Immobilized Pseudomonas Cepacia Lipase Research Articles

Enzymatic transesterification of Rubber Seed Oil Using Immobilized Pseudomonas Cepacia Lipase

Biodiesel or Fatty acid methyl ester (FAME) is a biofuel formed from transesterification of vegetable oil and fat. In this study, the source of vegetable oil is Rubber seed oil (RSO). The oil was extracted from rubber seed by solvent extraction, using n-hexane and methanol. The oil produced from both solvents’ extraction were used as feedstock. Biodiesel production from RSO was performed with methanol as acyl acceptor. using immobilized Pseudomonas cepacia lipase as catalyst. Three different parameters were studied, they are reaction time, lipase inhibition, and stepwise addition of methanol/oil molar ratio. The enzymatic transesterification with ultrasonic assisted process was utilized, as it enhances the miscibility between oil and methanol, which lead to reducing the reaction time and significantly increased the yield of biodiesel. The results indicated a significant biodiesel is produced with the highest yield of 76.55%, at 45 °C, with the lipase amount of 5 % (w/w), methanol to oil molar ratio of 3:1 within 6 hours. It is found that despite high content of free fatty acid of rubber seed oil, it has a potential as alternative feedstock for FAME production.

Effect of light irradiation on esterification of oleic acid with ethanol catalyzed by immobilized Pseudomonas cepacia lipase

The present study demonstrates the effect of light irradiation on the esterification of oleic acid catalyzed by immobilized Pseudomonas cepacia lipase. The reaction rates of all the experiments under light irradiation were found to be higher than dark conditions. The kinetics of reactions supported the Ping‐Pong Bi‐Bi mechanism with dead end inhibition by both the alcohol and acid substrates. Moreover, circular dichroism (CD) spectroscopy was used to analyze the effect of light on lipase enzyme. The CD spectroscopic studies confirmed that the conformational changes in the secondary structure of the lipase enzyme increased the reaction rate of light‐illuminated experiments, which might have opened up the active sites of enzymes and thus, resulted in higher reaction rates compared to dark reactions. These results have successfully demonstrated that the light illumination positively influenced the rate of P. cepacia enzyme‐catalyzed esterification reactions.

Enhanced biocatalytic activity of immobilized Pseudomonas cepacia lipase under sonicated condition.

The present work reports the use of biocatalyst and ultrasound for greener synthesis of cinnamyl propionate. The lipase Pseudomonas cepacia was immobilized on a copolymer of hydroxypropyl methyl cellulose and polyvinyl alcohol. This biocatalyst was u sed for ultrasound-assisted synthesis of cinnamyl propionate with the detailed optimization of various reaction parameters. Besides this, protocol was extended to synthesize various industrially important propionate esters. In addition to this, different enzyme-kinetic parameters such as r max and K m(vinyl propionate), K m(cinnamyl alcohol) and K i(cinnamyl alcohol) were studied which presented ordered bi-bi mechanism with an inhibition by cinnamyl alcohol. The developed biocatalyst demonstrated enhancement in catalytic activity and recyclability up to five recycles. Moreover, the biocatalyst was tested to investigate the effects of sonication via various characterization techniques such as scanning electron microscopy, thermogravimetry, and water content analysis.

Light Induced Esterification of Oleic Acid Catalyzed by Pseudomonas Cepacia Lipase

This paper reports on new approach of light illuminated esterification of oleic acid catalyzed by an enzymatic catalyst. Immobilized Pseudomonas cepacia lipase was used as a biocatalyst. Enzyme catalyzed esterification reaction was chosen due to the wide attention from chemical and biotechnology industry. The reaction was conducted under light illumination and dark condition at same experimental condition. The result was obtained in the form of changes in oleic acid concentration throughout 2h of reaction. The results indicate significant changes in rate of reaction for light illuminated reaction compare to reaction under dark condition. The initial rate of reaction for light induced and dark reaction are 1.2mol/L.m in , 1.1mol/L.min respectively. Influence of enzyme amount was experimented where it shows proportional increase with the amount of enzyme. Increasing the amount of enzyme also increases the availability of active sites of enzyme therefore the probability of light illumination activating the electron transfer in oxyanion hole increases with increase in energy level of electron lone pair. The results obtained indicate that light illuminating method for enzyme activation provide good alternative for future biotechnology industry.

