Oligosaccharide debranching enzyme mutant with weakened product inhibition and application of oligosaccharide debranching enzyme mutant

Abstract

The invention relates to an oligosaccharide debranching enzyme mutant with weakened product inhibition and application of the oligosaccharide debranching enzyme mutant, and belongs to the technical field of enzyme engineering. The 311th lysine and the 405th glutamic acid of the oligosaccharide debranching enzyme with the amino acid sequence as shown in SEQ ID NO.1 are found to be located at key positions of a substrate binding pocket through molecular docking, so that double mutants and three mutants are constructed, and the enzyme activity of the double mutants and the enzyme activity of the three mutants are respectively expressed and verified in escherichia coli; the three mutants V219A / K311M / E405A with the most obvious product inhibition effect weakening are obtained through screening, and the three mutants V219A / K311M / E405A are suitable for efficiently hydrolyzing various oligosaccharides such as maltose, isomaltose and panose to generate glucose. When glucose is produced by using an enzymic method, the oligosaccharide debranching enzyme mutant provided by the invention can further hydrolyze oligosaccharide in a starch saccharification liquid into glucose, so that the yield of glucose is improved, and the mutant has industrial production potential.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme engineering, in particular to an oligosaccharide debranching enzyme mutant with weakened product inhibition and application thereof. Background Art

[0002] In the enzymatic production process of glucose, starch is usually used as the substrate. After being liquefied into maltodextrin by α-amylase, glucoamylase and debranching enzyme are added for saccharification. Glucose accounts for the majority of the saccharification liquid, but it also contains small molecular miscellaneous sugars such as maltose, maltotriose, isomaltose, isomaltotriose, and panose.

[0003] Debranching enzymes are a class of enzymes that specifically and efficiently hydrolyze α-1,6 glycosidic bonds in starch and related polysaccharides. They are primarily classified as pullulanases, isoamylases, and oligosaccharide debranching enzymes. Oligosaccharide debranching enzymes, belonging to glycoside hydrolase family 13 (GH13), have a typical small substrate-binding pocket deeply embedded within their structure. These enzymes are suitable for small molecule substrates such as isomaltose and isomaltotriose, and can specifically generate glucose, making them more suitable for enzymatic glucose production processes.

[0004] Patent CN115851561A discloses a recombinant Escherichia coli expressing an oligosaccharide debranching enzyme (pspOG) derived from Paenibacillus sp. STB16 and its use in hydrolyzing maltodextrin products. Patent CN116426447A discloses a recombinant Bacillus subtilis expressing pspOG extracellularly and its use in hydrolyzing isomaltooligosaccharides. pspOG has high catalytic activity for α-1,6 glycosidic bonds and can specifically hydrolyze isomaltooligosaccharides containing only α-1,6 glycosidic bonds, such as isomaltose and isomaltotriose, into glucose, and panose (containing one α-1,6 glycosidic bond and one α-1,4 glycosidic bond) into glucose and maltose. Patent CN116064456A discloses a pspOG mutant and its application in glucose mother liquor. After modifying the wild-type enzyme, the mutant V219A has the ability to simultaneously hydrolyze α-1,4 and α-1,6 glycosidic bonds. It can further convert oligosaccharide impurities containing α-1,4 glycosidic bonds, such as maltose, maltotriose, and panose, into glucose, showing certain application potential. However, the wild-type pspOG and its mutant V219A suffer from severe product inhibition. At a glucose concentration of 100 mg / mL, the enzyme activities of the wild-type pspOG and its mutant V219A are only 7% and 40% of the initial enzyme activity (glucose concentration of 0 mg / mL). They are mainly used for the regeneration of glucose from crystallized glucose mother liquor (glucose concentration is relatively low), and are difficult to directly apply to glucose production processes.

