Novozym 435 displays very different selectivity compared to lipase from Candida antarctica B adsorbed on other hydrophobic supports

This paper shows that the properties of lipase B from Candida antarctica (CAL-B) may be easily modulated using different hydrophobic supports to immobilize it (octyl and butyl-agarose, octadecyl-Sepabeads or Lewatit). CAL-B could be fully desorbed from the supports by just incubating the biocatalyst with Triton X-100, although the concentration of detergent necessary was to fully desorb the enzyme varied with the support employed (from 1% for butyl-agarose to 4% for octadecyl-Sepabeads), suggesting that in all cases, the main reason for the enzyme immobilization was hydrophobic interactions. Lewatit VP OC 1600 yielded very different results in terms of activity, selectivity or enantioselectivity in the hydrolysis of rac-2-O-butyryl-2-phenylacetic acid (1) and 3-phenylglutaric acid dimethyl diester (3) compared to the other preparations. For example, in the hydrolysis of 1, Novozym 435 preferred the S-isomer (with an E value higher than 100) whereas all the other preparations preferred the R isomer (e.g. octyl-agarose-CAL-B with E value of 50). In the hydrolysis of 3, Novozym 435 gave S-3-phenylglutaric acid methyl ester with an ee higher than 99%, by coupling the first asymmetric hydrolysis to the enantiospecific hydrolysis of the monoester. CAL-B immobilized on Lewatit at low ionic strength not only behaved similarly to Novozym 435, but also presented some differences that should be due to the exact protocol of the enzyme immobilization in Novozym 435.     

Immobilization of porcine pancreatic lipase on poly-hydroxybutyrate particles for the production of ethyl esters from macaw palm oils and pineapple flavor

Poly-hydroxybutyrate particles (PHB) were used as support to immobilize porcine pancreatic lipase (PPL). The biocatalysts prepared were tested in the synthesis of pineapple flavor by esterification of butanol and butyric acid in heptane medium, and in the synthesis of ethyl esters by transesterification of macaw palm pulp (MPPO) and macaw palm kernel (MPKO) oils with ethanol in solvent-free systems. The effect of protein loading on the biocatalyst activity was assessed in olive oil hydrolysis. Maximum hydrolytic activity of 292.8 ± 8.60 IU/g was observed. Langmuir isotherm model was applicable to the adsorption of PPL on PHB particles. Maximum immobilized protein amount was 24.3 ± 1.70 mg/g. The optimal pH and temperature in hydrolysis reaction for the immobilized PPL were at pH 8.5 and 50 °C, while for the crude PPL extract were at pH 8.0 and 45 °C. Immobilized PPL exhibited full hydrolytic activity after 2 h of incubation in non-polar solvents. In esterification reaction, optimal conversion was around 93% after 2 h of reaction. After six esterification cycles, the biocatalyst retained 63% of its initial activity. The biocatalyst prepared attained transesterification yield of 50% after 48 h of reaction for MPKO and 35% after 96 h of reaction for MPPO.     

Lipase immobilisation: an equilibrium study of lipases immobilised on hydrophobic and hydrophilic/hydrophobic supports

This work investigates the enzyme-support equilibrium behaviour in immobilised lipase biocatalysts. Equilibrium data determines the maximum enzyme up-take by unit weight of support. Four lipases were immobilised on two polymeric supports, respectively. They were Lipase PS from Pseudomonas, Lipolase 100L from Humicola, SP871 from Rhizomucor miehel and QL from Alcaligenes. The supports were Accurel EP100 (a polypropylene material) and 45SAA (a polypropylene/silica composite). Experimentally, equilibrium was expressed in terms of lipase loading (LU/g support) versus residual lipase concentration (LU/dm³). Activity, efficiency and operational stability of the immobilised lipases were assayed by solvent-free esterification of oleic acid and octanol.Equilibrium data were modelled by the Langmuir, Freundlich and Redlich–Peterson formulae. It was found that Lipolase 100L/Accurel, PS/45SAA and SP871/45SAA systems conformed to the Langmuir behaviour, while Lipase PS/Accurel and SP871/Accurel systems followed the Freundlich behaviour and Lipolase 100L/45SAA, QL/45SAA and QL/Accurel EP100 resembled Redlich–Peterson behaviour. Whereas immobilisation on Accurel EP100 resulted in classical equilibrium isotherms with all four lipases, immobilisation on support 45SAA resulted in two-plateau equilibrium curves which included a step change in the isotherm for all lipases studied, except for SP871. Quantitatively, for 1 g lipase, Accurel and 45SAA had a maximum capacity of 140 and 260 kLU for PS, 112 and 550 kLU for Lipolase 100L, 320 and 800 kLU for SP871 and 18 and 29 kLU for QL, respectively.     

