CN114985010A

CN114985010A - Bionic protease and preparation method and application thereof

Info

Publication number: CN114985010A
Application number: CN202210538765.5A
Authority: CN
Inventors: 许宙; 许珂宇; 程云辉; 夏利伟; 陈茂龙; 文李; 丁利
Original assignee: Changsha University of Science and Technology
Current assignee: Changsha University of Science and Technology
Priority date: 2022-05-18
Filing date: 2022-05-18
Publication date: 2022-09-02
Anticipated expiration: 2042-05-18
Also published as: CN114985010B

Abstract

The invention discloses a bionic protease and a preparation method and application thereof, wherein the preparation method comprises the following steps: and (2) performing coordination reaction on a zirconium source and pyromellitic acid in a solvent to prepare the biomimetic protease. The biomimetic protease has excellent catalytic activity, and the preparation method has simple steps and mild conditions, so that the biomimetic protease can be applied to proteolysis.

Description

Bionic protease and preparation method and application thereof

Technical Field

The invention relates to protease, in particular to bionic protease and a preparation method and application thereof.

Background

Proteases catalyze the hydrolysis of peptide bonds to amino and carboxyl groups and have been widely used in the fields of food processing, biosensing, environmental protection, biomedicine, and the like. The catalytic condition of the protease is mild, and the protease is efficient and specific, so the protease has wide and huge application in industry. Statistically, the proportion of proteolytic enzymes is at a maximum of about 75% in all industrial enzyme preparations. However, the inherent disadvantages of natural enzymes, such as variability, high cost, laborious preparation and difficult recovery, have greatly limited their practical applications.

To overcome these disadvantages, researchers have been working on the search for artificial enzyme mimics. To date, hydrolysis of proteins using metal ions as lewis acid catalysts is the most common method for constructing artificial proteases. This approach is very limited, however, because many transition metals and lanthanides form gels under neutral and basic conditions. To avoid gel formation, organic ligands are often added to form metal complexes.

Wherein the Co (III) complex [ Co (trien) OH (H) ₂ O)] ²⁺ Is one of the most studied metal ion complexes capable of rapidly hydrolyzing peptide bonds. Metal-substituted Polyoxometalates (POMs) are also commonly reported to have protease-like activity, a class of polyoxometalate compounds formed by oxygen linkage of transition metal ions, which can be modified to have various chemical and physical properties. In addition, Zr (IV) -substituted POM (Zr-POMs) possess very good protease-like activity, because Zr (IV) has a large coordination number, flexible geometry, high oxophilicity and Lewis acidity.

In 2014 Li et al reported a Copper-based metal organic framework (Cu-MOF) material with protease-like activity, which can catalyze the hydrolysis of peptide bonds in Bovine Serum Albumin (BSA) and casein. Due to the large surface area and porous structure of MOF, Cu-MOF has a higher affinity for proteins than native trypsin and Cu (ii) complexes.

Recent studies have found that Zr-MOF has proteolytic-like enzyme activity, derived from Zr ₆ [Zr ₆ (μ ₃ -O) ₄ (μ ₃ -OH) ₄ ]Zr-MOF formed by self-assembling nodes and carboxylic acid ligands is one of the most widely used MOFs at present, has the function of selectively hydrolyzing dipeptides and proteins under physiological conditions as a heterogeneous and recyclable artificial enzyme, and has excellent recoverability and excellent stability. It has strong Zr (IV) -O bonds, thus having very excellent thermal and chemical stability. In addition, Zr-MOF is simple to prepare and low in price, so that the Zr-MOF is often used as a catalyst for some chemical reactions. However, the Zr-MOF catalytic activity is low, and the demand of industrial production cannot be met.

Disclosure of Invention

The invention aims to provide biomimetic protease and a preparation method and application thereof, wherein the biomimetic protease has excellent catalytic activity, and the preparation method has simple steps and mild conditions, so that the biomimetic protease and the biomimetic protease can be applied to proteolysis.

In order to achieve the above object, the present invention provides a method for preparing biomimetic protease, comprising: and (2) performing coordination reaction on a zirconium source and pyromellitic acid in a solvent to prepare the biomimetic protease.

