Lipase-mediated Resolution of Branched Chain Fatty Acids

Branched chain fatty acids (BCFAs) are fatty acids substituted with alkyl groups. Many of them are chiral and therefore occur in two enantiomeric forms. This review describes their occurrence in Nature, their biosynthesis, their properties as flavours, and their enzymatic kinetic resolution. Many lipases are able to separate the enantiomers of BCFAs, in hydrolysis, esterification or transesterification reactions. Very often, the stereoselectivity of these reactions is remarkably high, even when the chiral carbon atom is remote from the carboxylic acid group.     

Reactor Design for the Novozym 435 ® -catalysed Enantioselective Esterification of 4-methyloctanoic Acid

The bench scale Novozym 435 ® catalysed esterification of 4-methyloctanoic acid with ethanol was studied at 35°C. Esterification in a batch reactor (molar ratio of 1:8 (acid:EtOH)) resulted in the isolation of the enantiomerically enriched product (ee p =81%) and substrate (ee s =93%). In order to integrate reaction and separation, liquid-vapour equilibria calculations were performed showing that an excess of ethanol results in a very low ester fraction in the vapour phase. Since this is undesirable for an integrated process of reaction and product removal, a repeated batch reaction was performed using a molar ratio of 10:1 (acid:EtOH). After six cycles (45% conversion) the ee of 4-methyloctanoic acid ethyl ester turned out to be 80%. For different E values the ee p was calculated for batch and repeated batch reactions. It was shown that in all cases the ee p was higher for the repeated batch reaction. However, the product is not enantiopure since the E value of the reaction is rather low at the low ethanol concentration used. An alternative approach would be the continuous separation of the product during the reaction. A mathematical model was developed to describe esterification in a packed bed reactor integrated with product separation. This model shows that integration of reaction and product removal in advance is not suitable either to obtain an enantiomerically pure product. Since the optimal reaction conditions (high ethanol concentration) and the optimal separation system (low ethanol concentration) do not match in this reaction, the preference is given to the batch reaction at high ethanol concentrations because in that case the highest enantioselectivity of the enzyme is obtained.     

Reactor Design for the Novozym 435 ® -catalysed Enantioselective Esterification of 4-methyloctanoic Acid

The bench scale Novozym 435 ® catalysed esterification of 4-methyloctanoic acid with ethanol was studied at 35°C. Esterification in a batch reactor (molar ratio of 1:8 (acid:EtOH)) resulted in the isolation of the enantiomerically enriched product (ee p =81%) and substrate (ee s =93%). In order to integrate reaction and separation, liquid-vapour equilibria calculations were performed showing that an excess of ethanol results in a very low ester fraction in the vapour phase. Since this is undesirable for an integrated process of reaction and product removal, a repeated batch reaction was performed using a molar ratio of 10:1 (acid:EtOH). After six cycles (45% conversion) the ee of 4-methyloctanoic acid ethyl ester turned out to be 80%. For different E values the ee p was calculated for batch and repeated batch reactions. It was shown that in all cases the ee p was higher for the repeated batch reaction. However, the product is not enantiopure since the E value of the reaction is rather low at the low ethanol concentration used. An alternative approach would be the continuous separation of the product during the reaction. A mathematical model was developed to describe esterification in a packed bed reactor integrated with product separation. This model shows that integration of reaction and product removal in advance is not suitable either to obtain an enantiomerically pure product. Since the optimal reaction conditions (high ethanol concentration) and the optimal separation system (low ethanol concentration) do not match in this reaction, the preference is given to the batch reaction at high ethanol concentrations because in that case the highest enantioselectivity of the enzyme is obtained.     

