DESC:FIELD OF THE INVENTION
[0001] This invention relates to the field of Biology, Cell Biology Life Science, Computational Biology, Biocatalysis, Enzymes and Chemistry. The invention relates to a biotechnological approach where structural elements of cells are modified to perform new functions related to catalysis and support of enzymatic activity, leading to applications in biocatalysis, medicine, and industry.
BACKGROUND OF THE INVENTION
[0002] The invention is based on two Cytoskeletal Proteins namely F-Actin and FtsZ, a Tubulin homolog
F-Actin
[0003] Cytoskeleton proteins are involved in several biological processes related to the cell shape and movement, it also gives structural support to cells and assists in intracellular transport of organelles. One example of skeleton protein is actin filaments which are assembled and stabilized by accessory protein into higher order contractile structures such as stress fibres or contractile bundles. Filamentous actin (F-actin) exhibits a higher temperature and pressure stability (Rosin, Christopher, et al. 2014). While the biocatalysts, are delicate materials that hover close to the thermodynamic limit of stability. Actin has the ability to transition between monomeric (G-actin) and filamentous (F-actin). F-actin exhibits a higher temperature stability compared with G-actin and the thermal stability depends on the bound nucleotide and divalent cation (Rosin, Christopher, et al. 2014). The supramolecular assemblies of F-actin are stable enough to withstand the extreme conditions usually encountered on Earth. F-actin can stand upto ~1 and 2 kbar (Rosin, Christopher, et al. 2014). F-actin is thermally more stable than G-actin. The thermal denaturation of G-actin is shown around 60-62 °C, while for the F-actin it is shown around 65-70 °C. In case of F-actin bound with phalloidin have shown higher temperature stability at 75-80°C (Levitsky, Dmitrii I., et al., 2008).
[0004] The assembly of actin filaments is regulated by the ionic strength of actin solutions. In solutions of low ionic strength, actin filament depolymerizes to monomers. Actin then polymerizes spontaneously if the ionic strength is increased to physiological level. The actin monomers also bind ATP, which is hydrolysed to ADP following filament assembly. Although ATP is not required for polymerization, actin monomers to which ATP is bound polymerize more readily than those to which ADP is bound. Actin polymerization is reversible, filaments can depolymerize by the dissociation of actin subunits, allowing actin filaments to be broken down when necessary. Thus, an apparent equilibrium exists between actin monomers and filaments, which is dependent on the concentration of free monomers.
[0005] Actin polymerization is a reversible process, in which monomers both associate with and dissociate from the ends of actin filaments. The rate of subunit dissociation (koff) is independent of monomer concentration, while the rate of subunit association is proportional to the concentration of free monomers and given by C × kon (C = concentration of free monomers, kon = subunit association). An apparent equilibrium is reached at the critical concentration of monomers (Cc), where koff = Cc × kon. (Cooper et. al.,1987). Significance of ionic environment in actin polymerization is well established, Mg2+, K+ and Ca2+ ionic strength in actin assembly is also studied extensively (Maruyama, K. 1981, Cooper, John A., et al. 1983, Kang, Hyeran, et al. 2013, Hundt, Nikolas et al. 2022).
[0006] There are known compounds/molecules that stabilizes filaments, thus inducing actin polymerization, these compounds stabilize the assembled polymer by binding to F-actin which promote polymerization through a shifting of the equilibrium towards filaments (Ruggiero et. al., 2021). Phalloidin is one of the best-known compounds for promoting polymerization (Cooper et. al.,1987). Another molecule Jasplakinolide is cyclodepsipeptide promotes actin polymerization and binds F-actin competitively with phalloidin and induces actin filament stabilization (Bubb et. al., 1994). There are also F-actin assembly inducing-agents like Dolastatin 11 (Bai, Ruoli, et al. 2011), Hectochlorin (Marquez, Brian L., et al., 2002) and Doliculide (Bai et. al., 2002), which promotes/stimulate the formation of F-actin.
[0007] Nitrilases are known to be labile and unstable, which has significantly hindered process applications. The lack of purified enzyme preparations has prompted the use of whole cell biocatalysts (Asano et. al., 2012). Although, commercial nitrilase preparations have been developed recently, they are very expensive for synthetic applications (Asano et. al., 2012). Immobilization of the whole cells harbouring the ‘expensive’ nitrilase offers an ‘inexpensive’ alternative to improve its reusability and stability and, from the point of view of process economics, it renders the enzyme reusable for subsequent reactions, thereby reducing the cost price. Enzyme immobilization provides a feasible way to enhance their operational stability, since it provides an insoluble enzyme complex on a specialized module, allowing convenient reusability. To overcome the limitations of nitrilase reusability and stability, a structural protein F-actin is engineered to give a hydrolase activity.
FtsZ (Tubulin homolog)
[0008] Cytoskeleton proteins are known for their uses to maintain cell structure and their mechanical support to maintain cytoplasm organelles. In human cells, cytoskeletons are made up of three different major class of proteins which is microtubules, microfilaments, and intermediate filaments. Microtubules are the largest type of filaments usually made-up tubulin proteins. Microfilaments are necessary filaments made up of small proteins like actin. There are other mid-sized proteins like keratin, vimentin etc. Similar to this, bacteria contain cytokinesis proteins that are important in cell division, organelle activity, and structural alignment. The majority of the proteins that are involved in cell division are referred to as divisomes. These important protein complexes are found in bacteria and are in charge of constricting the inner and outer membranes during cell division. Tubulin homologs are an important component of divisomes, these homologs are known to be FtsZ proteins.
[0009] FtsZ is a protein which forms Z-ring structures found in helical form and is known to move important organelles of the cell during the cell division process. FtsZ is similar to eukaryotic tubulins sequentially and also structurally. Structurally, eukaryotic tubulin has additional C-terminal domain which is absent in the FtsZ. There are other small proteins such as FtsA, FtsN SepF, ZipA etc are required for forming and stabilizing the polymer of FtsZ and its functioning during cell division. FtsA is known to form an amphipathic helix that facilitates the binding of FtsZ to the membranes. During cell division, FtsA binds to FtsZ and interacts with the inner membrane of the cell wall. The FtsA binds to the Z-ring formed by the FtsZ and interacts with the membrane.
[0010] Cyanide compounds are health hazards for humans and also for the environment. Cyanide compounds can be seen in wastewaters, coal processing, ore leaching, pharmaceutical industry wastes, plastics, agrochemicals etc. There are many known chemical methods for eliminating the cyanide group but using a chemical process to come out of another chemical is not environmentally feasible, also these chemical processes are not cost-effective and take longer times (Martínková et al., 2015). A convenient method to overcome this drawback is only by using enzymes. The nitrilase enzymes are known for converting different molecules which has cyanide group into carboxylic acid and ammonia (shen et. all., 2020, Gong et. all 2012).
[0011] Applications for nitrilases in processes have been severely hampered by their labile and unstable nature. Whole-cell biocatalysts are used since refined enzyme preparations are lacking. Despite the recent development of commercial nitrilase preparations, these products are exceedingly versatile for synthetic applications (Asano et al., 2012). The 'inexpensive' method to increase the nitrilase's stability and reusability is to immobilise the entire cells containing the 'expensive' nitrilase. From the perspective of process economics, this makes the enzyme reusable for subsequent operations, lowering the cost price. Because it places an insoluble enzyme complex on a customised module and permits practical reusability, enzyme immobilisation offers a workable solution to improve its operational stability.
[0012] As explained above about nitrilase enzyme it is an opportunity to develop new or alternative methods as a solution to overcome industrial requirements and environmental aspects. FtsZ are structurally highly stable and known to form a Z-like ring structure which is a polymer that is highly strong and can act as an immobilization factor. Taking advantage of this higher stability, reusability, temperature and pressure tolerance, and aggregation property, we developed engineered FtsZ which can be used to perform nitrilase reaction mechanisms.
