D-amino acid oxidative enzyme mutant and application thereof

Abstract

Provided is a D-amino acid oxidative enzyme mutant. The sequence of the mutant comprises a sequence by mutating the 54.sup.th amino acid residue N, the 58.sup.th amino acid residue F, the 211.sup.th amino acid residue C, and the 213.sup.th amino acid residue M of the sequence shown in SEQ ID NO:1 or the sequence having at least 76% identity with SEQ ID NO:1. The D-amino acid oxidative enzyme mutant has a higher enzyme activity, enzyme activity stability and/or ammonium resistance than a mild D-amino acid oxidative enzyme mutant. Also provided is an application of the D-amino acid oxidative enzyme mutant in preparing 2-oxo-4-(hydroxymethylphosphinyl)butyric acid.

Claims

1. A D-amino acid oxidase mutant, wherein the amino acid sequence of the D-amino acid oxidase mutant is SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 75, SEQ ID NO: 79, SEQ ID NO: 82, SEQ ID NO: 90, SEQ ID NO: 100, or SEQ ID NO: 102.

2. An isolated nucleic acid, wherein the nucleic acid encodes the D-amino acid oxidase mutant of claim 1.

3. A recombinant expression vector comprising the nucleic acid of claim 2.

4. A transformant comprising the nucleic acid of claim 2.

5. A method for preparing 2-oxo-4-(hydroxymethylphosphinyl) butyric acid comprising: using the D-amino acid oxidase mutant of claim 1 to prepare the 2-oxo-4-(hydroxymethylphosphinyl) butyric acid.

6. The nucleic acid of claim 2, wherein the nucleic acid is SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 101 or SEQ ID NO: 103.

7. A transformant comprising the recombinant expression vector of claim 3.

8. The D-amino acid oxidase mutant of claim 1, wherein the amino acid sequence of the D-amino acid oxidase mutant is SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 75, SEQ ID NO: 79, SEQ ID NO: 82 or SEQ ID NO: 90.

9. The D-amino acid oxidase mutant of claim 8, wherein the amino acid sequence of the D-amino acid oxidase mutant is SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 72, SEQ ID NO: 79, SEQ ID NO: 82 or SEQ ID NO: 90.

10. The D-amino acid oxidase mutant of claim 1, wherein the amino acid sequence of the D-amino acid oxidase mutant is SEQ ID NO: 100 or SEQ ID NO: 102.

11. The nucleic acid of claim 6, wherein the nucleic acid is SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO: 24, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89 or SEQ ID NO: 91.

12. The nucleic acid of claim 6, wherein the nucleic acid is SEQ ID NO: 64, SEQ ID NO: 73, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 89 or SEQ ID NO: 91.

13. The nucleic acid of claim 6, wherein the nucleic acid is SEQ ID NO: 101 or SEQ ID NO: 103.

Description

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

(1) The present invention is further illustrated by the following examples, but is not thereby limited to the scope described hereto. The experimental methods for which specific conditions are not indicated in the following examples are usually selected in accordance with the conventional methods and conditions, or in accordance with the instruction of commodity.

(2) The experimental methods in the present invention are conventional methods unless otherwise specified, and specific gene cloning operations can refer to “Molecular Cloning: A Laboratory Manual” edited by J. Sambrook et al.

(3) The abbreviations of amino acids in the present invention are conventional in the art unless otherwise specified, and the amino acids corresponding to the specific abbreviations are shown in Table 1.

(4) TABLE-US-00001 TABLE 1 Name of amino acid Three-letter symbol One-letter symbol alanine Ala A arginine Arg R asparagine Asn N aspartic acid Asp D cysteine Cys C glutamine Gln Q glutamic acid Glu E Glycine Gly G histidine His H isoleucine Ile I leucine Leu L lysine Lys K methionine Met M phenylalanine Phe F proline Pro P serine Ser S threonine Thr T tryptophan Trp W tyrosine Tyr Y valine Val V

(5) The codons corresponding to the amino acids are also conventional in the art, and the correspondence between specific amino acids and codons is shown in Table 2.

