Amino acid modified chitosan beads: Improved polymer supports for immobilization of lipase from Candida rugosa

Amino acid modified chitosan beads (CBs) for immobilization of lipases from Candida rugosa were prepared by activation of a chitosan backbone with epichlorohydrin followed by amino acid coupling. The beads were analyzed by elemental analysis and solid state NMR with coupling yields of the amino acids ranging from 15 to 60%. The immobilized lipase on unmodified chitosan beads showed the highest immobilization yield (92.7%), but its activity was relatively low (10.4%). However, in spite of low immobilization yields (15–50%), the immobilized lipases on the amino acid modified chitosan beads showed activities higher than that of the unmodified chitosan beads, especially on Ala or Leu modified chitosan beads (Ala-CB or Leu-CB) with 49% activity for Ala-CB and 51% for Leu-CB. The immobilized lipases on Ala-CB improved thermal stability at 55 °C, compared to free and immobilized lipases on unmodified chitosan beads and the immobilized lipase on Ala-CB retained 93% of the initial activity when stored at 4 °C for 4 weeks. In addition, the activity of the immobilized lipase on Ala-CB retained 77% of its high initial activity after 10 times of reuse. The kinetic data (k_cat/K_m) supports that the immobilized lipase on Ala-CB can give better substrate specificity than the unmodified chitosan beads.     

Recombinant lipase from Candida rugosa for regioselective hydrolysis of peracetylated nucleosides. A comparison with commercial non-recombinant lipases

Abstract

Commercial lipases from the yeast Candida rugosa have been compared with two recombinant C. rugosa lipases, rCRL1 and rCRL1lid3, with respect to their immobilization and exploitation in biotransformations aimed at the synthesis of pyrimidine nucleosides. Immobilization on octyl-agarose and decaoctyl-Sepabeads but not on Eupergit^® C gave comparable results to commercial lipases for rCRL1, while only a low percentage (12%) of rCRL1lid3 was efficiently immobilized. When immobilized on decaoctyl-Sepabeads, rCRL1 showed a markedly higher stability to chemical inactivation, since it could maintain 100% activity after 180 h incubation in 30% (v/v) acetonitrile. Hydrolysis of peracylated uridine and cytidine and their fluorinated counterparts proceeded with high regioselectivity and good yield, and even improved when rCRL1 was immobilized on decaoctyl-Sepabeads.     

Immobilized, thermostable S- and F-forms of the extracellular invertase from Candida utilis can hydrolyse sucrose up to 100 °C

When grown on a sucrose-containing medium, Candida utilis synthesizes and secretes two invertases: one of molecular size of 280 kDa (the S-form – Slow-migrating) and a new form of Mr of 62 kDa (the F-form – Fast-migrating). Prior to immobilization, purification of S- and F-forms of invertase increased the immobilization yield to 89–100%, in comparison with that of crude invertase preparation (52%). The immobilized purified S- and F-form of invertase remained partially active after 15 min at 100 °C; the F-form retained almost 30% of its maximum activity. The immobilized S-form or F-form of invertase almost completely inverted (95% hydrolysis) 60% (w/v) sucrose over 5 h continuous reaction at 80 °C. Moreover, at 90 °C the immobilized F-form hydrolysed 70% of 60% (w/v) sucrose over 5 h, while the capability of the immobilized S-form of inverting sucrose over 5 h reaction decreased from 80% to 45%.     

Synthesis of calix[n]arene-based silica polymers for lipase immobilization

The objective of this study was to prepare new calix[n]arene-based silica polymers for immobilization of Candida rugosa lipase. The amino functionalized calix[4]arene (C4P), calix[6]arene (C6P) and calix[8]arene (C8P)-based silica polymers were used for the covalent attachment of C. rugosa lipase using glutaraldehyde as a coupling agent. The characterization of synthesized CnP polymers and immobilized lipases were made by Fourier transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA) and scanning electron microscope (SEM) techniques. The hydrolytic activities of immobilized lipases (CnP-L) were evaluated and compared with the free enzyme. The activity recovery of immobilized CRL (C. rugosa lipase) based on the carrier C4P, C6P and C8P reaches 74.6%, 68.5% and 51.4%, respectively. The optimal pH and temperature region of the immobilized lipases for the hydrolysis of p-NPP were 7.0 and 50 °C. Nevertheless, the immobilized lipase has good stability, adaptability and reusability in comparison with the free enzyme.     

