ENGINEERED POLYPEPTIDES AND THEIR APPLICATIONS IN THE SYNTHESIS OF BETA-HYDROXY-ALPHA-AMINO ACIDS

Abstract

The present invention provides engineered polypeptides that are useful for the asymmetric synthesis of β-hydroxy-α-amino acids under industrial-relevant conditions. The engineered polypeptides disclosed in this invention were developed through directed evolution based on the ability of catalytic synthesis of (2S, 3R)-2-amino-3-hydroxy-3-(4-nitrophenyl) propanoic acid. The present disclosure also provides polynucleotides encoding engineered polypeptides, host cells capable of expressing engineered polypeptides, and methods of producing β-hydroxy-α-amino acids using engineered polypeptides. Compared to other processes of preparation, the use of the engineered polypeptides of the present invention for the preparation of β-hydroxy-α-amino acids results in high purity of the desired stereoisomers, mild reaction conditions, low pollution and low energy consumption. So, it has good industrial application prospects.

Claims

1. An engineered aldolase polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO:2 that is, under suitable reaction conditions, capable of condensing p-nitrobenzaldehyde with glycine to produce (2S, 3R)-2-amino-3-hydroxy-3-(4-nitrophenyl) propanoic acid in a diastereomeric excess of at least 60%.

2. The aldolase polypeptide of claim 1, wherein said suitable reaction conditions include about 40 g/L p-nitrobenzaldehyde, about 178 g/L glycine, about 50 μM pyridoxal 5′-phosphate (PLP), and about 25% (v/v) ethanol, at about 30° C.

3. The aldolase polypeptide of claim 1, wherein the amino acid sequence comprises an amino acid sequence that differs from the sequence of SEQ ID NO:2 at one or more amino acid residues selected from among: 16, 17, 19, 26, 32, 33, 37, 42, 43, 44, 45, 46, 47, 48, 49, 91, 92, 118, 132, 134, 154, 164, 168, 176, 182, 185, 189, 191, 216, 217, 218, 227, 234, 237, 244, 247, 262, 282, 284, 285, 288, 291, 292, 293, 294, 295, 302, 305, 316, 318, 319, 320, 324, and 352, wherein the numbering refers to SEQ ID NO:2, and wherein the differing amino acid sequence encodes a polypeptide having aldolase activity.

4. The aldolase polypeptide of claim 3, wherein the amino acid sequence of the aldolase polypeptide comprises one or more of the following amino acid residues: X16 is E; X17 is G or E; X19 is W or N; X26 is V or L; X32 is V; X33 is N; X37 is T, M or K, X38 is D, P, S, E or A; X39 is L or A; X41 is Y; X42 is M; X43 is P or Y; X44 is D; X45 is I; X46 is H; X47 is H; X48 is L; X49 is S; X91 is H, S, L, K or N; X92 is W; X118 is R, I or G; X132 is S; X134 is Q; X154 is G, S, A, F or R; X164 is R; X168 is N; X176 is P; X182 is A; X185 is T; X189 is S; X191 is H; X216 is C; X217 is W; X218 is C or S, X227 is P; X234 is R; X237 is T; X244 is I or V; X247 is Y or H; X262 is I; X282 is R, Y or K; X284 is K, F, A or V; X285 is P, S or K; X288 is I or T; X291 is F, V, W or Y; X292 is K or V; X293 is P; X294 is K or M; X295 is G or Q; X302 is M; X305 is T or P; X316 is K, S, V or R; X318 is G or L; X319 is Y or V; X320 is K or E; X324 is L; or X352 is Y, Q, or A, wherein the numbering above refers to SEQ ID NO:2.

5. An engineered polypeptide, which is a polypeptide of (a) or (b) below: (a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 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, 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, 224, 226, 228, 230, 232, 234, and 236; (b) a polypeptide having aldolase activity, which comprises an amino acid sequence having (i) at least 80% sequence identity to one of the amino acid sequences recited in (a), and (ii) a substitution, deletion, addition or insertion of one or more amino acid residues relative to said one amino acid sequence recited in (a).

