Mutant, expression vector, host and application thereof

Abstract

The invention relates to the field of bioengineering, in particular to a mutant, an expression vector, a host and application thereof. The invention provides application of a mutation site as a target spot in preparation of alpha keto acid and / or L-glufosinate-ammonium. The mutation sites comprise one or more of the 52nd site, the 54th site, the 56th site, the 58th site, the 217th site, the 354th site and the 355th site of the D-amino acid oxidase. According to the invention, an active pocket of D-amino acid oxidase is modified, and a D-amino acid oxidase single-point or multi-point combined mutant with significantly enhanced catalytic activity on a catalytic substrate DL-glufosinate-ammonium is screened through multiple rounds of iterative saturated mutation. By utilizing the D-amino acid oxidase mutant provided by the invention, kinetic resolution of racemic glufosinate-ammonium is realized, and the D-amino acid oxidase mutant can be widely applied to production and application of L-glufosinate-ammonium.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering, and in particular to a mutant, an expression vector, a host and applications thereof. Background Art

[0002] Glufosinate (also known as Glufosinate), chemically known as 2-amino-4-(hydroxymethylphosphono)butyric acid, is the world's second-best-selling broad-spectrum contact herbicide. Upon entering the plant body, glufosinate inhibits glutamine synthetase activity by occupying the active site, thereby affecting plant nitrogen metabolism. Free ammonium cannot be metabolized, leading to excessive accumulation and ultimately plant death. Compared to other traditional lethal herbicides such as glyphosate and paraquat, glufosinate is a new natural herbicide product with advantages such as low toxicity, high efficacy, a broad spectrum of activity, and a long-lasting effect, gradually gaining market share.

[0003] Glufosinate is a racemate of D / L-glufosinate, with only L-glufosinate (fine glufosinate) having herbicidal activity. If the ineffective D-glufosinate can be removed and optically pure L-glufosinate can be produced, the amount of glufosinate used can be reduced by 50%, which can reduce pesticide costs and alleviate environmental pressure. At the same time, fine glufosinate is superior to glufosinate in terms of herbicidal speed, thoroughness, and stability, and is expected to gradually replace ordinary glufosinate in the future. To date, there have been many reports on the chemical synthesis of L-glufosinate, but the steps are lengthy, the synthetic route is complex, the yield is low, and the chiral resolution reagents are expensive. In contrast, biological methods have strict stereoselectivity, mild reaction conditions, high yield, and the product is easy to separate and purify. Exploring the biological synthesis of L-glufosinate has important commercial value and significant social benefits.

[0004] Amino acid oxidase (DAAO) is a typical flavin protease that uses flavin adenine dinucleotide (FAD) as a prosthetic group. This enzyme exhibits strong stereoisomer selectivity and a broad spectrum of substrates for catalytic reactions, making it widely used in qualitative and quantitative analysis of D-amino acids, biosensors, and the production of L-amino acids and α-keto acids. DAAO is widely present in the tissues and organs of vertebrates such as humans, mice, rabbits, and pigs, as well as in microorganisms such as algae, Neurospora, Aspergillus, bacteria, Trigonella protei, Saccharomyces cerevisiae, and Candida. Recent studies have shown that D-amino acid oxidase can catalyze the oxidation of D-glufosinate in the racemic glufosinate substrate to the corresponding α-keto acid, which is then converted to L-glufosinate by L-amino acid dehydrogenase or transaminase. Therefore, D-amino acid oxidase holds great promise for the production of L-glufosinate. However, currently available natural D-amino acid oxidases suffer from low or no catalytic activity. In view of this, further in-depth research is needed to solve the problem that the wild-type D-amino acid oxidase has no enzyme activity or low enzyme activity towards the substrate D-phosphinothricin through molecular modification. SUMMARY OF THE INVENTION

[0005] In view of this, the present invention provides mutants, expression vectors, hosts and their applications. By modifying the active pocket of D-amino acid oxidase and performing multiple rounds of iterative saturation mutagenesis, single-point or multi-point combinatorial mutants of D-amino acid oxidase with significantly enhanced catalytic activity towards the catalytic substrate DL-glufosinate are screened out. The kinetic resolution of racemic glufosinate is achieved using the D-amino acid oxidase mutants provided by the present invention, which can be widely applied to the production and application of L-glufosinate.

[0006] To achieve the above-mentioned invention objectives, the present invention provides the following technical solutions:

[0007] The present invention provides the application of the mutation site as a target in the preparation of L-glufosinate;

[0008] The mutation site includes one or more of the 52nd, 54th, 56th, 58th, 217th, 354th and 355th positions of D-amino acid oxidase.

[0009] The present invention also provides mutants of D-amino acid oxidase, and the mutations include: amino acid mutations and / or codon mutations;

[0010] The amino acid mutations are obtained by single-point or multi-point mutations at the 52nd, 54th, 56th, 58th, 217th, 354th and 355th positions of D-amino acid oxidase;

[0011] The codon mutations are obtained by mutations at one or more of the 154-156th, 160-162nd, 166-168th, 172-174th, 649-651st and 1060-1065th positions of the nucleic acid molecule encoding the D-amino acid oxidase.

[0012] In some embodiments of the present invention, in the above-mentioned mutant, the amino acid sequence of the D-amino acid oxidase is as shown in SEQ ID NO: 75: MAIDKRVVVLGTGVVGLSCGLVLSRQGYRVHFIARDLPEDSTSQG FASPWAGANWTPFYSRDEGPRQAKWEEATFARWVSLVPSGLAMWLNDTRRYADTDAGLLGHWYRDTVRNYRELPPSELPKGVAAGAAYDTLSVNAPLYCQALARELQTLGATFERRSVSSIEQVFEGQDDIALVVNATGLGAKSIAGIEDSACHPVRGQTVLVKSGCKRCTMDSSNPEAPAYIIPRPGGEVICGGTYLVDDWDLSPSASTAQRILTQCLALDPSISTDGTLDGIHILRHNVGLRPARTGGPRVEVGKLTLPLVRSTEPGTALALGTARPAPAGASSEAVGAPSEAVKREVTLVHAYGFSSAGYQQSWGVAQDVLGLVEGEIGPPRAWWTQRGKL. (Wp1DAAO from Rhodotorula gramini WP1)

[0013]

[0014] In some embodiments of the present invention, the mutant comprises: the G at position 52 of the D-amino acid oxidase is mutated to R; and / or

[0015] The G at position 52 of the D-amino acid oxidase is mutated to V; and / or

[0016] The N at position 54 of the D-amino acid oxidase is mutated to 1; and / or

[0017] The N at position 54 of the D-amino acid oxidase is mutated to S; and / or