[0005] Product inhibition is a common regulatory mechanism in enzyme-catalyzed reactions and a bottleneck that must be overcome in the application of industrial enzymes. It manifests itself as a significant decrease in enzyme catalytic efficiency with increasing product concentration, leading to a gradual slowing and stagnation of the reaction rate, and even the initiation of a reverse reaction. Because both wild-type pspOG and its mutant V219A exhibit severe product inhibition, they are unable to further convert oligosaccharide impurities into glucose in saccharification solutions with extremely high glucose concentrations, and may even trigger a reverse reaction, resulting in a reduced glucose yield. Summary of the Invention

[0006] To solve the above technical problems, the present invention discovered through molecular docking that the 311th lysine and the 405th glutamate of the oligosaccharide debranching enzyme amino acid sequence as shown in SEQ ID NO.1 are located at key positions in the substrate binding pocket. Combined with the known oligosaccharide debranching enzyme mutation site V219A, double mutants and triple mutants were constructed, which significantly reduced the product inhibition effect of the oligosaccharide debranching enzyme and are suitable for efficiently hydrolyzing oligosaccharides to produce glucose.

[0007] The first object of the present invention is to provide an oligosaccharide debranching enzyme mutant, wherein the oligosaccharide debranching enzyme mutant has the following mutations in the amino acid sequence of the parent sequence shown in SEQ ID NO.1:

[0008] (1) Valine at position 219 was mutated to alanine;

[0009] (2) Lysine at position 311 is mutated to methionine and / or glutamic acid at position 405 is mutated to alanine.

[0010] Furthermore, the nucleotide sequence of the parent oligosaccharide debranching enzyme is shown in SEQ ID NO.2.

[0011] Furthermore, in the oligosaccharide debranching enzyme mutant, the amino acid sequence of the parent sequence shown in SEQ ID NO.1 is mutated from valine at position 219 to alanine, lysine at position 311 to methionine, and glutamic acid at position 405 to alanine.

[0012] The second object of the present invention is to provide a gene encoding the above oligosaccharide debranching enzyme mutant.

[0013] The third object of the present invention is to provide an expression vector comprising the above gene.

[0014] Furthermore, the expression vector uses pET-28a(+) as a backbone.

[0015] The fourth object of the present invention is to provide a host cell comprising the above oligosaccharide debranching enzyme mutant, the above gene, or the above expression vector.

[0016] In one embodiment of the present invention, the host cell is Escherichia coli.

[0017] A fifth object of the present invention is to provide a preparation comprising the above host cells.

[0018] The sixth object of the present invention is to provide a method for reducing the inhibition of oligosaccharide debranching enzyme products, wherein the valine at position 219 of the oligosaccharide debranching enzyme shown in the nucleotide sequence of SEQ ID NO.1 is mutated to alanine, the lysine at position 311 is mutated to methionine, and the glutamic acid at position 405 is mutated to alanine.

[0019] A seventh object of the present invention is to provide use of the oligosaccharide debranching enzyme mutant, the host cell or the preparation in hydrolyzing oligosaccharides into glucose.

[0020] Furthermore, the oligosaccharide includes one or more of maltose, maltotriose, isomaltose, isomaltotriose and panose.

[0021] The eighth object of the present invention is to provide a method for hydrolyzing oligosaccharides into glucose, comprising adding the oligosaccharide debranching enzyme mutant, the host cell or the preparation to a reaction system with oligosaccharides as substrates.

[0022] Furthermore, the oligosaccharide includes one or more of maltose, maltotriose, isomaltose, isomaltotriose and panose.

[0023] A ninth object of the present invention is to provide a method for producing glucose, comprising the steps of:

[0024] Step S1, preparing starch milk;

[0025] Step S2, adding amylase to the starch milk to obtain a liquefied liquid;

[0026] Step S3, adding glucoamylase to the liquefied liquid to obtain a saccharified liquid;

[0027] Step S4: adding the oligosaccharide debranching enzyme mutant, the host cell or the preparation to the saccharification solution.

[0028] Furthermore, in step S4, the added amount is 10-500 U of the oligosaccharide debranching enzyme mutant per gram of dry substrate.

[0029] In one embodiment of the present invention, in step S4, the added amount is 200 U of the oligosaccharide debranching enzyme mutant per gram of dry substrate.

[0030] Furthermore, in step S4, the reaction time is 12-48 hours.

[0031] In one embodiment of the present invention, in step S4, the reaction time is 24 hours.