6. An engineered aldolase polypeptide, which is capable of condensing p-nitrobenzaldehyde with glycine to produce (2S, 3R)-2-amino-3-hydroxy-3-(4-nitrophenyl) propionic acid under suitable reaction conditions at greater stereoselectivity and/or activity than that of SEQ ID NO:2.

7. A polypeptide immobilized on a solid material by a chemical bond or a physical adsorption method, wherein the polypeptide comprises the engineered aldolase polypeptides according to claim 1.

8. A polynucleotide encoding the engineered aldolase polypeptide of claim 1.

9. The polynucleotide of claim 8, wherein the polynucleotide sequence is 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, 223, 225, 227, 229, 231, 233, and 235.

10. An expression vector comprising the polynucleotide of claim 8.

11. The expression vector of claim 10, which comprises a plasmid, a cosmid, a bacteriophage or a viral vector.

12. A host cell comprising the expression vector of claim 10.

13. A method of preparing an aldolase polypeptide, wherein said method comprises the steps of (i) culturing the host cell of claim 12 and (ii) obtaining an aldolase polypeptide from the culture.

14. An aldolase catalyst obtained by culturing the host cells of claim 12, wherein said aldolase catalyst comprises cells or culture fluid containing the aldolase polypeptides, or an article processed therewith, further wherein the article refers to an extract obtained from the culture of transformant cell, an isolated product obtained by isolating or purifying an aldolase from the extract, or an immobilized product obtained by immobilizing transformant cell, an extract thereof, or isolated product of the extract.

15. A process of preparing a β-hydroxy-α-amino acid of formula (I): ##STR00069## wherein the β-hydroxy-α-amino acid of formula (I) has the indicated stereochemical configuration at the chiral center marked with an*; further wherein: R.sup.1 is selected from among optionally substituted or unsubstituted aryl or heteroaryl and optionally substituted or unsubstituted C.sub.1-C.sub.8 hydrocarbyl; R.sup.2 is selected from among —H, —CH.sub.2OH, —CH.sub.2SH, —CH.sub.2SCH.sub.3, and optionally substituted or unsubstituted C.sub.1-C.sub.4 hydrocarbyl; wherein the process comprises the steps of: (a) contacting an aldehyde substrate of formula (II) and an amino acid substrate of formula (III) ##STR00070## with the engineered polypeptide of claim 1, under suitable reaction conditions; and (b) converting the aldehyde substrate and the amino acid substrate to produce the β-hydroxy-α-amino acid, further wherein the β-hydroxy-α-amino acid of formula (I) is obtained in diastereomeric excess.

16. The process of claim 15, wherein the β-hydroxy-α-amino acid of formula (I) is: ##STR00071## further wherein R.sup.2 is —H, —CH.sub.3, —CH.sub.2CH.sub.3j—CH(CH.sub.3).sub.2, —CH.sub.2OH, —CH.sub.2SH or —CH.sub.2SCH.sub.3; R.sup.3 is a C.sub.1-C.sub.4 hydrocarbyl, —H, a halogen selected from among —F, —Cl, —Br and —I, —NO.sub.2, —NO, —SO2R′, —SOR′, —SR′, —NR′R′, —OR′, —CO.sub.2R′, —COR′, —C(O)NR′, —SO.sub.2NH.sub.2, —SONH.sub.2, —CN, or —CF.sub.3, wherein each R′ is independently selected from —H or (C.sub.1-C.sub.4) hydrocarbyl; R.sub.3 can also be ##STR00072## R.sup.4 is a C.sub.1-C.sub.4 hydrocarbyl, —H, a halogen selected from among —F, —Cl, —Br and —I, —NO.sub.2, —NO, —SO.sub.2R, —SOR, —SR′, —NR′R—OR′, —CO.sub.2R′, —COR′, —C(O)NR′, —SO.sub.2NH.sub.2, —SONH.sub.2, —CN, or —CF.sub.3 wherein each R′ is independently selected From —H or (C.sub.1-C.sub.4) hydrocarbyl; and the aldehyde substrate of formula (II) is: ##STR00073##

17. The process of claim 16, wherein R.sup.3 is: in the para position of the phenyl ring; in the meta position of the phenyl ring; in the ortho position of the phenyl ring; in both the para position and the meta position of the phenyl ring; in both the para position and the ortho position of the phenyl ring; in both the meta position and the ortho position of the phenyl ring.

