Epimerases and variants thereof

Abstract

The present disclosure relates to epimerases and variants thereof, and the use of epimerases and variants thereof in the synthesis of allulose.

Description

Technical Field

[0001] This disclosure relates to epimerases and their variants, and the application of said epimerases and their variants in the preparation of allulose. Specifically, said epimerases and their variants exhibit high catalytic activity in the reaction of fructose to allulose. Background Technology

[0002] High-fructose corn syrup and its artificial sweetener alternatives (such as erythritol, aspartame, saccharin, sucralose, etc.) all have adverse health effects in both the short and long term. Therefore, there is a need for a safe and cost-effective alternative available in large quantities. Allulose (CAS 551-68-8) is such a molecule; it has been approved as GRAS (Generally Recognized As Safe) by the US FDA and is hailed as the "perfect sugar" (https: / / www.npr.org / sections / thesalt / 2015 / 08 / 25 / 434597445 / in-the-hunt-for-the-perfect-sugar-substitute-another-candidate-emerges).

[0003] Allulose is a "rare sugar" with a natural abundance of less than 0.2% of fructose. Although allulose can be obtained through chemical synthesis, this method has poor atom economy, low yield, and heavy environmental burden, making it unsuitable for large-scale production.

[0004] Allulose is a naturally occurring C-3 epimer of fructose, and wild-type (naturally occurring) enzymes (“epimerases”; EC5.1.3.x) can perform the interconversion of fructose and allulose. For allulose to be economically competitive with inexpensive and readily available fructose, it needs to be produced at similar environments and scales. Since current commercial fructose production involves the continuous action of immobilized glucose isomerases (e.g., Sweetzyme T) on high-concentration (>500-600 g / L) glucose solutions at high temperatures (>50-60°C) for several months, a competitive process for allulose production using epimerases must also meet the same stringent standards. However, most wild-type enzymes typically react under low substrate concentrations and environmental conditions.

[0005] Therefore, there is a need for wild-type epimerases or improved variants thereof that can efficiently catalyze the conversion of fructose to allulose under high substrate concentrations and high temperatures. Summary of the Invention

[0006] This invention creatively discovers an epimerase capable of catalyzing the conversion of fructose to allulose under high substrate concentrations and high temperatures. Furthermore, through multiple rounds of screening, several epimerase variants with improved epimerase activity were obtained. The epimerase and its variants of this invention can tolerate fructose substrate concentrations of at least 600 g / L at temperatures of at least 60°C.

[0007] Accordingly, in a first aspect, the present invention provides a method for preparing allulose, the method comprising causing fructose to undergo the following reaction in the presence of an epimerase:

[0008]

[0009] The epimerase comprises an amino acid sequence as shown in SEQ ID NO:2, or a fragment or variant thereof having epimerase activity.

[0010] In some embodiments of the invention, the epimerase variant has improved epimerase activity compared to the wild-type epimerase having the amino acid sequence shown in SEQ ID NO:2.

[0011] In some embodiments, the epimerase variant comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:2. In some preferred embodiments, the epimerase variant comprises an amino acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:2. In some preferred embodiments, the epimerase variant comprises an amino acid sequence having at least about 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:2.

[0012] In some embodiments, the epimerase variant has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, or 43 amino acid substitutions compared to the wild-type epimerase having the amino acid sequence shown in SEQ ID NO:2.

[0013] In the method described in this invention, the epimerase variant has one or more substitutions corresponding to the following positions in SEQ ID NO:2: K12, K27, Y30, I32, L33, T38, E49, L62, G65, S80, K83, V101, G105, D119, T123, N127, G132, K138, E142, K143, I145, V151, V180, L181, T200, G211, L223, A226, N233, D238, N239, P245, F246, I257, A267, Q270, D274, and Y285.

[0014] In some preferred embodiments, the epimerase variant has one or more of the following substitutions corresponding to the position of SEQ ID NO:2: K12D, K27E, K27A, Y30F, I32V, L33C, T38A, E49W, L62I, G65N, G65T, S80R, K83E, V101I, G105C, G105N, D119K, T123C, T123H, N127Q, N127M, G132S, K138L, E142K, E142R, K143A, I145L, V15 1C, V180L, L181M, T200A, T200Q, G211D, G211E, G211S, L223I, A226P, A226R, A226L, N233R, D238T, N239T, P245T, P245V, F246Y, I257A, A267D, Q270E, D274V, Y285R, Y285A, Y285S, Y285N, Y285K, Y285H, and Y285Q.

[0015] In some embodiments, the epimerase variant has an amino acid substitution corresponding to position E142 of SEQ ID NO:2. In some preferred embodiments, the epimerase variant has an E142K substitution corresponding to position E142 of SEQ ID NO:2.

[0016] In some embodiments, the epimerase variant, compared to SEQ ID NO:2, further has substitutions selected from the group consisting of:

[0017] Y30F+L181M+Y285R;

[0018] Y30F+L181M;

[0019] K12D+Y30F+L181M+Y285R;

[0020] Y30F+Y285R;

[0021] Y30F+L62I+V151C+Y285R;

[0022] Y30F+V151C+L181M;

[0023] Y30F+V151C;

[0024] Y30F+L62I+L181M;

[0025] K12D+Y30F+F246Y;

[0026] K12D+Y30F+L62I+V151C;

[0027] K12D+V151C;

[0028] Y30F+V151C+L181M+Y285R;

[0029] Y30F;

[0030] K12D+Y30F+V151C+L181M;

[0031] Y30F+I32V+V151C;

[0032] Y30F+I32V+V151C+L181M;

[0033] K12D+K142R;

[0034] K12D+Y30F+V151C;

[0035] Y30F+I32V;

[0036] K12D;

[0037] Y30F+L62I+V151C+L181M;

[0038] Y30F+I32V+L181M;

[0039] Y30F+G65N;

[0040] K12D+Y30F+I32V+L62I;

[0041] Y30F+I32V+K142R+V151C;

[0042] Y30F+I32V+L62I+L181M; and

[0043] K12D+Y30F+I32V+V151C+L181M.

[0044] In some preferred embodiments, the epiomerase variant has an E142K+Y30F substitution compared to SEQ ID NO:2.

[0045] In some embodiments, the epimerase variant, compared to SEQ ID NO:2, further has substitutions selected from the following: P245T; G211D; G211E; P245V; T200A; D238T; D274V; T123H; A226R; K138L; I145L; V180L; N239T; G105N; V101I; T200Q; K143A; A226P; G105C; G211S; Q270E; Y285H; E49W; T123C; A226L; K83E; S80R; Y285Q; and L33C.

[0046] In some preferred embodiments, the epiomerase variant has a substitution of E142K+Y30F+P245T compared to SEQ ID NO:2.

