Stabilised dry protein deamidase composition

Abstract

A technique of using a compositional innovation of a dry enzyme composition to improve the stability of a protein deamidase composition enables improvement of the stability of a dry protein deamidase composition by making both a protein deamidase and magnesium chloride coexist in a dry enzyme composition. The technique involves making adjustments so that the pH is at least 2 and less than 5 when the composition is dissolved in water at a concentration of 1 w/v %.

Claims

1. A dry stabilized composition comprising a dry protein deamidase and magnesium chloride, wherein the dry stabilized composition has a deamidation activity after storage for one month at 40° C. of at least 60% as compared to a deamidation activity of the dry stabilized composition stored for one month at −20° C., wherein an amino acid sequence of the protein deamidase has identity of 80% or more with the amino acid sequence represented by SEQ ID NO: 1 or 2, and wherein a content of the magnesium chloride in the dry stabilized composition is from 2.5% to 6.5% by weight.

2. The dry stabilized composition according to claim 1, wherein a content of the magnesium chloride is from 0.05 to 0.13 mg/u.

3. A stability-improving method for improving stability of a protein deamidase in a dry enzyme composition including the protein deamidase, wherein the protein deamidase coexists with the magnesium chloride in the dry enzyme composition, or a pH of the dry enzyme composition upon dissolution in water at 1 w/v % is adjusted to 2 or higher and lower than 5.

4. A dry stabilized composition comprising a dry protein deamidase having a pH of 2 or higher and 4 or lower upon dissolution in water at 1 w/v %, wherein the dry stabilized composition has a deamidation activity after storage for one month at 40° C. of at least 60% as compared to a deamidation activity of the dry stabilized composition stored for one month at −20° C., wherein an amino acid sequence of the protein deamidase has an identity of 80% or more with an amino acid sequence represented by SEQ ID NO: 1 or 2, and wherein a stabilizing agent other than an agent for adjusting the pH is not added.

Description

EXAMPLES

(1) Hereinafter, the present invention will be described further in detail by way of Examples. However, the present invention is not limited to the following Examples.

(2) [Preparation of Protein Deamidase]

(3) In the following Test examples, as the protein deamidase, a protein deamidase preparation “Protein-glutaminase “Amano” 500” (derived from Chryseobacterium genus) 500 u/g manufactured by Amano Enzyme Inc. was used. The “Protein-glutaminase “Amano” 500” is an enzyme preparation of which activity is adjusted to 500 u/g by including dextrin in addition to the protein deamidase. Note that the protein deamidase may also be acquired by the following method. Chryseobacterium sp. No. 9670 is cultured with shaking in an LB base medium (manufactured by Gibco) at 25° C. for 2 to 7 days in accordance with the method described in Patent document 2, and then a supernatant was obtained by centrifugation. After completing the culture, the culture liquid was subjected to centrifugal separation (12000 rpm, 4° C., 20 min.) to obtain a supernatant as a crude enzyme liquid. The supernatant was processed through ultra-filtration (UF) concentration (SEP-0013), salting out, Phenyl Sepharose, and Sephacryl S-100 to purify the enzyme.

(4) [Measuring Method of Protein Deamidase Activity in Composition Containing Protein Deamidase]

(5) In the following Test examples, the protein deamidase activity in the composition containing the protein deamidase was measured as follows. Note that, in the following description, a substrate Z-Gln-Gly represents N-benzyloxycarbonyl-L-glutaminylglycine.

(6) 1. To 1.0 ml of 0.2M phosphate buffer solution (pH 6.5) containing 34 mM Z-Gln-Gly, 0.1 ml of a sample solution appropriately diluted with 0.2M phosphate buffer solution (pH 6.5) containing 0.002% Triton-X was added, followed by incubation at 37° C. for 10 min. Then, the reaction was terminated by adding 1.0 ml of 0.4 M trichloroacetic acid.

(7) 2. The amount of free ammonia was quantified using an ammonia test kit (manufactured by Wako Pure Chemical Industries, Ltd.).

