Glycerol acyl-transfer kinetics of a circular permutated Candida antarctica lipase B

Triacylglycerols containing a high abundance of unusual fatty acids, such as γ-linolenic acid, or novel arylaliphatic acids, such as ferulic acid, are useful in pharmaceutical and cosmeceutical applications. Candida antarctica lipase B (CALB) is quite often used for non-aqueous synthesis, although the wild-type enzyme can be rather slow with bulky and sterically hindered acyl donor substrates. The catalytic performance of a circularly permutated variant of CALB, cp283, with various acyl donors and glycerol was examined. In comparison to wild-type CALB, butyl oleate and ethyl γ-linolenate glycerolysis rates were 2.2- and 4.0-fold greater, respectively. Cp283 showed substrate inhibition by glycerol, which was not the case with the wild-type version. With either ethyl ferulate or vinyl ferulate acyl donors, cp283 matched the performance of wild-type CALB. Changes in active site accessibility resulting from circular permutation led to increased catalytic rates for bulky fatty acid esters but did not overcome the steric hindrance or energetic limitations experienced by arylaliphatic esters.     

Synthesis of ethyl ferulate in organic medium using celite-immobilized lipase

In the present work we have evaluated synthesis of ethyl ferulate by the esterification reaction of ferulic acid and ethanol catalyzed by a commercial lipase (Steapsin) immobilized onto celite-545 in a short period of 6 h in DMSO. The immobilized lipase was treated with cross-linking agent glutaraldehyde (1%; v/v). The optimum synthesis of ethyl ferulate was recorded at 45 °C, pH 8.5 and 1:1 ratio of ethanol and ferulic acid. Co²⁺, Ba²⁺and Pb²⁺ ions enhanced the synthesis of ethyl ferulate Hg²⁺, Cd³⁺and NH⁴⁺ ions had mild inhibitory effect. The celite-bound lipase produced 68 mM of ethyl ferulate under optimized reaction conditions.     

Evaluation of immobilized lipases on poly-hydroxybutyrate beads to catalyze biodiesel synthesis

Five microbial lipase preparations from several sources were immobilized by hydrophobic adsorption on small or large poly-hydroxybutyrate (PHB) beads and the effect of the support particle size on the biocatalyst activity was assessed in the hydrolysis of olive oil, esterification of butyric acid with butanol and transesterification of babassu oil (Orbignya sp.) with ethanol. The catalytic activity of the immobilized lipases in both olive oil hydrolysis and biodiesel synthesis was influenced by the particle size of PHB and lipase source. In the esterification reaction such influence was not observed. Geobacillus thermocatenulatus lipase (BTL2) was considered to be inadequate to catalyze biodiesel synthesis, but displayed high esterification activity. Butyl butyrate synthesis catalyzed by BTL2 immobilized on small PHB beads gave the highest yield (≈90 mmol L(-1)). In biodiesel synthesis, the catalytic activity of the immobilized lipases was significantly increased in comparison to the free lipases. Full conversion of babassu oil into ethyl esters was achieved at 72 h in the presence of Pseudozyma antarctica type B (CALB), Thermomyces lanuginosus lipase (Lipex(?) 100 L) immobilized on either small or large PHB beads and Pseudomonas fluorescens (PFL) immobilized on large PHB beads. The latter preparation presented the highest productivity (40.9 mg of ethyl esters mg(-1) immobilized protein h(-1)).     

Synthetic resin-bound truncated Candida antarctica lipase B for production of fatty acid alkyl esters by transesterification of corn and soybean oils with ethanol or butanol

A gene encoding a synthetic truncated Candida antarctica lipase B (CALB) was generated via automated PCR and expressed in Saccharomyces cerevisiae. Western blot analysis detected five truncated CALB variants, suggesting multiple translation starts from the six in-frame ATG codons. The longest open reading frame, which corresponds to amino acids 35-317 of the mature lipase, appeared to be expressed in the greatest amount. The truncated CALB was immobilized on Sepabeads? EC-EP resin and used to produce ethyl and butyl esters from crude corn oil and refined soybean oil. The yield of ethyl esters was 4-fold greater from corn oil than from soybean oil and was 36% and 50% higher, respectively, when compared to a commercially available lipase resin (Novozym 435) using the same substrates. A 5:1 (v/v) ratio of ethanol to corn oil produced 3.7-fold and 8.4-fold greater yields than ratios of 15:1 and 30:1, respectively. With corn oil, butyl ester production was 56% higher than ethyl ester production. Addition of an ionic catalytic resin step prior to the CALB resin increased yields of ethyl esters from corn oil by 53% compared to CALB resin followed by ionic resin. The results suggest resin-bound truncated CALB has potential application in biodiesel production using biocatalysts.     

