Bioaffinity Based Immobilization of Enzymes

Saleemuddin, M.

doi:10.1007/3-540-49811-7_6

Cited by 68 publications

(57 citation statements)

References 135 publications

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“…The use of recombinant tags allows proteins to attach to a substrate in a defined orientation, but the interactions of the tags are reversible (e.g., glutathione S-transferase, oligohistidine) and, hence, are not stable over the course of subsequent assays or require large mediator proteins (e.g., biotin-streptavidin, antigen-antibody; refs. [7][8][9]. A further disadvantage with all of these methods is that they are not well suited to controlling the densities of immobilized proteins.…”

mentioning

confidence: 99%

Selective immobilization of proteins to self-assembled monolayers presenting active site-directed capture ligands

Hodneland

Lee

Min

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

330

291

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This paper describes a method for the selective and covalent immobilization of proteins to surfaces with control over the density and orientation of the protein. The strategy is based on binding of the serine esterase cutinase to a self-assembled monolayer presenting a phosphonate ligand and the subsequent displacement reaction that covalently binds the ligand to the enzyme active site. Surface plasmon resonance (SPR) spectroscopy showed that cutinase binds irreversibly to a monolayer presenting the capture ligand at a density of 1% mixed among tri(ethylene glycol) groups. The covalent immobilization is specific for cutinase, and the glycolterminated monolayer effectively prevents unwanted nonspecific adsorption of proteins. To demonstrate that the method could be used to immobilize proteins of interest, a cutinase-calmodulin fusion protein was constructed and immobilized to the monolayer. SPR showed that calcineurin selectively associated with the immobilized calmodulin. This capture ligand immobilization method combines the advantages that the immobilization reaction is highly selective for the intended protein, the tether is covalent and, hence, stable, and the method avoids the need for synthetic modification and rigorous purification of proteins before immobilization. These characteristics make the method well suited to a range of applications and, in particular, for constructing protein microarrays. Many experimental approaches in biology and applications in diagnostics and drug discovery require proteins immobilized on substrates (1-3). Of particular relevance is the recent introduction of protein microarrays-prepared by immobilizing hundreds or thousands of different proteins to a common substrate-that allow highly parallel experiments with small amounts of proteins and reagents. MacBeath and Schreiber (4), for example, immobilized a series of proteins on aldehyde-terminated glass slides and showed that these proteins were able to interact with other molecules in solution. More recently, Snyder and coworkers (5) immobilized a library of oligohistidine fusion proteins from Saccharomyces cerevisiae onto Ni-nitrilotriacetic acid (NTA) surfaces and used the array to identify several calmodulin-and phospholipid-binding motifs. These examples demonstrate the potential of protein arrays, but have also highlighted the need for methods that present proteins in a well defined environment and simultaneously prevent unwanted nonspecific protein interactions. This paper presents a method for the selective and irreversible immobilization of proteins to self-assembled monolayers of alkanethiolates on gold by using active site-directed ligands that first bind to a specific protein and subsequently react with the protein to give a covalent link.A variety of methods are commonly used for immobilizing proteins. The most widely used method relies on nonspecific adsorption of the protein to a solid support (6). Simple chemical couplings of reactive groups within proteins (amines, acids) also have been used to immobilize prot...

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confidence: 99%

Selective immobilization of proteins to self-assembled monolayers presenting active site-directed capture ligands

Hodneland

Lee

Min

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

330

291

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“…It is also nice to see Indian groups contributing to CLEA design. (Saleemuddin and Husain 1991;Saleemuddin 1999;Talekar et al, 2012) Use in low water media Very few groups work in this area. Parmar till recently had a group.…”

Section: Applied Bio-catalysis and "Make In India" Programmentioning

confidence: 99%

Skills in biocatalysis are essential for establishing a sound biotechnological base in India

