Ketose 3-epimerase with improved thermostability
阅读说明：本技术 热稳定性提高的酮糖3-差向异构酶 (Ketose 3-epimerase with improved thermostability ) 是由 大谷耕平 石川一彦 中村雅子 香月和敬 于 2019-01-24 设计创作，主要内容包括：本发明人发现了：在源自球形节杆菌(<I>Arthrobacter globiformis</I>)的酮糖3-差向异构酶的氨基酸序列中,通过取代特定的氨基酸,可得到热稳定性提高的突变酶,且发现了可有效地生产D-阿洛酮糖。(The inventor finds that: from Arthrobacter globiformis (A), (B), (C Arthrobacter globiformis ) In the amino acid sequence of the ketose 3-epimerase of (a), a mutant enzyme having improved thermostability can be obtained by substituting a specific amino acid, and it has been found that D-psicose can be efficiently produced.)
1. Ketose 3-epimerase mutant derived from Arthrobacter globiformis represented by SEQ ID NO. 1Arthrobacter globiformis) Has at least 1 amino acid mutation within the amino acid sequence of the wild-type ketose 3-epimerase of (1) the ratio of the enzymatic activity at 70 ℃ T70 to the enzymatic activity at 50 ℃ T50, T70/T50, shows a higher value than that of the wild-type ketose 3-epimerase T70/T50; or (2) the residual enzyme activity A at 50 ℃ of the same enzyme treated at 60 ℃ for 1 hour in a 50mM phosphate buffer suspension containing 2mM magnesium sulfate at pH8.0, in the case where the activity of the untreated enzyme at 50 ℃ is 100, shows a higher value than that of the wild-type ketose 3-epimerase.
2. The mutant ketose 3-epimerase according to claim 1, wherein T70/T50 is 1.0 or more.
3. The ketose 3-epimerase mutant according to claim 1 or 2, wherein the residual enzyme activity A is 40 or more.
4. The mutant ketose 3-epimerase according to any one of claims 1 to 3, wherein at least 1 amino acid in the amino acid sequence of the wild-type ketose 3-epimerase is mutated to an amino acid substitution at 1 to 10 positions.
5. The ketose 3-epimerase mutant according to claim 1, wherein at least 1 amino acid mutation within the amino acid sequence of the wild-type ketose 3-epimerase exists at any one of the following positions counted from the amino terminus of the amino acid sequence of SEQ ID NO: 1:
6. 14, 22, 26, 34, 67, 68, 69, 70, 75, 91, 95, 100, 101, 110, 122, 137, 144, 160, 173, 177, 200, 214, 222, 237, 261, 270, 271, 275, 278, 281, and 289.
6. A ketose 3-epimerase mutant wherein the ketose 3-epimerase mutant is selected from mutants having amino acid substitutions at the following positions counted from the amino terminus of the amino acid sequence of SEQ ID NO: 1:
。
7. The ketose 3-epimerase mutant according to claim 6, wherein the ketose 3-epimerase mutant is further selected from the following mutants:
。
8. The ketose 3-epimerase mutant according to claim 6, wherein the ketose 3-epimerase mutant is further selected from the following mutants:
。
9. The ketose 3-epimerase mutant according to any one of claims 1 to 8, which is immobilized on an immobilization carrier.
10. A process for producing D-ketose, which comprises: reacting a ketose 3-epimerase mutant according to any one of claims 1 to 8 or an immobilized ketose 3-epimerase mutant according to claim 9 with D-fructose.
Technical Field
The present invention relates to a novel ketose 3-epimerase with improved thermostability and a method for producing the same.
Background
D-psicose (D- プシコース, D-psicose) is known as one of rare sugars present only in a trace amount in nature, and has a sweetness of about 70% of granulated sugar, and thus is used as a sweetener. It is also known that D-psicose has a caloric value close to zero and various physiological functions such as suppression of an increase in blood glucose level, and is attracting attention as a functional food material. Due to such circumstances, there is an increasing demand for an efficient and safe production method of D-psicose in the food industry.
On the other hand, since D-psicose is an epimer of D-fructose, a production method in which D-psicose 3-epimerase reacts with D-fructose was established. For example, from Arthrobacter globiformis (A), (B) and (C)Arthrobacter globiformis) Ketose 3-epimerase of M30 (patent document 1) or Agrobacterium tumefaciens-derived enzyme(s) ((S))Agrobacterium tumefaciens) D-psicose 3-epimerase (patent document 2). In addition, psicose (サイコース/プシコース) epimerase mutant derived from Agrobacterium tumefaciens (patent document 3) or burkholderia (B.) (S.) having improved thermostability by suppressing enzyme denaturation due to temperature for maintaining production efficiencyBurkholderia) The D-psicose 3-epimerase mutant of (patent document 4) was used for the production of D-psicose.
Disclosure of Invention
Problems to be solved by the invention
As described above, various methods have been proposed for producing D-psicose by allowing an enzyme such as D-psicose 3-epimerase to act thereon, but no method for producing an enzyme having improved thermostability as a D-psicose-producing enzyme derived from a strain having approved food safety such as Arthrobacter globiformis has been established. In addition, although the optimum temperature of ketose 3-epimerase derived from Arthrobacter globiformis is magnesium (Mg)2+) The reaction temperature is 60 to 80 ℃ under the existing conditions, but when the reaction is carried out for a long time in a state that the reaction temperature exceeds 50 ℃, the enzyme activity gradually decreases, and the production of D-psicose is reduced.
