Specificity of papaya lipase in esterification of aliphatic alcohols a comparison with microbial lipases

Straight-chain saturated C4 to C18 alcohols and unsaturated C18 alcohols such as cis-9-octadecenyl (oleyl) cis-6-octadecenyl (petroselinyl), cis-9, cis-12-octadecadienyl (linoleyl), all-cis-9,12,15-octadecatrienyl (α-linolenyl), and all-cis-6,9,12-octadecatrienyl (γ-linolenyl) alcohols, were esterified with caprylic acid using papaya (Carica papaya) latex lipase (CPL) and immobilized lipase from Candida antarctica (Lipase B, Novozym, NOV) and Rhizomucor miehei (Lipozyme, LIP) as biocatalysts. With CPL, highest activity was found for octyl and decyl caprylate syntheses, whereas both NOV and LIP showed a broad chain-length specificity toward the alcohol substrates. CPL strongly discriminated against all C18 alcohols studied, relative to n-hexanol, whereas the microbial lipases accepted the C18 alcohols as substrates nearly as well as n-hexanol. Both petroselinyl and γ-linolenyl alcohol were very well accepted as substrates by NOV as well as LIP, although the corresponding fatty acids, i.e., petroselinic and γ-linolenic acid, are strongly discriminated against by several microbial and plant lipases, including LIP and CPL.     

Synthesis, purification, and characterization of structured lipids produced from chicken fat

A structured lipid (SL) was synthesized enzymatically from chicken fat by incorporating a medium-chain length fatty acid (caprylic acid) into chicken fat triacylglycerols. Carica papaya latex was used as the biocatalyst. The optimal substrate mole ratio found was 1∶2 (chicken fat fatty acids/caprylic acid). At this ratio of reactants, the incorporation of caprylic acid (C_8∶0) at 65°C was 23.4 mol%, whereas at 55°C the incorporation of caprylic acid was 17.6 mol%. A packed-bed column bioreactor was designed for the synthesis of SL from chicken fat. In using ground crude C. papaya latex (a _w <0.1), 7.1 mol% of caprylic acid was incorporated into the chicken fat triacylglycerols after 117 min of reactor residence time. After purification of the SL, the acyl positional distribution of fatty acids on the glycerol backbone was determined by ¹³C nuclear magnetic resonance (NMR) spectroscopy. From the NMR spectrum of the SL, it was determined that saturated fatty acyl residues at the 1,3-positions of the SL triacylglycerols increased to 62% over that of the starting chicken fat triacylglycerols, suggesting that caprylic acid was preferentially incorporated at the 1,3-positions. In addition, differential scanning calorimetry thermograms were obtained to compare the crystallization characteristics of the starting chicken fat with the SL prepared from it. This work was presented at the Biocatalysis Symposium in April 2000, held at the 91st Annual Meeting and Expo of the American Oil Chemists Society, San Diego, CA.     

Carica papaya latex-catalyzed synthesis of structured triacylglycerols

One impediment to the industrial use of enzymes in fat and oil transformations is the higher cost often associated with an enzymatic process compared with the corresponding chemical process. Processes that utilize plant enzymes, however, may have advantages because of their lower cost and ready availability. One example of such a plant-derived enzyme is Carica papaya latex (CPL), the principal source of the protease papain. Recently, it has been shown that this latex also catalyzes the lipolysis of triacylglycerols and that this latex lipase has a selectivity for short-chain acyl groups as well as a 1,3-glycerol selectivity. These selectivities can be used in the synthesis of structured triacylglycerols. In this paper we describe the utility of CPL in lipase-catalyzed reactions, specifically the synthesis of low-calorie triacylglycerol analogs. Presented in part at the American Oil Chemists’ Society’s 88th Annual Meeting & Expo, Seattle, WA, May 1997.     

