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WO2013016794A1 - Méthode de prévision de mutants augmentant l'indice d'hydrophobicité de la surface de protéines - Google Patents

Méthode de prévision de mutants augmentant l'indice d'hydrophobicité de la surface de protéines Download PDF

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WO2013016794A1
WO2013016794A1 PCT/BR2012/000260 BR2012000260W WO2013016794A1 WO 2013016794 A1 WO2013016794 A1 WO 2013016794A1 BR 2012000260 W BR2012000260 W BR 2012000260W WO 2013016794 A1 WO2013016794 A1 WO 2013016794A1
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protein
amino acids
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Goran NESIC
José Alberto JARDINE
Izabela AGOSTINHO PENA NESHICH
José Augusto SALIM
Ivan MANZONI
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Empresa Brasileira de Pesquisa Agropecuaria EMBRAPA
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B15/00—ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment

Definitions

  • the present invention relates to a protein engineering method in which surface regions of enzymes that would be mutable that have little influence on the physicochemical and structural properties of amino acids of the catalytic site are identified for the purpose of create mutants that have the most hydrophobic macromolecular surface.
  • this technology can be applied to the rational design of enzymes with greater solubility in hydrophobic media (vegetable oils, for example).
  • All vegetable or animal fat is composed mainly of triglycerides (one ester glycerol molecule (one th alcohol) with three fatty acid molecules), and free fatty acids (FFA).
  • triglycerides one ester glycerol molecule (one th alcohol) with three fatty acid molecules
  • FFA free fatty acids
  • the triglycerides present in the oil are transformed into smaller fatty acid ester (biodiesel) molecules from a transesterifying agent (primary alcohol) and a catalyst (base or acid).
  • Biodiesel is defined as a fatty acid ester obtained from the chemical reaction preferably catalyzed in a basic medium of vegetable or animal fats with a primary alcohol (ethyl or methyl). When obtained in this way the process is called transesterification. Biodiesel can also be obtained by utilizing free fatty acids by the preferably acidic esterification processes and by the cracking process.
  • Vegetable oils composed of short chain fatty acids ensure better process performance because interaction with the transesterifying agent and catalyst is more effective (RICACZESKI, CC; ZANCANARO, D .; ALZANI.A.; FERREIRA, EF Biodiesel, an expanding fuel Synergismus scyentifica UTFPR, Pato Branco, 01 (1, 2,3,4): 1-778 (2006).
  • the transesterification reaction is reversible and requires Excess alcohol in the reaction (1: 6 molar) is required to increase the yield of alkyl esters and allow the formation of a separate glycerol phase.
  • the most used alcohol to obtain biodiesel is methanol, which promotes better yields.
  • Brazil is one of the largest producers of ethyl alcohol (ethanol) in the world, there is a stimulus for the substitution of methanol for ethanol, generating an agricultural fuel totally independent of petroleum.
  • the difficulty in using ethanol is that water is one of the causative agents of parallel saponification reactions, consuming the catalyst and reducing the efficiency of the transesterification reaction.
  • the most widely used catalyst is sodium hydroxide (NaOH), widely known as caustic soda. Potassium hydroxide (KOH) may also be used. About 0.5% by weight of oil is indicated.
  • Basic catalysts such as those mentioned above, accelerate the reaction by 4000 times faster than acid catalysts such as hydrochloric acid (HCI) and are more economically viable.
  • HCI hydrochloric acid
  • the use of basic catalysts promotes a higher level of saponification in the process: the catalyst reacts with the oil-free fatty acids to form soap. Every 1% by weight of caustic soda used as catalyst, about 7% by weight of soap will be produced. Therefore, for the transesterification process to be satisfactory, vegetable oils must contain a maximum of 3% free fatty acid.
  • Lipases are enzymes that catalyze the hydrolysis and synthesis of acylglycerols in long chain fatty acids using triacylglycerol with the substrate when in aqueous medium.
  • the use of lipases in biodiesel production is relatively recent, but it has been very promising due to a number of advantages over chemical catalysts.
  • Commercial lipolytic enzymes were generally selected for purposes related to the food industry. In such processes, the reaction medium is emulsified through the use of detergents, which is an unfeasible situation for biodiesel production, as it adds steps to the process that increase the cost and production time.
