KR101100831B1 - Method for determining the cyanophycin synthetase activity using quantitative analysis of ATP - Google Patents

Abstract

In the present invention, in measuring the titer of cyanopysin synthetase, the amount of ATP used as an energy source in the reaction of cyanopysin synthase is measured to predict the degree of cyanopysin synthesis by the polymerization reaction and the enzyme titer therefrom. The present invention relates to a method for measuring the titer of cyanopysin synthetase, and by using a commercially available ATP quantitative reagent and instrument, the amount of ATP reduction can be quantitatively measured accurately, and thus the degree of polymerization of amino acids can be accurately calculated. In addition, it solves various problems that occur when using conventional radiation, and has the effect of accurately measuring the titer of enzyme.

Cyanopysin, ATP, Enzyme Titer Determination

Description

Method for determining the cyanophycin synthetase activity using quantitative analysis of ATP

The present invention relates to a method for measuring the titer of cyanopysin synthase by quantitative measurement of ATP, and more specifically, in measuring the titer of cyanopycin synthase, it is used as an energy source in the reaction of cyanopycin synthase. The present invention relates to a method for measuring the titer of cyanopysin synthetase, which measures the amount of reduction in ATP, predicts the degree of cyanophosphine synthesis by polymerization, and calculates the enzyme titer therefrom.

Cyanophycin is a multi-L-arginyl-poly-L-aspartic acid (CGP), a homodimer enzyme cyanopycin synthase It is synthesized by (Ziegler et al. 1998). Recently, there have been reports of the widespread development of cyanopysin synthase in eubacteria. Biosynthesis of CGP is independent of ribosomal protein synthesis inhibitors, but is dependent on the presence of monomers L-arginine and L-aspartic acid, ATP, thiol groups and preexisting cyanopysin primers (Simon, 1976). (Β-asp-arg) ₃ , a tripeptide of the CGP building block β-aspartyl arginine, was identified as the shortest primer for cyanophycin synthetase (CphA) reaction (Berg et al. 2000).

According to a conventionally proposed reaction model (Berg et al., 2000), CphA is mainly expressed in L at the C-terminus of the CGP primer through two steps, an acyl phosphorylation step (step 1), and an introduction or extension step (step 2). Catalyze the sequential mixing of arginine and L-aspartic acid. Step 1 can be described as follows:

Step 1

First, the free carboxylic acid group (β-carboxyl or α-carboxyl) of aspartic acid at the C-terminus of CGP is activated with a γ-phosphate residue at one of the ATP binding sites of CphA to generate acylphosphate intermediate complexes and ADP. Adenosine 5′-tetraphosphate is also formed by the method of Bouhss et al. (1999) on the reaction mechanism of the MurD enzyme:

As a second step, when β-acylphosphate is preformed as follows, the amino group of L-arginine is replaced with phosphate by nucleophilic attack and introduced into the CGP primer.

When α-acyl phosphate is synthesized, CphA catalyzes the reaction between acyl phosphate and L-aspartic acid and extends the aspartyl backbone. CphA contains two ATP binding sites (Berg et al. 2003) and ATP-dependent ligation similar to the thio-template mechanism of nonribosomal peptide synthesis of the ATP-occupying superfamily (Clifforf et al, 2003). As it catalyzes the reaction, acyl phosphate synthesis is the main reaction of CphA. By combining the phosphorylation step with the introduction step, the following scheme can be illustrated:

1 L-Asp + 1 L-Arg + 2 ATP + (Asp-Arg) _n

->

(Asp-Arg) _{n + 2} + 2 ADP + 2 Pi

Where (Asp-Arg) _n is the initial CGP primer block. Thus, the introduction of 1 mole of each amino acid monomer corresponds to the consumption of 2 moles of ATP.

To assess the activity of the CphA enzyme, so far it has been used with a slight modification of Simon's unique radiation method (Simon, 1976) (Ziegler et all 1998, Abboulmagd et al. 2000, Hai et al. 2008). The principle of the method is to quantitatively count L-aspartic acid or L-arginine, which is a titanium-labeled or ¹⁴ C-labeled monomer introduced into a CGP primer. In conclusion, radiolabeling methods are not used in many laboratories because they are expensive, incompatible, and require special stability and protection techniques. Radiolabeling assays are also time consuming because unreacted amino acid monomers must be removed from the synthesized CGP product during the wash step. Moreover, repeated washings resulted in loss of CGP products and often caused deviations in counting radioactivity. Thus, ATP consumption may serve as a non-radioactive method for assessing CphA activity. In conventional studies, ATP consumption has been used to measure various ATP hydrolase, such as kinase activity (Bakiet al. 2007) and ATPase activity (Beauge & Campos, 1986, Ahmad et al. 2006).