(6) TABLE-US-00002 TABLE 2 The first The second nucleotide The Third nucleotide T C A G nucleotide T phenylalanine F serine S tyrosine Y cysteine C T phenylalanine F serine S tyrosine Y cysteine C C leucine L serine S termination termination A codon codon leucine L serine S termination tryptophan W G codon 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

(7) Pet28a and bugbuster protein extraction reagent were purchased from Novagen; NdeI enzyme and HindIII enzyme were purchased from Thermo Fisher, BL21 competent cells were purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.; catalase was purchased from Shandong Fengtai Biotechnology Co., Ltd.

Example 1 Preparation of Wild-Type D-Amino Acid Oxidase (DAAO)

(8) Wild-type (wt) N.sub.2DAAO enzyme gene (with GenBank accession number KWU45700, from Rhodotorula sp. JG-1b) was fully synthesized by Suzhou GENEWIZ Biotechnology Co., Ltd. (Bio-Nano Technology Park Building B1 and C3, 218 Xinghu Road, Suzhou Industrial Park, Suzhou).

(9) Synthesized DAAO gene was ligated to pET28a (see J. Am. Chem. Soc., 2017, 139(32), 11241-11247 for specific methods), at restriction sites NdeI & HindIII, and the ligated vector was transformed into host E. coli BL21 (DE3) competent cells. The cells were inoculated in LB liquid medium and incubated in a shaker at 200 rpm, 37° C. When the OD.sub.600 reached about 0.8, the cells broth was added with sterile glycerin at a final concentration of 25%, numbered and stored in a low-temperature refrigerator at −80° C. for later use.

(10) The composition of LB liquid medium is as follows: peptone 10 g/L, yeast powder 5 g/L, NaCl 10 g/L, dissolved with deionized water and diluted to a final volume, sterilized at 121° C. for 20 minutes for later use.

(11) After resuscitating the engineered strain containing the enzyme gene which was store in a low-temperature refrigerator at −80° C. described above by streaking on a plate, a single colony was inoculated into 5 ml of LB liquid medium containing 50 μg/ml kanamycin, and incubated at 37° C. for 12 h with shaking. 2% of inoculum was transferred to 150 ml of fresh LB liquid medium containing 50 μg/ml kanamycin, then incubated at 37° C. with shaking until the OD.sub.600 value reached about 0.8, followed by cooling to 30° C. IPTG was added to a final concentration of 0.5 mM for induced culturing for 16 h. After cultivation, the culture solution was centrifuged at 10,000 rpm for 10 min, the supernatant was discarded, and the cells were collected and stored in an ultra-low temperature refrigerator at −20° C. for later use.

(12) After culturing, the cells were collected and washed twice with 50 mM phosphate buffer solution with pH 8.0, and then resuspended in phosphate buffer solution, pH 8.0, followed by lysing homogeneously. The lysis liquid was centrifuged to remove cell debris, thus obtaining a crude enzyme solution containing recombinant wtN.sub.2DAAO.

Example 2 Construction of D-Amino Acid Oxidase (DAAO) Mutant Library (Position 211, 213)

(13) After mutating positions 54 and 58 (specifically N54V, F58Q) on the wtN.sub.2DAAO sequence described in Example 1, a mutated D-amino acid oxidase sequence was obtained, and gene N.sub.2DAAO (N54V, F58Q) was synthesized according to the sequence by Suzhou GENEWIZ Biotechnology Co., Ltd. (Bio-Nano Technology Park Building B1 and C3, 218 Xinghu Road, Suzhou Industrial Park, Suzhou). Then the gene was ligated to plasmid pET28a at restriction sites NdeI and HindIII to construct plasmid pET28a-N.sub.2DAAO (see J. Am. Chem. Soc., 2017, 139(32), 11241-11247 for the plasmid construction method). The target band was amplified by PCR using plasmid pET28a-N2DAAO as template.

(14) Wherein, PCR primer sequences were designed for the construction of mutant libraries with mutations at positions 211 and 213 of the mutated D-amino acid oxidase sequence (N54V, F58Q), as shown in Table 3:

(15) TABLE-US-00003 TABLE 3 Mutation SEQ position ID No. and primer name Primer sequence NO: 1 211-213 forward AGCAACTGTAAACGCNNKAC 104 primer CNNKGACAGTAGTGACCCG 2 211-213 reverse CGGGTCACTACTGTCMNNGG 105 primer TMNNGCGTTTACAGTTGCT

(16) Wherein, N represents any of the nucleotides A, G, C and T, M represents A or C, and K represents G or T; it is selected according to the nucleotide encoding the amino acid to which the site needs to be mutated. For example, NNK in the A166-forward primer can represent AAG (lysine), AAT (aspartic acid), AGG (arginine) or AGT (serine), etc. The nucleotides corresponding to specific amino acids can be found in Table 2.