Preferably, the zirconium source is selected from ZrOCl ₂ ·8H ₂ O、ZrCl ₄ 、Zr(NO ₃ ) ₂ .6H ₂ At least one of O.

Preferably, the molar ratio of the zirconium source to the pyromellitic acid is 1: 08-1.2.

Preferably, the coordination reaction satisfies at least the following conditions: the reaction is carried out in a closed environment, the reaction temperature is 140-160 ℃, and the reaction time is 20-30 h.

Preferably, the molar ratio of the zirconium source to the solvent is 28 mmol: 350-400 mL.

Preferably, the solvent is selected from at least one of N, N-dimethylacetamide, formic acid, acetic acid, ethanol, and hydrochloric acid.

Preferably, the solvent comprises N, N-dimethylacetamide and formic acid in a volume ratio of 2-3: 1.

The invention also provides a biomimetic protease, which is prepared by the preparation method.

The invention also provides application of the biomimetic protease in catalyzing protein hydrolysis.

Preferably, the method of application is: taking the biomimetic protease as a catalyst, and performing hydrolysis reaction on the protein in a dispersing agent;

wherein the molar ratio of the protein to the biomimetic protease to the dispersant is 20 mmol: 1.5-2.5 mmol: 900-;

preferably, the hydrolysis reaction satisfies at least the following conditions: the reaction temperature is 50-70 ℃, and the reaction time is 3-6 h;

preferably, the protein is selected from at least one of soy protein isolate, surimi protein and casein;

preferably, the dispersant is at least one of water, phosphate buffer, tris solution and sodium trimethylsilylpropionate solution.

As described above, in recent years, with the rapid progress of nanotechnology, it has been found that various functional nanomaterials also have protease-like activities. However, synthetic nanoenzymes also have some problems and drawbacks, such as: the lower activity and specificity are two major obstacles for limiting the application of the nano-enzyme to replace the natural enzyme, and the targeted improvement of the selectivity is also the focus of research.

Because the catalytic reaction of the enzyme mainly occurs on the surface, the surface modification for improving the selectivity can possibly reduce the catalytic activity; there are many types of nanoenzymes, but the current research on enhancing the activity of nanoenzymes mainly focuses on the nanomaterials with peroxidase-like activity, and then on the oxidase activity, there are few researches on other types of enzyme activity enhancing strategies. The enhancement strategies of other enzyme activities should be further researched, so that the activity level of the nanometer enzyme is integrally improved; accurate regulation and control of the enhancement of various enzyme activities of the nano material: such as AuNP, have peroxidase, oxidase and catalase activities simultaneously. Hybridization of nanoenzymes also produces products with two or more enzyme activities. An activity enhancement strategy may have an effect on multiple enzyme activities, but often only a single catalytic activity is required for practical applications. Therefore, the single regulation and control property is a big defect of the nano enzyme while the enzyme activity is enhanced.

Therefore, the modification of the artificial synthetase has a plurality of difficulties, and under the background of the invention, as shown in figure 1-1 and figure 1-2, carboxyl is introduced on the basis of Zr-MOFs structure to simulate the catalytic activity center of natural zinc metalloprotease, and Lewis acidic Zr is used ⁴⁺ Sites to mimic Zn in native zinc metalloproteases ²⁺ The carboxyl group is used for simulating the action of glutamic acid at the active center of natural metalloprotease, and the biomimetic protease (Zr-MOF protease) is constructed, so that the biomimetic protease has a high-activity protease-like function, can be used for efficiently hydrolyzing various industrial proteins, and can be applied to the food industry.

Additional features and advantages of the invention will be set forth in the detailed description which follows.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:

FIG. 1-1 is a central diagram of the catalytic activity of a native zinc metalloprotease;

FIGS. 1-2 are central graphs of Zr-MOF catalytic activity in biomimetic design;

FIG. 2 is a representation of each of the Zr-MOF and biomimetic protease, wherein (A) is a PXRD representation of the Zr-MOF and biomimetic protease, (B) is an FTIR representation of the Zr-MOF and biomimetic protease, (C) is an SEM representation of the Zr-MOF, and (D) is an SEM representation of the biomimetic protease;

FIG. 3 is a high performance liquid chromatogram showing the hydrolysis of diglycine;

FIG. 4 is an SDS-PAGE pattern of hydrolyzed soy protein isolate;

FIG. 5 is an SDS-PAGE pattern of hydrolyzed surimi protein;

FIG. 6 is an SDS-PAGE pattern of hydrolyzed casein;

FIG. 7 is an SDS-PAGE pattern of the three proteins in the absence of biomimetic protease.

