Improvement of enzyme activity, stability and selectivity via immobilization techniques

In this tutorial review, an overview of the why, what and how of enzyme immobilisation for use in biocatalysis is presented. The importance of biocatalysis in the context of green and sustainable chemicals manufacture is discussed and the necessity for immobilisation of enzymes as a key enabling technology for practical and commercial viability is emphasised. The underlying reasons for immobilisation are the need to improve the stability and recyclability of the biocatalyst compared to the free enzyme. The lower risk of product contamination with enzyme residues and low or no allergenicity are further advantages of immobilised enzymes. Methods for immobilisation are divided into three categories: adsorption on a carrier (support), encapsulation in a carrier, and cross-linking (carrier-free). General considerations regarding immobilisation, regardless of the method used, are immobilisation yield, immobilisation efficiency, activity recovery, enzyme loading (wt% in the biocatalyst) and the physical properties, e.g. particle size and density, hydrophobicity and mechanical robustness of the immobilisate, i.e. the immobilised enzyme as a whole (enzyme + support). The choice of immobilisate is also strongly dependent on the reactor configuration used, e.g. stirred tank, fixed bed, fluidised bed, and the mode of downstream processing. Emphasis is placed on relatively recent developments, such as the use of novel supports such as mesoporous silicas, hydrogels, and smart polymers, and cross-linked enzyme aggregates (CLEAs).

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Enzyme immobilisation in biocatalysis : Why, what and how

Immobilization is often the key to optimizing the operational performance of an enzyme in industrial processes, particularly for use in non-aqueous media. Different methods for the immobilization of enzymes are critically reviewed. The methods are divided into three main categories, viz. (i) binding to a prefabricated support (carrier), (ii) entrapment in organic or inorganic polymer matrices, and (iii) cross-linking of enzyme molecules. Emphasis is placed on relatively recent developments, such as the use of novel supports, e.g., mesoporous silicas, hydrogels, and smart polymers, novel entrapment methods and cross-linked enzyme aggregates (CLEAs).

http://largens.com/biochemlab/peroxidase2.pdf

Enzyme immobilization: The quest for optimum performance

Enzyme biocatalysis plays a very relevant role in the development of many chemical industries, e.g., energy, food or fine chemistry. To achieve this goal, enzyme immobilization is a usual pre-requisite as a solution to get reusable biocatalysts and thus decrease the price of this relatively expensive compound. However, a proper immobilization technique may permit far more than to get a reusable enzyme; it may be used to improve enzyme performance by improving some enzyme limitations: enzyme purity, stability (including the possibility of enzyme reactivation), activity, specificity, selectivity, or inhibitions. Among the diverse immobilization techniques, the use of pre-existing supports to immobilize enzymes (via covalent or physical coupling) and the immobilization without supports [enzyme crosslinked aggregates (CLEAs) or crystals (CLECs)] are the most used or promising ones. This paper intends to give the advantages and disadvantages of the different existing immobilization strategies to solve the different aforementioned enzyme limitations. Moreover, the use of nanoparticles as immobilization supports is achieving an increasing importance, as the nanoparticles versatility increases and becomes more accessible to the researchers. We will also discuss here some of the advantages and drawbacks of these non porous supports compared to conventional porous supports. Although there are no universal optimal solutions for all cases, we will try to give some advice to select the optimal strategy for each particular enzyme and process, considering the enzyme properties, nature of the process and of the substrate. In some occasions the selection will be compulsory, for example due to the nature of the substrate. In other cases the optimal biocatalyst may depend on the company requirements (e.g., volumetric activity, enzyme stability, etc).

