is therefore proposed for enhancement of a reaction of lipase-catalyzed ester hydrolysis.

A Bi-Enzymatic Cascade Pathway towards Optically Pure FAHFAs*.

Recently discovered endogenous mammalian lipids, fatty acid esters of hydroxy fatty acids (FAHFAs), have been proved to have anti-inflammatory and anti-diabetic effects. Due to their extremely low abundancies in vivo, forging a feasible scenario for FAHFA synthesis is critical for their use in uncovering biological mechanisms or in clinical trials. Here, we showcase a fully enzymatic approach, a novel in vitro bi-enzymatic cascade system, enabling an effective conversion of nature-abundant fatty acids into FAHFAs. Two hydratases from Lactobacillus acidophilus were used for converting unsaturated fatty acids to various enantiomeric hydroxy fatty acids, followed by esterification with another fatty acid catalyzed by Candida antarctica lipase A (CALA). Various FAHFAs were synthesized in a semi-preparative scale using this bi-enzymatic approach in a one-pot two-step operation mode. In all, we demonstrate that the hydratase-CALA system offers a promising route for the synthesis of optically pure structure-diverse FAHFAs.

Enzymatic Synthesis of Estolides from Castor Oil

Estolides are fatty acid polyesters with applications in both industry and consumer products. Recently, reports have emerged detailing lipase-catalyzed synthesis of estolides from free hydroxy fatty acids. In this paper, we describe a simple alternative enzymatic process, in which castor oil is directly converted to an estolide mixture by Candida antarctica lipase A (CALA) catalyzed transesterification. The reaction mixture is analyzed by NMR to determine the estolide number (EN) and MALDI MS to identify individual components, in addition to titration to determine the acid value (AV). Estolide trimers and tetramers (EN 2–3) were formed over 24 h in a system with 2:1 (v/v) castor oil–water. Further, utilizing different lipase specificities, addition of Thermomyces lanuginosus lipase (TLL), allowed the CALA product mixture to be cleaned up by hydrolyzing attached glycerol. In addition, a three-enzyme process is suggested, in which a simple alcohol is added and Candida antarctica lipase B (CALB) is used to esterify carboxylic acids to lower AV.

Conversion of waste frying palm oil into biodiesel using free lipase A from Candida antarctica as a novel catalyst

Biodiesel, also known as fatty acid methyl ester (FAME), is usually prepared using edible vegetable oils by transesterification. In this work, a clean method for biodiesel production was developed using frying palm oil (FPO) and free Candida antarctica lipase A (CALA) as feedstock and catalyst, respectively. The effect of transesterification conditions (water content, lipase load, substrate ratio, time and temperature) on FAME yield was evaluated and optimized by response surface methodology. Results showed that free lipase CALA had the excellent methanol resistance. And lipase CALA can simultaneously catalyze the transesterification of FPO and the esterification of free fatty acid (FFA) in the presence of excess water. Low mass transfer limitation of the system using lipase CALA as catalyst was found. The effect of reaction conditions on FAME yield decreased in order of water content > reaction time > enzyme load. The optimal reaction conditions were 16.6 wt% water content and 5.5 wt% enzyme load with 7:1 molar ratio of methanol to FPO at 30 °C for 22 h. The maximum FAME yield (94.6 ± 1.4%) was achieved under the optimal conditions. Arrhenius equation and Ea of the transesterification were lnV0 = 9.45–3964.4/T and 32.96 kJ/mol, respectively. K’m and Vmax were 6.75 × 10−1 mol/L and 2.38 × 10−2 mol/(L·min), respectively. This work supplied a clean method to produce biodiesel using waste frying oil with high water and FFA contents as feedstock and a green method using cheap free liquid lipase as catalyst.

A Series of Novel Esters of Capsaicin Analogues Catalyzed by Candida antarctica Lipases

Capsaicin analogues are typically synthetized by condensation of the amide group of vanillylamine with a fatty acid derivative. The enzyme of choice to perform this reaction is Candida antarctica lipase B; however, this enzyme is unable to react with the phenolic substiruents of the vanillyl ring. So far, this can only be achieved chemically, resulting in capsaicin esters with higher lipophilicity and less irritation than the parent compound. In need of searching for new capsaicin derivatives, we investigated the transesterification of the phenolic OH group of capsaicin and several capsaicin analogues by Candida antarctica lipase A (CALA) with vinyl esters. Capsaicin esters were successfully synthetized with a reaction yield of 80.6% and 57.5% with vinyl butyrate and vinyl laurate, respectively. When the reactions were performed with capsaicin analogues of different acyl chain lengths, CALA exhibited a noticeable transesterification preference for medium-length capsaicin analogues. In an attempt to explore the catalytic limits of CALA, we performed the transesterification of capsaicin from Capsicum oleoresin. The results showed similar transesterification yields to those obtained with semi-pure capsaicin. Within our knowledge, this is the first report to achieve the synthesis of phenolic OH esters of capsaicin and capsaicin analogues by enzymatic means.

