TRANSAMINASE MUTANT, IMMOBILIZED TRANSAMINASE AND USE IN PREPARATION OF SITAGLIPTIN

Abstract

Provided is use of immobilized transaminase in preparation of sitagliptin and/or (R)-3-amino-1-morpholine-4-(2,4,5-trifluorophenyl)-1-butanone. The immobilized transaminase comprises resin and a transaminase mutant, the amino acid sequence of the transaminase mutant is as shown in SEQ ID NO: 3 or SEQ ID NO: 7. Also provided is an immobilized transaminase, a transaminase mutant, a preparation method therefor and use thereof. The enzyme activity of the transaminase mutant in the catalysis of a ketoamide substrate is high, and the enzyme activity is still high after the transaminase mutant is prepared into the immobilized transaminase. When the transaminase mutant is used for catalyzing the ketoamide substrate to produce sitagliptin or an intermediate thereof, a screened solvent reaction system is combined, the immobilized transaminase is high in conversion rate and good in stereoselectivity and stability, the repeatability rate is improved, and the operation is simpler, thereby reducing the cost of production, and it is beneficial to industrial production.

Claims

1. A transaminase mutant, wherein, the transaminase mutant has an amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 7; preferably, the transaminase mutant has a nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 8.

2. An immobilized transaminase, wherein, the immobilized transaminase comprises resin and the transaminase mutant of claim 1; preferably, the transaminase mutant is covalently bonded to the resin; or, the resin is preferably epoxy resin; more preferably, SEPABEADS.sup.®EC HFA, ReliZyme™ HFA403, ReliZyme™ EP113, ReliZyme™ EP403 or SEPABEADS.sup.®EC EP, e.g. SEPABEADS.sup.®EC HFA.

3. A method for preparing the immobilized transaminase of claim 2, which comprises: 1) contacting solution of the transaminase mutant with the resin to form the immobilized transaminase; 2) filtering and rinsing the immobilized transaminase.

4.4. A method for preparing sitagliptin or (R)-3-amino-1-morpholine-4-(2,4,5-trifluorophenyl)-1-butanone, which comprises a step of catalyzing ketoamide substrate with immobilized transaminase in the presence of amino donor in the reaction solvent to obtain sitagliptin or (R)-3-amino-1-morpholine-4-(2,4,5-trifluorophenyl)-1-butanone; wherein, the immobilized transaminase is as described in claim 2, or the reaction solvent is isopropanol aqueous solution; preferably, the ketoamide substrate is 4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazine-7-(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-ketone or 1-morpholine-4-(2,4,5-trifluorophenyl)-1,3-butanedione; or, the amino donor is isopropylamine; or, the molar ratio of the amino donor to the ketoamide substrate is 1:1-5:1; or, when the reaction solvent is isopropanol aqueous solution, the volume content of water is 2%-20%; or, the concentration of the ketoamide substrate is 20 g/L-200 g/L; or, the mass ratio of the immobilized transaminase to the ketoamide substrate is 1:1-6:1; or, the prepared reaction system further comprises a cofactor for transaminase, such as pyridoxal phosphate, and its concentration is preferably 0.5-5 mg/mL; or, the temperature of the reaction is 30-60° C., preferably 45° C.

5. A use of the immobilized transaminase of claim 2 in preparation of sitagliptin or (R)-3-amino-1-morpholine-4-(2,4,5-trifluorophenl)-1-butanone; preferably, the preparation uses a reaction solvent of isopropanol aqueous solution, more preferably, volume content of water in the isopropanol aqueous solution is 2%-20%, or, reaction system for the preparation further comprises a cofactor for transaminase, such as pyridoxal phosphate, preferably at a concentration of 0.5-5 mg/mL.

6. A polynucleotide encoding the transaminase mutant of claim 1.

7. A recombinant expression vector, which comprises the polynucleotide of claim 6; preferably, skeleton of the recombinant expression vector is plasmid pET28a.