Production of Tricarboxylic Acid Anhydrides from Fatty Acids by a Lipase-producing Strain of Pseudomonas cepacia

A lipase-producing strain of Pseudomonas cepacia isolated from a soil sample was found to produce five compounds when oleic acid was added to the culture medium as lipase inducer. The five compounds were isolated by solvent extraction, silicagel column chromatography and preparative HPLC, and their structural elucidation was performed by mass spectrometry, and infrared and nuclear magnetic resonance spectroscopies. The products were identified as dec-3-ene-1,3,4-tricarboxylic acid 3,4-anhydride (product 1 ), undec-3-ene-1,3,4-tricarboxylic acid 3,4-anhydride (product 2 ), dodec-3-ene-I,3,4-tricarboxylic acid 3,4-anhydride (product 3 ), dodec-3,8-diene-1,3,4-tricarboxylic acid 3,4-anhydride (product 4 ) and dodec-3,6-diene-I,3,4-tricarboxylic acid 3,4-anhydride (product 5 ). Accumulation of these compounds in the culture medium started after oleic acid consumption and followed a pattern similar to that found for cell growth and for lipase production. The five compounds were radioactively labeled when [U- 14 C]oleic acid was supplied to the culture medium, thus showing that they were produced by transformation of the acid. When isolated from cultures containing [1,2- 13 C]acetic acid and oleic acid as the sole sources of carbon, the compounds showed to contain the 13 C isotope only in the first five atoms of carbon of the molecule. Several long chain fatty acids also acted as precursors of these compounds, with maximal yields for chain lengths between 11 and 18 atoms of carbon. None of the five compounds acted as lipase inducer when added to the culture medium instead of oleic acid. The compounds showed moderate antibacterial and antifungal activities when tested in solid media bioassays.     

Production of Tricarboxylic Acid Anhydrides from Fatty Acids by a Lipase-producing Strain of Pseudomonas cepacia

A lipase-producing strain of Pseudomonas cepacia isolated from a soil sample was found to produce five compounds when oleic acid was added to the culture medium as lipase inducer. The five compounds were isolated by solvent extraction, silicagel column chromatography and preparative HPLC, and their structural elucidation was performed by mass spectrometry, and infrared and nuclear magnetic resonance spectroscopies. The products were identified as dec-3-ene-1,3,4-tricarboxylic acid 3,4-anhydride (product 1 ), undec-3-ene-1,3,4-tricarboxylic acid 3,4-anhydride (product 2 ), dodec-3-ene-I,3,4-tricarboxylic acid 3,4-anhydride (product 3 ), dodec-3,8-diene-1,3,4-tricarboxylic acid 3,4-anhydride (product 4 ) and dodec-3,6-diene-I,3,4-tricarboxylic acid 3,4-anhydride (product 5 ). Accumulation of these compounds in the culture medium started after oleic acid consumption and followed a pattern similar to that found for cell growth and for lipase production. The five compounds were radioactively labeled when [U- 14 C]oleic acid was supplied to the culture medium, thus showing that they were produced by transformation of the acid. When isolated from cultures containing [1,2- 13 C]acetic acid and oleic acid as the sole sources of carbon, the compounds showed to contain the 13 C isotope only in the first five atoms of carbon of the molecule. Several long chain fatty acids also acted as precursors of these compounds, with maximal yields for chain lengths between 11 and 18 atoms of carbon. None of the five compounds acted as lipase inducer when added to the culture medium instead of oleic acid. The compounds showed moderate antibacterial and antifungal activities when tested in solid media bioassays.     

The Comparative Discriminating Abilities of Lipases in Different Media and Their Application in Fatty Acid Enrichment

The generally held belief that the selectivity of lipase can be changed by changing the media from aqueous to non-aqueous was tested by monitoring the rates of hydrolysis, ester synthesis and transesterification with a range of fatty acid mono-esters. Although the absolute rates of reaction varied, hydrolysis was by far the most rapid of the three, the relative rates for the fatty acids used were similar in all three reaction types. The selectivity of the five enzymes used appeared to remain unchanged irrespective of the type of reaction, i.e. hydrolysis of p-nitrophenyl esters, direct ester synthesis with butanol and fatty acid or transesterification with butyl butyrate and fatty acid, and could not be changed by changing water activity. This principle was applied to screen for suitable lipases which could be used to increase the gamma linolenic acid content of a fatty acid mixture. Enzymes could be selected by measuring the rate of hydrolysis of a range of P-nitrophenyl esters.     

