Improvements in current strategies for carrier-based immobilisation have been developed using hetero-functionalised supports that enhance the binding efficacy and stability through multipoint attachment. New commercial resins (Sepabeads) exhibit improved protein binding capacity. Novel methods of enzyme self-immobilisation have been developed (CLEC, CLEA, Spherezyme), as well as carrier materials (Dendrispheres), encapsulation (PEI Microspheres), and entrapment. Apart from retention, recovery and stabilisation, other advantages to enzyme immobilisation have emerged, such as enhanced enzyme activity, modification of substrate selectivity and enantioselectivity, and multi-enzyme reactions. These advances promise to enhance the roles of immobilisation enzymes in industry, while opening the door for novel applications.

Advances in enzyme immobilisation

Endowed with unparalleled high catalytic activity and selectivity, enzymes offer enormous potential as catalysts in practical applications. These applications, however, are seriously hampered by enzymes’ low thermal and chemical stabilities. One way to improve these stabilities is the enzyme immobilization. Among various tested methods of this process that make use of different enzyme-carrier interactions, immobilization by adsorption on solid carriers has appeared most common. According to these findings, in this review we present a comparative analysis of the literature reports on the recent trends in the immobilization of the enzymes by adsorption. This thorough study was prepared in order to provide a deeper understanding of the process. Both carriers, carrier modifiers and procedures developed for effective adsorption of the enzymes are discussed. The review may thus be helpful in choosing the right adsorption scheme for a given enzyme to achieve the improvement of its stability and activity for a specific application.

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Enzyme immobilization by adsorption: a review

Immobilization of lipases on hydrophobic supports: immobilization mechanism, advantages, problems, and solutions.

Biodiesel production from waste cooking oil using bifunctional heterogeneous solid catalysts

Candida rugosa lipase was immobilized by covalent binding on controlled pore silica (CPS) using glutaraldehyde as cross-linking agent under aqueous and nonaqueous conditions. The immobilized C. rugosa was more active when the coupling procedure was performed in the presence of a nonpolar solvent, hexane. Similar optima pH (7.5–8.0) was found for both free and immobilized lipase. The optimum temperature for the immobilized lipase was about 10°C higher than that for the free lipase. The thermal stability of the CPS lipase was also greater than the original lipase preparation. Studies on the operational stability of CPS lipase revealed good potential for recycling under aqueous (olive-oil hydrolysis) and nonaqueous (butyl butyrate synthesis) conditions.

Characterization and utilization of Candida rugosa lipase immobilized on controlled pore silica.

Three different approaches for lipase catalysed synthesis of citronellyl acetate by a commercial available immobilized lipase have been studied: a) direct esterification reaction of citronellol with acetic acid; b) alcoholysis of butyl acetate with citronellol and c) transesterification of citronellyl butyrate with butylacetate. Heptane was used as solvent for all the experiments. The extent of reaction occurred in the order alcoholysis>transesterification>esterification. Substrate partitioning between the immobilization support and the organic medium seems to greatly influence the catalytic performance of this enzyme preparation. Production of citronellyl acetate, was found to be dependent on the partition coefficient of the acyl donor which account the greatest value for the acetic acid.

Evaluation of different approaches for lipase catalysed synthesis of citronellyl acetate

A lipase from Candida rugosa immobilized on styrene-divinylbenzene copolymer was used to catalyse the direct esterification of butanol and butyric acid. A factorial design was employed to evaluate the effects of temperature (37–50 °C), substrate molar ratio of butyric acid to butanol (0.6 to 2.0) and enzyme amount (0.2–0.4 g) on the ester yield. The main effects were fitted by multiple regression analysis to a linear model and maximum ester yield could be obtained working at 41 °C with 0.4 g of lipase. The mathematical model obtained, representing the ester yield has been found to describe adequately the experimental results. Under optimal conditions, concentration of 32.4 g butyl butyrate/l that corresponds to a yield of 75% was obtained.