Enzymatic production of (S)-3-cyano-5-methylhexanoic acid ethyl ester with high substrate loading by immobilized Pseudomonas cepacia lipase

(S)-3-Cyano-5-methylhexanoic acid ethyl ester is a valuable synthetic intermediate for pregabalin. Immobilized lipase PS from Pseudomonas cepacia was screened and shown to be the best biocatalyst for the enantioselective hydrolysis of 3-cyano-5-methylhexanoic acid ethyl ester, a racemic mixture involving a β-stereocenter. The optimum temperature and pH for the biocatalytic process were 35°C and 6.0, respectively. Lipase PS IM exhibited a strong tolerance toward high substrate concentrations of up to 2.0M (366g/l). In the scaled-up biotransformation, (S)-3-cyano-5-methylhexanoic acid ethyl ester was produced in 0.89M (162.9g/l), 99.2% ee, and 44.5% yield. These results indicated that lipase PS IM catalyzed the preparation of (S)-3-cyano-5-methylhexanoic acid ethyl ester and could be used as an efficient route for the large-scale production of pregabalin.

Effect of pretreatment by different organic solvents on esterification activity and conformation of immobilized Pseudomonas cepacia lipase

Abstract Experiments were carried out to investigate the effect of pretreatment by organic solvents with different hydrophobicities, functional groups and molecular constitutions as activation agents on initial esterification activity and secondary structure of immobilized Pseudomonas cepacia lipase. The results showed that esterification activity of immobilized P. cepacia lipase treated with organic solvents containing –C O– and –C N– functional groups was highest, followed by the one treated with –C–C– functional groups but the lowest with –OH and aromatics functional groups. An organic solvent with a branched structure was more favorable compared with a straight chain in terms of enhancing enzyme activity. Conformational studies via Fourier transform-infrared spectroscopy indicated that the catalytic activity variance was attributed to the secondary structure changes for immobilized P. cepacia lipase treated with organic solvents. Moreover, the effects of moisture, pH and temperature on the esterification activity of immobilized P. cepacia lipase were also addressed.

Improved Synthesis and Isolation of 2′‐O‐Methyladenosine: Effective and Scalable Enzymatic Separation of 2′/3′‐O‐Methyladenosine Regioisomers

AbstractAn efficient separation of a mixture of 2′/3′‐O‐methyladenosine regioisomers (1 + 2; 1:1) has been developed by selective enzymatic acylation using immobilized Pseudomonas cepacia lipase (PSL‐C) in combination with acetonoxime levulinate as acyl donor. The 3′‐hydroxy group of 2′‐O‐methyladenosine (1) was acylated with high selectivity (ca. 70 %), whereas an equal amount of 3′‐O‐methyladenosine (2) in the same solution resulted in minor acylation of 5′‐hydroxy group (ca. 8 %). The differential behavior of both regioisomers towards enzymatic acylation allowed to develop a separation protocol. Upon extraction of the acylated products, the 3′‐O‐methyladenosine was isolated in 81 % yield and 97 % purity from the aqueous layer. Hydrolysis of acylated products in organic layer furnished 2′‐O‐methyladenosine in 67 % yield and 99 % purity. The separation process was successfully applied to the crude reaction mixture of methylated products (ca. 3:1 of 1/2) on 5‐g scale. We also report on the use of methyl p‐toluenesulfonate as a safe reagent for 2′‐O‐methylation of adenosine.(© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)

Enzymatic Regioselective Levulinylation of 2′‐Deoxyribonucleosides and 2′‐O‐Methylribonucleosides