Effect of Dietary Mono- and Polyunsaturated Fatty Acids on the Fatty Acid Composition of Pigs' Adipose Tissues

In two experiments with growing-finishing pigs six different dietary fats were added to a conventional diet (control - C) to study the effects of dietary monounsaturated (MUFA) and polyunsaturated fatty acids (PUFA) on the fatty acid composition of backfat and kidney fat at similar amounts of double bonds in feed (Exp. 1:7% pork fat - PF, 4.95% olive oil - OO, 3.17% soybean oil - SO) or a constant amount of 5% of processed fats (Exp. 2: partially hydrogenated fat - SAT, fractionated pork fats: olein - OLE, stearin - STE). Compared with the control, PUFA were only slightly increased in backfat of pigs fed PF, OLE, STE or OO, although dietary PUFA intake was up to 70% higher. With SO PUFA were significantly increased in adipose tissues, predominantly at the expense of MUFA. Consequently, a non-linear relationship was found between PUFA intake and proportion in backfat. MUFA were incorporated at the expense of SFA, therefore, adipose tissues of OO fed animals were lowest in SFA. Despite comparable amounts of double bonds in feed (Exp. 1), the degree of unsaturation measured as fat score (sum of double bonds) was in the order SO > OO > PF > C. In contrast, the proportion of SFA was C > PF = SO > OO. Regarding the decisive role of SFA for fat consistency it may be concluded that MUFA should also be considered in feeding recommendations for pigs. Furthermore, in case of a high dietary supply of MUFA, a simple index of double bonds might not be sufficiently conclusive to judge pig fat quality.     

Effects of Feeding Pigs Increasing Levels of C 18:1 Trans Fatty Acids on Fatty Acid Composition of Backfat and Intramuscular Fat as well as Backfat Firmness

Forty Large White pigs were fed from 30kg to 103kg body mass on diets supplemented with 6% of pure high-oleic sunflower oil (HO) or HO plus increasing amounts of partially hydrogenated rape seed oil (HR; 1.85%, 3.70%, 5.55%), containing high levels of j 6 to j 11 C 18:1 trans fatty acid isomers. Increasing dietary C 18: trans fatty acids resulted in a linear increase in C 18:1 trans fatty acids and conjugated linoleic acid (cis-9, trans-11 CLA) in backfat (BF) as well as in neutral lipids (NL) and phospholipids (PL) of M. long. dorsi. Thus, the rate of bioconversion of trans vaccenic acid (TVA) into CLA and incorporation of C 18:1 trans and CLA into pig adipose tissue was not limited up to 25g total C 18:1 trans fatty acids including 3.3g of TVA perkg feed. BF was higher in C 18:1 trans fatty acids and CLA than M. long. dorsi NL and PL. In BF and NL the sum of saturated fatty acids (SFA) increased with increasing dietary amounts of HR, while in PL SFA were reduced. Thus, according to their physical properties, C 18:1 trans fatty acids partly replaced SFA in PL. Firmness of backfat was also significantly increased (P<0.05) with increasing amounts of HR in feed.     

Activation of the Protein Deacetylase SIRT6 by Long-chain Fatty Acids and Widespread Deacylation by Mammalian Sirtuins

Mammalian sirtuins (SIRT1 through SIRT7) are members of a highly conserved family of NAD⁺-dependent protein deacetylases that function in metabolism, genome maintenance, and stress responses. Emerging evidence suggests that some sirtuins display substrate specificity toward other acyl groups attached to the lysine ϵ-amine. SIRT6 was recently reported to preferentially hydrolyze long-chain fatty acyl groups over acetyl groups. Here we investigated the catalytic ability of all sirtuins to hydrolyze 13 different acyl groups from histone H3 peptides, ranging in carbon length, saturation, and chemical diversity. We find that long-chain deacylation is a general feature of mammalian sirtuins, that SIRT1 and SIRT2 act as efficient decrotonylases, and that SIRT1, SIRT2, SIRT3, and SIRT4 can remove lipoic acid. These results provide new insight into sirtuin function and a means for cellular removal of an expanding list of endogenous lysine modifications. Given that SIRT6 is a poor deacetylase in vitro, but binds and prefers to hydrolyze long-chain acylated peptides, we hypothesize that binding of certain free fatty acids (FFAs) could stimulate deacetylation activity. Indeed, we demonstrate that several biologically relevant FFAs (including myristic, oleic, and linoleic acids) at physiological concentrations induce up to a 35-fold increase in catalytic efficiency of SIRT6 but not SIRT1. The activation mechanism is consistent with fatty acid inducing a conformation that binds acetylated H3 with greater affinity. Binding of long-chain FFA and myristoylated H3 peptide is mutually exclusive. We discuss the implications of discovering endogenous, small-molecule activators of SIRT6.     