Preparation of a biocatalyst via physical adsorption of lipase from Thermomyces lanuginosus on hydrophobic support to catalyze biolubricant synthesis by esterification reaction in a solvent-free system

Lipase from Thermomyces lanuginosus (TLL) was immobilized on mesoporous hydrophobic poly-methacrylate (PMA) particles via physical adsorption (interfacial activation of the enzyme on the support). The influence of initial protein loading (5–200 mg/g of support) on the catalytic properties of the biocatalysts was determined in the hydrolysis of olive oil emulsion and synthesis of isoamyl oleate (biolubricant) by esterification reaction. Maximum adsorbed protein loading and hydrolytic activity were respectively ≈100 mg/g and ≈650 IU/g using protein loading of 150 mg/g of support. The adsorption process followed the Langmuir isotherm model (R² = 0.9743). Maximum ester conversion around 85% was reached after 30 min of reaction under continuous agitation (200 rpm) using 2500 mM of each reactant in a solvent-free system, 45 °C, 20% m/v of the biocatalyst prepared using 100 mg of protein/g of support. Apparent thermodynamic parameters of the esterification reaction were also determined. Under optimal experimental conditions, reusability tests of the biocatalyst (TLL-PMA) after thirty successive cycles of reaction were performed. TLL-PMA fully retained its initial activity up to twenty two cycles of reaction, followed by a slight decrease around 8.6%. The nature of the product (isoamyl oleate) was confirmed by attenuated total reflection Fourier transform infrared (ATR-FTIR), proton (¹H NMR) and carbon (¹³C NMR) nuclear magnetic resonance spectroscopy analyses.     

Laccase from Pleurotus sajor-caju on functionalised SBA-15 mesoporous silica: Immobilisation and use for the oxidation of phenolic compounds

Laccase from Pleurotus sajor-caju was immobilised on functionalised SBA-15 mesoporous silica. The immobilisation process reached the equilibrium after about 100 min. In order to study the effect of loading (L) on activity of the immobilised laccase, the adsorption isotherm was built and the activity of the corresponding immobilised biocatalysts was determined. The activity of the immobilised preparations reached a maximum at L = 217 kU g_SBA-15⁻¹, whereas higher loadings gave rise to a less-efficient biocatalyst. The immobilised laccase was used for the oxidation of a mixture of four phenolic compounds (protocatechuic acid, ferulic acid, sinapic acid and caffeic acid) chosen among those present in olive mill wastewaters (OMWs). These compounds determine the phytotoxicity of OMWs. Different reaction rates were observed for the oxidation of the examined phenolic compounds. The biocatalyst was recycled and a conversion of 84 mol% at the 10th reuse and of about 60 mol% after the 14th reuse was obtained. In conclusion, the laccase immobilised on SBA-15 is a potential biocatalyst for bioremediation of OMWs, which is an important environmental problem in the regions around the Mediterranean Sea.     

