Improvement of enzyme activity, stability and selectivity via immobilization techniques
TL;DR: In all cases, enzyme engineering via immobilization techniques is perfectly compatible with other chemical or biological approaches to improve enzyme functions and the final success depend on the availability of a wide battery of immobilization protocols.
About: This article is published in Enzyme and Microbial Technology.The article was published on 2007-05-02. It has received 3016 citations till now. The article focuses on the topics: Immobilized enzyme.
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TL;DR: In this tutorial review, some of the main reasons that may produce an improvement in enzyme activity, specificity or selectivity, either real or apparent, due to immobilization are listed.
Abstract: Immobilization of enzymes may produce alterations in their observed activity, specificity or selectivity. Although in many cases an impoverishment of the enzyme properties is observed upon immobilization (caused by the distortion of the enzyme due to the interaction with the support) in some instances such properties may be enhanced by this immobilization. These alterations in enzyme properties are sometimes associated with changes in the enzyme structure. Occasionally, these variations will be positive. For example, they may be related to the stabilization of a hyperactivated form of the enzyme, like in the case of lipases immobilized on hydrophobic supports via interfacial activation. In some other instances, these improvements will be just a consequence of random modifications in the enzyme properties that in some reactions will be positive while in others may be negative. For this reason, the preparation of a library of biocatalysts as broad as possible may be a key turning point to find an immobilized biocatalyst with improved properties when compared to the free enzyme. Immobilized enzymes will be dispersed on the support surface and aggregation will no longer be possible, while the free enzyme may suffer aggregation, which greatly decreases enzyme activity. Moreover, enzyme rigidification may lead to preservation of the enzyme properties under drastic conditions in which the enzyme tends to become distorted thus decreasing its activity. Furthermore, immobilization of enzymes on a support, mainly on a porous support, may in many cases also have a positive impact on the observed enzyme behavior, not really related to structural changes. For example, the promotion of diffusional problems (e.g., pH gradients, substrate or product gradients), partition (towards or away from the enzyme environment, for substrate or products), or the blocking of some areas (e.g., reducing inhibitions) may greatly improve enzyme performance. Thus, in this tutorial review, we will try to list and explain some of the main reasons that may produce an improvement in enzyme activity, specificity or selectivity, either real or apparent, due to immobilization.
1,487 citations
TL;DR: The advantages and disadvantages of the different existing immobilization strategies to solve the different aforementioned enzyme limitations are given and some advice to select the optimal strategy for each particular enzyme and process is given.
Abstract: 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).
1,378 citations
TL;DR: This tutorial review focuses on the understanding of enzyme immobilisation, which can address the issue of enzymatic instability.
Abstract: Enzymes are versatile catalysts in the laboratory and on an industrial scale. To broaden their applicability in the laboratory and to ensure their (re)use in manufacturing the stability of enzymes can often require improvement. Immobilisation can address the issue of enzymatic instability. Immobilisation can also help to enable the employment of enzymes in different solvents, at extremes of pH and temperature and exceptionally high substrate concentrations. At the same time substrate-specificity, enantioselectivity and reactivity can be modified. However, most often the molecular and physical–chemical bases of these phenomena have not been elucidated yet. This tutorial review focuses on the understanding of enzyme immobilisation.
1,115 citations
TL;DR: An overview of the denaturation mechanisms in aqueous and non-aqueous environment is given in this article, and various methods of enzyme stabilization with respect to their use in the aqueously and nonaqueous environments have been given.
1,009 citations
Cites background or methods from "Improvement of enzyme activity, sta..."
...Further, decreased inhibition by reaction products, selectivity towards non-natural substrates and better functional properties compared to the corresponding soluble enzymes, all make immobilization one of the most preferred method of enzyme improvement towards stabilization [32]....
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...[32] have given an excellent review of immobilization as a method for stabilization of multimeric enzymes, stabilization towards industrial application and storage stability....
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TL;DR: The aim of this review is to emphasise the importance of measure as well as possible, the last stage of the biodegradation, in order to certify the integration of new materials into the biogeochemical cycles.
