Method of stabilizing an aqueous solution or suspension of metalloprotease and stable aqueous solution or suspension of metalloprotease

 

(57) Abstract:

The invention relates to a method of stabilizing a solution or suspension of metalloprotease for better storage and transportation of such enzyme. N-blocked amino acid is added to aqueous solution or suspension of the protease. The invention concerns a composition with metalloproteases, stabilized with N-blocked amino acids. Adding N-benzyloxycarbonylamino to metalloprotease the enzyme is stable even in the absence of excess ions. Correspondingly storing metalloprotease for a long time, and transportation and storage of liquid enzyme correspondingly simplified. Regeneration metalloprotease improved in the presence of the NPAA, when the specified enzyme used in the reaction of binding between the N-blocked amino acid and amino acid ester. The effect is most noticeable when N-benzyloxycarbonylamino acid is used as the N-benzyloxycarbonylamino and phenylalaninamide ether used as the amino acid ester in the production of aspartame. The method according to the invention provides for inhibiting the deactivation of metalloprotease and uluchsheniya to a method of stabilizing an aqueous solution or suspension of metalloprotease, as well as to aqueous solution of metalloprotease stabilized to better use, storage and transportation of such enzyme.

Proteases are used in various fields of food industry and industrial detergents (surfactants). It is known that where the protease used in aqueous solution or in suspension, it loses its activity due to spontaneous autolysis or denaturation (pH, temperature, and so on).

Recently developed method of synthesis of the dipeptide of the sugar substitute aspartame, using the reaction of the merger, kataliziruemaya (neutral) metalloprotease such as "thermolysin" (produced by Daiwa Chemical Co.) (K. Oyama, Bioindustry, vol. 1, N 9, pp. 5 to 11, 1985). Therefore, proteases, especially metalloprotease, should become important as synthetases for peptides in the field of production. However, these enzymes are not stable when the enzymes are used or stored in aqueous solution or in suspension for a long period of time.

Protease (metalloprotease) are usually sold on the commercial market in the form of a powder. Powdered enzyme readily forms aerosols and, therefore, there is a problem bezopasnosti can be allergic phenomena. Recently, therefore, the enzymes protease (metalloprotease) is used in the form of liquid product or suspension, keeping in mind the safety in their use. However, since the stability of the enzyme with this liquid enzyme or suspension should apply at low temperature during use, transportation and storage, this usually leads to increased costs (storage at low temperature, lower activity and so on).

Moreover, when the enzyme metalloprotease use in binding assays between N-benzyloxycarbonyl-L-aspartic acid (hereinafter referred to as "Z-Asp) and L - or DL-phenylalaninamide ether (hereinafter referred to as "PM"), which is the process of production-L-aspartyl-L-phenylalaninamide ether (hereinafter referred to as "Aspartame" or "APM"), the aspect of reducing the concentration of active enzyme also occurs due to adsorption of the enzyme on the crystals by-product N-benzyloxycarbonylation-alaninemia ether (hereinafter referred to as "L-APM.PM", such as "Z-APM.DPM"), in addition to the problem of stabilization of the enzyme.

If metalloprotease, such as thermolysin" produced by Bacillus thermoteolyticus Rokko, stored or used in idealize in a certain concentration. However, in the presence of calcium ions may be fouling due to the formation of calcium carbonate in various parts of pipelines and tanks, which will often have a significant negative impact on the practical use of the enzyme.

Goal that must be achieved by the present invention is to provide methods of use, storage and transportation of metalloprotease or similar enzyme protease in a stable form without the formation of scale and the provision of such stabilized compositions containing the enzyme.

Another goal that must be achieved by the present invention is to provide a method of increasing the ratio of regeneration, which is achieved by suppressing the adsorption of the enzyme on which is formed during the reaction of the crystal (Z-APM.(D)PM).

In accordance with the invention, the presence or addition of N-blocked amino acids (hereinafter referred to as "NPAA) is effective to prevent the deactivation of enzymes metalloprotease, especially for a noticeable increase in thermostability of enzymes and their stability during storage. In particular, there has been a noticeable effect of N-benzyl-oxycarbonyl-which can be significantly stabilized even in the absence of an excess of calcium ions through the presence or addition of NPAA. The purpose of this invention, therefore, is achieved if the N-blocked amino acid is present or it is added in an aqueous solution or suspension containing the protease.

In addition, deactivation of the enzyme metalloprotease caused by mixing, in the presence of a suspension can also be significantly ingibirovany using presence or adding NPAA, which is another characteristic feature.

Moreover, in accordance with the invention, the enzyme metalloprotease can be significantly stabilized and the coefficient of regeneration can be significantly increased by the presence of the NPAA, if such an enzyme is used in binding assays between Z-Asp and PM.