[0047] In some embodiments, the epimerase variant, compared to SEQ ID NO:2, further has substitutions selected from the group consisting of:

[0048] G105C+T123H+G211D+A226R+T245P;

[0049] G105C+T200Q+G211D+A226R+T245P+D274V;

[0050] G105C+V180L+A226R+D238T+N239T;

[0051] G105C+L223I;

[0052] G105C+G211D+A226R+T245P+D274V;

[0053] S80R+G105C+V180L+A226P+D238T+N239T+Q270E;

[0054] G105N+V180L+G211D+A226R+T245P+D274V;

[0055] G105C+V180L+T200A+G211D+A226R+T245P+D274V;

[0056] G105C+V180L+T200Q+G211E+A226R+T245P;

[0057] G105C+G211D+L223I+T245P;

[0058] G105C+N127M+T200A+G211D+L223I+N233R+T245P;

[0059] G105N+T123C+V180L+T200A+A226R+D274V;

[0060] G105C+V180L+T200A+A226R;

[0061] G105C+T123C+A226R;

[0062] G105C+T123C+T200A+G211D+A226R+T245P;

[0063] G105C+T123C+V180L+A226R+N239T;

[0064] G105C+A226R+D274V;

[0065] G105C+T123H+G211E+A226R+T245P+D274V;

[0066] V180L+T200A+G211D+A226R+T245P+D274V;

[0067] G105C+T200Q+A226P;

[0068] G105C+N127M+G211E+N233R+T245P;

[0069] G105C+T123H+G211D+A226R+D274V;

[0070] G105C+T123C+V180L+T200Q+A226P;

[0071] G105C+T123C+G211D+A226R;

[0072] N127M+G211D+N233R+T245P;

[0073] D119K+N127M+G211D+L223I+N233R+T245P;

[0074] G105C+T200A+A226P+D274V;

[0075] G105C+D119K+L223I;

[0076] V180L+T200Q+A226R+D274V;

[0077] G105C+T200Q+G211E+A226R+T245P+D274V;

[0078] G105C+T200A+G211E+A226R+T245P+D274V;

[0079] G105C+V180L+A226P+D274V;

[0080] G105C+T123C+G211E+A226R+T245P; and

[0081] G105C+K143A+I145L+V180L+T200A+A226P.

[0082] In some preferred embodiments, the epimerase variant, compared to SEQ ID NO:2, has a substitution of E142K+Y30F+P245T+G105C+T123H+G211D+A226R+T245P or E142K+Y30F+P245T+G105C+T200Q+G211D+A226R+T245P+D274V.

[0083] In some preferred embodiments, the epimerase variant comprises the same variant as SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 1 38, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222 or 224 amino acid sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity.

[0084] In some preferred embodiments, the epimerase variant comprises or consists of an amino acid sequence selected from the group consisting of: SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 12 6, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222 and 224.

[0085] In the method described in this invention, the reaction conditions for preparing allulose involve fructose concentration, reaction temperature, pH value of the reaction system, and / or reaction time.

[0086] In some implementations, the fructose concentration is at least 100 g / L, at least 200 g / L, at least 300 g / L, at least 400 g / L, at least 500 g / L, at least 600 g / L, at least 700 g / L, at least 800 g / L, at least 900 g / L, or at least 1000 g / L fructose. In some implementation schemes, the substrate concentration is 100 g / L-1000 g / L, 200 g / L-1000 g / L, 300 g / L-1000 g / L, 400 g / L-1000 g / L, 500 g / L-1000 g / L, 600 g / L-1000 g / L, 700 g / L-1000 g / L, 800 g / L-1000 g / L, 900 g / L-1000 g / L, 100 g / L-900 g / L, 100 g / L-800 g / L, 100 g / L-700 g / L, 100 g / L-600 g / L. 0g / L, 100g / L-500g / L, 100g / L-400g / L, 100g / L-300g / L, 100g / L-200g / L, 200g / L-900g / L, 200g / L-800g / L, 300g / L-900g / L, 300g / L-800g / L, 400g / L-900g / L, 400g / L-800g / L, 500g / L-900g / L, 500g / L-800g / L, 600g / L-900g / L or 600g / L-800g / L. In some preferred embodiments, the fructose concentration is 500 g / L-900 g / L, 500 g / L-800 g / L, 600 g / L-900 g / L, or 600 g / L-800 g / L. In some more preferred embodiments, the fructose concentration is 600 g / L-800 g / L. In some most preferred embodiments, the fructose concentration is 600 g / L.

[0087] In some embodiments, the reaction temperature is at least 40°C, at least 45°C, at least 50°C, at least 55°C, at least 60°C, at least 65°C, at least 70°C, at least 75°C, or at least 80°C. In some embodiments, the reaction temperature is 40°C-80°C, 40°C-75°C, 40°C-70°C, 40°C-65°C, 40°C-60°C, 45°C-80°C, 50°C-80°C, 55°C-80°C, 60°C-80°C, 50°C-75°C, 50°C-70°C, 55°C-75°C, 55°C-70°C, 60°C-75°C, or 60°C-70°C. In some embodiments, the reaction temperature is 50°C-80°C, 55°C-80°C, 60°C-80°C, 50°C-75°C, 50°C-70°C, 55°C-75°C, 55°C-70°C, 60°C-75°C, or 60°C-70°C. In some preferred embodiments, the reaction temperature is 60°C-70°C, for example, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, or 70°C.

[0088] In some embodiments, the pH of the reaction system is 6-10, 6-9.5, 6-9, 6-8.5, 6-8, 6.5-10, 7-10, 7.5-10, 8-10, 8-9.5, 8-9, 7-9, 7-8.5, 7-8, 7.5-9, or 7.5-8.5. In some preferred embodiments, the pH of the reaction system is 7.5-8.5, for example, pH 8.

[0089] In some embodiments, the reaction time is at least 1 hour, at least 3 hours, at least 6 hours, at least 9 hours, at least 12 hours, at least 15 hours, at least 18 hours, at least 21 hours, at least 24 hours, at least 2 days, at least 5 days, or at least 10 days. In some preferred embodiments, the reaction time is at least 24 hours, for example, 24 hours.

[0090] In some embodiments, the reaction system for preparing allulose also contains MnSO4. In some preferred embodiments, the concentration of MnSO4 in the reaction system is 0.1-0.3 mM. In some more preferred embodiments, the concentration of MnSO4 in the reaction system is 0.2 mM.

[0091] In this invention, epimerase activity can be characterized by the conversion rate of fructose to allulose under certain reaction conditions. For example, allulose can be prepared using the epimerase or its variants described in this invention under reaction conditions of 600 g / L fructose concentration, reaction temperature of 60°C, 65°C, or 70°C, 0.2 mM MnSO4, and pH 8. The epimerase activity or its variant activity can be expressed by the conversion rate of fructose to allulose under these reaction conditions.