Immobilization of lipase B from Candida antarctica on porous styrene-divinylbenzene beads improves butyl acetate synthesis

A new biocatalyst of lipase B from Candida antarctica (MCI-CALB) immobilized on styrene-divinylbenzene beads (MCI GEL CHP20P) was compared with the commercial Novozym 435 (immobilized lipase) in terms of their performances as biocatalysts for the esterification of acetic acid and n-butanol. The effects of experimental conditions on reaction rates differed for each biocatalyst, showing different optimal values for water content, temperature, and substrate molar ratio. MCI-CALB could be used at higher acid concentrations, up to 0.5 M, while Novozym 435 became inactivated at these acid concentrations. Although Novozym 435 exhibited 30% higher initial activity than MCI-CALB for the butyl acetate synthesis, the reaction course was much more linear using the new preparation, meaning that the MCI-CALB allows for higher productivities per cycle. Both preparations produced around 90% of yield conversions after only 2 h of reaction, using 10% (mass fraction) of enzyme. However, the main advantage of the new biocatalyst was the superior performance during reuse. While Novozym 435 was fully inactivated after only two batches, MCI-CALB could be reused for six consecutive cycles without any washings and keeping around 70% of its initial activity. It is proposed that this effect is due to the higher hydrophobicity of the new support, which does not retain water or acid in the enzyme environment. MCI-CALB has shown to be a very promising biocatalyst for the esterification of small-molecule acids and alcohols.     

FTIR-ATR characterization of free Rhizomucor meihei lipase (RML), Lipozyme RM IM and chitosan-immobilized RML

The synthesis and characterization of biocatalysts based on lipase from Rhizomucor miehei (RML) immobilized on chitosan-based supports were investigated. The enzyme was immobilized on chitosan following two strategies: (i) physical adsorption; and (ii) covalent bonding using glutaraldehyde. The content of enzyme bound in the supports, as precipitable protein, was analyzed using UV/visible methods. FTIR-ATR spectroscopy was employed to characterize the prepared biocatalysts, as well the native enzyme and a commercial biocatalyst Lipozyme RM IM, used as reference materials. Analysis of the amide I′ signal allowed to follow the changes in the secondary structure of the enzyme after binding to the support and its thermal stability. The hydrolysis of ethyl stearate monitored in situ by FTIR-ATR was used as a test reaction. Results showed that RML was bound to Chit and Glut–Chit with minor changes in its secondary structure, thermal stability and enzymatic activity in a selected reaction test.     

Multipoint covalent immobilization of lipase on chitosan hybrid hydrogels: influence of the polyelectrolyte complex type and chemical modification on the catalytic properties of the biocatalysts

This work aimed at the production of stabilized derivatives of Thermomyces lanuginosus lipase (TLL) by multipoint covalent immobilization of the enzyme on chitosan-based matrices. The resulting biocatalysts were tested for synthesis of biodiesel by ethanolysis of palm oil. Different hydrogels were prepared: chitosan alone and in polyelectrolyte complexes (PEC) with κ-carrageenan, gelatin, alginate, and polyvinyl alcohol (PVA). The obtained supports were chemically modified with 2,4,6-trinitrobenzene sulfonic acid (TNBS) to increase support hydrophobicity, followed by activation with different agents such as glycidol (GLY), epichlorohydrin (EPI), and glutaraldehyde (GLU). The chitosan-alginate hydrogel, chemically modified with TNBS, provided derivatives with higher apparent hydrolytic activity (HA_app) and thermal stability, being up to 45-fold more stable than soluble lipase. The maximum load of immobilized enzyme was 17.5 mg g⁻¹ of gel for GLU, 7.76 mg g⁻¹ of gel for GLY, and 7.65 mg g⁻¹ of gel for EPI derivatives, the latter presenting the maximum apparent hydrolytic activity (364.8 IU g⁻¹ of gel). The three derivatives catalyzed conversion of palm oil to biodiesel, but chitosan-alginate-TNBS activated via GLY and EPI led to higher recovered activities of the enzyme. Thus, this is a more attractive option for both hydrolysis and transesterification of vegetable oils using immobilized TLL, although industrial application of this biocatalyst still demands further improvements in its half-life to make the enzymatic process economically attractive.     