OBJECTIVES OF THE INVENTION
Actin
[0013] The objective of the invention is an engineered structural protein F-actin having a hydrolase activity, where the recombinant protein catalyses the 1-(cyanomethyl) cyclohexane-1-carbonitrile to 2-(1-cyanocyclohexyl) acetic acid (an intermediate of gabapentin) and where the recombinant protein contains catalytic residues adapted from nitrilase enzyme. The engineered structural protein F-actin having the properties of surface adhesion (acting as a self-immobilized enzyme), improved biocatalytic reusability for hydrolysis as compared to nitrilases i.e., the engineered F-actin can be used in multiple cycles without the change in hydrolysis rate, selectivity, and yield for 1-(cyanomethyl) cyclohexane-1-carbonitrile. The making structural F-actin nitrilase gives the edge over nitrilase which is known to be labile, unstable and limitations of nitrilase reusability.
FtsZ
[0014] The objective of the invention is also an engineered, reusable, self-assembling and self-immobilizing FtsZ that can function as a nitrilase enzyme for the conversion of 1-(cyanomethyl)cyclohexane-1-carbonitrile into 2-(1-cyanocyclohexyl)acetic acid. The FtsZ are known to be involved in cellular functions and are highly stable for longer duration at varying temperatures and pH conditions. Engineering FtsZ to accommodate nitrilase active site or nitrilase reaction performing catalytic residues will improve the uses and industrial application of nitrilase functions and overcome many drawbacks. The incorporation of catalytic residues and converting FtsZ into a functional enzyme gives the advantages of reusability as FtsZ are long-living proteins, they form polymeric fibres or filamentous structures which act as self-immobilized proteins. The objective of this patent is to provide an engineered FtsZ to perform nitrilase function.
SUMMARY
Actin
[0015] Nitrilases are able to hydrolyse nitriles into their corresponding carboxylic acids and ammonia, but its applications in processes have been severely hampered by their labile and unstable nature (Asano et. al., 2012). The lack of purified enzyme preparations has prompted the use of whole cell biocatalysts leading to commercial nitrilase preparations which were developed recently are expensive for synthetic applications (Asano et. al., 2012). F-actin is reported for feature like rigid in tension and flexible in torsion (Viney et. al., 2001), hence nitrilase active site is engineered into F-actin to over the previously defined drawbacks of nitrilase enzyme. The supramolecular assemblies of F-actin exhibit a higher temperature stability compared with G-actin and the thermal stability depends on the bound nucleotide and divalent cation (Rosin, Christopher, et al. 2014), it can also withstand up to ~1 and 2 kbar (Rosin, Christopher, et al. 2014) of pressure. The thermal denaturation of G-actin is shown around 60-62 °C, while for the F-actin it showed around 65-70 °C. In case of F-actin it bound with phalloidin have shown higher temperature stability at 75-80°C (Levitsky, Dmitrii I., et al., 2008). This extended feature of F-actin was harnessed along with nitrilase hydrolysis capability to convert nitrile substrates namely 2-chloropyridine-3-carbonitrile, 2-(2-methylpropyl)butanedinitrile, 3-cyanopyridine, acrylonitrile, glycolonitrile, mandelonitrile and 1-(cyanomethyl)cyclohexane-1-carbonitrile into corresponding carboxylic acids 2-chloronicotinic acid, (S)-3-cyano-5-methylhexanoic acid, nicotinic acid, acrylic acid, glycolic acid, (R)-mandelic acid and 1-(1-cyanocyclohexyl)acetic acid respectively with an engineered F-actin (Figure 1). The F-actin is engineered to incorporate the four catalytic residues namely Cysteine (C), Lysine (K), and two Glutamic acid (E) giving a nitrile hydrolysis function. The incorporation of nitrilase catalytic residues were achieved using computational engineering method. The computational engineering method involves a series of steps to design a functional nitrilase active site in F-actin, which is illustrated below in stepwise manner (Figure 7). The first step involves the identification of target F-actin for engineering. The identified actin is originated from three different organism E. coli K12 (Uniprot ID: POA9X4), Komagataella phaffii (Uniprot ID: Q9P4D1) and Archea bacteria (Accession number: WP_231190500) (Oda et. al., 2009). The Cucurbitacin E binding pocket were identified in all the three F-actins (Figure 3), for these identified pockets nitrilase catalytic residues (C-E-E-K) were introduced and additional mutation was also introduced for better substrate affinity, these identified pockets were fitted with four catalytic residues (C-E-E-K) of nitrilase computationally and additional substitution for substrate fitting, using a defined geometry as shown in Figure 2A-B. The geometry is defined using three atoms of the catalytic residues, Cys sulphur, Glu oxygen and Lys nitrogen atom. The defined atoms were used to derive the appropriate angles and distances among each atoms. The derived distance and angles between the atoms were defined as a geometrical constraint for screening in each identified pocket (Figure 2A-B). The identified pockets which obey the criteria of defined angels and distances with defined atoms were mutated to corresponding residues in the F-actin (Figure 4). The engineered F-actin containing the nitrilase active site is simulated in aqueous environment to check the stability of the for catalytic residues as reference to nitrilase. The stable F-actin nitrilase were docked with 1-(cyanomethyl) cyclohexane-1-carbonitrile substrate to derive enzyme substrate complex. The derived enzyme-substrate complex is again simulated in the aqueous environment for stability of substrate (Figure 7). The unstable variants of F-actin were reengineered in the active site (apart from catalytic residues, or 6Å of catalytic residue/substrate) using site saturation mutagenesis (SSM) and re-simulated in the aqueous environment until the stable F-actin nitrilase substrate complex is achieved (Figure 5). The reaction feasibility study of engineered F-actin from E. coli K12 using QMD method using Car-Parrinello molecular dynamics (CPMD). The chemical reaction processed to converting nitrile to corresponding carboxylic acid product. The reaction relative energy versus reaction co-ordinate graph was plotted for the first step of the reaction (Figure 8). The catalytic residues of engineered F-actin are represented with purple, and substrate in orange colour. The stable F-actin Nitrilase is synthesized and inserted into pET-28a (+) and expressed in E. coli BL21 (DE3) (Figure 6).
FtsZ
[0016] Nitriles can be hydrolysed into the appropriate carboxylic acids and ammonia by nitrilase enzymes, these enzymes are labile and unstable characteristics have severely limited their use in processes. Since there aren't any commercially available pure enzyme preparations, whole-cell biocatalysts have been used instead. This has resulted in the recent development of commercial nitrilase preparations that are useful for a wide range of synthetic applications (Asano et al., 2012). Nitrilase is known for converting many commercially required substrates and 1-(cyanomethyl)cyclohexane-1-carbonitrile is one among them (Fig.9). There are many substrates with cyanide group that can converted using the nitrilase enzyme.
[0017] FtsZ are known to form a Z-ring-like structure which is in a helical shape, these Z-rings adhere to the membrane of the bacterial cell wall and generate force and provide support during the cell division process (Fig.10). The FtsZ shows multiple pockets available for engineering studies (Fig.11). Targeting these regions for engineering may provide the best results. The active site of any enzyme is a signature and a sensitive region where the reaction performs with the help of catalytic residues. The engineering process includes the incorporation of catalytic residues according to the architecture of the nitrilase native catalytic pocket (Fig.12). The nitrilase active site shows a hydrophilic nature and also requires two water molecules for the reaction to perform. The engineering process involves computational methods to engineer FtsZ to have nitrilase function which is diagrammatically explained (Fig.13). The engineering process starts by identifying the available pockets or cavities in the protein that are large enough to accommodate nitrilase active site residues and substrate molecule. The incorporation of residues maintains the criteria of catalytic architecture. Cys is an important amino acid to initiate the nitrilase reaction, in the second step we scanned if the pockets have Cys residues in them. If Cys is present, then we fix the Cys residue and extracts surrounding 12Å residues in the pocket. Once we fix Cys and extract the surrounding residues list, we mutate each residue in the pocket to Lys to match the architecture. A plain drawn between CB & CA of Cys (vector 1) & Lys (vector 2) in such a way that a vector is drawn between points towards the same point. A normalised plane is defined between Cys & Lys, the angle between vector1 and vector2 should match to be a satisfied criterion and to ensure Cys and Lys are pointed towards the same direction. If Cys is not present in the identified pockets, then all positions are mutated to Cys, later by following steps until finding Lys with a 12Å radius of Cys. In the second step of building the catalytic architecture, residues within 5Å around Cys and Lys mutated to Glu. There are two glutamic acids required for the reaction and stabilizing the active site. Two Glu residues are incorporated where Glu1 is present closer to Cys and Glu2 is closer to Lys. The architecture also holds stringent distance criteria of distance between Cys-SG to Lys-N, Cys-SG-Glu1, and Lys-N-Glu2-OD1. By defining this criteria we found two potential pockets where we incorporated nitrilase active site residues successfully.