Cyanopysin is a biotechnology because the poly-α-L aspartic acid obtained after reducing or eliminating arginine residues can be used not only for other industries but also for healthcare (Oppermann-Sanyo & Steinb, 2002). Of particular interest in the field.

The main structure of CphA represents two regions, each with a potential ATP-binding site (Berg et al. 2000) typical of the ATP-grasp fold superfamily. The N-terminal region introduces aspartate into CGP, while the C-terminal region links arginine to CGP, and it is believed that lysine residue K497 plays an important role for this. The substitution of Ala-497 with Lys-497 stopped the introduction of L-arginine but did not inhibit the introduction of L-aspartic acid. This is in contrast to wild type CphA (Berg et al. 2000), where activity is maintained only in the presence of each monomer. However, other reports on the role of lysine residue K197 in the highly conserved MurD family similar to K479 pointed to the major role of carbamoylated lysine residues in the Mg-ATP binding complex. Mutation can mainly affect the acyl phosphorylation stage 1 of the CphA response described above.

The ENTILENATP assay bioluminescence detection kit available from Promega (Madison, WI) quantifies the amount of ATP remaining in solution after enzyme reaction. The assay utilizes luciferase / D-luciferin interaction in the presence of oxygen, as described below:

ATP + D-Luciferin + Luciferase + O ₂

->

Oxyluciferin + AMP + Pi + CO ₂ + Light.

Here, the luminescence intensity is proportional to the concentration of ATP. Promega ENLITEN kits rapidly quantify ATP using luciferase / luciferin complex (L / L) and detect ATP up to 0.1 fmol or less. This is a non-radioactive assay suitable for testing the ATP-concentration consumed by the CphA enzymatic reaction.

Consumption of ATP (see step 1) for activation of the C-terminal carboxyl group of CGP is a major step in the total reaction catalyzed by CphA. Thus, the difference in ATP concentration (ΔATP) before and after the addition of cyanopysin synthase corresponds to the difference in relative light units (RLU). Relative optical units are consistent with CphA activity.

In order to apply ATP assays to measure CphA activity in CphA containing protein fractions as well as cell-free extracts, the present invention had to minimize and eliminate any ATP-hydrolysis factors, in particular ATPases and ribosomal protein synthesis pathways. ATPases activity was inhibited in the presence of vanadate and neomycin (Lipsky, JJ & PS Lietman 1980), sodium fluoride (Zulfiqar & Senior, 2006, Marquis, 1977), and the ribosomal protein was inhibited by chloramphenicol (Simon, 1976). It was inhibited by the addition of.

In the present invention, a high-speed for measuring the activity of cyanopysin synthase CphANE1bK497A mutated in crude cell extract and His-labeled CphANE1b to demonstrate the inactivation mechanism caused by the substitution of a carbamoylated lysine residue K497 with an alanine residue. Non-radiolabeled analysis was provided. It has been suggested that K497 (Accession No. EF660552) is a major residue available for arginine introduction into CGP (Berg, 2003).

It is therefore an object of the present invention to provide a method for measuring the titer of cyanopysin synthase simply by quantification of ATP without the use of radioactive material.

In order to achieve the above object, the present invention measures the amount of ATP used as an energy source in the reaction of cyanopysin synthetase in measuring the enzyme titer of cyanopycin, and thus the degree of cyanopysin synthesis by the polymerization reaction. And predicting the enzyme titer therefrom provides a method for measuring the enzyme titer of cyanopysin.

The method according to the present invention comprises the steps of using a solution obtained by breaking a cell having CphA, or using a purified CphA to perform cyanopysin synthesis reaction;

Measuring the degree of synthesis of cyanopycin by measuring the change in ATP content by measuring the amount of ATP before the synthesis reaction and the amount of ATP after the reaction; And

Calculating CphA Activity from the Measured Synthesis of Cyanopysin

.

The method for measuring the titer of cyanopysin synthase through the quantitative measurement of ATP according to the present invention can quantitatively accurately measure the amount of ATP decrease by using a commercially available ATP quantitative reagent and a device, and thus the degree of polymerization of amino acids. Accurate calculations can solve various problems that occur when using conventional radiation, and at the same time have the effect of accurately measuring enzyme titers.

