Enzymatic synthesis of ascorbyl ester derived from linoleic acid

Bioprocess and Biosystems Engineering - Antioxidants are substances that defend cells against damage, kidnapping and destroying free radicals. They have been largely used in the food industry due...     

New sensor allowing continuous water activity measurement of submerged or solid-substrate fermentations

Water activity of the substrate is an important and acute factor for growth as well as for metabolic production of microorganisms or for biocatalyst systems. A sensor has been designed in order to control this parameter on-line during submerged and solid-substrate fermentations. This sterilizable sensor allows the measurement of the relative humidity of the atmosphere in a small chamber by means of a capacitive element separated from the medium by a thin ethylenepolytetrafluoride membrane. For high a(w) values (>0.90) a sequential circulation of a dried gas prevents the sensor saturation. Measurements are rapid and accurate, and control of the water activity of a fermentation medium has been carried out for 5 days using this sensor.     

Enzymatic method for continuous monitoring of inorganic pyrophosphate synthesis

A sensitive method for the analysis of inorganic pyrophosphate (PPi) which utilizes the enzymes ATP sulfurylase and firefly luciferase is described. The assay is based on continuous monitoring of the ATP formed in the ATP sulfurylase reaction using purified firefly luciferase. The assay can be completed in less than 2 s and is not affected by inorganic phosphate. The method has been used for continuous monitoring of formation of PPi in Rhodospirillum rubrum chromatophores. The assay is extremely sensitive, the linear range of the assay being 1 X 10(-9) - 5 X 10(-7) M PPi. It is suitable for routine applications. It is also possible to use the method for determination of low amounts of adenosine 5'-phosphosulfate.     

Enzymatic synthesis of arbutin undecylenic acid ester and its inhibitory effect on melanin synthesis

Transesterification of arbutin and undecylenic acid vinyl ester was catalyzed by alkaline protease, Bioprase, in dimethylformamide to get arbutin derivative having undecylenic acid at 6-position of glucose moiety, 6-O-undecylenoyl p-hydroxyphenyl beta-D-glucopyranoside. The reaction rate increased with increase of arbutin concentration, and when its concentration was 0.9 M, the conversion rate was more than 90% under addition of 2 M undecylenic acid vinyl ester. The obtained arbutin ester significantly suppressed melanin production in murine B16 melanoma cells.     

Enzymatic method for continuous monitoring of DNA polymerase activity

A simple and rapid method for the assay of DNA polymerase activity has been developed. The PPi formation in the DNA polymerase reaction is continuously monitored by a coupled enzymatic method (P. Nyrén and A. Lundin, 1985, Anal. Biochem. 151, 504-509) utilizing the enzymes ATP-sulfurylase and firefly luciferase. The method has been used for continuous monitoring of DNA synthesis in vitro, and the effect of an inhibitor, adriamycin, on the polymerase activity was studied. The assay is very sensitive and yields linear responses between 1.5 and 30 micrograms/ml of DNA polymerase (Micrococcus luteus) (2-40 pmol of PPi generated per minute).     

Enzymatic synthesis of a catecholic polyphenol product with excellent antioxidant activity

Abstract

Polyphenols, especially catecholic stilbene derivatives, have attracted much attention due to the huge pharmacological effects and promising health benefits. However, their chemical synthesis via regioselective ortho-hydroxylation on aromatic rings is highly challenging. In this study, 3′-hydroxypterostilbene (HPS) is taken as a model product due to its strong potential as an antitumor agent. One-step enzymatic synthesis of HPS from pterostilbene (PS) was explored, with immobilised tyrosinase as catalyst. The impact of solvent, pH, temperature, oxygen and reductant concentration on the reaction was investigated, and the conversion was optimised by employing the response surface methodology (RSM). Finally, a high yield of 77.9% was obtained in 2.7?h. This study demonstrates the first successful use of a biotechnological strategy to synthesise HPS. The antioxidant activities of both PS and HPS were evaluated by using the DPPH assay, demonstrating that HPS is more potent than PS as a radical scavenger.     