In particular, this invention can be used with advantage for storage of a solution or suspension of metalloprotease and for dealing with them in the presence of NPAA or to use this stabilizing effect in enzymatic binding of amino acids, in particular when receiving aspartame. According to this invention, the storage stability of the enzymes metalloprotease and the recovery rate of enzymes metalloprotease can be significantly improved by using a small amount of NPAA. Accordingly, the e or storage of the enzyme over a long period of time becomes possible, but it can also be achieved by increasing the rate of regeneration enzymes metalloprotease. This is especially cost effective when dealing with expensive enzymes.

Below the invention will be explained in detail.

Metalloprotease used in the method according to the invention are not limited specifically, but proteases, such thermolysin (neutral), are the most amenable to effective action. As metalloprotease mentioned, for example, those produced by Bacillus proteolyticus or Bacillus stearothermophilus, or those produced by their expression of the gene in other organisms. The solution or suspension intended for use in this invention may be as crude, containing many impurities and purified.

NPAA intended for use as a stabilizing additive in the method of the present invention may be in the form of a salt such as sodium salt, NPAA or in the form of crystals NPAA. If desirable, the solution or suspension containing various kinds of NPAA, or a mixture containing different types of NPAA, may also be used. In any case, the NPAA is not limited specifically. The effects caused NPAA does not deteriorate in any way is in NPAA can be all natural L-amino acids, as well as D-amino acids; acid group in AA may also be in the form of ether groups, such as benzyl ether.

As the N-blocking group NPAA suitable are, for example, benzyloxycarbonyl (Z), tertbutyloxycarbonyl (BOC), formyl (F), p-methoxybenzenesulfonyl (pMZ). In particular, benzyloxycarbonyl-blocked amino acids (ZAA) show, as detected exclusively excellent stabilization effect. The percentage retained in the active form of the enzyme in the presence of NZAA can reach 90% and above, while in its absence it is about 32% when stored under the conditions temperature 50oC for 5 hours. As NZAA considered N-benzyloxycarbonyl-L-aspartic acid (also referred to as Z-Asp), where the amino acid is L-aspartic acid; N-benzyloxycarbonyl-L-glutamic acid (hereinafter referred to as Z-Glu), where the amino acid is L-glutamic acid; N-benzyloxycarbonyl-L-phenylalanine (hereinafter referred to as Z-Phe), where the amino acid is phenylalanine; N-benzyloxycarbonylglycine (hereinafter referred to as Z-Gly), where the amino acid is glycine.

In a system where there is an excess of calcium ions, the percentage withheld in auchenia 5 hours. In this same system, however, the deactivation of the enzyme metalloprotease markedly inhibited by the addition of NPAA. In particular, when NZAA adds, almost never happens inactivation of the protease. The results show that the solution of the enzyme metalloprotease is very stable during storage or can be used at room temperature or at temperatures above room temperature for a long period of time due to the presence of NPAA. Thus, adding NPAA is highly advantageous economically in the preparation and handling of expensive enzymes. According to this invention metalloprotease is quite stable even in the absence of excess calcium ions. Therefore, this invention also allows you to avoid problems that can be caused by calcium ions, such as the formation of scale. Here may be noted that in Japanese application 6226989 considers stabilization of alkalinities used in detergents by adding a specific amount of a reversible inhibitor, such as hemostatic or Z-Phe. The authors of the present invention, however, see that adding NPAA other enzymes are proteases, such as popes who re can be used in enzymatic APM-synthase, does not result in any significant stabilization of the enzyme.

Moreover, when the enzyme metalloprotease used for binding assays between Z-Asp and PM, lowering the regeneration of the enzyme can significantly offset, and very strongly, with the presence of the NPAA in aqueous solution or in suspension of the enzyme.

Molar concentration NPAA intended to be present in aqueous solution or in suspension of the enzyme metalloprotease for storage and transport of the enzyme in accordance with this invention should be greater than 30 times the concentration of the specified enzyme, preferably 50 times.

On the other hand, when the enzyme metalloprotease used in binding assays between Z-Asp and PM, and Z-Asp is used as NPAA, the molar concentration of Z-Asp, which must be present in aqueous solution or in suspension of the enzyme after binding assays, should exceed more than 500 times the concentration of the specified enzyme and preferably more than 1000 times. Typically, the concentration of Z-Asp must be greater than 15 mmol/l, preferably greater than 30 mmol/L.

In binding assays between Z-Asp and PM the recovery rate of the enzyme sustaiable binding, 50 mmol/l (molar concentration in 1724 times more than the concentration of enzyme).

On the other hand, the recovery rate of the enzyme is only 50%, if the concentration of Z-Asp, which is present in aqueous solution or in suspension of the enzyme after binding assays is 10 mmol/l (molar concentration of 345 times greater than the concentration of enzyme).

