Microbial production of short and medium chain esters: Enzymes,pathways, and applications

Sustainable production of bulk chemicals is one of the major challenges in the chemical industry, particularly due to their low market prices. This includes short and medium chain esters, which are used in a wide range of applications, for example fragrance compounds, solvents, lubricants or biofuels. However, these esters are produced mainly through unsustainable, energy intensive processes. Microbial conversion of biomass-derived sugars into esters may provide a sustainable alternative. This review provides a broad overview of natural ester production by microorganisms. The underlying ester-forming enzymatic mechanisms are discussed and compared, with particular focus on alcohol acyltransferases (AATs). This large and versatile group of enzymes condense an alcohol and an acyl-CoA to form esters. Natural production of esters typically cannot compete with existing petrochemical processes. Much effort has therefore been invested in improving in vivo ester production through metabolic engineering. Identification of suitable AATs and efficient alcohol and acyl-CoA supply are critical to the success of such strategies and are reviewed in detail. The review also focusses on the physical properties of short and medium chain esters, which may simplify downstream processing, while limiting the effects of product toxicity. Furthermore, the esters could serve as intermediates for the synthesis of other compounds, such as alcohols, acids or diols. Finally, the perspectives and major challenges of microorganism-derived ester synthesis are presented.     

Preparation of short chain esters by the lipase from mucor miehei using heptane and silica gel

Butyl acetate, isoamyl acetate and isoamyl valerate were prepared by Mucor miehei lipase catalyzed esterification of free acids and alcohols carried out in non-aqueous systems using heptane and silica gel which removes water formed in the reaction. For butyl and isoamyl acetate 1:3 and for isoamyl acetate 1:2 molar proportions of acid to alcohol were found to be optimal. Heptane(5 ml) and 0.01g silica gel per 0.1M acid were found to improve the yields. Under optimum conditions using 60°C, within 48 hours 40% butyl acetate, 53% isoamyl acetate and 61% isoamyl valerate conversions were observed.     

Effect of acyl donor chain length on isoquercitrin acylation and biological activities of corresponding esters

The enzymatic acylation of isoquercitrin with fatty acid esters of various carbon chain lengths was carried out in 2-methyl-2-butanol using Novozym 435^®. The conversion yield and the initial rate decreased from 66% to 38% and from 17.7 to 10.1 μmol/h respectively, as the carbon chain of the acyl donor increased from C4 to C18. Isoquercitrin acylated derivatives showed higher xanthine oxidase inhibition activities than isoquercitrin. This property increased with the lipophilicity of the derivative esters. The scavenging activity of isoquercitrin esters against ABTS and DPPH radicals decreased with the acyl chain length. Conversely, for esters from C6 to C18, a linear growing relationship can be established between the chain length and the superoxide radical scavenging activity. Furthermore, an improved antiproliferative effect on Caco2 cancer cells was induced by addition of isoquercitrin esters comparing with isoquercitrin.     

Comparative effects of dietary fish oil and carbohydrate on plasma lipids and hepatic activities of phosphatidate phosphohydrolase, diacylglycerol acyltransferase and neutral lipase activities in the rat

In rats fed a fish oil-enriched diet, plasma triacylglycerols were lowered 51%. At the same time there was a mean 45% reduction in Mg2+-dependent phosphatidate phosphohydrolase activity in liver microsomes and a mean 20% decrease in microsomal triacylglycerol (neutral) and diacylglycerol hydrolase activities, but not of diacylglycerol acyltransferase. These observations support the hypothesis that decreases in the activities of phosphatidate phosphohydrolase and of both lipases are involved in the expression of the inhibitory effects of fish oil feeding on hepatic lipoprotein triacylglycerol secretion. Conversely, the feeding of a sucrose-enriched diet resulted in a mean 39% rise in plasma triacylglycerols, a 19% increase in triacylglycerol hydrolase and a mean 45% increase in Mg2+-dependent microsomal phosphohydrolase activity. The effects of the two nutritional interventions on phosphatidate phosphohydrolase activity confirm a key function for this enzyme in triacylglycerol formation.     