Means for solving the problems
The present inventors have found, in various studies on a method for producing D-psicose by allowing an enzyme to act, that: in the amino acid sequence of ketose 3-epimerase derived from Arthrobacter globiformis, a mutant enzyme with improved thermostability can be obtained by substituting a specific amino acid, and it was found that D-psicose can be efficiently produced. More specifically, it was found that a ketose 3-epimerase mutant having at least 1 amino acid mutation within the amino acid sequence of the wild-type ketose 3-epimerase derived from Arthrobacter globiformis represented by SEQ ID NO: 1 was obtained, and that (1) the ratio of the enzyme activity at 70 ℃ (T70) to the enzyme activity at 50 ℃ (T50) T70/T50 showed a higher value than that of the wild-type ketose 3-epimerase T70/T50; or (2) the residual enzyme activity A at 50 ℃ of the same enzyme treated at 60 ℃ for 1 hour in a suspension of 50mM phosphate buffer (pH8.0 containing 2mM magnesium sulfate) with the activity of the untreated enzyme at 50 ℃ being 100 shows a higher value than that of the wild-type ketose 3-epimerase.
That is, the present invention has been completed based on the above findings, and is constituted by the following (1) to (9).
<1 > a ketose 3-epimerase mutant which has at least 1 amino acid mutation within the amino acid sequence of a wild-type ketose 3-epimerase derived from Arthrobacter globiformis represented by SEQ ID NO: 1, and (1) the ratio T70/T50 of the enzyme activity at 70 ℃ (T70) to the enzyme activity at 50 ℃ (T50) shows a higher value than T70/T50 of the wild-type ketose 3-epimerase; or (2) the residual enzyme activity A at 50 ℃ of the same enzyme treated at 60 ℃ for 1 hour in a suspension of 50mM phosphate buffer (pH8.0 containing 2mM magnesium sulfate) with the activity of the untreated enzyme at 50 ℃ being 100 shows a higher value than that of the wild-type ketose 3-epimerase.
<2 > the above-mentioned <1 > of the ketose 3-epimerase mutant, wherein T70/T50 is 1.0 or more.
< 3> the above-mentioned <1 > or <2 > of the ketose 3-epimerase mutant wherein the residual enzyme activity A is 40 or more.
< 4 > the ketose 3-epimerase mutant according to any one of the above <1 > - < 3>, wherein at least 1 amino acid in the amino acid sequence of the wild-type ketose 3-epimerase is mutated to an amino acid substitution at 1 to 10 positions.
< 5 > the ketose 3-epimerase mutant <1 > above, wherein at least 1 amino acid mutation in the amino acid sequence of the wild-type ketose 3-epimerase is present at any one of the following positions counted from the amino terminus of the amino acid sequence of SEQ ID NO: 1:
6. 14, 22, 26, 34, 67, 68, 69, 70, 75, 91, 95, 100, 101, 110, 122, 137, 144, 160, 173, 177, 181, 200, 203, 214, 222, 226, 237, 261, 270, 271, 275, 278, 281, and 289.
< 6 > the ketose 3-epimerase mutant, wherein the ketose 3-epimerase mutant is selected from mutants having mutations at the following positions from the number of amino terminals of the amino acid sequence of SEQ ID NO: 1:
ketose 3-epimerase mutants
1 from the amino terminus of the amino acid sequence of SEQ ID NO
1
144
2
137 and 173
3
26
4
214
5
14
6
122
7
271 and 278
8
67. 68, 69 and 70
9
22
10
160 and 289
11
160. 271, 278 and 289
12
22. 67, 68, 69, 70, 160, and 289
13
22. 67, 68, 69, 70, 160, 177, and 289
14
222
15
278
16
200
17
177. 181 and 203
18
160. 226 and 289
19
237
20
270 and 281
21
275 and 278
22
177. 200, 237, 275 and 278
23
75
24
100 and 101
25
261
26
75. 177 and 237
27
6 and 177
28
110 and 177
29
67. 69 and 70
30
22 and 34
31
91
32
95
33
137
34
270
< 7 > the above < 6 > the ketose 3-epimerase mutant, wherein the ketose 3-epimerase mutant is further selected from the following mutants:
ketose 3-epimerBroussonetia mutant
Mutations within the amino acid sequence of SEQ ID NO 1
PM3
S144C
PM4
S137C and N173C
PM5
T26C
PM9
L214P
PM12
F14W
PM14
L122Y
PM15
L122W
PM17
H271C and D278C
PM18
S67P, D68A, A69D and T70G
PM19
S22T
PM23
R160A and H289VSARHKP
PM26
R160A. H271C, D278C and H289VSARHKP
PM28
S22T, S67P, D68A, A69D, T70G, R160A and H289VSARHKP
PM29
S22T, S67P, D68A, A69D, T70G, R160A, H177M and H289VSARHKP
PM31
D222H
PM32
D278F
PM33
A200L
PM36
H177M, Y181F and Y203Q
PM37
R160A, H289VSARHKP and K226D
PM38
V237I
PM39
A200K
PM40
A270C and T281C
PM41
F275C and D278C
PM43
H177M, A200K, V237I, F275C and D278C
PM44
E75P
PM45
T100P and D101V
PM46
L261M
PM48
R160A and H289VSAR
PM51
E75P, H177M and V237I
PM55
R160A and H289VSARHK
PM56
H6Y and H177M
PM57
M110W and H177M
PM58
S67P, A69D and T70G
PM59
S22T and V34I
PM60
D278L
PM61
V237L
PM62
V91I
PM63
A95R
PM64
S137K
PM65
A270K
< 8 > the above < 6 > the ketose 3-epimerase mutant, wherein the ketose 3-epimerase mutant is further selected from the following mutants:
ketose 3-epimerase mutants
Mutations within the amino acid sequence of SEQ ID NO 1
PM4
S137C and N173C
PM17
H271C and D278C
PM18
S67P, D68A, A69D and T70G
PM23
R160A and H289VSARHKP
PM26
R160A, H271C, D278C and H289VSARHKP
PM29
S22T, S67P, D68A, A69D, T70G, R160A, H177M and H289VSARHKP
PM32
D278F
PM33
A200L
PM38
V237I
PM39
A200K
PM41
F275C and D278C
PM43
H177M, A200K, V237I, F275C and D278C
PM44
E75P
PM45
T100P and D101V
PM48
R160A and H289VSAR
PM51
E75P, H177M and V237I
PM55
R160A and H289VSARHK
PM57
M110W and H177M
PM58
S67P, A69D and T70G
PM60
D278L
PM61
V237L
PM63
A95R
PM65
A270K
< 9 > the ketose 3-epimerase mutant according to any one of the above <1 > to < 8 >, which is immobilized on an immobilization carrier.
