Industrial enzymes

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Industrial enzymes are enzymes that are commercially used in a variety of industries such as pharmaceuticals, chemical production, biofuels, food & beverage, and consumer products. Due to advancements in recent years, biocatalysis through isolated enzymes is considered more economical than use of whole cells. Enzymes may be used as a unit operation within a process to generate a desired product, or may be the product of interest. Industrial biological catalysis through enzymes has experienced rapid growth in recent years due to their ability to operate at mild conditions, and exceptional chiral and positional specificity, things that traditional chemical processes lack. [1] Isolated enzymes are typically used in hydrolytic and isomerization reactions. Whole cells are typically used when a reaction requires a co-factor. Although co-factors may be generated in vitro, it is typically more cost-effective to use metabolically active cells. [1]

Contents

Enzymes as a unit of operation

Immobilization

Despite their excellent catalytic capabilities, enzymes and their properties must be improved prior to industrial implementation in many cases. Some aspects of enzymes that must be improved prior to implementation are stability, activity, inhibition by reaction products, and selectivity towards non-natural substrates. This may be accomplished through immobilization of enzymes on a solid material, such as a porous support. [2] Immobilization of enzymes greatly simplifies the recovery process, enhances process control, and reduces operational costs. Many immobilization techniques exist, such as adsorption, covalent binding, affinity, and entrapment. [3] Ideal immobilization processes should not use highly toxic reagents in the immobilization technique to ensure stability of the enzymes. [4] After immobilization is complete, the enzymes are introduced into a reaction vessel for biocatalysis.

Adsorption

Enzyme adsorption onto carriers functions based on chemical and physical phenomena such as van der Waals forces, ionic interactions, and hydrogen bonding. These forces are weak, and as a result, do not affect the structure of the enzyme. A wide variety of enzyme carriers may be used. Selection of a carrier is dependent upon the surface area, particle size, pore structure, and type of functional group. [5]

Covalent binding

Example of Enzyme Immobilization through Covalent Binding Figure 1 Example of Enzyme Immobilization by Covalent Binding.png
Example of Enzyme Immobilization through Covalent Binding

Many binding chemistries may be used to adhere an enzyme to a surface to varying degrees of success. The most successful covalent binding techniques include binding via glutaraldehyde to amino groups and N-hydroxysuccinide esters. These immobilization techniques occur at ambient temperatures in mild conditions, which have limited potential to modify the structure and function of the enzyme. [6]

Affinity

Immobilization using affinity relies on the specificity of an enzyme to couple an affinity ligand to an enzyme to form a covalently bound enzyme-ligand complex. The complex is introduced into a support matrix for which the ligand has high binding affinity, and the enzyme is immobilized through ligand-support interactions. [3]

Entrapment

Immobilization using entrapment relies on trapping enzymes within gels or fibers, using non-covalent interactions. Characteristics that define a successful entrapping material include high surface area, uniform pore distribution, tunable pore size, and high adsorption capacity. [3]

Recovery

Enzymes typically constitute a significant operational cost for industrial processes, and in many cases, must be recovered and reused to ensure economic feasibility of a process. Although some biocatalytic processes operate using organic solvents, the majority of processes occur in aqueous environments, improving the ease of separation. [1] Most biocatalytic processes occur in batch, differentiating them from conventional chemical processes. As a result, typical bioprocesses employ a separation technique after bioconversion. In this case, product accumulation may cause inhibition of enzyme activity. Ongoing research is performed to develop in situ separation techniques, where product is removed from the batch during the conversion process. Enzyme separation may be accomplished through solid-liquid extraction techniques such as centrifugation or filtration, and the product-containing solution is fed downstream for product separation. [1]

Enzymes as a Unit Operation
EnzymeIndustryApplication
Palatase [7] FoodEnhance cheese flavor
Lipozyme TL IM [7] FoodInteresterification of vegetable oil
Lipase AK Amano [7] PharmaceuticalSynthesis of chiral compounds
Lipopan F [7] FoodEmulsifier
Cellulase [8] BiofuelClass of enzymes that degrade cellulose to glucose monomers
Amylase [9] Food/biofuelClass of enzymes that degrade starch to glucose monomers
Xylose isomerase [10] Food High-fructose corn syrup production
Resinase [7] PaperPitch control in paper processing
Penicillin amidase [11] PharmaceuticalSynthetic antibiotic production
Amidase ChemicalClass of enzymes used for non-proteinogenic enantiomerically pure amino acid production

Enzymes as a desired product

To industrialize an enzyme, the following upstream and downstream enzyme production processes are considered:

Upstream

Upstream processes are those that contribute to the generation of the enzyme.