  • lipases as bio-catalysts allows easy glycerol recovery without the need for purification or chemical waste production. Despite the advantages mentioned, the enzymes have a low process yield compared to inorganic catalysts.
  • a lipase enzyme with a higher surface hydrophobic would react better with the substrate in an environment free of polar solvents leading to a higher yield in the conversion of oil to biodiesel when compared to natural lipases.
  • Novozyme 435 was selected for the present study. This enzyme, marketed in the form immobilized by Novozymes (http://www.novozymes.com), is the Antarctic Candida fungus lipase B (NCBI gi: 576300), being a monomeric protein belonging to the family of folding hydrolases of the type ⁇ / ⁇ .
  • Patkar et al. 1998 (Patkar S, Vind J, Kelstrup E, Christensen MW, Svendsen A, Borch K, Kirk O. Effect of mutations in Antarctic Candida B lipase. Chem Phys Lipids. 1998 Jun; 93 (1-2) : 95-101.) Tested the effects of mutations on C. antarctic lipase B by means of residue mutations close to the active site and found that the T103G mutation that introduced the GXSXG consensus sequence (found in most lipases) led to an increase in term stability but reduced to half the enzyme-specific activity in ester formation. Another mutation studied, W104H drastically affected these two properties (term stability and activity), reducing them.
  • US 6,398,707 describes a technique for increasing the activity of an immobilized lipase enzyme and a technique for regeneration of deactivated immobilized lipase by the use of an alcohol with a carbon number of not less than three.
  • PI 0418062-3A describes a process for the production of biodiesel from renewable oil in the presence of lipase catalysis in an organic reaction system.
  • a short chain alcohol ROH is used as an acyl receptor, a relatively hydrophilic organic solvent having no negative effect on lipase reactivity being used as reaction medium, and renewable oil feedstock being catalyzed by a lipase for synthesis.
  • biodiesel by transesterification yielding biodiesel production in 94% and in reduced reaction time.
  • PI 0419166-8A describes a process for producing biodiesel from soap feedstock, ie any soap feedstock generated in the alkali refining process containing from 10 to 60% water, 0, 1 to 2.0% sterols, 35 to 85% fatty derivatives including partial glycerides. This process occurs by: neutralizing and separating the soaps with strong acids to pH 2-8, followed by esterification. enzymatic composition using Lipase with concentration ranging from 100ppm to 10 wt%, using C1 to C6 alkanol in a weight ratio of 5 to 100 wt% of the fatty components and using temperature of 15 to 70 ° C.
  • the description of the invention contained herein is a novel approach to protein engineering for lipases in order to optimize them into an alternative form for obtaining more fat soluble proteins.
  • the present methodology indicates that the exchange of amino acids located on the protein surface of C. antarctica lipase B would lead to a substantial increase in surface hydrophobicity (calculated from an index called the SHI ("Surface Hydrophobicity Index") and which postulates This property is important in improving the efficiency of biodiesel production by biocatalysis.
  • SHI Surface Hydrophobicity Index
  • the present invention relates to a protein engineering method in which surface regions of enzymes that would be mutable that have little influence on the physicochemical and structural properties of amino acids of the catalytic site are identified for the purpose of create mutants that have the most hydrophobic macromolecular surface.
  • step "c” Define limit values for each of the characteristics selected in step "c"; e) Select the amino acids whose characteristics met the values of each of the descriptors selected in step “c" through specific software;
  • a second embodiment of the invention relates to mutant proteins obtained by said method.
  • Figure 1 In silico mutant generation process steps of protein structures with more hydrophobic surface area. Steps: A) Selection of protein structure whose surface should be modified. B) Selection of descriptors and C) their value ranges. D) Selection of amino acids that have the characteristics chosen using the BluStar STING J PD tool. E) Construction of three-dimensional models by homology modeling followed by F) Molecular dynamics energy minimization refinement. G) The final models are classified according to the V4 score for the lowest interference in the catalytic site of the wt enzyme. H) The best single mutations according to the V4 score are chosen to compose the mutant multi-mutant enzymes which, I). are evaluated in terms of the V4 score and the J) hydrophobicity index calculation.
