Synthesis of functionalized polyethylenimine-grafted mesoporous silica spheres and the effect of side arms on lipase immobilization and application

In the present study, silicate mesoporous materials (MCM-41), MCM-41-grafted polyethylenimine (MCM-41@PEI), and succinated PEI containing amine, amide, and acid groups were successfully synthesized and characterized by Fourier transform infrared spectroscopy, thermogravimetric analysis, X-ray diffraction, scanning electron microscopy, transmission electron microscopy, Brunauer–Emmett–Teller analysis, and X-ray photoelectron spectroscopy. Thermomyces lanuginosa lipase (TLL) was then immobilized onto MCM-41 and polymer-grafted MCM-41 by physical adsorption. Besides, for enzyme immobilization via covalent bonding, glutaraldehyde (GLU), and hexamethylene diisocyanate (HMDI) were used as the bridges for binding the enzyme to supports. The best result was obtained with the immobilized lipase on MCM-41@PEI-GLU. In the study of the enzyme reusability, it was shown that about 83% of the initial activity could be retained after 12 cycles of uses. The immobilized lipase on the selected support was also applied for the synthesis of ethyl valerate. Following 24 h incubation in n-hexane and solvent free media, the esterification percentages were 79% and 67%, respectively.     

Simple and efficient immobilization of lipase B from Candida antarctica on porous styrene-divinylbenzene beads

Two commercial porous styrene-divinylbenzene beads (Diaion HP20LX and MCI GEL CHP20P) have been evaluated as supports to immobilize lipase B from Candida antarctica (CALB). MCI GEL CHP20P rapidly immobilized the enzyme, permitting a very high loading capacity: around 110 mg CALB/wet g of support compared to the 50 mg obtained using decaoctyl Sepabeads. Although enzyme specificity of the enzyme immobilized on different supports was quite altered by the support used in the immobilization, specific activity of the enzyme immobilized on MCI GEL CHP20P was always higher than those found using decaoctyl Sepabeads for all assayed substrates. Thus, a CALB biocatalyst having 3-8 folds (depending on the substrate) higher activity/wet gram of support than the commercial Novozym 435 was obtained. Half-live of CAL-Diaion HP20LX at 60 °C was 2-3 higher than the one of Novozym 435, it was 30-40 higher in the presence of 50% acetonitrile and it was around 100 folds greater in the presence of 10 M hydrogen peroxide.Results indicate that styrene-divinylbenzene supports may be promising alternatives as supports to immobilize CALB.     

The study on effective immobilization of lipase on functionalized bentonites and their properties

Three different functionalized bentonites including acid activated bentonite (B_a), organically modified bentonite with cetyltrimethyl ammonium bromide (B_CTMAB) and the composite by acid activation and organo-modification (B_a-CTMAB) were prepared, and used for immobilization of lipase from bovine pancreatic lipase by adsorption. The amount of lipase adsorbed on the functionalized bentonites was in the following sequence: B_a > B_CTMAB > B_a-CTMAB, showing the strongest affinity of B_a for lipase among the three supports. However, the immobilized lipase on B_a-CTMAB showed the highest activity in the hydrolysis of olive oil by 1.67 times of activity of free lipase due to the hydrophobically interfacial activation and enlarged catalytic interface. While, the activity of immobilized lipase on B_a was lower than 20% of free lipase’s activity due to the absence of hydrophobic activation and negative impact of excessive hydrogen ions on the surface. The K_m values for the immobilized lipase on B_a-CTMAB (0.054 g/mL) and B_CTMAB (0.074 g/mL) were both lower than that of free lipase (0.115 g/mL), and the V_max values were higher for the immobilized lipases, exhibiting a higher affinity of the immobilized lipase toward olive oil than free lipase. In comparison to free lipase, the better resistance to heating inactivation, storage stability and reusability of the immobilized lipases on B_a-CTMAB and B_CTMAB were also obtained. The results show that the efficient and stable biocatalysts for industrial application can be prepared by using the low-cost bentonite mineral as the supports.     

Sol–gels and cross-linked aggregates of lipase PS from Burkholderia cepacia and their application in dry organic solvents

Lipase PS from Burkholderia cepacia (formerly Pseudomonas cepacia) was successfully immobilized in sol–gels under low methanol conditions using lyophilization in order to dry the gel. The enzyme was also cross-linked with glutaraldehyde to CLEAs without any additives. These immobilized enzyme preparations were employed for the highly enantioselective acylations of 1-phenylethanol (1), 1-(2-furyl)ethanol (2) and N-acylated 1-amino-2-phenylethanol (3) with vinyl acetate in organic solvents. Enzymatic hydrolysis of the obtained ester product was observed as a side reaction of the acylation of 3 in the presence of lipase PS powder. Hydrolysis was suppressed when the immobilized preparations of lipase PS were used.     

