Display of Candida antarctica lipase B on Pichia pastoris and its application to flavor ester synthesis

Two alternative cell-surface display systems were developed in Pichia pastoris using the α-agglutinin and Flo1p (FS) anchor systems, respectively. Both the anchor cell wall proteins were obtained originally from Saccharomyces cerevisiae. Candida antarctica lipase B (CALB) was displayed functionally on the cell surface of P. pastoris using the anchor proteins α-agglutinin and FS. The activity of CALB displayed on P. pastoris was tenfold higher than that of S. cerevisiae. The hydrolytic and synthetic activities of CALB fused with α-agglutinin and FS anchored on P. pastoris were investigated. The hydrolytic activities of both lipases displayed on yeast cells surface were more than 200 U/g dry cell after 120 h of culture (200 and 270 U/g dry cell, respectively). However, the synthetic activity of CALB fused with α-agglutinin on P. pastoris was threefold higher than that of the FS fusion protein when applied to the synthesis of ethyl caproate. Similarly, the CALB displayed on P. pastoris using α-agglutinin had a higher catalytic efficiency with respect to the synthesis of other short-chain flavor esters than that displayed using the FS anchor. Interestingly, for some short-chain esters, the synthetic activity of displaying CALB fused with α-agglutinin on P. pastoris was even higher than that of the commercial CALB Novozyme 435.     

Retention factors and resolutions of amino benzoic acid isomers with some lonic liquids

Ionic liquids in the form of organic salts are being widely used as new solvent media. In this paper three positional isomers,o-amino benzoic acid,m-amino benzoic acid, andp-amino benzoic acids were separated with four different ionic liquids as mobile phase additives using high performance liquid chromatography (HPLC). The following ionic liquids were used: 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIm][BF₄]), 1-ethyl-3-methylimidazolium tetrafluoroborate ([EMIm][BF₄]), 1-ethyl-3-methylimidazolium methylsulfate ([EMIm][MS]), and 1-octyl-3-methylimidazolium methylsulfate ([OMIm][MS]). The effects of the alkyl group length on the imidazolium ring and its counterion, and the concentrations of the ionic liquids on the retention factors and resolutions of amino benzoic acid isomers were tested. The results of the separations with ionic liquids as the eluents were better than those without ionic liquids. Excellent separations of the three isomers were achieved using 2.0≈8.0 mM/L [OMIm][MS] and 1.0≈8.0 mM/L [EMIm][MS] as the eluent modifiers.     

Optimization of (R,S)-1-phenylethanol kinetic resolution over Candida antarctica lipase B in ionic liquids

The kinetic resolution of racemates constitutes one major route to manufacture optically pure compounds. The enzymatic kinetic resolution of (R,S)-1-phenylethanol over Candida antarctica lipase B (CALB) by using vinyl acetate as the acyl donor in the acylation reaction was chosen as model reaction. A systematic screening and optimization of the reaction parameters, such as enzyme, ionic liquid and substrates concentrations with respect to the final product concentration, were performed. The enantioselectivity of immobilized CALB commercial preparation, Novozym 435, was assayed in several ionic liquids as reaction media. In particular, three different ionic liquids: (i) 1-butyl-3-methylimidazolium hexafluorophosphate [bmim][PF₆], (ii) 1-butyl-3-methylimidazolium tetrafluoroborate [bmim][BF₄] and (iii) 1-ethyl-3-methylimidazolium triflimide [emim][NTf₂] were tested. At 6.6% (w/w) of Novozym 435, dispersed in 9.520 M of [bmim][PF₆] at 313.15 K, using an equimolar ratio of vinyl acetate/(R,S)-1-phenylethanol after 3 h of bioconversion, the highest possible conversion (50%) was reached with enantiomeric excess for substrate higher than 99%.     

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.     