[0018] The N at position 54 of the D-amino acid oxidase is mutated to L; and / or

[0019] The N at position 54 of the D-amino acid oxidase is mutated to G; and / or

[0020] The N at position 54 of the D-amino acid oxidase is mutated to V; and / or

[0021] The N at position 54 of the D-amino acid oxidase is mutated to Q; and / or

[0022] The N at position 54 of the D-amino acid oxidase is mutated to R; and / or

[0023] The T at position 56 of the D-amino acid oxidase is mutated to N; and / or

[0024] The T at position 56 of the D-amino acid oxidase is mutated to S; and / or

[0025] The T at position 56 of the D-amino acid oxidase is mutated to Q; and / or

[0026] The F at position 58 of the D-amino acid oxidase is mutated to K; and / or

[0027] The F at position 58 of the D-amino acid oxidase is mutated to H; and / or

[0028] The F at position 58 of the D-amino acid oxidase is mutated to R; and / or

[0029] The M at position 217 of the D-amino acid oxidase is mutated to I; and / or

[0030] The M at position 217 of the D-amino acid oxidase is mutated to T; and / or

[0031] The M at position 217 of the D-amino acid oxidase is mutated to V; and / or

[0032] The S at position 354 of the D-amino acid oxidase is mutated to G; and / or

[0033] The S at position 354 of the D - amino acid oxidase is mutated to P; and / or

[0034] The S at position 354 of the D - amino acid oxidase is mutated to A; and / or

[0035] The S at position 355 of the D - amino acid oxidase is mutated to G; and / or

[0036] The S at position 355 of the D - amino acid oxidase is mutated to A; and / or

[0037] The S at position 355 of the D - amino acid oxidase is mutated to P.

[0038] In some embodiments of the present invention, the above - mentioned mutants include: the C at position 156 of the gene encoding the D - amino acid oxidase is mutated to G; and / or

[0039] The C at position 156 of the gene encoding the D - amino acid oxidase is mutated to T; and / or

[0040] The C at position 1062 of the gene encoding the D - amino acid oxidase is mutated to T.

[0041] In some embodiments of the present invention, the above - mentioned mutants include: the C at position 156 of the gene encoding the D - amino acid oxidase is mutated to G; and

[0042] The N at position 54 of the D - amino acid oxidase is mutated to I; and

[0043] The T at position 56 of the D - amino acid oxidase is mutated to N; and

[0044] The F at position 58 of the D - amino acid oxidase is mutated to K; and

[0045] The M at position 217 of the D - amino acid oxidase is mutated to V; and

[0046] The S at position 354 of the D - amino acid oxidase is mutated to G; and

[0047] The S at position 355 of the D - amino acid oxidase is mutated to A.

[0048] In some embodiments of the present invention, the above - mentioned mutants have:

[0049] (1), an amino acid sequence as shown in any of SEQ ID NO:1 to SEQ ID NO:28; or

[0050] (2) An amino acid sequence obtained by substituting, deleting, or adding one or more amino groups to the amino acid sequence shown in (1), and having the same or similar function as the amino acid sequence shown in (1); or

[0051] (3) An amino acid sequence having at least 80% identity with the amino acid sequence shown in (1) or (2).

[0052] In some embodiments of the present invention, the sequence of SEQ ID NO:1 is: MAIDKRVVVLGTGVVGLSC GLVLSRQGYRVHFIARDLPEDSTSQGFASPWAGANWTPKYSRDEGPRQAKWEEATFARWVSLVPSGLAMWLNDTRRYADTDAGLLGHWYRDTVRNYRELPPSELPKGVAAGAAYDTLSVNAPLYCQALARELQTLGATFERRSVSSIEQVFEGQDDIALVVNATGLGAKSIAGIEDSACHPVRGQTVLVKSGCKRCTMDSSNPEAPAYIIPRPGGEVICGGTYLVDDWDLSPSASTAQRILTQCLALDPSISTDGTLDGIHILRHNVGLRPARTGGPRVEVGKLTLPLVRSTEPGTALALGTARPAPAGASSEAVGAPSEAVKREVTLVHAYGFSSAGYQQSWGVAQDVLGLVEGEIGPPRAWWTQRGKL. (Wp1DAAO-F58K)

[0053] In some embodiments of the present invention, the sequence of SEQ ID NO:2 is: MAIDKRVVVLGTGVVGLSC GLVLSRQGYRVHFIARDLPEDSTSQGFASPWAGANWTPHYSRDEGPRQAKWEEATFARWVSLVPSGLAMWLNDTRRYADTDAGLLGHWYRDTVRNYRELPPSELPKGVAAGAAYDTLSVNAPLYCQALARELQTLGATFERRSVSSIEQVFEGQDDIALVVNATGLGAKSIAGIEDSACHPVRGQTVLVKSGCKRCTMDSSNPEAPAYIIPRPGGEVICGGTYLVDDWDLSPSASTAQRILTQCLALDPSISTDGTLDGIHILRHNVGLRPARTGGPRVEVGKLTLPLVRSTEPGTALALGTARPAPAGASSEAVGAPSEAVKREVTLVHAYGFSSAGYQQSWGVAQDVLGLVEGEIGPPRAWWTQRGKL. (Wp1DAAO-F58H)

[0054] In some embodiments of the present invention, the sequence of SEQ ID NO:3 is: MAIDKRVVVLGTGVVGLSC GLVLSRQGYRVHFIARDLPEDSTSQGFASPWAGANWTPRYSRDEGPRQAKWEEATFARWVSLVPSGLAMWLNDTRRYADTDAGLLGHWYRDTVRNYRELPPSELPKGVAAGAAYDTLSVNAPLYCQALARELQTLGATFERRSVSSIEQVFEGQDDIALVVNATGLGAKSIAGIEDSACHPVRGQTVLVKSGCKRCTMDSSNPEAPAYIIPRPGGEVICGGTYLVDDWDLSPSASTAQRILTQCLALDPSISTDGTLDGIHILRHNVGLRPARTGGPRVEVGKLTLPLVRSTEPGTALALGTARPAPAGASSEAVGAPSEAVKREVTLVHAYGFSSAGYQQSWGVAQDVLGLVEGEIGPPRAWWTQRGKL. (Wp1DAAO-F58R)