Substrate fitting:
[0018] Once the pocket is matched with the architecture and given distance criteria further steps will be processed where induced fit mode of studies will be conducted. In the engineered pocket 1-(cyanomethyl)cyclohexane-1-carbonitrile molecule subjected to induced fit mode studies. The induced fit studies were conducted to check the feasibility of the pocket capable of fitting the substrate with feasible energy and distance between the substrate and catalytic residue. The distance between Cys-S and cyanide carbon of the substrate, Lys-N and nitrogen from the cyanide group of the substrate is set to be within 4Å.
[0019] Based on the above distance criteria it is possible to sample different substrates that have cyanide groups in them. The conformation of the substrate where it falls under the above-mentioned distance criteria and the conformation of the substrate with feasible energy will be considered for further steps (Fig.14).
[0020] The selected conformations were taken for Molecular dynamics simulations where engineered FtsZ is complexed with substrate from the induced fit studies. In the MD simulation, the FtsZ complex was placed in a water box, and the overall charge of the system was neutralised by adding NaCl ions with a concentration of 0.15 to mimic the natural conditions of a cell. The neutralised system is taken for the energy minimisation and equilibration steps before conducting the production run.
[0021] The production run simulation for the engineered FtsZ complex was conducted for 50ns *2 runs in that, the RMSD graph (Fig.15) shows the stability of the simulation across the time. Also, the distance criteria used in the simulations explain the stability at the molecular level, which is the distance between the Cys256-S and sub-C (cyanide carbon) was maintained at ~3.5Å +/- 0.2Å, similarly, the distance between Lys-N and Sub-N (Cyanide nitro group) is maintained at ~2.5Å +/- 0.22Å.
[0022] The MD simulations provide insight into the stability of the ES complex, a frame from the simulation taken for QM/MM studies to confirm the feasibility of the reaction happening in the pocket. The QM/MM simulations were conducted only for the first step of the nitrilase reaction. The Ground state energy is maintained as 0 Kcal/mol and Transition state (TS) and Intermediate state (IS) energies were calculated accordingly (Fig.16).
DETAILED DESCRIPTION OF THE FIGURES
[0023] Figure 1: Biocatalytic conversion of 1-(cyanomethyl) cyclohexane-1-carbonitrile to 2-(1-cyanocyclohexyl) acetic acid (an intermediate of gabapentin) using engineered F-Actin and substrate spectrum for the engineered F-Actin.
[0024]
[0025] Figure 2: (A) Geometrically defined, nitrilase catalytic residues (CYS, LYS, GLU, GLU). (B) Crucial catalytic distances defined for the binding mode analysis between the catalytic residues.
[0026] Figure 3: (A) Cucurbitacin E binding pocket of F-actin, (B) Identified Cucurbitacin E binding pocket of mreB protein from E. coli K12 strain (C) Identified Cucurbitacin E binding pocket of ACT1 protein from Komagataella phaffii GS115 strain (D) Identified Cucurbitacin E binding pocket of ACT1 protein from Archea bacteria; The identified CurE binding pockets were engineered with the incorporation of nitrilase catalytic residues (Cys, Lys, Glu, Glu) using computational method as defined in the methodology.
[0027] Figure 4: Engineered Cucurbitacin E binding pocket of F-actin, (A) Engineered CurE pocket in mreB of E.coli with the incorporation of catalytic residues Cys, Lys, Glu, and Glu. (B) Engineered CurE pocket in ACT1 of Komagataella phaffii with the incorporation of catalytic residues Lys, Glu, and Glu. (C) Engineered CurE pocket in ACT1 of Archea bacteria with the incorporation of catalytic residues Cys, Lys, Glu, and Glu; Incorporation of catalytic residues were performed using computational method as defined in the methodology. A QMD study was performed to check the feasibility of the reaction for the first step.
[0028] Figure 5: Additionally mutated residues of F-actin in Cucurbitacin E binding pocket to achieve nitrile hydrolysis of 1-(cyanomethyl) cyclohexane-1-carbonitrile to 2-(1-cyanocyclohexyl) acetic acid (an intermediate of gabapentin) of functions. Incorporation of additional mutation in F-actin of E.coli (A) Komagataella phaffi (B) and Archea bacteria (C); additional mutation are represented in pink colour and catalytic mutations are represented in cyan colour.
[0029] Figure 6: Schematic flow chart showing method of engineering for F-actin using
[0030] computational methods.
[0031] Figure 7: Catalytic distances of substrate 1-(cyanomethyl) cyclohexane-1-carbonitrile in engineered F-actins derived from 50ns of molecular dynamics simulations. (A) Schematic representation of catalytic distance of substrate to nitrilase catalytic residues (C-E-E-K). (B) Measured D1 distance between catalytic Glutamic acid and Lysine residue (C) Measured D2 distance between catalytic Lysine and substrate nitrile carbon. (D) Measured D3 distance between catalytic Cysteine to substrate nitrile carbon.
[0032] Figure 8: Thermodynamic profile for the reaction mechanism studied for engineered F-actin: thioimidate. Profile represents the relative energy when the Cysteine (purple) sulphur attacks the substrate (orange) nitrile carbon and nitrile abstracts hydrogen from water molecule. Energies are in kcal.mol-1.
[0033] Figure 9: General reaction mechanism by nitrilase enzyme where the 1-(cyanomethyl) cyclohexane-1-carbonitrile molecule holds cyanide group which will be converted into carboxyl group as it is shown in the reaction.
[0034] Figure 10: The FtsZ polymer formation during cell division where the FtsZ does not form filament as eucaryotic microtubules but forms a Z-ring structure which is also a polymer present in helical conformation. A) the representation of bacterial cells where the chromosome is shown in lines and the origin of replication is shown with a green circle, FtsZ proteins in purple circles inside the cell. B) Aggregation of FtsZ proteins, C) formation of small filaments and interaction with membranes, and D) formation of the Z-ring during the cell division which helps the movement of organelles.
[0035] Figure 11: In the pocket analysis conducted on FtsZ protein, the pockets were identified and considered for the incorporation of catalytic sites. The protein is shown in light pink and light green ribbons and the pockets are in different colors with surface representation.
[0036] Figure 12: The topology of catalytic residues from the nitrilase active site used to incorporate in the FtsZ protein. The required catalytic residues to perform nitrilase function are Cys, Glu, Lys, and Glu which are responsible for the reaction.
[0037] Figure 13: Process for explaining the stepwise method used for engineering of FtsZ where the starting structure was subjected to find different pockets that could be considered for the engineering studies. The pockets which have Cys in them were taken as a higher priority and engineered to accommodate other catalytic residues. Once the pocket is built according to geometry, the substrate molecule fits in the pocket by conducting induced fit mode studies. The complex where the substrate is bound in the engineered nitrilase pocket is further validated using MD simulations and QM studies. If the engineered pocket fails to fit the substrate in MD and unfavourable energy is taken in QM studies, then the pocket will be discarded and the pocket will be redesigned.
[0038] Figure 14: The near-attack conformation of the substrate in the engineered active site of the protein. The distance between the catalytic Cys184 and the carbon of the cyanide group is 2.81Å and the distance between Lys and N of the cyanide group is 2.05Å. The conformation was an output of induced fit studies which is further considered for MD and QM studies.