Enzymatic synthesis of ethyl esters from waste oil using mixtures of lipases in a plug‐flow packed‐bed continuous reactor

This work describes the continuous synthesis of ethyl esters via enzymatic catalysis on a packed‐bed continuous reactor, using mixtures of immobilized lipases (combi‐lipases) of Candida antarctica (CALB), Thermomyces lanuginosus (TLL), and Rhizomucor miehei (RML). The influence of the addition of glass beads to the reactor bed, evaluation of the use of different solvents, and flow rate on reaction conditions was studied. All experiments were conducted using the best combination of lipases according to the fatty acid composition of the waste oil (combi‐lipase composition: 40% of TLL, 35% of CALB, and 25% of RML) and soybean oil (combi‐lipase composition: 22.5% of TLL, 50% of CALB, and 27.5% of RML). The best general reaction conditions were found to be using tert‐butanol as solvent, and the flow rate of 0.08 mL min^?1. The combi‐lipase reactors operating at steady state for over 30 days (720 h), kept conversion yields of ～50%, with average productivity of 1.94 g_{ethyl esters} h^?1, regardless of the type of oil in use. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:952–959, 2018     

Cholinium carboxylate ionic liquids for pretreatment of lignocellulosic materials to enhance subsequent enzymatic saccharification

The present study is the first report demonstrating that ionic liquids consisting of cholinium cations and linear carboxylate anions ([Ch][CA] ILs) can be used for pretreatment of lignocellulosic materials to enhance subsequent enzymatic saccharification. Six variants of [Ch][CA] ILs were systematically prepared by combining cholinium cations with linear monocarboxylate anions ([C_nH_2n+1–COO]⁻, n = 0–2) or dicarboxylate anions ([HOOC–C_nH_2n+1–COO]⁻, n = 0–2). These [Ch][CA] ILs were analyzed for their toxicity to yeast cell growth and their ability to pretreat kenaf powder for subsequent enzymatic saccharification. When assayed against yeast growth, the EC₅₀ for choline acetate ([Ch][OAc]) was 510 mM, almost one order of magnitude higher than that for 1-ethyl-3-methylimidazolium acetate ([Emim][OAc]). The cellulose saccharification ratio after pretreatment at 110 °C for 16 h with [Ch][OAc] (100.6%) was almost comparable with that after pretreatment with [Emim][OAc]. Therefore, [Ch][OAc] is a biocompatible alternative to [Emim][OAc] for lignocellulosic material pretreatment.     

Cyclodextrins improve the antimicrobial activity of the chloride salt of Ruthenium(II) chloro-phenanthroline-trithiacyclononane

The complex [Ru([9]aneS₃)phenCl]Cl (phen = 1,10-phenanthroline) and its synthetic precursor [Ru([9]aneS₃)dmsoCl₂] were immobilized in permethylated β-cyclodextrin (TRIMEB). A new crystalline structure of the precursor, obtained from a batch ethanol solution at low temperature (4°C), is fully described from single-crystal X-ray diffraction data. [Ru([9]aneS₃)phenCl]Cl was also encapsulated in native β-cyclodextrin for comparison with the TRIMEB compound. All three compounds were obtained with a 1:1 host:guest stoichiometry and were studied by powder X-ray diffraction (including synchrotron radiation data), thermogravimetric analysis (TGA), ¹³C{¹H} CP/MAS NMR and FTIR spectroscopies. The bacterial growth inhibitory action of the complex [Ru([9]aneS₃)phenCl]Cl and its two cyclodextrin compounds was tested on Gram-negative (Salmonella, Escherichia) and Gram-positive strains (Bacillus, Listeria, Enterococcus and Staphilococcus) and results show a positive effect of cyclodextrin immobilization on the antimicrobial properties. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effect of high salt concentrations on the stability of immobilized lipases: Dramatic deleterious effects of phosphate anions