The preparation method of < 10> D-ketose is characterized in that: the ketose 3-epimerase mutant described in any one of the above <1 > -to < 8 > or the immobilized ketose 3-epimerase mutant described in < 9 > is allowed to act on D-fructose.
Effects of the invention
The present invention can provide: a ketose 3-epimerase mutant which has improved thermal stability compared with the wild type and is a production enzyme derived from D-psicose of Arthrobacter globiformis whose food safety has been approved.
Since the ketose 3-epimerase mutant derived from Arthrobacter globiformis with improved thermostability of the present invention can maintain the enzyme activity at a temperature higher than 50 ℃ for a longer time, D-psicose can be efficiently produced by allowing it to act on the substrate D-fructose as compared with the existing ketose 3-epimerase derived from Arthrobacter globiformis (wild type).
Drawings
FIG. 1 shows the residual activity of each immobilized enzyme on the 42 th day from the start of operation of the reaction column.
Detailed Description
(Arthrobacter globiformis)
The wild-type enzyme of the ketose 3-epimerase utilized in the present invention is derived from Arthrobacter globiformis. In the food industry, the safety of the bacteria has been confirmed. In the United states, the bacterium functions as a "Glucose isomerase from immobilized Arthrobacter globiformis" (Glucose isomerase)arthrobacter globiformis) "EAFUS (edible Added to Food in the United States Food additives) as recorded in FDA: food Additive Database (a Food Additive Database). Since the method of use is to directly immobilize the cells, it was demonstrated that the safety of the cells per se is very high.
In addition, in Europe, The gist of being used as "Citrus fermentation to remove limonin and reduce bitterness" (European food and feed bacteria Association) is described by EFFCA (The European food and feed bacteria Association) and IFD (International feed for The Roofing Trade, International roof Federation) "in The list of well-documented microorganisms for food use (Inventory of micro organismswith a filed family of use in food". This shows that the strain is used for fermentation as well as yeast and that the safety of the strain is very high.
In Japan, Arthrobacter (Arthrobacter) Belongs to the genus "alpha-amylase, isomaltose dextranaseisomaltodextrase)"etc. are included in the food additive list.
Thus, the bacterium has been used for a long time in Japan, Europe and America, and the genus Arthrobacter can be said to be a highly safe bacterium.
(ketose 3-epimerase)
(1) The wild-type ketose 3-epimerase used in the present invention is derived from Arthrobacter globiformis. Preferably derived from Arthrobacter globiformis M30 (accession number NITE BP-1111) and having the following amino acid sequence (SEQ ID NO: 1).
The wild-type ketose 3-epimerase of the present invention is preferably a ketose 3-epimerase having the substrate specificities of the following (a) and (B) and having the physicochemical properties of the following (a) to (f).
(SEQ ID NO: 1)
1 MKIGCHGLVW TGHFDAEGIR YSVQKTREAG FDLVEFPLMD PFSFDVQTAK；
51 SALAEHGLAA SASLGLSDAT DVSSEDPAVV KAGEELLNRA VDVLAELGAT；
101 DFCGVIYSAM KKYMEPATAA GLANSKAAVG RVADRASDLG INVSLEVVNR；
151 YETNVLNTGR QALAYLEELN RPNLGIHLDT YHMNIEESDM FSPILDTAEA；
201 LRYVHIGESH RGYLGTGSVD FDTFFKALGR IGYDGPVVFE SFSSSVVAPD；
251 LSRMLGIWRN LWADNEELGA HANAFIRDKL TAIKTIELH。
Substrate specificity of wild-type ketose 3-epimerase:
(A) epimerizing the 3-position of D-or L-ketose to produce the corresponding D-or L-ketose.
(B) Among D-or L-ketoses, the substrate specificity for D-fructose and D-psicose is highest.
Physicochemical properties of wild-type ketose 3-epimerase:
(a) molecular weight
The subunit has a molecular weight of about 32kDa as determined by SDS-PAGE, a molecular weight of 120kDa as determined by gel filtration, and a homotetramer (homotetramer) structure of 32kDa as determined by subunit.
(b) Optimum pH
Reaction at 30 ℃ for 30 minutes with 20mM magnesium (Mg)2+) Under the existing condition, the content is 6-11.
(c) Optimum temperature
Reaction at pH7.5 for 30 min with 20mM magnesium (Mg)2+) The temperature of the catalyst is 60-80 ℃ under the existing condition.
(d) Stability of pH
Is stable at least in a range of pH 5-11 at 4 ℃ for 24 hours.
(e) Thermal stability
4mM magnesium ion (Mg) at pH7.5 for 1 hour2+) Stable in the presence of a solvent at temperatures below about 50 ℃. In the presence of magnesium ions (Mg)2+) Stable in the absence of any other conditions below about 40 ℃.
(f) By activation of metal ions
By divalent manganese ions (Mn)2+) Divalent cobalt ion (Co)2+) Calcium (Ca)2+) And magnesium ion (Mg)2+) And (4) activating.