A chemo-biocatalytic approach in the synthesis of β-O-naphtylmethyl-N-peracetylated lactosamine

A chemo-enzymatic approach combining an enzymatic regioselective hydrolysis of peracetylated N-acetyl-α-d-glucosamine (1) with a mild controlled acyl migration led to 2-acetamido-2-deoxy-1,3,6-tri-O-acetyl-α-d-glucopyranose, which was further used in a glycosylation reaction in the synthesis of β-O-naphtylmethyl-N-peracetylated lactosamine.Candida rugose lipase (CRL) immobilized on octyl-agarose and modified by covering it with polyethyleneimine was the best catalyst in terms of activity, stability and regioselectivity in the hydrolysis of 1, producing the deacetylation in C-6 in 95% overall yield. Other immobilized lipases were not specific or with a very low activity towards the hydrolysis of 1.An acyl chemical migration by incubation of the deacetylated C-6 derivative at pH 8.5, 4 °C, and 10–20% acetonitrile permitted to obtain up to 75% overall yield of the 4-OH derivative product. This molecule was successfully applied in a glycosylation reaction to get the peracetylated α-d-lactosamine and finally, the peracetyl-β-O-naphtylmethyl-lactosamine derivative in 20% overall yield.     

Enhancing the thermal stability of lipases through mutagenesis and immobilization on zeolites

The hydrolysis reaction of p-nitrophenyl butyrate catalyzed by lipases was followed with in situ UV/vis diode array spectrophotometry. Five enzymes - Candida antarctica lipase B and Fusarium solani pisi cutinase wild-type and three single-mutation variants - were tested as catalysts in homogeneous conditions and immobilized on zeolite NaY, on a polyacrylate support and as cross-linked aggregates. Using deconvolution techniques and kinetic modeling, the thermal stability of the different biocatalysts was compared in operational conditions and the results were supported by steady-state enzyme fluorescence measurements. We concluded that both the mutagenesis and the immobilization on zeolite NaY had a positive effect on the thermal stability of F. solani pisi cutinase.     

Production of structured triacylglycerols by acidolysis catalyzed by lipases immobilized in a packed bed reactor

The aim of this work was to produce structured triacylglycerols (STAGs), with caprylic acid located at positions 1 and 3 of the glycerol backbone and docosohexaenoic acid (DHA) at position 2, by acidolysis of tuna oil and caprylic acid (CA) catalyzed by lipases Rd, from Rhizopus delemar, and Palatase 20000L from Mucor miehei immobilized on Accurel MP1000 in a packed bed reactor (PBR), working in continuous and recirculation modes. First, different lipase/support ratios were tested for the immobilization of lipases and the best results were obtained with ratios of 0.67 (w/w) for lipase Rd and 6.67 (w/w) for Palatase. Both lipases were stable for at least 4 days in the operational conditions. In the storage conditions (5 °C) lipases Rd and Palatase maintained constant activity for 5 months and 1 month, respectively.These catalysts have been used to obtain STAGs by acidolysis of tuna oil and CA in a PBR operating with recirculation of the reaction mixture through the lipase bed. Thus, STAGs with 52–53% CA and 14–15% DHA were obtained. These results were the basis for establishing the operational conditions to obtain STAGs operating in continuous mode. These new conditions were established maintaining constant intensity of treatment (IOT, lipase amount × reaction time/oil amount). In this way STAGs with 44–50% CA and 17–24% DHA were obtained operating in continuous mode. Although the compositions of STAGs obtained with both lipases were similar, Palatase required an IOT about four times higher than lipase Rd.To separate the acidolysis products (free fatty acids, FFAs, and STAGs) an extraction method of FFAs by water–ethanol solutions was tested. The following variables were optimized: water/ethanol ratio (the best results were attained with a water/ethanol ratio of 30:70, w/w), the solvent/FFA–STAG mixture ratio (3:1, w/w) and the number of extraction steps (3–5). In these conditions highly pure STAGs (93–96%) were obtained with a yield of 85%. The residual FFAs can be eliminated by neutralization with a hydroethanolic KOH solution to obtain pure STAGs. The positional analysis of these STAGs, carried out by alcoholysis catalyzed by lipase Novozym 435, has shown that CA represents 55% of fatty acids located at positions 1 and 3 and DHA represents 42% of fatty acids at position 2.     