We have analyzed the effects of the buffer nature on the stability of immobilized lipases. Commercial phospholipase Lecitase Ultra (LU), lipase B from Candida antarctica (CALB) and lipase from Thermomyces lanuginosus (TLL) have been immobilized on octyl-glyoxyl agarose beads. The enzymes were readily inactivated using 4 M sodium phosphate but 6 M NaCl did not inactivate them. Using 2 M of sodium phosphate, the inactivation of the 3 immobilized enzymes still was very significant even at 25 °C but at lower rate than with higher phosphate concentration. Thermal stress inactivations of the immobilized enzymes revealed that even 100 mM sodium phosphate produced a significant decrease in enzyme stability; this effect was less pronounced for Lecitase but dramatic for CALB. While 6 M NaCl presented slightly positive (LU) or negative (TLL) effects on their thermal stabilities of, CALB was thermally stabilized under the same conditions. Results were very different using free enymes. Fluorescence spectroscopy revealed dramatic structural rearrangements of the immobilized enzymes in the presence of high phosphate concentration. From these results, the use of sodium phosphate does not seem to be recommended for studies on thermal stability of lipases, although this should be verified for each enzyme and immobilized preparation.     

Enantioselective acylation of (RS)-phenylethylamine catalysed by lipases

The enzymatic acylation of (RS)-phenylethylamine with different acyl donors catalysed by lipases, was studied in organic solvents with different hydrophobicities and in mixtures with ionic liquids ((ILs); [BMIm][BF₄], [BMIm][SCN], [BMIm][Cl] and [BMIm][PF₆]). Using lipases from Candida antarctica B (CAL-B) and from Aspergillus niger higher conversion degrees and E-values were obtained with ethyl acetate as the acyl donor. When CAL-B was used as the biocatalyst, in a two-phase system formed by [BMIm][X]/dichloromethane or [BMIm][X]/chloroform, the selectivity was better than that obtained in pure organic solvents. The selectivity was found to be related to individual anions in ILs. In this reaction, the ion effectiveness in enhancing the enzyme selectivity followed the series: Cl⁻ > SCN⁻ > BF₄⁻ > PF₆⁻ in mixtures with dichloromethane, and PF₆⁻ > BF₄⁻ > SCN⁻ > Cl⁻ in mixtures with chloroform.     

A density functional theory study on the interactions between dibenzothiophene and tetrafluoroborate-based ionic liquids

The interactions between dibenzothiophene (DBT) and N-butyl-N-methylimidazolium tetrafluoroborate ([BMIM][BF₄]), N-butyl-N-methylmorpholinium tetrafluoroborate ([Bmmorpholinium][BF₄]), N-butyl-N-methylpiperdinium tetrafluoroborate ([BMPiper][BF₄]), N-butyl-N-methylpyrrolidinium tetrafluoroborate ([BMPyrro][BF₄]), and N-butylpyridinium tetrafluoroborate ([BPY][BF₄]) were investigated using density functional theory approach. Geometric, electron, and topological properties were analyzed using natural bond orbital, atoms in molecules theory, and noncovalent interaction methods in order to understand intermolecular interactions between DBT and ionic liquids. The result shows that hydrogen bond and van der Waals interactions are widespread in all the ionic liquids-DBT systems. Ion-π interactions between DBT and cation or anion are also observed, while π⁺-π interactions are only found in the [BMIM][BF₄]-DBT and [BPY][BF₄]-DBT systems. The order of interaction energy is [BPY][BF4]-DBT > [BMIM][BF₄]-DBT >> [BMPiper][BF₄]-DBT > [BMPyrro][BF₄]-DBT > [BMmorpholinum][BF₄]-DBT. The energies between DBT and the two ionic liquids containing aromatic cations are significantly higher.     

A Brønsted acidic ionic liquid as an efficient and environmentally benign catalyst for biodiesel synthesis from free fatty acids and alcohols

Biodiesel could be synthesized using Brønsted acidic ionic liquid N-methyl-2-pyrrolidonium methyl sulfonate ([NMP][CH₃SO₃]) as a catalyst, specially with free long-chain fatty acids or their mixtures, as well as with low-molecular weight alcohols as substrates. This catalyst showed good catalytic and reusable performance under mild conditions and without any additional organic solvent. The ionic liquid could be reused eight times after the water in the ionic liquid was removed. The yields of fatty acid alkyl esters could reach between 93.6% and 95.3% after the esterifications were carried out at 70 °C for 8 h. Therefore, an efficient and environmentally friendly catalyst was provided for the synthesis of biodiesel from low-cost feedstocks such as waste oils.     