Synthesis of flavor and fragrance esters using <Emphasis Type="Italic">Candida antarctica</Emphasis> lipase

Candida antarctica lipase fraction B (CAL-B) showed substrate specificity in the synthesis of esters in hexane involving reactions of short-chain acids having linear (acetic and butyric acids) and branched chain (isovaleric acid) structures, an unsaturated (tiglic acid) fatty acid, and phenylacetic acid with n-butanol and geraniol. The variation in the conversion to the esters was ca. 10%. Similar results were observed in a study of the alcohol specificity of the enzyme for esterification of acetic and butyric acids with four alcohols: n-butyl, isopentyl, 2-phenylethyl, and geraniol. Enantioselectivity of CAL-B in hexane with a range of chiral -substituted or -substituted carboxylic acids and n-butyl alcohol was analyzed. The results show that CAL-B can be employed as a robust biocatalyst in esterification reactions due to the high conversions obtained in the synthesis of short-chain flavor esters in an organic solvent, although this enzyme exhibited modest enantioselectivity with chiral short-chain carboxylic acids.     

Immobilized biocatalysts for detoxification of neurotoxic organophosphorous compounds

New biocatalysts were developed using organophosphorus hydrolase (OPH, EC 3.1.8.1) with a polyhistidine tag at the N-terminus of the protein (His₆-OPH). The use of His₆-OPH together with previously developed approaches for the entrapment of cells into poly(vinyl alcohol) cryogels and covalent immobilization of enzymes into porous fabric materials, impregnated with chemically cross-linked chitosan sulphate gel, enabled dramatic improvement of catalytic characteristics against various organophosphorous compounds (OPCs; Paraoxon, Coumaphos, Methyl parathion, etc.). The polyhistidine tag of OPH was used to create a new immobilized biocatalyst using metal-chelating carriers, such as Ni²⁺-nitrilotriacetic acid-agarose and Co²⁺-iminodiacetic acid-polyacrylamide cryogel. The latter biocatalyst had high activity and stability for the continuous hydrolysis of OPCs.     

Lipase-catalysed synthesis of ester oils from biodiesel by-products

Ester oils obtained from natural long-chain fatty acids and alcohols are versatile substitutes for many petroleum-based products. Their efficient synthesis with the solvent-free esterification of free fatty acids (FFA) from by-products of biodiesel fabrication and 2-ethyl-1-hexanol with immobilised lipase from Thermomyces lanuginosa was investigated. The immobilisation of the biocatalyst in static emulsion yielded a specific esterification activity that was higher by a factor of 4.9–9.4 than the activity of the native enzyme. Favourable properties of the silicone-based immobilisation matrix in terms of stability and immobilisation yield were observed. In biodiesel by-products, the immobilised lipase catalysed the esterification of FFA as well as the transesterification of residual fatty acid methyl esters (FAME) to the desired ester oils. A conversion of 90% FFA and 35% FAME gave a total yield of 60%. The inactivation coefficients during repeated use in a stirred-tank reactor with intermittent pressure reduction were exceptionally low.     

Evaluation and optimization of immobilized lipase for esterification of fatty acid and monohydric alcohol

Commercial available lipases viz. Lipozyme™, Novozyme-735 and Candida antartica lipase-B (CAL-B) were immobilized on seven different supports by simple adsorption process. The importance of suitable enzyme–support combination in esterification of lauric acid and iso-propanol was validated experimentally. Effect of long chain fatty acids (C4–C18) and small chain monohydric alcohols (C1–C6) on specific activities of different immobilized lipases were evaluated. Lauric acid (C12) was found to be the most preferred fatty acid and t-amyl alcohol (C5) being the best alcohol. CAL-B adsorbed on Lewatit was the most efficient immobilized enzyme for esterification reaction. Selectivity constant for lauric acid (3.4) was the highest among all fatty acids tested, whereas there was not much difference in selectivity between different alcohols. Furthermore, increase in fatty acid unsaturation leads to decrease catalytic efficiency of immobilized CAL-B. The optimum conditions for t-amyllaurate synthesis were as follows: lauric acid—0.5 M, t-amyl alcohol—0.3 M and amount of immobilized enzyme—150 mg. Finally, CAL-B adsorbed on Lewatit was reused for three consecutive cycles.     