8. A transformant, which is prepared by introducing the polynucleotide of claim 6 into a host; preferably, the host is Escherichia coli; preferably Escherichia coli BL21.

9. A use of the transaminase mutant of claim 1 in the preparation of sitagliptin or (R)-3-amino-1-morpholine-4-(2,4,5-trifluorophenyl)-1-butanone.

10. A use of reaction solvent in the preparation of sitagliptin or (R)-3-amino-1-morpholine-4-(2,4,5-trifluorophenyl)-1-butanone, wherein the reaction solvent is isopropanol aqueous solution; preferably, the use comprises a step of catalyzing ketoamide substrate with transaminase in isopropanol aqueous solution in the presence of amino donor to obtain sitagliptin or (R)-3-amino-1-morpholine-4-(2,4, 5-trifluorophenyl)-1-butanone; more preferably, the transaminase is the immobilized transaminase of claim 2 or, the ketoamide substrate is 4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazine-7-(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-ketone or 1-morpholine-4-(2,4,5-trifluorophenyl)-1, 3-butanedione; or, the amino donor is isopropylamine; or, the molar ratio of the amino donor to the substrate is 1:1-5:1; or, when the reaction solvent is isopropanol aqueous solution, the volume content of water is 2%-20%; or, the concentration of the ketoamide substrate is 20 g/L-200 g/L; or, the mass ratio of the immobilized transaminase to the substrate is 1:1-6:1; or, the prepared reaction system further comprises a cofactor for transaminase, such as pyridoxal phosphate, and its concentration is preferably 0.5-5 mg/mL; or, the temperature of the reaction is 30-60° C., preferably 45° C.

11. A method for preparing of sitagliptin phosphate, wherein, the method comprises the following steps: (1) preparing sitagliptin or (R)-3-amino-1-morpholine-4-(2,4,5-trifluorophenyl)-1-butanone according to the method of claim 4; (2) reacting sitagliptin or (R)-3-amino-1-morpholine-4-(2,4,5-trifluorophenyl)-1-butanone prepared in step (1) to obtain sitagliptin phosphate; preferably, the sitagliptin phosphate is sitagliptin phosphate monohydrate.

12. A transformant, which is prepared by introducing the recombinant expression vector of claim 7 into a host; preferably, the host is Escherichia coli; preferably Escherichia coli BL21.

13. A use of a reaction solvent in the preparation of sitagliptin or (R)-3-amino-1-morpholine-4-(2,4,5-trifluorophenyl)-1-butanone, wherein the reaction solvent is isopropanol aqueous solution; preferably, the use comprises a step of catalyzing ketoamide substrate with transaminase in isopropanol aqueous solution in the presence of amino donor to obtain sitagliptin or (R)-3-amino-1-morpholine-4-(2,4,5-trifluorophenyl)-1-butanone; more preferably, the transaminase is the transaminase mutant of claim 1; or, the ketoamide substrate is 4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazine-7-(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-ketone or 1-morpholine-4-(2,4,5-trifluorophenyl)-1,3-butanedione; or, the amino donor is isopropylamine; or, the molar ratio of the amino donor to the substrate is 1:1-5:1; or, when the reaction solvent is isopropanol aqueous solution, the volume content of water is 2%-20%; or, the concentration of the ketoamide substrate is 20 g/L-200 g/L; or, the mass ratio of the immobilized transaminase to the substrate is 1:1-6:1; or, the prepared reaction system further comprises a cofactor for transaminase, such as pyridoxal phosphate, and its concentration is preferably 0.5-5 mg/mL; or, the temperature of the reaction is 30-60° C., preferably 45° C.

Description

DETAILED DESCRIPTION

[0067] The present invention is further illustrated hereinafter by means of Examples, but the present invention is not limited to the scope of the described Examples. Experimental methods without specific conditions specified in the following Examples shall be selected in accordance with conventional methods and conditions or in accordance with commodity instructions.

[0068] The experimental methods used in the present invention are conventional methods unless otherwise specified. Specific gene cloning operations can be referred to the “Molecular Cloning: A Laboratory Manual” edited by J. Sambrook et al.