18. The process of claim 15, wherein the β-hydroxy-α-amino acid of formula (I) is selected from among: ##STR00074## ##STR00075##

19. A process for preparing a compound of formula A2 (2S, 3R)-2-amino-3-hydroxy-3-(4-nitrophenyl) propionic acid: ##STR00076## wherein the process comprises the steps of: (a) contacting p-nitrobenzaldehyde of formula A1 ##STR00077## with an engineered aldolase polypeptide of claim 1 in the presence of glycine, in a suitable solvent, under suitable reaction conditions; and (b) of converting the compound of formula A1 to the compound of formula A2.

20. The process of claim 15, wherein the β-hydroxy-α-amino acid product is present in diastereomeric excess of at least 60%.

21. The process of claim 15, wherein the reaction is carried out in a solvent comprising water, methanol, ethanol, propanol, isopropanol, isopropyl acetate, dimethylsulfoxide (DMSO) or dimethylformamide (DMF).

22. The process of claim 15, wherein the reaction conditions include a temperature of 10° C. to 60° C.

23. The process of claim 15, wherein the reaction conditions include pH 4.0 to pH 8.0.

24. The process of claim 15, wherein the aldehyde substrate is present at a loading of 5 g/L to 400 g/L.

25. The engineered aldolase polypeptide of claim 1, wherein said (2S, 3R)-2-amino-3-hydroxy-3-(4-nitrophenyl) propanoic acid is produced in a diastereomeric excess of at least 80%.

26. The engineered aldolase polypeptide of claim 1, wherein said (2S, 3R)-2-amino-3-hydroxy-3-(4-nitrophenyl) propanoic acid is produced in a diastereomeric excess of 95% or more.

27. The host cells of claim 12, wherein said host cell is E. coli.

28. An aldolase catalyst obtained by the method of claim 13, wherein said aldolase catalyst comprises cells or culture fluid containing the aldolase polypeptides, or an article processed therewith, wherein the article refers to an extract obtained from the culture of transformant cell, an isolated product obtained by isolating or purifying an aldolase from the extract, or an immobilized product obtained by immobilizing transformant cell, an extract thereof, or isolated product of the extract.

Description

3. EXAMPLES

[0158] The following examples further illustrate the present invention, but the present invention is not limited thereto. In the following examples, experimental methods with conditions not specified, were conducted at the commonly used conditions or according to the supplier's′ suggestion.

Example 1: Gene Cloning and Construction of Expression Vectors

[0159] The amino acid sequence of the wild-type aldolase from Pseudomonas putida can be retrieved from NCBI, and the corresponding nucleic acids were then synthesized by a vendor using conventional techniques in the art and cloned into the expression vector pACYC-Duet-1. The recombinant expression plasmid was transformed into E. coli BL21 (DE3) competent cells under the conditions of 42° C. and thermal shock for 90 seconds. The transformation solution was plated on LB agar plates containing chloramphenicol which was then incubated overnight at 37° C. Recombinant transformants were obtained.

Example 2: Recombinant Expression of Aldolase Polypeptides

[0160] The resulting transformant such as recombinant E. coli BL21 (DE3) from example 1 was inoculated into LB medium containing chloramphenicol (peptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, pH 7.0) which was then cultured in a shaking incubator at 30° C., 250 rpm overnight. The overnight culture was subcultured into a 1 L flask containing 250 mL of TB medium (tryptone 12 g/L, yeast extract 24 g/L, glycerol 4 mL/L, PBS) at 30° C., 250 rpm in a shaking incubator. When the OD.sub.600 of subculture broth reached 0.6˜0.8, IPTG was added to induce the expression of recombinant aldolase at a final concentration of 0.1 mmol/L. After expression overnight, the culture was centrifuged to get resting cells. The pelleted resting cells were suspended in a pH 7.4 buffer, and then sonicated in an ice bath to get cell lysate., The supernatant of cell lysate was collected by centrifugation as a crude enzyme solution of the recombinant aldolase, and the supernatant was further freeze-dried using a lyophilizer to obtain crude enzyme powder.