Catalytic activity of α-chymotrypsin in enzymatic peptide synthesis in ionic liquids

The catalytic activity of α-chymotrypsin in the enzymatic peptide synthesis of N-acetyl-l-tryptophan ethyl ester with glycyl glycinamide was examined in ionic liquids and organic solvents. The water content in 1-ethyl-3-methylimidazolium bis(fluorosulfonyl)imide ([emim][FSI]) affected the initial rates of peptide synthesis and hydrolysis. The activity of α-chymotrypsin was influenced by a kind of anions consisting of the same cation, [emim], when an ionic liquid was used as a solvent. The initial rate of peptide synthesis was improved 16-fold by changing from an organic solvent, acetonitrile, to an ionic liquid, [emim][FSI], at 25 °C. The activity of α-chymotrypsin in the peptide synthesis in [emim][FSI] was 17 times greater than that in acetonitrile at 60 °C, although the activity of α-chymotrypsin in the peptide synthesis gradually decreased with an increase in reaction temperature in [emim][FSI], similar to organic solvents. Moreover, α-chymotrypsin exhibited activity in [emim][FSI] and [emim][PF₆] at 80 °C.     

Ionic liquids as a reaction medium for lipase-catalyzed methanolysis of sunflower oil

Ionic liquids, 1-butyl-3-methyl imidazolium hexafluorophosphate ([BMIm][PF₆]) and 1-ethyl-3-methyl imidazolium hexafluorophosphate ([EMIm][PF₆]), were used for the methanolysis of sunflower oil using Candida antarctica lipase (Novozyme 435) and gave yields of fatty acid methyl esters at 98–99% within 10 h. The optimum conditions of methanolysis in hydrophobic ionic liquids are 2% (w/w) lipase, 1:1 (w/w) oil/ionic liquid and 1:8 (mol/mol) oil/methanol at 58–60°C. Methanolysis using hydrophilic ionic liquids, 3-methyl imidazolium tetrafluoroborate ([HMIm][BF₄]) and 1-butyl-3-methyl imidazolium tetrafluoroborate ([BMIm][BF₄]), gave very poor yields. A hydrophobic ionic liquid thus protects the lipase from methanol. Recovered ionic liquids and lipase were used for four successive reaction cycles without any significant loss of activity.     

Enzymatic hydrolysis of racemic ibuprofen esters using Rhizomucor miehei lipase immobilized on different supports

This research describes the immobilization of Rhizomucor miehei lipase (RML) and chemically aminated RML (NH₂-RML) on different supports including octyl-sepharose (octyl-RML), activated sepharose with cyanogen bromide (CNBr-RML and CNBr-NH₂-RML), glyoxyl sepharose (Gx-RML and Gx-NH₂-RML) and glyoxyl sepharose dithiothreitol (Gx-DTT-RML and Gx-DTT-NH₂-RML). The highest immobilization yield was achieved for octyl-RML (>98%) followed by CNBr-RML (88%). Octyl-RML had the most specific activity (13.6) among all derivatives. The other preparations had moderate activities likely because of chemical reaction during covalent attachment of the enzyme. The catalytic behavior of lipase immobilized in hydrolysis reactions was investigated using methyl, ethyl, propyl, butyl and isobutyl-ibuprofen esters and the influence of the alkyl chain and the alcoholic residue of the ester were studied. Butyl ester was the most interesting ester for carrying out hydrolysis. The highest enantioselectivity of enzyme (E = 8.8) was obtained with isooctane/sodium phosphate buffer pH 7.0 at temperature of 40 °C. Increasing temperature from 40 to 50 °C caused decreasing in enantioselectivities and conversions. Also esterification of ibuprofen was carried out in solvent systems containing isooctane and two ionic liquids (ILs); [BMIM][PF₆] and [BMIM][BF₄]. Poor conversions and enantioselectivities were observed during esterification in all solvents.     