Solvent-free synthesis of glyceryl ferulate using a commercial microbial lipase

A process was optimized for the enzymatic synthesis of glyceryl ferulate with a yield of up to 96% using a vacuum-rotary evaporation strategy under following conditions: 15 mmol glycerol, 1.5 mmol ethyl ferulate, 170 mg Candida antarctica lipase, at 60°C for 10 h and under a vacuum of 10 mm Hg. The immobilized lipase can be used 10 times.     

Decarboxylative aldol reaction catalysed by lipases and a protease in organic co-solvent mixtures and nearly anhydrous organic solvent media

Abstract

The effects of the choice of lipase, reaction medium, immobilization, presence of additives and temperature on conversion and stereoselectivity during a lipase catalysed decarboxylative aldol reaction were examined. It was shown that Candida antarctica lipase B (CALB) catalysed a decarboxylative aldol reaction between 4-nitrobenzaldehyde and ethyl acetoacetate in a 60% acetonitrile–40% aqueous buffer co-solvent mixture. Interestingly, free and immobilized forms of CALB showed opposite enantioselectivity in this media. The addition of 30 mol% imidazole increased the reaction rate from 8.5 to 55.7 μM min^??1 mg^??1. A 98% conversion could be achieved in 14 h (instead of 168 h) by adding imidazole. Other lipases also catalysed this reaction in different reaction media to a varying extent. With Mucor javanicus lipase in 30% DMSO, 20% enantiomeric excess (ee) of the (R)-product was observed. CALB also catalysed this reaction in nearly anhydrous acetonitrile. In the presence of cross-linked protein coated microcrystals of CALB, 90% conversion was obtained in this media in 24 h. A commercially available protease, alcalase, was also found to catalyse this reaction. While low water media gave poor conversion, the reaction in aqueous–60% acetonitrile co-solvent mixture gave 99% conversion in 72 h, provided imidazole was used as an additive.     

Lipase catalyzed reaction of L-ascorbic acid with cinnamic acid esters and substituted cinnamic acids

To perform the lipase-catalyzed synthesis of L-ascorbic acid derivatives from plant-based compounds such as cinnamic and ferulic acid under mild reaction conditions, the activities of immobilized Candida ntarctica lipase with different cinnamic acid esters and substituted cinnamic acids were compared. As a result, immobilized C. ntarctica lipase was found to prefer vinyl cinnamic acid to other esters such as allyl-, ethyl-, and isobutyl cinnamic acids as well as substituted cinnamic acids such as p-coumaric acid, caffeic acid, ferulic acid, and sinapic acid. Based on these results, large-scale synthesis of 6-O-cinnamyl-L-ascorbic acid ester was performed using immobilized C. ntarctica lipase in dry organic solvent, resulting in 68% yield (493 mg) as confirmed by ¹³C-NMR.     

A novel,two consecutive enzyme synthesis of feruloylated monoacyl- and diacyl-glycerols in a solvent-free system

Feruloylated mono- and di-acylglycerols were synthesized in a two step reaction: ethyl ferulate was first transesterified with glycerol and then this was esterified with oleic acid. The yield of the combined feruloylated mono- and di-acylglycerols in the second reaction reached 96% when esterification of 0.3 g transesterification products with 2.22 g oleic acid was catalyzed with 0.25 g Candida antarctica lipase at 60°C under a vaccum of 10 mmHg for 1.33 h.     

Synthesis of glyceryl ferulate by immobilized ferulic acid esterase

Glyceryl ferulate was synthesized by the condensation of ferulic acid with glycerol using Pectinase PL “Amano” from Aspergillus niger, which contained ferulic acid esterase, to improve the water-solubility of ferulic acid. The optimum reaction medium was glycerol/0.1 M acetate buffer, pH 4.0, (98:2 v/v). The enzyme immobilized onto Chitopearl BCW3003 exhibited the highest activity among the those immobilized onto various kinds of Chitopearl BCW resins. The optimum temperature for the immobilized enzyme was 50°C, and it could be reused at least five times without a significant loss in activity for the synthesis of glyceryl ferulate in batch reaction. Storage of the reaction mixture at 25°C improved the molar fraction of glyceryl ferulate relative to the dissolved ferulic residues.     