Potential of Different Enzyme Immobilization Strategies to Improve Enzyme Performance

Application of chitin- and chitosan-based materials for enzyme immobilizations: a review

Lactose-Derived Prebiotics: A Process Perspective

Optimization of reaction conditions and the donor substrate in the synthesis of hexyl-β-d-galactoside

Synthesis of cephalexin with immobilized penicillin acylase at high substrates concentration at an acyl donor to nucleophile molar ratio of 3 was comparatively evaluated in aqueous and ethylene glycol media using a statistical model Variables under study were temperature, pH and enzyme to substrate ratio and their effects were evaluated on cephalexin yield, ratio of initial rates of cephalexin synthesis to phenylglycine methyl ester hydrolysis, volumetric and specific productivity of cephalexin synthesis, that were used as response parameters Results obtained in both reaction media were modeled using surface of response methodology and optimal operation conditions were determined in terms of an objective function based on the above parameters At very high substrates concentrations the use of organic co-solvents was not required to attain high yields and actually almost stoichiometric yields were obtained in a fully aqueous media with the advantages of higher productivities than in an organic co-solvent media and compliance with the principles of green chemistry

Comparative study of the enzymatic synthesis of cephalexin at high substrate concentration in aqueous and organic media using statistical model

Synthesis of ascorbyl palmitate by enzymatic esterification of palmitic acid and ascorbic acid was conducted in an organic medium with Pseudomonas stutzeri lipase TL immobilized in different supports and its performance was compared with commercial Novozym 435 lipase used as a reference. The enzyme was immobilized in different supports and the best catalyst was selected in terms of immobilization yield and mass specific activity to perform the reactions of synthesis. Synthesis of ascorbyl palmitate was optimized considering temperature, substrate molar ratio and enzyme to limiting substrate mass ratio as variables, and substrate conversion and specific productivity as evaluation parameters. The best reaction conditions for immobilized lipase TL were 55 °C, 1:5 ascorbic to palmitic acid molar ratio, and 1:10 lipase to ascorbic acid mass ratio, obtaining 57 % substrate conversion and a specific productivity of 0.013 [g ascorbic acid/(g enzyme × min)]; the best conditions for Novozym 435 were 70 °C, ascorbic to palmitic acid molar ratio 1:10, and 1:10 lipase to ascorbic acid mass ratio, obtaining 51 % substrate conversion and a specific productivity of 0.016 [g ascorbic acid/(g enzyme × min)].

Synthesis of Ascorbyl Palmitate with Immobilized Lipase from Pseudomonas stutzeri

This work reports the study of ZnO-based anodes for the photoelectrochemical regeneration of the oxidized form of nicotinamide adenine dinucleotide (NAD+). The latter is the most important coenzyme for dehydrogenases. However, the high costs of NAD+ limit the use of such enzymes at the industrial level. The influence of the ZnO morphologies (flower-like, porous film, and nanowires), showing different surface area and crystallinity, was studied. The detection of diluted solutions (0.1 mM) of the reduced form of the coenzyme (NADH) was accomplished by the flower-like and the porous films, whereas concentrations greater than 20 mM were needed for the detection of NADH with nanowire-shaped ZnO-based electrodes. The photocatalytic activity of ZnO was reduced at increasing concentrations of NAD+ because part of the ultraviolet irradiation was absorbed by the coenzyme, reducing the photons available for the ZnO material. The higher electrochemical surface area of the flower-like film makes it suitable for the regeneration reaction. The illumination of the electrodes led to a significant increase on the NAD+ regeneration with respect to both the electrochemical oxidation in dark and the only photochemical reaction. The tests with formate dehydrogenase demonstrated that 94% of the regenerated NAD+ was enzymatically active.

Lorena Wilson

Papers

Lactose-Derived Prebiotics: A Process Perspective

Optimization of reaction conditions and the donor substrate in the synthesis of hexyl-β-d-galactoside

Comparative study of the enzymatic synthesis of cephalexin at high substrate concentration in aqueous and organic media using statistical model

Synthesis of Ascorbyl Palmitate with Immobilized Lipase from Pseudomonas stutzeri

ZnO Materials as Effective Anodes for the Photoelectrochemical Regeneration of Enzymatically Active NAD.