[0161] According to the recombinant expression process using shaking flasks as mentioned above, a miniaturized expression process in 96-well plate was performed by proportionally reducing the scale. The crude enzyme solution was obtained through chemical lysis rather than ultrasonication.

Example 3: Reaction Conditions and Analytical Methods for Measuring Activity and Stereoselectivity of Aldolase Polypeptides

[0162] p-nitrobenzaldehyde was added at a final concentration of 7.5 g/L in a 96-well plate, where p-nitrobenzaldehyde was dissolved in ethanol (EtOH) prior to its addition. The final concentration of ethanol in the system was 40% (v/v), while glycine was added at 10 times the molar amount of p-nitrobenzaldehyde (i.e., 37.4/L), and pyridoxal phosphate (PLP) was added at the final concentration of 0.05 mmol/L, and finally the crude enzyme solution was added. The total volume of the reaction was 200 μl. After the reaction was run for 4 hours, the reaction was quenched with 50% acetonitrile to inactivate aldolase polypeptides. The quenched reaction was centrifuged and resulting supernatant was diluted and then subjected to HPLC analysis to determine the substrate conversion and the de value for product A2.

[0163] Enzymatic reaction was scaled up to 5 mL of total reaction volume on the basis of the above 96-well microplate reaction. The loading of p-nitrobenzaldehyde was 40 g/L, the loading of glycine was 199.2 g/L, the final concentration of PLP was 0.05 mmol/L, concentration of ethanol in the system was 30% (v/v) and crude enzyme powder loading was 4 g/L.

[0164] The analytical method for the determination of the conversion and the de value of the product was as follows: the reaction solution was centrifuged and the supernatant was diluted with 50% acetonitrile to a product concentration of less than 1 g/L. 10 μl of this diluted sample was injected into an Agilent 1260 HPLC to analyze the conversion. The column was Phenomenex Chirex 3126 (D)-penicillamine 150*4.6 mm, mobile phase was 3 mM copper sulfate: methanol=90: 10, at a flow rate of 1 mL per minute, at a column temperature of 50° C., and the detection wavelength was 235 nm. The retention time of (2S, 3R)-2-amino-3-hydroxy-3-(4-nitrophenyl) propanoic acid was 22.53 min; the retention time of (2R, 3R)-2-amino-3-hydroxy-3-(4-nitrophenyl) propanoic acid was 24.17 minutes; the retention time of p-nitrobenzaldehyde was 28.65 minutes; the retention time of (2S, 3S)-2-amino-3-hydroxy-3-(4-nitrophenyl) propanoic acid and (2R, 3S)-2-amino-3-hydroxy-3-(4-nitrophenyl) propanoic acid was 64.19 minutes. The total analysis time was 70 minutes.

Example 4: Construction of Aldolase Mutant Library

[0165] Quikchange kit (supplier: Agilent) was preferably used here. The sequence design of the mutagenesis primers was performed according to the instructions of the kit. The PCR system consisted of 10 μl of 5× Buffer, 1 μl of 10 mM dNTP, 1 μl of plasmid DNA template (50 ng/μl), 0.75 μl (10 uM) each of the upstream and downstream primers, 0.5 μl of high fidelity enzyme and 36 μl of ddH2O, The PCR primer has a NNK codon at the mutation position.

[0166] PCR amplification steps: (1) 98° C. pre-denaturation 3 min; (2) 98° C. denaturation 10s; (3) annealing and extension 3 min at 72° C.; steps of (2)˜(3) repeated 25 times; (5) extension 10 min at 72° C.; (6) cooling to 4° C., 2 μl of DpnI was added to the PCR product and the plasmid template was eliminated by overnight digestion at 37° C. The digested PCR product was transformed into E. coli BL21 (DE3) competent cells and plated on LB agar plates containing chloramphenicol to obtain a site-saturation mutagenesis library.