911 citations
Cites background from "Improvement of enzyme activity, sta..."
...Moreover, they are also protected against autocatalytic denaturation (in particular proteases) (Mateo et al., 2007)....
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References
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TL;DR: An overview of glutaraldehyde as a crosslinking reagent is given by describing its structure and chemical properties in aqueous solution in an attempt to explain its high reactivity toward proteins, particularly as applied to the production of insoluble enzymes.
Abstract: Glutaraldehyde possesses unique characteristics that render it one of the most effective protein crosslinking reagents. It can be present in at least 13 different forms depending on solution conditions such as pH, concentration, temperature, etc. Substantial literature is found concerning the use of glutaraldehyde for protein immobilization, yet there is no agreement about the main reactive species that participates in the crosslinking process because monomeric and polymeric forms are in equilibrium. Glutaraldehyde may react with proteins by several means such as aldol condensation or Michael-type addition, and we show here 8 different reactions for various aqueous forms of this reagent. As a result of these discrepancies and the unique characteristics of each enzyme, crosslinking procedures using glutaraldehyde are largely developed through empirical observation. The choice of the enzyme-glutaraldehyde ratio, as well as their final concentration, is critical because insolubilization of the enzyme must result in minimal distortion of its structure in order to retain catalytic activity. The purpose of this paper is to give an overview of glutaraldehyde as a crosslinking reagent by describing its structure and chemical properties in aqueous solution in an attempt to explain its high reactivity toward proteins, particularly as applied to the production of insoluble enzymes.
1,515 citations
"Improvement of enzyme activity, sta..." refers methods in this paper
...The glutaraldehyde technique is very versatile and ay be used in very different fashions [37–39]....
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...[37] Migneault I....
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TL;DR: The X-ray structure of the Mucor miehei triglyceride lipase is reported and the atomic model obtained reveals a Ser .. His .. Asp trypsin-like catalytic triad with an active serine buried under a short helical fragment of a long surface loop.
Abstract: True lipases attach triacylglycerols and act at an oil-water interface; they constitute a ubiquitous group of enzymes catalysing a wide variety of reactions, many with industrial potential. But so far the three-dimensional structure has not been reported for any lipase. Here we report the X-ray structure of the Mucor miehei triglyceride lipase and describe the atomic model obtained at 3.1 A resolution and refined to 1.9 A resolution. It reveals a Ser..His..Asp trypsin-like catalytic triad with an active serine buried under a short helical fragment of a long surface loop.
1,149 citations
TL;DR: It is proposed that the structure of the enzyme in this complex of R. miehei lipase with n-hexylphosphonate ethyl ester is equivalent to the activated state generated by the oil–water interface.
Abstract: LIPASES are hydrolytic enzymes which break down triacylglycerides into free fatty acids and glycerols. They have been classified as serine hydrolases owing to their inhibition by diethyl p-nitrophenyl phosphate1. Lipase activity is greatly increased at the lipid-water interface2,3, a phenomenon known as interfacial activation. X-ray analysis has revealed the atomic structures of two triacylglycerol lipases, unrelated in sequence: the human pancreatic lipase (hPL)4, and an enzyme isolated from the fungus Rhizomucor (formerly Mucor) miehei5 (RmL). In both enzymes the active centres contain structurally analogous Asp-His-Ser triads (characteristic of serine proteinases), which are buried completely beneath a short helical segment, or 'lid'. Here we present the crystal structure (at 3 A resolution) of a complex of R. miehei lipase with n-hexylphosphonate ethyl ester in which the enzyme's active site is exposed by the movement of the helical lid. This movement also increases the nonpolarity of the surface surrounding the catalytic site. We propose that the structure of the enzyme in this complex is equivalent to the activated state generated by the oil–water interface.
1,068 citations
"Improvement of enzyme activity, sta..." refers background in this paper
...The closed orm, considered inactive, where the active site is isolated from he reaction medium by a polypeptide chain called lid, and the pen form, where this lid is displaced and the active site is fully xposed to the reaction medium [95–98]....
