Novel enzyme-polymer conjugates for biotechnological applications

Romero, Óscar; Rivero, Cintia W.; Guisán, José M.; Palomo, José M.

doi:10.7717/peerj.27

Cited by 21 publications

(18 citation statements)

References 23 publications

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“…33 When CCP was fused with the lipases, enzymatic activity increased by only 20% to 60%; however, in the case of both AP and CCP fused lipases, enzymatic activity increased by 300% ( Figure S3).…”

Section: Discussionmentioning

confidence: 86%

Recombinant Lipase Engineered with Amphipathic and Coiled-Coil Peptides

et al. 2015

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Lipases have been utilized industrially to produce biodiesel, oleochemicals, and pharmaceuticals. Many efforts such as metagenomics, directed evolution and enzyme immobilization have been devoted to enhance the lipase activity.Here, we designed a recombinant lipase, NKC-M37-MAT, that was generated by incorporating an N-terminal amphipathic peptide (NKC) and a C-terminal coiled-coil peptide (MAT) into Photobacterium lipolyticum M37 lipase. The hydrophobic face of NKC improve the accessibility (K m ), and catalytic efficiency (K cat /K m ) of the soluble lipase toward hydrophobic substrate and tetrameric MAT further enhanced lipase catalytic activity (U/mg) through cooperative binding to its substrate such that the catalytic activity (U/mg) of NKC-M37-MAT was increased by a maximum of 54-fold compared with the wildtype, which decreased the biodiesel production time 5-fold from 30 h to 6 h. This novel approach shows promise as a platform technology to increase lipase catalytic efficiency for industrial-scale production of biodiesel and biochemicals synthesized from hydrophobic substrates.Triacylglycerol lipase (E.C. 3.1.1.3), 1 which catalyzes the hydrolysis of triglycerides to fatty acids and glycerols, plays an important role in industries that produce products such as detergents, food, leather, paper, cosmetics, and bioenergy. 1-3 Because of their broad substrate specificity and enantioselectivity, lipases have been utilized by the pharmaceutical industry to synthesize pure R-and S-optical isomers. 4 The energy industry is also using lipases to produce biodiesel from vegetable oil as well. 1,3,5,6 Biodiesel products, an emerging renewable energy source, are produced through trans-esterification in the presence of a catalyst which could be basic (generally, NaOH), acid (usually, HCl or H 2 SO 4 ), or enzymatic. 7-9 However, base-catalyzed trans-esterification is not feasible since alkaline can react with free fatty acid to form unwanted soap and water, which would affect the biodiesel quality and require extra investment for downstream separation and purification. In acid-catalyzed esterification, large excess amount of alcohol is required in order to reach high biodiesel yield, and if sulfuric acid is utilized as catalysts, it is difficult to remove them after reaction. Moreover, chemical-catalyzed trans-esterification produces toxic wastewater and consumes high amounts of energy to maintain high reaction temperature. 8 To overcome these problems, enzymatic methods using lipase are receiving special attention as an attractive alternative approach for biodiesel production. The great advantages of lipases compared with chemical catalysts are as follows: 1) relatively low reaction temperatures (approximately 40°C), 6 2) fewer process steps that generate reduced amounts of hazardous materials, 3) improved product separation that generates higher purity glycerol. 5,6 Since the enzymatic synthesis of biodiesel occurs at the lipid-water interface, increasing the accessibility of water-soluble lipase such as M37 to hydro...

