Nanomaterials as Matrices for Enzyme Immobilization

Figures & data

Table 1. shows the immobilization of enzymes on different nanomaterials and their application.

Nature of the particle	Enzyme	Conjugation method	Applications/comments	Reference No.
(a) Superparamagnetic nanoparticles
Fe3O4 particles encapsulated in anhydrous and porous silica coating of 3.5 nm thickness.	Lactamase	Covalent coupling	Easy recovery, 99.5 % loss in enzyme activity.	42
γ-Fe2O3	Candida rugosa lipase	Covalent coupling with two different approaches.	46 % loss in hydrolytic activity of lipases. Improved storage stability.	43
Maghemite (iron oxide)	Candida rugosa lipase	Covalent coupling with 3-aminopropyltriethoxysilane and glutaraldehyde chemistry	High stereoselectivity in kinetic resolution of racemic carboxylates. Improved long-term stability.	44
Iron oxide in “siliceous mesocellular foam”	Pseudomonas cepacia lipase	Noncovalent coupling	Kinetic resolution of (±)-1-phenyl-ethanol. Seven times reusable.	45
Glycidyl methacrylate coated iron oxide nanoparticles	Chloroperoxidase (CPO)	Covalent coupling	Enhancement in the stability of nanobiocatalyst, with no loss of activity after recycling 11 times.	46
Silica coated magnetic nanoparticles	Alpha-chymotrypsin and lipase	Covalent immobilization of crosslinked enzyme clusters	Highly active and stable under harsh shaking for more than 15 days.	47
Magnetite (Mag NP)	Candida antarctica lipase	Immobilization via γ-aminopropyltriethoxysilane (APTS)	Easy recovery, reusable. Highly enantioselective for secondary alcohols.	48
Nature of silica	Enzyme entrapped	Type of immobilization	Comments	Reference No.
(b) Mesoporous Materials
Single enzyme nanoparticles (SENs)	alpha-chymotrypsin and trypsin	Covalent linkage of polymer to the surface of enzymes	Due to thin porous polymer network (1 nm), no mass transfer constraints, high stability and activity in organic solvents.	54
Biomodal mesoporous silica	Several enzymes with different molecular weights like catalase, protease, peroxidase and cytochrome C	Immobilization by adsorption followed by assembling an organic/inorganic nanocomposite shell on the particle surface	High enzymatic activity, stability and protection from proteolysis.	55
Nanoporous silica gel glass	alpha-chymotrypsin	Covalent binding	Half-life of entrapped enzyme was 1000 times higher than that of native alpha-chymotrypsin in organic solvents.	56
Type of carbon nanotube	Enzyme	Conjugation method	Applications/comments	Reference No.
c) Carbon nanotubes
Multi-walled carbon nanotubes	Soybean peroxidase, Subtilisin Carlsberg, Candida antartica lipase B (CALB) and Tyrosinase	Attachment via carbodiimide chemistry	Water soluble carbon nanotube enzyme conjugates. Low mass transfer resistance, high activity, stability and reusability.	60
Multi-walled carbon nanotubes	Candida rugosa lipase	Adsorption	Immobilization led to 2.2 and 14 fold increase in transesterification activity in n-hexane and [Bmim] [PF6] respectively. Also showed high enantioselectivity as determined by kinetic resolution of (±) 1-phenylethanol in [Bmim] [PF6].	61
Multi-walled carbon nanotubes	Xylanase	Adsorption	Refolding of 8 M urea denatured xylanase was done. 55 % activity could be regained with denatured form of purified form of xylanase and 92 % with commercial preparation of xylanase in 8 M urea.	62
Single-walled carbon nanotube	Soybean peroxidase, subtilisin Carlsberg, trypsin, proteinase K, and horseradish peroxidase	Adsorption	Stabilized proteins at elevated temperatures and in organic solvents to a greater extent than conventional flat supports.	63
Multi-walled Carbon nanotubes were solubilized via functionalization with poly(propionylethylenimine-co-ethylenimine).		Immobilization via diimide activated amidation reaction	The method for efficient preparation of solubilized carbon nanotubes was developed.	64
Type of nanoparticle	Enzyme	Conjugation method	Applications/comments	Reference No.
d) Some other less used nanomaterials
Polystyrene nanoparticles	Alpha-chymotrypsin	Covalent attachment	Effect of particle mobility on the catalytic efficiency of enzyme was studied.	69
Modified (by grafting with carboxylic surfactant) zirconia nanoparticles.	Lipases from Candida rugosa and Pseudomonas cepacia	Immobilization of enzymes by adsorption	Both lipases gave significantly higher activity and enantioselectivity compared with crude lipase powders. Also, these nanohybrid could be reused for eight cycles	70
Titanium oxide	Alpha-chymotrypsin, RNase A, Papain, Lysozyme, Subtilisin A, GFPuv, Pseudomonas cepacia lipase	Adsorption	Refolding of heat denatured enzymes carrying positive charges was carried out.	71
Silica nanoparticles	Chicken egg lysozyme	Adsorption	Effect of nanoparticle size on structure and enzymatic activity was studied.	72

Figure 1. Preparation procedure for the magnetically separable biocatalysts. (1) Fe(NO₃)₃·9H₂O, methanol; propionic acid; calcination; (2) octyltrimethoxylsilane, toluene, 120 °C for 24 h; (3) 0.2 g of magnetic foam, 20 mL of 50 mg/mL, 6 h at rt; (4) 0.2 g of hydrophobic magnetic foam, 20 mL of 50 mg/mL, 6 h at rt. (Reproduced from [45] with the permission of Copyright Clearance Center's Rightslink service, Elsevier (http://s100.copyright.com/CustomerAdmin/PLF.jsp?lID=2010051_1274898046362).

Figure 2. TEM images of: (a) MWNTs; (b) Candida rugosa lipase CRL absorbed on MWNTs. The diameters of the nanotubes without enzyme were 20 ± 5 nm and with enzyme 30 ± 5 nm (the diameter values represent the average of 10 TEM images in each case). Reproduced from [61] with the permission of BioMed Central (http://creativecommons.org/licenses/by/2.0).

Figure 3. TiO₂ nanoparticles as synthetic chaperones. Thermally denatured basic proteins interacted with TiO₂ nanoparticles. This interaction prevented protein aggregation and facilitated correct refolding of the protein molecules. Reproduced from [71] with the permission of Royal Society of Chemistry, London.

Figure 4. Affinity layering by bioaffinity immobilization. The lectin concanavalin A (Con A) was bound to its affinity matrix Sephadex G-100. Horseradish Peroxidase (HRP) is a glycoenzyme with known affinity for the lectin and hence was immobilized on Con A-sephadex G-100 by bioaffinity immobilization. In subsequent steps (in which Con A acted as the affinity ligand), alternate layers of Con A and HRP were created. This “going vertical” of affinity layering is a promising strategy for depositing large amount of biological activity on a small surface (for further details about this work, please see reference [82]).

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