  • Figure 2 Three-dimensional cartoon-like structure of Antarctic Candida Lipase B protein (UCB.pdb) showing its secondary structure consisting of three beta leaves and six alpha-helices (a).
  • the colored protein surface according to electrostatic potential shows the amount of polar amino acids (gray and dark gray) present in the enzyme surface studied - the blank area shows the area occupied by hydrophobic amino acids (b).
  • Figure 3 Comparison between calculated parameters for amino acid Ser-105 (one of the three amino acids member of the lipase catalytic triad) in protein containing mutations and in native structure of the Antarctic Candida Lipase B enzyme.
  • the figure is showing for Ser-105 the variation of the values of the parameters listed in table 3, found in models where amino acids cited in table 1 were replaced by Val.
  • the descriptors "solvent accessibility” and “Cross Presence Order” are presented. as main introducing factors of variability among the studied models.
  • the parameters pertinent to the native structure and the different models change in color, starting with native protein (in black) and mutant "Ser_3" (in dark gray).
  • Figure 4 Comparison between calculated parameters for the amino acid Asn-187 (one of the three amino acids of the lipase catalytic triad members) in mutants, relative to the values obtained for the native structure of the Antarctic Candida Lipase B enzyme.
  • the figure is showing for Asn-187 the variation of the values of the parameters listed in table 3, found in models where amino acids cited in table 1 were replaced by Val.
  • the descriptor "solvent accessibility" is the main factor introducing variability. among the models studied.
  • the parameters pertaining to the native structure and the different models change in color, starting with native protein (in black) and mutant "Ser_3" (in dark gray).
  • Figure 5 Comparison between calculated parameters of the His-224 (one of three amino acid lipid catalytic triad member amino acids) in mutants, relative to the values obtained for the native structure of the Antarctic Candida Lipase B enzyme.
  • the figure is showing for His-224 the variation of the values of the parameters listed in table 3, found in models where amino acids cited in table 1 were replaced by Val.
  • the descriptor "solvent accessibility" is presented as the main introducing factor. of variability between the studied models.
  • the parameters pertaining to the native structure and the different models change in color, starting with native protein (in black) and mutant "Ser_3" (in dark gray).
  • Figure 6 Comparison between the calculated parameters for the amino acid Ser-105 (one of the three amino acids of the lipase catalytic triad members) in mutants, following an additional step of molecular dynamics energy minimization in the models obtained in the Modeller output, relative to values obtained for the native structure of the Antarctic Candida Lipase B enzyme.
  • the figure is showing for Ser-105 the variation of the values of the parameters listed in table 3, found in models where amino acids cited in table 1 were replaced by Val.
  • the descriptors "solvent accessibility” and "Cross Presence Order” are presented. as main introducing factors of variability among the models studied.
  • the parameters pertinent to the native structure and the different models change in color, starting with native protein (in black) and mutant "Ser_3" (in dark gray).
  • Figure 7 Comparison between the calculated parameters for the amino acid Asn-187 (one of three lipid catalytic triad member amino acids) in mutants, following an additional step of molecular dynamics energy minimization in the models obtained in the Modeller output, relative to values obtained for the native structure of the Antarctic Candida Lipase B enzyme.
  • the figure is showing for Asn-187 the variation of the values of the parameters listed in table 3, found in models where amino acids cited in table 1 were replaced by Val.
  • the descriptor "solvent accessibility" is the main introducer factor of variability. - among the studied models.
  • the parameters pertaining to the native structure and the different models change in color, starting with native protein (in black) and mutant "Ser_3" (in dark gray).
  • Figure 8 Comparison between calculated parameters of the amino acid His-224 (one of the three amino acids of the lipase catalytic triad members) in mutants, following an additional step of molecular dynamics energy minimization in the Modeller output models, relative to the values obtained for the native structure of the Antarctic Candida Lipase B enzyme.
  • the figure is showing for His-224 the variation of the values of the parameters listed in table 3, found in models where amino acids cited in table 1 were replaced by Val.
  • the descriptor "solvent accessibility" is the main factor introducing variability. among the models studied. The parameters pertaining to the native structure and the different models change in color, starting with native protein (in black) and mutant "Ser_3" (in dark gray).
