Amano Lipase Research Articles

Amano Lipase A grafting onto a silica surface

Since their discovery, enzymes have fulfilled an important role as catalysts of many chemical reactions. Their high selectivity, efficiency and their rate of catalyzing are the reasons for interest in the industrial utilization of biocatalysts. However, due to the low stability and resistance of enzymes in process conditions, this task proved to be very difficult to accomplish. Previous studies have resulted in the development of a number of methods, the aim of which has been the improvement of enzyme properties, and in particular immobilization that is based on the decreasing mobility of enzymes (Synowiecki and Wolowska, 2007). Substances obtained via this method can be characterized not only by an improved chemical and thermal stability, but they also show a slower decrease in catalytic activity in comparison to free enzymes. An additional advantage of immobilized biocatalysts is the possibility of performing catalyzed reactions in polar as well as non-polar solvents (Tischer and Wedekind, 1999). Few immobilization methods are commonly applied, and they differ in terms of the supports utilized, the type and energy of interactions between the matrix and enzyme, and the type of compound participating in the formation of the interaction. Often, the type of reaction and the enzyme that catalyzes a given chemical change determine the choice of the immobilization method. Solutions already developed can be divided into immobilization with covalent bond formation, immobilization through encapsulation, entrapment and cross-linking (Kalantari et al., 2012). The most popular and commonly used method of enzyme immobilization is adsorption on a carrier. This procedure is based on the creation of specific interactions between the biocatalyst and the support, which usually are hydrogen bonds or van der Waals forces (Cao et al., 2003). Formation of these relations enables the application of an intermediate compound which most often is a representative of biofunctional carbonyl compounds, for example, glutaraldehyde. The advantages of this method are the lack of interference in the enzyme internal structure, the rate of the process and its low cost; however, the drawbacks include weak biocatalyst connections with the support, which may result in fast elution of the enzyme from the support, being a consequence of a loss of the catalytic activity of the substance. This paper presents the results of studies undertaken to develop an effective adsorption immobilization procedure for Amano Lipase A on an inorganic support with the use of a glutar aldehyde as an intermediate compound in the creation of interactions between the silica support and the biocatalyst.

Enantioselective preparation of (R) and (S)-3-hydroxycyclopentanone by kinetic resolution

Abstract A straightforward approach to enantiomerically enriched ( R ) and ( S )-3-hydroxycyclopentanone is described. The key step involves a kinetic resolution of racemic 3-hydroxycyclopentanone using commercial Pseudomonas cepacia lipase immobilized on diatomite (Amano lipase PS-DI). The absolute stereochemistry of the product was determined by derivatization into ( R )-3-(benzyloxy)cyclopentanone.

Enabling the utilization of wool as an enzyme support: Enhancing the activity and stability of lipase immobilized onto woolen cloth

An improved, simple, effective and superior protocol has been developed to immobilize amano lipase from Pseudomonas fluorescens on woolen cloth using polyethyleneimine (PEI) with glutaraldehyde (GA) cross-linking. The success of immobilization was confirmed by FTIR and confocal laser scanning microscope (CLSM), the latter proving that enzyme is well distributed across the wool fiber surfaces throughout the cloth. Woolen cloth therefore provides a large outer and inner fiber surface area for immobilization with minimal mass transfer resistances during immobilization. The optimal protocol (GA at 0.5% and pH 6, lipase solution pH 6) gave an enzyme load of 46.6mgg−1dry cloth with expressed activity of 178.3U, 46.8% immobilization yield and 30.2% retained activity. Zeta potential measurements showed that PEI significantly enhanced the positive charge on woolen cloth and shifted the isoelectric point to approximately 7. Therefore at a lipase solution pH of around 6, the wool–PEI and lipase are oppositely charged, leading to a maximal adsorption of lipase to the wool surface. The immobilized lipase also had a good stability and 81% of its original activity was maintained after 10 runs in tributyrin emulsion hydrolysis. This protocol provides a significant improvement in terms of retained activity and lipase stability compared to previous immobilizations on wool and opens up the possibility of using wool as a cheap and effective lipase support material for continuous lipase reactions/reactors and possibly enzyme enhanced woolen fabrics.