Detailed Description

The following describes in detail specific embodiments of the present invention. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.

The invention provides a preparation method of biomimetic protease, which comprises the following steps: and (2) performing coordination reaction on a zirconium source and pyromellitic acid in a solvent to prepare the biomimetic protease.

In the above-mentioned production method, the kind of the zirconium source is not particularly required, but in order to further improve the catalytic activity of the produced biomimetic protease, preferably, the zirconium source is selected from ZrOCl ₂ ·8H ₂ O、ZrCl ₄ 、Zr(NO ₃ ) ₂ .6H ₂ At least one of O.

In the above preparation method, the amount of each material is not specifically required, but in order to further improve the catalytic activity of the produced biomimetic protease, the molar ratio of the zirconium source to the pyromellitic acid is preferably 1: 08-1.2.

In the above preparation method, the conditions of the coordination reaction are not particularly required, but in order to further improve the catalytic activity of the produced biomimetic protease, it is preferable that the coordination reaction at least satisfies the following conditions: the reaction is carried out in a closed environment, the reaction temperature is 140-160 ℃, and the reaction time is 20-30 h.

In the above-mentioned production method, the amount of the solvent to be used may be selected within a wide range, but in order to enable sufficient dispersion of the respective materials and thereby improve the reaction yield, it is preferable that the molar ratio of the amount of the zirconium source to the amount of the solvent to be used is 28 mmol: 350-400.

On the basis of the above embodiment, the kind of the solvent may also be selected from a wide range, but in order to further improve the reaction yield, it is preferable that the solvent is selected from at least one of N, N-dimethylacetamide, formic acid, acetic acid, ethanol, and hydrochloric acid; further preferably, the solvent comprises N, N-dimethylacetamide and formic acid in a volume ratio of 2-3: 1.

In the above application, the specific method of catalytic application of the biomimetic protease may be various, but in order to further improve the catalytic effect, it is preferable that the method of application is: taking the biomimetic protease as a catalyst, and performing hydrolysis reaction on the protein in a dispersing agent; wherein the molar ratio of the protein to the biomimetic protease to the dispersant is 20 mmol: 1.5-2.5 mmol: 900-.

In the above application, the conditions of the hydrolysis reaction may be selected within a wide range, but in order to further enhance the effect of hydrolysis, it is preferable that the hydrolysis reaction satisfies at least the following conditions: the reaction temperature is 50-70 ℃, and the reaction time is 3-6 h;

in the above-mentioned application, the kind of the protein may be selected from a wide range, but in view of the market application prospect, it is preferable that the protein is selected from at least one of soybean protein isolate, surimi protein and casein.

In the above application, the kind of the dispersant may be selected from a wide range, but in order to further enhance the effect of hydrolysis, it is preferable that the dispersant is at least one of water, a phosphate buffer, a tris solution, and a sodium trimethylsilylpropionate solution.

The present invention will be described in detail below by way of examples. In the following examples, proteins and their degradation products were separated by SDS-polyacrylamide gel electrophoresis in ultrapure water, and the separated bands were finally stained with Coomassie Brilliant blue, thereby analyzing the degradation of proteins by biomimetic proteases.

Diglycine and its hydrolysate were determined by PITC pre-column derivatization liquid chromatography.

Derivation of amino acids: accurately measuring 200 mu L of sample solution, and placing the sample solution in a 1.5mL centrifuge tube; respectively adding 100 mu L of triethylamine acetonitrile solution and 100 mu L of PITC acetonitrile solution, uniformly mixing, and standing for 1 hour at 25 ℃; adding 400 mu L of n-hexane into a centrifuge tube, uniformly mixing, and standing at 25 ℃ for 10 min; taking the lower PITC-AA solution, and passing through a 0.45 mu m water film; 200 mu L of filtrate is taken, diluted by adding 800 mu L of water, shaken up and 10 mu L of sample is injected.