[0069] The abbreviated symbols of amino acids in the present invention are conventional in the art unless otherwise specified, and the amino acids corresponding to the specific abbreviated symbols are shown in Table 1.

TABLE-US-00001 Amino acid name Three-letter symbol Single letter symbol Amino acid name Three-letter symbol Single letter symbol Alanine Ala A Leucine Leu L Arginine Arg R Lysine Lys K Asparagine Asn N Methionine Met M Aspartic acid Asp D Phenylalanine Phe F Cysteine Cys C Proline Pro P Glutanine Gln Q Serine Ser S Glutamic acid Glu E Threonine Thr T Glycine Gly G Tryptophan Trp W Histidine His H Tyrosine Tyr Y Isoleucine Ile I Valine Val V

[0070] The codon corresponding to the amino acid is also conventional in the art, and the corresponding relationship between the specific amino acid and the codon is shown in Table 2.

TABLE-US-00002 First nucleotide Second nucleotide Third nucleotide T C A G T Phenylalanine F Serine S Tyrosine Y Cysteine C T Phenylalanine F Serine S Tyrosine Y Cysteine C C Leucine L Serine S Termination codon Termination codon A Leucine L Serine S Termination codon Tryptophan W G C Leucine L Proline P Histidine H Arginine R T Leucine L Proline P Histidine H Arginine R C Leucine L Proline P Glutamine Q Arginine R A Leucine L Proline P Glutamine Q Arginine R G A Isoleucine I Threonine T Asparagine N Serine S T Isoleucine I Threonine T Asparagine N Serine S C Isoleucine I Threonine T Lysine K Arginine R A Methionine M Threonine T Lysine K Arginine R G G Valine V Alanine A Aspartic acid D Glycine G T Valine V Alanine A Aspartic acid D Glycine G C Valine V Alanine A Glutamic acid E Glycine G A Valine V Alanine A Glutamic acid E Glycine G G

[0071] Pet28a was purchased from Novagen; NdeI enzyme and HindIII enzyme were purchased from Thermo Fisher, and BL21 competent cells were purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.

Example 1. Preparation of a Transaminase Mutant Enzyme Solution

[0072] The genes shown in the nucleotide sequences of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8 were synthesized by Suzhou Genewiz Biotechnology Co., Ltd. (C3 building, Bio-Nanotechnology Park, No. 218, Xinghu street, Suzhou Industrial Park). The above genes encode transaminases shown in SEQ ID NO: 1 (i.e. SEQ ID NO: 110 in US8293507), SEQ ID NO: 3, SEQ ID NO: 5 (i.e. SEQ ID NO: 130 in US9617573) and SEQ ID NO: 7 in the Sequence Listing, respectively.

TABLE-US-00003 Enzyme number Amino acid sequence number Nucleotide sequence number Enz.1 1 2 Enz.1-M122Q-P223T 3 4 Enz.2 5 6 Enz.2-M122F 7 8

[0073] Then, the synthetic genes were enzymatically ligated to pET28a, with restriction sites NdeI & HindIII, and the enzymatically ligated vector was transformed into the host E. coli BL21 competent cells. The constructed strains were inoculated in TB culture, and incubated at37° C. and 200 rpm in a shaker, for inducing overnight with IPTG at a concentration of 0.1 mM, and then the stains were harvested to obtain the engineered bacteria containing transaminase genes.

[0074] The engineered bacteria containing transaminase gene were activated by plate streaking, and then a single colony was picked and inoculated into 5 mL LB liquid medium containing 50 .Math.g/mL kanamycin, and cultured under shaking at 37° C. for 12 h. At a rate of 2% (v/v), the medium was transferred to 150 mL fresh LB liquid medium containing 50 .Math.g/mL kanamycin. After shaking at 37° C. until OD600 reached 0.8, IPTG was added at a final concentration of 0.5 mM, and the bacteria was induced at 18° C. for 16 h. After cultivation, the culture medium was centrifuged at 10,000 rpm for 10 min, then the supernatant was discarded, and the cells were collected and stored in a -80° C. ultra-low temperature refrigerator for further use.