Selection of a suitable enzyme

An enzyme must be selected based upon the desired reaction. The selected enzyme defines the required operational properties, such as pH, temperature, activity, and substrate affinity. [12]

Identification and selection of a suitable source for the selected enzyme

The choice of a source of enzymes is an important step in the production of enzymes. It is common to examine the role of enzymes in nature and how they relate to the desired industrial process. Enzymes are most commonly sourced through bacteria, fungi, and yeast. Once the source of the enzyme is selected, genetic modifications may be performed to increase the expression of the gene responsible for producing the enzyme. [12]

Process development

Process development is typically performed after genetic modification of the source organism, and involves the modification of the culture medium and growth conditions. In many cases, process development aims to reduce mRNA hydrolysis and proteolysis. [12]

Large scale production

Scaling up of enzyme production requires optimization of the fermentation process. Most enzymes are produced under aerobic conditions, and as a result, require constant oxygen input, impacting fermenter design. Due to variations in the distribution of dissolved oxygen, as well as temperature, pH, and nutrients, the transport phenomena associated with these parameters must be considered. The highest possible productivity of the fermenter is achieved at maximum transport capacity of the fermenter. [12] [13]

Downstream

Downstream processes are those that contribute to separation or purification of enzymes.

Removal of insoluble materials and recovery of enzymes from the source

The procedures for enzyme recovery depend on the source organism, and whether enzymes are intracellular or extracellular. Typically, intracellular enzymes require cell lysis and separation of complex biochemical mixtures. Extracellular enzymes are released into the culture medium, and are much simpler to separate. Enzymes must maintain their native conformation to ensure their catalytic capability. Since enzymes are very sensitive to pH, temperature, and ionic strength of the medium, mild isolation conditions must be used. [12]

Concentration and primary purification of enzymes

Depending on the intended use of the enzyme, different levels purity are required. For example, enzymes used for diagnostic purposes must be separated to a higher purity than bulk industrial enzymes to prevent catalytic activity that provides erroneous results. Enzymes used for therapeutic purposes typically require the most rigorous separation. Most commonly, a combination of chromatography steps is employed for separation. [12]

The purified enzymes are either sold in pure form and sold to other industries, or added to consumer goods.

Enzymes as a Desired Product
EnzymeIndustryApplication
Novozym-435 [7] Consumer Goods Isopropyl myristate production (Cosmetic)
Bromelain [14] FoodMeat tenderizer
Noopazyme [7] FoodImprove noodle quality
Asparaginase [15] PharmaceuticalLymphatic cancer therapeutic
Ficin [16] PharmaceuticalDigestive aid
Urokinase [17] PharmaceuticalAnticoagulant
β-Lactamase PharmaceuticalPenicillin allergy treatment
Subtilisin [18] Consumer GoodsLaundry detergent

See also

Related Research Articles

In chemical analysis, chromatography is a laboratory technique for the separation of a mixture into its components. The mixture is dissolved in a fluid solvent called the mobile phase, which carries it through a system on which a material called the stationary phase is fixed. Because the different constituents of the mixture tend to have different affinities for the stationary phase and are retained for different lengths of time depending on their interactions with its surface sites, the constituents travel at different apparent velocities in the mobile fluid, causing them to separate. The separation is based on the differential partitioning between the mobile and the stationary phases. Subtle differences in a compound's partition coefficient result in differential retention on the stationary phase and thus affect the separation.

ELISA Method to detect an antigen using an antibody and enzyme

The enzyme-linked immunosorbent assay (ELISA) is a commonly used analytical biochemistry assay, first described by Eva Engvall and Peter Perlmann in 1971. The assay uses a solid-phase type of enzyme immunoassay (EIA) to detect the presence of a ligand in a liquid sample using antibodies directed against the protein to be measured. ELISA has been used as a diagnostic tool in medicine, plant pathology, and biotechnology, as well as a quality control check in various industries.

Chemical specificity is the ability of binding site of a macromolecule to bind specific ligands. The fewer ligands a protein can bind, the greater its specificity.

Binding site Molecule-specific coordinate bonding area in biological systems

In biochemistry and molecular biology, a binding site is a region on a macromolecule such as a protein that binds to another molecule with specificity. The binding partner of the macromolecule is often referred to as a ligand. Ligands may include other proteins, enzyme substrates, second messengers, hormones, or allosteric modulators. The binding event is often, but not always, accompanied by a conformational change that alters the protein's function. Binding to protein binding sites is most often reversible, but can also be covalent reversible or irreversible.