Gupta¹

2015

Proceedings of the Indian National Science Academy

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Techniques developed by enzymologists and protein chemists have been indispensible in many areas of biotechnology. Bio-separation of proteins, biocatalyst engineering and medium engineering are important examples of this. Our understanding of enzyme specificity has helped us in gaining insight into catalytic promiscuity. This, along with the possibility of carrying out biocatalysis in organic solvents has widened the scope of using enzymes for organic synthesis.Reestablishing a strong base in enzymology is crucial towards creating a strong base for the field of biotechnology in India. Keywords IntroductionQuite a few aspects of biotechnology involve production and applications of enzymes. In the old days, most of these aspects were covered under industrial enzymology (Godfrey and Reichelt, 1983). Gradually, a more broad based term 'applied biocatalysis' came into vogue (Straathof and Adlercreutz, 2000) which was an interface between enzymology and biochemical engineering. This phase was characterized by few new developments: (1) As a result of the rDNA technology, our capability to produce mutant properties grew exponentially. There was a scramble to produce proteins at a lower cost. The stakes were no longer limited to low cost industrial grade enzymes. Costlier proteins used in molecular biology, diagnostics and biosensors and pharmaceutical proteins/enzymes could be produced by cloning and their properties tailored to suit the applications. The result was a paradigm shift in the downstream processing methods. Shorter and efficient protocols emerged. Affinity chromatography, rather than being a 'polishing' step at the end, was promoted to the earlier steps of purification. In fact, upstream and downstream processing steps were integrated (Gupta, 2002;Przybycien et al., 2004;Mondal et al., 2006). (2) It was realized that enzymes could be used in non aqueous media (Carrea and Riva 2000; Gupta 2000;Patel 2000; Vulfson et al., 2001). This opened up novel applications; especially in the synthesis of chiral compounds. Table 1 lists some of the milestones in the area of applied biocatalysis.It is remarkable how much enzymology contributed to all this.The present review attempts to emphasize the importance of the skills which are part of the toolbox Published Online on 10 December 2015 1114Munishwar Nath Gupta and Joyeeta Mukherjee et al., 1973; Mosbach 1976 Mosbach , 1980 Nilsson and Mosbach 1981; Mattiasson 1983;Mattiasson 1988) Berezin's group in Moscow was the third one (Berezin 1978; Mozhaev et al., 1987;Martinek and Mozhaev 1993). Current work is adequately discussed at many places ( Enzyme deactivation models Most people wrongly give half lives without analysing the kinetics of deactivation. Sadana's work needs to be known more widely. (Sadana 1991(Sadana , 1993 Enzymes as reverse micelles This is another approach in low water enzymology. (Luisi 1985; Larsson et al., 1987; Adlercreutz et al., 1988) Enzyme use in biosensors Immobilization allows enzymes to interface with transducers. (Yarapolov et al...

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“…This immobilized d-hydantoinase can be used for the production of d-amino acids from the corresponding hydentoins and may therefore be of use in the chemical and pharmaceutical industries. Saleemuddin (1999) advocated that bioaffinity based immobilizations are usually reversible facilitating the reuse of support matrix, orient the enzymes favourably and offer the possibility of enzyme immobilization directly from partially pure enzyme preparations or even cell lysates. Enzymes lacking innate ability to bind various affinity supports can be made to bind them by chemically or iaenetically linking the enzymes with appropriate polypeptides domains like cellulose binding domain, histidine rich peptides etc.…”

Section: Introductionmentioning

confidence: 99%

Immobilization of the Proteases of Euphorbia Royleana

Nawaz¹,

Khan²

2000

Pakistan J. of Biological Sciences

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Immobilization of the protease of Euphorbia royleana on DEAE-A50 cellulose was investigated. The percentage of immobilization was found to be 31%. A continuous immobilized enzyme proteolytic system was developed and tested for its hydrolyzing tendency which was found to be significantly effective for continuous proteolysis. The life span of the enzyme immobilized on DEAE-A50 cellulose was 22 days. Thus it is a successful attempt towards the development lit an immobilized enzyme system to preserve enzyme in certain modified forms.

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Bioaffinity Based Immobilization of Enzymes

Cited by 68 publications

References 135 publications

Selective immobilization of proteins to self-assembled monolayers presenting active site-directed capture ligands

Selective immobilization of proteins to self-assembled monolayers presenting active site-directed capture ligands

Skills in biocatalysis are essential for establishing a sound biotechnological base in India

Immobilization of the Proteases of Euphorbia Royleana

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