Optimization of Lipase-catalysed Synthesis of Butyl Butyrate Using a Factorial Design

The technique based on sol–gel approach was used to generate silica matrices derivatives by hydrolysis of silane compounds. The present work evaluates a hybrid matrix obtained with tetraethoxysilane (TEOS) and polyvinyl alcohol (PVA) on the immobilization yield of lipase from Pseudomonas fluorescens. The resulting polysiloxane–polyvinyl alcohol (POS–PVA) matrix combines the property of PVA as a suitable polymer to retain proteins with an excellent optical, thermal and chemical stability of the host silicon oxide matrix. Aiming to render adequate functional groups to the covalent binding with the enzyme the POS–PVA matrix was chemically modified using epichlorohydrin. The results were compared with immobilized derivative on POS–PVA activated with glutaraldehyde. Immobilization yield based on the recovered lipase activity depended on the activating agent and the highest efficiency (32%) was attained when lipase was immobilized on POS–PVA activated with epichlorohydrin, which, probably, provided more linkage points for the covalent bind of the enzyme on the support. This was confirmed by determining the morphological properties using different techniques as X-ray diffraction and scanning electron microscopy (SEM). Comparative studies were carried out to attain optimal activities for free lipase and immobilized systems. For this purpose, a central composite experimental design with different combinations of pH and temperature was performed. Enzymatic hydrolysis with the immobilized enzyme in the framework of the Michaelis–Menten mechanism was also reported. Under optimum conditions, the immobilized derivative on POS–PVA activated with epichlorohydrin showed to have more affinity for the substrate in the hydrolysis of olive oil, with a Michaelis–Menten constant value (Km) of 293 mM, compared to the value of 401 mM obtained for the immobilized lipase on support activated with glutaraldehyde. Data generated by DSC showed that both immobilized derivatives have similar thermal stabilities.

Pseudomonas fluorescens lipase immobilization on polysiloxane–polyvinyl alcohol composite chemically modified with epichlorohydrin

The boiling point and volatility are important properties for fuels, as it is for quality control of the industry of petroleum diesel and biofuels. In addition, through the volatility is possible to predict properties, such as vapor pressure, density, latent heat, heat of vaporization, viscosity, and surface tension of biodiesel. From thermogravimetry analysis it is possible to find the kinetic parameters (activation energy, pre-exponential factor, and reaction order), of thermally simulated processes, like volatilization. With the kinetic parameters, it is possible to obtain the thermodynamic parameters by mathematical formula. For the kinetic parameters, the minor values of activation energy were found for mineral diesel (E = 49.38 kJ mol−1), followed by babassu biodiesel (E = 76.37 kJ mol−1), and palm biodiesel (E = 87.00 kJ mol−1). Between the two biofuels studied, the babassu biodiesel has the higher minor value of activation energy. The thermodynamics parameters of babassu biodiesel are, ΔS = −129.12 J mol−1 K−1, ΔH = +80.38 kJ mol−1 and ΔG = +142.74 kJ mol−1. For palm biodiesel ΔS = −119.26 J mol−1 K−1, ΔH = + 90.53 kJ mol−1 and ΔG = +141.21 kJ mol−1, and for diesel ΔS = −131.3 J mol−1 K−1, ΔH = +53.29 kJ mol−1 and ΔG = +115.13 kJ mol−1. The kinetic thermal analysis shows that all E, ΔH, and ΔG values are positive and ΔS values are negative, consequently, all thermodynamic parameters indicate non-spontaneous processes of volatilization for all the fuels studied.

Kinetic and thermodynamic parameters of volatilization of biodiesel from babassu, palm oil and mineral diesel by thermogravimetric analysis (TG)

Candida rugosa lipase was immobilized on poly(N-methylolacrylamide) by physical adsorption. The biocatalyst performance (immobilized lipase) was evaluated in both aqueous (hydrolysis) and organic (butyl butyrate synthesis) media. In the first case, a comparative study between free and immobilized derivatives was provided in terms of pH, temperature and thermal stability following the olive oil hydrolysis, establishing new optimum values. In the second case, the influence of temperature, biocatalyst concentration and acid/alcohol molar ratio was simultaneously studied according to a 23 full experimental design. The highest molar conversion (96 %), volumetric productivity (1.73 g L–1 h–1) and specific esterification activity (1.00 μM mg–1 min–1) were obtained when working at the lowest level of temperature and butyric acid in excess. Under these conditions, repeated batch use of the immobilized enzyme was performed and half-life time (t1/2) was found to be 145 h.

H. F. de Castro

Papers

Evaluation of different approaches for lipase catalysed synthesis of citronellyl acetate

Optimization of Lipase-catalysed Synthesis of Butyl Butyrate Using a Factorial Design

Pseudomonas fluorescens lipase immobilization on polysiloxane–polyvinyl alcohol composite chemically modified with epichlorohydrin

Kinetic and thermodynamic parameters of volatilization of biodiesel from babassu, palm oil and mineral diesel by thermogravimetric analysis (TG)

Characterization of Candida rugosa Lipase Immobilized on Poly(N‐methylolacrylamide) and Its Application in Butyl Butyrate Synthesis