The levulinyl-protected nucleosides are key building blocks for the solution-phase synthesis of oligonucleotides. Short and efficient syntheses of 3'- and 5'-O-levulinylated 2'-deoxyribonucleosides and 2'-O-methylribonucleosides have been developed from the corresponding nucleosides by enzyme-catalyzed regioselective acylation in organic solvents or from 3',5'-di-O-levulinyl derivatives by regioselective enzymatic hydrolysis. Lipase-mediated levulinylation of various nucleosides has been accomplished with acetonoxime levulinate as an acyl donor. Use of immobilized Pseudomonas cepacia lipase (PSL-C) has furnished 3'-O-levulinylated 2'-deoxyribonucleosides in excellent yields. Similarly, Candida antarctica lipase B (CAL-B) has provided 5'-O-levulinylated nucleosides in high yields. 3'-O-Levulinylated 2'-deoxyribonucleosides and 2'-O-methylribonucleosides were prepared via selective hydrolysis of 3',5'-di-O-levulinate esters using CAL-B.

First Regioselective Enzymatic Alkoxycarbonylation of Primary Amines. Synthesis of Novel 5′‐ and 3′‐Carbamates of Pyrimidine 3′,5′‐Diaminonucleoside Derivatives Including BVDU Analogues.

AbstractFor Abstract see ChemInform Abstract in Full Text.

First regioselective enzymatic alkoxycarbonylation of primary amines. Synthesis of novel 5'- and 3'-carbamates of pyrimidine 3',5'-diaminonucleoside derivatives including BVDU analogues.

The first regioselective enzymatic alkoxycarbonylation of primary amino groups has been achieved in pyrimidine 3',5'-diaminonucleoside derivatives. Thus, Candida antarctica lipase B (CAL-B) catalyzed this reaction with nonactivated homocarbonates allowing the selective synthesis of several N-5' carbamates, including (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) analogues, with moderate-high yields, whereas immobilized Pseudomonas cepacia lipase (PSL-C) afforded mixtures of alkoxycarbonylated regioisomers. To obtain N-3' carbamates selectively, a short and efficient chemoenzymatic route was used employing some of the N-5'-protected derivatives previously synthesized.

Synthesis, Protonation Behavior, Conformational Analysis, and Regioselective Enzymatic Acylation of the Novel Diamino Analogue of (E)‐5‐(2‐Bromovinyl)‐2′‐deoxyuridine (BVDU).

AbstractFor Abstract see ChemInform Abstract in Full Text.

Synthesis, Protonation Behavior, Conformational Analysis, and Regioselective Enzymatic Acylation of the Novel Diamino Analogue of (E)-5-(2-Bromovinyl)-2′-deoxyuridine (BVDU)

(E)-3′,5′-Diamino-5-(2-bromovinyl)-2′,3′,5′-trideoxyuridine (5), the diamino analogue of BVDU (1), was synthesized from BVDU. The protonation behavior of 5 has been studied by means of pH-metric measurements and NMR spectroscopy. This study allows the determination of the basicity constants and the stepwise protonation sites. Thus, the main species at physiological pH is the monoprotonated form. The conformational analysis of this nucleoside analogue was also carried out through 1H NMR spectroscopy. In addition, a convenient synthesis of N-3′ and N-5′ acylated derivatives was developed by regioselective enzymatic acylation. Thus, Candida antarctica lipase B (CAL-B) selectively acylated the 5′-amino group, thus furnishing nucleosides 8. On the other hand, immobilized Pseudomonas cepacia lipase (PSL-C) exhibited the opposite selectivity, conferring acylation at the 3′-amino group, thus affording derivatives 9.