Hereinafter, preferred embodiments of the present invention will be described in more detail with reference to Examples. However, the scope of the present invention is not limited to these examples.

Example 1 Bacteria Strain Culture

Escherichia coli from the recombinant strain Invitrogen (San Diego, CA, USA) DH5α (F araD139 Δ ( ara , leu ) 7697 lacX74 galU galK rpsL (Strr) deoR Φ80dlacZ Δ M15 endA1 nupG recA1 mcrA Δ ( mrr hsdRMS mcrBC ) and BL21 ( S3F ) of Novazen (Madison, WI, USA) , ompT, gal (dcm), (DE3), (Ion), hsdS B (rB ^- mB ^-) were respectively used in the cloning and expression of His- cover CphA protein. Bacteria were incubated at 37 ° C. in LB medium with shaking at 220 rpm in 50 mL or 500 mL of LB medium supplemented with 75 μg mL ⁻¹ aspirin or 50 μg mL ⁻¹ kanamycin. To induce His-labeled protein, 1 mM of isopropyl-ß-D-thiogalactoside (IPTG) was added when cell growth reached OD ₆₀₀ = 0.3.

Example 2 DNA Isolation and Genetic Engineering

Plasmids were purified using HiYield ™ Plasmid Mini Kit (Real Genomics, Taipei, Taiwan). Enzymatic digestion and ligation of DNA molecules was performed using the indicated restriction endonucleases and T4 DNA ligase (Takara, Shiga, Japan).

Example 3 Site-directed mutagenesis of CphA gene

Site specific mutagenesis was performed according to the method of Kunkel (1987). To demonstrate the role of Lys in the enzymatic activity of CphA _NE1b , lysine was substituted with alanine to cause site specific mutagenesis resulting in mutant K497A.

Forward primer (K497A-F) with oligonucleotide sequence GGCACTAACGGTGCNACCACCACTACC and reverse primer (K497A-R) with nucleotide sequence GGTAGTGGTGGTNGCACCGTTAGTGCC were applied. Plasmid pUCbm20 :: cphA NE1b _served as DNA template. QuikChange site specific mutagenesis kits (Stratagene, La Jolla, Calif., USA) were used to induce such mutations. For plasmid propagation, the amplified PCR product was digested with DpnI to remove the methylated plasmid template. The unmethylated PCR product containing nicked form pUCbm20 :: cphA _NE1bK497A was then transformed into E. coli XL-10 Gold. Mutated genes were identified by DNA sequencing (GGBioCom Sequencing Service Center, Yongin, Korea). For expression and purification of His-labeled wild type and mutated enzymes, the cphA gene was acloned to the NdeI / BamHI position of the expression pET28a + and transformed with E. coli BL21 (DE3) pLysS according to standard methods.

Example 4 Site-Specific Mutant CphA _NE1bK497A Overproduction, Enzyme Purification and Western Blot

Purification of His-labeled CphA on Ni-NTA affinity chromatography and overproduction of His-labeled CphA _NE1bK497A protein in E. coli BL-21 (DE3) p LysS according to the recently disclosed method (Hai et al. , 2008) . Was performed. Polyvinylidene Fluoride (PVDF) Membrane and Semidry-TransBlot SD-apparatus; Western hybridization was performed using Bio-Rad Laboratories, Hercules, CA, USA. To identify His-labeled protein, the No. 70796-3dnk His-labeled monoclonal antibody kit from Novagen (Darmstadt, Germany) was used. Blot using p-nitro blue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) obtained from Roche Diagnostics GmbH (Mannheim, Germany) The signal was developed. The hybridization process was according to the manufacturer's instructions.

Example 5: Determination of ATP affinity in CGP primers and ATP affinity in CphA containing cell eluate

Aspirase (Biolabs) was used to demonstrate the presence of free and / or bound forms of ATP in cyanopycin preparations. In particular, cyanopycin used as a primer in the CphA reaction was resuspended in a reaction mixture of 100 μL total volume without CphA enzyme and ATP and incubated with 0.2 U.ml ⁻¹ aspirase for 4 hours. 100 μL of 7% Trichloracetic acid (TCA) was added to stop the reaction. For control, the reaction mixture without aspirase was applied. After vortexing for 1 minute and centrifugation at 14,000 g for 20 minutes at 4 ° C., the ATP concentration in the clean supernatant was measured using a luciferase kit (Promega) as described above.