Discovery and characterization of a stable lipase with preference toward long-chain fatty acids.

To identify novel lipases with stability and long-chain fatty acids preference by phylogenetic evolution analysis methods from database. Thermo-stable Candida antarctica Lipase-A (CALA) was set as a template for gene mining by PSI-BLAST. Based on phylogenetic analysis, three candidate lipases exhibiting 97%, 55%, and 35% identities with CALA, respectively, were selected for overexpression and characterization. Lipase, PhLip from Pseudozyma hubeiensis SY62 showed highest activity towards long-chain fatty acids, and showed maximum activity at pH 9.0 and 60 °C, and stability between 40 and 50 °C for 4 h and at pH 7-10 for 12 h. Enzymatic hydrolysis of Mucor circinelloides WJ11 oils by PhLip was about twofold higher than that by CALA, with respect to hydrolysis of long-chain fatty acids. Besides, fatty acids with 18 carbons, including oleic acid, linoleic acid, and linolenic acid, were preferred as substrates. The current investigation discovered a stable lipase PhLip with long-chain fatty acids preference. PhLip may be a potential candidate for producing polyunsaturated fatty acids from natural oils.

Lipase-Catalyzed Production of (S)-Carprofen Enhanced by Hydroxyethyl-β-cyclodextrins: Experiment and Optimization

Stereoselective resolution of (R,S)-carprofen methyl ester (CPOMe) by lipase-catalyzed hydrolysis to (S)-carprofen (CP) was investigated in an aqueous medium. With the highest catalytic activity, Candida antarctica Lipase A (CALA) was selected as catalyst compared with eight other lipases. Hydroxyethyl-β-cyclodextrin (HE-β-CD) was added to enhance the solubility of (R,S)-CPOMe, which significantly raised the conversion of substrate from 11.12% to 30.84%. Response surface methodology (RSM) was adopted to evaluate the influence of factors on the substrate conversion (c) and enantiomeric excess of product (eep), such as pH, concentrations of enzyme and HE-β-CD, temperature, substrate loading, and reaction time. The optimal conditions were obtained, including pH 6.0, 40 mg/mL CALA, 0.05 mmol substrate, 35 mmol/L HE-β-CD, agitation speed of 600 rpm, temperature of 76 °C, and reaction time of 30 h. Under the above conditions, (S)-CP as the desired product was obtained with an enantiomeric excess of 96.24% and o...

Lipase-catalyzed hydrolysis of (R,S)-2,3-diphenylpropionic methyl ester enhanced by hydroxypropyl-β-cyclodextrin.

The enantioselective hydrolysis of (R,S)-2,3-diphenylpropionic methyl ester ((R,S)-2,3-2-PPAME) catalyzed by lipase to (R)-2,3-diphenylpropionic acid ((R)-2,3-2-PPA) was studied in an aqueous system. The catalytic effects of different types of lipase were compared, and Candida antarctica lipase A (CALA) with higher catalytic activity and enantioselectivity was selected. Hydroxypropyl-β-cyclodextrin (HP-β-CD) was added to the aqueous system to increase the solubility of 2,3-2-PPAME, which resulted in an increase of 35.56% in substrate conversion remaining the high enantiomeric excess. The factors influencing the substrate conversion and the optical purity of product such as temperature, pH, concentrations of CALA and HP-β-CD, substrate loading, and reaction time were optimized. The optimal conditions for this reaction were obtained, including pH of 5.5, 30 mg/mL CALA, 25 mmol/L HP-β-CD, 0.12 mmol substrate, temperature at 60 °C, agitation speed at 400 rpm, and 48 h for reaction time. Under these optimal conditions, the substrate conversion was up to 44.70% and the optical purity of the product (R)-2,3-2-PPA was up to 98.20%. This work provides an efficient alternative method for lipase-catalyzed enantioselective hydrolysis of 2,3-2-PPAME to (R)-2,3-2-PPA by β-cyclodextrin inclusion in an aqueous reaction system of hydrolysis. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1355-1362, 2018.