Effects of cholinium-based ionic liquids on Aspergillus niger lipase: Stabilizers or inhibitors

Lipases are well-known biocatalysts used in several industrial processes/applications. Thus, as with other enzymes, changes in their surrounding environment and/or their thermodynamic parameters can induce structural changes that can increase, decrease, or even inhibit their catalytic activity. The use of ionic compounds as solvents or additives is a common approach for adjusting reaction conditions and, consequently, for controlling the biocatalytic activity of enzymes. Herein, to elucidate the effects of ionic compounds on the structure of lipase, the stability and enzymatic activity of lipase from Aspergillus niger in aqueous solutions (at 0.05, 0.10, 0.50, and 1.00 M) of six cholinium-based ionic liquids (cholinium chloride [Ch]Cl; cholinium acetate ([Ch][Ac]); cholinium propanoate ([Ch][Prop]); cholinium butanoate ([Ch][But]); cholinium pentanoate ([Ch][Pent]); and cholinium hexanoate ([Ch][Hex])) were evaluated over 24 hr. The enzymatic activity of lipase was maintained or enhanced in the lower concentrations of all the [Ch]⁺-ILs (below 0.1 M). [Ch][Ac] maintained the biocatalytic behavior of lipase, independent of the IL concentration and incubation time. However, above 0.1 M, [Ch][Pent] and [Ch][Hex] caused complete inhibition of the catalytic activity of the enzyme, demonstrating that the increase in the anionic alkyl chain length strongly affected the conformation of the lipase. The hydrophobicity and concentration of the [Ch]⁺-ILs play an important role in the enzyme activity, and these parameters can be controlled by adjusting the anionic alkyl chain length. The inhibitory effects of [Ch][Pent] and [Ch][Hex] may be of great interest to the pharmaceutical industry to induce pharmacological inhibition of gastric and pancreatic lipases.     

Stereoselective synthesis of (R)-1-chloro-3(3,4-difluorophenoxy)-2-propanol using lipases from Pseudomonas aeruginosa in ionic liquid-containing system

Direct transesterification of (R,S)-1-chloro-3-(3,4-difluorophenoxy)-2-propanol (rac-CDPP) (a key intermediate in the synthesis of the chiral drug (S)-lubeluzole) with vinyl butyrate by lipases from Pseudomonas aeruginosa (P. aeruginosa) MTCC 5113 was performed in hexane with ionic liquids (ILs) 1-butyl-3-methyl imidazolium hexafluorophosphate [BMIm][PF₆] and 1-butyl-3-methyl imidazolium tetrafluoroborate [BMIm][BF₄] as co-solvents. The maximum conversion (>49%) and enantiomeric excess (ee > 99.9%) was achieved in 6 h of incubation at 30 °C with [BMIm][PF₆] as co-solvent in a two-phase system. The enzyme was able to perform with the same specificity even at 60 °C in the presence of ILs. It was possible to use lipases repeatedly for more than 10 times while still maintaining absolute enantioselectivity and reactivity. Stability studies on lipases from P. aeruginosa in ILs revealed the fact that the enzyme constancy and the reactivity in catalyzing transesterification of rac-CDPP into (S)-1-chloro-3-(3,4-difluorophenoxy)-2-butanoate was of the order of [BMIm][PF₆] > [BMIm][BF₄] in two-phase system.     

Surface charge engineering of Thermomyces lanuginosus lipase improves enzymatic activity and biodiesel synthesis

Objectives

This study was aimed at engineering charged residues on the surface of Thermomyces lanuginosus lipase (TLL) to obtain TLL variant with elevated performance for industrial applications.