Enzymatic synthesis of betulinic acid ester as an anticancer agent: Optimization study

Abstract

Immobilized Candida antarctica lipase, Novozym 435, was used to catalyze the esterification reaction between betulinic acid and phthalic anhydride to synthesize 3-O-phthalyl betulinic acid in n-hexane/chloroform. Response surface methodology based on a five-level, four-variable central composite rotatable design was employed to evaluate the effects of synthesis parameters such as reaction time, reaction temperature, enzyme amount and substrate molar ratio on the yield of ester. Based on the response surface model, the optimal enzymatic synthesis conditions were predicted to be: reaction time 20.3 h, reaction temperature 53.9°C, enzyme amount 145.6 mg, betulinic acid to phthalic anhydride molar ratio 1:1.11. The predicted yield was 65.8% and the actual yield was 64.7%.     

Enzymatic synthesis of glycerides from DHA-enriched PUFA ethyl ester by glycerolysis under vacuum

Pseudomonas lipase immobilized on CaCO₃ powder was used for the glycerolysis of n−3 polyunsaturated fatty acid ethyl esters to prepare nutritionally valuable glycerides. The initial ethyl ester contained 65 or 99% docosahexaenoic acid ethyl ester (DHAEE) which is very unstable and readily oxidized. The process performance was intensified by: (1) using Pseudomonas lipase which has good specificity for docosahexaenoic acid (DHA), (2) shifting the reaction equilibrium by evaporation of the resulted ethanol under vacuum and (3) enhancing the enzyme operational stability by immobilization on CaCO₃ powder. Under these conditions, over 90% conversion of DHAEE was achieved in 5 h and the oxidative deterioration of DHA was avoided. The final product contained 53% partial glyceride and, thus, had good emulsifying power. The catalyst was reused 5 times showing a very good stability in this system. Other lipases were tried for this reaction and different glyceride compositions were obtained depending on the enzyme specificity for the 1(3)-position of glycerol.     

Effect of thermodynamic water activity on amino-acid ester synthesis catalyzed by agarose-chymotrypsin in 3-pentanone.

Chymotrypsin linked to agarose beads by multi-point covalent attachment catalyzes synthesis of Ac-Trp-OEt in 3-pentanone even when the thermodynamic water activity (aw) of the system is reduced to as low as 0.4. If fully hydrated catalyst is added to the reaction mixture before removal of water, product is formed linearly once aw has stabilized. The initial rate is reduced from that if aw is kept close to 1 (0.47 mmol s-1 (kg enzyme)-1), to 50% (aw 0.9), 25% (aw 0.4) and < 1% (aw 0.25). The large drop between aw of 1 and 0.9 probably reflects the effects of water removal on the agarose gel structure. Catalyst partly dried (even only to aw 0.86) before adding to the organic phase is inactive. At reduced aw, the equilibrium (when reached) is shifted in favor of the ester, as expected.     

Novel water soluble 2,6-dimethoxyphenyl ester derivatives with intravenous anaesthetic activity

A number of water soluble bis-amino-2,6-dimethoxyphenyl ester derivatives were found to exhibit improved anaesthetic activity in mice relative to propofol 1. Of the analogues disclosed, 44 was further profiled in rodents and found to be a superior agent to propofol for the induction and maintenance of anaesthesia.     

Sugar ester synthesis by a mycelium-bound Mucor circinelloides lipase in a micro-reactor equipped with water activity sensor

The mycelium-bound Mucor circinelloides lipase was used for the synthesis of esters of saccharides and fatty acids in 37 ml reactor equipped with magnetic stirrer and water activity sensor. Either di-n-pentyl ether or the mixture of di-n-pentyl and petroleum ethers were applied as reaction media. Water activity sensor provided on-line monitoring of this parameter and control of continuous processes of ester synthesis. It was found that two natural antioxidants, i.e. carotene and astaxanthin activated this lipase in organic solvents that could be beneficial for the synthesis of esters of compounds sensitive to oxidation, e.g. polyunsaturated fatty acids.     