(17) PCR amplification system is as follows:

(18) TABLE-US-00004 Reagent Volume (μL) 2 × PCR buffer (Contains high-fidelity enzyme) 25 forward primer 1 reverse primer 1 template 1 deionized water 22

(19) PCR amplification procedure is as follows:

(20) TABLE-US-00005 95° C. 5 min 95° C. 30 s 50° C. 30 s {close oversize brace} 30 cycles 72° C. 5 min 72° C. 10 min 12° C. ∞

(21) PCR products were digested with DpnI at 37° C. for 2 h. After that, the digested product was transformed into BL21 competent cells, which were then spread on LB medium containing 50 μg/mL kanamycin, and incubated overnight at 37° C. Cells were harvested and transformants containing the mutant library were obtained.

Example 3 High-Throughput Screening of Mutant Libraries

(22) Screening was performed according to the following experimental steps:

(23) The transformants obtained in Example 2 were inoculated and incubated in 96-wells plate, inducing with IPTG at 30° C. overnight. The cells were then harvested, and lysed by bugbuster protein extraction reagent, thus obtaining the enzyme solution by centrifugation.

(24) Detection method of microplate reader is as follows: 100 μL of 100 mM substrate (racemic glufosinate, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) with a pH of 8.0 was added with 50 μL of chromogenic substrate solution (containing 60 mg/mL TBHBA (3-hydroxy-2,4,6-tribromobenzoic acid), 100 mg/mL 4-AAP (4-aminoantipyrine) and 25 μL HRP (horseradish peroxidase, 0.1 mg/mL), and finally added with 25 μL of the DAAO mutant enzyme solution described above, to obtain a 200 μL microplate reaction system. It was analyzed at 30° C., pH 8.0. The absorbance at 510 nm at 0 min and 20 min was recorded respectively, and the difference between the two absorbance values was taken to screen positive clones using wild type as a reference.

(25) The selected positive clones were cultured as follows:

(26) The composition of LB liquid medium is as follows: 10 g/L of peptone, 5 g/L of yeast powder and 10 g/L of NaCl were dissolved in deionized water and diluted to a final volume, and sterilized at 121° C. for 20 minutes for later use.

(27) A single colony was inoculated into 5 ml LB liquid medium containing 50 μg/ml kanamycin, and incubated at 37° C. for 12 h with shaking. 2% of inoculum was transferred to 150 ml fresh LB liquid medium containing 50 μg/ml kanamycin and incubated at 37° C. with shaking until OD.sub.600 value reached about 0.8. IPTG was added to a final concentration of 0.5 mM for induced culturing at 30° C. for 16 h. After cultivation, the culture solution was centrifuged at 10,000 rpm for 10 min, the supernatant was discarded, and the cells were collected and stored in an ultra-low temperature refrigerator at −20° C. for later use.

(28) The collected cells were washed twice with 50 mM phosphate buffer solution, pH 8.0, and then resuspended in phosphate buffer solution, pH 8.0, followed by lysing homogeneously at low temperature and high pressure. The lysis liquid was centrifuged to remove cell debris, thus obtaining a crude enzyme solution containing recombinant DAAO mutant.

(29) Detection method of enzyme activity for mutant re-screening is as follows:

(30) 1 mL of 500 mM D, L-Glufosinate ammonium salt, 0.25 mL of the preliminary crude enzyme solution containing recombinant DAAO mutant described above, 1.25 mL of horseradish peroxidase (HRP), and 2.5 mL of chromogenic substrate solution (containing 60 mg/mL TBHBA and 100 mg/mL 4-AAP) were added to form a 5 mL reaction system, with disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, pH 8.0, as the reaction medium. The reaction was performed in a shaker at 30° C. Every 2 minutes, the reaction solution was scanned at 510 nm for absorbance value, and the kinetic curve of the enzyme reaction was plotted for absorbance and time (min), thus calculating the enzyme activity according to the slope of the curve. The results are shown in Table 4.