(ketose 3-epimerase mutant)
The ketose 3-epimerase mutant of the present invention has at least 1 amino acid mutation within the amino acid sequence of wild-type ketose 3-epimerase derived from Arthrobacter globiformis represented by SEQ ID NO: 1, and (1) the ratio T70/T50 of the enzyme activity at 70 ℃ (T70) to the enzyme activity at 50 ℃ (T50) shows a higher value than T70/T50 of the wild-type ketose 3-epimerase; or (2) the residual enzyme activity A at 50 ℃ of the same enzyme treated at 60 ℃ for 1 hour in a suspension of 50mM phosphate buffer (pH8.0 containing 2mM magnesium sulfate) with the activity of the untreated enzyme at 50 ℃ being 100 shows a higher value than that of the wild-type ketose 3-epimerase.
The mutant ketose 3-epimerase of the present invention is obtained by substituting a part of the amino acid sequence of a wild-type ketose 3-epimerase with a specific amino acid sequence.
The enzyme mutants were obtained by taking the amino acids representing each position shown in 1, and each mutant was tested for whether the thermostability was actually improved.
As a result, a mutant having at least 1 amino acid mutation within the amino acid sequence of the wild-type ketose 3-epimerase derived from Arthrobacter globiformis represented by SEQ ID NO: 1 and showing (1) a higher value of T70/T50 than that of the wild-type ketose 3-epimerase in the ratio of the enzyme activity at 70 ℃ (T70) to the enzyme activity at 50 ℃ (T50) T70/T50; or (2) the residual enzyme activity A at 50 ℃ of the same enzyme treated at 60 ℃ for 1 hour in a suspension of 50mM phosphate buffer (pH8.0 containing 2mM magnesium sulfate) with the activity of the untreated enzyme at 50 ℃ being 100 shows a higher value than that of the wild-type ketose 3-epimerase.
(1) The ratio T70/T50 of the enzyme activity at 70 ℃ (T70) to the enzyme activity at 50 ℃ (T50) can be determined as follows: for example, with D-psicose (containing 2mM of Mg)2SO450mM phosphate buffer solution (pH8.0) as a substrate, the ketose 3-epimerase was reacted at 50 ℃ or 70 ℃ for 10 minutes, and D-fructose as a reaction product was measured to determine the epimerase activity. More specifically, after the reaction was stopped, the reaction mixture was subjected to HPLC, and the enzyme activity was calculated from the peak area ratio of D-psicose as a substrate to D-fructose as a reaction product at 50 ℃ and 70 ℃.
The T70/T50 of the mutant ketose 3-epimerase calculated in this manner is preferably 0.70 or more, more preferably 0.80 or more, still more preferably 1.0 or more, and still more preferably 1.5 or more.
(2) The residual enzyme activity A at 50 ℃ of the same enzyme treated at 60 ℃ for 1 hour in a suspension of 50mM phosphate buffer (pH8.0) containing 2mM magnesium sulfate, with the activity of the untreated enzyme at 50 ℃ being 100, can be determined as follows: for example, the residual enzyme activity A can be determined by suspending the enzyme in a 50mM phosphate buffer (pH8.0, containing 2mM magnesium sulfate), treating the suspension at 60 ℃ for 1 hour, reacting the treated suspension at 50 ℃ for 10 minutes using D-psicose as a substrate, and measuring the epimerase activity. More specifically, after the reaction was stopped, the reaction mixture was subjected to HPLC, and the enzyme activities of the untreated enzyme and the enzyme treated at 60 ℃ for 1 hour were calculated from the peak area ratio of D-psicose as the substrate to D-fructose as the reaction product.
When the enzyme activity of the wild-type ketose 3-epimerase of SEQ ID NO. 1 is 100, the residual enzyme activity A of the mutant ketose 3-epimerase is preferably 40 or more, more preferably 50 or more, still more preferably 60 or more, and still more preferably 70 or more.
At least 1 amino acid mutation in the amino acid sequence of the wild-type ketose 3-epimerase derived from Arthrobacter globiformis represented by SEQ ID NO. 1 may be at least 1 mutation as long as the above-mentioned activity is exhibited, and preferably 1 to 10 amino acid substitutions, and more preferably 1 to 8 amino acid substitutions.
Furthermore, at least 1 amino acid mutation within the amino acid sequence of the wild-type ketose 3-epimerase is preferably present at any one of the following positions counted from the amino terminus of the amino acid sequence of SEQ ID NO: 1.
Position: 6. 14, 22, 26, 34, 67, 68, 69, 70, 75, 91, 95, 100, 101, 110, 122, 137, 144, 160, 173, 177, 181, 200, 203, 214, 222, 226, 237, 261, 270, 271, 275, 278, 281, and 289.
Furthermore, at least 1 amino acid mutation within the amino acid sequence of the wild-type ketose 3-epimerase is more preferably present at any one of the following positions counted from the amino terminus of the amino acid sequence of SEQ ID NO: 1.
Position: 6. 14, 22, 26, 34, 67, 68, 69, 70, 75, 91, 95, 100, 101, 110, 122, 137, 144, 160, 173, 177, 200, 214, 222, 237, 261, 270, 271, 275, 278, 281, and 289.
The amino acid to be inserted by substitution at each position may be any amino acid as long as it satisfies T70/T50 or the residual enzyme activity A described above. In general, 1 amino acid is preferably substituted with another amino acid, but 1 to 10 consecutive amino acids can be substituted at the carboxyl terminal.
The ketose 3-epimerase mutant is more preferably selected from mutants having amino acid substitutions at the positions described below.