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Section: Discussionmentioning

confidence: 86%

Recombinant Lipase Engineered with Amphipathic and Coiled-Coil Peptides

et al. 2015

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“…These protocols improved enzyme specificity and regio‐ or enantioselectivity in different biotransformations. Recently, different tailor‐made thiol ionic polymers were prepared and Cys introduced into the desired positions was activated with 2,2‐dithiodipyridine 62. Some of the modifications permitted greatly improved enzyme performance in the production of interesting drugs.…”

Section: Modification Of Immobilized Proteinsmentioning

confidence: 99%

Chemical Modification in the Design of Immobilized Enzyme Biocatalysts: Drawbacks and Opportunities

Rueda

Santos

Ortíz

et al. 2016

The Chemical Record

192

106

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Chemical modification of enzymes and immobilization used to be considered as separate ways to improve enzyme properties. This review shows how the coupled use of both tools may greatly improve the final biocatalyst performance. Chemical modification of a previously immobilized enzyme is far simpler and easier to control than the modification of the free enzyme. Moreover, if protein modification is performed to improve its immobilization (enriching the enzyme in reactive groups), the final features of the immobilized enzyme may be greatly improved. Chemical modification may be directed to improve enzyme stability, but also to improve selectivity, specificity, activity, and even cell penetrability. Coupling of immobilization and chemical modification with site-directed mutagenesis is a powerful instrument to obtain fully controlled modification. Some new ideas such as photoreceptive enzyme modifiers that change their physical properties under UV exposition are discussed.

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“…One of the most interesting modification of enzymes has been achieved using polymers [22]. For example, the use of tailor-made dextran polymers showed interesting results, obtaining novel and selective biocatalysts [7][8][9][10]23].…”

Section: Introductionmentioning

confidence: 99%

Chemical Modification of Novel Glycosidases from Lactobacillus plantarum Using Hyaluronic Acid: Effects on High Specificity against 6-Phosphate Glucopyranoside

et al. 2019

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Three novel glycosidases produced from Lactobacillus plantarum, so called Lp_0440, Lp_2777, and Lp_3525, were isolated and overexpressed on Escherichia coli containing a His-tag for specific purification. Their specific activity was evaluated against the hydrolysis of p-nitrophenylglycosides and p-nitrophenyl-6-phosphate glycosides (glucose and galactose) at pH 7. All three were modified with hyaluronic acid (HA) following two strategies: A simple coating by direct incubation at alkaline pH or direct chemical modification at pH 6.8 through preactivation of HA with carbodiimide (EDC) and N-hydroxysuccinimide (NHS) at pH 4.8. The modifications exhibited important effect on enzyme activity and specificity against different glycopyranosides in the three cases. Physical modification showed a radical decrease in specific activity on all glycosidases, without any significant change in enzyme specificity toward monosaccharide (glucose or galactose) or glycoside (C-6 position free or phosphorylated). However, the surface covalent modification of the enzymes showed very interesting results. The glycosidase Lp_0440 showed low glycoside specificity at 25 °C, showing the same activity against p-nitrophenyl-glucopyranoside (pNP-Glu) or p-nitrophenyl-6-phosphate glucopyranoside (pNP-6P-Glu). However, the conjugated cHA-Lp_0440 showed a clear increase in the specificity towards the pNP-Glu and no activity against pNP-6P-Glu. The other two glycosidases (Lp_2777 and Lp_3525) showed high specificity towards pNP-6P-glycosides, especially to the glucose derivative. The HA covalent modification of Lp_3525 (cHA-Lp_3525) generated an enzyme completely specific against the pNP-6P-Glu (phosphoglycosidase) maintaining more than 80% of the activity after chemical modification. When the temperature was increased, an alteration of selectivity was observed. Lp_0440 and cHA-Lp_0440 only showed activity against p-nitrophenyl-galactopyranoside (pNP-Gal) at 40 °C, higher than at 25 °C in the case of the conjugated enzyme.

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Novel enzyme-polymer conjugates for biotechnological applications

Cited by 21 publications

References 23 publications

Recombinant Lipase Engineered with Amphipathic and Coiled-Coil Peptides

Recombinant Lipase Engineered with Amphipathic and Coiled-Coil Peptides

Chemical Modification in the Design of Immobilized Enzyme Biocatalysts: Drawbacks and Opportunities

Chemical Modification of Novel Glycosidases from Lactobacillus plantarum Using Hyaluronic Acid: Effects on High Specificity against 6-Phosphate Glucopyranoside

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