Cross-linked enzyme aggregate: Difference between revisions

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In [[biochemistry]], a '''cross-linked enzyme aggregate''' is an [[immobilized enzyme]] prepared via [[crosslinkingcross-link]]ing of the physical enzyme aggregates with a difunctional cross-linker. They can be used as [[stereoselective]] industrial [[biocatalyst]]s.

==Background==

Enzymes are [[proteins]] that catalyze (''i.e.'' accelerate) [[chemical reactionsreaction]]s. They are Nature’snatural [[catalystscatalyst]]s and are ubiquitous, in plants, animals and [[microorganismsmicroorganism]]s where they catalyze processes that are vital to living organisms. They are intimately involved in numerous biotechnological processes, such as cheese making, beer brewing and winemaking, that date back to the dawn of civilization. Recent advances in [[biotechnology]], particularly in genetic and [[protein engineering]], and [[genetics]] have provided the basis for the efficient development of [[enzymesenzyme]]s with improved properties for established applications and novel, tailor-made [[enzymesenzyme]]s for completely new applications where [[enzymes]] were not previously used.

NowadaysToday, [[enzymes]] are widely applied in many different industries and the number of applications continues to increase. Examples include [[food]] (baking, dairy products, starch conversion) and [[beverage]] (beer, wine, fruit and vegetable juices) processing, [[compound feed|animal feed]], [[textilestextile]]s, [[wood pulp|pulp]] and [[paper]], [[detergentsdetergent]]s, [[biosensorsbiosensor]]s, [[cosmetics]], [[health care]] and [[nutrition]], [[Sewage treatment|waste water treatment]], [[pharmaceuticalspharmaceutical]] and [[chemicalschemical]] manufacture and, more recently, [[biofuelsbiofuel]]s such as [[biodiesel]]. The main driver for the widespread application of enzymes is their small environmental footprint.

Many traditional chemical conversions used in various industries suffer from inherent drawbacks from both an economic and environmental viewpoint. Non-specific reactions can afford low product yields, copious amounts of waste and impure products. The need for elevated temperatures and pressures leads to high energy consumption and high [[capital investment]] costs. Disposal of unwanted by-products may be difficult and/or expensive and hazardous solvents may be required. In stark contrast, enzymatic reactions are performed under mild conditions of temperature and pressure, in water as solvent, and exhibit very high rates and are often highly specific. Moreover, they are produced from renewable raw materials and are [[biodegradable]]. In addition, the mild operating conditions of enzymatic processes mean that they can be performed in relatively simple equipment and are easy to control. In short, they reduce the environmental footprint of manufacturing by reducing the consumption of energy and chemicals and concomitant generation of waste.

In the production of [[fine chemicals]], [[flavorsFlavoring|flavor]]s and [[fragrancesAroma compound|fragrance]]s, [[agrochemicalsagrochemical]]s and [[pharmaceuticalspharmaceutical]]s an important benefit of [[enzymesenzyme]]s is the high degree of [[chemoselectivity]], [[regioselectivity]] and [[enantioselectivity]] which they exhibit. Particularly, their ability to catalyze the formation of products in high [[enantiopurity]], by an exquisite stereochemical control, is of the utmost importance in these industries.

Notwithstanding all these desirable characteristic features of enzymes, their widespread industrial application is often hampered by their lack of long term operational stability and shelf-storage life, andas well as by their cumbersome recovery and re-use. These drawbacks can be generally be overcome by immobilizationenzyme ofimmobilization. the enzymeA andmajor a majorpresent challenge in industrial biocatalysis is the development of stable, robust and preferably insoluble biocatalysts.

==ImmobilisationImmobilization==

:''See [[Immobilized enzyme]] for more information.''

There are several reasons for immobilizing an enzyme. In addition to more convenient handling of the enzyme, it provides for its facile separation from the product, thereby minimizing or eliminating protein contamination of the product. Immobilization also facilitates the efficient recovery and re-use of costly enzymes, in many applications a conditio sine qua non for economic viability, and enables their use in continuous, fixed-bed operation. A further benefit is often enhanced stability, under both storage and operational conditions, e.g. towards [[Denaturation (biochemistry)|denaturation]] by heat or organic solvents or by [[Autolysis (biology)|autolysis]]. Enzymes are rather delicate molecules that can easily lose their unique three -dimensional structure, essential for their activity, by [[Denaturation (biochemistry)|denaturation]] (unfolding). Improved enzyme performance via enhanced stability, over a broad pH and temperature range as well as tolerance towards organic solvents, coupled with repeated re-use is reflected in higher catalyst productivities (kg product/kg enzyme) which, in turn, determine the enzyme costs per kg product.