Degradation of n-alkanes by Candida parapsilosis and Penicillium frequentans immobilized on granular clay and aquifer sand

Summary The immobilization intensity of cells of Penicillium frequentans and Candida parapsilosis on materials such as granular clay, granular clay + aquifer sand and aquifer sand alone, was followed by scanning electron microscopy (SEM). The results demonstrate that the granular clay was the best adsorbent for both organisms, followed by the mixture of both granular clay and aquifer sand. Poor adhesion of cells was detected on using aquifer sand alone with C. parapsilosis.The highest degree of degradation of the alkane mixture (C₁₂–C₁₈) used was achieved by cells immobilized on granular clay, followed by those cells adsorbed on clay and sand. The weakest degradation was observed with cells immobilized on the sand alone.     

Regioselective enzymatic hydrolysis of acetylated pyranoses and pyranosides using immobilised lipases. An easy chemoenzymatic synthesis of alpha- and beta-D-glucopyranose acetates bearing a free secondary C-4 hydroxyl group

Protected sugars with only one free hydroxyl group are useful building blocks for the synthesis of a large number of glycoderivatives. In order to avoid the problems of the classical chemical synthesis, we studied the regioselective enzymatic hydrolysis of different fully acetylated glycopyranoses and glycopyranosides. The main challenge was to obtain the hydrolysis of the substrates in only one position, with high regioselectivity, while avoiding any further hydrolysis towards partially acetylated sugars. Candida rugosa (CRL) and Pseudomonas fluorescens (PFL) lipases (EC 3.1.1.3) immobilised on octyl agarose afforded regioselective hydrolysis only in the 6- and 1-positions, respectively. Furthermore, a new one-pot chemoenzymatic approach has been developed in order to obtain alpha- and beta-protected glucopyranoses bearing a free secondary C-4 hydroxyl group. For instance, 1,2,3,6-tetra-O-acetyl-alpha-D-glucopyranose was easily synthesised in good overall yield (70%) starting from 1,2,3,4,6-penta-O-acetyl-alpha-D-glucopyranose by regioselective enzymatic hydrolysis in the 6-position, catalysed by CRL, followed by a temperature- and pH-controlled acyl migration.     

Enhancement of activity and selectivity of Candida rugosa lipase and Candida antarctica lipase A by bioimprinting and/or immobilization for application in the selective ethanolysis of fish oil

Candida rugosa lipase (CRL) and Candida antarctica lipase A (CALA) with improved activity and selectivity were prepared for use in organic solvent media. CRL bioimprinted with fatty acids exhibited eightfold enhanced transesterification activity in hexane. Combination of bioimprinting and coating with lecithin or with immobilization did not improve the activity further. CALA was immobilized with and without bioimprinting, none of which improved the activity. All modified lipases were tested for selective ethanolysis of fish oil to concentrate omega-3 polyunsaturated fatty acids (PUFA). None of the preparations, except the immobilized ones catalysed ethanolysis. Immobilized CRL-catalyzed ethanolysis giving 27% (v/v) ethyl esters (EE) in 48 h, of which 43 mol% was oleic acid but no PUFA was detected in the EE fraction. Fatty acid selectivity of CALA was significantly improved by immobilization combined with bioimprinting, resulting in 5.5-fold lower omega-3 PUFA in EE.     