[0055] 在本发明的一些实施方案中,SEQ ID NO:4的序列为:MAIDKRVVVLGTGVVGLSC GLVLSRQGYRVHFIARDLPEDSTSQGFASPWAGANWNPKYSRDEGPRQAKWEEATFARWVSLVPSGLAMWLNDTRRYADTDAGLLGHWYRDTVRNYRELPPSELPKGVAAGAAYDTLSVNAPLYCQALARELQTLGATFERRSVSSIEQVFEGQDDIALVVNATGLGAKSIAGIEDSACHPVRGQTVLVKSGCKRCTMDSSNPEAPAYIIPRPGGEVICGGTYLVDDWDLSPSASTAQRILTQCLALDPSISTDGTLDGIHILRHNVGLRPARTGGPRVEVGKLTLPLVRSTEPGTALALGTARPAPAGASSEAVGAPSEAVKREVTLVHAYGFSSAGYQQSWGVAQDVLGLVEGEIGPPRAWWTQRGKL。(Wp1DAAO-T56N / F58K)

[0056] 在本发明的一些实施方案中,SEQ ID NO:5的序列为:MAIDKRVVVLGTGVVGLSC GLVLSRQGYRVHFIARDLPEDSTSQGFASPWAGANWSPKYSRDEGPRQAKWEEATFARWVSLVPSGLAMWLNDTRRYADTDAGLLGHWYRDTVRNYRELPPSELPKGVAAGAAYDTLSVNAPLYCQALARELQTLGATFERRSVSSIEQVFEGQDDIALVVNATGLGAKSIAGIEDSACHPVRGQTVLVKSGCKRCTMDSSNPEAPAYIIPRPGGEVICGGTYLVDDWDLSPSASTAQRILTQCLALDPSISTDGTLDGIHILRHNVGLRPARTGGPRVEVGKLTLPLVRSTEPGTALALGTARPAPAGASSEAVGAPSEAVKREVTLVHAYGFSSAGYQQSWGVAQDVLGLVEGEIGPPRAWWTQRGKL。(Wp1DAAO-T56S / F58K)

[0057] In some embodiments of the present invention, the sequence of SEQ ID NO:6 is: MAIDKRVVVLGTGVVGLSC GLVLSRQGYRVHFIARDLPEDSTSQGFASPWAGANWQPKYSRDEGPRQAKWEEATFARWVSLVPSGLAMWLNDTRRYADTDAGLLGHWYRDTVRNYRELPPSELPKGVAAGAAYDTLSVNAPLYCQALARELQTLGATFERRSVSSIEQVFEGQDDIALVVNATGLGAKSIAGIEDSACHPVRGQTVLVKSGCKRCTMDSSNPEAPAYIIPRPGGEVICGGTYLVDDWDLSPSASTAQRILTQCLALDPSISTDGTLDGIHILRHNVGLRPARTGGPRVEVGKLTLPLVRSTEPGTALALGTARPAPAGASSEAVGAPSEAVKREVTLVHAYGFSSAGYQQSWGVAQDVLGLVEGEIGPPRAWWTQRGKL. (Wp1DAAO-T56Q / F58K)

[0058] In some embodiments of the present invention, the sequence of SEQ ID NO:7 is: MAIDKRVVVLGTGVVGLSC GLVLSRQGYRVHFIARDLPEDSTSQGFASPWAGANWNPKYSRDEGPRQAKWEEATFARWVSLVPSGLAMWLNDTRRYADTDAGLLGHWYRDTVRNYRELPPSELPKGVAAGAAYDTLSVNAPLYCQALARELQTLGATFERRSVSSIEQVFEGQDDIALVVNATGLGAKSIAGIEDSACHPVRGQTVLVKSGCKRCTIDSSNPEAPAYIIPRPGGEVICGGTYLVDDWDLSPSASTAQRILTQCLALDPSISTDGTLDGIHILRHNVGLRPARTGGPRVEVGKLTLPLVRSTEPGTALALGTARPAPAGASSEAVGAPSEAVKREVTLVHAYGFSSAGYQQSWGVAQDVLGLVEGEIGPPRAWWTQRGKL. (Wp1DAAO-T56N / F58K / M217I)

[0059] In some embodiments of the present invention, the sequence of SEQ ID NO:8 is: MAIDKRVVVLGTGVVGLSC GLVLSRQGYRVHFIARDLPEDSTSQGFASPWAGANWNPKYSRDEGPRQAKWEEATFARWVSLVPSGLAMWLNDTRRYADTDAGLLGHWYRDTVRNYRELPPSELPKGVAAGAAYDTLSVNAPLYCQALARELQTLGATFERRSVSSIEQVFEGQDDIALVVNATGLGAKSIAGIEDSACHPVRGQTVLVKSGCKRCTMDSSNPEAPAYIIPRPGGEVICGGTYLVDDWDLSPSASTAQRILTQCLALDPSISTDGTLDGIHILRHNVGLRPARTGGPRVEVGKLTLPLVRSTEPGTALALGTARPAPAGASSEAVGAPSEAVKREVTLVHAYGFSGAGYQQSWGVAQDVLGLVEGEIGPPRAWWTQRGKL. (Wp1DAAO-T56N / F58K / S354S / S355G)

[0060] In some embodiments of the present invention, the sequence of SEQ ID NO:9 is: MAIDKRVVVLGTGVVGLSC GLVLSRQGYRVHFIARDLPEDSTSQGFASPWAGANWNPKYSRDEGPRQAKWEEATFARWVSLVPSGLAMWLNDTRRYADTDAGLLGHWYRDTVRNYRELPPSELPKGVAAGAAYDTLSVNAPLYCQALARELQTLGATFERRSVSSIEQVFEGQDDIALVVNATGLGAKSIAGIEDSACHPVRGQTVLVKSGCKRCTMDSSNPEAPAYIIPRPGGEVICGGTYLVDDWDLSPSASTAQRILTQCLALDPSISTDGTLDGIHILRHNVGLRPARTGGPRVEVGKLTLPLVRSTEPGTALALGTARPAPAGASSEAVGAPSEAVKREVTLVHAYGFGAAGYQQSWGVAQDVLGLVEGEIGPPRAWWTQRGKL. (Wp1DAAO-T56N / F58K / S354G / S355A)

[0061] In some embodiments of the present invention, the sequence of SEQ ID NO:10 is: MAIDKRVVVLGTGVVGLS CGLVLSRQGYRVHFIARDLPEDSTSQGFASPWAGANWNPKYSRDEGPRQAKWEEATFARWVSLVPSGLAMWLNDTRRYADTDAGLLGHWYRDTVRNYRELPPSELPKGVAAGAAYDTLSVNAPLYCQALARELQTLGATFERRSVSSIEQVFEGQDDIALVVNATGLGAKSIAGIEDSACHPVRGQTVLVKSGCKRCTMDSSNPEAPAYIIPRPGGEVICGGTYLVDDWDLSPSASTAQRILTQCLALDPSISTDGTLDGIHILRHNVGLRPARTGGPRVEVGKLTLPLVRSTEPGTALALGTARPAPAGASSEAVGAPSEAVKREVTLVHAYGFGGAGYQQSWGVAQDVLGLVEGEIGPPRAWWTQRGKL. (Wp1DAAO-T56N / F58K / S354G / S355G)