Immobilization of Candida rugosa lipase on hexagonal mesoporous silicas and selective esterification in nonaqueous medium

Candida rugosa lipases (CRLs) immobilized by physical adsorption, cross-linking and covalent binding methods on a MSU-H type mesoporous silica previously modified organically by different strategies, respectively, were examined as biocatalysts for esterification of conjugated linoleic acid (CLA) and ethanol in nonaqueous medium. MSU-H silica was modified by nonionic surfactant of triblock copolymer Pluronic P123, amino-functionalization and glutaraldehyde-grafting and confirmed by FT-IR analysis. Interaction mechanisms of CRLs and supports involve covalent and non-covalent interactions including electrostatic repulsion and hydrophobic interaction at pH 7. The immobilized CRLs containing surfactant were prepared by cross-linking via entrapping CRL aggregates inside the pores of silicas. The surfactant located inside the silicas could interfacially activate the immobilized CRLs and favored catalytic esterification. The biocatalyst containing 38 wt.% of surfactant afforded 1111.1 U/mg of specific activity about eight times higher than soluble CRL, and maximal 56.7% of total CLA esterification with 96.5% of 9c, 11t-CLA isomer esterification degree. The immobilized-CRL with 64.5 mg/g of loading amount of protein exhibited maximal hydrolytic activity of 2945.3 U/g-support for grafting glutaraldehyde. This derivative showed a high level of esterification activity and operational stability and remained 43.2–46.9% of total esterification for 32 h consecutive four runs.     

Spherezymes: A novel structured self-immobilisation enzyme technology

Background  

Enzymes have found extensive and growing application in the field of chemical organic synthesis and resolution of chiral intermediates. In order to stabilise the enzymes and to facilitate their recovery and recycle, they are frequently immobilised. However, immobilisation onto solid supports greatly reduces the volumetric and specific activity of the biocatalysts. An alternative is to form self-immobilised enzyme particles.     

New active site oriented glyoxyl-agarose derivatives of <Emphasis Type="Italic">Escherichia coli</Emphasis>penicillin G acylase

Background  

Immobilized Penicillin G Acylase (PGA) derivatives are biocatalysts that are industrially used for the hydrolysis of Penicillin G by fermentation and for the kinetically controlled synthesis of semi-synthetic β-lactam antibiotics. One of the most used supports for immobilization is glyoxyl-activated agarose, which binds the protein by reacting through its superficial Lys residues. Since in E. coli PGA Lys are also present near the active site, an immobilization that occurs through these residues may negatively affect the performance of the biocatalyst due to the difficult diffusion of the substrate into the active site. A preferential orientation of the enzyme with the active site far from the support surface would be desirable to avoid this problem.     

Catalytic properties of lipase adsorbed on nanocarbon-containing mesoporous silica in esterification and transesterification reactions

Nanocarbon-containing mesoporous silica covered with a varying amounts of nanostructured carbon of different morphologies were used as supports to immobilize Thermomyces lanuginosus lipase. The catalytic properties of the prepared biocatalysts were studied in both the transesterification of vegetable (linseed) oil in the presence of ethyl acetate and the esterification of the fatty acid (capric C10:0) in the presence of secondary (isopropyl or isoamyl) alcohols. The physico-chemical characteristics, such as the amount of adsorbed lipase, its specific activity, and the dependence of the activity and stability of the prepared biocatalysts on the support type were evaluated. The Michaelis-Menten kinetics was studied in the esterification of capric acid with isoamyl alcohol. The prepared biocatalysts were shown to retain up to 90% activity for >1000 h in the synthesis of isoamyl caprate. The half-time of the biocatalysts inactivation in the transesterification of linseed oil was found to be more than 700 h at 40°C.     

Operational stabilities of different chemical derivatives of Novozym 435 in an alcoholysis reaction