[0092] In a second aspect, the present invention provides an epimerase variant that has improved epimerase activity compared to a wild-type epimerase having the amino acid sequence shown in SEQ ID NO:2.

[0093] In some embodiments, the epimerase variant has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, or 43 amino acid substitutions compared to the wild-type epimerase having the amino acid sequence shown in SEQ ID NO:2.

[0094] In some embodiments, the epimerase variant comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:2. In some preferred embodiments, the epimerase variant comprises an amino acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:2. In some preferred embodiments, the epimerase variant comprises an amino acid sequence having at least about 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:2.

[0095] In some embodiments, the epimerase variant has one or more substitutions corresponding to the following positions of SEQ ID NO:2: K12, K27, Y30, I32, L33, T38, E49, L62, G65, S80, K83, V101, G105, D119, T123, N127, G132, K138, E142, K143, I145, V151, V180, L181, T200, G211, L223, A226, N233, D238, N239, P245, F246, I257, A267, Q270, D274, and Y285.

[0096] In some preferred embodiments, the epimerase variant has one or more of the following substitutions corresponding to the position of SEQ ID NO:2: K12D, K27E, K27A, Y30F, I32V, L33C, T38A, E49W, L62I, G65N, G65T, S80R, K83E, V101I, G105C, G105N, D119K, T123C, T123H, N127Q, N127M, G132S, K138L, E142K, E142R, K143A, I145L, V15 1C, V180L, L181M, T200A, T200Q, G211D, G211E, G211S, L223I, A226P, A226R, A226L, N233R, D238T, N239T, P245T, P245V, F246Y, I257A, A267D, Q270E, D274V, Y285R, Y285A, Y285S, Y285N, Y285K, Y285H, and Y285Q.

[0097] In some embodiments, the epimerase variant has an amino acid substitution corresponding to position E142 of SEQ ID NO:2. In some preferred embodiments, the epimerase variant has an E142K substitution corresponding to position E142 of SEQ ID NO:2.

[0098] In some embodiments, the epimerase variant, compared to SEQ ID NO:2, further has substitutions selected from the group consisting of:

[0099] Y30F+L181M+Y285R;

[0100] Y30F+L181M;

[0101] K12D+Y30F+L181M+Y285R;

[0102] Y30F+Y285R;

[0103] Y30F+L62I+V151C+Y285R;

[0104] Y30F+V151C+L181M;

[0105] Y30F+V151C;

[0106] Y30F+L62I+L181M;

[0107] K12D+Y30F+F246Y;

[0108] K12D+Y30F+L62I+V151C;

[0109] K12D+V151C;

[0110] Y30F+V151C+L181M+Y285R;

[0111] Y30F;

[0112] K12D+Y30F+V151C+L181M;

[0113] Y30F+I32V+V151C;

[0114] Y30F+I32V+V151C+L181M;

[0115] K12D+K142R;

[0116] K12D+Y30F+V151C;

[0117] Y30F+I32V;

[0118] K12D;

[0119] Y30F+L62I+V151C+L181M;

[0120] Y30F+I32V+L181M;

[0121] Y30F+G65N;

[0122] K12D+Y30F+I32V+L62I;

[0123] Y30F+I32V+K142R+V151C;

[0124] Y30F+I32V+L62I+L181M; and

[0125] K12D+Y30F+I32V+V151C+L181M.

[0126] In some preferred embodiments, the epiomerase variant has an E142K+Y30F substitution compared to SEQ ID NO:2.

[0127] In some embodiments, the epimerase variant, compared to SEQ ID NO:2, further has substitutions selected from the following: P245T; G211D; G211E; P245V; T200A; D238T; D274V; T123H; A226R; K138L; I145L; V180L; N239T; G105N; V101I; T200Q; K143A; A226P; G105C; G211S; Q270E; Y285H; E49W; T123C; A226L; K83E; S80R; Y285Q; and L33C.

[0128] In some preferred embodiments, the epiomerase variant has a substitution of E142K+Y30F+P245T compared to SEQ ID NO:2.

[0129] In some embodiments, the epimerase variant, compared to SEQ ID NO:2, further has substitutions selected from the group consisting of:

[0130] G105C+T123H+G211D+A226R+T245P;

[0131] G105C+T200Q+G211D+A226R+T245P+D274V;

[0132] G105C+V180L+A226R+D238T+N239T;

[0133] G105C+L223I;

[0134] G105C+G211D+A226R+T245P+D274V;

[0135] S80R+G105C+V180L+A226P+D238T+N239T+Q270E;

[0136] G105N+V180L+G211D+A226R+T245P+D274V;

[0137] G105C+V180L+T200A+G211D+A226R+T245P+D274V;

[0138] G105C+V180L+T200Q+G211E+A226R+T245P;

[0139] G105C+G211D+L223I+T245P;

[0140] G105C+N127M+T200A+G211D+L223I+N233R+T245P;

[0141] G105N+T123C+V180L+T200A+A226R+D274V;

[0142] G105C+V180L+T200A+A226R;

[0143] G105C+T123C+A226R;

[0144] G105C+T123C+T200A+G211D+A226R+T245P;

[0145] G105C+T123C+V180L+A226R+N239T;

[0146] G105C+A226R+D274V;

[0147] G105C+T123H+G211E+A226R+T245P+D274V;

[0148] V180L+T200A+G211D+A226R+T245P+D274V;

[0149] G105C+T200Q+A226P;

[0150] G105C+N127M+G211E+N233R+T245P;

[0151] G105C+T123H+G211D+A226R+D274V;

[0152] G105C+T123C+V180L+T200Q+A226P;

[0153] G105C+T123C+G211D+A226R;

[0154] N127M+G211D+N233R+T245P;

[0155] D119K+N127M+G211D+L223I+N233R+T245P;

[0156] G105C+T200A+A226P+D274V;

[0157] G105C+D119K+L223I;

[0158] V180L+T200Q+A226R+D274V;

[0159] G105C+T200Q+G211E+A226R+T245P+D274V;

[0160] G105C+T200A+G211E+A226R+T245P+D274V;

[0161] G105C+V180L+A226P+D274V;

[0162] G105C+T123C+G211E+A226R+T245P; and

[0163] G105C+K143A+I145L+V180L+T200A+A226P.

[0164] In some preferred embodiments, the epimerase variant, compared to SEQ ID NO:2, has a substitution of E142K+Y30F+P245T+G105C+T123H+G211D+A226R+T245P or E142K+Y30F+P245T+G105C+T200Q+G211D+A226R+T245P+D274V.

[0165] In some preferred embodiments, the epimerase variant comprises the same variant as SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 1 38, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222 or 224 amino acid sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity.

[0166] In some preferred embodiments, the epimerase variant comprises or consists of an amino acid sequence selected from the group consisting of: SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 12 6, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222 and 224.

[0167] The epimerase variants of the present invention have improved enzyme properties compared with wild-type epimerases, such as improved activity, stability and stereoselectivity.

