Candida antarctica lipase B catalyzed enantioselective acylation of pyrimidine acyclonucleoside

Abstract

The influence of solvent and acyl group donor on selectivity of the transesterification reaction of 1-[1′,3′-dihydroxy-2′-propoxymethyl]-5-methyluracil, a structural analogue of ganciclovir was examined. Lipase (EC 3.1.1.3) B from Candida antarctica (CALB) enabled desymmetrization of prochiral hydroxyl groups when 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim][PF₆]) was used as a reaction medium. It was observed that CALB was up to 2.7–4 times more enantioselective in the ionic liquid [Bmim][PF₆] than in conventional organic solvents.     

Over-stabilization of Candida antarctica lipase B by ionic liquids in ester synthesis

Four different ionic liquids, based on dialkylimidazolium cations associated with perfluorinated and bis(trifluoromethyl)sulfonyl amide anions were used as reaction media for butyl butyrate synthesis catalyzed by free Candida antarctica lipase B at 2% (v/v) water content and 50 °C. Lipase had enhanced synthetic activity in all ionic liquids in comparison with two organic solvents (hexane, and 1-butanol), the enhanced activity being related to the increase in polarity of ionic liquids. The continuous operation of lipase with all the assayed ionic liquids showed over-stabilization of the enzyme. The reuse of free lipase in 1-butyl-3-methylimidazolium hexafluorophosphate in continuous operation cycles showed a half-life time 2300 times greater than that observed when the enzyme was incubated in the absence of substrate (3.2 h), and a selectivity higher than 90%.     

Ester synthesis from trimethylammonium alcohols in dry organic media catalyzed by immobilized Candida antarctica lipase B

Twenty-one different organic solvents were assayed as possible reaction media for the synthesis of butyryl esters from trimethylammonium alcohols in dry conditions catalyzed by immobilized Candida antarctica lipase B. The reactions were carried out following a transesterification kinetic approach, using choline and L-carnitine as primary and secondary trimethylammonium alcohols, respectively, and vinyl butyrate as acyl donor. The synthetic activity of the enzyme was strictly dependent on the water content, the position of the hydroxyl group in the trimethylammonium molecule, and the Log P parameter of the assayed solvent. Anhydrous conditions and a high excess of vinyl butyrate over L-carnitine were necessary to synthesize butyryl-L-carnitine. The synthetic reaction rates of butyryl choline were practically 100-fold those of butyryl-L-carnitine with all the assayed solvents. In both cases, the synthetic activity of the enzyme was dependent on the hydrophobicity of the solvent, with the optimal reaction media showing a Log P parameter of between -0.5 and 0.5. In all cases, 2-methyl-2-propanol and 2-methyl-2-butanol were shown to be the best solvents for both their high synthetic activity and negligible loss of enzyme activity after 6 days.     

Regioselective acylation of flavonoids catalyzed by immobilized Candida antarctica lipase under reduced pressure

A single-step acylation of rutin and naringin, catalyzed by immobilized Candida antarctica lipase B in 2-methyl-2-butanol, occurred preferentially on the primary hydroxyl group. Using palmitic methyl ester as acyl donor, the acylation rate of naringin was 10-fold higher than that of rutin. Under optimal conditions, i.e. a molar ratio acyl donor/naringin of 7:1 and 200 mbar, 92% naringin was acylated.     

Kinetic model of biodiesel production using immobilized lipase Candida antarctica lipase B

We have designed a kinetic model of biodiesel production using Novozym 435 (Nz435) with immobilized Candida antarctica lipase B (CALB) as a catalyst. The scheme assumed reversibility of all reaction steps and imitated phase effects by introducing various molecular species of water and methanol. The global model was assembled from separate reaction blocks analyzed independently. Computer simulations helped to explore behavior of the reaction system under different conditions. It was found that methanolysis of refined oil by CALB is slow, because triglycerides (T) are the least reactive substrates. Conversion to 95% requires 1.5–6 days of incubation depending on the temperature, enzyme concentration, glycerol inhibition, etc. Other substrates, free fatty acids (F), diglycerides (D) and monoglycerides (M), are utilized much faster (1–2 h). This means that waste oil is a better feedstock for CALB. Residual enzymatic activity in biodiesel of standard quality causes increase of D above its specification level because of the reaction 2M ↔ D + G. Filtration or alkaline treatment of the product prior to storage resolves this problem. The optimal field of Nz435 application appears to be decrease of F, M, D in waste oil before the conventional alkaline conversion. Up to 30-fold reduction of F-content can be achieved in 1–2 h, and the residual enzyme (if any) does not survive the following alkaline treatment.     