[0032] Beneficial effects of the present invention:

[0033] The present invention

[0034] The present invention significantly reduces the product inhibition effect of oligosaccharide debranching enzyme by three-point mutations of V219A, K311M and E405A, and improves the enzyme's hydrolysis efficiency of oligosaccharides such as maltose and isomaltose. The obtained oligosaccharide debranching enzyme mutant can be directly added after the saccharification stage of enzymatic production of glucose, is compatible with traditional processes, and can improve glucose yield, shorten reaction time or reduce enzyme dosage without changing the existing starch liquefaction-saccharification process. The mutant enzyme is efficiently expressed using engineered host cells (such as Escherichia coli) based on the pET-28a (+) vector to ensure the feasibility of industrial production, and the stability of the preparation meets the needs of large-scale application. It breaks through the limitations of traditional glucose amylase on α-1,6 glycosidic bonds, reduces residual difficult-to-degrade oligosaccharides in the saccharification solution, improves the adaptability of oligosaccharide debranching enzyme in the glucose production process, and has strong industrial application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0036] Figure 1 This is a diagram of molecular docking results in an embodiment of the present invention;

[0037] Figure 2 This is the effect of glucose concentration on the enzymatic activity of oligosaccharide debranching enzyme and its mutants in the examples of the present invention. DETAILED DESCRIPTION

[0038] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0039] The culture medium involved in the following examples is as follows:

[0040] LB liquid medium: yeast powder 5 g / L, tryptone 10 g / L, NaCl 10 g / L, pH 7.0.

[0041] LB solid medium: 1.5% (w / v) agar powder was added to LB medium.

[0042] TB medium: yeast powder 24 g / L, tryptone 12 g / L, KH2PO4 2.32 g / L, K2HPO4·3H2O 16.43 g / L, glycerol 5 g / L, pH 7.0.

[0043] The detection methods involved in the following embodiments are as follows:

[0044] (1) Determination of oligosaccharide debranching enzyme activity

[0045] The activity of the mutants was determined using 10 mM pNPG as a substrate. The reaction system consisted of 150 μL of 500 mM phosphate buffer (pH 6.0), 800 μL of pNPG solution, and 50 μL of enzyme. The reaction was incubated at 50°C for 5 minutes, then terminated by the addition of 1 mL of 1 M sodium carbonate. The released p-nitrophenyl group (pNP) was determined spectrophotometrically by measuring absorbance at 410 nm. One unit (U) of hydrolytic activity was defined as the amount of enzyme that produces 1 μmol of pNP per minute under the assay conditions.

[0046] (2) Effect of product concentration on oligosaccharide debranching enzyme activity

[0047] Using 10 mM pNPG as a substrate, different amounts of glucose (0-500 mg / mL) were added to the substrate. The enzyme activity was measured according to the assay method (1). The inhibitory effect of glucose on the enzyme was evaluated by the change in enzyme activity. The relative activity at different glucose addition levels was calculated, with the enzyme activity without exogenous glucose added as 100%.

[0048] (3) Analysis of saccharification reaction products

[0049] The oligosaccharide debranching enzyme mutant V219A / K311M / E405A was used in an enzymatic glucose production process, and the content of the product components was analyzed by high-performance liquid chromatography (HPLC). The analysis conditions were: a ChromCore normal phase / HILIC column, a 70% acetonitrile-water mobile phase, a flow rate of 1.0 mL / min, a column temperature of 30°C, and an injection volume of 10 μL.

[0050] Example 1: Construction of recombinant plasmid containing mutants

[0051] Molecular docking revealed that Figure 1), the product glucose molecule will form a stable hydrogen bond interaction with the 311th lysine and the 405th glutamate of the oligosaccharide debranching enzyme (nucleotide sequence as shown in SEQ ID NO.2) with an amino acid sequence as shown in SEQ ID NO.1, thereby binding to these two amino acids. Since these two amino acids are located in the key position of the substrate binding pocket and the side chain volume is relatively large, these two amino acids will not only bind to glucose through hydrogen bond interactions, hindering the release of glucose, but also hinder the release of glucose due to steric hindrance. In order to reduce steric hindrance and weaken hydrogen bond interactions, while not affecting the stability of the enzyme as much as possible, according to the results of FoldX simulation mutation, the present invention mutates lysine at position 311 to methionine and glutamate at position 405 to alanine, respectively obtaining oligosaccharide debranching enzyme mutants V219A / K311M, V219A / E405A and V219A / K311M / E405A.