[0062] In some embodiments of the present invention, the sequence of SEQ ID NO:11 is: MAIDKRVVVLGTGVVGLS CGLVLSRQGYRVHFIARDLPEDSTSQGFASPWAGANWNPKYSRDEGPRQAKWEEATFARWVSLVPSGLAMWLNDTRRYADTDAGLLGHWYRDTVRNYRELPPSELPKGVAAGAAYDTLSVNAPLYCQALARELQTLGATFERRSVSSIEQVFEGQDDIALVVNATGLGAKSIAGIEDSACHPVRGQTVLVKSGCKRCTMDSSNPEAPAYIIPRPGGEVICGGTYLVDDWDLSPSASTAQRILTQCLALDPSISTDGTLDGIHILRHNVGLRPARTGGPRVEVGKLTLPLVRSTEPGTALALGTARPAPAGASSEAVGAPSEAVKREVTLVHAYGFGPAGYQQSWGVAQDVLGLVEGEIGPPRAWWTQRGKL. (Wp1DAAO-T56N / F58K / S354G / S355P)

[0063] In some embodiments of the present invention, the sequence of SEQ ID NO:12 is: MAIDKRVVVLGTGVVGLS CGLVLSRQGYRVHFIARDLPEDSTSQGFASPWAGANWNPKYSRDEGPRQAKWEEATFARWVSLVPSGLAMWLNDTRRYADTDAGLLGHWYRDTVRNYRELPPSELPKGVAAGAAYDTLSVNAPLYCQALARELQTLGATFERRSVSSIEQVFEGQDDIALVVNATGLGAKSIAGIEDSACHPVRGQTVLVKSGCKRCTMDSSNPEAPAYIIPRPGGEVICGGTYLVDDWDLSPSASTAQRILTQCLALDPSISTDGTLDGIHILRHNVGLRPARTGGPRVEVGKLTLPLVRSTEPGTALALGTARPAPAGASSEAVGAPSEAVKREVTLVHAYGFPPAGYQQSWGVAQDVLGLVEGEIGPPRAWWTQRGKL. (Wp1DAAO-T56N / F58K / S354P / S355P)

[0064] In some embodiments of the present invention, the sequence of SEQ ID NO:13 is: MAIDKRVVVLGTGVVGLS CGLVLSRQGYRVHFIARDLPEDSTSQGFASPWAGAIWNPKYSRDEGPRQAKWEEATFARWVSLVPSGLAMWLNDTRRYADTDAGLLGHWYRDTVRNYRELPPSELPKGVAAGAAYDTLSVNAPLYCQALARELQTLGATFERRSVSSIEQVFEGQDDIALVVNATGLGAKSIAGIEDSACHPVRGQTVLVKSGCKRCTMDSSNPEAPAYIIPRPGGEVICGGTYLVDDWDLSPSASTAQRILTQCLALDPSISTDGTLDGIHILRHNVGLRPARTGGPRVEVGKLTLPLVRSTEPGTALALGTARPAPAGASSEAVGAPSEAVKREVTLVHAYGFSSAGYQQSWGVAQDVLGLVEGEIGPPRAWWTQRGKL. (Wp1DAAO-G52G / N54I / T56N / F58K)

[0065] In some embodiments of the present invention, the sequence of SEQ ID NO:14 is: MAIDKRVVVLGTGVVGLS CGLVLSRQGYRVHFIARDLPEDSTSQGFASPWAGASWNPKYSRDEGPRQAKWEEATFARWVSLVPSGLAMWLNDTRRYADTDAGLLGHWYRDTVRNYRELPPSELPKGVAAGAAYDTLSVNAPLYCQALARELQTLGATFERRSVSSIEQVFEGQDDIALVVNATGLGAKSIAGIEDSACHPVRGQTVLVKSGCKRCTMDSSNPEAPAYIIPRPGGEVICGGTYLVDDWDLSPSASTAQRILTQCLALDPSISTDGTLDGIHILRHNVGLRPARTGGPRVEVGKLTLPLVRSTEPGTALALGTARPAPAGASSEAVGAPSEAVKREVTLVHAYGFSSAGYQQSWGVAQDVLGLVEGEIGPPRAWWTQRGKL. (Wp1DAAO-G52G / N54S / T56N / F58K)

[0066] In some embodiments of the present invention, the sequence of SEQ ID NO:15 is: MAIDKRVVVLGTGVVGLS CGLVLSRQGYRVHFIARDLPEDSTSQGFASPWAGALWNPKYSRDEGPRQAKWEEATFARWVSLVPSGLAMWLNDTRRYADTDAGLLGHWYRDTVRNYRELPPSELPKGVAAGAAYDTLSVNAPLYCQALARELQTLGATFERRSVSSIEQVFEGQDDIALVVNATGLGAKSIAGIEDSACHPVRGQTVLVKSGCKRCTMDSSNPEAPAYIIPRPGGEVICGGTYLVDDWDLSPSASTAQRILTQCLALDPSISTDGTLDGIHILRHNVGLRPARTGGPRVEVGKLTLPLVRSTEPGTALALGTARPAPAGASSEAVGAPSEAVKREVTLVHAYGFSSAGYQQSWGVAQDVLGLVEGEIGPPRAWWTQRGKL. (Wp1DAAO-G52G / N54L / T56N / F58K)

[0067] In some embodiments of the present invention, the sequence of SEQ ID NO:16 is: MAIDKRVVVLGTGVVGLS CGLVLSRQGYRVHFIARDLPEDSTSQGFASPWAGAGWNPKYSRDEGPRQAKWEEATFARWVSLVPSGLAMWLNDTRRYADTDAGLLGHWYRDTVRNYRELPPSELPKGVAAGAAYDTLSVNAPLYCQALARELQTLGATFERRSVSSIEQVFEGQDDIALVVNATGLGAKSIAGIEDSACHPVRGQTVLVKSGCKRCTMDSSNPEAPAYIIPRPGGEVICGGTYLVDDWDLSPSASTAQRILTQCLALDPSISTDGTLDGIHILRHNVGLRPARTGGPRVEVGKLTLPLVRSTEPGTALALGTARPAPAGASSEAVGAPSEAVKREVTLVHAYGFSSAGYQQSWGVAQDVLGLVEGEIGPPRAWWTQRGKL. (Wp1DAAO-G52G / N54G / T56N / F58K)