Silanized palygorskite for lipase immobilization

Lipase from Candida lipolytica has been immobilized on 3-aminopropyltriethoxysilane-modified palygorskite support. Scanning electron micrographs proved the covalently immobilization of C. lipolytica lipase on the palygorskite support through glutaraldehyde. Using an optimized immobilization protocol, a high activity of 3300 U/g immobilized lipase was obtained. Immobilized lipase retained activity over wider ranges of temperature and pH than those of the free enzyme. The optimum pH of the immobilized lipase was at pH 7.0–8.0, while the optimum pH of free lipase was at 7.0. The retained activity of the immobilized enzyme was improved both at lower and higher pH in comparison to the free enzyme. The immobilized enzyme retained more than 70% activity at 40 °C, while the free enzyme retained only 30% activity. The immobilization stabilized the enzyme with 81% retention of activity after 10 weeks at 30 °C whereas most of the free enzyme was inactive after a week. The immobilized enzyme retains high activity after eight cycles. The kinetic constants of the immobilized and free lipase were also determined. The K_m and V_max values of immobilized lipase were 0.0117 mg/ml and 4.51 μmol/(mg min), respectively.     

Transformation of arachidonic acid to 19-hydroxy- and 20-hydroxy-eicosatetraenoic acids using Candida bombicola

Candida bombicola (ATCC 22214) and C. apicola (ATCC 96134), grown on glucose (100 g l^–1) and arachidonic acid (5Z, 8Z, 11Z, 14Z-eicosatetraenoic acid; AA), 1.25 g l^–1, synthesized sophorolipid up to 0.93 g l^–1. Acid hydrolysis of sophorolipid yielded 19-hydroxy-5Z, 8Z, 11Z, 14Z-eicosatetraenoic acid (19-HETE) and 20-hydroxy-5Z, 8Z, 11Z, 14Z-eicosatetraenoic acid (20-HETE) which were identified by TLC and GC-MS; the ratio of synthesis was 73:27, respectively. Conversion of AA by immobilized Candida bombicola, suspended in beads of 2% (w/v) calcium alginate for 96 h, gave an 83% conversion of 1 g AA l^–1 to 19- and 20-HETE. There was no significant loss in the efficiency of the immobilized cells after ten uses.     

Meetings & Symposia

Abstract

Porcine pancreatic lipase (PPL) and Candida cylindracea lipase (CCL) were immobilized on Celite and Amberlite IRA 938 by deposition from the aqueous solution by the addition of hexane. The influence of the immobilization on the activities of the immobilized lipase derivatives has been studied. The immobilized lipases were used in synthesis of pentyl isovalerates. Various reaction parameters affecting the synthesis of pentyl isovalerates were investigated. The reaction rates were compared with the rates of esterification with free lipases. The immobilized lipases were found to be very effective in the esterification reaction. The lipases immobilized on Celite 545 exhibited better operational stabilities than that of immobilized on Amberlite IRA‐938.     

Immobilization of lipases on alkyl silane modified magnetic nanoparticles: effect of alkyl chain length on enzyme activity

Background

Biocatalytic processes often require a full recycling of biocatalysts to optimize economic benefits and minimize waste disposal. Immobilization of biocatalysts onto particulate carriers has been widely explored as an option to meet these requirements. However, surface properties often affect the amount of biocatalysts immobilized, their bioactivity and stability, hampering their wide applications. The aim of this work is to explore how immobilization of lipases onto magnetite nanoparticles affects their biocatalytic performance under carefully controlled surface modification.

Methodology/Principal Findings

Magnetite nanoparticles, prepared through a co-precipitation method, were coated with alkyl silanes of different alkyl chain lengths to modulate their surface hydrophobicity. Candida rugosa lipase was then directly immobilized onto the modified nanoparticles through hydrophobic interaction. Enzyme activity was assessed by catalytic hydrolysis of p-nitrophenyl acetate. The activity of immobilized lipases was found to increase with increasing chain length of the alkyl silane. Furthermore, the catalytic activities of lipases immobilized on trimethoxyl octadecyl silane (C18) modified Fe₃O₄ were a factor of 2 or more than the values reported from other surface immobilized systems. After 7 recycles, the activities of the lipases immobilized on C18 modified nanoparticles retained 65%, indicating significant enhancement of stability as well through hydrophobic interaction. Lipase immobilized magnetic nanoparticles facilitated easy separation and recycling with high activity retaining.