[0075] 2 g of the cells were collected after the cultivation was washed twice with 50 mM phosphate buffer (pH 7.4), the cells were then resuspended in 20 mL of phosphate buffer (pH 7.4) and sonicated, followed by centrifuging the solution to remove the precipitate. The obtained supernatant was the crude enzyme solution containing transaminase mutant. 10 mL of crude enzyme solution was purified by nickel column (Bestchrom Shanghai Biosciences Co., Ltd.) to obtain a pure enzyme solution, and the protein concentration was kept at about 4.5 mg/mL.

[0076] Ketoamide substrate 2 (morpholinedione) and ketone amide substrate 1 (sitadione) were used as substrates to determine enzyme activity, respectively. The determination method is as follows: 0.1 g of sitadione or morpholinedione was added with 2 mL of anhydrous ethanol, 6.4 mL of 0.1 mol/L triethanolamine buffer (pH 8.5), 1.6 mL of 2 mol/L isopropylamine hydrochloride solution (pH 8.5) and 0.003 g of pyridoxal phosphate (PLP) powder were added. The shaker was preheated at 37° C. for 30 min, and then 0.1 mL of pure enzyme solution placed on the shaker, the solution then reacted at 37° C. for 30 min. Next, 0.5 mL of 6 N hydrochloric acid was added quickly and the solution was continuously shaken for 30 seconds to inactivate the enzyme. The samples were diluted with acetonitrile and detected by HPLC. The enzyme activity was calculated according to the standard curve. The results of enzyme activities of different mutants are shown in Table 4 below.

[0077] HPLC method: chromatographic column: C18 4.6×250 mm, 5 .Math.m; detector: UV268 nm; column temperature: 40° C.; flow rate: 0.8 mL/min; injection volume: 20 .Math.L; mobile phase A: water:acetonitrile:formic acid:ammonia water = 950:50:0.5:0.5, pH should be between 3.60 and 3.80, if the pH cannot be maintained in such range, 10% ammonia water or 10% formic acid can be used to adjust the pH to 3.70; ammonium formate can also be used to adjust pH to 3.70; mobile phase B:water:acetonitrile = 20:80; gradient elution: 100% A (0.01 min), 60% A+40% B (20 min), 60% A+40% B (40 min), 100% A (50 min), 100% A (60 min).

[0078] Retention time: morpholinedione: 27.828 min; (R)-3-amino-1-morpholine-4-(2,4,5-trifluorophenyl)-1-butanone: 14.835 min;

[0079] Retention time: sitadione: 34.811 min; sitagliptin: 17.489 min.

[0080] Retention time of morpholinedione control: 27.820 min;

[0081] Retention time of (R)-3-amino-1-morpholine-4-(2,4,5-trifluorophenyl)-1-butanone control: 14.856 min;

[0082] Retention time of sitadione control: 34.715 min;

[0083] Retention time of sitagliptin control (purchased from Beijing Yingxiang Technology Co., Ltd.): 17.705 min.

[0084] Wherein, morpholinedione substrate raw material and control (R)-3-amino-1-morpholine-4-(2,4,5-trifluorophenyl)-1-butanone were synthesized by the present company; the method refers to WO2019011236A1; the racemate of 3-amino-1-morpholine-4-(2,4,5-trifluorophenyl)-1-butanone was synthesized in the laboratory by ammoniation and catalytic hydrogenation of morpholinedione. Determination of the configuration of control (R)-3-amino-1-morpholine-4-(2,4,5-trifluorophenyl)-1-butanone: (R)-3-amino-1-morpholine-4-(2,4,5-trifluorophenyl)-1-butanone can be further reacted to prepare (3R)-N-tert-butoxycarbonyl-3-amino-4-(2,4,5-trifluorophenyl)-butyric acid (refer to WO2019011236A1); the R configuration could be determined by using the (3R)-N-tert-butoxycarbonyl-3-amino-4-(2,4,5-trifluorophenyl)-butyric acid standard (purchased from Anhui Haikang Pharmaceutical Co., Ltd.) as the control.