Influence of self-assembled monolayer surface chemistry on Candida antarctica lipase B adsorption and specific activity

Immobilization of Candida antarctica B lipase was examined on gold surfaces modified with either methyl- or hydroxyl-terminated self-assembled alkylthiol monolayers (SAMs), representing hydrophobic and hydrophilic surfaces, respectively. Lipase adsorption was monitored gravimetrically using a quartz crystal microbalance. Lipase activity was determined colorimetrically by following p-nitrophenol propionate hydrolysis. Adsorbed lipase topography was examined by atomic force microscopy. The extent of lipase adsorption was nearly identical on either surface (approximately 240 ng cm⁻²), but its specific activity was sixfold higher on the methyl-terminated SAM, showing no activity loss upon immobilization. A uniform, 5.5 nm high, highly packed monolayer of CALB formed on the methyl-terminated SAM, while the adsorbed protein was disordered on the hydroxyl-terminated SAM. Hydrophobic surfaces thus may specifically orient the lipase in a highly active state.     

Thermostabilization of Candida antarctica lipase B by double immobilization: Adsorption on a macroporous polyacrylate carrier and R1 silaffin-mediated biosilicification

A large improvement in the thermostability of Candida antarctica lipase B (CALB) was achieved through double immobilization, i.e., physical adsorption and R1 silaffin-mediated biosilicification. The C-terminus of CALB was fused with the R1 silaffin peptide for biosilicification. The CALB-R1 fusion protein was adsorbed onto a macroporous polyacrylate carrier and then subsequently biosilicified with tetramethyl orthosilicate (TMOS). After R1 silaffin-mediated biosilicification, the double-immobilized CALB-R1 exhibited remarkable thermostability. The T₅₀⁶⁰ of the double-immobilized CALB-R1 increased dramatically from 45 to 72 °C and that was 27, 13.8, 9.8 and 9.9 °C higher than the T₅₀⁶⁰ values of free CALB-R1, CALB-R1 adsorbed onto a resin, commercial Novozym 435, and Novozym 435 treated with TMOS, respectively. In addition, the time required for the residual activity to be reduced to half (t_1/2) of the double immobilized CALB-R1 elevated from 12.2 to 385 min, which is over 30 times longer life time compared free CALB-R1. The optimum pH for biosilicification was determined to be 5.0, and the double-immobilized enzyme showed much better reusability than the physically adsorbed enzyme even after 6 repeated reuses. This R1-mediated biosilicification approach for CALB thermostabilization is a good basis for the thermostabilization of industrial enzymes that are only minimally stabilized by protein engineering.     

Immobilisation procedure and reaction conditions for optimal performance of Candida antarctica lipase B in transesterification and hydrolysis

Abstract

The reaction kinetics of Candida antarctica lipase B (CalB) in the commercially available preparation Novozym^® 435 (N435) were compared to those of preparations of CalB immobilised on Accurel^® MP1000 (porous polypropylene). Two polypropylene preparations were made using enzyme loadings of 0.2% and 2% (w/w). All three preparations were used in hydrolysis as well as transesterification of two substrates, ethyl acrylate and ethyl methacrylate with octanol. Reactions carried out at water activity levels from 0.06 to 0.96 and at octanol concentrations between 25 and 500 mM showed that both water and octanol can inhibit CalB. Pronounced mass transfer limitations were also observed, which were more pronounced for N435 than for the two MP1000 preparations. The MP1000 preparations could thus use the lipase more efficiently in these reactions, achieving a specific activity (per g enzyme) between 5 and 20 times that of N435. To achieve high rates in the transesterification reaction, it is recommended to use low water activity and moderate alcohol concentration. In order to carry out a hydrolysis reaction, an intermediate water activity should be used to balance the effects of water as a limiting substrate and as a competitive inhibitor.     