As indicated above, metalloprotease can be significantly stabilized through the presence of the NPAA in aqueous solution or in suspension of this enzyme, and the recovery rate of this enzyme also increases significantly with the presence of the NPAA in aqueous solution or in suspension of the enzyme when the enzyme is used in binding assays between Z-Asp and PM.

Using metalloprotease in binding assays for the production of aspartame, therefore, is much more effective, if the molar concentration of the N-blocked amino acids, such as Z-Asp supports approximately 500 times greater than the concentration of the enzyme by adding N-blocked amino acids in the system, where the binding reaction during or in the final phase binding assays.

Zapravleny narrowing the scope of the invention.

EXAMPLE 1

5 g of powdered Termez (crude thermolysin, trade name of product of the company Daiwa Chemical Co.; the purification efficiency of about 20%) are suspended in one liter of water containing CaCl22H2O (6.8 mmol/l) (concentration Termez is 0.029 mmol/l), and to this add Z-Asp up until a certain concentration (2.62 mmol/l or 24.7 mmol/l, or, in other words, from exceeding 900 times up to 850 times, respectively, the concentration of enzyme) will not be achieved when the pH of the target solution, which was adjusted to 5.0 by adding an aqueous solution of 1 N. NaOH. To test the concentration of the enzyme is retained in the active form (i.e., the concentration of active enzyme), a solution of enzyme left in the flask with tap for sampling, having a volume of 2 liters at a controlled temperature of 70oC and under stirring with a speed of 240 rpm at regular intervals of time selected samples and the number of remaining enzyme in each sample was measured using HPLC (high performance liquid chromatography). The results obtained are presented in table. 1.

As noted in the table. 1, the enzyme is markedly stabilized by adding Z-Asp.

EXAMPLE 2

To 10 ml of water containing 0.029 mmol/l peredelennaya concentration (10 mmol/l) is not reached, and gradually add the water solution of 1 N. NaOH until pH 6.0. Thus prepared enzyme solution was placed into a water bath with controlled temperature 70oC. at regular intervals of time take samples and measure the number of remaining enzyme in each sample using HPLC. The results obtained are presented in table. 2.

As noted in the table. 2, the enzyme is markedly stabilized by adding NPAA, especially NZAA (Z-Asp, Z-Asp-OBzl, Z-Glu, Z-Phe -, and Z-Gly).

EXAMPLE 3

5 g of powdered Thermoase dissolved in one liter of water containing CaCl22H2O (concentration of CaCl2equal to 0.6 mmol/l) or water containing Z-Asp (concentration of Z-Asp is equal to 30 mmol/l), each with a concentration of Thermoase 0.029 mmol/l, and set a target pH of solutions 6.0 by adding an aqueous solution of 1 N. NaCl. The solution of enzyme left in the flask with tap for sampling, having a volume of 2 liters, temperature-controlled 40oC under stirring speed of 200 rpm

Samples are taken at regular intervals of time and the number of remaining enzyme in each sample was measured using HPLC. The results obtained are presented in table. 3.

The experiments in table. 3UB>; without such an excess of Ca2+will produce smaller scale.

As noted in the table. 3, the enzyme is markedly stabilized by adding Z-Asp, which is one of the NZAA, even in the absence of excess calcium ions.

EXAMPLE 4

A number NPAA added to 100 ml of an aqueous solution containing 0.029 mmol/l powdered Termez (not containing an excess of Ca), until, until you reached a certain concentration (10 mmol/l), in the same way as in example 2, bring the pH of the system to 6. All this is left in a water bath with controlled temperature 40oC.

From solutions of enzymes at regular intervals of time take samples and measure the number of remaining enzyme using HPLC. The results obtained are presented in table. 4.

As noted in the table. 4, the enzyme is markedly stabilized by adding NPAA, especially NZAA (Z-Asp, Z-Asp-OBzl, Z-Glu, Z-Phe -, and Z-Gly).

EXAMPLE 5

5 g of powdered Termez (degree of purification is equal to about 20%) are suspended in 750 g of water containing 0.1% CaCl22H2O in the flask with tap for sampling, having a volume of 2 liters, and added to a suspension of 250 g of celite (production Johns Manvill Corp. purity standard Super-Cel) to obrazky - equal to 0.029 mmol/L.

Z-Asp is added to the suspension until then, until it reaches a certain concentration (30 mmol/l), then the pH is brought to a value of 7.3 with 1 N. aqueous solution of NaOH. The suspension is stirred with a mixer equipped with a motor when the rotation speed of 240 rpm and take samples at regular intervals of time. The percentage of enzyme remaining in the active form, measured by HPLC. The results obtained are presented in table. 5.