[0168] In a third aspect, the present invention provides a fusion protein comprising an epimerase variant of the second aspect of the present invention.

[0169] In a fourth aspect, the present invention provides a polynucleotide that encodes an epimerase variant of the second aspect of the present invention or a fusion protein of the third aspect of the present invention.

[0170] In some preferred embodiments, the polynucleotide comprises the same as SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 13 7, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221 or 223 nucleotide sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity.

[0171] In some preferred embodiments, the polynucleotide comprises or consists of nucleotide sequences selected from the group consisting of: SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221 and 223.

[0172] In a fifth aspect, the present invention provides a nucleic acid construct comprising the polynucleotides of the fourth aspect of the present invention.

[0173] In a sixth aspect, the present invention provides a vector comprising the polynucleotide of the fourth aspect of the present invention or the nucleic acid construct of the fifth aspect of the present invention.

[0174] In a seventh aspect, the present invention provides a host cell comprising the polynucleotide of the fourth aspect of the present invention, the nucleic acid construct of the fifth aspect of the present invention, or the vector of the sixth aspect of the present invention.

[0175] In an eighth aspect, the present invention provides a composition comprising an epimerase variant of the second aspect of the present invention, a fusion protein of the third aspect of the present invention, a polynucleotide of the fourth aspect of the present invention, a nucleic acid construct of the fifth aspect of the present invention, a vector of the sixth aspect of the present invention, and / or a host cell of the seventh aspect of the present invention.

[0176] In a ninth aspect, the present invention provides the use of epimerase variants of the second aspect, fusion proteins of the third aspect, polynucleotides of the fourth aspect, nucleic acid constructs of the fifth aspect, vectors of the sixth aspect, host cells of the seventh aspect, or compositions of the eighth aspect in the preparation of allulose. Detailed Implementation

[0177] In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0178] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly specifies otherwise.

[0179] In this document, the terms “comprising,” “having,” “including,” and “containing” should be interpreted as open-ended terms (i.e., meaning “including but not limited to”).

[0180] In this document, “and / or” means and includes any and all possible combinations of one or more of the associated listed items. For example, “the composition contains A and / or B” can be interpreted as the composition contains A, the composition contains B, or the composition contains both A and B.

[0181] All numerical names, such as pH, temperature, time, concentration, and molecular weight, including ranges, are approximate values, appropriately varying in increments of 1.0 or 0.1, or optionally changing (+) or (-) by variations of + / - 15%, 10%, 5%, or 2%. It should be understood that all numerical names are preceded by the term "about." It should also be understood that the reagents described herein are exemplary only, and their equivalents are known in the art. When referring to measurable values ​​such as amount or concentration, the term "about" as used herein means a variation within 20%, 10%, 5%, 1%, 0.5%, or 0.1% of the specified amount.

[0182] In this document, the terms “nucleic acid,” “nucleic acid molecule,” “nucleic acid sequence,” “nucleotide sequence,” and “polynucleotide” are used interchangeably and refer to polymeric forms of nucleotides (ribonucleotides or deoxyribonucleotides) of any length. Therefore, the term includes, but is not limited to, single-stranded, double-stranded, or mixed-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising, consisting of, or substantially consisting of purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derived nucleotide bases.

[0183] The terms “protein,” “peptide,” “polypeptide,” and “amino acid sequence” are used interchangeably and, in their broadest sense, refer to a polymer of two or more amino acid subunits, amino acid analogs, or peptide mimics. “Protein,” “peptide,” “polypeptide,” and “amino acid sequence” contain at least two amino acids, and there is no limit to the maximum number of amino acids. As used herein, the term “amino acid” refers to natural and / or non-natural or synthetic amino acids, including D and L optical isomers and amino acid analogs.

[0184] Equivalents having one or more amino acid modifications compared to the protein or amino acid sequence described herein are also covered within the scope of this invention, provided that such modification does not affect or substantially does not affect the activity of the protein or amino acid sequence. In this document, amino acid modification can be amino acid substitution, amino acid deletion, or amino acid insertion. Amino acid substitution can be conserved or non-conserved. A conserved substitution (also called a conserved mutation, conserved replacement, or conserved variation) is an amino acid substitution in a protein that changes a given amino acid to a different amino acid having similar biochemical properties (e.g., charge, hydrophobicity, or size). In this document, "conserved substitution" means that an amino acid residue is replaced by another biologically similar residue. Examples of conserved substitution include one hydrophobic residue such as isoleucine, valine, leucine, or methionine replacing another; or one charged or polar residue replacing another, such as arginine replacing lysine, glutamic acid replacing aspartic acid, glutamine replacing asparagine, etc. Other exemplary examples of conservative substitutions include the following changes: alanine to serine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glycine to proline; histidine to asparagine or glutamine; lysine to arginine, glutamine, or glutamic acid; phenylalanine to tyrosine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and so on.

[0185] When referring to specific molecules, biological materials, or cellular substances, the terms "equivalent" and "functional variant" may be used interchangeably and refer to those that have minimal homology while still retaining the desired structure or function. Non-limiting examples of equivalent polypeptides include polypeptides having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with a reference polypeptide (e.g., a nucleotide sequence encoding an epimerase as described in this invention); or polypeptides encoded by a polynucleotide having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with a reference polynucleotide (e.g., a nucleotide sequence encoding an epimerase as described in this invention).

[0186] In this article, "expression" refers to the process by which a nucleic acid sequence is transcribed into mRNA and / or the transcribed mRNA is subsequently translated into peptides, polypeptides, amino acid sequences, or proteins. If the nucleic acid sequence originates from genomic DNA, expression may include the splicing of mRNA in eukaryotic cells.

[0187] When the term "encoding" is applied to a nucleic acid sequence, it refers to a nucleic acid sequence that, if in its natural state or when manipulated by methods well known to those skilled in the art, can be transcribed to produce mRNA and / or translated to produce a polypeptide, is called "encoding" a polypeptide. The antisense strand is the complement of such a nucleic acid, from which the coding sequence can be deduced.

[0188] "Homology" or "identity" refers to the sequence similarity between two polypeptides or two nucleic acid sequences. The percentage of identity can be determined by comparing positions in each sequence, which can be aligned for comparison purposes. When a position in the compared sequences is occupied by the same base or amino acid, the molecules are identical at that position. The degree of identity between sequences depends on the number of shared matching positions. "Irrelevant" or "non-homologous" sequences share less than 40% identity and less than 25% identity with one of the sequences of this invention. Tools for comparing sequence similarity are well known to those skilled in the art; for example, the alignment and percentage of sequence identity of the nucleic acid or amino acid sequences provided herein can be determined by importing the nucleic acid or amino acid sequence into ClustalW (available from https: / / genome.jp / tools bin / clustalw / ) and using ClustalW.