Kinetic resolution of drug intermediates catalyzed by lipase B from Candida antarctica immobilized on immobead‐350

Novozyme 435, which is a commercial immobilized lipase B from Candida antarctica (CALB), has been proven to be inadequate for the kinetic resolution of rac‐indanyl acetate. As it has been previously described that different immobilization protocols may greatly alter lipase features, in this work, CALB was covalently immobilized on epoxy Immobead‐350 (IB‐350) and on glyoxyl‐agarose to ascertain if better kinetic resolution would result. Afterwards, all CALB biocatalysts were utilized in the hydrolytic resolution of rac‐indanyl acetate and rac‐(chloromethyl)‐2‐(o‐methoxyphenoxy) ethyl acetate. After optimization of the immobilization protocol on IB‐350, its loading capacity was 150 mg protein/g dried support. Furthermore, the CALB‐IB‐350 thermal and solvent stabilities were higher than that of the soluble enzyme (e.g., by a 14‐fold factor at pH 5–70°C and by a 11‐fold factor in dioxane 30%–65°C) and that of the glyoxyl‐agarose‐CALB (e.g., by a 12‐fold factor at pH 10–50°C and by a 21‐fold factor in dioxane 30%–65°C). The CALB‐IB‐350 preparation (with 98% immobilization yield and activity versus p‐nitrophenyl butyrate of 6.26 ± 0.2 U/g) was used in the hydrolysis of rac‐indanyl acetate using a biocatalyst/substrate ratio of 2:1 and a pH value of 7.0 at 30°C for 24 h. The conversion obtained was 48% and the enantiomeric excess of the product (e.e._p) was 97%. These values were much higher than the ones obtained with Novozyme 435, 13% and 26% of conversion and e.e.p, respectively. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:878–889, 2018     

Modulation of lipase properties in macro-aqueous systems by controlled enzyme immobilization: enantioselective hydrolysis of a chiral ester by immobilized Pseudomonas lipase

Lipase from Pseudomonas fluorescens (PFL) has been immobilized by using different immobilization protocols. The catalytic behavior of the different PFL derivatives in the hydrolytic resolution of fully soluble (R,S) 2-hydroxy 4-phenyl butanoic acid ethyl ester (HPBE) in aqueous medium was analyzed. The soluble enzyme showed a significant but low enantioselectivity, hydrolyzing the S isomer more rapidly than the R-isomer (E = 7). The enzyme, immobilized via a limited attachment to a long and flexible spacer arm, showed almost identical activity and specificity to the soluble enzyme. However, other derivatives, e.g. PFL adsorbed on supports covered by hydrophobic moieties (octyl, decaoctyl), exhibited significant hyperactivation on immobilization (approximately 7-fold). Simultaneously, the enantioselectivity of the PFL-immobilized enzyme was significantly improved (from E = 7 to E = 80). By using such derivatives, almost pure R ester isomer (e.e. > 99%) has been obtained after 55% hydrolysis of the racemic mixture of a solution of 10% (w/v) (R,S) HPBE. The derivatives could be used for 10 cycles without any significant decrease in the activity of the biocatalyst.     

Chemoselective acylation of (hydroxyalkyl)phenols catalyzed by Candida antarctica lipase B

Acylation of (hydroxyalkyl)phenols with vinyl esters by lipase B from Candida antarctica proceeded smoothly in a highly chemoselective manner, affording their alkyl esters exclusively or at least predominantly. The enzyme therefore discriminates between an alcoholic hydroxyl from a phenolic hydroxyl in addition to having versatile catalytic abilities for organic synthesis.     

南极假丝酵母脂肪酶B在离子液体/超临界流体体系中的稳定性模拟

脂肪酶在离子液体/超临界流体体系中的结构稳定性是影响其活性的重要因素。本文采用分子动力学方法分别研究了南极假丝酵母脂肪酶B(CALB)在离子液体CYPHOS IL-201/极性超临界流体CHF_3两相体系和离子液体CYPHOS IL-201/非极性超临界流体CO_2两相体系中的结构稳定性,揭示影响CALB结构稳定性的因素。研究结果表明,在超临界CHF_3中,CHF_3破坏蛋白维持α螺旋结构的氢键是蛋白结构不稳定的主要原因;在超临界CO_2中,CALB蛋白的结构紧密性降低,有序二级结构发生了变化,导致稳定性下降。离子液体和两种超临界流体均形成了两相体系,蛋白处于离子液体相中,离子液体不溶于超临界流体,但超临界流体部分进入离子液体相,降低了离子液体相的黏度。其中,相比于CYPHOS IL-201/CO_2体系,CYPHOS IL-201/CHF_3体系的黏度降低多。在离子液体CYPHOS IL-201与超临界流体(CHF_3、CO_2)形成的两相体系中,离子液体CYPHOS IL-201具有保护蛋白结构的作用,使CALB蛋白结构更加稳定。     