[0039] Figure 15: A) The Root mean square deviation (RMSD) plot of c-alpha atoms over simulation. The RMSD plot indicates that the system attains stability during the simulation. B) The plot represents the distance between the active site residues and the substrate molecule. The distance between catalytic Cys184 and the carbon of the cyanide group and Lys and N of the cyanide group was maintained at ~ 2.22 +/- 1.02Å and 2.24 +/- 1.04Å respectively, across the simulation.
[0040] Figure 16: Car-Parrinello molecular dynamics (CPMD) for studying the mechanism of nitrilase in FtsZ. For QM calculations using CPMD, with the consideration of both the accuracy and the computing resources the GGA DFT method BLYP. CYS256, LYS407 and GLU257 were defined as a QM region for calculating the CPMD. The nitrilase mechanism involves the catalytic triad of CYS, LYS and GLU, the CYS plays the important role where it covalently binds to the carbon of the cyanide group of the substrate, reaction intermediate forms by transferring the proton of CYS to the water molecule and a proton transfer to cyanide group’s nitrogen from water. The proton transfer is termed as TS-1 (transition state-1) and the covalent bound of CYS is termed as the 1st intermediate (I-1) step. QM/MM calculations were conducted only for first step of the nitriliase reaction.
DESCRIPTION OF INVENTION IN DETAIL
Terminologies Explained / Abbreviations
Molecular dynamics simulation
[0041] Molecular Dynamics (MD) simulation is a method performed to study dynamical behaviour of any biological system. The atomic coordinates in the given biological entity such as protein or enzyme allows to show dynamical changes based on predefined force filed. In this invention, the MD simulation refers to the study the stability of engineered F-actin for nitrialse activity of mutated protein and its stability of mutated residues.
CPMD
[0042] Car-Parrinello molecular dynamics (CPMD) for studying the mechanism of nitrilase in F-actin. For QM calculations using CPMD, with the consideration of both the accuracy and the computing resources the GGA DFT method BLYP. CYS256, LYS407 and GLU257 were defined as a QM region for calculating the CPMD. The reaction follows by forming the covalent bond between the CYS256 and the nitrile carbon hydride shift will happen from CYS sulphur to the oxygen of water molecule, this initiates the formation of imine intermediate. The oxygen of water molecule will form the bond to the same imine carbon by donating its proton to the imine nitrogen and forms the primary amine by transfer of proton, The next step (step 2), nitrogen will break by forming ammonia and another water molecule donates its proton to CYS sulphur and the final product carboxylic acid is formed by hydroxyl bonding with the carbon.
Pockets
[0043] Pocket is a cavity found in the protein with the specific amino acid residues arranged that possesses the properties to bind the substrate. The functionality of protein is determined by the physiochemical properties, shape, and location within the protein. In this invention the pockets are referred to the available dents and cavities in the Actin protein.
Active site
[0044] Active site is the cavity in the protein where the substrate binds with high affinity and specificity and undergo chemical catalysis. The term active site in this invention refers to the active site of the nitrilase enzyme.
Catalytic residues
[0045] Catalytic residues are the amino acid residues that reside within the pocket of the protein which involve in chemical catalysis of the substrate. The catalytic residues facilitate the chemical catalysis by providing the functional groups that helps in stabilization of reaction intermediate within the active site of the protein. In this context the catalytic residues are of Nitrilase enzyme.
E-S Complex
[0046] Enzyme Substrate complex is abbreviated as E-S complex, which is a molecular association formed between the enzyme and substrate upon binding of substrate in the active site. The association between the Enzyme and substrate is temporary which is formed during an enzyme catalysed reaction. In this invention the E-S complex refers to the substrate binding of the Nitrilase enzyme in the engineered pocket of Actin protein.
Polymer
[0047] Protein Polymer is known as polypeptide composed of 20 different amino acids linked by peptide bonds. The sequential arrangement of amino acids in a polymer determines the primary structure of the protein polymer which then folds and forms secondary and tertiary structures upon interacting with itself.
Mutation/substitution
[0048] Mutation is a permanent change in the nucleotide sequence which can occur spontaneously or induced by external factors that result in change in the amino acid of the protein. This change alters the shape of the protein and has a positive or negative impact on the function of the protein depending upon the location of the mutation in the protein.
Geometric stabilization
[0049] Geometric stabilization is the arrangement of amino acid residues three dimensionally within the active site of the protein to facilitate the proper binding of substrate which results in the formation of Enzyme-Substrate complex allowing it for efficient chemical catalysis to form the product.
Binding Mode
[0050] The binding mode in this invention refers to how a substrate interacts with the enzyme in the active site. This is defined by the types of molecular interactions between the enzyme and the substrate during a chemical reaction.
Induced fit Docking (IFD)
[0051] Induced fit docking is used to predict the binding interaction between a ligand and a target protein. It accounts the dynamic changes in both the ligand and the protein conformations that occur.
Reusability
[0052] Here reusability of enzyme refers to the ability of enzyme to be used repeatedly in multiple catalytic cycles without undergoing significant degradation or loss of activity.
Site saturation mutagenesis (SSM)
[0053] Site saturation mutagenesis involves the introduction of all amino acid substitutions at a particular position within a protein or enzyme through a series of mutagenesis steps.
Thermal denaturation
[0054] Thermal denaturation is a process in which enzyme losses it’s biological activity due to exposure to high temperature.
Activation energy
[0055] Activation energy is defined as the minimum amount of kinetic energy that reactant molecules must possess to initiate a chemical reaction by overcoming the energy barrier associated with the transition state. It is also the energy difference between the ground state and transition state.
Centrifugation
[0056] A purification technique which can separate the particles or components based on their density using a high-speed rotating device called Centrifuge. In this invention the Centrifugation refers to the separation of recombinant engineered F-actin nitrilase from the substrates for reusability after multiple centrifugation cycle.
Angle
[0057] Angle in protein refers to the angle formed between the atoms of two residues in the protein. In this invention, the Angle directs to the angle between the catalytic residues of Nitrilase enzyme in the engineered pocket of FtsZ.
Place between residues
[0058] Place between residues refers to the distance between the side chain of any two amino acid residues in the protein. This refers to the distance between the catalytic residues of Nitrilase enzyme in the engineered pocket of FtsZ.
Hydrolysis
[0059] Hydrolysis in protein refers to the process of breaking down a molecule into two or more smaller molecules using water as a catalyst. Hydrolysis refers to GTP hydrolysis in this invention.
Substrate binding affinity
[0060] Substrate binding affinity is the strength of interaction between the enzyme and substrate upon binding. This binding interaction involves bonding and nonbonding interactions. The substrate binding affinity in this invention directs to the complementary binding interaction substrate and the engineered FtsZ protein.
Favourable and unfavourable interactions
[0061] Favourable and unfavourable interactions in the protein refer to the non-covalent interactions between the substrate and the amino acid residues of the active site in the protein. The favourable interactions contribute to the substrate binding having a complementary fit in the active site of the protein. The unfavourable interactions are the steric interactions occurring when nonbonding atoms come in proximity. The favourable and unfavourable interactions in this invention refer to the nonbonding interaction between the substrate and the amino acid residues of the engineered FtsZ protein.
Induced fit modes
[0062] The induced fit mode is the way the flexible active site of the enzyme accommodates the substrate through conformational changes. The conformational changes of the enzyme will be complementary to the substrate in shape and chemical composition. In this invention the Induced fit mode refers to the conformational changes in the engineered FtsZ protein to accommodate the substrate.
METHODOLOGY FOR ENGINEERING F-ACTIN TO HAVE NITRILASE FUNCTION
Methods in steps
1. Filamentous actin (F-actin) is a key cytoskeletal component of cells. Individual actin molecules are globular protein, each actin monomer (globular [G] actin) has tight binding site that mediate head to tail interactions with two actin monomers, so actin monomers polymerize to form filaments (filamentous [F] actin). The small molecule like Cucurbitacin E (CurE) and Lantrunculin A (LanA) binds to actin in different pockets and acts as inhibitor and of actin depolymerization and sequestration of monomeric actin respectively.