HPLC Method for Configuration Determination

[0085] Chromatographic column: Daicel Chiralpak AD-H (4.6 mm×250 mm, 5 .Math.m); mobile phase: n-hexane:isopropanol = 90:10; detection wavelength: 210 nm; flow rate: 1.0 mL/min; injection volume: 10 .Math.L; column temperature: 25° C.; running time: 40 min.

[0086] The raw material of sitadione substrate was synthesized by the present laboratory, the synthesis method refers to CN100430397C. The racemate of sitagliptin was synthesized by the present laboratory, which is prepared by ammoniation and catalytic hydrogenation of sitadione.

TABLE-US-00004 Enz.2 Enz.2-M122F Enz.1 Enz.1-M122Q-P223T Enzyme activity (U/mL) with sitadione as substrate 439 812 512 789 Enzyme activity (U/mL) with morpholinedione as substrate 57 121 75 243

[0087] As shown in Table 4, the enzyme activities of Enz.2-M122F and Enz.1-M122Q-P223T were significantly increased when sitadione or morpholinedione was used as the substrate. Wherein, the enzyme activity of Enz.2-M122F was about twice that of Enz.2. When Enz.1-M122Q-P223T catalyzed sitadione, the enzyme activity of Enz.1-M122Q-P223T increased by 50% compared with that of Enz.1. When Enz.1-M122Q-P223T catalyzed morpholinone, its enzyme activity was more than three times that of Enz.1.

Example 2. Preparation of Immobilized Transaminase Mutants

[0088] 12 g of transaminase mutant bacterial sludge was mixed with 120 mL of 100 mM phosphate buffer. The cells were homogenized under high pressure, and the supernatant liquid containing enzyme was collected by centrifugation. 22 g of K.sub.2HPO.sub.4•3H.sub.2O, 2.2 g of KH.sub.2PO.sub.4 and 0.024 g of PLP were added and stirred to dissolve. 10 g of resin was then added (see Table 5 for specific resin models, the resins were purchased from Suzhou Huitong Chromatography Separation and Purification Co., Ltd.) for fixation, followed by shaking the solution at 25° C. and 180 rpm for 20-25 h, and then subjected to suction filtration, deionization washing and suction filtration to obtain the immobilized enzyme. The immobilized enzyme activity was detected by using morpholinedione and sitadione as substrate, respectively. The enzyme activity detection method is as follows:

[0089] 1.2 g of morpholinedione or sitadione substrate was placed into a 50 mL triangular flask, 19 mL of isopropanol, 0.6 mL of 16 mg/mL PLP solution and 0.4 mL of isopropylamine were added, the mixture was shaken at 45° C. until suspended solids were completely dissolved, and then was cooled to 37° C. for 30 min. 0.5 g of immobilized enzyme was added into a preheated substrate for reaction in a shaker at 200 rpm, 37° C. for 20 min. The reaction solution was sampled, and the sample was diluted with acetonitrile and detected by HPLC. The standard concentration curve of standard sitagliptin (purchased from Beijing Yingxiang Technology Co., Ltd.)/standard sitagliptin intermediate (R)-3-amino-1-morpholine-4-(2,4,5-trifluorophenyl)-1-butanone (synthesized by Applicant, the synthesis method refers to WO2019011236A1) was used to calculate the concentration of product sitagliptin (when morpholinedione was used as the substrate, the calculated product was sitagliptin intermediate (R)-3-amino-1-morpholine-4-(2,4,5-trifluorophenyl)-1-butanone), and then the immobilized enzyme activities were calculated. The results are shown in Table 5 below.

[0090] HPLC detection method is the same that in Example 1.