(31) Definition of unit enzyme activity: under specific reaction conditions (30° C.), the amount of enzyme required to generate 1 μmol H.sub.2O.sub.2 per minute, wherein the unit of enzyme activity is U.

(32) Specific enzyme activity is the number of activity unit per milligram of enzyme protein, which is equal to enzyme activity/protein content, and the unit is U/mg or U/g.

(33) TABLE-US-00006 TABLE 4 Number of Specific enzyme Amino acid Nucleotide enzymes Mutation position activity (U/mg) SEQ ID NO: SEQ ID NO: 1 wt(N.sub.2DAAO) * 1 2 2 N.sub.2DAAO(N54V-F58Q-M213S) ** 5 6 3 N54V-F58Q-C211S-M213T ** 7 8 4 N54V-F58Q-C211A-M213T *** 9 10 5 N54V-F58Q-C211G-M213F *** 11 12 6 N54V-F58Q-C211M-M213R *** 13 14 7 N54V-F58Q-C211G-M213T *** 15 16 8 N54V-F58Q-C211A-M213R ** 17 18 9 N54V-F58Q-C211G-M213S *** 19 20 10 N54V-F58Q-C211D-M213V *** 21 22 11 N54V-F58Q-C211L-M213T **** 23 24 12 N54V-F58Q-C211A-M213C ** 25 26 13 N54V-F58Q-C211A-M213L ** 27 28 14 N54V-F58Q-C211G-M213L ** 29 30 15 N54V-F58Q-C211M-M213S *** 31 32 16 N54V-F58Q-C211A-M213S *** 33 34 17 N54V-F58Q-C211H-M213L *** 35 36 18 N54V-F58Q-C211S-M213C *** 37 38 19 N54V-F58Q-C211M-M213L *** 39 40 20 N54V-F58Q-C211S-M213L *** 41 42 21 N54V-F58Q-C211Y-M213F *** 43 44 22 N54V-F58Q-C211S-M213V *** 45 46 23 N54V-F58Q-C211M-M213T *** 47 48 24 N54V-F58Q-C211G-M213W *** 49 50 25 N54V-F58Q-C211A-M213A *** 51 52 26 N54V-F58Q-C211A-M213V *** 53 54 Note: in the above Table, * represents specific enzyme activity between 0-0.10 U/mg, ** represents specific enzyme activity between 0.1-1.0 U/mg, *** represents specific enzyme activity between 1.0-20 U/mg, **** represents specific enzyme activity between 20-50 U/mg.

(34) It can be seen from Table 4 that the specific enzyme activities of the obtained mutants were all higher than that of wild-type N.sub.2DAAO. Among them, the DAAO oxidase mutant 11 has the highest specific enzyme activity, which has such mutation: N at position 54 was mutated to V, F at position 58 was mutated to Q, C at position 211 was mutated to L and M at position 213 was mutated to T.

Example 4 Construction of a Mutant Library for Mutations at Positions 54, 56 and 58 of DAAO Oxidase Mutant 11 in Example 3

(35) The primer sequences designed for the construction of mutant library with mutations at positions 54, 56 and 58 of DAAO oxidase mutant 11 are shown in Table 5.

(36) TABLE-US-00007 TABLE 5 Mutation SEQ positions and ID No. name of primers Primer sequences NO: 1 54-56-58 CCCTTGGGCCGGTGCNNKTTGNN 106 forward primer KCCCNNKGATGAGCAAAGAAGC 2 54-56-58 GCTTCTTTGCTCATCMNNGGGMN 107 reverse primer NCAAMNNGCACCGGCCCAAGGG

(37) Wherein, N represents any of nucleotide A, G, C and T, M represents A or C, and K represents G or T; it is selected according to the nucleotide encoding the amino acid to which the site needs to be mutated. For example, NNK in the A166-forward primer can represent AAG (lysine), AAT (aspartic acid), AGG (arginine) or AGT (serine), etc. The nucleotides corresponding to specific amino acids can be found in Table 2.

(38) The plasmid template pET28a-DAAO oxidase mutant 12 was constructed according to the method disclosed in J. Am. Chem. Soc, 2017, 139(32), 11241-11247. The target band was amplified by PCR using plasmid pET28a-DAAO oxidase mutant 11 as template.