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...As stated above, lipases suffer very large conformational hanges during catalysis [95–98]....
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...[96] Brzozowski AM, Derewenda ZS, Dodson GG, Lawson DM, Turkenburg...
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Book•
01 Jan 1994
TL;DR: Part 1 general aspects: rate acceleration in enzyme-catalyzed reactions Michaelis-Menten kinetics enzyme inhibition specificity improvement or alteration of enzyme specifity enzyme stabilization and reactor configuration cofactor regeneration enzyme catalysis in organic solvents multienzyme systems and metabolic engineering rational design of new enzymatic catalysts references.
Abstract: Part 1 General aspects: rate acceleration in enzyme-catalyzed reactions Michaelis-Menten kinetics enzyme inhibition specificity improvement or alteration of enzyme specifity enzyme stabilization and reactor configuration cofactor regeneration enzyme catalysis in organic solvents multienzyme systems and metabolic engineering rational design of new enzymatic catalysts references. Part 2 Use of hydrolytic enzymes - amidases, proteases, esterases, lipases, nitrilases, phosphatases, epoxide hydrolases: amidases protease-catalyzed peptide synthesis proteases that act as esterases acetylcholine esterase pig liver esterase phospholipases cholesterol esterase lipases nitrile hydrolysis enzymes epoxide hydrolase phosphatase references. Part 3 Oxidoreductions: nicotinamide cofactor dependent oxidoreductions dehydrogenases which utilize ketoacids as substrates other NAD(P)-dependent dehydrogenases oxidoreductases that are metalloenzymes references. Part 4 C-C bond formation: aldol condensation ketol and aldol transfer reaction addition of HCN to aldehydes acyloin condensation C-C bond forming reactions involving acetyl coA isoprenoid and steroid synthesis replacement of chloroalanine C-C bond formation catalyzed by vitamin B[12] references. Part 5 Synthesis of glycoside bonds: background glycosyltransferases of the Leloir pathway substrate specificity and synthetic applications of glycosyltransferases non-leloir glycosyltransferases glycosidases transglycosidases synthesis of N-glycosides biological applications of synthetic glycoconjugates future opportunities references. Part 6 Addition, elimination and other group transfer reactions (phosphoryl-, methyl-,sulpho-and amino-transfer reactions): addition of water to alkenes - fumarase addition of ammonia to double bonds - ammonia lyases transamination - aminotransferases addition and elimination of carboxyl group nucleoside triphosphate requiring enzymatic reactions preparation of ATP chiral at -, - or - phosphorous phosphorothioate-containing DNA and RNA DNA and RNA oligomers incorporation of modified or unnatural bases into DNA or RNA dehalogenation synthesis of chiral methyl groups S-adenosylmethionine and transmethylation sulfate activation and transfer reactions.
812 citations
"Improvement of enzyme activity, sta..." refers background in this paper
...[2] Wong C-H, Whitesides GM....
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...Enzymes are catalysts bearing some excellent properties high activity, selectivity and specificity) that may permit to erform the most complex chemical processes under the most enign experimental and environmental conditions [1,2]....
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TL;DR: Enzyme-catalysed chemical transformations are now widely recognized as practical alternatives to traditional (non-biological) organic synthesis, and as convenient solutions to certain intractable synthetic problems.
Abstract: New catalytic synthetic methods in organic chemistry that satisfy increasingly stringent environmental constraints are in great demand by the pharmaceutical and chemical industries. In addition, novel catalytic procedures are necessary to produce the emerging classes of organic compounds that are becoming the targets of molecular and biomedical research. Enzyme-catalysed chemical transformations are now widely recognized as practical alternatives to traditional (non-biological) organic synthesis, and as convenient solutions to certain intractable synthetic problems.
800 citations
"Improvement of enzyme activity, sta..." refers background in this paper
...[1] Koeller KM, Wong CH....
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...Enzymes are catalysts bearing some excellent properties high activity, selectivity and specificity) that may permit to erform the most complex chemical processes under the most enign experimental and environmental conditions [1,2]....
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