Example 6 ATP Binding Assay

The ATP binding activity of CphA protein and CGP primer was measured as follows. 2 μg of CphA or 100 μg of CGP at 30 ° C. in 2 mM ATP 100 μL of reaction buffer A [20 mM Tris / HCl, pH 8.0, 5 mM magnesium chloride, 5 mM dithiothreitol (DTT), 10 mM KCl and 0.005% Triton X -100] in 2 mM ATP. Samples (10 μL each) were removed at 0, 1, 2, 3, 5 and 15 minutes and ATP binding assays for CGP and CphA were stopped by adding 90 μL of 5% trichloracetic acid in water without ATP, respectively. ATP bound to CGP and CphA was separated by centrifugation at 14,000 g for 20 minutes at 4 ° C. A dilution series of up to 10 ⁵ for unbound ATP in the supernatant was established to reduce its concentration to about 10 ⁻⁸ M, which is the optimal concentration for the ATP assay kit used. The ATP concentration applied to the reaction at zero point is the total ATP or [ATPtot]. The ATP concentration measured after incubation is the residual ATP or [ATPrem]. Therefore, the initial ATP binding activity [ATPbin] of CphA or CGP was calculated according to the following formula (1).

<Equation 1>

[ATPbin] = [ATPtot] [ATPrem]

[ATPtot] and [ATPrem] correspond to the relative light emitting units [RLUtot] and [RLUrem].

Thus [ATPbin] corresponds to [ΔRLU], which is calculated as follows.

[ΔRLU] = [RLUtot]-[RLUrem]

Factors affecting Equation 1 above are the initial ATP concentrations previously present in CGP and CphA containing cell eluate. To determine [ATPini], ATP was extracted from CGP and CphA with 3.5% TCA at room temperature. Since TCA inhibited the luciferase enzyme test, more than 10,000 dilutions were needed.

Example 7 Determination of Cyanopysin Synthetase Activity: Comparison of ATP Consumption Assay Using Conventional Radiolabeling Methods

Radiolabeling analysis for cyanopysin synthase activity was performed by modifying the method of Simon (Simon, 1976) (Aboulmagd et al., 2000, Hai et al. 2008). The introduction of L- [ ¹⁴ C] arginine was measured by counting on an LS 6500 liquid scintillation counter (Beckman Instruments GmbH, Munich, Germany).

Example 8 Non-Radio Label Analysis Using Luciferase Kit

100 μg CGP, 500 μM L-arginine, 5 mM L-aspartate, 10 mM MgCl ₂ , 20 mM KCl, 50 mM Tris / HCl (pH 8.2), 10 mM 2-mercaptoethanol, 2-4 μg purified A reaction volume of 100 μL containing cyanopysin synthetase (or 40 μg CphA containing cell eluate CL-CphA) was applied. The reaction was initiated by adding ATP at a final concentration of 500 μL, except for experiments and ATP saturation studies to determine Km for ATP consumption with ATP concentrations ranging from 10 ⁻⁸ M to 8.10 ⁻³ M. After 5 minutes of incubation at 30 ° C., 900 μL of cold 3.5% TCA solution was added to stop the reaction, and centrifuged to recover primers as well as CGP products, and the dilution process was performed as described above. ATP concentration [ATPsup] in the supernatant was measured on a luminometer as described above. The actual ATP amount [ATPcon] consumed by CphA during the cultivation corresponds to the enzyme activity, which was calculated by the following equation.

<Equation 2>

[ATPcon] = [ATPtot]-[ATPsup], similarly [ΔRLU] = [RLUtot]-[RLUsup]

Since the concentration of ATP in the reaction mixture was saturated (2 mM) (see Krehenbrink and Steinb, 2005), [ATPcon] is a linear function of the reaction time. Therefore, the volume activity of CphA was measured according to the following Equation 3.

<Equation 3>

U = [ATPac] / t (nmoles ATP. Min ⁻¹ ).

From this, the specific activity of CphA was measured by the following equation.

<Equation 4>

A (spec) = U / mg protein (nmoles ATP. Min ^-1 . Mg ^-1 protein)

For aliphatic acid protection assays, 0.5 M each of acetate or propionate was added to a reaction mixture containing _N -NTA purified His-labeled CphA _NE1b or CphA _NE1bK497A (minus ATP) and incubated on ice for 60 minutes. As described above, the reaction was initiated by adding ATP at a final concentration of 2 mM.