Industrial use of Novozym 435 in synthesis of structured lipids and biodiesel via alcoholysis is limited by mass transfer effects of the glycerides through immobilized enzymes and its low operational stability under operation conditions. To better understand this, differently modified Novozym 435 preparations, differing in their surface nature and in their interactions with reactants, have been compared in the alcoholysis of Camelina sativa oil. The three modifications performed have been carried out under conditions where all exposed groups of the enzyme have been modified. These modifications were: 2,4,6-trinitrobenzensulfonic acid (Novo-TNBS), ethylendiamine (Novo-EDA) and polyethylenimine (Novo-PEI). Changes in their operational performance are analyzed in terms of changes detected by scan electron microscopy in the support morphology.The hydrophobic nature of the TNBS accelerates the reaction rate; t-ButOH co-solvent swells the macroporous acrylic particles of Lewatit VP OC 1600 in all biocatalysts, except in the case of Novo-PEI. This co-solvent only increases the maximal conversions obtained at 24 h using the modified biocatalysts. t-ButOH reduces enzyme inactivation by alcohol and water. In a co-solvent system, these four biocatalysts remain fully active after 14 consecutive reaction cycles of 24 h, but only Novo-TNBS yields maximal conversion before cycle 5. Some deposits on biocatalyst particles could be appreciated during reuses, and TNBS derivatization diminishes the accumulation of product deposits on the catalyst surface. Most particles of commercial Novozym^® 435 are broken after operation for 14 reaction cycles. The broken particles are fully active, but they cause problems of blockage in filtration operations and column reactors. The three derivatizations studied make the matrix particles more resistant to rupture.     

Stability of lipase immobilized on <Emphasis Type="Italic">O</Emphasis>-pentynyl dextran

O-Pentynyl dextran (PyD), an amphiphilic polysaccharide derivative with a degree of substitution (DS) of 0.43 was compared with ion exchange resins Lewatit VP OC 1600, Amberlite XAD 761 and Duolite A568 for immobilization of Lipase from Rhizopus arrhizus by adsorption method. The immobilized enzymes were employed for esterification of octanoic acid with geraniol in n-hexane as model reaction. PyD showed higher lipase adsorption and with 249 μmol min⁻¹ g⁻¹ significant higher esterification activity than the other supports (67–83 μmol min⁻¹ g⁻¹). Biocatalysts from all types of supports except PyD became completely inactive within 8 weeks storing at −10 °C while lipase immobilized on PyD retained its full esterification activity for at least 14 weeks. In repeated use, yield decreased rapidly after two cycles for all supports except for PyD. For this biopolymeric support, constantly 90% yield was achieved even after eight cycles, when the biocatalyst was washed with n-hexane and water and then freeze-dried. To achieve this yield, prolonged reaction times were required, partly on the account of an increasing delay period, probably to adapt active conformation, until the reaction starts.     

Lipase-catalyzed esterification of conjugated linoleic acid with <Emphasis Type="SmallCaps">l</Emphasis>-carnitine in solvent-free system and acetonitrile

Lipase-catalyzed esterification of conjugated linoleic acid (CLA) with l-carnitine in solvent-free system and acetonitrile was studied. Three lipases (Novzym 435, Amamo AY30 and Amano AYS) have been assayed as suitable biocatalysts in the reaction. It was found that Amano AY30 was the most effective biocatalyst in both solvent-free system and acetonitrile. The conversion rate varied from 8.05 to 60.9% in terms of reaction conditions such as the amount of lipase, the presence of water, the amount of molecular sieves and reaction time. The conversions of substrate in solvent-free system were higher than that in acetonitrile. When the substrates were 1 mmol CLA and 1 mmol l-carnitine, the maximum conversion (60.9%) was obtained in solvent-free system with 150 mg lipase AY30, 50% water content and 150 mg molecular sieves at the reaction time of 24 h. A novel CLA ester product was successfully isolated and characterized by ESI-MS and ¹H NMR.     

Synthesis of enantiopure (5R)-hydroxyhexane-2-one with immobilised whole cells of Lactobacillus kefiri

Whole-cell reduction of (2,5)-hexanedione to yield highly enantiopure (5R)-hydroxyhexane-2-one (enantiomeric excess >99%) with Lactobacillus kefiri DSM 20587 was investigated. Cell immobilisation with sodium cellulose sulphate was chosen as the most suitable encapsulation matrix, giving an immobilisation yield of 40%. Despite the lowered biocatalytic activity from cell immobilisation, the bioreduction process was vastly improved with the help of reaction engineering techniques (batch to a plug flow reactor set-up). High selectivity (95%) and space–time yield (87 g L⁻¹ day⁻¹) were achieved in the plug flow reactor. The biocatalyst remained active (68% residual activity) after 6 days of operation.