Unsaturated Long Chain Free Fatty Acids Are Input Signals of the Salmonella enterica PhoP/PhoQ Regulatory System

The Salmonella enterica serovar Typhimurium PhoP/PhoQ system has largely been studied as a paradigmatic two-component regulatory system not only to dissect structural and functional aspects of signal transduction in bacteria but also to gain knowledge about the versatile devices that have evolved allowing a pathogenic bacterium to adjust to or counteract environmental stressful conditions along its life cycle. Mg²⁺ limitation, acidic pH, and the presence of cationic antimicrobial peptides have been identified as cues that the sensor protein PhoQ can monitor to reprogram Salmonella gene expression to cope with extra- or intracellular challenging conditions. In this work, we show for the first time that long chain unsaturated free fatty acids (LCUFAs) present in Salmonella growth medium are signals specifically detected by PhoQ. We demonstrate that LCUFAs inhibit PhoQ autokinase activity, turning off the expression of the PhoP-dependent regulon. We also show that LCUFAs exert their action independently of their cellular uptake and metabolic utilization by means of the β-oxidative pathway. Our findings put forth the complexity of input signals that can converge to finely tune the activity of the PhoP/PhoQ system. In addition, they provide a new potential biochemical platform for the development of antibacterial strategies to fight against Salmonella infections.     

Fatty Acids Bind Tightly to the N-terminal Domain of Angiopoietin-like Protein 4 and Modulate Its Interaction with Lipoprotein Lipase

Angiopoietin-like protein 4 (Angptl4), a potent regulator of plasma triglyceride metabolism, binds to lipoprotein lipase (LPL) through its N-terminal coiled-coil domain (ccd-Angptl4) inducing dissociation of the dimeric enzyme to inactive monomers. In this study, we demonstrate that fatty acids reduce the inactivation of LPL by Angptl4. This was the case both with ccd-Angptl4 and full-length Angptl4, and the effect was seen in human plasma or in the presence of albumin. The effect decreased in the sequence oleic acid > palmitic acid > myristic acid > linoleic acid > linolenic acid. Surface plasmon resonance, isothermal titration calorimetry, fluorescence, and chromatography measurements revealed that fatty acids bind with high affinity to ccd-Angptl4. The interactions were characterized by fast association and slow dissociation rates, indicating formation of stable complexes. The highest affinity for ccd-Angptl4 was detected for oleic acid with a subnanomolar equilibrium dissociation constant (K(d)). The K(d) values for palmitic and myristic acid were in the nanomolar range. Linoleic and linolenic acid bound with much lower affinity. On binding of fatty acids, ccd-Angptl4 underwent conformational changes resulting in a decreased helical content, weakened structural stability, dissociation of oligomers, and altered fluorescence properties of the Trp-38 residue that is located close to the putative LPL-binding region. Based on these results, we propose that fatty acids play an important role in modulating the effects of Angptl4.     

Identification of Amino Acids Conferring Chain Length Substrate Specificities on Fatty Alcohol-forming Reductases FAR5 and FAR8 from Arabidopsis thaliana