[0068] In some embodiments of the present invention, the sequence of SEQ ID NO:17 is: MAIDKRVVVLGTGVVGLS CGLVLSRQGYRVHFIARDLPEDSTSQGFASPWAGAVWNPKYSRDEGPRQAKWEEATFARWVSLVPSGLAMWLNDTRRYADTDAGLLGHWYRDTVRNYRELPPSELPKGVAAGAAYDTLSVNAPLYCQALARELQTLGATFERRSVSSIEQVFEGQDDIALVVNATGLGAKSIAGIEDSACHPVRGQTVLVKSGCKRCTMDSSNPEAPAYIIPRPGGEVICGGTYLVDDWDLSPSASTAQRILTQCLALDPSISTDGTLDGIHILRHNVGLRPARTGGPRVEVGKLTLPLVRSTEPGTALALGTARPAPAGASSEAVGAPSEAVKREVTLVHAYGFSSAGYQQSWGVAQDVLGLVEGEIGPPRAWWTQRGKL. (Wp1DAAO-G52G / N54V / T56N / F58K)

[0069] In some embodiments of the present invention, the sequence of SEQ ID NO:18 is: MAIDKRVVVLGTGVVGLS CGLVLSRQGYRVHFIARDLPEDSTSQGFASPWAGAQWNPKYSRDEGPRQAKWEEATFARWVSLVPSGLAMWLNDTRRYADTDAGLLGHWYRDTVRNYRELPPSELPKGVAAGAAYDTLSVNAPLYCQALARELQTLGATFERRSVSSIEQVFEGQDDIALVVNATGLGAKSIAGIEDSACHPVRGQTVLVKSGCKRCTMDSSNPEAPAYIIPRPGGEVICGGTYLVDDWDLSPSASTAQRILTQCLALDPSISTDGTLDGIHILRHNVGLRPARTGGPRVEVGKLTLPLVRSTEPGTALALGTARPAPAGASSEAVGAPSEAVKREVTLVHAYGFSSAGYQQSWGVAQDVLGLVEGEIGPPRAWWTQRGKL. (Wp1DAAO-G52G / N54Q / T56N / F58K)

[0070] In some embodiments of the present invention, the sequence of SEQ ID NO:19 is: MAIDKRVVVLGTGVVGLS CGLVLSRQGYRVHFIARDLPEDSTSQGFASPWARAVWNPKYSRDEGPRQAKWEEATFARWVSLVPSGLAMWLNDTRRYADTDAGLLGHWYRDTVRNYRELPPSELPKGVAAGAAYDTLSVNAPLYCQALARELQTLGATFERRSVSSIEQVFEGQDDIALVVNATGLGAKSIAGIEDSACHPVRGQTVLVKSGCKRCTMDSSNPEAPAYIIPRPGGEVICGGTYLVDDWDLSPSASTAQRILTQCLALDPSISTDGTLDGIHILRHNVGLRPARTGGPRVEVGKLTLPLVRSTEPGTALALGTARPAPAGASSEAVGAPSEAVKREVTLVHAYGFSSAGYQQSWGVAQDVLGLVEGEIGPPRAWWTQRGKL. (Wp1DAAO-G52R / N54V / T56N / F58K)

[0071] In some embodiments of the present invention, the sequence of SEQ ID NO:20 is: MAIDKRVVVLGTGVVGLS CGLVLSRQGYRVHFIARDLPEDSTSQGFASPWAVARWNPKYSRDEGPRQAKWEEATFARWVSLVPSGLAMWLNDTRRYADTDAGLLGHWYRDTVRNYRELPPSELPKGVAAGAAYDTLSVNAPLYCQALARELQTLGATFERRSVSSIEQVFEGQDDIALVVNATGLGAKSIAGIEDSACHPVRGQTVLVKSGCKRCTMDSSNPEAPAYIIPRPGGEVICGGTYLVDDWDLSPSASTAQRILTQCLALDPSISTDGTLDGIHILRHNVGLRPARTGGPRVEVGKLTLPLVRSTEPGTALALGTARPAPAGASSEAVGAPSEAVKREVTLVHAYGFSSAGYQQSWGVAQDVLGLVEGEIGPPRAWWTQRGKL. (Wp1DAAO - G52V / N54R / T56N / F58K)

[0072] In some embodiments of the present invention, the sequence of SEQ ID NO:21 is: MAIDKRVVVLGTGVVGLS CGLVLSRQGYRVHFIARDLPEDSTSQGFASPWAGAIWNPKYSRDEGPRQAKWEEATFARWVSLVPSGLAMWLNDTRRYADTDAGLLGHWYRDTVRNYRELPPSELPKGVAAGAAYDTLSVNAPLYCQALARELQTLGATFERRSVSSIEQVFEGQDDIALVVNATGLGAKSIAGIEDSACHPVRGQTVLVKSGCKRCTTDSSNPEAPAYIIPRPGGEVICGGTYLVDDWDLSPSASTAQRILTQCLALDPSISTDGTLDGIHILRHNVGLRPARTGGPRVEVGKLTLPLVRSTEPGTALALGTARPAPAGASSEAVGAPSEAVKREVTLVHAYGFSSAGYQQSWGVAQDVLGLVEGEIGPPRAWWTQRGKL. (Wp1DAAO - G52G / N54I / T56N / F58K / M217T)

[0073] In some embodiments of the present invention, the sequence of SEQ ID NO:22 is: MAIDKRVVVLGTGVVGLS CGLVLSRQGYRVHFIARDLPEDSTSQGFASPWAGAIWNPKYSRDEGPRQAKWEEATFARWVSLVPSGLAMWLNDTRRYADTDAGLLGHWYRDTVRNYRELPPSELPKGVAAGAAYDTLSVNAPLYCQALARELQTLGATFERRSVSSIEQVFEGQDDIALVVNATGLGAKSIAGIEDSACHPVRGQTVLVKSGCKRCTVDSSNPEAPAYIIPRPGGEVICGGTYLVDDWDLSPSASTAQRILTQCLALDPSISTDGTLDGIHILRHNVGLRPARTGGPRVEVGKLTLPLVRSTEPGTALALGTARPAPAGASSEAVGAPSEAVKREVTLVHAYGFSSAGYQQSWGVAQDVLGLVEGEIGPPRAWWTQRGKL. (Wp1DAAO-G52G / N54I / T56N / F58K / M217V)