Conclusions/Significance

The activity of immobilized lipases increased with increasing alkyl chain length of the alkyl trimethoxy silanes used in the surface modification of magnetite nanoparticles. Lipase stability was also improved through hydrophobic interaction. Alkyl silane modified magnetite nanoparticles are thus highly attractive carriers for enzyme immobilization enabling efficient enzyme recovery and recycling.     

Novel chitosan membranes as support for lipases immobilization: Characterization aspects

Membranes of chitosan (QS), chitosan treated with glutaraldehyde (QGA) and chitosan crown ether (QCE) were utilized as carriers for immobilization of Candida antarctica and Candida rugosa lipases. Membrane supports were characterized by several techniques (Raman spectroscopy, elemental analysis by CHN determination and Energy Dispersive X-ray (EDX), water sorption isotherms, and surface area from nitrogen sorption data). To verify the presence of enzymes, some of these techniques were also used for lipase on chitosan biocatalytic systems. Measurements of protein load from Biuret assays and catalytic activity in esterification in nonaqueous media were also made for the immobilized enzymes. Sorption isotherms at 20, 30, 40 and 50 °C for QS, QGA and QCE supports were fitted to the Guggenheim, Anderson and Böer model. GAB monolayer moisture parameter, Xm, varied between 0.029 and 0.051 for QS, 0.039 and 0.058 for QGA and 0.039–0.075 g of water g⁻¹ s.s. for QCE membranes. Elemental analysis and Raman spectra measurements of the lipase, supports and immobilized lipase systems gave evidence of the presence of enzymes on supports. Chitosan supports with internal surface area (m2 g⁻¹) among 3.31 and 1.26 were obtained. Regardless of these low values, acceptable protein load (0.61 to 3.21%) and esterification initial rates were achieved (0.88–2.75 mmol min⁻¹ g of protein⁻¹).     

Screening of lipases for regioselective hydrolysis of peracetylated β-monosaccharides

The monodeacetylation of peracetylated-β-d-galactose (1) and peracetylated N-acetyl-β-d-glucosamine (2) by different lipases is here described. Lipases from different sources in an immobilized form were evaluated to find those that offer the higher activity and regioselectivity in the reactions. In the hydrolysis of 1, the lipase from Aspergillus niger was the most active one, although it hydrolyzed the anomeric position. Using the lipase from Candida rugosa, 30% yield of the corresponding 6-OH isomer was achieved. On the other hand, in the hydrolysis of 2, the lipase from A. niger was the most active and regioselective catalyst, producing more than 75% of the 6-OH derivative product.     

Enhancement of the activity and enantioselectivity of lipase in organic systems by immobilization onto low-cost support

Lipase from Arthrobacter sp. was immobilized onto low-cost diatomite materials using different protocols for the resolution of 4-hydroxy-3-methyl-2-(2-propenyl)-2-cyclopenten-1-one (HMPC) by asymmetric acylation. The support surface was grafted various functional groups including methacryloxypropyl, vinyl, octyl, dodecyl and γ-(aminopropyl)-glutaraldehyde. These modifications resulted in various mechanisms during the immobilization and thus introduced different characteristics to the prepared lipases. The interfacially adsorbed lipase onto dodecyl-modified support exhibited both higher activity and stability among these immobilized preparations. The modified enzyme-aggregate coating method was performed based on interfacial adsorption in our work, and the characteristics of this immobilized lipase were investigated and compared with those by cross-linking and interfacial adsorption methods. It was shown that the enzyme-aggregate coated lipase yielded the highest activity with a recovered activity of 8.5-fold of the free enzyme, and the highest operational stability with 85% of initial activity remained after 10 recycles. Excellent enantioselectivity (E ≥ 400, with e.e. = 99% of S-HMPC) was obtained for most lipase preparations in our paper (E = 85 for the free enzyme).     