Ketose 3-epimerase mutants
1 from the amino terminus of the amino acid sequence of SEQ ID NO
1
144
2
137 and 173
3
26
4
214
5
14
6
122
7
271 and 278
8
67. 68, 69 and 70
9
22
10
160 and 289
11
160. 271, 278 and 289
12
22. 67, 68, 69, 70, 160, and 289
13
22. 67, 68, 69, 70, 160, 177, and 289
14
222
15
278
16
200
17
177. 181 and 203
18
160. 226 and 289
19
237
20
270 and 281
21
275 and 278
22
177. 200, 237, 275 and 278
23
75
24
100 and 101
25
261
26
75. 177 and 237
27
6 and 177
28
110 and 177
29
67. 69 and 70
30
22 and 34
31
91
32
95
33
137
34
270
The ketose 3-epimerase mutant is further preferably selected from mutants having amino acid substitutions at the following positions.
Ketose 3-epimerase mutants
1 from the amino terminus of the amino acid sequence of SEQ ID NO
1
144
2
137 and 173
5
14
7
271 and 278
8
67. 68, 69 and 70
10
160 and 289
11
160. 271, 278 and 289
12
22. 67, 68, 69, 70, 160, and 289
13
22. 67, 68, 69, 70, 160, 177, and 289
15
278
16
200
19
237
21
275 and 278
22
177. 200, 237, 275 and 278
23
75
24
100 and 101
26
75. 177 and 237
27
6 and 177
28
110 and 177
29
67. 69 and 70
30
22 and 34
31
91
32
95
33
137
34
270
Further, it is preferable that the mutant showing improved thermostability compared with the wild type has the amino acid substitution shown below. In the present specification, for example, R160A represents a substitution of arginine (R) at position 160 from the amino terminus of SEQ ID NO: 1 with alanine (A). H289VSARHKP indicates that histidine (H) at position 289 from the amino-terminus of SEQ ID NO: 1 is substituted by the sequence of VSARHKP.
Ketose 3-epimerase mutants
Mutations within the amino acid sequence of SEQ ID NO 1
PM3
S144C
PM4
S137C and N173C
PM5
T26C
PM9
L214P
PM12
F14W
PM14
L122Y
PM15
L122W
PM17
H271C and D278C
PM18
S67P, D68A, A69D and T70G
PM19
S22T
PM23
R160A and H289VSARHKP
PM26
R160A, H271C, D278C and H289VSARHKP
PM28
S22T, S67P, D68A, A69D, T70G, R160A and H289VSARHKP
PM29
S22T, S67P, D68A, A69D, T70G, R160A, H177M and H289VSARHKP
PM31
D222H
PM32
D278F
PM33
A200L
PM36
H177M, Y181F and Y203Q
PM37
R160A, H289VSARHKP and K226D
PM38
V237I
PM39
A200K
PM40
A270C and T281C
PM41
F275C and D278C
PM43
H177M, A200K, V237I, F275C and D278C
PM44
E75P
PM45
T100P and D101V
PM46
L261M
PM48
R160A and H289VSAR
PM51
E75P, H177M and V237I
PM55
R160A and H289VSARHK
PM56
H6Y and H177M
PM57
M110W and H177M
PM58
S67P, A69D and T70G
PM59
S22T and V34I
PM60
D278L
PM61
V237L
PM62
V91I
PM63
A95R
PM64
S137K
PM65
A270K
Among the above mutants, examples of more preferable ketose 3-epimerase mutants include: PM3, PM4, PM5, PM9, PM12, PM14, PM17, PM18, PM19, PM23, PM26, PM28, PM29, PM31, PM32, PM33, PM38, PM39, PM41, PM43, PM44, PM45, PM46, PM48, PM51, PM55, PM56, PM57, PM58, PM59, PM60, PM61, PM62, PM63, PM64, and PM 65.
Among the above mutants, a more preferable ketose 3-epimerase mutant includes: PM4, PM17, PM18, PM23, PM26, PM29, PM32, PM33, PM38, PM39, PM41, PM43, PM44, PM45, PM48, PM51, PM55, PM57, PM58, PM60, PM61, PM63, and PM 65.
Among the above mutants, particularly preferred are: PM17, PM23, PM26, PM29, PM32, PM38, PM39, PM41, PM43, PM51, PM57, PM60, PM61, and PM 65.
The ketose 3-epimerase mutant of the present invention can be substituted at any one position of at least 1 amino acid (more specifically, at least 1 amino acid in the amino acid sequence of SEQ ID NO: 1) in the amino acid sequence of the wild-type ketose 3-epimerase by appropriately using a known method. For example, a mutant in which an amino acid substitution at a target position is performed by a known genetic engineering method can be obtained.
In addition, the ketose 3-epimerase mutant of the present invention can be prepared as follows: for example, the objective transformant can be prepared by using a nucleic acid sequence encoding the amino acid sequence of each mutant or by introducing a plasmid vector into which a DNA fragment into which a ketose 3-epimerase mutation has been introduced into Escherichia coli (host) or the like. The type of the recombinant vector to be used is not particularly limited, and may be one commonly used in the art. In addition, recombinant producing ketose 3-epimerase may use Escherichia coli, Bacillus (Bacillus) ((R))BacillusBacillus), yeast or agrobacterium.
The ketose 3-epimerase mutant of the present invention can be used as a liquid enzyme, or as an immobilized enzyme by known immobilization means such as carrier binding method, crosslinking method, gel encapsulation method, or the like.
In the case of using the carrier binding method, a known immobilization carrier such as ion exchange resin, synthetic adsorbent, activated carbon, porous glass or silica gel may be used. In the case of using an ion exchange resin as the immobilization carrier, for example, a phenol-based gel type weakly basic ion exchange resin or a styrene type macroporous type weakly basic ion exchange resin can be used. In the present invention, commercially available products of these ion exchange resins can be used, and as commercially available products of the phenolic gel type weakly basic ion exchange resin, there can be mentioned: duolite A561, Duolite A568, Duolite PWA7 (supra, manufactured by Dow DuPont corporation), and the like. Further, commercially available products of styrene-based macroporous weakly basic ion exchange resins include Amberlite FPA95, Amberlite IRA904, Amberlite XE583 (manufactured by Dow DuPont Co., Ltd.), Purolite A111S, Purolite A103S (manufactured by Purolite Co., Ltd.), DIAION WA20 and DIAION WA30 (manufactured by Mitsubishi chemical Co., Ltd.).