[0074] In some embodiments of the present invention, the sequence of SEQ ID NO:23 is: MAIDKRVVVLGTGVVGLS CGLVLSRQGYRVHFIARDLPEDSTSQGFASPWAGAIWNPKYSRDEGPRQAKWEEATFARWVSLVPSGLAMWLNDTRRYADTDAGLLGHWYRDTVRNYRELPPSELPKGVAAGAAYDTLSVNAPLYCQALARELQTLGATFERRSVSSIEQVFEGQDDIALVVNATGLGAKSIAGIEDSACHPVRGQTVLVKSGCKRCTIDSSNPEAPAYIIPRPGGEVICGGTYLVDDWDLSPSASTAQRILTQCLALDPSISTDGTLDGIHILRHNVGLRPARTGGPRVEVGKLTLPLVRSTEPGTALALGTARPAPAGASSEAVGAPSEAVKREVTLVHAYGFSSAGYQQSWGVAQDVLGLVEGEIGPPRAWWTQRGKL. (Wp1DAAO-G52G / N54I / T56N / F58K / M217I)

[0075] In some embodiments of the present invention, the sequence of SEQ ID NO:24 is: MAIDKRVVVLGTGVVGLS CGLVLSRQGYRVHFIARDLPEDSTSQGFASPWAGAIWNPKYSRDEGPRQAKWEEATFARWVSLVPSGLAMWLNDTRRYADTDAGLLGHWYRDTVRNYRELPPSELPKGVAAGAAYDTLSVNAPLYCQALARELQTLGATFERRSVSSIEQVFEGQDDIALVVNATGLGAKSIAGIEDSACHPVRGQTVLVKSGCKRCTMDSSNPEAPAYIIPRPGGEVICGGTYLVDDWDLSPSASTAQRILTQCLALDPSISTDGTLDGIHILRHNVGLRPARTGGPRVEVGKLTLPLVRSTEPGTALALGTARPAPAGASSEAVGAPSEAVKREVTLVHAYGFGPAGYQQSWGVAQDVLGLVEGEIGPPRAWWTQRGKL. (Wp1DAAO-G52G / N54I / T56N / F58K / S354G / S355P)

[0076] In some embodiments of the present invention, the sequence of SEQ ID NO:25 is: MAIDKRVVVLGTGVVGLS CGLVLSRQGYRVHFIARDLPEDSTSQGFASPWAGAIWNPKYSRDEGPRQAKWEEATFARWVSLVPSGLAMWLNDTRRYADTDAGLLGHWYRDTVRNYRELPPSELPKGVAAGAAYDTLSVNAPLYCQALARELQTLGATFERRSVSSIEQVFEGQDDIALVVNATGLGAKSIAGIEDSACHPVRGQTVLVKSGCKRCTMDSSNPEAPAYIIPRPGGEVICGGTYLVDDWDLSPSASTAQRILTQCLALDPSISTDGTLDGIHILRHNVGLRPARTGGPRVEVGKLTLPLVRSTEPGTALALGTARPAPAGASSEAVGAPSEAVKREVTLVHAYGFGAAGYQQSWGVAQDVLGLVEGEIGPPRAWWTQRGKL. (Wp1DAAO-G52G / N54I / T56N / F58K / S354G / S355A)

[0077] In some embodiments of the present invention, the sequence of SEQ ID NO:26 is: MAIDKRVVVLGTGVVGLS CGLVLSRQGYRVHFIARDLPEDSTSQGFASPWAGAIWNPKYSRDEGPRQAKWEEATFARWVSLVPSGLAMWLNDTRRYADTDAGLLGHWYRDTVRNYRELPPSELPKGVAAGAAYDTLSVNAPLYCQALARELQTLGATFERRSVSSIEQVFEGQDDIALVVNATGLGAKSIAGIEDSACHPVRGQTVLVKSGCKRCTMDSSNPEAPAYIIPRPGGEVICGGTYLVDDWDLSPSASTAQRILTQCLALDPSISTDGTLDGIHILRHNVGLRPARTGGPRVEVGKLTLPLVRSTEPGTALALGTARPAPAGASSEAVGAPSEAVKREVTLVHAYGFAAAGYQQSWGVAQDVLGLVEGEIGPPRAWWTQRGKL. (Wp1DAAO-G52G / N54I / T56N / F58K / S354A / S355A)

[0078] In some embodiments of the present invention, the sequence of SEQ ID NO:27 is: MAIDKRVVVLGTGVVGLS CGLVLSRQGYRVHFIARDLPEDSTSQGFASPWAGAIWNPKYSRDEGPRQAKWEEATFARWVSLVPSGLAMWLNDTRRYADTDAGLLGHWYRDTVRNYRELPPSELPKGVAAGAAYDTLSVNAPLYCQALARELQTLGATFERRSVSSIEQVFEGQDDIALVVNATGLGAKSIAGIEDSACHPVRGQTVLVKSGCKRCTTDSSNPEAPAYIIPRPGGEVICGGTYLVDDWDLSPSASTAQRILTQCLALDPSISTDGTLDGIHILRHNVGLRPARTGGPRVEVGKLTLPLVRSTEPGTALALGTARPAPAGASSEAVGAPSEAVKREVTLVHAYGFGAAGYQQSWGVAQDVLGLVEGEIGPPRAWWTQRGKL. (Wp1DAAO-G52G / N54I / T56N / F58K / M217T / S354G / S355A) In some embodiments of the present invention, the sequence of SEQ ID NO:28 is: MAIDKRVVVLGTGVVGLS CGLVLSRQGYRVHFIARDLPEDSTSQGFASPWAGAIWNPKYSRDEGPRQAKWEEATFARWVSLVPSGLAMWLNDTRRYADTDAGLLGHWYRDTVRNYRELPPSELPKGVAAGAAYDTLSVNAPLYCQALARELQTLGATFERRSVSSIEQVFEGQDDIALVVNATGLGAKSIAGIEDSACHPVRGQTVLVKSGCKRCTVDSSNPEAPAYIIPRPGGEVICGGTYLVDDWDLSPSASTAQRILTQCLALDPSISTDGTLDGIHILRHNVGLRPARTGGPRVEVGKLTLPLVRSTEPGTALALGTARPAPAGASSEAVGAPSEAVKREVTLVHAYGFGAAGYQQSWGVAQDVLGLVEGEIGPPRAWWTQRGKL. (Wp1DAAO-G52G / N54I / T56N / F58K / M217V / S354G / S355A) The present invention also provides a nucleic acid molecule encoding the above-mentioned mutant, characterized in that the nucleic acid molecule has:

[0079] (4) A nucleotide sequence as shown in any of SEQ ID NO:29 to SEQ ID NO:56; or

[0080] (5) A nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence as shown in (4), and having the same or similar function as the nucleotide sequence as shown in (4); or

[0081] (6) A nucleotide sequence having at least 80% identity with the nucleotide sequence as shown in (4) or (5).