TABLE-US-00005 Enzyme number Resin model Isopropanol system U/g (morpholinedione as substrate) Isopropanol system U/g (sitadrone as substrate) Enz.1-M122Q-P233T SEPABEADS.sup.®EC HFA 273 375 Enz.1-M122Q-P233T ReliZyme™HFA403 224 377 Enz.1-M122Q-P233T ReliZyme™EP113 258 353 Enz.1-M122Q-P233T ReliZyme™EP403 183 286 Enz.1-M122Q-P233T SEPABEADS.sup.®EC EP 200 324 Enz.2-M122F SEPABEADS.sup.®EC HFA 188 954

[0091] According to the immobilization effects of different resins as shown in Table 5, the immobilized enzyme prepared with SEPABEADS.sup.®EC HFA resin was selected for catalytic reaction.

Example 3. Catalysis of Morpholinedione by Immobilized Enzyme in Isopropanol System (Resin: SEPABEADS.SUP.®.EC HFA)

[0092] 25 mL of isopropanol, 2.5 g of morpholinedione, 2 mL of water, 32 mg of PLP, 1.135 mL of isopropylamine and 10 g of immobilized enzyme prepared in Example 2 were added into a triangle flask, and the reaction was performed at 45° C. and 200 rpm in a shaker. After 24 hours of reaction, a sample was taken to detect the conversion rate. The immobilized enzyme was collected by filtering the reaction solution, then 25 mL of isopropanol, 2.5 g of morpholinedione, 2 mL of water, 32 mg of PLP and 1.135 mL of isopropylamine were added, and the reaction was performed at 45° C. and 200 rpm in a shaker. After 24 hours of reaction, a sample was taken to detect the conversion rate. The immobilized enzyme was reused according to the method described above. The results are shown in Table 6, which indicate that after the immobilized enzyme was applied for 10 batches of reaction, the conversion rates of Enz.1-M122Q-P233T and Enz.2-M122F remained above 85%, the ee value was >99.9%, and the immobilized enzyme was stable. Enz.1 and Enz.2 (not effective after 3 batches of application) had low conversion rates and were not suitable for catalyzing morpholinedione.

[0093] Wherein, the HPLC method for detecting the conversion rate is the same as that in Example 1.

[0094] Retention time: morpholinedione: 27.831 min; (R)-3-amino-1-morpholine-4-(2,4,5-trifluorophenyl)-1-butanone: 14.811 min.

[0095] Chiral HPLC method for detecting the ee value of the product is as follows:

[0096] chromatographic column: Daicel ChiralpakAD-H column 4.6 mm×250 mm, 5 .Math.m; mobile phase: n-hexane:isopropanol:diethylamine = 40:60:0.1; detector: UV268 nm; column temperature: 25° C.; flow rate: 0.8 mL/min; injection volume: 10 .Math.L. Retention time: (S)-3-amino-1-morpholine-4-(2,4,5-trifluorophenyl)-1-butanone: 10.295 min, (R)-3-amino-1-morpholine-4-(2,4,5-trifluorophenyl)-1-butanone: 28.091 min.

[0097] Retention times of racemate control: 10.290 min and 28.087 min.

[0098] Retention time of (R)-3-amino-1-morpholine-4-(2,4,5-trifluorophenyl)-1-butanone control: 28.093 min.

TABLE-US-00006 Batch Reaction time/h Conversion rate/% Enz.1-M122Q-P233T Conversion rate/% Enz.2-M122F Conversion rate/% Enz.1 Conversion rate/% Enz.2 1 24 89.25 86.26 45.24 47.55 2 24 88.4 91.52 46.67 48.34 3 24 87.53 93.43 45.43 46.58 4 24 87.31 93.59 / / 5 24 89.75 93.47 / / 6 24 91.75 94.07 / / 7 24 87.85 93.91 / / 8 24 87.14 93.77 / / 9 24 87.24 94.48 / / 10 24 87.30 93.92 / / average value 88.352 92.842 45.780 47.490 standard deviation 1.496 2.443 0.777 0.882 p-value 0.00027* 0.00299# / / *: compared to the conversion of Enz.1; #: compared to the conversion of Enz.2; /: not applicable.