Epoxidation of oleic acid catalyzed by PSCI-Amano lipase optimized by experimental design

The present work focuses on the oleic acid epoxide production by using PSCI Amano Lipase as biocatalyst in the reaction. An experimental design (central composite design – CCD) adopting surface response was applied to this purpose. Reactions were performed in a shaker equipment and different variables were investigated, such as temperature (25–55 °C), enzyme load (10–20 wt% of oleic acid mass), hydrogen peroxide load (0.1–0.2%) and reaction time. PSCI-Amano enzyme showed its best behavior as biocatalyst after 3 h of reaction at 55 °C, 10% enzyme load, 0.2% hydrogen peroxide and, applying 150 rpm as stirring. On these conditions, the epoxide yield was around 88%.

Chemical Development of an α2δ Ligand, (3S,5R)-3-(Aminomethyl)-5-methyloctanoic Acid

Three synthetic approaches, suitable for the large scale manufacture of the α2δ-ligand, (3S,5R)-3-(aminomethyl)-5-methyloctanoic acid 3, have been evaluated. The selected seven step manufacturing process has then been optimized and used to deliver over 20 kg of API; salient features of the synthesis include the use of 4,4,4-trimethoxybutyronitrile as an efficient four carbon amino acid equivalent. Highly selective kinetic resolution of the C3 stereocentre was accomplished via diastereoselective hydrolysis of a cyanoester intermediate using Amano Lipase PS-SD. Extensive process optimisation of the route starting from (R)-2-methylpentanol, led to significant improvements through telescoping, with less than 62 kg of solvent being needed to produce 1 kg of API.

Synthesis of new ( R)-secondary carbinols with different structures via enzymatic resolution

The present study deals with the biocatalytic enantioselective synthesis of 19 new chiral alcohols with alkyl (C 11–C 19) and phenyl, substituted phenyl, heteroaromatic, and naphthyl groups 4a– 4z with an ( R)-configuration and high enantiomeric excess (∼100%). The corresponding ketones 1a– 1z were synthesized and then reduced with NaBH 4 to their racemic alcohols 2a– 2z, which were converted into their racemic acetyl derivatives 3a– 3z. Enzymes of four different types were used for the enantioselective hydrolysis of these acetyl compounds 3a– 3z. The optimal reaction conditions for these four enzymes were established by investigating the enantiomeric excesses by chiral HPLC. Amano lipase from Burkholderica cepacia ( Pseudomonas cepacia) AL-PS was determined as the best enzyme in this work. This study presents an environmentally friendly and green chemistry method for the synthesis of these new ( R)-chiral carbinols 4a– 4z.

Miniemulsion as efficient system for enzymatic synthesis of acid alkyl esters

The aim of this work is to devise an efficient enzymatic process for the production of linear alkyl esters in aqueous miniemulsion systems. The esterification reactions of linear alcohols and carboxylic acids were performed with three different enzymes, commercial Amano lipase PS from Pseudomonas cepacia, Lipase type VII from Candida rugosa, and lyophilized Fusarium solani pisi cutinase expressed in Saccharomyces cerevisiae SU50. The miniemulsion system shows a high potential for the synthesis of linear alkyl esters, for example, hexyl octanoate, which could be synthesized with an ester yield of 94% using Amano lipase PS. Even with hydrophilic alcohols as ethanol, ethyl decanoate could be obtained with a concentration of 0.45 M and a yield of 62% using F. s. pisi cutinase as catalyst. High esterification rates for ethyl- and hexyloleate in miniemulsion showed a significant shift in cutinase selectivity towards longer chain length carboxylic acids. The stepwise addition of the alcohol led to an increase of the esterification yield. Moreover, increasing the amount of dispersed organic phase, mainly consisting of the substrates, led to a significant increase of the final ester concentration (e.g., concentration of 1.4 M for ethyl decanoate for the esterification with Amano Lipase PS).