2. Cucurbitacin E specifically binds to filamentous actin forming a covalent bond at residue Cys257, but not to monomeric actin (G-actin) (Sorensen et. al., 2013, Roopa et. al., 2019). The identified CurE binding pocket with the Cys257 was used for the incorporation the nitrilase catalytic residues.
3. The Cys257 of F-actin was considered as a catalytic Cys of nitrilase (Figure 2), considering the Cys257 as centre points within the 6 Å, the selected feasible hotspots were mutated to corresponding nitrilase catalytic residues (C-E-E-K)
4. The incorporated nitrilase catalytic residues were validated with defined catalytic architecture derived from the nitrilase enzyme. The defined catalytic architecture was based on the distance and angle between the catalytic residues. The unsatisfied catalytic architecture was re-engineered using computational method, with different hotspot positions.
5. Computationally filtered variants, which were fit according to catalytic architecture validation were docked with nitrile substrates using induced fit docking (IFD) method.
6. The docked complexes are extracted and assessed for binding affinity and feasible catalytic distance of the substrate for the catalysis. The considered distances were, D1, D2 and D3 as shown in the Figure 2. Favourable binding modes of the substrates were defined with certain conditions like, minimum distance for D1 = 3.0 Å, D2 = 3.5 Å and D3 = 3.0 Å. Also, the substrates should not show any geometrical or Van der Waals clashes with neighbouring residues. The unsatisfied binding mode variants were reengineered using computational method.
7. Screened favourable binding mode of nitrile substrates complexed with F-actin nitrilase were simulated in aqueous environment for 50ns. The stable E-S complex variant of F-actin were assessed based on distance of D1, D2, D3, RD1, RD2 and RD3 as shown in Figure 2. These distances were assessed across all F-actin variants during complete course of simulations. The unstable variants were sent for re-engineering for further additional mutations for packing of the substrate, while the stable variants are analysed for QM studies.
8. The QM studies were performed for the molecular simulations stable variants using CPMD program, for the first step of the reaction (Figure 2). The activation energy was calculated for the formation of bond between nitrile carbon and sulphur.
9. The lower activation energy F-actin variants were considered for the E. coli expression, while the high activation energy F-actin variants were re-engineered.
10. The E. coli expressed variants were screened against the nitrile substrates for the efficient hydrolysis of nitriles.
METHODOLOGY FOR ENGINEERING FtsZ TO HAVE NITRILASE FUNCTION

[0063] In the engineering process of FtsZ to accommodate nitrilase active site residues we developed an algorithm that considers the available pockets present FtsZ which is large enough to accommodate catalytic residues and substrate of interest which is 1-(cyanomethyl) cyclohexane-1-carbonitrile.
Methods in steps
1. Cavity identification to build the nitrilase pocket
Protein is scanned for cavities/pockets that are large enough to fit nitrilase catalytic architecture (NAC), these cavities are considered as potential cavities (PCs) to fit/engineer NAC, critical residues and their immediate surroundings are excluded if they are present in PCs. Pockets and their volume in an FtsZ are identified and calculated using caver tool, pockets with a volume more than the defined NAC volume (1.4Å of radius) are chosen as PCs to engineer novel NAC into FtsZ. The whole protein structure is comprehensively analysed to identify residues involved in salt bridge and disulfide formation; these residues are considered as critical residues since these interactions play a pivotal role in maintaining the structural integrity of the protein. Salt bridges refer to electrostatic interactions between oppositely charged amino acids within a protein, while disulfide bonds are covalent bonds formed between two Cysteine residues. These interactions safeguard protein against unfolding and denaturation. So, Immediate residues of salt bridge and disulfide-forming residues are deliberately excluded from the engineering process. To ensure that no alterations are made that might compromise the structural stability of the engineered protein.
2. Filtering the pockets to incorporate catalytic residues
Cys serves as a nucleophilic amino acid which attacks the cyanide carbon of the substrate and initiates a nitrilase reaction. Hence Cys is considered as the position of initiation (PoI) to engineer NAC. PCs are first scanned for any native Cys residues, PCs with existing Cys residues are prioritized for engineering. If PCs possess only one native Cys are identified, then this Cys residue is used as PoI to build NAC, if PCs with more than one Cys is found then, all native Cys positions are used as point of initiation to build NAC considering one Cys as PoI at a time, while mutating the rest of Cys residue in PCs to Ala.

3. Engineering method to incorporate catalytic residues
3.1) A zone of 12 Å residues around the PoI (Cys) will be considered for engineering, this threshold zone (TZ) size is calculated based on the geometric analysis of NAC from native nitrilase enzyme, 12 Å is considered because any residue mutated to Lys beyond this distance ( C alpha distance) will not have very less probability of desired of side-chain orientation i.e, Lys side chain will be far from nitrile group of the substrate that will be interacting with PoI Cys.
3.2) Each residue in TZ is mutated to Lys keeping Cys as a reference position, every introduced Lys is geometrically evaluated in such a way that the vector drawn between CB & CA of Cys (vector 1) & Lys (vector 2) directs towards a same point and the angle between vector1 and vector2 a normalised plane drawn defined between Cys & Lys are satisfying the geometric criteria predefined for NAC.
3.3) If Cys is not present in the PCs then all residue positions of PCs are treated as PoI, every residue in PCs is mutated to Cys followed by steps 3 and 4. This step is done to build/fit NAC with all possible PoI present in a PCs.
3.4) Residues within 5Å around Cys and Lys is mutated in all combination to introduce Two Glu residues necessary for the catalytic interaction. Two glutamic acids are required for the reaction and the active site stability, Glu residues are incorporated in a fashion where Glu1 is present closer to Cys and Glu2 is closer to Lys. Newly introduced Glu’s which will not affect the substrate binding cavity volume is most preferred.
3.5) The engineered PCs with introduced NAC architecture satisfying the stringent geometric criteria of distance between Cys-S to Lys-N, Cys-S-Glu1, and Lys-N-Glu2-OD1 are only recorded/passed on for visual inspection and then passed to further analysis and engineering.
4. Induced fit mode studies to check the feasibility of substrate fitting
The validated catalytic active site with the desired architecture was selected and taken for the induce fit analysis where the substrate is fitted to the active site of the protein. The induced fit mode of studies generates the best conformation with feasible non-covalent interaction to the protein active site by applying the Lamarckian genetic algorithm and setting the grid around the active site of the protein with 60, 60 and 60 at x, y and z directions respectively with 0.375 Å grid space around the active site residues CYS, GLU, LYS and GLY.
5. Selection of energetically feasible conformation
Post-induced fit analysis of the Enzyme-Substrate complex (E-S complex) by induce fit analysis, the structure with low energy value will be selected. Distance analysis between the CYS-SG (Cysteine’s sulphur) and the substrate’s nitrile carbon in the range of 3Å-3.5Å which according to the mechanism the CYS-SG forms the covalent bond with the substrate’s nitrile carbon and the nitrile nitrogen should be near to the LYS nitrogen with desired non-covalent interaction. The complex with undesired interaction and the complex with distance not in the range of 3-3.5Å move to redesign of the pocket where the undesired interacting residues mutate to make the desired interaction and again the pocket will undergo the induced fit analysis.
6. Validation 1: Molecular dynamics simulations
The complex with desired energy and distance in the range of 3-3.5Å, Molecular Dynamics (MD) is performed for the complex using GROMACS with force field AMBER99SB. For the preparation of the system for MD, the system was placed in the triclinic box at 0.4Å and the overall charge of the system was neutralised by adding NaCl ions with a concentration of 0.15 to mimic the natural conditions of a cell. The neutralised system is taken for the energy minimisation and equilibration steps before conducting the production run.