A single step purification, immobilization, and hyperactivation of lipases via interfacial adsorption on strongly hydrophobic supports

A number of bacterial lipases can be immobilized in a rapid and strong fashion on octyl-agarose gels (e.g., lipases from Candida antarctica, Pseudomonas fluorescens, Rhizomucor miehei, Humicola lanuginosa, Mucor javanicus, and Rhizopus niveus). Adsorption rates in absence of ammonium sulfate are higher than in its presence, opposite to the observation for typical hydrophobic adsorption of proteins. At 10 mM phosphate, adsorption of lipases is fairly selective allowing enzyme purification associated with their reversible immobilization. Interestingly, these immobilized lipase molecules show a dramatic hyperactivation. For example, lipases from R. niveus, M. miehei, and H. lanuginosa were 6-, 7-, and 20-fold more active than the corresponding soluble enzymes when catalyzing the hydrolysis of a fully soluble substrate (0.4 mM p-nitrophenyl propionate). Even higher hyperactivations and interesting changes in stereospecificity were also observed for the hydrolysis of larger soluble chiral esters (e.g. (R,S)-2-hydroxy-4-phenylbutanoic ethyl ester). These results suggest that lipases recognize these "well-defined" hydrophobic supports as solid interfaces and they become adsorbed through the external areas of the large hydrophobic active centers of their "open and hyperactivated structure". This selective interfacial adsorption of lipases becomes a very promising immobilization method with general application for most lipases. Through this method, we are able to combine, via a single and easily performed adsorption step, the purification, the strong immobilization, and a dramatic hyperactivation of lipases acting in the absence of additional interfaces, (e.g., in aqueous medium with soluble substrate). Copyright 1998 John Wiley & Sons, Inc.     

Fermentation of xylose and rice straw hydrolysate to ethanol byCandida shehatae NCL-3501

Candida shehatae NCL-3501 utilized glucose and xylose efficiently in batch cultures. The specific rate of ethanol production was higher with mixtures of glucose and xylose (0.64–0.83 g g^–1 cells d^–1) compared to that with individual sugars (0.38–0.58 g g^–1 cells d^–1). Although the optimum temperature for growth was 30°C, this strain grew and produced appreciable levels of ethanol at 45°C. A stable ethanol yield (0.40–0.43 g g^–1 substrate utilized) was obtained between 10 g L^–1 and 80 g L^–1 of initial xylose concentration. Conversion efficiency was further improved by immobilization of the cells in calcium alginate beads. Free or immobilized cells ofC. shehatae NCL-3501 efficiently utilized sugars present in rice straw hemicellulose hydrolysate, prepared by two different methods, within 48 h. Ethanol yields of 0.45 g g^–1 and 0.5 g g^–1 from autohydrolysate, and 0.37 g g^–1 from acid hydrolysate were produced by free and immobilized cells, respectively.     

Surface modification of nanofibrous poly(acrylonitrile-co-acrylic acid) membrane with biomacromolecules for lipase immobilization

In this work, poly(acrylonitrile-co-acrylic acid) (PANCAA) was electrospun into nanofibers with a mean diameter of 180 nm. To create a biofriendly microenvironment for enzyme immobilization, collagen or protein hydrolysate from egg skin (ES) was respectively tethered on the prepared nanofibrous membranes in the presence of 1-ethyl-3-(dimethyl-aminopropyl) carbodiamine (EDC)/N-hydroxyl succinimide (NHS). Confocal laser scanning microscopy (CLSM) was used to verify the surface modification and protein density on the nanofibrous membranes. Lipase from Candida rugosa was then immobilized on the protein-modified nanofibrous membranes by covalent binding using glutaraldehyde (GA) as coupling agent, and on the nascent PANCAA nanofibrous membrane using EDC/NHS as coupling agent, respectively. The properties of the immobilized enzyme were assayed. It was found that different pre-tethered biomacromolecules had distinct effects on the immobilized enzyme. The activity retention of the immobilized lipase on ES hydrolysate-modified nanofibrous membrane increased from 15.0% to 20.4% compared with that on the nascent one, while it was enhanced up to more than quadrupled (activity retention of 61.7%) on the collagen-modified nanofibrous membrane. The kinetic parameter, K_m and V_max, were also determined for the free and immobilized lipases. Furthermore, the stabilities of the immobilized lipases were obviously improved compared with the free one.