Subscriber access provided by READING UNIV
Article
Thermal Stabilization of Enzymes with Molecular Brushes Nataraja Sekhar Sekhar Yadavalli, Nikolay Borodinov, Chandan Kumar Choudhury, Tatiana QuiñonesRuiz, Amine Mohamed Laradji, Sidong Tu, Igor K. Lednev, Olga Kuksenok, Igor Luzinov, and Sergiy Minko ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.7b03138 • Publication Date (Web): 03 Nov 2017 Downloaded from http://pubs.acs.org on November 3, 2017
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
ACS Catalysis is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 29
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Catalysis
Thermal Stabilization of Enzymes with Molecular Brushes Nataraja S. Yadavalli,1 Nikolay Borodinov,2 Chandan K. Choudhury,2 Tatiana Quiñones-Ruiz,3 Amine M. Laradji,1 Sidong Tu,2 Igor K. Lednev,3 Olga Kuksenok,2* Igor Luzinov,2* and Sergiy Minko1*
1
Nanostructured Materials Laboratory, The University of Georgia, Athens, Georgia, 30602 USA
2
Department of Materials Science and Engineering, Clemson University, Clemson, South
Carolina 29634, USA 3
Department of Chemistry, University at Albany, State University of New York, Albany, New
York 12222, USA
1 ACS Paragon Plus Environment
ACS Catalysis
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 2 of 29
ABSTRACT Herein, we report a conjugation strategy, where we utilize a polyethylene oxide cylindrical molecular brush architecture to design a self-assembled structure for thermal stabilization of enzymes. We demonstrate that the proposed architecture of the moderately stiff polymer ligand results in a significant improvement of biocatalytic activity and thermal stability of lysozyme and trypsin that retain their activity even upon heating to 100oC and above. The molecular brush is bound via epoxy functional groups to lysine’s amino groups on the surface of the enzyme globule, promoting the formation of stiff and crowded cages around the enzymes and preventing the water molecules access to the enzyme and enzymes agglomeration. The molecular dynamic simulations show that the high concentration of polyethylene oxide in the vicinity of the enzyme is critical for their stability. Monitoring of lysozyme-molecular brush conjugates for 6 and 12 months in lyophilized form and in solution, respectively has shown that the conjugation does not compromise shelf life of the enzyme.
KEYWORDS Enzyme catalysis; Molecular dynamics; Nanostructures; Polymers; Self-assembly
2 ACS Paragon Plus Environment
Page 3 of 29
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Catalysis
INTRODUCTION The unparalleled catalytic efficiency and substrate specificity are placing enzymes among the major commercial catalysts for the use in sustainable chemical technologies, the food industry, for the deactivation of toxic materials and chemical weapons, in laundry, for the cleaning of oil spills, and for medical biosensing applications.1-5 Thermal stability of enzymes is also essential for such applications as deconstruction of biomass for biofuel industry6 and enzyme enhanced oil recovery applications.7-9 However, the main limitation of many biocatalysts is a loss of their activity in
harsh operating conditions (e.g. variations in temperature, pH, solvents, and a high concentration of reactants).10,11 Ensuring the thermal stability of these enzymes is vital for harnessing their catalytic performance beyond their natural environment.12-15 It is known that most mesophilic enzymes lose their catalytic activity at above 50-60 oC significantly due to unfolding of the protein molecule.16,17 However, natural enzymes produced by thermophiles can function effectively at elevated temperatures up to 130
o
C.16,18 These enzymes synthesized by
thermophilic microbes has motivated researchers for decades to discover new methods of stabilizing “conventional” enzymes. Two major research thrusts for enzyme stabilization are currently being pursued: i) the use of chimeric DNA genetic materials that carry codes for preprogrammed enhanced enzyme stability and ii) the conjugation of natural mesophilic enzymes with other molecules or solid carriers using intermolecular interactions and chemical bonds to strengthen the enzyme’s structure. Although the
genetic mutation is a proven method for generating highly thermostable
recombinant enzymes,19-21 it remains challenging to engineer enzymes that combine high biocatalytic activity and thermal stability. Immobilization of enzyme is well known approach to encapsulation, storage, delivery, separation and biocatalytic synthesis.22 A number of synthetic methods have been developed to protect the 3 ACS Paragon Plus Environment
ACS Catalysis
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 4 of 29