Synthesis and enantioselective rearrangement of (Z)‐4‐triphenylmethoxy‐2,3‐epoxybutan‐1‐ol enantiomers

Efficient enzyme catalyzed kinetic resolutions of a synthetically useful chiral building block, (Z)-4-triphenylmethoxy-2,3-epoxybutan-1-ol, are reported. The highest selectivities were achieved by Lipozyme TL IM and Amano Lipase PS enzymes in the presence of vinyl acetate. Enantiomeric enrichment of the optically active acetate isomer was accomplished by selective crystallization of the racemic part of the enantiomeric mixture. Enzyme catalyzed hydrolysis of the acetate also provided an optically pure epoxybutanol derivative. O-Benzylation of (+)-(Z)-1-hydroxy-4-triphenylmethoxy-2,3-epoxybutane followed by super base promoted diastereo- and enantio-selective rearrangement resulted in (+)-(2R,3R,1'R)-3-[1-hydroxy-2-(triphenylmethoxy)ethyl]-2-phenyloxetane in >98% ee and de. Configurations of the new optically active products were determined by chemical correlation.

Biodegradation of Polycaprolactone Powders Proposed as Reference Test Materials for International Standard of Biodegradation Evaluation Method

Polycaprolactone (PCL) powders were prepared from PCL pellets using a rotation mechanical mixer. PCL powders were separated by sieves with 60 and 120 meshes into four classes; 0–125 μm, 125–250 μm, 0–250 μm and 250–500 μm. Biodegradation tests of PCL powders and cellulose powders in an aqueous solution at 25°C were performed using the coulometer according to ISO 14851. Biodegradation tests of PCL powders and cellulose powders in controlled compost at 58°C were performed by the Mitsui Chemical Analysis and Consulting Service, Inc. according to ISO 14855-1 and by using the Microbial Oxidative Degradation Analyzer (MODA) instrument according to ISO/DIS 14855-2. PCL powders were faster biodegraded than cellulose powders. The reproducibility of biodegradation of PCL powders is excellent. Differences in the biodegradation of PCL powders with different class were not observed by the ISO 14851 and ISO/DIS 14855-2. An enzymatic degradation test of PCL powders with different class was studied using an enzyme of Amano Lipase PS. PCL with smaller particle size was faster degraded by the enzyme. PCL powders with regulated sizes from 125 μm to 250 μm are proposed as a reference material for the biodegradation test.

Biobased Contents of Biodegradable Poly(ε‐caprolactone) Composites Polymerized and Directly Molded Using Aluminium Triflate from Caprolactone with Cellulose and Inorganic Filler

Poly(epsilon-caprolactone) (PCL) composite samples were prepared by polymerization and direct molding. The starting compound was epsilon-caprolactone monomer liquid combined with cellulose and inorganic fillers, using aluminium triflate as a catalyst at 80 degrees C, for 6 or 24 h. Cylinder-shaped PCL composite samples with a homogeneously dispersed cellulose filler were prepared with (-)M(n) = 4 600 ((-)M(w)/(-)M(n) = 2.9). The mechanical properties of the PCL composite samples were studied using compression test methods. The strength of a PCL composite with 50 wt.-% cellulose filler (10.8 MPa) was found to be lower than the PCL sample without fillers (19.2 MPa). The biobased content of the PCL composite with 50 wt.-% cellulose filler (51.67%) measured using accelerated mass spectrometry (AMS) was slightly higher than the carbon ratio of cellulose in the starting powder samples (41.3 mol-%). The biobased content of the polymer composite powders by AMS was found not to be affected by the presence of inorganic fillers, such as talc. The rate and extent of biodegradation, caused by Amano Lipase PS, of the PCL composite sample with cellulose filler (40% degradation in 4 d) was the same as that of a PCL sample without the cellulose filler.