(39) The amplification reaction system is as follows:

(40) TABLE-US-00008 Reagent Volume (μL) 2 × PCR buffer (Contains high-fidelity enzyme) 25 forward primer 1 reverse primer 1 template 1 deionized water 22

(41) The amplification procedure is as follows:

(42) TABLE-US-00009 95° C. 5 min 95° C. 30 s 50° C. 30 s {close oversize brace} 30 cycles 72° C. 5 min 72° C. 10 min 12° C. ∞

(43) PCR products were digested with DpnI at 37° C. for 2 h. After the reaction, the digested product was transformed into BL21 competent cells, which was then spread on LB medium containing 50 μg/mL kanamycin. After incubating overnight at 37° C., the cells were harvested, and transformants containing the mutant library were obtained.

Example 5 Detections of the Stability of Enzyme Activity and the Influence of NH.SUB.4..SUP.+ Ion Concentration

(44) The transformants obtained in Example 4 was inoculated and incubated in a 96-well plate, inducing by IPTG at 30° C. overnight. After completing the induction, the cells were harvested and lysed with bugbuster protein extraction reagent, and centrifuged to obtain enzyme solution of the mutants.

(45) The enzyme solution of the mutants was treated at 50° C. for 2 h. According to the detection method of microplate reader described in Example 3, the thermostability effect of the mutations of the positive clones was analyzed for screening of the positive clones.

(46) Selected positive clones were cultured as follows:

(47) The composition of LB liquid medium is as follows: 10 g/L of peptone, 5 g/L of yeast powder and 10 g/L of NaCl were dissolved in deionized water and diluted to a final volume, and sterilized at 121° C. for 20 minutes for later use.

(48) A single colony was picked and inoculated into 5 ml LB liquid medium containing 50 μg/ml kanamycin, and incubated with shaking at 37° C. for 12 h. 2% of inoculum was transferred to 150 ml fresh LB liquid medium containing 50 μg/ml kanamycin and incubated with shaking at 37° C. until OD.sub.600 value reached about 0.8. IPTG was added to a final concentration of 0.5 mM for induced culturing at 30° C. for 16 h. After cultivation, the culture solution was centrifuged at 10,000 rpm for 10 min, the supernatant was discarded, and the cells were collected and stored in an ultra-low temperature refrigerator at −20° C. for later use.

(49) After cultivation, cells were collected and washed twice with 50 mM phosphate buffer solution, pH 8.0, and then resuspended in phosphate buffer solution, pH 8.0, followed by lysing homogeneously at low temperature and high pressure. The lysis liquid was centrifuged to remove cell debris, thus obtaining a crude enzyme solution containing recombinant DAAO mutant.

(50) The enzyme activity of the obtained crude enzyme solution containing DAAO mutant was detected for mutant re-screening by the same method described in Example 3. The results obtained are shown in Table 6, wherein the % value of remaining enzyme activity after 2 h reflects the stability of the mutant enzyme activity.