Carica papaya latex lipase:sn-3 stereoselectivity or short-chain selectivity? Model chiral triglycerides are removing the ambiguity

Short-chain fatty acids are usually located at positionsn-3 in natural triglycerides, particulary in dairy fats. As a result, it is extremely difficult to differentiate betweensn-3 stereospecificity and short-chain typoselectivity in many lipases and acyltransferases that perform in this way. This ambiguity can be removed through successive use of a chiral triglyceride with a short fatty acid in positionsn-1 and of its racemic in controlled hydrolysis reactions. After checking that the proposed method effectively confirmed the type of activity of control biocatalysts (Candida cylindracea nonspecific lipase andMucor miehei 1–3 regiospecific lipase), we confirmed thatCarica papaya latex has a strictsn-3 stereospecificity.     

Reactivity of medium-chain substrates in the interesterification of tripalmitin catalyzed by papaya lipase

Reactivity of different medium-chain substrates, i.e., n-octanol, caprylic acid, and its alkyl (methyl, ethyl, n-propyl, and n-butyl) esters, was assessed in the interesterification of tripalmitin catalyzed by papaya (Carica papaya) lipase. Alcoholysis with n-octanol was the fastest reaction leading to the highest conversion of tripalmitin to n-octyl palmitate and concomitant formation of di- as well as monopalmitoylglycerols. This was followed by transesterification of tripalmitin with n-butyl and n-propyl caprylates, which in turn were faster than transesterification with ethyl and methyl caprylates, yielding in each case the corresponding alkyl palmitates and triacylglycerols containing palmitoyl and capryloyl moieties as the major reaction products. Acidolysis of tripalmitin with caprylic acid yielded palmitic acid and triacylglycerols containing palmitoyl and capryloyl moieties as the major reaction products, however, with the lowest conversion among the three interesterification reactions studied. In each case, interesterification was accompanied by some hydrolysis of tripalmitin.     

Investigation of crude latex from various Carica papaya varietices for lipid bioconversions

The protein contents in crude latices from various varieties of papaya (Carica papaya) and their catalytic activities in proteolysis, lipolysis, and interesterification reactions were studied with regard to the variety, the geographic location of cultures, and the frequency of fruit tapping. Biocatalytic activities of these raw materials were compared to several commercially available crude and purified preparations of papain. These investigations were carried out in order to have a better physicochemical characterization of these raw materials, to select the adequate papaya latex for protein or lipid bioconversions, and to valorize them on an industrial scale. For the purified preparations of papain, only proteolytic activity was obtained. All crude papaya latices exhibit proteolytic, lipolytic, and interesterification activities, and no relationship between the proteolytic and lipolytic activities was observed. The high multiple correlation coefficient (R) on the order of R=0.93–0.99, obtained from the regression analysis for the lipolytic and interesterification activities for all crude papaya latices investigated suggested that there was a correlation between these enzyme activities. However, for the same lipase preparation, the interesterification activity differed substantially depending on the type of interesterification reaction.     

Two-step enzymatic synthesis of docosahexaenoic acid-rich symmetrically structured triacylglycerols via 2-monoacylglycerols

Symmetrically structured triacylglycerols (TG) rich in docosahexaenoic acid (DHA) with caprylic acid (CA) at the outer positions were synthesized enzymatically form bonito oil in a two-step process: (i) ethanolysis of bonito oil TG to 2-monoacylglycerols (2-MG) and fatty acid ethyl esters, and (ii) reesterification of 2-MG with ethyl caprylate. Ethanolysis catalyzed by immobilized Candida antarctica lipase (Novozym 435) yielded 92.5% 2-MG with 43.5% DHA content in 2 h. The 2-MG formed were reesterified with ethyl caprylate by immobilized Rhizomucor miehei lipase (Lipozyme IM) to give structured TG with 44.9% DHA content [based on fatty acid composition with caprylic acid (CA) excluded] in 1 h. The final structured lipids comprised 85.3% TG with two CA residues and one original fatty acid residue, 13% TG with one CA residue and two original fatty acid residues, and 1.7% tricaprylolglycerol (weight percent). The amount of TG with two CA residues and one C₂₂ residue (22∶6=DHA, 22∶5, and 22∶4) was 51 wt%. The 1,3-dicapryloyl-2-docosahexaenoylglycerol to 1,2(2,3)-dicapryloyl-3 (1)-docosahexaenoylglycerol ratio (based on high-performance liquid chromatography peak area percentages) was greater than 50∶1. The recovery of TG as structured lipids after silica gel column purification was approximately 71%. Ethyl esters and 2-MG formed at 2 h of ethanolysis could be used to determine the positional distribution of fatty acids in the intial TG owing to the high 1,3-regiospecificity of Novozym 435 and the reduced acyl migration in the system.     