Results

Site-directed mutagenesis of eight charged amino acids on the TLL surface were conducted and substitutions on the negatively charged residues D111, D158, D165, and E239 were identified with elevated specific activities and biodiesel yields. Synergistic effect was not discovered in the double mutants, D111E/D165E and D165E/E239R, when compared with the corresponding single mutants. One TLL mutant, D165E, was identified with increased specific activity (456.60 U/mg), catalytic efficiency (k_cat/K_m: 44.14 s^?1 mM^?1), the highest biodiesel conversion yield (93.56%), and comparable thermostability with that of the TLL.

Conclusions

Our study highlighted the importance of surface charge engineering in improving TLL activity and biodiesel production, and the resulting TLL mutant, D165E, is a promising candidate for biodiesel industry.

     

Stability and kinetic behavior of immobilized laccase from Myceliophthora thermophila in the presence of the ionic liquid 1‐ethyl‐3‐methylimidazolium ethylsulfate

The use of ionic liquids (ILs) as reaction media for enzymatic reactions has increased their potential because they can improve enzyme activity and stability. Kinetic and stability properties of immobilized commercial laccase from Myceliophthora thermophila in the water‐soluble IL 1‐ethyl‐3‐methylimidazolium ethylsulfate ([emim][EtSO₄]) have been studied and compared with free laccase. Laccase immobilization was carried out by covalent binding on glyoxyl–agarose beads. The immobilization yield was 100%, and the activity was totally recovered. The Michaelis‐Menten model fitted well to the kinetic data of enzymatic oxidation of a model substrate in the presence of the IL [emim][EtSO₄]. When concentration of the IL was augmented, the values of V_max for free and immobilized laccases showed an increase and slight decrease, respectively. The laccase–glyoxyl–agarose derivative improved the laccase stability in comparison with the free laccase regarding the enzymatic inactivation in [emim][EtSO₄]. The stability of both free and immobilized laccase was slightly affected by small amounts of IL (<50%). A high concentration of the IL (75%) produced a large inactivation of free laccase. However, immobilization prevented deactivation beyond 50%. Free and immobilized laccase showed a first‐order thermal inactivation profile between 55 and 70°C in the presence of the IL [emim][EtSO₄]. Finally, thermal stability was scarcely affected by the presence of the IL. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:790–796, 2014     

Effect of mobile phase additives on resolution of some nucleic compounds in high performance liquid chromatography

Here we investigate the chromatographic behavior, with reversed-phase high performance liquid chromatography (RP-HPLC) of nucleic compounds (nucleobases, nucleosides, and nucleotides) on a C₁₈ column in several different mobile phase additives, including1-butyl-3-methylimidazolium tetrafuloroborate ([BMIm][BF₄]), 1-ethyl-3-methylimidazolium methylsulfate ([EMIm][MS]) ionic liquids, ammonium formate, and potassium phosphate. The effect of the alkyl group length, the imidazolium ring, and the ionic liquid's counterions on retention and resolution of the samples were tested. The results show the potential application of a used buffer system, ion pairing system, and ionic liquid as mobile phase additives in liquid chromatography resolution of nucleic compounds.     

Optimization of lipase-catalyzed glucose ester synthesis in ionic liquids

Lipase-catalyzed esterification of glucose with fatty acids in ionic liquids (ILs) mixture was investigated by using supersaturated glucose solution. The effect of ILs mixture ratio, substrate ratio, lipase content, and temperature on the activity and stability of lipase was also studied. The highest yield of sugar ester was obtained in a mixture of 1-butyl-3-methylimidazolium trifluoromethanesulfonate ([Bmim][TfO]) and 1-methyl-3-octylimidazolium bis[(trifluoromethyl)-sulfonyl]amide ([Omim][Tf₂N]) with a volume ratio of 9:1, while Novozym 435 (Candida antarctica type B lipase immobilized on acrylic resin) showed the optimal stability and activity in a mixture of [Bmim][TfO] and [Omim][Tf₂N] with a 1:1 volume ratio. Reuse of lipase and ILs was successfully carried out at the optimized reaction conditions. After 5 times reuse of Novozym 435 and ILs, 78% of initial activity was remained.