D-psicose can be efficiently produced using the ketose 3-epimerase mutant of the present invention or the immobilized ketose 3-epimerase mutant of the present invention.
More specifically, for example, in the case of using an immobilized ketose 3-epimerase mutant of the present invention, an aqueous solution of D-fructose may be continuously passed through a reaction tower packed with the mutant to obtain a mixed aqueous solution of D-fructose and D-psicose in which a part of D-fructose is converted into D-psicose, and then decolorization/desalting/chromatographic separation (chromatographic fractionation) may be performed by a known method to produce a high-purity aqueous solution of D-psicose. Thereafter, crystalline D-psicose may be further produced using a known method.
When the D-fructose aqueous solution is passed through the reaction tower, the D-fructose aqueous solution is desirably heated to 50 to 80 ℃ and the concentration thereof is desirably 30 to 70wt%, and is desirably adjusted to a pH of 6 to 9 with a pH adjusting agent such as sodium hydroxide. Further, the flow rate of the aqueous D-fructose solution passing through the reaction column is desirably adjusted so that the conversion rate of D-fructose into D-psicose becomes 20% or more close to the reaction equilibrium of the enzyme. In addition, various ions, for example, 10 to 100ppm of magnesium ion or 50 to 500ppm of sulfite ion, may be included as an enzyme activator and stabilizer.
The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.
(examples)
< selection of substitution site in amino acid sequence of ketose 3-epimerase >
First, in order to select an amino acid sequence related to the activity and thermostability of ketose 3-epimerase, a primary structure (amino acid sequence) having high homology with ketose 3-epimerase derived from the strain was searched by Blast. As a result, the primary structural information of 12 kinds of enzymes having high homology with the ketose 3-epimerase was obtained. Since the three-dimensional structure of the enzyme derived from this strain has not been determined, the information thereof cannot be utilized. Then, an enzyme species whose three-dimensional structure (three-dimensional structure) is known is selected from the 12 types described above, and a MODEL of an inferred three-dimensional structure of the target enzyme is constructed using SWISS-MODEL and energy minimization calculation. By comparing the model structure with the structure of a similar enzyme, a site contributing to structural stability in the enzyme was searched/extracted, and an amino acid change which is considered to be able to thermally stabilize the steric structure of the enzyme in physico-chemical terms was made.
Each of the positions shown in Table 1 was selected as a specific amino acid sequence position expected to improve thermostability, and substituted with a specific amino acid sequence.
The conversion to a specific amino acid sequence is, for example, an amino acid sequence predicted to be effective in improving hydrophobic interaction, stabilizing a loop structure of a peptide main chain, introducing a covalent bond between amino acids by a disulfide bond, improving space density, stabilizing an association at the C-terminal end of an elongated protein, and the like (table 1).
< TABLE 1 >
Ketose 3-epimerase mutants
Mutations within the amino acid sequence of SEQ ID NO 1
PM3
S144C
PM4
S137C and N173C
PM5
T26C
PM9
L214P
PM12
F14W
PM14
L122Y
PM15
L122W
PM17
H271C and D278C
PM18
S67P, D68A, A69D and T70G
PM19
S22T
PM23
R160A and H289VSARHKP
PM26
R160A, H271C, D278C and H289VSARHKP
PM28
S22T, S67P, D68A, A69D, T70G, R160A and H289VSARHKP
PM29
S22T, S67P, D68A, A69D, T70G, R160A, H177M and H289VSARHKP
PM31
D222H
PM32
D278F
PM33
A200L
PM36
H177M, Y181F and Y203Q
PM37
R160A, H289VSARHKP and K226D
PM38
V237I
PM39
A200K
PM40
A270C and T281C
PM41
F275C and D278C
PM43
H177M, A200K, V237I, F275C and D278C
PM44
E75P
PM45
T100P and D101V
PM46
L261M
PM48
R160A and H289VSAR
PM51
E75P, H177M and V237I
PM55
R160A and H289VSARHK
PM56
H6Y and H177M
PM57
M110W and H177M
PM58
S67P、A69D、T70G
PM59
S22T and V34I
PM60
D278L
PM61
V237L
PM62
V91I
PM63
A95R
PM64
S137K
PM65
A270K
< preparation of expression vector and transformation of Escherichia coli >
First, mutation introduction was performed by a PCR method or a gene synthesis service (Thermo Fisher Scientific) using a DNA of ketose 3-epimerase containing an amino acid sequence in which 1 or 2 or more substitutions described in table 1 were performed. The DNA fragment into which the mutation had been introduced was inserted into pQE60 (Qiagen) vector for expression in E.coli, and E.coli (host) was transformed. The site of mutation introduction was confirmed by sequence analysis.
< preparation of crude enzyme solution >
First, a transformant (E.coli) expressing a ketose 3-epimerase mutant enzyme was inoculated (cultivated) in a medium containing 100μAmpicillin and 50 g/mlμIn g/ml LB medium (culture broth) containing kanamycin, the cells were pre-cultured at 37 ℃ for 16 hours. The preculture solution was added to a 10-100 fold amount of main culture solution containing 0.1mM IPTG, and cultured at 20 ℃ for 24 hours to induce the expression of the target enzyme. Next, the whole culture broth was centrifuged at 5,500 Xg at 4 ℃ for 20 minutes using a centrifuge. After centrifugation, the supernatant was removed, and the cells were recovered. The collected cells were suspended in an appropriate amount of 50mM phosphate buffer (pH8.0, containing 2mM magnesium sulfate), and subjected to 6 cycles of 15-second ultrasonic pulverization at 45-second intervals. Thereafter, the mixture was centrifuged at 5,500 Xg for 20 minutes at 4 ℃ to obtain a supernatant, thereby obtaining a crude enzyme solution. A crude enzyme solution of each mutant enzyme shown in Table 1 was obtained by the above-mentioned method and used for the evaluation of the enzyme activity.