[0099] The above results indicate that the transaminase mutation of the present invention shows a better effect in the experiment of catalysis of morpholinedione by immobilized enzyme as compared with that of Enz.1 and Enz.2 in the prior art, and the difference was statistically significant (p-values were all less than 0.05, wherein, p-values were calculated using a two-tailed t-test).

Example 4. Catalysis of Sitadione by Immobilized Enzyme in Isopropanol System

[0100] 25 mL of isopropanol, 3.75 g of sitadione, 2 mL of water, 32 mg of PLP, 1.7 mL of isopropylamine, and 10 g of immobilized enzyme prepared in Example 2 were added into a triangle flask, and the reaction was performed at 45° C. and 200 rpm in a shaker. After 24 hours of reaction, a sample was taken to detect the conversion rate. The immobilized enzyme was collected by filtering the reaction solution, then 25 mL of isopropanol, 3.75 g of sitadione, 2 mL of 16 mg/mL PLP aqueous solution, 1.7 mL of isopropylamine were added, and the reaction was performed at 45° C. and 200 rpm in a shaker. After 24 hours of reaction, a sample was taken to detect the conversion rate. The immobilized enzyme was reused according to the method described above. The results show that after the immobilized enzyme was applied for 10 batches of reaction, the conversion rate remained above 85%, the ee value was >99.9%, and the immobilized enzyme was stable. The specific application results are shown in Table 7 below.

[0101] Wherein, the HPLC method for detecting the conversion rate is the same as that in Example 1.

[0102] Retention time: sitadione: 34.827 min; sitagliptin: 17.394 min.

[0103] Chiral HPLC method for detecting the ee value of the product is as follows:

[0104] chromatographic column: Daicel ChiralpakAD-H column 4.6 mm×250 mm, 5 .Math.m; mobile phase: n-heptane:ethanol:diethylamine = 40:60:0.1; detector: UV268 nm; column temperature: 25° C.; flow rate: 0.8 mL/min; injection volume: 10 .Math.L. retention time: (S)-enantiomer: 14.181, sitagliptin: 17.580.

[0105] Retention times of racemate control: 14.172 min and 17.702 min;

[0106] retention time of sitagliptin control: 17.665 min.

TABLE-US-00007 Batch Reaction time/h Conversion rate/% Enz.1-M122Q-P233T Conversion rate/% Enz.2-M122F 1 24 86.25 92.11 2 24 85.16 91.52 3 24 85.94 90.61 4 24 86.01 89.55 5 24 87.04 91.32 6 24 84.96 90.00 7 24 85.27 89.25 8 24 86.21 89.28 9 24 86.69 91.27 10 24 86.03 91.56 *: compared to the conversion of Enz.1; #: compared to the conversion of Enz.2; /: not applicable.

[0107] Compared with the following Comparative Example 1, combined with the result of catalytic reaction of morpholinedione in embodiment 3 above, it can be seen that the reaction solvent isopropanol of the present invention is more suitable for catalyzing sitardione and/or morpholinedione (lower standard deviation which means higher stability) than IPAc.

Example 5. Preparation of Sitagliptin Phosphate Monohydrate

[0108] After the reaction of Example 4 was completed, filtration was performed to obtain the immobilized enzyme and filtrate, and the filtrate was concentrated and dried at 60° C. to obtain a concentrate. 100 mL of dichloromethane was added to dissolve the concentrate, then 100 mL of purified water was added, and the mixture was stirred, followed by adjusting the pH of the solution to 2-3 with 30% concentrated hydrochloric acid, and the mixture was placed for stratification. Next, 100 mL of dichloromethane was added to the aqueous phase and stirred, followed by adding 30% sodium hydroxide solution to adjust the pH to 11, and the mixture was placed for stratification. 100 mL of dichloromethane was added to the aqueous phase again, and the mixture was stirred for extraction, and placed for stratification. The organic phases of the two alkaline extractions were combined and concentrated at 60° C., 120 mL of isopropanol was then added to the concentrate and stirred to dissolve; 10.6 g of 85% phosphoric acid was added. The mixture was heated up to 75° C. and the mixture was stirred to dissolve, and then was slowly cooled down to precipitate sitagliptin phosphate. After being stored at 5° C. for 2 hours, the mixture was filtered, and the filter cake was dried at 60° C. to obtain sitagliptin phosphate monohydrate.