Basically, three traditional methods of [[enzyme immobilization]] can be distinguished: binding to a support(carrier), entrapment (encapsulation) and cross-linking. Support binding can be physical, [[Ionic bond|ionic]], or [[covalent]] in nature. However, physical bonding is generally too weak to keep the enzyme fixed to the carrier under industrial conditions of high reactant and product concentrations and high ionic strength. The support can be a synthetic [[resin]], a [[biopolymer]] or an inorganic [[polymer]] such as (mesoporous) silica or a [[zeolite]]. Entrapment involves inclusion of an enzyme in a polymer network (gel lattice) such as an organic polymer or a silica [[sol-gel]], or a [[Membrane (selective barrier)|membrane]] device such as a hollow fiber or a microcapsule. Entrapment requires the synthesis of the polymeric network in the presence of the enzyme. The third category involves cross-linking of enzyme aggregates or crystals, using a bifunctional reagent, to prepare carrier-free macroparticles.

The use of a carrier inevitably leads to ‘dilution of activity’, owing to the introduction of a large portion of non-catalytic ballast, ranging from 90% to >99%, which results in lower space-time yields and productivities. Moreover, immobilization of an enzyme on a carrier often leads to a substantial loss of activity, especially at high enzyme loadings. Consequently, there is an increasing interest in carrier-free immobilized enzymes, such as cross-linked enzyme crystals (CLECs) and cross-linked enzyme aggregates (CLEAs) that offer the advantages of highly concentrated enzyme activity combined with high stability and low production costs owing to the exclusion of an additional (expensive) carrier. <ref>Cao, L.; van Langen, L.; [[Roger A. Sheldon|Sheldon, R.A.]]; Immobilised enzymes: carrier-bound of carrier-free?; Curr. Opin. Biotechnol., 2003, 14, 387-394. {{doi|10.1016/S0958-1669(03)00096-X}}</ref>

==Cross-Linked Enzyme Aggregates (CLEAs)==

The use of cross-linked enzyme crystals (CLECs) as industrial [[biocatalysts]] was pioneered by Altus Biologics in the 1990s. CLECs proved to be significantly more stable to [[Denaturation (biochemistry)|denaturation]] by heat, organic solvents and [[proteolysis]] than the corresponding soluble enzyme or lyophilized (freeze-dried) powder. CLECs are robust, highly active immobilized enzymes of controllable particle size, varying from 1 to 100 micrometer. Their operational stability and ease of recycling, coupled with their high catalyst and volumetric productivities, renders them ideally suited for industrial biotransformations.

However, CLECs have an inherent disadvantage: enzyme [[crystallization]] is a laborious procedure requiring enzyme of high purity, which translates to prohibitively high costs. The more recently developed cross-linked enzyme aggregates (CLEAs) , on the other hand, are produced by simple precipitation of the enzyme from aqueous solution, as physical aggregates of protein molecules, by the addition of salts, or water miscible organic solvents or non-ionic polymers.<ref>[[Roger A. Sheldon|Sheldon, R.A.]]; Schoevaart, R.; van Langen, L.; A novel method for enzyme immobilization; Biocat. Biotrans, 2005, 23(3/4), 141-147. </ref><ref>[[Roger A. Sheldon|Sheldon, R.A.]]; Enzyme immobilisation: The quest for optimum performance; Adv. Synth. Catal., 2007, 349, 387-394. </ref> The physical aggregates are held together by non-covalent bonding without perturbation of their tertiary structure, that is without denaturation. Subsequent cross-linking of these physical aggregates renders them permanently insoluble while maintaining their pre-organized superstructure, and, hence their catalytic activity. This discovery led to the development of a new family of immobilized enzymes: cross-linked enzyme aggregates (CLEAs). Since precipitation from an aqueous medium, by addition of [[ammonium sulfate]] or [[polyethylene glycol]], is often used to purify enzymes, the CLEA methodology essentially combines purification and immobilization into a single unit operation that does not require a highly pure enzyme. It could be used, for example, for the direct isolation of an enzyme, in a purified and immobilized form suitable for performing biotransformations, from a crude [[Fermentation (biochemistry)|fermentation]] broth.