three-dimensional conformation of the enzymes via immobilization/encapsulation or conjugation23 on solid surfaces, in porous structures, ceramic and polymer matrixes,24-28 particles,29,30, capsules,31 imprints,32 inorganic hybrids,33 nanofibers,34,35 by cyclization of protein backbone,36 and through various protein-polymer conjugation techniques.37-39 Encapsulation of enzymes by tethering a single polymer molecule shell has advantages due to a combination of some conformational freedom and at the same time structural stabilization with a moderately stiff polymer shell.40-43 The mechanism of molecular crowding for thermal stabilization of enzymes has recently attracted great attention.44-49 The experimentally supported hypothesis is based on the study of natural environment of proteins in cell milieu. It was proposed that the excluded volume effect leads to an overall stabilizing effect.50 The experiments evidenced changes of both unfolding free energy and unfolding kinetics due to the molecular crowding effect.51 However, there is not yet well developed understanding of the effect because of many complications associated with experimental and modeling studies including the role of various kinds of interactions between the crowder and protein.52-55 The most extensively studied crowder is poly(ethylene glycol) (PEG). The literature reports show that the conjugation of PEG and proteins could have both positive and negative outcomes for their thermal stability.56-61 Additionally to mechanical stabilization of proteins, PEG-conjugates form a barrier for interactions of surrounding molecules of water with the protein.62 The latter may substantially contributes to protein stability. Recent studied demonstrated importance of molecular dimensions of PEG crowders, emphasizing that the pronounced stabilizing effect is achieved when the crowder's size is closer to that of the protein.63
4 ACS Paragon Plus Environment
Page 5 of 29
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Catalysis
Herein, we report an original enzyme-polymer conjugation strategy, which is based on a specific architecture of the cylindrical molecular brush copolymer ligand. The major hypothesis of this research refers to the moderate stiffness of the cylindrical brush structure that provides i) caging of the enzyme and slowing down unfolding kinetics and ii) reducing of the interaction of the enzyme molecule with water. The discussed in the paper results demonstrate a strong stabilizing effect. While the conjugation with polymers or particles is a well-known approach to improve stability of enzymes64-73, our results show the stability of the conjugates for up to 115150oC, which significantly exceeds thermal stability limits achieved with previously proposed stabilizing methods16,40,74 including the examples of enzyme conjugation methods tested on lysozyme75, trypsin38, organophosphorus hydrolase39, human carbonic anhydrase II76, and sarcosine oxidase77.
RESULTS Structure of the polymer ligand The basic architecture of the enzyme-polymer conjugate is explained in Figure 1(a-c). The enzyme is fortified by conjugation with a water-soluble polymer ligand (PL) poly(GMA-ranOEGMA), a random copolymer of 0.34 mol glycidyl methacrylate (GMA) and 0.66 mol oligo(ethylene glycol) methyl ether methacrylate (OEGMA, Mn 950) with an average 21.5 ethylene oxide monomeric units in OEGMA side groups (Figure 1d).
A number average
molecular mass of the copolymer is Mn=1.3×106 g/mol. Polymer chains of the PL copolymer are moderately stiff because of the excluded volume effect of the bulky side groups forming a cylindrical molecular brush-like structure (bottle-brush).78 Details of the synthesis and
5 ACS Paragon Plus Environment
ACS Catalysis
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 6 of 29
characterization of PL are provided in Methods with further details in Supplementary Information (SI-2). Copolymer chains visualized with AFM for a dry sample appeared as 0.5 nm thick wormlike structures79 (Figure 2a) when the chain conformation is not fully described by the Gaussian chain (random walk) statistics because of the bending elasticity. The adsorbed PL on the mica substrate is characterized with a 26 nm persistence length. This value of persistence length provides evidence that the side OEGMA chains are strongly stretched in the brush regime and contribute to the chain stiffnes.80,81 The cylindrical brush geometry is characterized by: the degree of polymerization of side OEGMA groups n=21.5; the monomer size in the side group a=0.25nm; the average distance between side groups estimated based on the copolymer composition (66 %mol OEGMA) as h=0.25 nm/0.66=0.38 nm; the linear grafting density σ=1/h=2.63 nm-1; and the brush thickness D. The copolymer molecule forms a cylindrical brush structure if h is much smaller than the side chain coil size which is the case, since h=0.38 nm