Example 9 Inhibition of ATP Hydrolytic Activity

Sodium fluoride (mw: 41.99; Sigma-Aldrich), Na ₃ VO ₄ (mw: 183.9) to prevent ATP hydrolytic activity of ATPases and alkaline phosphatase and to inhibit ribosomal protein synthesis in cell eluates ; Sigma) 100 μM each, neomycin. An inhibitor cocktail consisting of 50 μg / mL and chloramphenicol 15 μg / mL was used. ATP hydrolysis was assessed in the absence or presence of the selected inhibitor 60 minutes after preincubation of the CphA reaction mixture on ice. All other genuine chemicals and drugs used were purchased from Sigma-Aldrich Chemical Company (St. Louis, Mo.).

ATP consumption by the CphA enzyme in the CL may be interfered by other ATP hydrolase enzymes such as alkaline phosphatase and ATPase present in some ribosomes or membrane particles of the CL. It has been found that hydrolysis of ATP with or without an ATPase inhibitor cocktail is similar to the ATP consumption rate of the CphA _NE1b enzyme.

Example 10 Measurement of Bioluminescence Kinetics Associated with CphA Activity

Kinetic constants of CphA related to ATP consumption at 24 ° C. in 96-well plates were performed. 10 μL of reaction product of each reaction was mixed with 50 μL of luciferase kit (Promega) with an automatic injector with at least 400 luminometer remission records. The kinetic constants Vmax, Km were derived using the highest luminescence intensity.

Example 11 Reactivation of CphANE1bK497A Mutation with Acetate

Enzyme activity protected by acetate against recombinant E. coli DH5α bearing wild-type cphA _NE1b and mutant cphA _NE1bK497A was tested in vivo by adding IPTG as described above with feeding 6 mM sodium acetate (pH7.0) to the growth medium. . For the in vtro protection test, CphA and CphA containing cell eluate were precultured at 4 ° C. for 60 minutes with different concentrations of sodium acetate (100 mM or less).

Through the process of Examples 1 to 11 as described above was obtained as follows.

1. Factors Affecting ATP Consumption in CphA Catalysis

Affinity of ATP in CphA-containing Cell Eluates and CGP Primers

Since the actual ATP consumption by CphA can be simulated by the preference of ATP, the ATP content of CGP primers in reactive components such as CGP primers and CphA solutions were measured and shown in Table 1.

TABLE 1

ATP content in the reaction mixture and ATP binding capacity of CGP and CphANE1b

The meanings of the abbreviations used in Table 1 above are as follows:

a) CL-wt, wild type free cell eluate; CL-K497A, site-specific mutation derived cell _eluate of CphA _K497A ; + Ac, acetate added

b) addition of ATP at a concentration of 2 mM, if necessary, as a complete reaction mixture as described above

c) Inhibitor cocktails are described above

d) 6 nM concentration of acetate is added to the growth medium and enzyme activity is measured using standard reaction buffer.

e) In vitro CphA protection experiment was performed by adding 20 mM acetate to standard reaction buffer.

f) The relative light unit (RLU) of the starting concentration ATP is 554558.

g) Specific activity: 1 unit consumed 1 nmol.mg ^-1 prot. corresponds to min ⁻¹ ATP.

TCA terminal concentrations of 3.5% have been used to extract ATP from soil and biomass and to pellet proteins and CGP. Since CGP is insoluble in TCA solution (Simon, 1976), the data shows that CGP produced by strain MA19 and Nostoc ellipsosporum NE1 was obtained from cyanobacterial Synechococcus sp. It indicates the fact that it contains a greater amount of ATP than the product purified from the recombinant E. coli strain containing the nobacterial cphA gene. By apiase treatment, the content of [ATPini] was significantly reduced. Thus, as mentioned in the prior art (Simon 1973, Hai et al., 1999), the presence of small amounts of ATP in the CGP product is due to phosphate impurities. Remarkably, the presence of ATP in the CphA enzyme preparation was visually low, up to 120 ng, and their content was not affected by the apyase (see Table 1). Co-extracted ATP concentrations are believed to have been reduced by some ATP hydrolysis systems such as alkaline phosphatase, ATPase or kinases.