Example 5: High-Throughput Screening of Aldolase Mutant Libraries

[0167] Mutant colonies were picked from the LB agar plates, inoculated into 200 μl of LB medium (containing chloramphenicol) in a 96-well shallow plate and cultured overnight at 30° C. 20 μl of the above culture was used to inoculate 400 μl of TB medium (including chloramphenicol) in a deep-well plate. When OD.sub.600 of deep-well culture reached 0.6˜0.8, and IPTG was added to induce expression at a final concentration of 1 mM, and the expression undertook at 30° C. overnight. Once the overnight expression was done, the culture was centrifuged at 4000 rpm for 10 minutes to obtain cell pellets to which 200 μl of a chemical lysis reagent (1 g/L lysozyme, 0.5 g/L PMBS) was added to break the cells. Then cell lysate was centrifuged at 4000 rpm for 10 minutes, and subsequently 60 μl of supernatant per well were transferred into a deep well plate containing the reaction solution described in Example 3. The reaction was shaken at 30˜50° C. for desired time, and finally quenched with 50% acetonitrile. Samples were taken for analysis.

Example 6: Fermentation Process for the Expression of Engineered Aldolase

[0168] A single microbial colony of E. coli containing a plasmid bearing the target aldolase gene was inoculated into a 50 mL LB broth containing 30 μg/mL chloramphenicol (5.0 g/L Yeast Extract, 10 g/L Tryptone, 10 g/L sodium chloride). Cells were incubated overnight (at least 16 hours) with shaking at 250 rpm in a 30° C. shaker. When the OD600 of the culture reached 1.6 to 2.2, the culture was used to inoculate medium in fermentor.

[0169] A 5 L fermentor containing 2.0 L of growth medium was sterilized in a 121° C. autoclave for 30 minutes. The fermentor was inoculated with the abovementioned culture. Temperature of fermentor was maintained at 37° C. The growth medium in fermentor was agitated at 200-800 rpm and air was supplied to the fermentation vessel at 2-8 L/min to maintain the dissolved oxygen level at 30% saturation or greater. The culture was maintained at pH 7.0 by addition of 25-28% v/v ammonium hydroxide. Cell growth was maintained by feeding a feed solution containing 500 g/L of dextrose glucose monohydrate, 12 g/L ammonium chloride, and 5 g/L magnesium sulfate heptahydrate. After the OD.sub.600 of culture reached 25±5, the temperature of fermentor was decreased and maintained at 30° C., and the expression of aldolase polypeptides was induced by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. Fermentation process then continued for additional 18 hours. After the fermentation process was complete, cells were harvested using a Thermo Multifuge X3R centrifuge at 8000 rpm for 10 minutes at 4° C. Harvested cells were used directly in the downstream recovery process or stored frozen at −20° C.

[0170] 6 g of cell pellet was resuspended in 30 mL of 100 mM potassium phosphate buffer containing 250 μM pyridoxal 5′-phosphate (PLP), pH 7.5 at 4° C. The cells were then homogenized into cell lysate using a homogenizer. The cell lysate was clarified using a Thermo Multifuge X3R centrifuge at 8000 rpm for 10 minutes at 4° C. The clarified supernatant was dispensed into a shallow container, frozen at −20° C. and lyophilized to a enzyme powder. The aldolase enzyme powder was stored frozen at −20° C.

Example 7: Asymmetric Synthesis of (2s, 3r)-2-amino-3-hydroxy-3-(4-nitrophenyl) Propanoic Acid from Aldehydes and Amino Acids Catalyzed by Aldolase Polypeptides

[0171] Taking a total volume of 1.0 L as an example, the following items were added to the reaction vessel: 178 g of glycine, 30 g of p-nitrobenzaldehyde, 942 mL of a 25% (v/v) aqueous ethanol solution, 4 g of enzyme powder of SEQ ID NO: 6, 5 mL of PLP stock solution (10 mM). The reaction temperature was set at 30° C. and the stirring speed was 400 rpm. After 8 hours of reaction, the total conversion of the substrate was ≥20% and de ≥95% for the product A2. Supernatant was obtained by filtration of the reaction, and the supernatant was concentrated to precipitate a solid crude product. The crude solid was washed with 300 mL of pure water for 30 minutes by stirring at 25° C. Filtration was applied, and the filter cake was vacuum dried to obtain pure product (2S, 3R)-2-amino-3-hydroxy-3-(4-nitrophenyl) propanoic acid (chemical purity 99.5%, de ≥99%).