[0189] The term "nucleic acid construct" refers to a nucleotide sequence containing a complete expression cassette capable of expressing a target gene or protein. In addition to the coding sequence encoding the target gene or protein, a nucleic acid construct may also contain genetic elements such as promoters, enhancers, terminators, and / or polyadenylation signals for regulating gene expression. Those skilled in the art will understand that the genetic elements contained in a nucleic acid construct are combined in a "functionally linked" or "operably linked" manner to enable the target gene or protein to be expressed in a desired manner.

[0190] As used herein, the term "promoter" refers to an expression control sequence that controls the initiation and rate of transcription of a gene or transgene. Promoters can be, for example, constitutive, inducible, repressive, or tissue-specific. Promoters may contain genetic elements that regulate proteins and molecules such as RNA polymerases and transcription factors to which they can bind.

[0191] In this invention, "effective ligation" or "operationally ligation" refers to the connection of nucleic acid sequences, such that one sequence provides the function required for the linked sequences. In this invention, "operationally ligation" can refer to linking a promoter to a sequence of interest, such that the transcription of the sequence of interest is controlled and regulated by the promoter. When the sequence of interest encodes a protein and its expression is desired, "operationally ligation" means that a promoter is linked to the sequence in a manner that enables efficient transcription and translation of the sequence.

[0192] The term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host or capable of inserting a target gene fragment into the host genome, thereby transferring the carried target gene fragment into host cells and / or between host cells. The vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The vector also includes vectors having multiple of the above-described functions. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing suitable host cells containing the vector, the vector can produce the desired expression product.

[0193] The term "host cell" refers to a cell in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include "transformers" and "transformed cells," encompassing primary transformed cells and their derived progeny (regardless of passage number). The nucleic acid content of the progeny may not be entirely identical to that of the parent cells, and may contain mutations. This document includes mutant progeny that possess the same function or biological activity as those screened or selected in the original transformed cells.

[0194] "Diastereomers" refer to stereoisomers containing multiple chiral centers, where only one chiral center has a different configuration, while the configurations of the others are the same.

[0195] Epimerases are a class of enzymes that catalyze configurational changes in one of the asymmetric carbon atoms in a monosaccharide molecule (containing two or more asymmetric carbon atoms).

[0196] "Improved enzyme properties" refers to any enzyme property that is better or more desirable for a specific purpose compared to the properties exhibited by a reference enzyme. For the engineered epimerase variants described herein, this comparison is generally made against a wild-type epimerase; however, in some embodiments, the reference epimerase may be another improved engineered epimerase. Improveable enzyme properties include, but are not limited to, enzyme activity (which can be expressed as a percentage or rate of substrate conversion over a period of time), thermal stability, solvent stability, pH activity profile, coenzyme requirement, reluctance to inhibitors (e.g., product inhibition), stereospecificity, and stereoselectivity (including enantioselectivity). Therefore, "improved enzyme activity" can refer to an increase in the percentage or rate of substrate conversion over a period of time compared to a reference epimerase (e.g., a wild-type epimerase, or another improved engineered epimerase).

[0197] In some embodiments of this application, "episomerase activity" refers to the ability of the epimerase to convert a substrate (e.g., fructose) into its epimer (e.g., allulose), an ability that can be expressed as substrate conversion. Accordingly, an epimerase variant having "improved epimerase activity" means that the epimerase variant exhibits increased substrate conversion compared to a parental epimerase (e.g., a wild-type epimerase, or another improved engineered epimerase).

[0198] In some specific implementations, "epomerase activity" refers to the ability of the epimerase to convert fructose into allulose, which can be characterized by substrate conversion. After the catalytic reaction occurs under suitable reaction conditions, the contents of fructose and allulose in the reaction system can be determined by a high-performance liquid chromatography system combined with a refractive index-refractive index detector (UPLC-RID). The substrate conversion rate is calculated as [allulose UPLC-RID response / (fructose UPLC-RID response + allulose UPLC-RID response) * 100%], thereby reflecting the activity level of the epimerase.

[0199] Those skilled in the art will understand that the epimerase activity (or substrate conversion) measured under different reaction conditions varies. For example, for the wild-type epimerase (SEQ ID NO:2) described in this application, in a reaction system of 600 g / L fructose and 0.2 mM MnSO4 (pH 8), reacted at 65°C for 24 hours, its substrate conversion, measured and calculated by UPLC-RID, is approximately 24%. Since the substrate conversion rates measured and obtained under different reaction conditions vary, the relative activities of different epimerase variants and the parent epimerase can be calculated under the same reaction conditions, and the relative activity can characterize the activity of the epimerase variants. In some specific embodiments, the relative activity can be calculated as [epimerase variant substrate conversion / epimerase parent substrate conversion].

[0200] "Breatibility" refers to the preferential formation of one stereoisomer over another in a chemical or enzymatic reaction. Stereoselectivity can be partial, where one stereoisomer is formed more preferentially than the other, or it can be complete, where only one stereoisomer is formed. When the stereoisomers are enantiomers, stereoselectivity is called enantiomer selectivity, which is the fraction (usually reported as a percentage) of one enantiomer in the sum of the two enantiomers. It is typically reported in the art, optionally, as (usually as a percentage) an enantiomer excess (ee) calculated from the formula [major enantiomer - minor enantiomer] / [major enantiomer + minor enantiomer]. When the stereoisomers are diastereomers, stereoselectivity is called diastereomer selectivity, which is the fraction (usually reported as a percentage) of one diastereomer in a mixture of two diastereomers, typically reported, optionally, as a diastereomer excess (de). Enantiomer excess and diastereomer excess are types of stereoisomer excess.

[0201] "High stereoselectivity" refers to a chemical or enzymatic reaction that can convert a substrate into its corresponding product with an excess of at least about 85% stereoisomers.

[0202] Example

[0203] method

[0204] 1. Construction and expression of epimerase libraries

[0205] The coding sequence of the wild-type enzyme (SEQ ID NO:2, Uniprot: A0A2N2BKV3_9FIRM) from Firmicutes bacterium HGW-Firmicutes-3 was codon-optimized and expressed in *E. coli*. The optimized coding sequence is shown in SEQ ID NO:1. This gene was cloned into the expression vector pET-28a(+) driven by the T7 promoter. This expression vector contains the colE1 origin of replication and the kanamycin resistance gene. The vector was transformed into *E. coli* C43(DE3), and plasmid DNA was prepared from it using standard methods.