  • FIG 9 Surface hydrophobicity index (SHI) values for the native protein (Itcb.pdb) and the ten mutants modeled with the aid of Modeller software (shown in black). DmSHI (represented in gray) indicates the SHI variation after mutation. All mutants have positive DmSHI, ie all studied mutations increased the surface region composed of hydrophobic amino acids. Among the mutants, the Combl and Comb2 models (both with multiple mutations) stand out among the models with the highest SHI.
  • SHI Surface hydrophobicity index
  • the invention relates to the identification of amino acids with specific properties on protein surfaces whose mutation does not alter the physicochemical and structural properties of amino acids considered catalytic.
  • the applicability of the technique with the Antarctic Candida Lipase B enzyme an important alternative for biodiesel production without addition of inorganic catalysts, bringing other environmental benefits, such as energy saving compared to the current production method with the transesterification reaction, and also without the need for glycerol purification, one of the byproducts of the current method.
  • the present invention further relates to a method for predicting mutants that increases the protein surface hydrophobicity index and the proteins obtained by that method.
  • Java Protein Dossier (Neshich, G, Rocchia) , W., Mancini, AL, Yamagishi, ME, Kuser, PR, Fileto, R., Baudet, C, Pinto, IP, Montagner, AJ, Palandrani, JR, Krauchenco, JN, Torres, RC, Souza, S., Togawa, RC, Higa, RH 2004.
  • JavaProtein Dossier A Novel Web-Based Data Visualization Tool for Comprehensive Analysis of Protein Structure Nucleic Acids Res. 2004 Jul 1; 32 (Web Server issue): W595-601
  • the databases for selecting proteins can be various. More specifically the present invention utilizes the public database called Protein Data Bank (PDB, Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, Bourne PE. "The Protein Data Bank.” Nucleic Acids Res. 2000 Jan 1; 28 (1): 235-42).
  • PDB Protein Data Bank
  • the characteristics of the protein to be selected may be, but are not limited to, distance from the catalytic site, internal contact density, hydrophobicity, electrostatic potential, amino acid sequential position conservation, cross link order, cross presence order, density, sponticity, temperature factor and others from a list of 711 physicochemical and structural parameters stored in STING DB, duly listed and described by Neshich G, Rocchia W, Mancini AL, Yamagishi MEB, Kuser PR, Fileto R, Baudet C, Pinto I, AJ Montagner, Palandrani JF, Kraucenco JN, Torres RC, Souza S, Togawa RC and Higa RH "JavaProtein Dossi- er: a novel web-based data visualization tool for comprehensive analysis of protein structure. "Nucleic Acids Res. 2004 Jul 1; 32 (Web Server Issue): W595-601.
  • Amino acid selection can be done through a specific module of the software used, for example, but not limited to the Blue Star STING J PD module.
  • Amino acid modeling can be performed by modeling software such as, but not limited to, Swiss-Model software (Arnold K., Bordoli L, Kopp J., and Schwede T. (2006).
  • the SWISS MODEL Workspace A web-based environment for protein structure homology modeling. Bioinformatics, 22,195-201) and Modeller (A. Sali & T L. Blondell. "Comparative protein modeling by satisfaction of spatial restraints” J. Mol. Biol. 234, 779-815, 1993).
  • the Modeller software was used.
  • the protein lipase was used as an example and in this case the selected amino acids were replaced by the Alanine residues as this amino acid retains the main chain structural characteristics (secondary structure formation) or preferably by a Valine, which has a higher hydropathy index than Alanine (both hydrophobic).
  • Monitoring of modeled amino acids can be performed in software such as, but not limited to, Blue Star STING and stored in TGZ format files (unique format designed to optimize the performance of STING server and STING software).
  • the comparison made in the software may involve several descriptors that may be but are not limited to: Unused contacts energy; Unused contacts; Density; Solvent accessibility area; Sponge; Cross Link Order) (grain of amino acid interconnection); Cross Presence Order (grain of co-location between amino acids); Hydrophobicity; Local curvature (choice can be from 711 parameters available in STING DB).