Effects of diet and age on lipoprotein lipase and hepatic triglyceride lipase activities in the rat

Plasma clearance of triglyceride-rich lipoproteins appears decreased in aged humans and rats and may be due to lowered activities of the lipases responsible for lipid degradation. This study was designed to examine differential effects of age and diet on lipoprotein lipase (LPL) activity of adipose and heart tissue and hepatic triglyceride lipase (HTGL) activity. LPL and HTGL activities were examined in 3- and 13-month-old Sprague-Dawley rats after they had consumed either a high-carbohydrate or a high-fat diet for 14 days. The data were analyzed for age and diet differences by two-way analysis of variance. Although animals in the two age groups consumed diets of equal caloric content, the older rats gained less weight. Rats on the high-carbohydrate diet consumed less calories and gained less weight than the fat fed rats in both age groups. Neither heart nor adipose tissue LPL activity differed when examined for age or diet. HTGL activity levels, while not affected by age, were higher in the carbohydrate fed rats (P = 0.014). Regardless of age group, fasting plasma cholesterol levels were significantly higher in the carbohydrate-fed rats than fat-fed rats (P = 0.002). Thus, the diet effect was much stronger than the age effect for HTGL and plasma cholesterol levels.     

Effect of dietary carbohydrate type on lipoprotein lipase, hepatic lipase, and lecithin:cholesterol acyltransferase activities in cynomolgus monkeys

The effects of dietary sucrose and starch with and without exogenous cholesterol on postheparin plasma lipoprotein lipase (PHLA) and hepatic lipase (HLA) were studied in cynomolgus monkeys. Serum triglyceride levels were higher in sucrose-fed animals than starch and exogenous cholesterol lowered serum triglyceride levels when added to sucrose diet but not starch diets. Sucrose markedly increased insulin levels, more so than starch; however, dietary cholesterol lowered insulin levels in sucrose diet but increased the levels in starch diet. PHLA activity was increased two- to threefold greater in sucrose than in starch diets. Exogenous cholesterol lowered PHLA activity in sucrose diet but increased PHLA activity in starch diet. HLA activity was increased with sucrose more than starch. Lecithin:cholesterol acyltransferase (LCAT) activity was significantly higher in sucrose diets than in the starch diet. Addition of cholesterol to either of these diets lowered the LCAT activity. These results indicate that PHLA, HLA, and LCAT activities not only are affected by the nature of carbohydrates, but also are related to triglyceride metabolism. The interaction of carbohydrates and cholesterol in the diet by influencing these selected enzymes plays an integrated role in lipoprotein particle interconversion processes.     

Comparative study of the enzymatic,hemorrhagic, procoagulant and anticoagulant activities of some animal venoms

1. The enzymatic, hemorrhagic, procoagulant and anticoagulant activities of venoms of some animals including snakes, lizards, toads, scorpions, spider, wasps, bees and ants were compared.2. Snake venom was the richest source of enzymes among the animal venoms. Most other animal venoms were devoid of phosphodiesterase, l-amino acid oxidase, alkaline phosphomonoesterase and acetylcholinesterase activities and only a few exhibited arginine ester hydrolase activity. These venoms, however, exhibited wide ranges of protease, 5'-nucleotidase and hyaluronidase activities. Most of the animal venoms examined exhibited some phospholipase A activity.3. Other than snake venoms, only venoms of the toad Bufo calamita and the lizards were hemorrhagic, and only venoms of the social wasps, social bees and harvester ant exhibited strong anticoagulant activity. Procoagulant activity occurs only in snake venoms.     

Interactive effects of APOE haplotype, sex, and exercise on postheparin plasma lipase activities