Two-step sequential synthesis of pyrimidine derivatives containing a sugar branch via combining of enzymatic Michael addition/acylation

A new strategy for the enzymatic synthesis of pyrimidine derivatives containing a sugar branch was developed via combining of Michael addition and acylation. The first-step reaction of pyrimidines and vinyl 3-propionyloxy propionate was catalyzed by Amano lipase M from Mucor javanicus in DMSO. The initial reaction rates of different pyrimidines decreased in the order of fluorouracil, uracil, thymine, in agreement with their nucleophilicity. The succeeding regioselective acylation of d-glucose and d-mannose with the Michael adducts was catalyzed by alkaline protease from Bacillus subtilis in pyridine. The d-glucose and d-mannose were all acylated at C-6 position. Moderate yield was obtained for each step.

Enzymatic resolution of 2,2,2-trifluoro-1-arylethylamine derivatives by Pseudomonas fluorescens lipase in organic solvents

Amination of phenyl trifluoromethyl ketone with ammonium formate (Leuckart-Wallach reaction) in a one-pot reaction gave 2,2,2-trifluoro-1-phenylethylamine ( 3a) in high yield (81%). Other fluorinated 1-arylethylamine derivatives were obtained from their corresponding aryl ketones in moderate to good yields (55–81%). Enzymatic resolution of racemic 3a was done with Pseudomonas fluorescens lipase (Amano lipase AK) via enantioselective alcoholysis of its chloroacetamide ( 4a) with n-amyl alcohol in diisopropyl ether giving good enantioselectivity ( E-value = 44). Other amines also were resolved by lipase AK under similar conditions ( E-values = 25 to >100).

Synthesis of (R)-4,4,4-trifluoro-2-mercaptobutyric acid

Syntheses of (R)-4,4,4-trifluoro-2-mercaptobutyric acid from (S)-malic acid via a Mitsunobu reaction and from (rac)-thiomalic acid on enzymatic resolution, using Pseudomonas cepacia (Amano lipase PS), are described. A new method for direct determination of ees for (R)- and (S)-4,4,4-trifluoro-2-mercaptobutyric acid derivatives by HPLC on a polysaccharide phase is disclosed.

Empirical rules for the enantiopreference of lipase from Aspergillus niger toward secondary alcohols and carboxylic acids, especially α-amino acids

Lipase from Aspergillus niger (ANL, Amano lipase AP) catalyzes enantioselective hydrolysis and acylation reactions. To aid in the design of new applications of this lipase, we propose two empirical rules that predict which enantiomer reacts faster. For secondary alcohols, a rule proposed previously for other lipases also works for ANL, but with lower reliability (77%, 37 of 48 examples). For carboxylic acids, we examined both crude and partially-purified ANL because commercial ANL contains contaminating hydrolases. Partial purification removed a contaminating amidase and increased the enantioselectivity of ANL toward many α-amino acids, including cyclic amino acids. Unlike other lipases, ANL readily accepts positively-charged substrates and shows the highest enantioselectivity toward α-amino acids. Although a rule based on the sizes of the substituents could not predict the fast-reacting enantiomer, a rule limited to α-amino acids did predict the fast-reacting enantiomer. We estimate that the charged α-amino group contributes a factor of 40–100 ( ΔΔ G ‡=2.2–2.7 kcal/mol) to the enantioselectivity of ANL towards carboxylic acids.

Enzymatic Desymmetrization of Prochiral 2-Substituted-1,3-propanediols: A Practical Chemoenzymatic Synthesis of a Key Precursor of SCH51048, a Broad-Spectrum Orally Active Antifungal Agent

Two examples of a practical enzymatic desymmetrization of a 2-substituted-1,3-propanediol and their application to the synthesis of SCH51048, a broad-spectrum orally active antifungal, are described. In each case, enzymatic catalysis under both hydrolytic and transesterification conditions is described. In the first example the key intermediate, the R,S-monoester of triol 6, was obtained via Amano Lipase AK catalyzed hydrolysis of the dibutyrate 11b, or Novo SP435 catalyzed acetylation of triol 6. In the second example, desymmetrization of diol 13a using Novo SP435 or of dibutyrate 13c using Amano Lipase CE furnished the S-monoester (S)-14b,c, a key intermediate in a new efficient synthesis of SCH51048. Optimization of the Novo SP435 acetylation of diol 13a and the scaleup of the reaction is also described.