[0052] Using the expression vector pET-28a(+) as a template, complementary primer chains were designed. The primer sequences are shown in Table 1.

[0053] Site-directed mutagenesis was performed according to the instructions of the TaKaRa STAR Primer GXL kit. The PCR system was: 5× PrimeSTAR GXL Buffer (Mg 2+ Add 10 μL of PCR primer (Plus), 4 μL of dNTP Mixture (2.5 mM each), 1 μL of forward primer (10 μM), 1 μL of reverse primer (10 μM), 1 μL of template DNA, and 1 μL of PrimeSTAR GXL DNA Polymerase (1.25 U / μL). Add double-distilled water to 50 μL. PCR amplification conditions were: initial denaturation at 98°C for 3 minutes, followed by 30 cycles (98°C for 10 seconds, 60°C for 15 seconds, and 68°C for 7 minutes), and finally incubation at 68°C for 10 minutes.

[0054] Table 1 Site-directed mutagenesis primers

[0055]

[0056] Note: The underlined bases correspond to the corresponding mutated amino acids

[0057] Example 2: Construction of genetically engineered bacteria

[0058] The PCR product obtained in Example 1 was treated with Dpn I at 37°C. The Dpn I digestion system consisted of: 10 μL of PCR product, 7 μL of double-distilled water or Milli-Q water, 2 μL of 10X Buffer Y, and 1 μL of Dpn I. The treated PCR product was then transformed into E. coli JM109 competent cells. The resulting transformation product was then plated onto LB agar solid medium containing 100 μg / mL kanamycin and cultured overnight in a 37°C incubator for 12 hours. A single colony was then selected and inoculated into LB liquid medium containing 100 μg / mL kanamycin and cultured overnight at 37°C at 200 rpm. Plasmids were then extracted and sequenced according to the instructions in the plasmid extraction kit.

[0059] The correctly sequenced plasmid was transformed into the competent expression host E. coli BL21 (DE3), and finally the genetically engineered bacteria E. coli-V219A / K311M, E. coli-V219A / E405A, and E. coli-V219A / K311M / E405A were obtained.

[0060] Example 3: Expression of oligosaccharide debranching enzyme mutants

[0061] Seed culture: 100 μL of the preserved genetically engineered bacteria E. coli-V219A / K311M, E. coli-V219A / E405A, and E. coli-V219A / K311M / E405A were inoculated into LB medium containing the corresponding antibiotics, and cultured at 37°C and 200 rpm for 8-10 h.

[0062] Fermentation culture: inoculate the seed culture solution into the fermentation medium containing the corresponding antibiotics at an inoculum volume of 4% (v / v), and culture in a shaking incubator at 37°C and 200 rpm until the OD 600 When the value reached 0.6, IPTG was added to make the final concentration 0.05 mM, and the expression was induced at 25°C and 200 r / min for 48 h.

[0063] Crude enzyme acquisition: The fermentation broth was centrifuged at 4°C and 10,000 rpm for 20 min, the supernatant was discarded, and the residual cells were resuspended in 10 mM phosphate buffer (pH 6.0). The cells were then disrupted with an ultrasonic cell disruptor. After centrifugation, the enzyme solution was present in the supernatant.

[0064] Enzyme purification: Oligosaccharide debranching enzyme was purified using a nickel column. The equilibration solution (liquid A, pH = 7.5) was 500mM NaCl, 50mM Tris-HCl, and 20mM imidazole, and the eluent (liquid B, pH = 7.5) was 500mM NaCl, 50mM Tris-HCl, and 500mM imidazole. The crude enzyme solution was filtered through a 0.45μm aqueous membrane. The nickel column was first equilibrated with buffer A at a flow rate of 2mL / min, and then the sample was loaded at a rate of 1.5mL / min to allow the target protein to bind to the nickel column. After loading, the nickel column was equilibrated with buffer A again. After equilibration, the sample was eluted with 60% eluent B at a flow rate of 1mL / min, and the corresponding eluent was collected according to the elution peak.