Lipase-catalyzed synthesis of structured triacylglycerides from 1,3-diacylglycerides

A new method for the lipase-catalyzed synthesis of structured TAG (ST) is described. First, sn1,3-dilaurin or-dicaprylin were enzymatically synthesized using different published methods. Next, these were esterified at the sn2-position with oleic acid or its vinyl ester using different lipases. Key to successful enzymatic synthesis of ST was the choice of a lipase with appropriate FA specificity, i.e., one that does not act on the FA already present in the sn1,3-DAG, but that at the same time exhibits high selectivity and activity toward the FA to be introduced. Reactions were performed in the presence of organic solvents or in solvent-free systems under reduced pressure. With this strategy, mixed ST containing the desired compounds 1,3-dicaprylol-2-oleyl-glycerol or 1,3-dilauroyl-2-oleyl-glycerol (CyOCy or LaOLa) were obtained at 87 and 78 mol% yield, respectively, using immobilized lipases from Burkholderia cepacia (Amano PS-D) in n-hexane at 60°C. However, regiospecific analysis with porcine pancreatic lipase indicated that in CyOCy, 25.7% caprylic acid and in LaOLa 11.1% lauric acid were located at the sn2-position. Oleic acid vinyl ester was a better acyl donor than oleic acid. Esterification of sn1,3-DAG and free oleic acid gave very low yield (<20%) of ST in a solvent system and moderate yield (>50%) in a solvent-free system under reduced pressure.     

Thiyl radical-catalyzed isomerization of oils: An entry to the trans lipid library

The unsaturated fatty acyl moieties of TAG present in natural oils of borage, olive, and rice were converted to their corresponding geometrical trans isomers by thiyl radical-catalyzed isomerization. Thiyl radicals were generated from 2-mercaptoethanol under photolytic or thermal conditions. A relevant feature of this method is the absence of double-bond shifts, so that no positional trans isomers or conjugated polyenes are formed. Oils obtained after the isomerization were winterized to further increase their trans fatty acid content. Methanolysis and hydrolysis of the trans oil mixtures using an enzymatic method (lipase B from Candida antarctica) gave good conversions to the corresponding trans FAME and fatty acids, respectively. These results are relevant for the studies of lipid isomerism and trans fatty acid recognition, which is a growing concern in biochemistry and nutrition, and open new perspectives for the synthesis of glycerides and studies of their structure-activity relationships.     

Lipase-catalyzed modification of borage oil: Incorporation of capric and eicosapentaenoic acids to form structured lipids

Two immobilized lipases, nonspecific SP435 from Candida antarctica and sn-1,3 specific IM60 from Rhizomucor miehei, were used as biocatalysts for the restructuring of borage oil (Borago officinalis L.) to incorporate capric acid (10:0, medium-chain fatty acid) and eicosapentaenoic acid (20:5n-3) with the free fatty acids as acyl donors. Transesterification (acidolysis) reactions were carried out in hexane, and the products were analyzed by gas-liquid chromatography. The fatty acid profiles of the modified borage oil were different from that of unmodified borage oil. Higher incorporation of 20:5n-3 (10.2%) and 10:0 (26.3%) was obtained with IM60 lipase, compared to 8.8 and 15.5%, respectively, with SP435 lipase. However, SP435 lipase was able to incorporate both 10:0 and 20:5n-3 fatty acids at the sn-2 position, but the IM60 lipase did not. Solvents with log P values between 3.5 and 4.5 supported the acidolysis reaction better than those with log P values between −0.33 and 3.0.     