The chromatographic column adopts Venusil of Agela company

Amino acid analytical column (4.6X 250mm,5 μm), mobile phase A is 0.1mol/L acetic acid-sodium acetate buffer solution with pH6.5, and mobile phase B is pure acetonitrile solution. The elution flow rate was set at 1.0mL/min, the detection wavelength was 254nm, the column temperature was 40 ℃ and the loading volume was 10. mu.L. Adopting a gradient elution mode, wherein the elution condition is 0-2 min and 100% A; 2-15 min, 85% A; 15-17 min, 100% A; 17-18 min, 100% A.

Example 1

Bionic protease (Zr-MOF- (COOH) ₂ ) Preparation of

Taking equimolar amount of ZrOCl ₂ 8H2O and pyromellitic acid ligand 27.9mmol, added to a mixture of 270mL of N, N-Dimethylacetamide (DMA) and 108mL of formic acid. After magnetic stirring for one hour, the solution is transferred to a 500mL inner container of a polytetrafluoroethylene-lined reaction kettle, the inner container of the reaction kettle is placed in a high-pressure reaction kettle, a cover is screwed, and the reaction kettle is heated at 150 ℃ for 24 hours. After the reaction is finished, the obtained precipitate is washed with DMF and acetone respectively for three times and is stored for standby.

Example 2

The procedure is as in example 1, except that: ZrOCl ₂ ·8H ₂ O is replaced by equimolar amount of ZrCl ₄ 。

Example 3

The procedure is as in example 1, the only difference being: ZrOCl ₂ ·8H ₂ O is replaced by an equimolar amount of Zr (NO) ₃ ) ₂ .6H ₂ O。

Comparative example 1

Preparation of ZR-MOF:

weighing 5.20mmol of zirconium tetrachloride, adding the zirconium tetrachloride into 300mL of DMF, adding 9mL of acetic acid after magnetic stirring for 1h, adding 5.18mmol of terephthalic acid after continuously stirring for 1h, transferring the solution into a 500mL polytetrafluoroethylene-lined reaction kettle inner container after magnetic stirring for 2h until the solution is uniformly dispersed, placing the reaction kettle inner container into a high-pressure reaction kettle, screwing a cover, and heating at 120 ℃ for 24 h. After completion of the reaction, it was cooled to room temperature, the supernatant was discarded, and the resulting precipitate was washed three times with methanol and three times with DMF.

Test example 1

1) PXRD characterization is carried out on the products of example 1 and comparative example 1, the characterization results are shown in part A of figure 2, and the X-ray diffraction (XRD) pattern and the simulation pattern of the biomimetic protease of the ZR-MOF and the carboxylic acid functional derivative thereof are matched, so that the ZR-MOF and the biomimetic protease are confirmed to have the same crystal structure.

2) FTIR characterization of the products of example 1 and comparative example 1 is shown in section B of FIG. 2, from which it can be seen that the absorption bands of ZR-MOF and biomimetic protease are mostly concentrated at 1400cm ^-1 And 1584cm ^-1 This can be attributed to the stretching vibration of the coordinated terephthalic acid linker molecules. 1740-1700 cm ^-1 The broad bands of (a) correspond to symmetric and asymmetric stretches of C ═ O groups, indicating the presence of free-COOH groups in the biomimetic protease, successfully demonstrating the success of MOF preparation. Furthermore, the absorption peak of the biomimetic protease is blue-shifted compared to ZR-MOF, which may also be due to free-COOH groups.

3) SEM characterization is carried out on the products of example 1 and comparative example 1, the characterization graph of the product of example 1 is shown in a part D of a figure 2, the characterization graph of the product of comparative example 1 is shown in a part C of a figure 2, and the ZR-MOF is shown to be in a more regular pentagon shape and is about 100nm long; the prepared bionic protease is round, the surface is rough, and the average particle size under an electron microscope is about 150 nm.

4) The products of examples 2-3 were characterized in the same manner as described above, and the characterization results were substantially identical to those of the product of example 1.