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     

南极假丝酵母脂肪酶B的酿酒酵母表面展示及其催化己酸乙酯的合成

从南极假丝酵母(Candida antarctica)基因组克隆得到南极假丝酵母脂肪酶B(Candida antarctica Lipase B, CALB)全基因片段, 利用连接肽celA Linker将CALB与酿酒酵母细胞表面展示蛋白a-凝集素的C端连接融合, 构建表面展示载体pICAS-celAL-CALB, 转化酵母后获得重组酵母菌Saccharomyces cerevisiae pICAS-celAL-CALB。该重组酵母菌经葡萄糖诱导表达及分析, 表明CALB已在酿酒酵母细胞表面成功展示, 水解活力达26.26 u/(g·dry cell)。重组酵母菌经冻干能有效地实现在非水相中全细胞催化己酸和乙醇酯化合成己酸乙酯。反应物己酸与乙醇的摩尔比为1:1.25, 己酸乙酯的产率为98.0%, 具有较好的操作稳定性。     

Continuous synthesis of alkyl ferulate by immobilized Candida antarctica lipase at high temperature

1-Pentyl, 1-hexyl and 1-heptyl ferulates were continuously synthesized at 60–90°C using a reactor system in which a column packed with ferulic acid powders and another column packed with immobilized Candida antarctica lipase particles were connected in series. Conversions greater than 0.9 were achieved for the synthesis of the 1-hexyl and 1-heptyl ferulates at 90°C. The system could be stably operated for the 1-heptyl ferulate synthesis at 90°C for at least two weeks.     

Development of an amphiphilic matrix for immobilization of <Emphasis Type="Italic">Candida antartica</Emphasis> lipase B for biodiesel production

Biodiesel has been greatly interested as an alternative fuel and is produced by a transesterification reaction of oil with alcohol. Recently, microbial lipases have been used for biodiesel production. Among the microbial lipase, immobilized Candida antartica lipase B (CALB) is the most widely used. However, CALB is unstable and shows low catalytic efficiency in the reaction media because the reaction media contains a high concentration of methanol and the lipase is also inhibited by the by-product glycerol. In this study, to overcome these limitations, we developed an amphiphilic matrix to immobilize CALB. The immobilized lipase in an amphiphilic matrix with 80% ethyltrimethoxysilane (ETMS) in tetramethoxysilane (TMOS) and pretreated with oil showed the highest specific activity and biodiesel conversion ratio; about 90% biodiesel conversion in 24 h at an initial molar ratio of 1: 1 (oil: methanol) with stepwise methanol feeding in order to adjust the net molar ratio to be 1: 3.     

Lipase-catalyzed synthesis of vitamin C fatty acid esters

Fatty acid esters of vitamin C (ascorbic acid) where synthesized in a mainly solid-phase system in the presence of small amounts of organic solvent (acetone or t-butanol) catalyzed by immobilized lipase B from Candida antarctica.Highest reaction rates and yields of isolated products were obtained using fatty acid vinyl esters, e.g., 6-O-palmitoyl-l-ascorbic acid was obtained in 91% isolated yield after 48 h. As vitamin C and its esters are very sensitive to oxidation, a mild extraction method for the isolation of reaction products was developed.     

Displaying Candida antarctica lipase B on the cell surface of Aspergillus niger as a potential food-grade whole-cell catalyst

Aspergillus niger is a recognized workhorse used to produce food processing enzymes because of its extraordinarily high protein-producing capacity. We have developed a new cell surface display system de novo in A. niger using expression elements from generally recognized as safe certified microorganisms. Candida antarctica lipase B (CALB), a widely used hydrolase, was fused to an endogenous cell wall mannoprotein, CwpA, and functionally displayed on the cell surface. Localization of CALB was confirmed by enzymatic assay and immunofluorescence analysis using laser scanning confocal microscopy. After induction by maltose for 45 h, the hydrolytic activity and synthesis activity of A. niger mycelium-surface displayed CALB (AN-CALB) reached 400 and 240 U/g dry cell, respectively. AN-CALB was successfully used as a whole-cell catalyst for the enzymatic production of ethyl esters from a series of fatty acids of different chain lengths and ethanol. In a solvent-free system, AN-CALB showed great synthetic activity and afforded high substrate mole conversions, which amounted to 87 % for ethyl hexanoate after 2 h, 89 % for ethyl laurate after 2 h, and 84 % for ethyl stearate after 3 h. These results suggested that CwpA can act as an efficient anchoring motif for displaying enzyme on A. niger, and AN-CALB is a robust, green, and cost-effective alternative food-grade whole-cell catalyst to commercial lipase.     