Comparison of physical and covalent immobilization of lipase from Candida antarctica on polyamine microspheres of alkylamine matrix

Abstract

Polyamine microspheres (PA-M) prepared using polyethyleneimine as matrix were used for the immobilization of Candida antarctica lipase. The isoelectric point of PA-M is 10.6, and the hydrophobicity of PA-M was indicated using naphthalene. Optimization of conditions showed that the maximal loading of lipase on PA-M reached 230.2 mg g^{? 1} at pH 9.0 and 35°C. An increased buffer concentration had no effect on the activity of lipase but decreased the amount of lipase adsorbed. Simulation with Langmuir and Freundlich isotherms demonstrated that the adsorption of lipase on PA-M was thermodynamically favorable. Covalent crosslinking of the lipase adsorbed extended the pH range and increased the optimal temperature of the lipase activity. The physically adsorbed lipase (P-lipase) and the covalently immobilized derivative (C-lipase) retained more than 75% and 85% of their initial activity, respectively, after 10 cycles of usage. The half-lives of P-lipase and C-lipase at 50°C were 15.70 and 27.67 times higher than that of the free enzyme, respectively. Compared to P-lipase, covalent immobilization obviously reduced the catalytic efficiency and activation energy of the enzyme.     

Heterocycles 32. Efficient kinetic resolution of 1-(2-arylthiazol-4-yl)ethanols and their acetates using lipase B from Candida antarctica

In this paper we describe the chemoenzymatic synthesis of new enantiomerically enriched (R)- and (S)-1-(2-arylthiazol-4-yl)ethanols and their acetates by enzymatic enantioselective acetylation of the racemic alcohols rac-2a–d and by methanolysis of the corresponding racemic esters rac-3a–d mediated by lipase B from Candida antarctica (CaL-B) in non-aqueous media. In terms of stereoselectivity and activity, both procedures, acylation and alcoholysis, gave similar good results (50% conversion, E ≫ 200). The absolute configuration of the kinetic resolution products was determined by a detailed ¹H NMR study of the Mosher's derivatives of (S)-2b.     

Immobilized Candida antarctica lipase B on ZnO nanowires/macroporous silica composites for catalyzing chiral resolution of (R,S)-2-octanol

ZnO nanowires were successfully introduced into a macroporous SiO₂ by in situ hydrothermal growth in 3D pores. The obtained composites were characterized by SEM and XRD, and used as supports to immobilize Candida antarctica lipase B (CALB) through adsorption. The high specific surface area (233 m²/g) and strong electrostatic interaction resulted that the average loading amount of the composite supports (196.8 mg/g) was 3–4 times of that of macroporous SiO₂ and approximate to that of a silica-based mesoporous material. Both adsorption capacity and the activity of the CALB immobilized on the composite supports almost kept unchanged as the samples were soaked in buffer solution for 48 h. The chiral resolution of 2-octanol was catalyzed by immobilized CALB. A maximum molar conversion of 49.1% was achieved with 99% enantiomeric excess of (R)-2-octanol acetate under the optimal condition: a reaction using 1.0 mol/L (R,S)-2-octanol, 2.0 mol/L vinyl acetate and 4.0 wt.% water content at 60 °C for 8 h. After fifteen recycles the immobilized lipase could retain 96.9% of relative activity and 93.8% of relative enantioselectivity.     

Immobilization of Candida antarctica lipase B (CALB) on surface-modified rice husk ashes (RHA) via physical adsorption and cross-linking methods

In the present study, the recovery of activity of Candida antarctica lipase B (CALB) immobilized onto surface-modified rice husk ash (RHA) was 90% for both cross-linking and adsorption methods. Both cross-linked and adsorbed immobilized preparations were very stable, retaining more than 48% of their activity over the range of temperatures studied. The optimum temperature and optimum pH values were 37?°C and 7.0, respectively for both immobilized preparations, while the relative activities after storage at 4.0?°C for 60 days were 55% and 65% using cross-linking and adsorption methods, respectively. Also, the activity of the immobilized lipase began to decrease after 10 cycles, more than 58% of the initial activities were still retained after 10 cycles for both immobilization methods. These results indicated that lipase immobilized by cross-linking and adsorption not only effected activity recovery, but also remarkably effected stability, reusability and application adaptability. It can be concluded that, surface-modified RHA can be used as alternative supports for immobilization of CALB for polymerization reactions.     

Function of C-terminal peptides on enzymatic and interfacial adsorption properties of lipase from Gibberella zeae

Background

The crystal structure of lipase from Gibberella zeae (GZEL) indicates that its C-terminal extension is composed of a loop and a α-helix. This structure is unique, possibly providing novel evidence on lipase mechanisms.