  • Mutants are monitored based on the developed V 4 score and explained in the example with the Antarctic Candida Lipase B enzyme. The lower the score value, the lower the influence of the substituted residues on the amino acids of the catalytic site.
  • step T Perform the energy minimization by molecular dynamics of the 3D models generated in step T; h) Monitor the variation of the physicochemical and / or structural properties of the amino acids that make up the catalytic site by comparing the descriptors generated by the specific software server;
  • the method of the present invention utilizes the Blue Star STING software (Neshich, G., Togawa, R., Mancini, A.L., Kuser, R.R., Yamagishi, MEB, Pappas Jr., G., Torres, WV, Campos. , T. R, Ferreira, L. L, Luna, R. M., Oliveira, A. G, Miura, RT, Inoue, MK, Horita, L. G, de Souza, D. R, Dominiquin, R, ⁇ lvaro, A., Lima, CS, Ogawa, R. O., Gomes, B. G, Palandrani, JC R., Santos, G.
  • Blue Star STING software Neshich, G., Togawa, R., Mancini, A.L., Kuser, R.R., Yamagishi, MEB, Pappas Jr., G., Torres, WV, Campos. ,
  • hydrophilic amino acids found on the protein surface were selected to be mutated in silico by hydrophobic amino acids.
  • a set of characteristics has been selected to monitor how the singular mutations affect the amino acid descriptors of the catalytic site. Mutations have been shown to generate minimal variations of these characteristics even in models containing multiple mutations, thus resulting in models with a surface area consisting of a larger number of hydrophobic amino acids, thereby increasing solubility in hydrophobic media, such as media. vegetable oils.
  • the present invention also relates to the mutant proteins obtained by the mutant prediction method described herein.
  • the first step in developing possible lipase protein mutants is choosing a base three-dimensional structure (template).
  • template a base three-dimensional structure
  • amino acids that are found on the protein surface are chosen, and thus promote interactions with the solvent.
  • polar amino acids will be targets of possible mutations in the protein surface.
  • Another option used was the replacement of low value supporting amino acids on the hydropathy scale with residues with higher values on the same scale.
  • Modeller (A. Sali & TL Blundell. "Comparative protein modeling by the satisfaction of spatial restraints” J. Mol. Biol. 234, 779-815, 1993) for the generation of mutant models in relation to the base structure by the homology modeling method. Modeller software performs model generation using the template structure as a generator of spatial constraints applied to the structure to be modeled.
  • the input file consists of alignment between the template amino acid sequence (present in the PDB) and the amino acid sequence you wish to model, as shown in Table 1 below.
  • the underlined amino acids represent those residues that have replaced the template originals. In this case it is a multiple mutant that we named: "combol" and that contains all the residues listed in table 1.
  • Table 1 Polar, low hydrophobic protein surface amino acids selected as targets for mutation based on their distance from the catalytic site of the Antarctic Candida Lipase B enzyme (UCB.pdb) and at the same time containing no internal contact with others amino acids.
  • the highlighted amino acids listed are slightly hydrophobic and are therefore considered as suitable candidates for substitution with more hydrophobic Valine.
  • the force field used was Gromos96 (van Gunsteren, W. R; Billeter, SR; Eising, AA; Hunenberger, R; Kruger, R; Mark, AE; Scott, WR R; Tironi, IG "Biomolecular Simulation: The GROMOS96 Manual and User Guide “vdf Hochschulverlag AG an der ETH Ziirich and BIOMOS bv: Zurich, Groningen, 1996.), with existing parameters for amino acids and nucleic acids, as well as ions such as calcium, chlorine and also water molecules , as the protocol used uses explicit solvent. Table 2 below summarizes the sequence of commands used in model generation with this additional refinement step:
  • the first command line uses the "pdb” format file as the input and converting it to the gromacs (.gro) and topology (.top) coordinate file using the force field (which contains parameters for all system atoms) G53a6, which is code for the Gromos96 field.
  • the topology files and coordinates are joined by generating the output file in "tpr” format with parameters defined by the minim.mdp file, which contains information about which library and algorithms to use, and limit values on how much power the minimization should be run. .
  • minim.mdp file which contains information about which library and algorithms to use, and limit values on how much power the minimization should be run.