Enzymatic synthesis of galactosyl lactic ethyl ester and its polymer for use as biomaterials

Lactate-based chemicals and polymers including poly(lactic acid) (PLA) are highly valuable materials for biomedical, food and general-purpose applications. Chemical synthesis, albeit the high reaction velocities achieved with it, often leaves chemical residues that are subject to health and safety concerns. Alternative biosynthesis is preferred in order to overcome these problems. Herein we report a novel enzymatic synthesis for the preparation of beta-d-galactosyl-l-lactic acid ethyl ester (GLAEE). Such a product, which may find applications in food and personal care products, is generally difficult to synthesize via traditional chemical routes because the reactions have to be highly selective due to the multiple hydroxyl groups of sugars. We further explore the enzymatic polymerization of GLAEE to form a unique biopolymer, poly(beta-d-galactoside-co-l-lactic acid) (PGLA). Novozyme 435 was found efficient in catalyzing the polymerization reaction in acetone with a conversion yield of 60% within 100 h. The molecular weight of the polymer product ranged from about 800-2000 as analyzed by using ESI-MS. It is expected that a variety of sugar-hydroxyl acids copolymers can be prepared through the same approach and a new class of biomaterials can thus be developed.     

Enzymatic synthesis of arbutin undecylenic acid ester and its inhibitory effect on mushroom tyrosinase

A novel tyrosinase inhibitor, an arbutin derivative having undecylenic acid at the 6-position of its glucose moiety, was enzymatically synthesized. Its inhibitory activity was studied in vitro by using catechol and phenol as substrates. The IC₅₀ value of the arbutin ester on tyrosinase using catechol (4 × 10⁻⁴ M) was 1% of that when arbutin (4 × 10⁻² M) was used. Using phenol, IC₅₀ of the arbutin ester (3 × 10⁻⁴ M) as substrate was 10% of that of arbutin (3 × 10⁻³ M). These results suggest that the arbutin ester inhibits the latter part of the tyrosinase reaction, which consists of hydroxylation and oxidation.     

Enzymatic measurement of saccharopine with saccharopine dehydrogenase

We have developed an enzymatic method for measuring saccharopine, a key intermediate in lysine metabolism. With the enzyme saccharopine dehydrogenase, saccharopine can be oxidized to lysine and alpha-ketoglutarate with the corresponding conversion of NAD to NADH. The natural equilibrium favors saccharopine formation, but using hydrazine to trap one of the products, alpha-ketoglutarate, shifts the reaction toward quantitative oxidation of saccharopine. A stable endpoint is reached in 15-20 min, and although high concentrations of alpha-ketoglutarate slow the reaction, the end product is fully recovered. Unlike previous assays this technique is specific, convenient, and capable of measuring saccharopine directly in protein-free biological fluids or extracts.     

Enzymatic continuous production of N-acetyl-l-methionine from N-acetyl-dl-methioninamide with Erwinia carotovora containing a new amidase activity

A procedure for production of $\text{N-}\text{acetyl-}\text{l}\text{-methionine}$ ($\text{N-}\text{Ac-}\text{l}\text{-Met}$) from its racemic mixture by enzymatic continuous hydrolysis of the l-enantiomer of $\text{N-}\text{acetyl-}\text{dl}\text{-methioninamide}$ ($\text{N-Ac-}\text{dl}\text{-Met · NH}{}_2$) is described. For this hydrolysis, whole cells of Erwinia carotovora immobilized by κ-carrageenan gel were employed. The complete conversion of $\text{N-}\text{Ac-}\text{dl}\text{-Met · NH}{}_2$ to $\text{N-}\text{Ac-}\text{l}\text{-Met}$ was achieved by using a column packed with the immobilized cells. The half-life of the enzyme activity of the column was calculated to be ～100 days. Pure crystalline $\text{N-}\text{Ac-}\text{l}\text{-Met}$ was readily isolated from the effluent with >87.5% yield. $\text{N-}\text{Ac-}\text{d}\text{-Met · NH}{}_2$ was easily racemized by incubating in ethanol containing 0.1 m NaOH for 30 min at 80°C, and used again for substrate.     