(8) Specifically, after mixing 0.2 ml of the reaction liquid with 0.8 ml of purified water, 1.0 ml of a chromogen reagent A (a solution containing 0.43 M phenol and 0.5 M sodium pentacyanonitrileferrate (III) dihydrate) was added to the mixture and mixed. Further, 0.5 ml of a chromogen reagent B (0.89 M potassium hydroxide aqueous solution) was added to the mixture and mixed. After 1.0 ml of a chromogen reagent C (an aqueous solution containing potassium carbonate and sodium hypochlorite) was added to the mixture and mixed, the mixture was incubated at 37° C. for 20 min. Subsequently, absorbance (A1) at a wavelength of 630 nm was measured.

(9) 3. As a blank. 1.0 ml of 0.4 M trichloroacetic acid was added to 0.1 ml of the sample solution and then 1.0 ml of 0.2 M phosphate buffer solution (pH 6.5) containing 30 mM Z-Gln-Gly was added to the mixture and mixed. The resulting mixture solution was also subjected to the color developing operation in the same manner and absorbance (A2) was measured.

(10) 4. The amount of enzyme required to produce 1 μmol ammonia per minute was defined as 1 u, and the protein deamidase activity (PG activity) was calculated by the following formula.
PG activity (u/g)=F=(A1 −A2)×(1/17.03)×(2.1/0.1)×(1/10)×N

(11) F: constant calculated by creating calibration curve of ammonia

(12) 17.03: molecular weight of ammonia

(13) 2.1: total amount of reaction liquid

(14) 0.1: adding amount of sample

(15) 10: reaction time (min.)

(16) N: dilution factor

(17) [Test Example 1: Verification of Stability-Improving Effect by Magnesium Chloride-1]

(18) (1) Preparation of Dry Protein Deamidase Composition

(19) The enzyme liquid was obtained by dissolving “Protein-glutaminase “Ammo” 500” (manufactured by Amano Enzyme Inc.) (500 u/g) in water and also dissolving therein magnesium chloride in accordance with formulation described in Table 1 (a weight ratio with respect to the total weight (expressed in terms of dry weight) of the protein deamidase preparation including the protein deamidase, dextrin, and magnesium chloride and a weight of the protein deamidase preparation per unit activity of the protein deamidase). Further, as Comparative example, the enzyme liquid without magnesium chloride (no addition) and the enzyme liquid in which magnesium chloride was replaced with cysteine or calcium chloride were prepared in the same manner in accordance with formulation described in Table 1 (the weight ratio with respect to the total weight (expressed in terms of dry weight) of the protein deamidase preparation including the protein deamidase and dextrin or the protein deamidase preparation including the protein deamidase, dextrin, and cysteine or calcium chloride, and the weight per unit activity of the protein deamidase). These enzyme liquids were adjusted to pH 6.0 (25° C.) using 1M hydrochloric acid and 1M sodium hydroxide.

(20) The enzyme liquid (after pH adjustment) was powdered using a spray dryer (EYELA SPRAY DRYER SD-1000) to obtain the dry pr in deamidase composition as a spray-dried product.

(21) (2) Evaluation of Stability

(22) The spray-dried products thus obtained were each put into a GX film bag (manufactured by TOPPAN PRINTING CO., LTD.), and the bags were sealed by heat sealing and then stored for 2 days at temperature conditions of −20° C. and 60° C. The protein deamidase activity after storage was measured and the remaining activity (%) was derived as a percentage of the protein deamidase activity at 60° C. with respect to the protein deamidase activity at −20° C.

(23) The result is shown in Table 1. The stability was improved in the case of including magnesium chloride (Example 1) as compared with the case of not including magnesium chloride (Comparative example 1), the case of including cysteine instead of magnesium chloride (Comparative example 2), and the case of including calcium chloride instead of magnesium chloride (Comparative example 3). This confirmed that magnesium chloride exhibited an excellent stabilizing effect on the dry protein deamidase composition.