Hepatic lipase (HL) and lipoprotein lipase (LPL) activities (HLA, LPLA) modify lipoproteins and facilitate their binding to hepatic receptors. Apolipoprotein E (APOE) physically interacts with the lipases, and the three common haplotypes of the APOE gene (ε2, ε3, and ε4) yield protein isoforms (E2, E3, and E4, respectively) that are functionally different. Lipase activities themselves differ by sex and exercise training status. The interaction of APOE genotype, exercise training, and sex effects on lipase activities has not been studied. We measured postheparin plasma lipase activities in normolipidemic men and women with the three most common APOE genotypes, which are the haplotype combinations ε2/ε3 (n = 53 ), ε3/ε3 (n = 62), and ε4/ε3 (n = 52), enrolled in 6 mo of aerobic exercise training. These haplotype combinations comprise an estimated 11.6, 62.3, and 21.3% of the population, respectively. Baseline HLA was 35% lower in women than in men (P < 0.0001). In men but not women, HLA was higher in ε2/ε3 group compared with ε4/ε3 (P = 0.01) and ε3/ε3 (P = 0.05). Neither sex nor APOE genotype affected baseline LPLA. Training decreased HLA by 5.2% (P = 0.018) with no APOE effect. The apparent increase in LPLA following exercise was significant and APOE dependent only when corrected for baseline insulin (P < 0.05). Exercise decreased LPLA by 0.8 μmol free fatty acid (FFA)·ml?1·h?1 (-6%) in ε3/ε3 compared with the combined increases of 6.6% in ε2/ε3 and 12% in ε4/ε3 (P = 0.018 vs. ε3/ε3). However, these differences were statistically significant only after correcting for baseline insulin. We conclude that common APOE genotypes interact with 1) sex to modulate HLA regardless of training status, with ε2/ε3 men demonstrating higher HLA than ε3/ε3 or ε4/ε3 men, and 2) aerobic training to modulate LPLA, regardless of sex, with ε3/ε3 subjects showing a significant decrease compared with an increase in ε2/ε3 and ε3/ε4 after controlling for baseline insulin.     

Comparative NMR study on the impact of point mutations on protein stability of Pseudomonas mendocina lipase

In this work we compare the dynamics and conformational stability of Pseudomonas mendocina lipase enzyme and its F180P/S205G mutant that shows higher activity and stability for use in washing powders. Our NMR analyses indicate virtually identical structures but reveal remarkable differences in local dynamics, with striking correspondence between experimental data (i.e., (15)N relaxation and H/D exchange rates) and data from Molecular Dynamics simulations. While overall the cores of both proteins are very rigid on the pico- to nanosecond timescale and are largely protected from H/D exchange, the two point mutations stabilize helices alpha1, alpha4, and alpha5 and locally destabilize the H-bond network of the beta-sheet (beta7-beta9). In particular, it emerges that helix alpha5, undergoing some fast destabilizing motions (on the pico- to nanosecond timescale) in wild-type lipase, is substantially rigidified by the mutation of Phe180 for a proline at its N terminus. This observation could be explained by the release of some penalizing strain, as proline does not require any "N-capping" hydrogen bond acceptor in the i+3 position. The combined experimental and simulated data thus indicate that reduced molecular flexibility of the F180P/S205G mutant lipase underlies its increased stability, and thus reveals a correlation between microscopic dynamics and macroscopic thermodynamic properties. This could contribute to the observed altered enzyme activity, as may be inferred from recent studies linking enzyme kinetics to their local molecular dynamics.     

Studies on the substrate specificity of a carboxyl ester hydrolase from human pancreatic juice. I. Action on carboxyl esters,glycerides and phospholipids

Purified carboxyl ester hydrolase (carboxylic-ester hydrolase, EC 3.1.1.1) from human pancreatic juice was found to hydrolyze triacetin, methyl butyrate and glycerides solubilized by bile salts. It has no activity on substrate presented as emulsion or monomolecular films.The human enzyme was found to deacylate phospholipids and lysophospholipids at different rates. The hydrolysis of short-chain phospholipids and lysophospholipids at different rates. The hydrolysis of short-chain phosphatidylcholines was dependent of substrate solubility and dioctanoyl phosphatidylcholine was deacylated with the highest rate. Long-chain phosphatidylcholines and lysophosphatidylcholines present in microsomal membranes were deacylated with very low rates, only lysophosphatidylcholine deacylation was faster. Evidence is presented that human carboxyl ester hydrolase is the lyophosphatidylcholine-hydrolyzing enzyme corresponding to bovine lysophospholipase.Bile salts play an important part on the activity of human carboxyl ester hydrolase, in addition to the role of detergent that they have on insoluble substrates.     