Improving ascorbyl oleate synthesis catalyzed by Candida antarctica lipase B in ionic liquids and water activity control by salt hydrates

Lipase catalyzed-synthesis of ascorbyl oleate was performed in ionic liquids with addition of salt hydrate pairs for water activity control. The highest yield in the synthesis of ascorbyl oleate (72%) was obtained in [BMIM][BF₄] in the presence of the salt pair NaI 2/0, which corresponds to a water activity of 0.3. Purity and chemical identity of 6-O-ascorbyl oleate was confirmed by ¹H and ¹³C NMR analysis.     

Buffer-mediated activation of Candida antarctica lipase B dissolved in hydroxyl-functionalized ionic liquids

Ionic-liquid buffer having phosphate anion was synthesized for the development of buffered enzymatic ionic liquid systems. Both the conformation and transesterification activity of Candida antarctica lipase B (CALB) dissolved in the hydroxyl-functionalized ionic liquids were buffer dependent. Intrinsic fluorescence studies indicated that the CALB possessed a more compact conformation in the medium consisted of ionic liquid buffer having phosphate anion and hydroxyl-functionalized ionic liquids like 1-(1-hydroxyethyl)-3-methyl-imidazolium tetrafluoroborate and 1-(1-hydroxyethyl)-3-methyl-imidazolium nitrate. High activity and outstanding stability could be obtained with the CALB enzyme in the buffered ionic liquids for the transesterification.     

Aminolysis of 2-hydroxy esters catalyzed by Candida antarctica lipase

Candida antarctica lipase catalyzed the aminolysis of 2-hydroxy esters with amines in organic solvents to yield the corresponding 2-hydroxy amides. The reactions proceeded at 28–30 °C in dioxane for 6 h with 3 mM substrates with yields ranging between 45% (w/w) (for branched substrates) to 88% (w/w) (for linear substrates). Although the reaction was not enantioselective, because of its simplicity it represents an alternative method for the synthesis of functionalised amides.     

Green enzymatic production of glyceryl monoundecylenate using immobilized Candida antarctica lipase B

Enzymatic synthesis of glyceryl monoundecylenate (GMU) was performed using indigenously immobilized Candida anatarctica lipase B preparation (named as PyCal) using glycerol and undecylenic acid as substrates. The effect of molar ratio, enzyme load, reaction time, and organic solvent on the reaction conversion was determined. Both batch and continuous processes for GMU synthesis with shortened reaction time were developed. Under optimized batch reaction conditions such as 1:5 molar ratio of undecylenic acid and glycerol, 2?h of reaction time at 30% substrate concentration in tert-butyl alcohol, conversion of 82% in the absence of molecular sieve, and conversion of 93% in the presence of molecular sieve were achieved. Packed bed reactor studies resulted in high conversion of 86% in 10-min residence time. Characterization of formed GMU was performed by FTIR, MS/MS. Enzymatic process resulted in GMU as a predominant product in high yield and shorter reaction time periods with GMU content of 92% and DAG content of 8%. Optimized GMU synthesis in the present study can be used as a useful reference for industrial synthesis of fatty acid esters of glycerol by the enzymatic route.     

Kinetic resolution of rac-2-pentanol catalyzed by Candida antarctica lipase B in the ionic liquid, 1-butyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]amide

The ionic liquid, l-butyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]amide ([Bmim] [NTf2]), was used as a reaction medium for the kinetic resolution of rac-2-pentanol catalyzed by free Candida antarctica lipase B, using vinyl propionate at 2% (v/v) water content. The synthetic activity of lipase in [Bmim] [NTf2] was up 2.5-times greater than in hexane, and showed high enantioselectivity (ee > 99.99%). The optimal temperature and pH were 60 degrees C and 7, respectively. A decrease in water activity (aw) produced a decay in synthetic activity, and an exponential increase in selectivity.     