Structured lipids: Lipase-catalyzed interesterification of tricaproin and trilinolein

Structured lipids were synthesized by interesterification of trilinolein and tricaproin with sn-1,3-specific (IM 60) and nonspecific (SP 435) lipases. The interesterification reaction was performed by incubating a 1:2 mole ratio of trilinolein and tricaproin in 3 mL hexane at 45°C for the IM 60 lipase from Rhizomucor miehei, and at 55°C for the SP 435 lipase from Candida antarctica. Reaction products were analyzed by reverse-phase high-performance liquid chromatography with an evaporative light-scattering detector. The fatty acids at the sn-2 position were identified after pancreatic lipase hydrolysis and analysis with a gas chromatograph. IM 60 lipase produced 53,5 mol% dicaproyllinolein (total carbon number = C33) and 22.2% monocaproyldilinolein (C45). SP 435 lipase produced 41% C33 and 18% C45. When caproic acid was used in place of tricaproin as the acyl donor, the IM 60 lipase produced 62.9% C33. The effects of variation in mole ratio, temperature, added water, solvent polarity, and time course on the interesterification reaction were also investigated. In the absence of organic solvent, IM 60 lipase produced 52.3% C33.     

Modification of butterfat by selective hydrolysis and interesterification by lipase: Process and product characterization

Butterfat was chemically modified via combined hydrolysis and interesterification, catalyzed by a commercial lipase immobilized onto a bundle of hydrophobic hollow fibers. The main goal of this research effort was to engineer butterfat with improved nutritional properties by taking advantage of the sn-1,3 specificity and fatty acid specificity of a lipase in hydrolysis and ester interchange reactions, and concomitantly decrease its level of long-chain saturated fatty acid residues (viz., lauric, myristic, and palmitic acids) and change its melting properties. All reactions were carried out at 40°C in a solvent-free system under controlled water activity, and their extent was monitored via chromatographic assays for free fatty acids, esterified fatty acid moieties, and triacylglycerols; the thermal behavior of the modified butterfat was also assessed via calorimetry. Lipase-modified butterfat possesses a wider melting temperature range than regular butterfat. The total saturated triacylglycerols decreased by 2.2%, whereas triacylglycerols with 28–46 acyl carbons (which contained two or three lauric, myristic, or palmitic acid moieties) decreased by 13%. The total monoene triacylglycerols increased by 5.4%, whereas polyene triacylglycerols decreased by 2.9%. The triacylglycerols of interesterified butterfat had ca. 10.9% less lauric, 10.7% less myristic, and 13.6% less palmitic acid residues than those of the original butterfat.     

Use of <Emphasis Type="Italic">Rhizopus delemar</Emphasis> lipase as compared with other lipases for determination of <Emphasis Type="Italic">sn</Emphasis>-2 fatty acids in triacylglycerol

The FA composition in the sn-2 position of TAG is routinely determined after porcine pancreatic lipase hydrolysis. However, the content of saturated FA increased when a pancreatic lipase preparation with higher specific activity was used. Lipase from Rhizopus delemar was selected as a potential replacement lipase for the following reasons: (i) The FA specificity is nearly equivalent in hydrolysis activity toward FA such as lauric, myristic, palmitic, palmitoleic, stearic, oleic, linoleic, and α-linolenic acids; and (ii) lipase from R. delemar hydrolyzes fatty acyl residues at the sn-1,3 positions of TAG. Acyl migration products were present at less than 0.8% in lipase hydrolysates containing 6–14% of sn-2 MAG. A reproducibility CV of less than 5% was obtained in a collaborative study in which the compositions of the main FA at the sn-2 position in olive oil were determined using lipase from R. delemar. This article was presented in part at the Biocatalysis Symposium, 94th AOCS Annual Meeting & Expo, Kansas City, Missouri, May 2003.     