Simultaneous monitoring of the enantiomeric purities of both substrate and product in the enzymatic resolution of (R,S)‐2‐acetoxy‐3‐bromopropyl para‐toluenesulfonate by HPLC chiral column chromatography

AbstractThe enantiomeric purities of both substrate, 2‐acetoxy‐3‐bromopropyl para‐toluenesulfonate (I), and product, 2‐hydroxy‐3‐bromopropyl p‐toluenesulfonate (II) were examined in one analysis. The enzymatic resolution was conducted by Amano lipase AK and the enantiomeric excess as well as the conversion rate were monitored by HPLC analysis utilizing a Chiralcel OD column. After 7 h of reaction, HPLC results indicated that the enantiomeric purities of both substrate (I) and product (II) were greater than 95% and the conversion rate was around 55%. © 1995 Wiley‐Liss, Inc.

Isolation of racemic 2,4-pentanediol and 2,5-hexanediol from commercial mixtures of racemic and meso isomers by way of cyclic sulfites.

Enantiomerically pure diols with C 2 symmetry such as 23-butanediol, 1, 2,4-pentanediol, 2, and 2,5-hexanediol, 3, are useful chiral auxiliaries, but they are expensive because chemists lack good synthetic routes that eliminate both the meso isomer and one enantiomer. Enzymic resolutions efficiently separate the enantiomer, but do not remove the meso isomer. To simplify enzymic resolutions of 2 and 3, we developed simple methods to isolate the racemic isomer from commercial mixtures of racemic and meso isomers. For 2, the meso isomer selectively reacted with SOCl 2 to give a cyclic sulfite that was removed by column chromatography to leave (±)- 2,92% de, 1.4 g, 55% yield. For 3, both meso and racemic isomers reacted with SOCl 2 to give cyclic sulfites, but the sulfite derived from the meso isomer rearranged to trans-2,5-dimethyltetrahydrofuran under acidic conditions. Hydrolysis of the remaining sulfite gave (±)-3,84% de, 1.1 g, 37% yield. Resolution of (±)- 2 and (±)-3 using lipase from Pseudomonas cepacia yielded (2R,4R)-2-diacetate, 78% ee, > 97% de.40% of theory and(2R,5R)-3-diacetate, 94% ee. > 97% de, 47% of theory. Previously reported acetylations of 2 and 3 by lipase from Candida antarctica (CAL) or by lipase from Pseudomonas sp. (Amano lipase AK) are more enantioselective and thus, the best route to enantiomerically and diastereomerically pure 2 and 3 is removal of the meso isomer by way of cyclic sulfites followed by resolution with CAL or Amano lipase AK,

Triglyceride interesterification by lipases. 3. Alcoholysis of pure triglycerides

Lipase-catalyzed alcoholysis of triolein dissolved in ethanol or isopropanol for the formation of ethyl and isopropyl esters was investigated. Of 16 lipases screened, Amano lipase from P. fluorescens was selected for investigation of the effects of basic reaction conditions on alcoholysis yields. Ethanolysis yields were only slightly affected by water additions to immobilized lipase preparations. Isopropyl ester yields decreased with water addition. Good operational stability was observed over 17 days. Changes in initial triolein concentration in the range 5–50 mM had very little effect on ester yields. The ionic strength of the phosphate buffer used in lipase immobilization affected ethanolysis and isopropanolysis yields in opposite ways. The highest ethanolysis yields were obtained with lipases immobilized from 250 mM buffer, while isopropyl ester yields were highest with lipases immobilized from water. In addition, the quantities and isomers of monoglyceride intermediates in ethanolysis were affected by the immobilization buffer strength. Larger quantities of 2-monoglycerides were formed in ethanolysis reactions with lipase preparations immobilized from water.