7. Validation 2: QM/MM simulations
Quantum mechanics/molecular mechanics is a simulation method where ab initio quantum mechanics calculations and molecular mechanics force fields are used to study any given chemical/biological reaction. The QM studies were conducted on engineered nitrilase FtsZ complex, and compared with native nitrilase active site. The unfavourable energy forming engineered
EXPERIMENTS AND RESULTS of ENGINEERING of F-ACTIN TO HAVE NITRILASE FUNCTION
Expression Protocol for Engineered F-actin Nitrilase Polypeptides
[0064] A full-length clone of actin was subcloned into bacterial expression vector, pET-28 a (+) (pZL100) in both orientations; the correct orientation encodes full-length, wild type actin. pET-28 a (+) vector has T7 promoter and inserted between EcoRI and BamHI restriction site. The growth of cultures was started from glycerol stocks and were grown in LB broth at 37 °C. Bulk cultures were seeded with a 1/100 innoculum of overnight culture. Unless otherwise stated, bulk cultures were in 1 liter of M10 + medium+ 40µg/ml ampicillin, incubated at 37 °C in a 2 liter baffled flask with vigorous shaking. When the cultures reached an OD550 of 0.2-0.3, they were induced with 4mM IPTG. When cells reached an OD550 of 0.9 -1.0, the culture flasks were plunged into an ice-water bath for 20-30 min. There was a low level of actin synthesis prior to induction in LB broth, but none in M10 + medium. The use of glycerol as a carbon source avoided catabolite repression. The other changes in M10 medium were necessary to increase the growth rate of our strains.
French Press Lysis
[0065] 1.0 liter of actin containing was grown in M10 +medium, induced and harvested at an OD550 of 1.35. At this point all procedures were done at 4°C or on ice. The bacteria were washed with low salt buffer (10 mM triethanolamine, pH 8 at 4°C, 0.5mM ATP, 0.5mM DTT, 0.1 mM CaCl2), and resuspended in 20 ml of low salt buffer + protease inhibitors (20µg/ml aprotinin, 10µg/ml leupeptin, 2.5 µg/ml pepstatin, 2.5µg/ml chymostatin). Lysis was in the French pressure cell at 1000 lb/in2 , running the lysate through twice. The lysate was centrifuged at 116,500 x g for 8 min.
Sarkosyl Lysis
[0066] All procedure were at 4°C or on ice. Bacteria from 1 liter of culture were sedimented as washed with a buffer containing 20mM Tris, pH 8 at 4°C and 50mM NaCl. Washed cells were resuspended in 15 ml of STE (10% sucrose, 100mM Tris, pH 8 at 4° C, 1.5 EDTA) and lysozyme was added to 100µg/ml. The cells were incubated on ice 10 -15 min, then added to 132 ml of lysis buffer. While stirring, 3ml of 10% sarkosyl was added (0.2% final concentration), increasing the rate of stirring to compensate for the increased viscosity, but avoiding turbulence. The lysis of E. coli cells is performed in sarkosyl detergent due to co-aggregation with bacterial outer membrane components. Sarkosyl detergent is a relatively mild chaotrope with a specific effect upon this co-aggregation process. After the addition of sarkosyl, the lysate contained: 15 mM triethanolamine (pH 8 at 4°C), 50mM NaCl, 2.5 mM ATP, 1.0 mM GDP, 1.0mM DTT, 20µg/ml aprotinin, 10µg/ml leupeptin, 5µg/ml pepstatin, 2.5µg/ml chymostatin, 0.43 mM PMSF, 0.43 mM phenanthroline, 10mM Tris, 0.16 mM EDTA, 1.0% sucrose. After 2 min, the lysate was mildly sonicated to reduce viscosity: 7 - 10 second bursts at 90 watts. The lysate was centrifuged at 32,000 x g for 11 min. The supernatant was collected and octylglucoside was added to a concentration of 2% to sequester sarkosyl in nonionic detergent micelles. After stirring for 5min, MgCl2, and CaCl2 were added to a concentration of 1.25 mM and 1.06 mM respectively. The estimated free concentration of each divalent cation was 0.1 mM. After stirring for 20 min, this fraction was centrifuged at 60,000 x g for 12hr to pellet 30s ribosome subunits. The accumulation of actin in E.coli strains harbouring is shown in Fig. 6 (SDS Page ). When bacteria were lysed directly into a solution containing 0.2% sarkosyl, large amount of actin species were soluble. Divalent cations were also added; this was necessary for maintaining actin in its native form.
Biocatalytic Assay of Engineered F-actin Nitrilase
[0067] The standard assays were carried out by mixing the substrate 1-(cyanocyclohexyl) acetonitrile (200 mM, final concentration) with the purified enzyme in phosphate buffer (100 mM, pH 7.0). The reaction mixture (10 mL final volume) was incubated at 45°C for 10 min with shaking at 150 rpm in the reciprocal shaker. Aliquots (500 µL) were sampled, and the reactions were quenched by the addition of 500 µL HCl (2 M). The conversion was determined by measuring the amount of 1-(cyanocyclohexyl) acetic acid formed in the reaction. One unit of enzyme activity was designed as the amount of enzyme producing 1 µmol of 1-(cyanocyclohexyl) acetic acid per minute under the standard assay conditions. The concentration of 1-(cyanocyclohexyl) acetic acid was determined by a C18 (5 µm × 250 mm × 4.6 mm) column (Elite Analytical Instruments Co., Ltd.). The parameters used for detection of compounds by a UV detector were set at a wavelength of 215 nm, and each sample (20 µL) was eluted at 40°C with 5 mM NH4H2PO4/15 mM sodium perchlorate (pH 1.8) treated with perchloric acid: acetonitrile = 76:24 (v/v, 1.0 mL/min) as mobile phase. The reaction mixture contained cell suspension (200 mg/mL) in 50 mM phosphate buffer (pH 7.8) and 120 mM substrate dissolved in DMSO. The biocatalysis reaction was carried out in an incubator shaker (37 °C, 200 rpm) for 24 h. The cells were removed by centrifugation (7000x g) and the supernatant was extracted with ethyl acetate to recover the product.
EXPERIMENTS AND RESULTS of ENGINEERING of FtsZ TO HAVE NITRILASE FUNCTION
Expression
[0068] engineered FtsZ were overexpressed in milligram quantities from pET11 vectors in E. coli BL21(DE3), isolated as inclusion bodies from sonicated cells, dissolved in 0.02 M Tris, pH 7.5, 10 mM dithiothreitol, 7.5 M urea (Buffer A), concentrated to about 35 mg/mL by the use of a centrifugal ultrafilter (Ultrafree30, Millipore Corp.), and stored at -80 °C until use.
Purification
[0069] Dialyzing against 20 mM Tris/HCl (pH 7.9), 50 mM KCl, 1 mM EGTA, 2.5 mM MgAc, and 10% glycerol was used to purify untagged FtsZ proteins. The resultant product was kept at -80 °C. The protein can be kept for more than two years under those circumstances without seeing a noticeable decline in activity. To prevent the sample from thawing and freezing, the protein needs to be stored in 100 µl aliquots. The sample can be stored at 4 °C for up to a week after it has thawed. High quantities of FtsZ can be dissolved and kept at 7–10 mg/ml. Maintaining a high protein concentration is crucial to prevent unintended effects of storage buffer components in subsequent studies. In order to reduce the concentration of the other dialysis buffer ingredients in the reaction mix and to lower the glycerol concentration below 1%, the storage concentration should thus permit a dilution of FtsZ of at least 10x. High amounts of imidazole or salt can also interfere with FtsZ polymerization; therefore, these ingredients need to be eliminated from the polymerization buffer. Following the instructions, SepF was purified and kept at -80 °C in an elution buffer.
Assay for folding
[0070] In tubes suitable for high-speed centrifugation, prepare the reaction mix by adding MgCl2 and FtsZ to one of the polymerization buffers. With MgCl2 at 10 mM and FtsZ at 12 µM at 50 µl final volume, the total volume should be 49 µl. Put the tube in a shaking incubator and shake it for two minutes at 30 °C and 300 rpm (you can also gently flick the sample and microfuge it for a short while before prewarming it at 30 °C). Add 1 µl of GTP or GDP stock solution to the same buffer used in the experiment to begin polymerization (final concentration of 2 mM). For buffers with 50 mM and 300 mM KCl, respectively, incubate for 10 or 2 minutes.