As noted in the table. 5, an enzyme that is subject to change and is deactivated in suspension, significantly stabilized by adding Z-Asp.

EXAMPLE 6

5 g of powdered Protease-TD (product produced by Amano Pharmaceutical Co.; metalloprotease produced by Bacillus Stearothermophilus; the degree of purification is about 20%) are suspended in one liter of water containing 0.1% CaCl22H2O, when the concentration of the Protease-TD 0.029 mmol/l Z-Asp add until then, until it reaches a certain concentration (10 mmol/l), then bring the pH to 5.0 using 1 N. aqueous solution of NaOH. The resulting solution was allowed to stand in the flask with tap for sampling, having a volume of 2 liters, maintaining a controlled temperature of 70oC under stirring with shaida sample measured by HPLC. The results obtained are presented in table. 6.

As noted in the table. 6, the enzyme is markedly stabilized by adding Z-Asp.

EXAMPLE 7

943 g of an aqueous solution of Z-Asp (number of Z-Asp is 1.0 mol) and 1258 g of an aqueous solution of DL-PM (PM number is 2.0 mol) are mixed, heated to 40oC, and the pH of the aqueous solution to be installed using 25% NaOH solution, and this solution is used as the substrate solution.

Separately dissolved 1.28 g CaCl22H2O and 127 g of NaCl in 1242 g of purified water, and 45 g Termez dissolved in this aqueous solution, and an aqueous solution is used as an aqueous solution of the enzyme.

The substrate solution and the aqueous solution are mixed in a flask with tap for sampling a volume of 5 liters, which is placed in a water bath maintained during temperature 40oC, and the pH of this mixed solution was adjusted to a value of 6.0, and starts the reaction of binding of Z-Asp and PM.

Concentration Termez is 0.029 mmol/l as in the previous examples.

Stirring the mixed solution is carried out with a stirring speed of 130 rpm until then, until 3.5 hours after the start of the binding assays, and spend mixing with skorosti the initial number of Z-Asp, is 80.5% after 7 hours, and the concentration of Z-Asp, which remains after the reaction link is 55.9 mmol/l

Termbase, which regenerates after binding assays, is 41.4 g, and the recovery rate of approximately 92%.

Comparative example

943 g of an aqueous solution of Z-Asp (number of Z-Asp is 1.0 mol) and 1572 aqueous solution of DL-PM mixed, heated to 40oC, and set the pH of the aqueous solution with 25% NaOH solution, and this solution is used as the substrate solution.

Separately dissolved 1.28 g CaCl22H2O and 127 g of NaCl in 1242 g of purified water, and dissolved 45 g Termez in this aqueous solution, and this aqueous solution is used as an aqueous solution of the enzyme.

In this comparative example, a greater number of PM compared with example 7 results in a smaller number of Z-Asp, which remains after the tie. You can see that this results in a significantly reduced recovery rate of Termez.

The substrate solution and the aqueous solution are mixed in a flask with tap for sampling a volume of 5 liters, which is placed in a water bath maintained during temperature 40oC, and the pH of this mixed the aqueous solution is carried out with a stirring speed of 130 rpm until until 3.5 hours after the start of the binding assays, and carry out stirring with a stirring speed of 30 rpm for 3.5 hours, until only 7 hours.

The output of Z-APM. (D)PM, based on the original number of Z-Asp is 95.2% after 7 hours, and the concentration of Z-Asp, which remains after the binding reaction was 10.5 mol/L.

Termbase, which regenerates after binding assays, is 19.5 g, and the recovery rate of approximately 44%.

1. Method of stabilizing an aqueous solution or suspension of metalloprotease, characterized in that an aqueous solution or suspension of metalloprotease given N-protected amino acid in a molar concentration in excess of more than 30 times the molar concentration of metalloprotease.

2. The method according to p. 1, characterized in that the aqueous solution or suspension containing metalloprotease and N-protected amino acid, stored or transported without additional processing.

3. The method according to p. 1 or 2, characterized in that the N-protected amino acid is N-benzyloxycarbonylamino.

4. The method according to any of paragraphs.1 to 3, characterized in that metalloprotease is thermolysin-like, neutral met the first amino acid of more than 500 times the concentration of metalloprotease in binding assays N-protected amino acids and amino acid ester.

6. The method according to p. 5, characterized in that the specified molar concentration of N-protected amino acids in excess of more than 500 times the concentration of metalloprotease provide by adding N-protected amino acid to the reaction system during the specified binding assays or in its final stages.

7. The method according to p. 5 or 6, characterized in that the binding reaction is a reaction of binding of N-benzyloxycarbonylamino acid and phenylalaninamide ether.

8. The stabilized aqueous solution or suspension of metalloprotease containing N-protected amino acid in a molar concentration in excess of more than 30 times the molar concentration of metalloprotease.

 

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