Application example 1

Hydrolysis of diglycine:

the biomimetic protease (0.20mmol) prepared in example 1 was taken into a centrifuge tube containing 950mL of ultrapure water, and stirred (shaken by a magnetic stirrer) at 25 ℃ for 30 minutes to uniformly disperse the MOF particles. Then 50mL of 4mmol/L diglycine (2.0mmol) was added. The reaction mixture was kept at 60 ℃ for reaction at pH 7.0, and after completion of the reaction, Zr-MOF (i.e., biomimetic protease) was removed by centrifugation at 15000rpm for 20 minutes.

Detecting the hydrolysis of diglycine in different reaction time periods, specifically referring to FIG. 3, wherein part A represents the HPLC chromatogram after the hydrolysis of diglycine in different time periods, part B represents the graph of change of diglycine concentration and glycine concentration with hydrolysis time, and part C represents the graph of ln [ GG ] as a function of reaction time; gly represents glycine, and Gly-Gly and GG both represent diglycine.

As can be seen from fig. 3: at 60 ℃ and pH 7.0, the data show that the glycylglycine is hydrolyzed rapidly within 3h, and the reaction rate gradually slows down as the concentration of glycylglycine decreases. The reaction rate constant of the bionic protease for hydrolyzing diglycine is known to be 2.57 multiplied by 10 through the part C in the graph ^-5 s ^-1 。

The biomimetic protease prepared in example 1 was changed to the products of examples 2 and 3 according to the same method, and the reaction rate constant for hydrolyzing diglycine was determined to be substantially identical to that of the biomimetic protease prepared in example 1.

According to the same method, the biomimetic protease is respectively removed, or the biomimetic protease is replaced by equimolar amount of ZrCl ₄ Or MOF-808, and the rate constant of the hydrolysis reaction was finally determined, and the specific results are shown in Table 1.

In addition, the uncatalyzed hydrolysis reaction rate constant of glycylglycine was 7.4X 10 at 60 ℃ ^-9 s ^-1 This indicates that the reaction rate of the hydrolysis of diglycine is increased by 3.47X 10 when the biomimetic protease is used ³ And (4) doubling.

TABLE 1

Catalyst and process for preparing same	Without catalyst	ZrCl ₄	MOF-808	Biomimetic proteases
					Constant of reaction rate	7.4×10 ^-9 s ^-1	5.55×10 ^-7 s ^-1	2.63×10 ^-5 s ^-1	2.57×10 ^-5 s ^-1

Application example 2

The biomimetic protease (2.0mmol) prepared in example 1 was added to a beaker containing 1L of ultrapure water and stirred (shaken by a magnetic stirrer) at room temperature for 30 minutes to uniformly disperse the MOF particles. Then a 20mmol sample of soy protein isolate was added. The reaction mixture was kept at 60 ℃ for reaction, and after the reaction was completed, Zr-MOF was removed by centrifugation at 15000rpm for 20 minutes. The hydrolysis of the protein samples was examined at different reaction time periods, as shown in FIGS. 4-6. FIG. 4 is an SDS-PAGE pattern of hydrolyzed soy protein isolate; FIG. 5 is an SDS-PAGE pattern of hydrolyzed surimi protein; FIG. 6 is an SDS-PAGE pattern of hydrolyzed casein; FIG. 7 is an SDS-PAGE pattern of the three proteins in the absence of biomimetic protease.

The results shown in fig. 4-6 indicate that the biomimetic protease can simultaneously catalyze the hydrolysis of three proteins and shows a broad-spectrum catalytic activity, wherein the biomimetic protease has high catalytic activity on soybean protein and surimi protein and low catalytic activity on casein. As shown in FIG. 4, as the reaction proceeded, the bands of the soy protein isolate became gradually lighter and almost disappeared by 24 hours, indicating that hydrolysis occurred. No new bands with lower molecular weight appeared during the reaction, probably because the biomimetic protease had a broad spectrum of cleavage sites, directly hydrolyzing the long peptide of soy protein isolate into many shorter fragments, which could not be observed (because the short polypeptides would leak out of the SDS PAGE gel). As can be seen from FIG. 5, after 3h of incubation, a new band with a molecular weight of more than 96kDa was generated in the electrophoretogram of surimi protein, and the concentration of surimi protein increased and then decreased as the reaction proceeded, which is very good evidence that surimi protein is hydrolyzed in the presence of SDS-PAGE patterns and that the bionic protease has a selective cleavage site for this band. Similarly, the casein band also becomes lighter with increasing hydrolysis time, with bands with a molecular weight of about 26kDa hydrolyzing faster and bands with a molecular weight of about 23kDa hydrolyzing slower (see FIG. 6). Control experiments showed that hydrolysis of the three proteins was not observed in the absence of the biomimetic protease at 60 ℃ (see figure 7), confirming the catalytic role of SDS-PAGE images in protein hydrolysis.