[0071] Spoon the tubes into an ultracentrifuge rotor and spin down for 10 minutes at 350,000 x g (89,700 rpm for TLA 120.1 rotor) at 25 °C. Transfer the supernatant into a fresh tube as soon as possible after carefully removing the tubes from the rotor when they have finished spinning. Put 20 µl of supernatant into 20 µl of 2x sample buffer to get samples ready for SDS-PAGE. Cook at 98 °C for 10 minutes. The pellet fraction containing FtsZ polymers should be mixed with 50 µl of 2x sample buffer. Put the 2 ml tube inside the ultracentrifuge tube. To achieve the same 2x dilution as the supernatant sample, boil the pellet again for 10 minutes at 98°C. Then, add 50 µl of demineralized water. Spin the tube for the ultracentrifuge. After boiling the pellet for 10 minutes at 98°C to resuspend it, dilute the sample 2 times with 50 µl of demineralized water. Spin down the ultracentrifuge tube for five minutes at 18,000 x g in an Eppendorf centrifuge after turning it upside down in the 2 ml tube. From the 2 ml tube, remove the ultracentrifuge tube. Place 10 µl of the pellet sample and each supernatant side by side on a 10% SDS-PAGE gel. Utilize 150 V for the gel. Use Coomassie Brilliant Blue G-250 to stain the gel, then store it for measurement.
Analysis of Z-ring and Proteins from Z-ring
[0072] Using 90° angle light scattering, assess the polymerization efficiency of FtsZ in various buffers for protein analysis. Switch on a flowing water bath to keep the temperature of the cuvette chamber at 30 °C and switch on a fluorescence spectrometer. Let the lamp warm up for a few minutes to prevent temperature swings. High voltage 300 V detector, 350 nm emission and excitation wavelength, and 4 nm slit width. It should be noted that a lot of spectrometers automatically set the signal to a specific percentage (like 60%) of the maximum at the beginning of an experiment. This means that as soon as GTP is added to polymerize FtsZ, a scatter signal will go out of range. Before beginning a series of studies, it is advisable to first determine the proper signal amplification—detector high voltage—for optimum polymerization. Design an acquisition protocol for data. Select a 3,600-second time-based acquisition method.
[0073] Clean a fluorescence cuvette carefully with water and ethanol. If needed, sonicate it for five minutes at room temperature in a water bath. Cuvettes with a 200 µl volume and a 1 cm route length were employed in this protocol and kept in a storage solution (0.5-1%). Single-use plastic cuvettes that are compatible with UV light can be used as a substitute. As the final concentration calculated for 300 µl, prepare 294 µl of a master mix of the polymerization buffer with 10 mM MgCl2 and 12 µM FtsZ, and vortex. After filling the cuvette with 196 µl of the polymerization buffer, put it in the spectrometer. At 30 °C, incubate for 2 minutes. To ensure the stability of the signal, begin the data-gathering process and wait 90 seconds. To get a final reaction volume of 200 µl, add 4 µl of 100 mM GDP or GTP 2 mM. pipette up and down with a larger volume pipette to mix and resume acquisition.
Biocatalytic Assay of Engineered FtsZ Nitrilase
[0074] The standard assays were carried out by mixing the substrate 1-(cyanocyclohexyl)acetonitrile (200 mM, final concentration) with the purified FtsZ in phosphate buffer (100 mM, pH 7.0). The reaction mixture (10 mL final volume) was incubated at 45°C for10 min with shaking at 150 rpm in the reciprocal shaker. Aliquots (500 µL) were sampled, and the reactions were quenched by the addition of 500 µL HCl (2 M). The conversion was determined by measuring the amount of 1-(cyanocyclohexyl) acetic acid formed in the reaction. One unit of FtsZ activity was designed as the amount of enzyme producing 1 µmol of 1-(cyanocyclohexyl) acetic acid per minute under the standard assay conditions. The concentration of 1-(cyanocyclohexyl) acetic acid was determined by a C18 (5 µm × 250 mm × 4.6 mm) column. The parameters used for detection of compounds by a UV detector were set at a wavelength of 215 nm, and each sample (20 µL) was eluted at 40°C with 5 mM NH4H2PO4/15 mM sodium perchlorate (pH 1.8) treated with perchloric acid: acetonitrile = 76:24 (v/v, 1.0 mL/min) as mobile phase. The reaction mixture contained cell suspension (200 mg/mL) in 50 mM phosphate buffer (pH 7.8) and 120 mM substrate dissolved in DMSO. The biocatalysis reaction was carried out in an incubator shaker (37 °C, 200 rpm) for 24 h. The cells were removed by centrifugation (7000x g) and the supernatant was extracted with ethyl acetate to recover the product.
Example 1 - F-Actin Nitrilase
Relative Activity of F-Actin Nitrilase after different centrifugation cycles with different RPM, concentrations of cation, ATP and stabilizing agent Phalloidin.

S. No Centrifugation Cycles Relative Activity (%)
01 1 100
02 2 100
03 3 100
04 4 80
05 5 80
06 6 70
07 7 70
08 8 70
09 9 70
10 10 70
Table 1. Relative activity of engineered F-actin nitrilase, after different cycles. The centrifugation rpm was kept at 7000 rpm for 10min, constant concentration of ATP, Mg2+ ions and temperature.
S. No Centrifugation Cycles Relative Activity (%)
01 1 100
02 2 100
03 3 90
04 4 80
05 5 80

Table 2. Relative activity of engineered F-actin nitrilase, after different cycles. The centrifugation rpm was kept at 10000 rpm for 5min, with constant concentration of ATP, Mg2+ ions and temperature.
S. No Centrifugation Cycles Relative Activity (%)
01 1 10
02 2 10
03 3 10
04 4 5
05 5 5
06 6 5
07 7 ND
08 8 ND
09 9 ND
10 10 ND

Table 3. Relative activity of engineered F-actin nitrilase, after different cycles. The centrifugation rpm was kept at 7000 rpm for 10min, without ATP and Mg2+ ions. The centrifugation temperature was kept constant for each cycle. ND: not detected.
S. No Centrifugation Cycles Conc. of Mg2+ (mg/l) Relative Activity (%)
01 1 2 60
02 2 5 60
03 3 7 80
04 4 10 80
05 5 12 100
06 6 15 100
07 7 17 100
08 8 21 100
09 9 24 100
10 10 27 100

Table 4. Relative activity of engineered F-actin nitrilase, after different cycles. The centrifugation rpm was kept at 7000 rpm for 10min, without ATP and varying concentrations Mg2+ ions. The centrifugation temperature was kept constant for each cycle.
S. No Centrifugation Cycles Conc. of Mg2+ (mg/l) Conc. of Phalloidin (mg/l) Relative Activity (%)
01 1 2 3 80
02 2 5 6 80
03 3 7 9 90
04 4 10 12 90
05 5 12 15 100
06 6 15 18 100
07 7 17 21 100
08 8 21 24 120
09 9 24 27 120
10 10 27 30 120

Table 5. Relative activity of engineered F-actin nitrilase, after different cycles. The centrifugation rpm was kept at 7000 rpm for 10min, without ATP and varying concentrations Mg2+ ions and Phalloidin. The centrifugation temperature was kept constant for each cycle.
S. No Accession Number Organisms Active Site Mutations Additional Mutations Binding Free Energy (kcal/mol) Relative Activity (%)
01 Q9P4D1 Komagataella pastoris E254K, I213E, L194E F307G, K214G, L190G, G183R -5.99 100
02 POA9X4 Escherichia coli (K12) L300C, I256K, I198E, A225E K219G, H220G -5.26 100
03 WP231190500 Archaea K215C, L191K, Y308E, P260E C259G, C219G
-3.96 100
Table 6. Catalytic and additional substitutions incorporation in F-actin from different organisms for nitrilase function, with 1-(cyanocyclohexyl)acetonitrile binding affinity and their relative activity.