< evaluation of Activity of crude enzyme solution >
(i) Comparison of the ratio of the enzyme Activity at 70 ℃ (T70) to the enzyme Activity at 50 ℃ (T50) T70/T50
The enzymatic reaction of ketose 3-epimerase is an equilibrium reaction in which approximately D-psicose: d-fructose = 30: 70, the amount of D-fructose produced can be easily measured when D-psicose is used as a substrate. Therefore, evaluation of the ketose 3-epimerase activity was carried out by measuring the amount of D-fructose produced in the case of carrying out an enzyme reaction using D-psicose as a substrate.
Specifically, the enzyme reactions were carried out under the reaction conditions shown in table 2 using the respective crude enzyme solutions obtained by the above-described methods. After the reaction was stopped, the reaction solution was purified by desalting with an ion exchange resin, filtered, and subjected to HPLC (HPLC system (Tosoh corporation), MCIGEL CK08EC (mitsubishi chemical corporation)). The peak area of D-fructose measured by HPLC was calculated, and the respective enzyme activities were calculated from the peak area ratios of 50 ℃ and 70 ℃.1 unit of enzyme activity is defined as: under each condition, D-psicose is subjected to epimerization to generate 1 in 1 minuteμThe amount of D-fructose in mol. The results are shown in Table 4.
< TABLE 2 >
As a result of the measurement, it was found from Table 4 that T70/T50 was higher in activity value (0.7. + -. 0.1) than the wild-type mutant enzyme. These mutant enzymes suggest an improvement in thermostability.
As described above, the amino acid sequence substitutions were made by selecting each position shown in Table 1 as a specific amino acid sequence position expected to improve thermostability, but the thermostability of the mutants produced was not changed much or sometimes decreased, and the results contrary to the expectation were obtained.
(ii) Comparison of the residual enzyme Activity A at 50 ℃ of the same enzyme treated at 60 ℃ for 1 hour in a suspension of 50mM phosphate buffer (pH8.0 containing 2mM magnesium sulfate) with the untreated enzyme having an activity at 50 ℃ of 100
Next, the residual activity of the enzyme after heat treatment was measured by leaving it at 60 ℃ for 1 hour. The crude enzyme solution obtained as described above was used to react the enzyme after heat treatment with D-psicose as a substrate under the conditions shown in Table 3 below. After the enzyme reaction was stopped, the reaction solution cooled in ice water for 10 minutes was purified by desalting with an ion exchange resin and subjected to HPLC. The peak area of D-psicose measured by HPLC was calculated, and the enzyme activities at 50 ℃ of the enzyme after heat treatment and the enzyme without treatment were calculated. The residual enzyme activity after the heat treatment was calculated as the relative activity when the untreated enzyme activity was taken as 100. The results are shown in Table 4.
< TABLE 3>
As a result, it was found from Table 4 that the residual enzyme activity A was higher than that of the wild-type enzyme. Suggesting that these enzymes have high thermostability.
< TABLE 4 >
T70/T50
Residual enzyme activity
Wild type
0.7±0.1
35.1
PM3
0.77
37.6
PM4
0.82
49.8
PM5
0.77
33.5
PM9
0.71
29.6
PM12
0.77
37.5
PM14
0.73
31.2
PM15
0.57
29.6
PM17
1.25
92.9
PM18
0.91
48.4
PM19
0.78
22.9
PM23
1.52
69.9
PM26
1.59
93.0
PM28
0.93
22.1
PM29
1.50
50.0
PM31
0.73
30.9
PM32
1.60
100.0
PM33
0.92
60.0
PM36
0.47
19.1
PM37
0.41
25.4
PM38
1.66
90.9
PM39
1.01
63.2
PM40
0.38
22.8
PM41
1.08
83.9
PM43
1.41
86.0
PM44
0.84
52.9
PM45
0.77
53.0
PM46
0.76
27.6
PM48
0.52
46.1
PM51
1.32
83.6
PM55
1.24
62.9
PM56
0.81
33.5
PM57
2.54
60.9
PM58
0.90
43.7
PM59
0.99
20.8
PM60
2.53
100.0
PM61
1.67
90.8
PM62
0.85
30.9
PM63
0.84
40.0
PM64
0.92
38.4
PM65
1.46
86.4
Among the above mutants, in particular, T70/T50 or residual enzyme activity a of PM3, PM4, PM5, PM9, PM12, PM14, PM17, PM18, PM19, PM23, PM26, PM28, PM29, PM31, PM32, PM33, PM38, PM39, PM41, PM43, PM44, PM45, PM46, PM48, PM51, PM55, PM56, PM57, PM58, PM59, PM60, PM61, PM62, PM63, PM64 and PM65 is excellent as compared with the wild type.
Among these, in particular, compared with the wild type, PM4, PM17, PM18, PM23, PM26, PM29, PM32, PM33, PM38, PM39, PM41, PM43, PM44, PM45, PM48, PM51, PM55, PM57, PM58, PM60, PM61, PM63, and PM65 have T70/T50 of 1.0 or more or have a residual enzyme activity a of 40 or more, and are excellent.
In addition, PM17, PM23, PM26, PM29, PM32, PM38, PM39, PM41, PM43, PM51, PM57, PM60, PM61, and PM65 exhibit 2-fold or more of activity, and are particularly excellent, relative to the wild-type value of T70/T50 or residual enzyme activity a.