  • all files generated under the name lip1-EM-vacuum are used in the molecular dynamics round having as output file a file in "pdb" format with the coordinates of the system atoms with minimized global energy.
  • the 17 generated models and the Antarctic Candida Lipase B enzyme were analyzed by the Blue Star STING software, which stores in its database (STING_DB) more than 700 physicochemical, structural and evolutionary descriptors.
  • the values for each of the descriptors for the Antarctic Candida Lipase B enzyme are precalculated and stored in the STING DB, while the descriptors for the Modeller-generated models can be calculated by the Blue Star STING server, generating TGZ format files.
  • the descriptors used in the comparative analysis are listed in table 3.
  • mutant mutated structures were generated in a total of 4 models of which two were created using Modeller and two with a sequence of mutations. Modeller and Energy Minimization performed by Gromacs, and finally followed by comparative analysis with the native structure of Antarctic Candida Lipase B, since combined mutations should influence the hydrophobic solubility of the enzyme in question more than Simple mutations.
  • Figures 3 to 5 show the results of each of the parameters Table 3 (except for the "Unused Contact Energy” and “Unused Contacts” parameters) comparatively to the values given by the native structure ITCB.pdb for the three amino acids of the catalytic site: Ser-105, Asp -187 and His-224, respectively.
  • ⁇ represents the variation of the Ser-105 residue parameters of each mutant relative to the Ser-105 residue parameters of the native protein ITCB.pdb.
  • V 2 and V 3 represent the same variation score for residues Asn-187 and His-224, respectively.
  • Variation scores were calculated, generating a list of best mutations in the sense of least influence on the catalytic triad parameters indicated by lower V 4 values, shown in table 4. Table 4 shows that the variation of the monitored parameters for all mutants was minimal and therefore all substitutions could be used for multiple mutant generation.
  • Table 4 - V A variation score values for each mutant.
  • V ⁇ V j
  • f ⁇ P 1TCB ⁇ / - ⁇ ⁇ *, ⁇ ⁇
  • SHI Surface Hydrophobicity Index
  • ANHI Aminoacid Normalized Hydrophobicity Index
  • JavaProtein Dossier a novel web-based data visualization tool for comprehensive analysis of protein structure Nucleic Acids Res. 2004 Jul 1; 32 (Web Server issue): W595-601) .
  • the hydrophobicity index value of "i" is extracted from scales The present study used the Radicka scale (Radzicka, A. & Wolfen- den, R. (1988). Comparing the polarities of the amino acids - side-chain distribution coefficients between the vapor-phase , cyclohexane, 1-octanol, and neutral aqueous-solution (Biochemistry 27, 1664-1670).
  • the isolated chain SHI is calculated as the sum of all hydrophobic residue (HB) ANHIs of the chain in question divided as the sum of all hydrophilic residue (HL) ANHIs of this same chain, as shown below:
  • this index reflects a measure of surface hydrophobicity, the higher the higher the SHI. If the surface contains more hydrophilic amino acids, the lower the SHI.
  • the SHI was then calculated for all mutants and compared to the Lipase B SHI used as template (1TCB). To compare them, the "DmSHI" (difference of the mutants in relation to the native protein SHI) was defined, being the Itcb.pdb SHI minus the mutant SHI.
  • Figure 9 presents the values for the single mutants and also the values found for the two multiple mutants.
  • Figure 10 we illustrate the mutations chosen in the three-dimensional structure of the protein, as well as the region of the catalytic site.

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Abstract

La présente invention concerne une méthode de génie protéique consistant à identifier des régions de la surface d'enzymes susceptibles de subir des mutations influençant de manière réduite les propriétés physico-chimiques et structurales des acides aminés du site catalytique, avec pour objectif de créer des mutants présentant une surface macromoléculaire plus hydrophobe. Cette technologie peut être appliquée à la conception rationnelle d'enzymes présentant une plus grande solubilité dans des milieux hydrophobes tels que les huiles végétales.
PCT/BR2012/000260 2011-08-04 2012-08-01 Méthode de prévision de mutants augmentant l'indice d'hydrophobicité de la surface de protéines Ceased WO2013016794A1 (fr)

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