Modification of fatty acid selectivity of Candida antarctica lipase A by error-prone PCR

ObjectiveTo generate Candida antarctica lipase A (CAL-A) mutants with modified fatty acid selectivities and improved lipolytic activities using error-prone PCR (epPCR).ResultsA Candida antarctica lipase A mutant was obtained in three rounds of epPCR. This mutant showed a 14 times higher ability to hydrolyze triacylglycerols containing conjugated linoleic acids, and was 12 and 14 times more selective towards cis-9, trans-11 and trans-10, cis-12 isomers respectively, compared to native lipase. Lipolytic activities towards fatty acid esters were markedly improved, in particular towards butyric, lauric, stearic and palmitic esters.ConclusionDirected molecular evolution is an efficient method to generate lipases with desirable selectivity towards CLA isomers and improved lipolytic activities towards esters of fatty acids.

Candida antarctica lipase A effectively concentrates DHA from fish and thraustochytrid oils.

The fatty acid selectivity of Candida antarctica lipase A (CAL-A) was applied to produce DHA concentrate by controlling the rate and extent of hydrolysis. Calcium was utilized to achieve a higher degree of hydrolysis. CAL-A was not regioselective but rather fatty acid selective, showing sequential selectivity for saturated, monounsaturated and polyunsaturated fatty acids in the order of increasing double bonds. Based on its strong initial preference for saturates, CAL-A was used to concentrate 82% docosahexaenoic acid (DHA) and 11% omega-6 docosapentaenoic acid (DPA-n6) after partial hydrolysis of algal oil. Thermomyces lanuginosus (TL 100L) lipase was used to partially remove DPA-n6, further concentrating DHA to 89%. CAL-A was immobilized on octadecyl-activated resin without altering its fatty acid selectivity.

Rationale behind the near-ideal catalysis of Candida antarctica lipase A (CAL-A) for highly concentrating ω-3 polyunsaturated fatty acids into monoacylglycerols.

Dramatic decline in the quality and quantity of ω-3 PUFAs from marine resource demands new environmental-friendly technology to produce high quality ω-3 PUFAs concentrates in a better bioavailable form. Accordingly this work demonstrated an exceptionally highly efficient non-aqueous approach that non-regiospecific and non ω-3 PUFAs preferential Candida antarctica lipase A (CAL-A), functioning as a near-ideal biocatalyst, is capable to directly concentrate ω-3 PUFAs from 20% to 30% in oils to up to >90% in monoacylglycerols form through one step reaction. The rationale behind the experimental observation is justified and the catalytic property and specificity of an ideal enzyme tackling this task are defined. High selectivity and efficiency, excellent reusability of biocatalyst, general applicability for concentrating ω-3 PUFAs from both fish and microalgae oils, simple process for product recovery (e.g. by short path distillation), make this novel approach a highly industrially relevant and with potential application in food and drug industries.

The near-ideal catalytic property of Candida antarctica lipase A to highly concentrate n-3 polyunsaturated fatty acids in monoacylglycerols via one-step ethanolysis of triacylglycerols

Declining quantity/quality of available n-3 polyunsaturated fatty acids (n-3 PUFAs) resources demand innovative technology to concentrate n-3 PUFAs from low quality oils into value-added products/health-beneficial ingredients rich in n-3 PUFAs. This work proposed the catalytic property and specificity of an ideal enzyme required to tackle this task and identified Candida antarctica lipase A (CAL-A) is such a near-ideal enzyme in practice, which concentrates n-3 PUFAs from 25% to 27% in oils to a theoretically closer value 90% in monoacylglycerols (MAGs) via one-step enzymatic ethanolysis. Non-regiospecificity and high non-n-3 PUFAs preference of CAL-A are the catalytic feature to selectively cleave non-n-3 PUFAs in all 3 positions of triacylglycerols (TAGs); while high ethanol/TAGs ratio, low operation temperature and high tolerance to polar ethanol are essential conditions beyond biocatalyst itself. C-13 Nuclear magnetic resonance (13C NMR) analysis and competitive factor estimation verified the hypothesis and confirmed the plausible suggestion of catalytic mechanism of CAL-A.