(51) TABLE-US-00010 TABLE 6 % value Enzyme of remaining No. of activity enzyme activity Amino acid Nucleotide enzyme Mutation position (U/ml) after 2 h SEQ ID NO: SEQ ID NO: 1 WT(N.sub.2DAAO) * # 1 2 11 N54V- T56T- F58Q- *** # 23 24 C211L-M213T 27 N54C- T56T- F58G- *** # 55 56 C211L-M213T 28 N54S- T56T- F58H- *** ## 57 58 C211L-M213T 29 N54G- T56T- F58K- *** ### 59 60 C211L-M213T 30 N54A- T56T- F58H- *** ### 61 62 C211L-M213T 31 N54A- T56T- F58K- ** ### 63 64 C211L-M213T 32 N54C- T56T- F58A- *** # 65 66 C211L-M213T 33 N54A- T56T- F58H- ** ### 61 67 C211L-M213T 34 N54G- T56T- F58A- * # 68 69 C211L-M213T 35 N54V- T56T- F58H- *** # 70 71 C211L-M213T 36 N54T- T56T- F58Q- **** ### 72 73 C211L-M213T 37 N54A- T56T- F58H- ** ### 61 74 C211L-M213T 38 N54A- T56T- F58Q- ** ## 75 76 C211L-M213T 39 N54A- T56T- F58K- ** ### 63 77 C211L-M213T 40 N54A- T56T- F58K- ** ### 63 78 C211L-M213T 41 N54A- T56N- F58H- **** ## 79 80 C211L-M213T 42 N54A- T56N- F58H- **** ### 79 81 C211L-M213T 43 N54A- T56S- F58H- **** ### 82 83 C211L-M213T 44 N54A- T56S- F58H- **** # 82 84 C211L-M213T 45 N54A- T56S- F58H- **** ### 82 85 C211L-M213T 46 N54A- T56T- F58H- **** ### 61 86 C211L-M213T 47 N54A- T56T- F58H- *** ## 61 87 C211L-M213T 48 N54A- T56S- F58H- *** ## 82 88 C211L-M213T 49 N54A- T56N- F58H- **** ### 79 89 C211L-M213T 50 N54A- T56L- F58H- **** ## 90 91 C211L-M213T Note: In the column of enzyme activity in the above Table, * represents the enzyme activity between 0-0.10 U/mg, ** represents the specific enzyme activity between 0.1-2.0 U/mg, *** represents the specific enzyme activity between 2.0-3.5 U/mg, and **** represents the specific enzyme activity between 3.5-10 U/mg. In the column of remaining enzyme activity after 2 h, # represents the remaining enzyme activity after 2 h is between 0-50%, ## represents the remaining enzyme activity after 2 h is between 50-80%, ### represents the remaining enzyme activity after 2 h is between 80-100%.

(52) Some mutants in the above Table (such as mutants 39 and 40, mutants 43-45, etc.) have the same amino acid sequence, but different DNA sequence, since their codons are not the same. The relevant amino acid sequences and DNA sequences are listed in the Sequence Listing.

(53) The results showed that all the above mutants except mutant 34 have higher enzyme activity and stability of enzyme activity than those of the wild type. Wherein, the enzyme activity of mutant 36, mutant 41, mutant 42, mutant 43, mutant 45, mutant 46, mutant 49 and mutant 50 is much greater than that of mutant 11, and the stability of enzyme activity improved a lot. In addition, it can be seen from the Table that although the mutants described above have the same amino acid sequence, their ammonium ions tolerance is different due to different nucleotide sequences.

(54) Then the mutants obtained above were tested for the influence of NH.sub.4.sup.+ ion concentration as follows:

(55) NH.sub.4Cl with a final concentration of 2 M was added into the 200 μL microplate reaction system in the detection method of microplate reader described in Example 3, and the enzyme activity before and after the addition of NH.sub.4Cl was analyzed to determine the influence of the NH.sub.4Cl concentration on the enzyme activity. The obtained enzyme activity data are shown in Table 7:

(56) TABLE-US-00011 TABLE 7 Ratio of remaining enzyme activity after No. of adding 2M Amino acid Nucleotide enzyme Mutation position NH.sub.4Cl SEQ ID NO: SEQ ID NO: 1 WT(N.sub.2DAAO) + 1 2 27 54C-56T-58G-C211L-M213T +++ 55 56 28 54S-56T-58H-C211L-M213T +++ 57 58 29 54G-56T-58K-C211L-M213T ++ 59 60 30 54A-56T-58H-C211L-M213T + 61 62 31 54A-56T-58K-C211L-M213T ++++ 63 64 32 54C-56T-58A-C211L-M213T ++ 65 66 33 54A-56T-58H-C211L-M213T + 61 67 35 54V-56T-58H-C211L-M213T + 70 71 36 54T-56T-58Q-C211L-M213T +++ 72 73 37 54A-56T-58H-C211L-M213T + 61 74 39 54A-56T-58K-C211L-M213T ++++ 63 77 40 54A-56T-58K-C211L-M213T ++++ 63 78 41 54A-56N-58H-C211L-M213T +++ 79 80 42 54A-56N-58H-C211L-M213T ++++ 79 81 43 54A-56S-58H-C211L-M213T +++ 82 83 44 54A-56S-58H-C211L-M213T ++++ 82 84 45 54A-56S-58H-C211L-M213T +++ 82 85 46 54A-56T-58H-C211L-M213T ++ 61 86 47 54A-56T-58H-C211L-M213T ++ 61 87 48 54A-56S-58H-C211L-M213T ++ 82 88 49 54A-56N-58H-C211L-M213T ++++ 79 89 50 54A-56L-58H-C211L-M213T ++ 90 91 Note: In the above table, + represents the ratio of remaining enzyme activity between 0-0.30, ++ represents the ratio of remaining enzyme activity between 0.3-0.5, +++ represents the ratio of remaining enzyme activity between 0.5-0.6, and ++++ represents the ratio of remaining enzyme activity between 0.6-1.