Novel enzymatic synthesis of levulinyl protected nucleosides useful for solution phase synthesis of oligonucleotides

An efficient synthesis of 3′- and 5′- O-levulinyl-2′-deoxy- and 2′- O-alkylribonucleosides has been developed from appropriate nucleosides by enzyme-catalyzed regioselective acylation in organic solvents. Several lipases were screened in combination with acetonoxime levulinate as an acylating agent. Immobilized Pseudomonas cepacia lipase (PSL-C) was selected for acylation of the 3′-hydroxyl group in nucleosides, furnishing 3′- O-levulinylated products 1 and 3 in excellent yields. Similarly, Candida antarctica lipase B (CAL-B) provided 5′- O-levulinyl nucleosides 2 and 4 in high yields. Base-protected cytidine and adenosine analogs were found to be good substrates for lipase-mediated acylations. To demonstrate the industrial utility of this method, 3′- O-levulinyl thymidine and N 2-Ibu-5′- O-levulinyl-2′deoxyguanosine were synthesized on a 25 g scale. Additionally, PSL-C was reused to make the processes further economical.

Efficient Chemoenzymatic Synthesis of (S)‐ and (R)‐5‐(1‐Aminoethyl)‐2‐(cyclohexylmethoxy)benzamide: Key Intermediate for Src‐SH2 Inhibitor.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Building blocks for the solution phase synthesis of oligonucleotides: regioselective hydrolysis of 3',5'-Di-O-levulinylnucleosides using an enzymatic approach.

A short and convenient synthesis of 3'- and 5'-O-levulinyl-2'-deoxynucleosides has been developed from the corresponding 3',5'-di-O-levulinyl derivatives by regioselective enzymatic hydrolysis, avoiding several tedious chemical protection/deprotection steps. Thus, Candida antartica lipase B (CAL-B) was found to selectively hydrolyze the 5'-levulinate esters, furnishing 3'-O-levulinyl-2'-deoxynucleosides 3 in >80% isolated yields. On the other hand, immobilized Pseudomonas cepacia lipase (PSL-C) and Candida antarctica lipase A (CAL-A) exhibit the opposite selectivity toward the hydrolysis at the 3'-position, affording 5'-O-levulinyl derivatives 4 in >70% yields. A similar hydrolysis procedure was successfully extended to the synthesis of 3'- and 5'-O-levulinyl-protected 2'-O-alkylribonucleosides 7 and 8. This work demonstrates for the first time application of commercial CAL-B and PSL-C toward regioselective hydrolysis of levulinyl esters with excellent selectivity and yields. It is noteworthy that protected cytidine and adenosine base derivatives were not adequate substrates for the enzymatic hydrolysis with CAL-B, whereas PSL-C was able to accommodate protected bases during selective hydrolysis. In addition, we report an improved synthesis of dilevulinyl esters using a polymer-bound carbodiimide as a replacement for dicyclohexylcarbodiimide (DCC), thus considerably simplifying the workup for esterification reactions.

Efficient chemoenzymatic synthesis of ( S)- and ( R)-5-(1-Aminoethyl)-2-(cyclohexylmethoxy)benzamide: key intermediate for Src-SH2 inhibitor

A facile chemoenzymatic synthesis of both the S and R forms of 5-(1-aminoethyl)-2-(cyclohexylmethoxy)benzamide a key intermediate of non-peptidic Src SH2 inhibitors is described. Both the enantiomers were synthesized in high optical purity (>99% ee) by reduction followed by lipase-mediated acylation of the precursor 6 in one-pot. Immobilized Pseudomonas cepacia lipase offered high degree of enantioselectivity with spontaneity.

Synthesis of enantiomers of indanoxazolidinone based on the lipase-catalyzed resolution of the corresponding N-carbamylamino derivative

Abstract Enantiomerically enriched (4R,5S)- and (4S,5R)-indano[1,2-d]oxazolidinones were enzymatically prepared from (±)-1-amino-2-indanol. Racemic 1-(N′-chloroacetyl-N-carbamylamino)-2-indanol O-chloroacetate was hydrolyzed with immobilized Pseudomonas cepacia lipase in the presence of β-cyclodextrin in acetone-buffer solution, to afford (1S,2R)-1-(N′-chloroacetyl-N-carbamylamino)-2-indanol (90%e.e.) and the unreacted (1R,2S)-substrate (97%e.e.), in nearly quantitative yields. The deprotection provided enantiomers of 1-N-carbamylamino-2-indanol, the precursor of indanoxazolidinone, via nitrosation-deaminocyclization reaction.