(24) TABLE-US-00001 TABLE 1 Weight ratio with respect to total Stabilizing weight (in terms Weight per Remaining agent of dry weight) unit activity activity Comparative No addition — — 40% example 1 Comparative Cysteine 6.5% 0.13 mg/u 38% example 2 Comparative Calcium 6.5% 0.13 mg/u 62% example 3 chloride Example 1 Magnesium 6.5% 0.13 mg/u 72% chloride
[Test Example 2: Verification of Stability-Improving Effect by Magnesium Chloride 2]
(1) Preparation of Dry Protein Deamidise Composition

(25) The dry protein deamidase composition (Example 2) was prepared as a spray-dried product in the same a as in Test example 1 except that the weight ratio of magnesium chloride was changed to 2.5% by weight, and the composition was subjected to the following stability evaluation test. Further, the dry protein deamidase composition with no addition (Comparative example 1) and the dry protein deamidase composition having the weight ratio of 6.5% by weight (Example 1), obtained in Test example 1, were also subjected to the following stability evaluation test.

(26) (2) Evaluation of Stability

(27) The spray-dried products thus obtained were each put into a GX film bag (manufactured by TOPPAN PRINTING CO., LTD.), and the bags were sealed by heat sealing and then stored for 1 month at temperature conditions of −20° C. and 40° C. The protein deamidase activity after storage was measured and the remaining activity (%) was derived as a percentage of the protein deamidise activity at 40° C. with respect to the protein deamidase activity at −20° C.

(28) Table 2 shows the results of the stability evaluation of the dry protein deamidase composition with no addition (Comparative example 1) and the dry protein deamidase composition having the weight ratio of 2.5% by weight (Example 2) or 6.5% by weight (Example 1). As shown in Table 2, the stabilizing effect of the dry protein deamidase composition was confirmed in both Examples. This confirmed that magnesium chloride exhibited an excellent stabilizing effect on the dry protein deamidase composition

(29) TABLE-US-00002 TABLE 2 Weight ratio with respect to total Stabilizing weight (in terms Weight per Remaining agent of dry weight) unit activity activity Comparative No addition — — 50% example 1 Example 2 Magnesium 2.5% 0.05 mg/u 66% chloride Example 1 Magnesium 6.5% 0.13 mg/u 92% chloride
[Test Example 3: Verification of Powder Characteristics-Improving Effect by Using Both Magnesium Chloride and Calcium Chloride]ps (1) Preparation of Dry Protein Deamidase Composition

(30) The enzyme liquid was obtained by dissolving “Protein-glutaminase “Ammo” 500” (manufactured by Amano Enzyme Inc.) (500 u/g) in water and also dissolving therein magnesium chloride, or magnesium chloride and calcium chloride in accordance with formulation described in Table 3 (the weight ratio with respect to the total weight (expressed in terms of dry weight) of the protein deamidase preparation including the protein deamidase, dextrin, magnesium chloride, and with or without calcium chloride, and the weight of the protein deamidase preparation per unit activity of the protein deamidase). The enzyme liquids were adjusted to pH 6 (25° C.) using 1M hydrochloric acid and 1M sodium hydroxide.

(31) The enzyme liquid (after pH adjustment) was powdered using a spray dryer (EYELA SPRAY DRYER SD-1000) to obtain the dry protein deamidase composition as a spray-dried product.

(32) (2) Evaluation of Stability

(33) The spray-dried products thus obtained were each put into a GX film bag (manufactured by TOPPAN PRINTING CO., LTD.), and the bags were sealed by heat sealing and then stored for 1 month at temperature conditions of −20° C. and 40° C. The protein deamidase activity after storage was measured and the remaining activity (%) was derived as a percentage of the protein deamidase activity at 40° C. with respect to the protein deamidase activity at −20° C.

(34) (3) Evaluation of Powder Characteristics

(35) The spray-dried products thus obtained were each put into a GX film bag (manufactured by TOPPAN PRINTING CO., LTD.), and the bags were sealed by heat sealing and then stored for 1 month at a temperature condition of 40° C. After that, the appearance of the powders was visually observed to evaluate powder characteristics on the basis of the following criteria.