[0082]

[0083]

[0084]

[0085]

[0086]

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[0090]

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[0101]

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[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110] The present invention also provides a primer set for amplifying the above nucleic acid molecule, and the primer set includes one or more of the following combinations:

[0111] Combination X:

[0112] (7), the upstream primer has a nucleotide sequence shown in SEQ ID No: (2X - 1); and

[0113] (8), the downstream primer has a nucleotide sequence shown in SEQ ID No: (2X); or

[0114] (9), a nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence shown in (7) or (8), and a nucleotide sequence having the same or similar function as the nucleotide sequence shown in (7) or (8); or

[0115] (10), a nucleotide sequence having at least 80% homology with the nucleotide sequence shown in (7) or (8);

[0116] wherein, X is any integer selected from 29 to 37.

[0117] The present invention also provides an expression vector, including: the above nucleic acid molecule and an acceptable element.

[0118] The present invention also provides a host, which is transformed and / or transfected with the above expression vector.

[0119] The present invention also provides the use of the above mutant, the above nucleic acid molecule, the above primer set, the above expression vector and / or the above host in the preparation of the α-keto acid and / or L-glufosinate.

[0120] The present invention also provides a method for preparing α-keto acid and / or L-glufosinate. Using D-amino acid or racemic amino acid as a substrate, the above mutant or the above host is subjected to an oxidation reaction to obtain the α-keto acid and / or the L-glufosinate.

[0121] In some embodiments of the present invention, in the above method, the concentration of the substrate is 0.2 mol / L.

[0122] In some embodiments of the present invention, in the above method, the temperature of the oxidation reaction is 30 °C, the time is 2 h, and the pH value is 8.0.

[0123] The present invention provides the use of the mutation site as a target in the preparation of α-keto acid and / or L-glufosinate;

[0124] The mutation sites include one or more of the 52nd, 54th, 56th, 58th, 217th, 354th, and 355th positions of D - amino acid oxidase.

[0125] The beneficial effects of the present invention include:

[0126] (1) Based on the D - amino acid oxidase (Wp1DAAO, NCBI accession number: XP_018272978.1) derived from Rhodotorula graminis WP1, the present invention solves the problem of its low enzyme activity towards the substrate D - glufosinate by a rational design - based molecular modification method, and obtains mutants that can catalyze the preparation of α - keto acid and D - glufosinate.

[0127] (2) The rational design method used in the present invention can quickly obtain D - amino acid oxidase mutants with high catalytic activity towards the D - glufosinate substrate through screening with a relatively small mutant library.

[0128] (3) Using D - glufosinate as the substrate, the method of the present invention utilizes D - amino acid oxidase for oxidation reaction and kinetic resolution to obtain the corresponding keto acid and L - glufosinate. The process is simple, the reaction conditions are mild, the catalyst cost is low, and the process is highly efficient and green. It is an ideal scheme for the preparation of L - glufosinate and can be widely applied in the market. Description of the Drawings

[0129] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art.

[0130] Figure 1 Showing the high - performance liquid chromatography (HPLC) spectrum of the 2 - oxo - 4 - (hydroxymethylphosphinyl) butyric acid (PPO) standard.

[0131] Figure 2 Showing the HPLC spectra of the D,L - glufosinate standard and the L - glufosinate standard.

[0132] Figure 3 Showing the protein electrophoresis patterns of the wild - type and mutant D - amino acid oxidase gene - engineered bacteria.

[0133] Figure 4 Showing the protein electrophoresis patterns of the purified wild - type and mutant D - amino acid oxidase.

[0134] Figure 5 Showing the technical route diagram for the preparation of L - glufosinate by multi - enzyme cascade reaction.

[0135] Figure 6 Showing the changes in the contents of D - glufosinate and L - glufosinate and the ee value during the multi - enzyme cascade reaction. Detailed Embodiments

[0136] The present invention discloses mutants, expression vectors, hosts and their applications.

[0137] It should be understood that the expression "one or more of..." individually includes each of the objects recited after the expression and various different combinations of two or more of the recited objects, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited objects should be understood to have the same meaning, unless otherwise understood from the context.

[0138] The terms "comprising", "having" or "containing", including the use of their grammatical synonyms, should generally be understood as open-ended and non-limiting, e.g., not excluding other unrecited elements or steps, unless specifically stated otherwise or understood from the context.

[0139] It should be understood that the order of steps or the order of performing certain actions is not important as long as the present invention remains operable. In addition, two or more steps or actions can be performed simultaneously.

[0140] The use of any and all examples or exemplary language such as "for example" or "including" in this document is merely intended to better illustrate the present invention and does not limit the scope of the present invention unless a claim is made. No language in this specification should be construed as indicating that any unclaimed element is essential for the practice of the present invention.

[0141] In addition, the numerical ranges and parameters used to define the present invention are approximate values. The relevant values in the specific embodiments have been presented as precisely as possible herein. However, any numerical value inherently and inevitably contains standard deviations resulting from individual testing methods. Therefore, unless otherwise clearly stated, it should be understood that all ranges, quantities, numerical values and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0142] The present invention provides a D - amino acid oxidase variant, which is obtained by single - point mutation or multi - point combined mutation at positions 52, 54, 56, 58, 217, 354, and 355 of the amino acid sequence shown in SEQ ID NO.1.

[0143] The present invention performs homology modeling on D - amino acid oxidase (Wp1DAAO, NCBI accession number: XP_018272978.1, the amino acid sequence is shown in SEQ ID NO:75, and the nucleotide sequence is shown in SEQ ID NO:76) derived from Rhodotorula graminis WP1, conducts molecular docking with the substrate D - glufosinate, and selects 13 key amino acid residues within the side chain group of D - glufosinate to mutate them into alanine or the smaller glycine to expand its pocket volume or change the pocket electrostatic property, constructs a D - amino acid oxidase mutant library by iterative saturation mutagenesis, and screens the optimal combination mutant strains of D - amino acid oxidase mutants for catalytic preparation of PPO.

[0144] Furthermore, the D - amino acid oxidase variant is one of the following single - point mutations or multi - point combination mutations:

[0145] The present invention provides nucleic acids encoding D - amino acid oxidase, which have at least one of the following sequences:

[0146] (Ⅰ) F58K, F58H, F58R;

[0147] (Ⅱ) T56N / F58K, T56S / F58K, T56Q / F58K, T56N / F58K / M217I, T56N / F58K / S354S / S355G, T56N / F58K / S354G / S355A, T56N / F58K / S354G / S355G, T56N / F58K / S354G / S355P, T56N / F58K / S354P / S355P, G52G / N54I / T56N / F58K, G52G / N54S / T56N / F58K, G52G / N54L / T56N / F58K, G52G / N54G / T56N / F58K, G52G / N54V / T56N / F58K, G52G / N54Q / T56N / F58K, G52R / N54V / T56N / F58K, G52V / N54R / T56N / F58K;

[0148] (Ⅲ) G52G / N54I / T56N / F58K / M217T, G52G / N54I / T56N / F58K / M217V, G52G / N54I / T56N / F58K / M217I, G52G / N54I / T56N / F58K / S354G / S355P, G52G / N54I / T56N / F58K / S354G / S355A, G52G / N54I / T56N / F58K / S354A / S355A;

[0149] (Ⅳ) G52G / N54I / T56N / F58K / M217T / S354G / S355A, G52G / N54I / T56N / F58K / M217V / S354G / S355A.