Comparative study of the effects of 2 types of protein-energy malnutrition followed by a balanced refeeding, on the lipase, phospholipase A2 and amylase activities of the pancreas and pancreatic juice in the growing rat

The intake of 2 p. 100 casein diet as only protein source decreased overall phospholipase A2 activity. Amylase activity was more affected than lipase one. The effects of intake of 5 p. 100 gluten diet as only protein source were less important than 2 p. 100 casein diet. Refeeding on 20 p. 100 or 15 p. 100 casein diet caused a considerable increase of phospholipase A2, lipase and amylase activities.     

福寿螺和田螺消化酶活性比较

为比较福寿螺(Pomacea canaliculata(Lamarck,1828))和当地中国圆田螺(Cipangopaludina chinensis(Gray,1832))消化能力的差异,探索福寿螺成功入侵的机制,以田螺为对照,测定了1—4龄的福寿螺和田螺的胃和肝脏的消化酶——纤维素酶(羧甲基纤维素法)、淀粉酶(3,5-二硝基水杨酸法)和脂肪酶(滴定法)的活性。结果表明:1)相同年龄的福寿螺胃和肝脏中的消化酶活性明显高于田螺。其中,纤维素酶活性分别高出1.00—2.11倍、1.66—2.84倍;淀粉酶活性分别高出1.53—3.47倍、1.47—1.80倍;脂肪酶活性分别高出2.07—4.73倍、6.13—9.93倍。2)在生长发育过程中,福寿螺胃和肝脏中的消化酶活性变化幅度(51.2%—131.2%)明显高于田螺(23.3%—47.1%)。3)福寿螺的各种消化酶之间存在协同作用。如福寿螺的淀粉酶活性与脂肪酶活性呈极显著正相关(胃中r=0.736**、肝脏中r=0.867**)。此外,胃中的淀粉酶活性还与纤维素酶活性呈显著正相关关系(r=0.696*)。相应地,田螺胃中的淀粉酶和脂肪酶之间也存在显著的正相关关系(r=0.706*),而肝脏中的纤维素酶与脂肪酶活性呈显著负相关(r=-0.593*)。4)福寿螺对纤维素类和淀粉类物质都有较强的消化能力,且能较好地消化脂肪类物质,而田螺能消化纤维素类和淀粉类物质,对脂肪的消化能力却很弱。福寿螺的纤维素酶和淀粉酶活性分别是田螺的2.42和1.88倍,脂肪酶活性达到了5.66倍。可见,福寿螺具有较高的消化酶活性,且各消化酶之间存在正协同性。这可能是导致福寿螺食量大、食性杂,使其能快速生长和成功入侵的重要原因之一。     

Characterization of mono-, di- and triacylglycerol lipase activities in the isolated rat heart

The lipolytic activities of heart tissue towards full and partial acylglycerols were characterized. Tissue lysosomal, acid lipase activity (pH 4.8) was inhibited by high salt, protamine sulfate, NaF, MgATP, Triton X-100, serum and the esterase-inhibitor diethylparanitrophenyl phosphate. The tissue neutral triacylglycerol lipase activity (pH 7.4) was recovered predominantly in the microsomal and soluble fractions and exhibited essentially identical properties towards activators (serum, apolipoprotein C-II) and reagents (NaCl, Triton X-100, NaF, MgATP and diethylparanitrophenyl phosphate) relative to vascular lipoprotein lipase, except for protamine sulfate which increased the serum-stimulated neutral triacylglycerol lipase activity. Triacylglycerol hydrolysis at acid pH was incomplete, whereas at neutral pH full hydrolysis occurred. Myocardial mono- and diacylglycerol lipase activities, with pH optima of 8.0 and 7.4, respectively, were recovered in the microsomal fraction. They differed immunologically from neutral lipase and lipoprotein lipase and did not bind to heparin-Sepharose 4B. They were kinetically different, partially inhibited by NaCl and differentially affected by protamine sulfate. NaF, Triton X-100 and diethylparanitrophenyl phosphate. Our data suggest that endogenous hydrolytic activity against full and partial acylglycerols is mediated by separate enzymes.     