[0206] Epiomerase libraries, including single-site saturation mutant libraries and combinatorial mutant libraries, were constructed using both MegaWHOP and SOE (overlapping extension splicing / hanging extension splicing PCR) methods. These libraries were then cloned into the pET-28a(+) vector and transformed and expressed in *E. coli* C43(DE3). For the single-site saturation mutant library, degenerate primers were designed using NNK to introduce saturation mutations at each desired location. Each degenerate primer was used to generate a large primer via normal PCR, and the entire plasmid with the single-site saturation mutation was produced using MegaWHOP. All MegaWHOP products were then mixed and transformed to generate a pooled site-saturated library. Each primer contained one or more mutations or no mutations in each desired region for mutation introduction. Multiple primers for each mutation region were mixed in equimolar amounts for the initial PCR. Further SOE PCR was performed to fuse all initial PCR products to generate a combinatorial library with all desired mutations. The SOE product was then assembled into the pET-28a(+) vector using the Gibson method, and then transformed into E. coli C43(DE3) to generate a combined library.

[0207] 2. Obtaining cryoprecipitates containing epimerase genes

[0208] Single colonies of recombinant *E. coli* containing the target gene were picked and placed into 200 μL of LB or TB 96-well plates containing 1% glucose and 0.1% kanamycin. The plates were incubated overnight in a shaking incubator at 85% humidity, 30°C, and 250 rpm. 10 μL of the bacterial culture was then inoculated into a deep-well plate containing 380 μL of TB medium and 0.1% kanamycin. After sealing, the plate was incubated for 2-3 hours in a shaking incubator at 85% humidity, 30°C, and 250 rpm. Then, 10 μL of 40 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added for induction culture overnight. The cells were centrifuged at 4000 rpm for 20 minutes at 4°C, and the supernatant was discarded. The bacterial pellet was stored at -80°C for later use.

[0209] 3.96-well plate enzyme activity determination

[0210] Thaw the frozen cell pellet in a 96-well plate to room temperature. Add 200 μL of 100 mM triethanolamine sulfate buffer (pH 8 or pH 9) containing 0.5 g / L polymixin B sulfate (PMBS), 1 g / L lysozyme, and 0.1% v / v DNAase to each well and incubate at room temperature for 90 min. Then centrifuge the lysate at 4000 rpm / 4℃ for 20 min and collect the supernatant (i.e., crude enzyme lysate) into a deep-well plate containing 600-800 g / L fructose and 0.2 mM manganese sulfate (pH 8). Seal the plate and place it in a constant-temperature shaker at 600 rpm at 60℃-70℃ for 24 hours. After that, add 800 μL of 0.5 M H2SO4 to terminate the reaction and centrifuge at 4000 rpm at room temperature for 10 min. Analyze the supernatant directly or dilute it further for analysis.

[0211] The Agilent 1290 high-performance liquid chromatography (UPLC) system is equipped with a 1260 refractive index detector (RID) and a Carbomix Ca-NP5 column for the separation and detection of target products and substrates. Isocratic elution was performed using 100% deionized water at a flow rate of 0.5 mL / min for 2 min per injection. The column temperature was maintained at 80 °C, and the detector temperature at 45 °C.

[0212] 4. Preparation of enzyme powder

[0213] A single colony of *E. coli* containing a plasmid encoding the target enzyme was inoculated into a 15 mL culture tube containing 7 mL of TB medium (containing 1% glucose and 0.1% kanamycin). The culture tube was incubated at 250 rpm / 30°C for 16-24 hours. Then, 6 mL of the bacterial culture was inoculated into a 1000 mL shake flask containing 250 mL of TB medium (containing 0.1% kanamycin). The shake flask was incubated at 250 rpm / 30°C until the OD600 reached 0.6-0.8 (usually requiring 2-4 hours). Then, 250 μL of IPTG was added, and the culture was continued overnight. Centrifuge at 4000 rpm for 20 min at 4°C, discard the supernatant and collect the bacterial cells. Resuspend the cells in 35 mL of 50 mM triethanolamine sulfate buffer (pH 8 or 9). Centrifuge again at 4000 rpm for 20 min at 4°C, discard the supernatant, and resuspend in 4-5 mL of 50 mM triethanolamine sulfate buffer (pH 8 or 9) containing 0.2 g / L pyridoxal-phosphate (PLP). Disrupt the cell resuspension using a Constant System “one-shot” at 35-40 kpsi. Centrifuge the disrupted cell mixture at 7830 rpm for 1 hour at 4°C. Freeze the supernatant at -80°C for at least 1 hour, then freeze-dry to obtain the enzyme powder.

[0214] 5.96-well plate assay for enzyme activity

[0215] Dissolve the enzyme powder in a 600-800 g / L crystalline fructose solution containing 0.2 mM manganese sulfate at pH 8, and then add the solution to a 96-well plate. Seal the plate and place it in a constant-temperature shaker at 600 rpm for 24 hours at 60°C or 70°C. Stop the reaction by adding 800 μL of 0.5 M H₂SO₄ to each well. Centrifuge at 4000 rpm for 10 minutes at room temperature. Use the supernatant for direct testing or dilute before testing.

[0216] 6. Determine enzyme activity in vials

[0217] Dissolve the enzyme powder in a 600-800 g / L crystalline fructose solution containing 0.2 mM MnSO4 at pH 8 and dilute to the required concentration for the reaction. Pipette 10 mL of the reaction solution into a 20 mL reaction vial, add a stir bar, and place the vial in a preheated magnetic stirrer at 60°C for 4 hours. After the reaction is complete, transfer 100 μL to a 1.5 mL centrifuge tube; repeat this process three times for each sample. Stop the reaction by adding 500 μL of 0.5 M H2SO4 and shake at 600 rpm for 10 minutes on a constant-temperature shaker. After the reaction, centrifuge at 4000 rpm at room temperature for 10 minutes. Use the supernatant directly for testing or dilute it before testing.

[0218] Example 1. Identification of the first generation epimerase “Gen 1” (SEQ ID NO:14)

[0219] A site-saturated mutant library was constructed based on the amino acid sequence of the wild-type epimerase, SEQ ID NO:2, whose coding sequence is shown in SEQ ID NO:1. Enzyme activity was measured in 96-well plates by mixing the crude enzyme lysate with a solution containing 600 g / L fructose and 0.2 mM MnSO4 (pH 8) and reacting at 60 °C for 24 hours. After terminating the reaction, the reaction mixture was analyzed by UPLC (RID).

[0220] The activities of the parental epimerase and its variants were characterized by substrate conversion, calculated as: allulose UPLC-RID response / (fructose UPLC-RID response + allulose UPLC-RID response) * 100%. The relative activity of the epimerase variant relative to the parent was calculated as: epimerase variant substrate conversion / epimerase parent substrate conversion.

[0221] The relative activities of epimerase variants relative to the parental epimerase are shown in Table 1. In Tables 1-4, "nt" represents the coding sequence of the corresponding variant, and "aa" represents the amino acid sequence of the corresponding variant.

[0222] Table 1

[0223]

[0224] "++" indicates that the relative activity is greater than 2;

[0225] "++" indicates that the relative activity is between 1.5 and 2;

[0226] "+" indicates that the relative activity is between 1 and 1.5.