Enzymatic enrichment of arachidonic acid from Mortierella single-cell oil

An attempt was made to enrich arachidonic acid (AA) from Mortierella single-cell oil, which had an AA content of 25%. The first step involved the hydrolysis of the oil with Pseudomonas sp. lipase. A mixture of 2.5 g oil, 2.5 g water, and 4000 units (U) Pseudomonas lipase was incubated at 40°C for 40 h with stirring at 500 rpm. The hydrolysis was 90% complete after 40 h, and the resulting free fatty acids (FFA) were extracted with n-hexane (AA content, 25%; recovery of AA, 91%). The second step involved the selective esterification of the fatty acids with lauryl alcohol and Candida rugosa lipase. A mixture of 3.5 g fatty acids/lauryl alcohol (1:1, mol/mol), 1.5 g water, and 1000 U Candida lipase was incubated at 30°C for 16 h with stirring at 500 rpm. Under these conditions, 55% of the fatty acids were esterified, and the AA content in the FFA fraction was raised to 51% with a 92% yield. The long-chain saturated fatty acids in the FFA fraction were eliminated as urea adducts. This procedure raised the AA content to 63%. To further elevate the AA content, the fatty acids were esterified again in the same manner with Candida lipase. The repeated esterification raised the AA content to 75% with a recovery of 71% of its initial content.     

Measuring stereoselectivity in lipase-catalyzed acidolysis reactions by ultra-high resolution 13C nuclear magnetic resonance

Elucidating the stereoselectivity of lipases in synthetic reactions of triacylglycerols has hitherto been carried out using traditional analytical techniques to determine the composition of the reaction products. These methods are laborious and are not always appropriate for analysis of certain triacylglycerol types. A direct method, utilizing a stereospecific deuterium-labeled triacylglycerol substrate, has been developed where the stereoisomeric composition of the reaction product is determined by ultra-high resolution ¹³C nuclear magnetic resonance (NMR) spectroscopy. Through lipase-catalyzed transesterification of deuterium-labeled trilauroylglycerol with oleic acid, chemical shifts were induced in the ¹³C NMR spectrum by the deuterium atom and olefinic double bonds, enabling unambiguous stereospecific assignment of triacylglycerol species. By this method of analysis, we found an effect of the degree of reaction conversion on the extent of stereoisomerism in the triacylglycerol product. Stereoselectivity was greatest (for sn-1) with lipase from Rhizomucor miehei. Lipases from Rhizopus niveus, Candida rugosa, Carica papaya, and the cutinase from Fusarium sp. were also found to exhibit stereoselectivity, with preference for either sn-1 or sn-3 acyl exchange.     

Enzymatic enrichment of arachidonic acid from Mortierella single-cell oil

An attempt was made to enrich arachidonic acid (AA) from Mortierella single-cell oil, which had an AA content of 25%. The first step involved the hydrolysis of the oil with Pseudomonas sp. lipase. A mixture of 2.5 g oil, 2.5 g water, and 4000 units (U) Pseudomonas lipase was incubated at 40°C for 40 h with stirring at 500 rpm. The hydrolysis was 90% complete after 40 h, and the resulting free fatty acids (FFA) were extracted with n-hexane (AA content, 25%; recovery of AA, 91%). The second step involved the selective esterification of the fatty acids with lauryl alcohol and Candida rugosa lipase. A mixture of 3.5 g fatty acids/lauryl alcohol (1:1, mol/mol), 1.5 g water, and 1000 U Candida lipase was incubated at 30°C for 16 h with stirring at 500 rpm. Under these conditions, 55% of the fatty acids were esterified, and the AA content in the FFA fraction was raised to 51% with a 92% yield. The long-chain saturated fatty acids in the FFA fraction were eliminated as urea adducts. This procedure raised the AA content to 63%. To further elevate the AA content, the fatty acids were esterified again in the same manner with Candida lipase. The repeated esterification raised the AA content to 75% with a recovery of 71% of its initial content.     

Carica papaya latex: A low‐cost biocatalyst for human milk fat substitutes production