(36) ∘: neither caking nor deliquescence was observed

(37) Δ: mild caking or deliquescence was observed x: severe caking or deliquescence was observed

(38) The result is shown in Table 3. In the case of magnesium chloride alone (Example 1), the mild caking that was not observed in the case of no addition (Comparative example 1) was observed. However, the level of the caking was acceptable as the dry enzyme composition. In the case of using both magnesium chloride and calcium chloride (Examples 3 to 5), the stability was on a declining trend as compared with the case of magnesium chloride alone (Example 1). However, the stability was still maintained at an excellent level, and, furthermore, it was confirmed that the caking of the dry protein deamidase composition was reduced and the powder characteristics were improved.

(39) TABLE-US-00003 TABLE 3 Weight ratio with respect to total Stabilizing weight (in terms Weight per Remaining Powder agent of dry weight) unit activity activity characteristics Comparative No addition — — 50% ◯ example 1 Example 1 Magnesium chloride 6.5% 0.13 mg/u 92% Δ Example 3 Magnesium chloride 4.0% 0.08 mg/u 84% ◯ Calcium chloride 2.5% 0.05 mg/u Example 4 Magnesium chloride 2.5% 0.05 mg/u 81% ◯ Calcium chloride 4.0% 0.08 mg/u Example 5 Magnesium chloride 1.0% 0.02 mg/u 82% ◯ Calcium chloride 5.5% 0.11 mg/u
[Test Example 4: Verification of Effect of pH Upon Dissolution on Stability of Dry Composition]
(1) Preparation of Dry Protein Deamidase Composition

(40) The enzyme liquid was obtained by dissolving “Protein-glutaminase “Amano” 500” (manufactured by Amano Enzyme Inc.) (500 u/g) in water and also dissolving therein magnesium chloride and calcium chloride in accordance with formulation described in Table 4 (the weight ratio with respect to the total weight (expressed in terms of dry weight) of the protein deamidase preparation including the protein deamidase, dextrin, magnesium chloride, and calcium chloride and the weight of the protein deamidase preparation per unit activity of the protein deamidase). Note that, in an example described as “No addition” in Table 4, the enzyme liquid containing neither magnesium chloride nor calcium chloride was obtained. The pH (25° C.) of these enzyme liquids were adjusted to 2, 3, 4, 5, 6, 7, or 8 using 1M hydrochloric acid and/or 1M sodium hydroxide.

(41) The enzyme liquid (after pH adjustment) was powdered using a spray dryer (EYELA SPRAY DRYER SD-1000) to obtain the dry protein deamidase composition as a spray-dried product.

(42) Note that, when the obtained dry protein deamidase compositions (each obtained from the enzyme liquids at pH 2, 3, 4, 5, 6, 7, and 8) were redissolved in water at 1 w/v % again, the pH (25° C.) of the compositions after redissolution remained the same at 2, 3, 4, 5, 6, 7, and 8.

(43) (2) Evaluation of Stability

(44) The spray-dried products thus obtained were each put into a GX film bag (manufactured by TOPPAN PRINTING CO., LTD.), and the bags were sealed by heat sealing and then stored for 1 month at temperature conditions of −20° C. and 40° C. The protein deamidase activity after storage was measured and the remaining activity (%) was derived as a percentage of the protein deamidase activity at 40° C. with respect to the protein deamidase activity at −20° C.

(45) The result is shown in Table 4. As for the dry protein deamidase compositions containing neither magnesium chloride nor calcium chloride, it was confirmed that the dry protein deamidase compositions having a pH of from 2 to 4 at the time of the enzyme liquid (Examples 6 to 8) had the higher stability than the dry protein deamidase compositions having a pH of from 5 to 8 at the time of the enzyme liquid (Comparative examples 4 to 7). In particular, it was confirmed that the dry protein deamidase compositions having a pH of from 2 to 3 at the time of the enzyme liquid (Examples 6 and 7) had the significantly high stability. On the other hand, when the dry protein deamidase compositions containing magnesium chloride and calcium chloride (Examples 9 to 15) were compared with the dry protein deamidase compositions with no addition (Examples 6 to 8 and Comparative examples 4 to 7) at the same pH levels at the time of the enzyme liquid, the former stability was improved in all cases. The degree of improvement in the former stability in the comparison with the dry protein deamidase compositions with no addition was particularly significant at pH 4 to 8.