Development of a sensor allowing the continuous measurement of the water activity value of a liquid medium

Summary An experimental sensor allows continuous measurement and regulation of the water activity of a liquid medium, by measuring the relative humidity of a stream of air equilibrated with the medium. The measurements were precise (± 0.007 unities of water activity), and the small size of the sensor makes it practical for use in a standard fermentor.     

A comparison of lipase-catalysed ester and lactone synthesis in low-water systems: analysis of optimum water activity.

We investigated the effects of the lyophilisation medium (enzyme plus buffer salt and additives) and of water activity (a(w)) on the catalytic properties of lipase from Chromobacterium viscosum (lipase CV) in organic solvents; catalysis of ester and lactone synthesis were compared and, despite the similarities of the reactive groups involved in these reactions, some interesting differences were observed. Including 2-[N-morpholino]ethanesulfonic acid (MES) buffer in the lyophilisation medium of lipase CV increased its catalytic activity in transesterification and lactonisation, although the buffer salt requirement for maximal activity differed between the two reactions. Sorbitol, glucose, lactose, 18-crown-6 (crown ether 18-C-6), beta-cyclodextrin and bovine serum albumin were employed as alternative additives in the transesterification reaction, but were not as effective as MES buffer. Salt hydrates were used to investigate the effect of a(w) on esterification and lactonisation reactions catalysed by lipase CV. The maximum rate of hexadecanolide synthesis in toluene occurred at a(w) = 0.48. The optimum a(w) for the transesterification reaction in heptane/alcohol mixtures depended on the alcohol substrate employed (1-heptanol, 2-heptanol, or 3-methyl-3-hexanol) but not on the acyl donor (p-NP acetate or caprylate). The optimum a(w) values for both reactions were unchanged when a common solvent system (toluene/1-heptanol) was employed, indicating that the dependence of enzyme activity on a(w) is an intrinsic property of the enzyme-catalysed reaction and not a function of the solvent or other additives.     

Enzymatic synthesis of farnesyl laurate in organic solvent: initial water activity, kinetics mechanism, optimization of continuous operation using packed bed reactor and mass transfer studies

The influence of water activity and water content was investigated with farnesyl laurate synthesis catalyzed by Lipozyme RM IM. Lipozyme RM IM activity depended strongly on initial water activity value. The best results were achieved for a reaction medium with an initial water activity of 0.11 since it gives the best conversion value of 96.80%. The rate constants obtained in the kinetics study using Ping-Pong-Bi-Bi and Ordered-Bi-Bi mechanisms with dead-end complex inhibition of lauric acid were compared. The corresponding parameters were found to obey the Ordered-Bi-Bi mechanism with dead-end complex inhibition of lauric acid. Kinetic parameters were calculated based on this model as follows: V _max = 5.80 mmol l⁻¹ min⁻¹ g enzyme⁻¹, K _m,A = 0.70 mmol l⁻¹ g enzyme⁻¹, K _m,B = 115.48 mmol l⁻¹ g enzyme⁻¹, K _i = 11.25 mmol l⁻¹ g enzyme⁻¹. The optimum conditions for the esterification of farnesol with lauric acid in a continuous packed bed reactor were found as the following: 18.18 cm packed bed height and 0.9 ml/min substrate flow rate. The optimum molar conversion of lauric acid to farnesyl laurate was 98.07±0.82%. The effect of mass transfer in the packed bed reactor has also been studied using two models for cases of reaction limited and mass transfer limited. A very good agreement between the mass transfer limited model and the experimental data obtained indicating that the esterification in a packed bed reactor was mass transfer limited.