< preparation of immobilized enzyme >
1. A crude enzyme solution of PM 38/41/43/51/wild type (hereinafter referred to as WT) was obtained in the same manner as described in "preparation of crude enzyme solution".
The enzyme activity of each crude enzyme solution was measured according to the methods and conditions described in < evaluation of activity of crude enzyme solution > and < Table 2 > above.
2. The immobilized carrier (ion exchange resin Amberlite FPA95 or Duolite A568 (both manufactured by Dow DuPont Co.) washed and equilibrated with 50mM phosphate buffer solution (pH8.0) containing 2mM magnesium sulfate and the crude enzyme solution were mixed in a vial in a ratio of 630U of the enzyme activity to 1mL of the crude enzyme solution of the immobilized carrier, and mixed by inversion for 24 hours or more.
3. The immobilized enzyme prepared by the above method was washed with 50mM phosphate buffer (pH8.0) containing 2mM magnesium sulfate, and then stored in a refrigerator at 4 ℃ until use.
< continuous reaction >
The following experimental procedures were carried out according to JIS K7002: 1988 (Industrial glucose isomerase).
1. In a reaction column heated to 55 ℃: (φ18 mm. times.L 400mm) was packed with 29mL of the immobilized enzyme, and the reaction raw material (containing 50wt% of fructose and 240ppm of MgSO 2) was supplied to the reaction column4And 150ppm of Na2SO3)。
2. The effluent from the reaction column was stored every 1 day, and subjected to HPLC analysis (in the same manner as < evaluation of activity of crude enzyme solution > to calculate the area A of psicose and fructose in the HPLC chromatogramPsiAnd AFru。
3. The flow rate of the reaction raw material is appropriately adjusted so that the isomerization rate x represented by the formula (1) becomes 20% or more.
x=APsi/(APsi＋AFru) (1)
4. The enzymatic activity a of the immobilized enzyme was calculated by the formula (2).
a=F/VCSxeln(xe/(xe﹣x)) (2)
Wherein F is the flow rate of the substrate, CSAs substrate concentration, xeThe apparent equilibrium reactivity (0.29) was obtained, and V was the bed volume of the immobilized enzyme.
Enzyme Activity a at day 42 from the beginning of the operation of the reaction column42And the enzyme activity a on day 1 from the start of operation1The ratio of the amounts (hereinafter, referred to as the residual activity on day 42) is shown in FIG. 1. The residual activity of WT on day 42 was about 60%, while the residual activity of PM38/41/43/51 on day 42 was about 80%, and the decrease in enzyme activity was small after 42 days of operation. This indicates a slow decline in activity.
In the actual production of psicose, the reaction tower is often operated at a temperature of 50 ℃ or higher for a long time (several months to several years or so). In this case, the enzyme activity gradually decreased, and the yield of psicose decreased. Since the mutant used in this example had a slow decrease in activity, the yield of psicose was not easily decreased, and it was industrially useful.
From the above results, it can be seen that: compared with the existing wild enzyme, the mutant enzyme of the invention can maintain the enzyme activity at the optimal temperature by improving the thermal stability, and can effectively produce the D-psicose.
<110> Songgu chemical industry Co., Ltd
<120> ketose 3-epimerase with improved thermostability
<130>OP18170
<160>1
<170>PatentIn version 3.1
<210>1
<211>289
<212>PRT
<213> wild-type psicose epimerase derived from Arthrobacter globiformis
<400>1
Met Lys Ile Gly Cys His Gly Leu Val Trp Thr Gly His Phe Asp Ala
1 5 10 15
Glu Gly Ile Arg Tyr Ser Val Gln Lys Thr Arg Glu Ala Gly Phe Asp
20 25 30
Leu Val Glu Phe Pro Leu Met Asp Pro Phe Ser Phe Asp Val Gln Thr
35 40 45
Ala Lys Ser Ala Leu Ala Glu His Gly Leu Ala Ala Ser Ala Ser Leu
50 55 60
Gly Leu Ser Asp Ala Thr Asp Val Ser Ser Glu Asp Pro Ala Val Val
65 70 75 80
Lys Ala Gly Glu Glu Leu Leu Asn Arg Ala Val Asp Val Leu Ala Glu
85 90 95
Leu Gly Ala Thr Asp Phe Cys Gly Val Ile Tyr Ser Ala Met Lys Lys
100 105 110
Tyr Met Glu Pro Ala Thr Ala Ala Gly Leu Ala Asn Ser Lys Ala Ala
115 120 125
Val Gly Arg Val Ala Asp Arg Ala Ser Asp Leu Gly Ile Asn Val Ser
130 135 140
Leu Glu Val Val Asn Arg Tyr Glu Thr Asn Val Leu Asn Thr Gly Arg
145 150 155 160
Gln Ala Leu Ala Tyr Leu Glu Glu Leu Asn Arg Pro Asn Leu Gly Ile
165 170 175
His Leu Asp Thr Tyr His Met Asn Ile Glu Glu Ser Asp Met Phe Ser
180 185 190
Pro Ile Leu Asp Thr Ala Glu Ala Leu Arg Tyr Val His Ile Gly Glu
195 200 205
Ser His Arg Gly Tyr Leu Gly Thr Gly Ser Val Asp Phe Asp Thr Phe
210 215 220
Phe Lys Ala Leu Gly Arg Ile Gly Tyr Asp Gly Pro Val Val Phe Glu
225 230 235 240
Ser Phe Ser Ser Ser Val Val Ala Pro Asp Leu Ser Arg Met Leu Gly
245 250 255
Ile Trp Arg Asn Leu Trp Ala Asp Asn Glu Glu Leu Gly Ala His Ala
260 265 270
Asn Ala Phe Ile Arg Asp Lys Leu Thr Ala Ile Lys Thr Ile Glu Leu
275 280 285
His
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