(46) TABLE-US-00004 TABLE 4 Weight ratio with pH at the respect to total time of Stabilizing weight (in terms Weight per enzyme Remaining agent of dry weight) unit activity liquid activity Example 6 No addition — — 2 99% Example 7 3 93% Example 8 4 72% Comparative 5 57% example 4 Comparative 6 50% example 5 Comparative 7 42% example 6 Comparative 8 38% example 7 Example 9 Magnesium 2.5% 0.05 mg/u 2 102%  Example 10 chloride + (Magnesium chloride) (Magnesium chloride) 3 94% Example 11 Calcium 4.0% 0.08 mg/u 4 95% Example 12 chloride (calcium chloride) (Calcium chloride) 5 84% Example 13 6 73% Example 14 7 73% Example 15 8 70%
[Test Example 5: Liquid Product]
(1) Preparation of Liquid Protein Deamidase Composition

(47) The enzyme liquid was obtained by dissolving “Protein-glutaminase “Amano” 500” (manufactured by Amano Enzyme Inc.) (500 u/g) in water at a Brix value of 25% and also dissolving therein magnesium chloride and calcium chloride in the amounts described in Table 5 (the amount with respect to the total weight (expressed in terms of dry weight) of the protein deamidase, dextrin, magnesium chloride, and calcium chloride and the weight of the dry preparation per unit activity of the protein deamidase Note that, in an example described as “No addition” in Table 6, the enzyme liquid containing neither magnesium chloride nor calcium chloride was obtained. The pH (25° C.) of the enzyme liquids were adjusted to 6 or 7 using 1M hydrochloric acid and/or 1M sodium hydroxide to obtain liquid protein deamidase compositions.

(48) (2) Evaluation of Stability

(49) The obtained liquid products were put into polypropylene tubes, covered with lids, and then stored for 1 month at 40° C. The protein deamidase activity after storage was measured and the remaining activity (%) was derived as a percentage of the protein deamidase activity after storage with respect to the protein deamidase activity before storage.

(50) The result is shown in Table 5. As for the liquid compositions, the stability of both the liquid compositions with no addition (Comparative examples 8 and 9) and the liquid compositions containing magnesium chloride and calcium carbonate (Comparative examples 10 and 11.) was reduced at low pH. On the other hand, as shown in the above Table 4, the stability of the powder compositions was, conversely, increased with a lower pH at the time of the enzyme liquid. That is, it was confirmed that the effect of the pH on the stability was completely different between the liquid compositions and the powder compositions.

(51) Further, when the liquid compositions containing magnesium chloride and calcium chloride (Comparative examples 10 and 11) were compared with the liquid compositions with no addition (Comparative examples 8 and 9) at the same pH levels, the former stability was reduced in both cases. On the other hand, as shown in the above Table 1 to Table 4, the stability of the powder composition containing magnesium chloride, or magnesium chloride and calcium chloride was, conversely, higher than that of the powder composition with no addition. That is, it was confirmed that the effect of magnesium chloride and calcium chloride on the stability was completely different between the liquid compositions and the dry compositions.

(52) TABLE-US-00005 TABLE 5 Weight ratio with pH at the respect to total Weight per unit time of Stabilizing weight (in terms activity (in terms enzyme Remaining agent of dry weight) of dry weight) liquid activity Comparative No addition — — 6 54% example 8 Comparative 7 99% example 9 Comparative Magnesium 2.5% 0.05 mg/u 6 48% example 10 chloride + (Magnesium chloride) (Magnesium chloride) Comparative Calcium 4.0% 0.08 mg/u 7 61% example 11 chloride (Calcium chloride) (Calcium chloride)

(53) The preferable embodiments of the present invention have been described above. The present invention is not limited to these embodiments and the embodiments may be modified in various ways without departing from the gist of the present invention.