Cross-linked enzyme aggregates of recombinant Candida antarctica lipase B for the efficient synthesis of olvanil,a nonpungent capsaicin analogue

Despite the proven therapeutic role of capsaicin in human health, its usage is still hampered by its high pungency. In this sense, nonpungent capsaicin analogues as olvanil are a feasible alternative to the unpleasant sensations produced by capsaicin while maintaining a similar pharmacological profile. Olvanil can be obtained by a lipase-catalyzed chemoenzymatic process. In the present work, recombinant Candida antarctica lipase B (CALB) was expressed in Pichia pastoris and subsequently immobilized by cross-linked enzyme aggregate (CLEA) methodology for the synthesis of olvanil. The CALB-CLEAs were obtained directly from the fermentation broth of P. pastoris without any purification step in order to assess the role of the contaminant proteins of the crude extract as co-feeders. The CALB-CLEAs were also bioimprinted to enhance the catalytic performance in olvanil synthesis. When CALB was precipitated with isopropanol, the obtained CALB-CLEAs exhibited the highest activity in the synthesis of olvanil, regardless of the glutaraldehyde concentration. The maximum product synthesis was found at 72 hr obtaining 6.8 g L⁻¹ of olvanil with a reaction yield of 16%. When CALB was bioimprinted with olvanil, the synthesis was enhanced 1.3 times, reaching 10.7 g L⁻¹ of olvanil at 72 hr of reaction with a reaction yield of 25%. Scanning electron microscopy images indicated different morphologies of the CLEAs depending on the precipitating agent and the template used for bioimprinting. Recombinant CALB-CLEAs obtained directly from the fermentation broth are a suitable alternative to commercial enzymatic preparations for the synthesis of olvanil in organic medium.     

Novel selective biocatalytic deacetylation studies on dihydropyrimidinones

Five racemic ethyl 4-(3/4-acetoxyphenyl)-6-methyl-3,4-dihydropyrimidin-2-one-5-carboxylates have been synthesized by acetylation of corresponding hydroxy analogues, which in turn have been synthesized under microwave condition by multi-component Biginelli cyclocondensation of ethyl acetoacetate, urea and the corresponding hydroxybenzaldehydes in the presence of ferric chloride. These dihydropyrimidinones have been subjected to biocatalytic resolution using acetoxyl group on the phenyl ring as remote handle; Novozyme^®-435, an immobilized lipase from Candida antarctica in THF:DIPE has been found to catalyze the deacetylation reactions in an enantioselective fashion.     

Multipoint covalent immobilization of microbial lipase on chitosan and agarose activated by different methods

In this work Candida antarctica lipase type B (CALB) was immobilized on agarose and chitosan. The influence of activation agents (glycidol, glutaraldehyde and epichlorohydrin) and immobilization time (5, 24 and 72 h) on hydrolytic activity, thermal and alkaline stabilities of the biocatalyst was evaluated. Protein concentration and enzymatic activity in the supernatant were determined during the immobilization process. More active derivatives were attained when the enzymatic extract was first purified through dialysis. The highest activities achieved were: for agarose-glyoxyl (with glycidol), 845 U/g of gel, after 72 h of immobilization; for chitosan-glutaraldehyde and agarose-glutaraldehyde, respectively, 1209 U/g of gel and 2716 U/g of gel, after 5 h of immobilization. Thermal stability was significantly increased, when compared to the soluble enzyme: 20-fold for agarose-glyoxyl (with glycidol)-CALB, 18-fold for chitosan-glutaraldehyde-CALB and 21-fold for agarose-glutaraldehyde. The best derivative, 58-fold more stable than the soluble enzyme, was obtained when CALB was immobilized on chitosan activated in two steps, using glycidol and glutaraldehyde, 72 h immobilization time. The stabilization degree of the derivative increased with the immobilization time, an indication that a multipoint covalent attachment between enzyme and the support had really occurred.     

Enzymatic Synthesis of Cinnamic Acid Derivatives

Using Novozym 435 as catalyst, the syntheses of ethyl ferulate (EF) from ferulic acid (4-hydroxy 3-methoxy cinnamic acid) and ethanol, and octyl methoxycinnamate (OMC) from p-methoxycinnamic acid and 2-ethyl hexanol were successfully carried out in this study. A conversion of 87% was obtained within 2 days at 75 °C for the synthesis of EF. For the synthesis of OMC at 80 °C, 90% conversion can be obtained within 1 day. The use of solvent and high reaction temperature resulted in better conversion for the synthesis of cinnamic acid derivatives. Some cinnamic acid esters could also be obtained with higher conversion and shorter reaction times in comparison to other methods reported in the literature. The enzyme can be reused several times before significant activity loss was observed. Revisions requested 10 January 2006; Revisions received 17 January 2006     

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.