The catalytic hydrolysis effects of the products of examples 2 and 3 on soy protein isolate, surimi protein and casein were substantially equivalent to those of the biomimetic protease prepared in example 1, performed in the same manner.

According to the tests, the bionic protease prepared by the method can be used for hydrolyzing protein paper commonly used in the food industry; the method has feasibility of being applied to the food industry, selects several proteins (casein, soy protein and surimi protein) commonly used in the food industry, incubates the three proteins with the biomimetic protease at 60 ℃, and the nano-enzyme can simultaneously catalyze the hydrolysis of the three proteins within 3 hours and shows a broad-spectrum catalytic activity, wherein the biomimetic protease has higher catalytic activity on the soy protein and the surimi protein and lower catalytic activity on the casein. In conclusion, the biomimetic protease has good capability of catalyzing the hydrolysis of the extremely stable peptide bonds in the protein.

Application example 3

The Zr-MOFs obtained after the end of the experiment in application example 1 were stirred in methanol for 1 day to exchange water (during the experiment Zr-MOFs were dispersed in water so that there was water in the material after the experiment, and after stirring in methanol, the water therein could be exchanged for methanol), this process was repeated twice, and the used MOFs were air dried and activated at 150 ℃ for 20 hours. Subsequently, the diglycine was hydrolyzed as described above in application example 1. The catalytic hydrolysis was repeated 5 times, and the reaction rate constant was recorded for each time, and the results are shown in Table 2.

TABLE 2

As can be seen from Table 2, the biomimetic protease prepared by the invention can be repeatedly catalyzed, and the catalytic efficiency is not obviously reduced after 5 times of repeated hydrolysis, so that the biomimetic protease can be repeatedly recycled for at least 5 times.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.

Claims

1. A preparation method of biomimetic protease is characterized by comprising the following steps: and (2) performing coordination reaction on a zirconium source and pyromellitic acid in a solvent to prepare the biomimetic protease.

2. The method of claim 1, wherein the zirconium source is selected from ZrOCl ₂ ·8H ₂ O、ZrCl ₄ 、Zr(NO ₃ ) ₂ .6H ₂ At least one of O.

3. The preparation method according to claim 1, wherein the molar ratio of the zirconium source to pyromellitic acid is 1: 08-1.2.

4. The production method according to claim 1, wherein the coordination reaction satisfies at least the following condition: the reaction is carried out in a closed environment, the reaction temperature is 140-160 ℃, and the reaction time is 20-30 h.

5. The preparation method according to claim 1, wherein the molar ratio of the zirconium source to the solvent is 28 mmol: 350-400 mL.

6. The production method according to any one of claims 1 to 5, wherein the solvent is at least one selected from the group consisting of N, N-dimethylacetamide, formic acid, acetic acid, ethanol, and hydrochloric acid.

7. The production method according to claim 6, wherein the solvent comprises N, N-dimethylacetamide and formic acid in a volume ratio of 2-3: 1.

8. A biomimetic protease, wherein the biomimetic protease is prepared by the preparation method of any one of claims 1-7.

9. Use of a biomimetic protease according to claim 8 for catalyzing proteolysis.

10. The application of claim 9, wherein the method of applying is: taking the biomimetic protease as a catalyst, and performing hydrolysis reaction on the protein in a dispersing agent;

wherein the molar ratio of the protein to the biomimetic protease to the dispersant is 20 mmol: 1.5-2.5 mmol: 900-1100 mL;