Using examples of firefly luciferase gene for the study of biological activities (estrogens, retinoids, phorbol esters)]

Some possible applications of chimeric cellular models, specifically responding to an effector through firefly luciferase induction are presented with the help of examples in relation with the biological activity of estradiol or retinoic acid, or phorbol ester. A comparison of experiments on either chimeric or natural responses shows that: i) the responses of both type of cellular models are effector concentration-dependent; ii) these concentrations are in the same order of magnitude; partial agonist compounds and antagonist compounds; iv) potencies (EC50) of test-compounds are similarly classified. Moreover we show that a chimeric cellular model allows the observation of interactions between hormone or effector pathways: it allows readily performed kinetic studies and long-term experiments in intact cells that permit to investigate the effect of a given effector, its reversibility and time-dependent action. Therefore, various steps of a cellular signalling pathway involved in the action of an effector may be observed with such a valuable tool.     

Hydrolysis of prostaglandin glycerol esters by the endocannabinoid-hydrolyzing enzymes, monoacylglycerol lipase and fatty acid amide hydrolase

Cyclooxygenase-2 (COX-2) can oxygenate the endocannabinoids, arachidonyl ethanolamide (AEA) and 2-arachidonylglycerol (2-AG), to prostaglandin-H2-ethanolamide (PGH2-EA) and -glycerol ester (PGH2-G), respectively. Further metabolism of PGH2-EA and PGH2-G by prostaglandin synthases produces a variety of prostaglandin-EA's and prostaglandin-G's nearly as diverse as those derived from arachidonic acid. Thus, COX-2 may regulate endocannabinoid levels in neurons during retrograde signaling or produce novel endocannabinoid metabolites for receptor activation. Endocannabinoid-metabolizing enzymes are important regulators of their action, so we tested whether PG-G levels may be regulated by monoacylglycerol lipase (MGL) and fatty acid amide hydrolase (FAAH). We found that PG-Gs are poor substrates for purified MGL and FAAH compared to 2-AG and/or AEA. Determination of substrate specificity demonstrates a 30-100- and 150-200-fold preference of MGL and FAAH for 2-AG over PG-Gs, respectively. The substrate specificity of AEA compared to those of PG-Gs was approximately 200-300 fold higher for FAAH. Thus, PG-Gs are poor substrates for the major endocannabinoid-degrading enzymes, MGL and FAAH.     

Effect of copper deficiency on the lymphatic absorption of cholesterol, plasma chylomicron clearance, and postheparin lipase activities

The lymphatic absorption of cholesterol and plasma clearance of chylomicrons were investigated in Cu-deficient rats (CuD) fed 0.5 mg Cu/kg diet, as compared with Cu-adequate control rats (CuA) fed 7.5 mg/kg diet. Cholesterol absorption was measured by the 14C-radioactivity appearing in the mesenteric lymph at hourly intervals for 8 hr after an intraduodenal dose of [14C]cholesterol. The plasma clearance of chylomicrons was measured at 3, 6, and 10 min after an intravenous dose of chylomicrons labeled in vivo with [3H]retinyl ester. Cumulative [14C]cholesterol absorption and total lymphatic output of cholesterol were significantly decreased in CuD at 4 hr and thereafter, with no change in percentage distribution of free and esterified cholesterol. Over an 8-hr period, 7.3% of the dose was absorbed by CuD and 9.2% by CuA. When [3H]chylomicrons, obtained from a CuD or CuA donor rat, were injected into CuD and CuA recipient rats, the label was cleared faster in CuD during the first 3 min. At 6 and 10 min, however, no significant difference in percentage clearance of the dose was observed between the groups. The half-life (t1/2) of [3H]chylomicrons and the total 3H-radioactivity taken up by the liver during the entire 10-min period did not differ between the groups, regardless of the source of chylomicrons. The activities of both endothelial lipoprotein lipase (LPL) and hepatic lipase (HL) in postheparin plasma were markedly lower in CuD. As expressed in micromoles fatty acid released/hr/ml plasma, the activities of LPL in CuD and CuA were 32.6 +/- 1.9 and 45.6 +/- 1.3, respectively. A similar magnitude of difference was also observed in HL activity. The data provide evidence that copper deficiency impairs the intestinal transport of cholesterol and the peripheral lipolysis of chylomicrons. The data, however, strongly suggest that the hepatic uptake of chylomicron remnants via the apo-E-dependent mechanism may not be impaired in Cu deficiency.