[0065] Example 4: Product inhibition analysis of oligosaccharide debranching enzyme mutants

[0066] The activity of the mutants was determined using 10 mM pNPG as a substrate. The reaction system consisted of 150 μL of 500 mM phosphate buffer (pH 6.0), 800 μL of pNPG solution, and 50 μL of enzyme. The reaction was incubated at 50°C for 5 minutes, then terminated with 1 mL of 1 M sodium carbonate. The released p-nitrophenyl group (pNP) was determined spectrophotometrically by measuring absorbance at 410 nm.

[0067] Using pNPG as a substrate, varying amounts of glucose (0-500 mg / mL) were added to the substrate. Enzyme activity was measured using the enzyme activity assay method. The inhibitory effect of glucose on the enzyme was assessed by changes in enzyme activity. Relative activity was calculated at different glucose addition levels, with the enzyme activity in the absence of exogenous glucose as 100%.

[0068] The results are as follows Figure 2 As shown, when the glucose concentration was 100 mg / mL, the relative enzyme activity of the wild-type enzyme was 7%, the relative enzyme activity of the single mutant V219A was about 40%, the relative enzyme activity of the double mutant V219A / E405A was about 80%, the relative enzyme activity of the double mutant V219A / K311M was about 73%, and the relative enzyme activity of the triple mutant V219A / K311M / E405A was about 90%.

[0069] When the glucose concentration was 500 mg / mL, the wild-type enzyme was basically inactivated, the relative enzyme activity of the single mutant V219A was about 12%, the relative enzyme activity of the double mutant V219A / E405A was about 50%, the relative enzyme activity of the double mutant V219A / K311M was about 40%, and the relative enzyme activity of the triple mutant V219A / K311M / E405A was about 60%.

[0070] Example 5: Effect of glucose on the efficiency of oligosaccharide debranching enzyme mutants in hydrolyzing different oligosaccharides

[0071] Common byproducts of glucose production, including maltose, maltotriose, isomaltose, isomaltotriose, and panose, were prepared into standard solutions containing 100 mg / mL glucose (500 mM phosphate buffer, pH 6.0, oligosaccharide concentration 5 mg / mL). Wild-type oligosaccharide debranching enzyme or its mutants were added at 10 U / g of enzyme. After incubation at 50°C for 12 hours, the glucose content produced was determined using a glucose assay, with the 100 mg / mL glucose present in the substrate subtracted. The glucose assay reaction system consisted of 1.5 mL of the assay solution and 50 μL of the reaction solution.

[0072] The glucose yields of different oligosaccharides hydrolyzed by the wild-type oligosaccharide debranching enzyme and its mutants are shown in Table 2. In the presence of 100 mg / mL glucose in the system, the wild-type enzyme and the single mutant V219A had very low hydrolysis abilities for maltose, maltotriose, isomaltose, isomaltotriose, and panose, with the final glucose yield being below 30%. However, V219A / K311M / E405A could almost completely convert isomaltose, isomaltotriose, and panose into glucose, with glucose yields exceeding 80% and approaching 100%. Most maltose and maltotriose could be converted into glucose, which was significantly improved compared to both the wild-type enzyme and the single mutant V219A.

[0073] Table 2 Effect of glucose on the hydrolysis efficiency of oligosaccharide debranching enzyme mutants

[0074]

[0075] Example 6: Application of mutants in enzymatic production of glucose

[0076] (1) Slurry preparation: 30% corn starch milk by mass was used as the substrate, kept at 60°C for 15 min, the pH was adjusted to 6.0, and a thermostable α-amylase was added at an enzyme dosage of 12 U / g;

[0077] (2) Liquefaction: The starch emulsion was liquefied twice by jet liquefaction: the first jet was at 110°C and the second jet was at 140°C. The liquefied liquid was kept in a laminar flow tank for 30 min until the DE value (glucose value, representing the degree of starch hydrolysis) reached 15;

[0078] (3) Saccharification: The liquefied liquid was cooled at 60°C for 15 min, the pH was adjusted to 5.0, glucoamylase was added at a dosage of 60 U / g, and the reaction was maintained at a constant temperature of 60°C for 48 h.