Comparative Example 1. Catalysis of Sitadione by Immobilized Resin in IPAc (Isopropyl Acetate) System

[0109] 25 mL of isopropyl acetate, 1.25 g of sitadione, 2 mL of 16 mg/mL PLP aqueous solution, 0.95 mL of isopropylamine, and 3.75 g of immobilized enzyme prepared in Example 2 were added into a triangle flask, and the reaction was performed at 45° C. and 200 rpm in a shaker. After 24 hours of reaction, a sample was taken to detect the conversion rate. The immobilized enzyme was collected by filtering the reaction solution, then 25 mL of isopropyl acetate, 1.25 g of sitadione, 2 mL of water and 32 mg of PLP, 0.95 mL of isopropylamine were added, and the reaction was performed at 45° C. and 200 rpm in a shaker. After 24 hours of reaction, a sample was taken to detect the conversion rate. The immobilized enzyme was reused according to the method described above. The results are shown in Table 8, which indicate that the stability of immobilized enzyme was poor (i.e. high standard deviation) and the conversion rate is low, after the immobilized enzyme is applied for three batches of reaction.

TABLE-US-00008 Batch Conversion rate/% Enz.1-M122Q-P233T-SEPABE ADS.sup.®EC HFA Conversion rate/% Enz.2-SEPABEA DS.sup.®EC HFA Conversion rate/% Enz.1-SEPABEA DS.sup.®EC HFA Reaction time/h 1 71.54 90.1 73.43 24 2 61.62 84.52 67.25 24 3 59.45 77.18 62.18 24 average value 64.203 83.933 67.62 / standard deviation 6.446 6.480 5.634 / /: not applicable.

Comparative Example 2. Catalysis of Morpholinedione by Immobilized Resin in IPAc (Isopropyl Acetate) System

[0110] 25 mL of isopropyl acetate, 1.25 g of morpholinedione, 2 mL of water, 32 mg of PLP, 0.95 mL of isopropylamine, and 5 g of immobilized enzyme prepared in Example 2 were added into a triangle flask, and the reaction was performed at 45° C. and 200 rpm in a shaker. After 24 hours of reaction, a sample was taken to detect the conversion rate. The immobilized enzyme was collected by filtering the reaction solution, then 25 mL of isopropyl acetate, 1.25 g of morpholinedione, 2 mL of 16 mg/mL PLP aqueous solution and 0.95 mL of isopropylamine were added, and the reaction was performed at 45° C. and 200 rpm in a shaker. After 24 hours of reaction, a sample was taken to detect the conversion rate. The immobilized enzyme was reused according to the method described above. The results are shown in Table 9.

TABLE-US-00009 Batch Conversion rate/% Conversion rate/% Conversion rate/% Reaction time/h Enz.1-M122Q-P233T-SEPABE ADS.sup.®EC HFA Enz.2-SEPABEA DS.sup.®EC HFA Enz.1-SEPABEA DS.sup.®EC HFA 1 90.39 46.37 45.12 24 2 75.61 42.43 43.39 24 3 71.45 38.78 39.24 24 average value 79.150 42.527 42.583 / standard deviation 9.954 3.796 3.022 / /: not applicable.

[0111] Tables 8 and 9 show that in the water-saturated IPAc (isopropyl acetate) solvent system, after different immobilized enzymes catalyzed sitadione and morpholinedione for 3 batches, respectively, the stability of the immobilized enzyme was relatively poor and the conversion rate was low, wherein the conversion rate is significantly different from that of the immobilized enzyme of the present invention, which shows that the isopropyl acetate system is not suitable for the application of the immobilized enzyme.