(57) The above results show that the above mutants have higher ammonium ions tolerance than the wild type. Among them, the enzyme activity, the stability of enzyme activity, and the ammonium ions tolerance of mutant 42 and mutant 49 were greatly improved compared with mutant 11. In addition, it can be seen from the Table that although the mutants described above have the same amino acid sequence, their ammonium ions tolerance is different due to different nucleotide sequences.

Example 6 Enzyme Activity of Other DAAO Enzymes with Combined Mutations at Positions 211 and 213

(58) The rtDAAO enzyme from Rhodosporidium toruloides UniProtKB/Swiss-Prot P80324 has 76% identity to the sequence of GenBank accession number KWU45700 (SEQ ID NO: 1), and the sequence of which is shown in SEQ ID NO:3. After mutating positions 54, 58 and 213 (specifically N54V, F58Q, M213 S) on the basis of this sequence, a mutated D-amino acid oxidase sequences was obtained, and the gene rtDAAO (N54V-F58Q-M213S) was synthesized according to this sequence. Gene was synthesized by the same company described above. Point mutation was performed in this rtDAAO (N54V-F58Q-M213S) enzyme, wherein position 54 was mutated to A, position 56 was mutated to N, position 58 was mutated to H, position 211 was mutated to L, and position 213 was mutated to T, thus obtaining mutants shown in Table 8. These obtained mutants were subjected to detection of enzyme activity for mutant re-screening, and the method was the same as that in Example 3. The calculated enzyme activity and specific enzyme activity data are shown in Table 8.

(59) The ammonium tolerance of the mutants was tested using the 200 μL microplate reaction system according to the detection method of microplate reader described in Example 3. NH.sub.4Cl was added to a final concentration of 2 M, and the enzyme activity before and after the addition of NH.sub.4Cl was analyzed to determine the influence of the NH.sub.4Cl concentration on the enzyme activity. The obtained enzyme activity data are shown in Table 9.

(60) TABLE-US-00012 TABLE 8 Specific Enzyme enzyme No. of activity activity Amino acid Nucleotide enzyme Mutation position (U/ml) (U/mg) SEQ ID NO: SEQ ID NO: 51 wt(rtDAAO) 0.015 0.009046 3 4 52 rtDAAO(N54V-F58Q-M213S) 20.49 3.21 92 93 53 rtDAAO(N54A-F58H) 2.96 3.69 94 95 54 rtDAAO(N54A-56N-F58H) 13.33 4.29 96 97 55 rtDAAO(C211L-M213T) 6.30 2.21 98 99 56 rtDAAO(N54A-F58H-C211L-M213T) 3.46 0.75 100 101 57 rtDAAO(N54A-T56N-F58H-C211L-M213T) 12.96 2.99 102 103

(61) TABLE-US-00013 TABLE 9 Ratio of remaining enzyme activity after No. of adding 2M Amino acid Nucleotide enzyme Mutation position NH.sub.4Cl SEQ ID NO: SEQ ID NO: 51 wt(rtDAAO) 0 3 4 52 rtDAAO(N54V-F58Q-M213S) 0.14 92 93 53 rtDAAO(N54A-F58H) 0.98 94 95 54 rtD AAO(N54A-T56N-F58H) 0.29 96 97 55 rtDAAO(C211L-M213T) 0.10 98 99 56 rtDAAO(N54A-F58H-C211L-M213T) 0.78 100 101 57 rtDAAO(N54A-T56N-F58H-C211L-M213T) 0.43 102 103

(62) It can be seen from the above Table that the ammonium tolerance of mutant 56 (N54A-F58H-C211L-M213T) is improved; the ammonium tolerance of mutant 57 (N54A-T56N-F58H-C211L-M213T) is improved with little change in enzyme activity.