Regioselective acylation of disaccharides in tert-butyl alcohol catalyzed by Candida antarctica lipase

The acylation of several disaccharides by ethyl butanoate and ethyl dodecanoate was catalyzed by Candida antarctica lipase in tert-butyl alcohol, at temperatures ranging from 40 degrees to 82 degrees C (reflux temperature). The relative reaction rates of the various disaccharides were directly related to their solubility. The primary products were the monoesters derived from acylation of the primary alcohol groups. At higher conversions diesters were formed, and the ratio of diester to monoester was markedly dependent on the structure of the disaccharide. Thus, reaction of maltose with ethyl dodecanoate in refluxing tert-butyl alcohol afforded the 6'-monododecanoate even at high conversions. Trehalose, in contrast, afforded the 6,6'-diester. Acylation of the less soluble sucrose and lactose was much slower, but a moderate (37%) conversion of sucrose was observed after a prolonged reaction time (7 days). A number of other lipases and proteases were tested but C. antarctica lipase was unique in catalyzing the acylation of sucrose in refluxing tert-butyl alcohol. (c) 1996 John Wiley & Sons, Inc.     

The effect of ionic liquid media on activity,selectivity and stability of Candida antarctica lipase B in transesterification reactions

Nineteen different 1,3-dialkylimidazolium-based ionic liquids (ILs) were used as reaction media for the synthesis of butyl butyrate by transesterification from vinyl butyrate and 1-butanol catalyzed by Candida antarctica lipase B (CaLB). The reaction was also carried out in hexane as a reference solvent. In all the water-immiscible ILs assayed, the enzymatic activity and selectivity were higher than that obtained in hexane. However, in water-miscible ILs, the activity was lower than in the reference solvent, although they showed >99.99% selectivity. Two solvent properties, hydrophobicity and nucleophilicity, were considered key parameters for analyzing the behavior of CaLB in ILs. In the case of ILs based on the same anion, the synthetic activity was gradually enhanced by increasing cation hydrophobicity. Furthermore, the activity of CaLB was greater in ILs containing anions of lower nucleophilicity. Stability studies indicate that CaLB exhibited greater stability in water-immiscible ILs than in water-miscible ILs.     

Engineering of Candida antarctica lipase B for hydrolysis of bulky carboxylic acid esters

Candida antarctica lipase B (CALB) is a widely used biocatalyst with high activity and specificity for a wide range of primary and secondary alcohols. However, the range of converted carboxylic acids is more narrow and mainly limited to unbranched fatty acids. To further broaden the biotechnological applications of CALB it is of interest to expand the range of converted carboxylic acid and extend it to carboxylic acids that are branched or substituted in close proximity of the carboxyl group. An in silico library of 2400 CALB variants was built and screened in silico by substrate-imprinted docking, a four step docking procedure. First, reaction intermediates of putative substrates are covalently docked into enzyme active sites. Second, the geometry of the resulting enzyme-substrate complex is optimized. Third, the substrate is removed from the complex and then docked again into the optimized structure. Fourth, the resulting substrate poses are rated by geometric filter criteria as productive or non-productive poses. Eleven enzyme variants resulting from the in silico screening were expressed in Escherichia coli BL21 and measured in the hydrolysis of two branched fatty acid esters, isononanoic acid ethyl ester and 2-ethyl hexanoic acid ethyl esters. Five variants showed an initial increase in activity. The variant with the highest wet mass activity (T138S) was purified and further characterized. It showed a 5-fold increase in hydrolysis of isononanoic acid ethyl ester, but not toward sterically more demanding 2-ethyl hexanoic acid ethyl ester.     

Enzymatic synthesis of sialic acid derivative by immobilized lipase from Candida antarctica

The effects of important reaction parameters on the enhancement of sialic acid derivative lipophilic properties through the lipase-catalyzed esterification of N-acetyl neuraminic acid methyl ester are investigated in this study. It is found that the lipase Novozym 435 from Candida antarctica is particularly useful in the preparation of sialic acid methyl ester monononanoate (SAMEMN). The optimum temperature for the SAMEMN synthesis reaction using Novozym 435 is 60 °C, and nonanoic anhydride is found to be the best substrate among all acyl donors. The Novozym 435-catalyzed esterification of N-acetyl neuraminic acid methyl ester gave a maximum yield of 87.7% after 6 h in acetonitrile at 60 °C. Because the novel method developed is simple, yet effective, it could potentially be used industrially for the production of sialic acid derivatives.     

Synthesis of 2-ethylhexyl oleate catalyzed by Candida antarctica lipase immobilized on a magnetic polymer support in continuous flow

Bioprocess and Biosystems Engineering - This study investigated the synthesis of 2-ethylhexyl oleate catalyzed by Candida antarctica lipase immobilized on magnetic poly(styrene-co-divinylbenzene)...