[0079] (4) Oligosaccharide debranching enzyme conversion: After the liquefaction and saccharification reactions were completed, the saccharification solution was cooled at 60°C for 15 min, the pH was adjusted to 6.0, and the oligosaccharide debranching enzyme mutant V219A / K311M / E405A was added at an enzyme dosage of 200 U / g, and the reaction was continued for 24 h.

[0080] The product analysis results of the saccharification reaction are shown in Table 3. In the enzymatic production process for glucose, after the liquefaction and saccharification reactions were completed, the glucose yield in the saccharified liquid was 95.0%. Adding the triple mutant V219A / K311M / E405A to continue the conversion further increased the glucose yield, reaching a final glucose yield of 98.1%. In contrast, the wild-type enzyme and the single mutant V219A were severely inhibited by the product, undergoing a reverse reaction, reducing the glucose yield to 88.2% and 90.6%, respectively. This suggests that the triple mutant V219A / K311M / E405A is more adaptable to the enzymatic production process for glucose, helping to improve substrate conversion and product purity during glucose production, thereby reducing energy consumption and wastewater generation in the subsequent glucose crystallization process.

[0081] Table 3 Analysis of saccharification reaction products

[0082]

[0083] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. An oligosaccharide debranching enzyme mutant, characterized in that: The oligosaccharide debranching enzyme mutant makes the following mutations to the parent sequence of the amino acid sequence shown in SEQ ID NO.1: (1) Valine at position 219 was mutated to alanine; (2) Lysine at position 311 is mutated to methionine and / or glutamic acid at position 405 is mutated to alanine.

2. The oligosaccharide debranching enzyme mutant according to claim 1, characterized in that: In the oligosaccharide debranching enzyme mutant, the amino acid sequence of the parent sequence shown in SEQ ID NO. 1 is mutated from valine at position 219 to alanine, lysine at position 311 to methionine, and glutamic acid at position 405 to alanine.

3. A gene encoding the oligosaccharide debranching enzyme mutant according to claim 1 or 2.

4. An expression vector comprising the gene according to claim 3.

5. The expression vector according to claim 4, characterized in that: The expression vector uses pET-28a(+) as the backbone.

6. A host cell comprising the oligosaccharide debranching enzyme mutant according to claim 1 or 2, the gene according to claim 3, or the expression vector according to claim 4 or 5.

7. A preparation comprising the host cell according to claim 6.

8. A method for reducing the inhibition of oligosaccharide debranching enzyme products, characterized in that: The valine at position 219 of the oligosaccharide debranching enzyme with a nucleotide sequence as shown in SEQ ID NO.1 is mutated to alanine, the lysine at position 311 is mutated to methionine, and the glutamic acid at position 405 is mutated to alanine.

9. Use of the oligosaccharide debranching enzyme mutant according to claim 1 or 2, the host cell according to claim 6 or the preparation according to claim 7 in hydrolyzing oligosaccharides into glucose.

10. The use according to claim 10, characterized in that: The oligosaccharide comprises one or more of maltose, maltotriose, isomaltose, isomaltotriose and panose.

11. A method for hydrolyzing oligosaccharides into glucose, characterized in that: The oligosaccharide debranching enzyme mutant according to claim 1 or 2, the host cell according to claim 6 or the preparation according to claim 7 is added to a reaction system with oligosaccharides as substrates.

12. The method according to claim 11, wherein: The oligosaccharide comprises one or more of maltose, maltotriose, isomaltose, isomaltotriose and panose.

13. A method for producing glucose, characterized in that: The following steps are involved: Step S1, preparing starch milk; Step S2, adding amylase to the starch milk to obtain a liquefied liquid; Step S3, adding glucoamylase to the liquefied liquid to obtain a saccharified liquid; Step S4: adding the oligosaccharide debranching enzyme mutant according to claim 1 or 2, the host cell according to claim 6, or the preparation according to claim 7 to the saccharification solution.

14. The method according to claim 13, wherein: In step S4, the added amount is 10-500 U of the oligosaccharide debranching enzyme mutant per gram of dry substrate.

15. The method according to claim 13, wherein: In step S4, the reaction time is 12-48 hours.

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