CLEAs are very attractive biocatalysts, owing to their facile, inexpensive and effective production method. They can readily be reused and exhibit improved stability and performance. The methodology is applicable to essentially any enzyme, including [[Cofactor (biochemistry)|cofactor]] dependent oxidoreductases.<ref>[[Roger A. Sheldon|Sheldon, R.A.]]; Sorgedrager, M.J.; Janssen, M.H.A.; Use of cross-linked enzyme aggregates (CLEAs) for performing biotransformations; Chemistry Today, 2007, 25, 62-67. </ref> Application to penicillin acylase used in [[antibiotic]] synthesis showed large improvements over other type of biocatalysts.<ref>Illanes, A.; Wilson, L.; Caballero, E.; Fernandez-Lafuente, R.; Guisan, J.M.; Crosslinked penicillin acylase aggregates for synthesis of beta-lactam antibiotics in organic medium; Appl. Biochem. Biotechnol, 2006, 133, 189-202. </ref>

The potential applications of CLEAs are numerous and include:

1. # Synthesis of [[pharmaceuticals]], [[flavorsFlavoring|flavor]]s and [[fragrancesAroma compound|fragrance]]s, [[agrochemicalsagrochemical]]s, [[nutraceuticalsnutraceutical]]s, [[fine chemicals]], bulk monomers and [[biofuelsbiofuel]]s.

# [[Compound feed|Animal feed]], ''e.g.'' [[phytase]] for utilization of organically bound [[phosphate]] by pigs and poultry.

2. # [[Animalfood feedprocessing|Food]] and beverage processing, ''e.g.'' [[phytaselipases]] forin utilizationcheese ofmanufacture organically boundand [[phosphatelaccase]] byin pigs[[wine and poultryclarification]].

# [[Cosmetics]], ''e.g.'' in skin care products

3. Food# [[Oils]] and beverage[[fat]]s processing, ''e.g.'' in bio[[lipaseslubricants]], in cheese manufacture and bio[[laccaseemulsifiers]], inbio-derived wine clarification[[emollients]].

6. # [[Carbohydrate]] processing, ''e.g.'' [[laccase]] in carbohydrate oxidations.

4. # [[CosmeticsPulp and paper]], ''e.g.'' in skinpulp care productsbleaching.

8. # [[Detergents]], ''e.g.'' [[proteases]], [[amylases]] and [[lipases]] for removal of protein, carbohydrate and fat stains.

5. [[Oils]]# andWaste [[fats]]water processingtreatment, ''e.g.'' infor removal of bio[[lubricantsphenols]], bio[[emulsifiersdyes]], bioand [[emollientsendocrine disruptor|endocrine disrupters]].

10. # [[Biosensors]]/[[diagnostics]], ''e.g.'' [[glucose oxidase]] and cholesterol oxidase biosensors.

11. # Delivery of proteins as therapeutic agents or nutritional/digestive supplements ''e.g.'' [[beta-galactosidase]] for digestive hydrolysis of [[lactose]] in dairy products to alleviate the symptoms of [[lactose intolerance]].

6. [[Carbohydrate]] processing, ''e.g.'' [[laccase]] in carbohydrate oxidations.

7. Pulp and paper, ''e.g.'' in pulp bleaching.

8. [[Detergents]], ''e.g.'' [[proteases]], [[amylases]] and [[lipases]] for removal of protein, carbohydrate and fat stains.

9. Waste water treatment, ''e.g.'' for removal of [[phenols]], [[dyes]], and endocrine disrupters.

10. [[Biosensors]]/[[diagnostics]], ''e.g.'' [[glucose oxidase]] and cholesterol oxidase biosensors.

11. Delivery of proteins as therapeutic agents or nutritional/digestive supplements ''e.g.'' [[beta-galactosidase]] for digestive hydrolysis of [[lactose]] in dairy products to alleviate the symptoms of [[lactose intolerance]].

==References==

{{reflist}}

==External links==

* [http://www.cleatechnologies.com/technology.html Description of the Methodology]

{{Chiral synthesis}}

[[Category:Organic chemistryCatalysis]]

[[Category:Enzymes]]

[[Category:Stereochemistry]]