Protein purification is a series of processes intended to isolate one or a few proteins from a complex mixture, usually cells, tissues or whole organisms. Protein purification is vital for the specification of the function, structure and interactions of the protein of interest. The purification process may separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins. Separation of one protein from all others is typically the most laborious aspect of protein purification. Separation steps usually exploit differences in protein size, physico-chemical properties, binding affinity and biological activity. The pure result may be termed protein isolate.

Bioreactor

A bioreactor refers to any manufactured device or system that supports a biologically active environment. In one case, a bioreactor is a vessel in which a chemical process is carried out which involves organisms or biochemically active substances derived from such organisms. This process can either be aerobic or anaerobic. These bioreactors are commonly cylindrical, ranging in size from litres to cubic metres, and are often made of stainless steel. It may also refer to a device or system designed to grow cells or tissues in the context of cell culture. These devices are being developed for use in tissue engineering or biochemical/bioprocess engineering.

Affinity chromatography is a method of separating a biomolecule from a mixture, based on a highly specific macromolecular binding interaction between the biomolecule and another substance. The specific type of binding interaction depends on the biomolecule of interest; antigen and antibody, enzyme and substrate, receptor and ligand, or protein and nucleic acid binding interactions are frequently exploited for isolation of various biomolecules. Affinity chromatography is useful for its high selectivity and resolution of separation, compared to other chromatographic methods.

Molecular imprinting Technique in polymer chemistry

Molecular imprinting is a technique to create template-shaped cavities in polymer matrices with predetermined selectivity and high affinity. This technique is based on the system used by enzymes for substrate recognition, which is called the "lock and key" model. The active binding site of an enzyme has a shape specific to a substrate. Substrates with a complementary shape to the binding site selectively bind to the enzyme; alternative shapes that do not fit the binding site are not recognized.

Biocatalysis Use of natural catalysts to perform chemical transformations

Biocatalysis refers to the use of living (biological) systems or their parts to speed up (catalyze) chemical reactions. In biocatalytic processes, natural catalysts, such as enzymes, perform chemical transformations on organic compounds. Both enzymes that have been more or less isolated and enzymes still residing inside living cells are employed for this task. Modern biotechnology, specifically directed evolution, has made the production of modified or non-natural enzymes possible. This has enabled the development of enzymes that can catalyze novel small molecule transformations that may be difficult or impossible using classical synthetic organic chemistry. Utilizing natural or modified enzymes to perform organic synthesis is termed chemoenzymatic synthesis; the reactions performed by the enzyme are classified as chemoenzymatic reactions.

Schild regression

In pharmacology, Schild regression analysis, named for Heinz Otto Schild, is a tool for studying the effects of agonists and antagonists on the response caused by the receptor or on ligand-receptor binding.

Immobilized enzyme

An immobilized enzyme is an enzyme attached to an inert, insoluble material—such as calcium alginate. This can provide increased resistance to changes in conditions such as pH or temperature. It also lets enzymes be held in place throughout the reaction, following which they are easily separated from the products and may be used again - a far more efficient process and so is widely used in industry for enzyme catalysed reactions. An alternative to enzyme immobilization is whole cell immobilization.

Biomolecular engineering is the application of engineering principles and practices to the purposeful manipulation of molecules of biological origin. Biomolecular engineers integrate knowledge of biological processes with the core knowledge of chemical engineering in order to focus on molecular level solutions to issues and problems in the life sciences related to the environment, agriculture, energy, industry, food production, biotechnology and medicine.

Molecular binding is an attractive interaction between two molecules that results in a stable association in which the molecules are in close proximity to each other. It is formed when atoms or molecules bind together by sharing of electrons. It often, but not always, involves some chemical bonding.

In biochemistry, a cross-linked enzyme aggregate is an immobilized enzyme prepared via cross-linking of the physical enzyme aggregates with a difunctional cross-linker. They can be used as stereoselective industrial biocatalysts.

An enzymatic biofuel cell is a specific type of fuel cell that uses enzymes as a catalyst to oxidize its fuel, rather than precious metals. Enzymatic biofuel cells, while currently confined to research facilities, are widely prized for the promise they hold in terms of their relatively inexpensive components and fuels, as well as a potential power source for bionic implants.