This work aims at evaluating the potential of Carica papaya lipase (CPL) self‐immobilized in papaya latex as a biocatalyst for the synthesis of human milk fat substitutes (HMFS), to be used as a low‐cost alternative to commercial lipases. Two different CPL preparations, one extracted from the papaya fruit (CPL I) and the other from petiole leaves (CPL II) of papaya tree, were tested as catalysts for the acidolysis between tripalmitin and (i) oleic acid or (ii) omega‐3 PUFA, batchwise, at 60°C, in solvent‐free media. After 24 h, molar incorporation was higher for oleic acid (22.1 mol%) when CPL I was used. This biocatalyst was selected for further studies. RSM was used to model reaction conditions: medium formulation (molar ratio oleic acid/tripalmitin, MR, 1.2:1–6.8:1) and temperature (58–72°C). Acyl migration decreased with MR increase. In batch operational stability assays at 60°C, using MR of 2:1 and 6:1, the highest stability was observed for a MR of 2:1. Practical applications: The use of this biocatalyst is a feasible way to valorize papaya agro‐residues which represent an important environmental problem in the producing countries. The obtained results were rather promising since, with this almost zero‐cost biocatalyst, it was possible to produce a high added‐value product (HMFS). Under optimized conditions, the obtained results were comparable with those obtained with expensive immobilized commercial lipases.     

Enzymatic synthesis of steryl esters of polyunsaturated fatty acids

Steryl esters of long-chain fatty acids have water-holding properties, and polyunsaturated fatty acids (PUFA) have various physiological functions. Because steryl ester of PUFA can be expected to have both features, we attempted to synthesize steryl esters of PUFA by enzymatic methods. Among lipases used, Pseudomonas lipase was the most effective for the synthesis of cholesteryl docosahexaenoate. When a mixture of cholesterol/docosahexaenoic acid (3:1, mol/mol), 30% water, and 3000 units/g of lipase was stirred at 40°C for 24 h, the esterification extent attained 89.5%. Under the same reaction conditions, cholesterol, cholestanol, and sitosterol were also esterified efficiently with docosahexaenoic, eicosapentaenoic, arachidonic, and γ-linolenic acids.     

Isolation of erucic acid from rapeseed oil by lipase-catalyzed hydrolysis

Three lipases were compared for their ability to hydrolyze high erucic acid rapeseed oil, with the objective of concentrating the erucic acid in a single glyceride fraction. Lipase fromPseudomonas cepacia released all fatty acids rapidly and did not result in selective distribution of erucic acid.Geotrichum candidum lipase released C20 and C22 fatty acids extremely slowly, resulting in their accumulation in the di- and triglyceride fractions. Less than 2% of the total erucic acid was found in the free fatty acid (FFA) fraction. Lipase fromCandida rugosa released erucic acid more slowly than C20 and C18 fatty acids at 35°C but only resulted in a limited accumulation of the erucic acid in the di- and triglyceride fractions. However, when hydrolysis catalyzed byC. rugosa lipase was carried out below 20°C, the reaction mixture solidified and was composed solely of FFAs and diglycerides. The diglyceride fraction contained approximately 95% erucic acid while about 20% of the total erucic acid was found in the FFA fraction. It is concluded that hydrolysis at low temperature withC. rugosa lipase results in a higher purity of erucic acid in the glyceride fraction than can be obtained withG. candidum lipase, but with considerable loss of erucic acid to the FFA fraction.     

Neutral Lipid Characterization of Non-Water-Soluble Fractions of <Emphasis Type="Italic">Carica Papaya</Emphasis> Latex

The non-water-soluble fraction of Carica papaya latex (CPL) constitutes a waste material from papain production; very little information exists regarding its chemical composition. The non-water-soluble fraction of CPL was fractionated by liquid chromatography into neutral lipids, glycolipids and phospholipids. The most abundant compounds were found to be the polar lipids, accounting for 79.2% (w/w) of the total extractible matter, while the total amount of neutral lipids was only around 20%. It was composed of free fatty acids, sterols and triterpenic alcohols, but no glycerides were detected. A high content of saturated fatty acids was measured; these saturated fatty acids were represented by very long chains with C24:0, C26:0 and C28:0 accounting for 6.3, 11.0 and 6.3%, respectively, in the total extractible matter and 7.3, 9.0 and 3.9% in the FFA fraction. The monounsaturated fatty acids were about 23–25% in both samples, with oleic acid (C18:1) being the most abundant. The polyunsaturated fatty acids that were 25.1% in the total matter and 21.6% in the FFA fraction were mainly represented by linoleic acid (C18:2n-6). Finally, a very interesting characteristic of the FA composition of this latex concerns the presence of odd-numbered fatty acids in significant amounts (around 22% in the total extract and 24.3% in the FFA fraction).