Fatty alcohols play a variety of biological roles in all kingdoms of life. Fatty acyl reductase (FAR) enzymes catalyze the reduction of fatty acyl-coenzyme A (CoA) or fatty acyl-acyl carrier protein substrates to primary fatty alcohols. FAR enzymes have distinct substrate specificities with regard to chain length and degree of saturation. FAR5 (At3g44550) and FAR8 (At3g44560) from Arabidopsis thaliana are 85% identical at the amino acid level and are of equal length, but they possess distinct specificities for 18:0 or 16:0 acyl chain length, respectively. We used Saccharomyces cerevisiae as a heterologous expression system to assess FAR substrate specificity determinants. We identified individual amino acids that affect protein levels or 16:0-CoA versus 18:0-CoA specificity by expressing in yeast FAR5 and FAR8 domain-swap chimeras and site-specific mutants. We found that a threonine at position 347 and a serine at position 363 were important for high FAR5 and FAR8 protein accumulation in yeast and thus are likely important for protein folding and stability. Amino acids at positions 355 and 377 were important for dictating 16:0-CoA versus 18:0-CoA chain length specificity. Simultaneously converting alanine 355 and valine 377 of FAR5 to the corresponding FAR8 residues, leucine and methionine, respectively, almost fully converted FAR5 specificity from 18:0-CoA to 16:0-CoA. The reciprocal amino acid conversions, L355A and M377V, made in the active FAR8-S363P mutant background converted its specificity from 16:0-CoA to 18:0-CoA. This study is an important advancement in the engineering of highly active FAR proteins with desired specificities for the production of fatty alcohols with industrial value.     

Human Fatty Acid Transport Protein 2a/Very Long Chain Acyl-CoA Synthetase 1 (FATP2a/Acsvl1) Has a Preference in Mediating the Channeling of Exogenous n-3 Fatty Acids into Phosphatidylinositol

The trafficking of fatty acids across the membrane and into downstream metabolic pathways requires their activation to CoA thioesters. Members of the fatty acid transport protein/very long chain acyl-CoA synthetase (FATP/Acsvl) family are emerging as key players in the trafficking of exogenous fatty acids into the cell and in intracellular fatty acid homeostasis. We have expressed two naturally occurring splice variants of human FATP2 (Acsvl1) in yeast and 293T-REx cells and addressed their roles in fatty acid transport, activation, and intracellular trafficking. Although both forms (FATP2a (M_r 70,000) and FATP2b (M_r 65,000 and lacking exon3, which encodes part of the ATP binding site)) were functional in fatty acid import, only FATP2a had acyl-CoA synthetase activity, with an apparent preference toward very long chain fatty acids. To further address the roles of FATP2a or FATP2b in fatty acid uptake and activation, LC-MS/MS was used to separate and quantify different acyl-CoA species (C14–C24) and to monitor the trafficking of different classes of exogenous fatty acids into intracellular acyl-CoA pools in 293T-REx cells expressing either isoform. The use of stable isotopically labeled fatty acids demonstrated FATP2a is involved in the uptake and activation of exogenous fatty acids, with a preference toward n-3 fatty acids (C18:3 and C22:6). Using the same cells expressing FATP2a or FATP2b, electrospray ionization/MS was used to follow the trafficking of stable isotopically labeled n-3 fatty acids into phosphatidylcholine and phosphatidylinositol. The expression of FATP2a resulted in the trafficking of C18:3-CoA and C22:6-CoA into both phosphatidylcholine and phosphatidylinositol but with a distinct preference for phosphatidylinositol. Collectively these data demonstrate FATP2a functions in fatty acid transport and activation and provides specificity toward n-3 fatty acids in which the corresponding n-3 acyl-CoAs are preferentially trafficked into acyl-CoA pools destined for phosphatidylinositol incorporation.     

Specificities of Human and Rat Brain Enzymes of Cholesterol Ester Metabolism Toward Very Long Chain Fatty Acids: Implication for Biochemical Pathogenesis of Adrenoleukodystrophy

Abstract: Specificities of the cholesterol-esterifying enzyme and the three cholesterol esterases in rat brain with respect to the chain length of fatty acids were examined. For each of the hydrolases, activities toward cholesteryl lignocerate and cerotate were generally less than 1% of that toward cholesteryl oleate. However, both lignoceric and cerotic acids were esterified at rates approximately 10% of that for oleic acid. In postmortem human control and adrenoleukodystrophy brains, the esterifying activity toward cerotic acid was on the average 25% of that toward oleic acid. The abnormal accumulation of cholesterol esters with very long chain fatty acids observed in adrenoleukodystrophy can therefore occur in the absence of deficient activities of the cholesterol esterases, if the free fatty acid pool of the brain contains an abnormal amount of very long chain fatty acids.