[0227] Enzyme powders were prepared for the first seven variants in Table 1 and the parent enzyme SEQ ID NO:2, and their activity was measured. The variant with the best enzyme powder activity was selected as the parent enzyme for the next round of enzyme engineering. Based on the comparison of enzyme powder activities, the epimerase variant shown in SEQ ID NO:14 (whose encoding nucleic acid sequence is SEQ ID NO:13) was designated as the first generation epimerase (Gen1) and used as the parent enzyme for the next round of enzyme engineering.

[0228] Example 2. Identification of the second-generation epimerase “Gen 2” (SEQ ID NO:70)

[0229] A site-saturated mutant library was constructed based on the amino acid sequence SEQ ID. NO. 14 of the epimerase. Enzyme activity was measured in 96-well plates by mixing crude enzyme lysate with a solution containing 600 g / L fructose and 0.2 mM MnSO4 (pH 8) and reacting at 60 °C for 24 h. After terminating the reaction, the reaction mixture was analyzed by UPLC (RID). The results are shown in Table 2.

[0230] Table 2

[0231]

[0232] "++" indicates relative activity > 1.2;

[0233] "+" indicates that the relative activity is between 1 and 1.2.

[0234] Enzyme powders were prepared for the first 15 variants in Table 2 and the parent enzyme SEQ ID NO:14, and their activity was measured. The variant with the best enzyme powder activity was selected as the parent enzyme for the next round of enzyme engineering. Based on the comparison of enzyme powder activities, the epimerase variant shown in SEQ ID NO:70 (whose encoding nucleic acid sequence is SEQ ID NO:69) was designated as the second-generation epimerase (Gen1) and used as the parent enzyme for the next round of enzyme engineering.

[0235] Example 3. Identification of the third-generation epimerase “Gen 3” (SEQ ID NO:100)

[0236] A site-saturated mutant library was constructed based on the amino acid sequence SEQ ID. NO. 70 of the epimerase. Enzyme activity was measured in 96-well plates by mixing the crude enzyme lysate with a solution containing 600 g / L fructose and 0.2 mM MnSO4 (pH 8) and reacting at 70 °C for 24 h. After terminating the reaction, the reaction mixture was analyzed by UPLC (RID). The results are shown in Table 3.

[0237] Table 3

[0238]

[0239] "++" indicates that the relative activity is greater than 2;

[0240] "++" indicates that the relative activity is between 1.5 and 2;

[0241] "+" indicates that the relative activity is between 1 and 1.5.

[0242] Enzyme powders were prepared for the first seven variants in Table 3 and the parent enzyme SEQ ID NO:70, and their activity was measured. The variant with the best enzyme powder activity was selected as the parent enzyme for the next round of enzyme engineering. Based on the comparison of enzyme powder activities, the epimerase variant shown in SEQ ID NO:100 (whose encoding nucleic acid sequence is SEQ ID NO:99) was designated as the third generation epimerase (Gen3) and used as the parent enzyme for the next round of enzyme engineering.

[0243] Example 4. Identification of the fourth generation epimerase "Gen 4"

[0244] A site-saturated mutant library was constructed based on the amino acid sequence SEQ ID. NO. 100 of the epimerase. Enzyme activity was measured in 96-well plates by mixing crude enzyme lysate with a solution containing 600 g / L fructose and 0.2 mM MnSO4 (pH 8) and reacting at 70 °C for 24 h. After terminating the reaction, the reaction mixture was analyzed by UPLC (RID). The results are shown in Table 4.

[0245] Table 4

[0246]

[0247]

[0248] "++" indicates that the relative activity is greater than 2;

[0249] "++" indicates that the relative activity is between 1.5 and 2;

[0250] "+" indicates that the relative activity is between 1 and 1.5.

[0251] As shown in Table 4, variants with the amino acid sequences of SEQ ID NO:158 or SEQ ID NO:160 exhibited the highest relative activity. Therefore, variants with the amino acid sequences of SEQ ID NO:158 or SEQ ID NO:160 can be used as parents for further enzyme engineering screening.

Claims

1. A method for preparing allulose, the method comprising reacting fructose in the presence of an epimerase or an epimerase variant as follows: ， in, The amino acid sequence of the epimerase is shown in SEQ ID NO: 2, and the amino acid sequence of the epimerase variant has an E142K substitution at position E142 corresponding to SEQ ID NO: 2, compared to SEQ ID NO:

2.

2. The method of claim 1, wherein the epimerase variant, compared to SEQ ID NO: 2, further has substitutions selected from the group consisting of: Y30F+L181M+Y285R; Y30F+L181M; K12D+Y30F+L181M+Y285R; Y30F+Y285R; Y30F+L62I+V151C+Y285R; Y30F+V151C+L181M; Y30F+V151C; Y30F+L62I+L181M; K12D+Y30F+F246Y; K12D+Y30F+L62I+V151C; K12D+V151C; Y30F+V151C+L181M+Y285R; Y30F; K12D+Y30F+V151C+L181M; Y30F+I32V+V151C; Y30F+I32V+V151C+L181M; K12D+Y30F+V151C; Y30F+I32V; K12D; Y30F+L62I+V151C+L181M; Y30F+I32V+L181M; Y30F+G65N; K12D+Y30F+I32V+L62I; Y30F+I32V+L62I+L181M; and K12D+Y30F+I32V+V151C+L181M.

3. The method of claim 2, wherein the epimerase variant has an E142K+Y30F substitution compared to SEQ ID NO:

2.

4. The method of claim 3, wherein the epimerase variant, compared to SEQ ID NO: 2, further has substitutions selected from: P245T; G211D; G211E; P245V; T200A; D238T; D274V; T123H; A226R; K138L; I145L; V180L; N239T; G105N; V101I; T200Q; K143A; A226P; G105C; G211S; Q270E; Y285H; E49W; T123C; A226L; K83E; S80R; Y285Q; and L33C.

5. The method of claim 4, wherein the epimerase variant has a substitution of E142K+Y30F+P245T compared to SEQ ID NO:

2.