Example 8: Preparation of D-(−)-threo-2-amino-1-(4-nitrophenyl)-1,3-propanediol From (2S, 3R)-2-amino-3-hydroxy-3-(4-nitrophenyl) Propanoic Acid

[0172] 1000 mL of anhydrous methanol was added to a reaction vessel with ice bath, and it was stirred for 1 hour. The temperature of the reaction vessel was maintained at 5° C., and within 1 hour, 128 mL of thionyl chloride was slowly added dropwise into the reaction vessel. After the addition of thionyl chloride was completed, the reaction mixture was stirred in an ice bath for 1 hour. Then 100 g of (2S, 3R)-2-amino-3-hydroxy-3-(4-nitrophenyl) propanoic acid was added into the reaction, followed by raising reaction temperature to 25° C. and stirring for 3 hours. Then the reaction temperature was slowly raised to 65° C. (reflux). Reflux reaction was carried out for about 24 hours. After etherification was complete, SO.sub.2, HCl, and methanol were removed by depressurization at 40° C. until no liquid flew out. Then 1000 mL ice water were added to cool down the reaction. At the same time, KOH solution was added dropwise to adjust the pH of reaction to about 8.0, and the reaction was stirred for 1 hour. Finally, the reaction was filtered, and the filtered cake was washed with water. 80 g of a white solid substance was obtained after drying the filter cake which was the ester product.

[0173] In order to obtain D-(−)-threo-2-amino-1-(4-nitrophenyl)-1,3-propanediol by reducing the ester product, 1200 mL THF, 75 g ester product were added into a reaction vessel, and it was stirred for 30 min. Then 12 g NaBH4 slowly added into the reaction which was stirred for 1 hour and then heated to reflux (50-55° C.). Subsequently, 160 mL methanol were slowly added dropwise into the reaction within 30 min, followed by stirring for 3 hours to finish the reaction. Concentrated hydrochloric acid were used to adjust the pH of finished reaction to ≤2, and it was stirred overnight. The reaction was filtered, and THF and methanol were removed from the filtrate under reduced pressure. 500 mL of pure water were then added to the filtrate, and KOH were added to adjust pH ≥10. The filtrate was kept at 4° C. for crystallization to occur. The crystallized substance was recovered by filtration, and filter cake was dried to get about 55 g D-(−)-threo-2-amino-1-(4-nitrophenyl)-1,3-propanediol.

Example 9 Asymmetric Synthesis of (2S, 3R)-3-[p-(Methyl sulfonyl) phenyl]-3-hydroxy-2-amino-propanoic Acid Catalyzed by Aldolase Polypeptides

[0174] ##STR00067##

[0175] Taking a total volume of 1.0 L as an example, the following items were added to the reaction vessel: 178 g of glycine, 40 g of p-methylsulfonylbenzaldehyde, 958 mL of a 40% (v/v) aqueous ethanol solution, 4 g of the enzyme powder of SEQ ID NO: 18, 5 mL of PLP stock solution (10 mM). The reaction temperature was set at 30° C. and the stirring speed was 400 rpm. After 6 hours of reaction, the conversion of p-methylsulfonylbenzaldehyde was ≥40%. The de for product (2S, 3R)-3-[p-(methylsulfonyl) phenyl]-hydroxy-2-amino-propanoic acid was ≥90%.

Example 10 Asymmetric Synthesis of (2S, 3R)-2-amino-3-(3,4-dihydroxybenzene)-3-hydroxypropanoic Acid Catalyzed by Aldolase Polypeptides

[0176] ##STR00068##

[0177] Taking a total reaction volume of 1.0 L for example, the following items were added to the reaction vessel: 55 g of glycine, 10 g of 3,4-dihydroxybenzaldehyde, 960 mL of deionized water, 10 g of enzyme powder of SEQ ID NO: 44, 5 mL of PLP stock solution (10 mM). The reaction temperature was set at 30° C., stirring speed was 400 rpm. After 2 hours of reaction, the total conversion of the substrate 3,4-dihydroxybenzaldehyde was ≥40%.

[0178] It should be understood that after reading the above contents of the present invention, those skilled in the art may make various modifications or changes to the present invention. And these equivalent forms also fall within the scope of the appended claims of the present invention.