Example 2 – FtsZ Nitrilase
Relative Activity of FtsZ Nitrilase after different centrifugation cycles with different RPM, concentrations of cation, GTP.
S. No Centrifugation Cycles Relative Activity (%)
01 1 100
02 2 100
03 3 100
04 4 80
05 5 80
06 6 70
07 7 65
08 8 60
09 9 60
10 10 60
Table 1. Relative activity of engineered FtsZ nitrilase, after different cycles. The centrifugation rpm was kept at 7000 rpm for 10min, with constant concentration of GTP, Mg2+ ions and temperature.
S. No Centrifugation Cycles Relative Activity (%)
01 1 100
02 2 100
03 3 95
04 4 85
05 5 80

Table 2. Relative activity of engineered FtsZ nitrilase, after different cycles. The centrifugation rpm was kept at 10000 rpm for 5min, with constant concentration of GTP, Mg2+ ions and temperature.
S. No Centrifugation Cycles Relative Activity (%)
01 1 10
02 2 10
03 3 10
04 4 8
05 5 8
06 6 5
07 7 5
08 8 5
09 9 ND
10 10 ND
Table 3. Relative activity of engineered FtsZ nitrilase, after different cycles. The centrifugation rpm was kept at 7000 rpm for 10min, without GTP and Mg2+ ions. The centrifugation temperature was kept constant for each cycle.
S. No Centrifugation Cycles Conc. of Mg2+ (mg/l) Relative Activity (%)
01 1 2 60
02 2 5 60
03 3 7 80
04 4 10 80
05 5 12 80
06 6 15 90
07 7 17 90
08 8 21 100
09 9 24 100
10 10 27 100

Table 4. Relative activity of engineered FtsZ nitrilase, after different cycles. The centrifugation rpm was kept at 7000 rpm for 10min, without GTP and varying concentrations Mg2+ ions. The centrifugation temperature was kept constant for each cycle.
S. No Centrifugation Cycles Conc. of Mg2+ (mg/l) Conc. of Taxol (mg/l) Relative Activity (%)
01 1 2 3 70
02 2 5 6 70
03 3 7 9 80
04 4 10 12 80
05 5 12 15 90
06 6 15 18 90
07 7 17 21 100
08 8 21 24 100
09 9 24 27 100
10 10 27 30 100

Table 5. Relative activity of engineered FtsZ nitrilase, after different cycles. The centrifugation rpm was kept at 7000 rpm for 10 minutes, without GTP and varying concentrations of Mg2+ ions and Taxol. The centrifugation temperature was kept constant for each cycle.
ADVANTAGES/SIGNIFICANCE OF THE INVENTION
[0075] The current invention unveils a method of engineering of F-actin into a functional nitrilase and their uses. The recombinant engineered F-actin nitrilase can covert nitrile substrates to corresponding carboxylic acids with much better efficiency with any reported nitrilases in terms of activity. The engineered F-actin nitrilase also possess higher enzyme reusability feature as compared to the nitrilase. The engineered F-actin can be used for up to 10 cycles of biocatalysis without losing the activity function. The present invention also unveils a method of engineering FtsZ proteins as functional enzymes. FtsZ forms Z-ring fibers which are highly stable and known for their function inside the cell. The FtsZ proteins are highly stable when they form long filaments. In this invention, FtsZ is engineered to have nitrile-degrading functions. Nitriles are common in many hazardous places, industrial wastes, agrochemical wastes etc., and engineered FtsZ are found forming Z-ring helical structures and acting as an immobilised enzyme. This invention helps to overcome many industrial applications where FtsZ holds higher temperature, pressure and substrate tolerance, and better activity than known nitrilase enzymes. The FtsZ are also known for their better reusability up to 10-15 times. The reusability helps to improve industrial biocatalysis functions with better activity.
OTHER PUBLICATIONS
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,CLAIMS:We claim,

1. A filamentous actin that is engineered to have nitrilase activity wherein the engineered filamentous actin has integrated catalytic residues Cys-Glu-Glu-Lys within the Cucurbitacin E binding site of the filamentous actin, enabling the transformation of specific nitrile substrates into their corresponding carboxylic acid products.
2. The filamentous actin of claim 1 has origins traced to E. coli K12, Komagataella phaffii, and Archea bacteria, holding sequence identities of SEQ ID 4, 5, and 6 respectively.
3. The nitrile substrates of claim 1 which includes 2-chloropyridine-3-carbonitrile,2-(2-methylpropyl) butanedinitrile and others and resultant transformed carboxylic acid products of claim 1 which includes 2-chloronicotinic acid, (S)-3-cyano-5-methylhexanoic acid, and more respectively.
4. The filamentous actin polypeptide of claim 2 derived from SEQ ID 4 exhibit features such as a cysteine at X300, a lysine at X256, and others and the filamentous actin polypeptide of claim 2 derived from SEQ ID 5 exhibit features including a lysine at X254, glutamic acid at X213, and more and the filamentous actin polypeptide of claim 2 derived from SEQ ID 6 exhibit features like a cysteine at X215, a lysine at X191, among others.
5. The filamentous actin of Claim 4 can be in a refined state or a crude extract.
6. The filamentous actin of Claim 5 displays enhanced qualities such as better thermostability, solvent stability, or enzymatic action compared to native nitrilase.
7. The filamentous actin of Claim 5 is recognized as immobilized nitrilase and offers heightened enzyme reusability compared to the native nitrilase.
8. The filamentous actin of Claim 5 operates at a pH range of 7 to 10 and at temperatures between 25°C to 55°C.
9. The polynucleotide of filamentous actin of Claim 4 is functionally associated with specific promoters, fostering its production in a host cell such as E. coli and the F-actin nitrilase is expressed using the vector pET28a (+).
10. The filamentous actin of Claim 4 possessing nitrilase activity exhibits extended reusability over multiple cycles, with the application of a specified washing buffer post-utilization to maintain its enzymatic performance.
11. A filamentous or Z-ring-shaped FtsZ that is engineered to possess nitrilase activity wherein the engineered filamentous or Z-ring-shaped FtsZ has integrated catalytic residues Cys-Glu-Glu-Lys, enabling the transformation of specific nitrile substrates into their corresponding carboxylic acid products.
12. The FtsZ of claim 11 has origins traced to E. coli, Bacillus subtilis, and Mycobacterium tuberculosis holding sequence identities of SEQ ID 4, 5, and 6 respectively.
13. The nitrile substrates of claim 11 include 2-chloropyridine-3-carbonitrile,2-(2-methylpropyl) butanedinitrile, and others and the resultant carboxylic acid products of claim 11 include 2-chloronicotinic acid, (S)-3-cyano-5-methylhexanoic acid, and more respectively
14. The FtsZ polypeptide of claim 12 derived from SEQ ID 4 exhibits features such as a cysteine at X184, a lysine at X220, and others and the FtsZ polypeptide of claim 12 derived from SEQ ID 5 shows features including a lysine at X228, glutamic acid at X180, and more
15. The FtsZ enzyme of claim 14 can be present in a refined state or as a crude extract.
16. The engineered FtsZ of claim 14 displays enhanced qualities such as better thermostability, solvent stability, or enzymatic action compared to native nitrilase.
17. The FtsZ of claim 15 is recognized as an immobilized nitrilase and offers heightened enzyme reusability compared to the native nitrilase.
18. The FtsZ of claim 15 operates at a pH range of 7 to 10 and at temperatures ranging between 25°C to 55°C.
19. The polynucleotide of FtsZ of claim 14 is functionally associated with specific promoters, fostering its production in a host cell such as E. coli and this FtsZ nitrilase is expressed using the vector pET28a (+).
20. The engineered FtsZ of claim 15 possessing nitrilase activity exhibits extended reusability over multiple cycles, with the application of a specified washing buffer post-utilization to maintain its enzymatic performance.