6. The method of claim 5, wherein the epimerase variant, compared to SEQ ID NO: 2, further has substitutions selected from the group consisting of: G105C+T123H+G211D+A226R+T245P; G105C+T200Q+G211D+A226R+T245P+D274V; G105C+V180L+A226R+D238T+N239T; G105C+L223I; G105C+G211D+A226R+T245P+D274V; S80R+G105C+V180L+A226P+D238T+N239T+Q270E; G105N+V180L+G211D+A226R+T245P+D274V; G105C+V180L+T200A+G211D+A226R+T245P+D274V; G105C+V180L+T200Q+G211E+A226R+T245P; G105C+G211D+L223I+T245P; G105C+N127M+T200A+G211D+L223I+N233R+T245P; G105N+T123C+V180L+T200A+A226R+D274V; G105C+V180L+T200A+A226R; G105C+T123C+A226R; G105C+T123C+T200A+G211D+A226R+T245P; G105C+T123C+V180L+A226R+N239T; G105C+A226R+D274V; G105C+T123H+G211E+A226R+T245P+D274V; V180L+T200A+G211D+A226R+T245P+D274V; G105C+T200Q+A226P; G105C+N127M+G211E+N233R+T245P; G105C+T123H+G211D+A226R+D274V; G105C+T123C+V180L+T200Q+A226P; G105C+T123C+G211D+A226R; N127M+G211D+N233R+T245P; D119K+N127M+G211D+L223I+N233R+T245P; G105C+T200A+A226P+D274V; G105C+D119K+L223I; V180L+T200Q+A226R+D274V; G105C+T200Q+G211E+A226R+T245P+D274V; G105C+T200A+G211E+A226R+T245P+D274V; G105C+V180L+A226P+D274V; G105C+T123C+G211E+A226R+T245P; and G105C+K143A+I145L+V180L+T200A+A226P.

7. The method of claim 6, wherein the epimerase variant, compared to SEQ ID NO: 2, has a substitution of E142K+Y30F+P245T+G105C+T123H+G211D+A226R+T245P or E142K+Y30F+P245T+G105C+T200Q+G211D+A226R+T245P+D274V.

8. The method of any one of claims 1-7, wherein the epimerase variant consists of an amino acid sequence selected from the group consisting of: SEQ ID NO: 14, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222 and 224.

9. An epimerase variant having improved epimerase activity compared to a wild-type epimerase with the amino acid sequence shown in SEQ ID NO: 2, and wherein the amino acid sequence of the epimerase variant has a substitution of E142K at position E142 corresponding to SEQ ID NO: 2 compared to SEQ ID NO:

2.

10. The epimerase variant of claim 9, wherein the epimerase variant further comprises, compared to SEQ ID NO: 2, a substitution selected from the group consisting of: Y30F+L181M+Y285R; Y30F+L181M; K12D+Y30F+L181M+Y285R; Y30F+Y285R; Y30F+L62I+V151C+Y285R; Y30F+V151C+L181M; Y30F+V151C; Y30F+L62I+L181M; K12D+Y30F+F246Y; K12D+Y30F+L62I+V151C; K12D+V151C; Y30F+V151C+L181M+Y285R; Y30F; K12D+Y30F+V151C+L181M; Y30F+I32V+V151C; Y30F+I32V+V151C+L181M; K12D+Y30F+V151C; Y30F+I32V; K12D; Y30F+L62I+V151C+L181M; Y30F+I32V+L181M; Y30F+G65N; K12D+Y30F+I32V+L62I; Y30F+I32V+L62I+L181M; and K12D+Y30F+I32V+V151C+L181M.

11. The epimerase variant of claim 10, wherein the epimerase variant has an E142K+Y30F substitution compared to SEQ ID NO:

2.

12. The epimerase variant of claim 11, wherein the epimerase variant further comprises, compared to SEQ ID NO: 2, a substitution selected from: P245T; G211D; G211E; P245V; T200A; D238T; D274V; T123H; A226R; K138L; I145L; V180L; N239T; G105N; V101I; T200Q; K143A; A226P; G105C; G211S; Q270E; Y285H; E49W; T123C; A226L; K83E; S80R; Y285Q; and L33C.

13. The epimerase variant of claim 12, wherein the epimerase variant has the substitution of E142K+Y30F+P245T compared to SEQ ID NO:

2.

14. The epimerase variant of claim 13, wherein the epimerase variant, compared to SEQ ID NO: 2, further has substitutions selected from the group consisting of: G105C+T123H+G211D+A226R+T245P; G105C+T200Q+G211D+A226R+T245P+D274V; G105C+V180L+A226R+D238T+N239T; G105C+L223I; G105C+G211D+A226R+T245P+D274V; S80R+G105C+V180L+A226P+D238T+N239T+Q270E; G105N+V180L+G211D+A226R+T245P+D274V; G105C+V180L+T200A+G211D+A226R+T245P+D274V; G105C+V180L+T200Q+G211E+A226R+T245P; G105C+G211D+L223I+T245P; G105C+N127M+T200A+G211D+L223I+N233R+T245P; G105N+T123C+V180L+T200A+A226R+D274V; G105C+V180L+T200A+A226R; G105C+T123C+A226R; G105C+T123C+T200A+G211D+A226R+T245P; G105C+T123C+V180L+A226R+N239T; G105C+A226R+D274V; G105C+T123H+G211E+A226R+T245P+D274V; V180L+T200A+G211D+A226R+T245P+D274V; G105C+T200Q+A226P; G105C+N127M+G211E+N233R+T245P; G105C+T123H+G211D+A226R+D274V; G105C+T123C+V180L+T200Q+A226P; G105C+T123C+G211D+A226R; N127M+G211D+N233R+T245P; D119K+N127M+G211D+L223I+N233R+T245P; G105C+T200A+A226P+D274V; G105C+D119K+L223I; V180L+T200Q+A226R+D274V; G105C+T200Q+G211E+A226R+T245P+D274V; G105C+T200A+G211E+A226R+T245P+D274V; G105C+V180L+A226P+D274V; G105C+T123C+G211E+A226R+T245P; and G105C+K143A+I145L+V180L+T200A+A226P.

15. The epimerase variant of claim 14, wherein the epimerase variant, compared to SEQ ID NO: 2, has a substitution of E142R+Y30F+P245T+G105C+T123H+G211D+A226R+T245P or a substitution of E142K+Y30F+P245T+G105C+T200Q+G211D+A226R+T245P+D274V.

16. The epimerase variant of any one of claims 9-15, wherein the epimerase variant comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 14, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222 and 224.

17. A fusion protein comprising any epimerase variant according to any one of claims 9-16.

18. A polynucleotide encoding an epimerase variant of any one of claims 9-16 or a fusion protein of claim 17.

19. The polynucleotide of claim 18, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of: SEQ ID NO: 13, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 95, 97, 99, 1 01, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221 and 223.

20. A nucleic acid construct comprising the polynucleotide of claim 18 or 19.

21. A vector comprising the polynucleotide of claim 18 or 19 or the nucleic acid construct of claim 20.

22. A host cell comprising the polynucleotide of claim 18 or 19, the nucleic acid construct of claim 20, or the vector of claim 21.

23. A composition comprising the epimerase variant of any one of claims 9-16, the fusion protein of claim 17, the polynucleotide of claim 18 or 19, the nucleic acid construct of claim 20, the vector of claim 21, and / or the host cell of claim 22.

24. Use of any epimerase variant of claims 9-16, the fusion protein of claim 17, the polynucleotide of claim 18 or 19, the nucleic acid construct of claim 20, the vector of claim 21, the host cell of claim 22, or the composition of claim 23 in the preparation of allulose.

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