Affinity electrophoresis

Affinity electrophoresis is a general name for many analytical methods used in biochemistry and biotechnology. Both qualitative and quantitative information may be obtained through affinity electrophoresis. The methods include the so-called electrophoretic mobility shift assay, charge shift electrophoresis and affinity capillary electrophoresis. The methods are based on changes in the electrophoretic pattern of molecules through biospecific interaction or complex formation. The interaction or binding of a molecule, charged or uncharged, will normally change the electrophoretic properties of a molecule. Membrane proteins may be identified by a shift in mobility induced by a charged detergent. Nucleic acids or nucleic acid fragments may be characterized by their affinity to other molecules. The methods have been used for estimation of binding constants, as for instance in lectin affinity electrophoresis or characterization of molecules with specific features like glycan content or ligand binding. For enzymes and other ligand-binding proteins, one-dimensional electrophoresis similar to counter electrophoresis or to "rocket immunoelectrophoresis", affinity electrophoresis may be used as an alternative quantification of the protein. Some of the methods are similar to affinity chromatography by use of immobilized ligands.

A ligand binding assay (LBA) is an assay, or an analytic procedure, which relies on the binding of ligand molecules to receptors, antibodies or other macromolecules. A detection method is used to determine the presence and extent of the ligand-receptor complexes formed, and this is usually determined electrochemically or through a fluorescence detection method. This type of analytic test can be used to test for the presence of target molecules in a sample that are known to bind to the receptor.

Chemoproteomics entails a broad array of techniques used to identify and interrogate protein-small molecule interactions. Chemoproteomics complements phenotypic drug discovery, a paradigm that aims to discover lead compounds on the basis of alleviating a disease phenotype, as opposed to target-based drug discovery, in which lead compounds are designed to interact with predetermined disease-driving biological targets. As phenotypic drug discovery assays do not provide confirmation of a compound's mechanism of action, chemoproteomics provides valuable follow-up strategies to narrow down potential targets and eventually validate a molecule's mechanism of action. Chemoproteomics also attempts to address the inherent challenge of drug promiscuity in small molecule drug discovery by analyzing protein-small molecule interactions on a proteome-wide scale. A major goal of chemoproteomics is to characterize the interactome of drug candidates to gain insight into mechanisms of off-target toxicity and polypharmacology.

HaloTag

HaloTag is a self-labeling protein tag. It is a 297 residue peptide derived from a bacterial enzyme, designed to covalently bind to a synthetic ligand. The bacterial enzyme can be fused to various proteins of interest. The synthetic ligand is chosen from a number of available ligands in accordance with the type of experiments to be performed. This bacterial enzyme is a haloalkane dehalogenase, which acts as a hydrolase and is designed to facilitate visualization of the subcellular localization of a protein of interest, immobilization of a protein of interest, or capture of the binding partners of a protein of interest within its biochemical environment. The HaloTag is composed of two covalently bound segments including a haloalkane dehalogenase and a synthetic ligand of choice. These synthetic ligands consist of a reactive chloroalkane linker bound to a functional group. Functional groups can either be biotin or can be chosen from five available fluorescent dyes including Coumarin, Oregon Green, Alexa Fluor 488, diAcFAM, and TMR. These fluorescent dyes can be used in the visualization of either living or chemically fixed cells.

Dye-ligand affinity chromatography is one of the Affinity chromatography techniques used for protein purification of a complex mixture. Like general chromatography, but using dyes to apply on a support matrix of a column as the stationary phase that will allow a range of proteins with similar active sites to bind to, refers to as pseudo-affinity. Synthetic dyes are used to mimic substrates or cofactors binding to the active sites of proteins which can be further enhanced to target more specific proteins. Follow with washing, the process of removing other non-target molecules, then eluting out target proteins out by changing pH or manipulate the salt concentration. The column can be reused many times due to the stability of immobilized dyes. It can carry out in a conventional way by using as a packed column, or in high-performance liquid chromatography (HPLC) column.