2. Evidence of ATP-hydrolysis activity involving CphA containing CL.

Inhibitory Effects of VO4, NaF and Neomycin

E. coli strains contain dimeric alkaline phosphatase with varying molecular weights from 80 kDA to 89 kDA [Taylor & Coleman (1972), Lazdunski and Lazdunski (1969). 89,000 Anderson et al . (1975), because it is optimal at pH 8.0 similarly to the CphA enzyme (Garen and Levinthal 1960), its activity in CL, along with ATPase activity of cell membrane particles, did not interfere with ATP-consuming by the CphA reaction. 3, the neomycin of a 20 μM concentration is more renal sodium potassium ATPase Full disabled (Lipsky, JJ & PS Lietman, 1980) , and sketch Ria coli (Escherichia coli) Escherichia coli 70S and the 30S particle ribosome ATPase in Activity (Ganoza & Kiel, 2001)

3. ATP Binding Activity and ATP Saturation Studies

Advantages of ATP consumption analysis over radiolabeling for CphA.

Important Kinetic Parameters of ATP Km

The ATP consumption method for measuring CphA activity was highly reproducible with a standard deviation of ± 5% compared to the deviation typically obtained with conventional radiolabeling methods (Simon, 1976).

TABLE 2

Comparison of Titer Measurements of Cyanopysin Synthetase

enzyme Titer a)
(Existing radiation method) Titer b)
(Method according to the present invention) CphA (Cell Extract)  1.5 ± 0.5   4.5 ± 0.2 CphA _K497A (mutant protein, cell extract)   0.2 ± 0.1   0.0 ± 0.0 His-tagged CphA (tablet protein)  18.0 ± 1.0  33.0 ± 0.5 His-tagged CphA _K497A (mutant protein, tablet protein)   0.1 ± 0.1   0.0 ± 0.0

a) Titer: 1 mg of protein per minute polymerizes 1 nmol of carbon 14 labeled arginine

b) Titer: Activity with 1 mg of protein using 1 nmol of ATP per minute

3 replicates

4. Inactivation of Aph binding capacity of CphA caused by K497A mutation.

Reactivation of enzymes in vitro and in vivo with acetate

In vivo study results are shown in Figure 2 A (accumulation CGP), B and C (enzyme purification). As shown in FIG. 2, (A) is a graph showing the ATP consumption of His-labeled purified cyanopysin synthase CphANE1b at log ₁₀ (RLU) as a function of incubation time (min), (B) ATP of CphANE1b This is a graph showing the saturation curve, (C) is a graph showing the Michaelis constant measurement for [ATP] (Km ATP) through the Lineweaver-Burk graph showing the relationship between 1 / V and 1 / [ATP].

As shown in FIG. 2, the activity of CphANE1b was measured by the difference before and after 5 min incubation of 2 μg purified enzyme in a reaction mixture containing various ATPs at concentrations from 0.1 μM to 8.10 ³ μM. In reaction buffer containing excess L-arginine, L-aspartic acid and CGP primer, the Km of [ATP] was 241.

As shown in FIG. 3, the expression and elution profile of His-labeled CphANE1bK497A was shown in comparison to the wild type enzyme His-labeled CphANE1b obtained in the previous study (Hai et al. 2008a), and the purified site-specific mutation and The results of western hybrid studies to provide expression levels of wild type CphANE1b enzyme are shown (B).

As shown in FIG. 4, inactivation of the acetyl phosphorylation step via site-specific mutation of Lys-497 by alanine substitution to obtain CphANE1bK497A is protected in vivo by adding 6 mM acetate to the culture medium, or reaction The mixture was protected in vitro by adding up to 100 mM sodium acetate. Higher concentrations of acetate or propionate (up to 0.5 M) inhibited the activity of wild type and mutant CphANE1b.

1 is a diagram illustrating a cyanopysin synthesis process.

FIG. 2 is a graph showing (A) ATP consumption of His-tagg purified cyanopysin synthase CphANE1b at log ₁₀ (RLU) as a function of incubation time (min), and (B) ATP saturation curve of CphANE1b. (C) A graph showing the Michaelis constant measurement for [ATP] (Km ATP) through a LineWeaver-Burk graph showing the relationship between 1 / V and 1 / [ATP].

Figure 3 is a diagram showing the results of mutated His-tagg CphaNE1bK497A purification using Ni-NTA affinity chromatography.

4 is a diagram showing a result of the mutation the activity CphANE1bK497A by the acetate protection in in vivo and in vitro.

Claims (2)

Cyanopysin synthesis comprising measuring the amount of ATP used as an energy source in the reaction of cyanopysin synthetase (CphA) to predict the degree of cyanopycin synthesis by the polymerization reaction, and calculating the titer of the enzyme therefrom. In the method for measuring the titer of an enzyme, A method for measuring the titer of cyanopysin synthase, characterized by using His-tagged CphA purified protein as the cyanopysin synthase. delete

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