References

  1. 1 2 3 4 Schmid, A.; Dordick, J. S.; Hauer, B.; Kiener, A.; Wubbolts, M.; Witholt, B. (2001). "Industrial biocatalysis today and tomorrow". Nature. 409 (6817): 258–268. doi:10.1038/35051736. PMID   11196655.
  2. ↑ Mateo, Car; Fernandez-Lorente, Gloria; Guisan, Jose; Fernandez-Lafuente, Roberto (2007). "Improvement of enzyme activity, stability and selectivity via immobilization techniques". Enzyme and Microbial Technology. 40 (6): 1451–1463. doi:10.1016/j.enzmictec.2007.01.018.
  3. 1 2 3 Datta, Sumitra; Christena, L. Rene; Rajaram, Yamuna Rani Sriramulu (2017-04-17). "Enzyme immobilization: an overview on techniques and support materials". 3 Biotech. 3 (1): 1–9. doi:10.1007/s13205-012-0071-7. ISSN   2190-5738. PMC   3563746 . PMID   28324347.
  4. ↑ Guisan, Jose (2006). Immobilization of Enzymes and Cells. Springer Science & Business Media.
  5. ↑ Jesionowski, Teofil; Zdarta, Jakub; Krajewska, Barbara (2014-08-01). "Enzyme immobilization by adsorption: a review". Adsorption. 20 (5–6): 801–821. doi: 10.1007/s10450-014-9623-y . ISSN   0929-5607.
  6. ↑ Walker, John (1988). Methods in Molecular Biology - New Protein Techniques. Humana Press. pp. 495–499.
  7. 1 2 3 4 5 6 7 Houde, Alain; Kademi, Ali; Leblanc, Danielle (2004-07-01). "Lipases and their industrial applications: an overview". Applied Biochemistry and Biotechnology. 118 (1–3): 155–170. doi:10.1385/ABAB:118:1-3:155. ISSN   0273-2289. PMID   15304746.
  8. ↑ Sun, Ye; Cheng, Jiayang (2002-05-01). "Hydrolysis of lignocellulosic materials for ethanol production: a review". Bioresource Technology. Reviews Issue. 83 (1): 1–11. doi:10.1016/S0960-8524(01)00212-7. PMID   12058826.
  9. ↑ van der Maarel, Marc J. E. C; van der Veen, Bart; Uitdehaag, Joost C. M; Leemhuis, Hans; Dijkhuizen, L (2002-03-28). "Properties and applications of starch-converting enzymes of the α-amylase family". Journal of Biotechnology. 94 (2): 137–155. doi:10.1016/S0168-1656(01)00407-2. PMID   11796168.
  10. ↑ Bhosale, S. H.; Rao, M. B.; Deshpande, V. V. (1996-06-01). "Molecular and industrial aspects of glucose isomerase". Microbiological Reviews. 60 (2): 280–300. ISSN   0146-0749. PMC   239444 . PMID   8801434.
  11. ↑ Buchholz, Klaus (2016-05-01). "A breakthrough in enzyme technology to fight penicillin resistance—industrial application of penicillin amidase". Applied Microbiology and Biotechnology. 100 (9): 3825–3839. doi:10.1007/s00253-016-7399-6. ISSN   0175-7598. PMID   26960323.
  12. 1 2 3 4 5 6 Sharma, Kumar; Beniwal, Vikas (2014). Industrial Enzymes: Trends, Scope, and Relevance. Nova Science Publishers, Inc.
  13. ↑ Taherzadeh, Madhavan; Nampoothiri, Christian (2015). Industrial Biorefineries and White Biotechnology. Elsevier B.V. ISBN   978-0-444-63453-5.
  14. ↑ Bekhit, Alaa A.; Hopkins, David L.; Geesink, Geert; Bekhit, Adnan A.; Franks, Philip (2014-01-01). "Exogenous Proteases for Meat Tenderization". Critical Reviews in Food Science and Nutrition. 54 (8): 1012–1031. doi:10.1080/10408398.2011.623247. ISSN   1040-8398. PMID   24499119.
  15. ↑ Lanvers-Kaminsky, Claudia (2017-03-01). "Asparaginase pharmacology: challenges still to be faced". Cancer Chemotherapy and Pharmacology. 79 (3): 439–450. doi:10.1007/s00280-016-3236-y. ISSN   0344-5704. PMID   28197753.
  16. ↑ González-Rábade, Nuria; Badillo-Corona, Jesús Agustín; Aranda-Barradas, Juan Silvestre; Oliver-Salvador, María del Carmen (2011-11-01). "Production of plant proteases in vivo and in vitro — A review". Biotechnology Advances. 29 (6): 983–996. doi:10.1016/j.biotechadv.2011.08.017. PMID   21889977.
  17. ↑ Kotb, Essam (2014-05-01). "The biotechnological potential of fibrinolytic enzymes in the dissolution of endogenous blood thrombi". Biotechnology Progress. 30 (3): 656–672. doi:10.1002/btpr.1918. ISSN   1520-6033. PMID   24799449.
  18. ↑ "Spar Bio Laundry Tablets" . Retrieved 2017-04-18.
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