[0150] In the above text, " / " means "and", that is, the two sites before and after " / " mutate simultaneously; for example: T56N / F58K means that the 56th threonine mutates to asparagine, and the 58th amino acid mutates from phenylalanine to lysine; G52V / N54R / T56N / F58K means that the 52nd glycine mutates to valine, the 54th amino acid mutates from asparagine to arginine, the 56th amino acid mutates from threonine to asparagine, and the 58th amino acid mutates from phenylalanine to lysine.

[0151] The present invention also provides a coding gene for the D - amino acid oxidase variant described in any one of the above.

[0152] The present invention also provides an expression vector containing the coding gene described above. Preferably, the original expression vector is pET - 28a(+).

[0153] The present invention also provides a genetically engineered bacterium containing the coding gene described above. Preferably, the host cell of the genetically engineered bacterium is E.coli BL21(DE3).

[0154] The present invention also provides the use of the D - amino acid oxidase variant described above in the preparation of α - keto acid and L - glufosinate.

[0155] The present invention also provides the use of the genetically engineered bacterium described above in the preparation of α - keto acid and L - glufosinate.

[0156] The present invention also provides a preparation method of α - keto acid and L - glufosinate, including using the D - amino acid oxidase described in the present invention to carry out an oxidation reaction with D - amino acid or racemic amino acid as the substrate. The D - amino acid oxidase is obtained by fermenting and culturing a host that integrates the nucleic acid or expression unit of the D - amino acid oxidase gene of the present invention into its genome, or by fermenting and culturing a host transfected or transformed with the plasmid vector or plasmid combination described in the present invention.

[0157] Specifically, in the preparation process of the α - keto acid, the substrate concentration in the oxidation reaction system is 0.2 mol / L, the reaction temperature is 30 °C, the time is 2 h, and the pH value of the reaction solution is 8.0.

[0158] Specifically, in the preparation process of L-glufosinate, the substrate concentration in the reaction system is 0.2 mol / L, the reaction temperature is 40 °C, the isopropanol concentration is 0.3 mol / L, the NADP+ concentration is 0.2 mMol / L, the dry cell weight of glutamate dehydrogenase Pp GluDH is 0.1 g / L, the dry cell weight of alcohol dehydrogenase BsADH is 0.2 g / L, the dry cell weight of D-amino acid oxidase and catalase MaCAT is 10 g / L, and the pH value of the reaction solution is 8.0.

[0159] The experimental methods in the present invention are all conventional methods unless otherwise specified. For gene cloning operations, reference can be specifically made to "Molecular Cloning: A Laboratory Manual" edited by J. Sambrook et al.

[0160] Reagents used in upstream genetic engineering: DPNI used in the examples of the present invention was purchased from TaKaRa, Takara Bio Inc. (Dalian); plasmid extraction kits and DNA recovery and purification kits were purchased from Axygen Hangzhou Co., Ltd.; E. coli BL21(DE3), plasmid pET-28a(+), etc. were purchased from Novagen; DNA marker, low molecular weight standard protein, and agarose electrophoresis reagents were purchased from Beijing TransGen Biotech Co., Ltd.; primer synthesis and sequence sequencing work were completed by Tsingke Biotechnology Co., Ltd. The usage methods of the above reagents refer to the product manuals. In the text of this application, the three-letter or single-letter expressions of amino acids adopt the amino acid codes specified by IUPAC (Eur. J. Biochem., 138: 9-37, 1984).

[0161] The concentration of PPO was detected by high performance liquid chromatography (HPLC). The specific method was as follows: Chromatographic conditions: Column model: P QS-C18, 5 μm, 4.6 mm × 250 mm. Mobile phase: 50 mM diammonium hydrogen phosphate solution, added with 0.9% of 10% tetrabutylammonium hydroxide aqueous solution, adjusted to pH 3.6 with 50% phosphoric acid solution (mass fraction), and added with 8% acetonitrile. Detection wavelength: 205 nm. Flow rate: 0.8 mL / min. Column temperature: 40 °C.

[0162] The optical purity of glufosinate was detected by high performance liquid chromatography (HPLC). The specific method was as follows: Pre-column derivation was used for determination. Derivatization reaction and determination: Take 100 μL of the sample and add 100 μL of the derivatization reagent, mix well and incubate at 25 °C for 5 min. Chromatographic conditions: QS-C18; Detection wavelength: 338 nm; Column temperature: 30 °C; Injection volume: 20 μL; Mobile phase: 50 mM sodium acetate aqueous solution: acetonitrile = 9:0.5; Flow rate: 1 mL / min.

[0163] 0.2 M Boric Acid Buffer: Weigh 7.62 g of sodium tetraborate decahydrate, and make up to 100 mL with deionized water for later use.

[0164] Derivatization Reagent: Weigh 0.03 g of o-phthalaldehyde and 0.1 g of N-acetyl-L-cysteine, add 400 μL of absolute ethanol and 4 mL of boric acid buffer, and ultrasonically dissolve it completely. Prepare it immediately before use.

[0165] In Examples 1 to 6 and the comparative example of the present invention, the raw materials and reagents used can all be purchased from the market.

[0166] The present invention will be further described below in conjunction with examples:

[0167] Construction and Screening of an Iterative Saturation Mutation Library for Substrate D-GLUFOSINATE in Example 1

[0168] Thirteen sites close to the target sites of Wp1DAAO were divided into a group, with a total of 8 groups, namely G52 / N54, T56 / F58, M217, Y227 / I229, T241 / Y242, R289, S354 / S355, Q395, and one-point or two-point saturation mutations were carried out.

[0169] 1. Whole Plasmid PCR

[0170] Using the pET-28a(+)-Wp1DAAO plasmid as a template, design upstream and downstream primers covering the mutation points (Table 1) for whole plasmid PCR

[0171] Table 1 Primers Required for the Construction of a Single-Point or Two-Point Combined Saturation Mutation Library

[0172]

[0173] PCR Amplification System:

[0174] DNA polymerase 25 μL;

[0175] Upstream primer (10 pmol / μL) 1.5 μL;

[0176] Downstream primer (10 pmol / μL) 1.5 μL;

[0177] Template 1.0 μL;

[0178] ddH2O 21 μL.

[0179] PCR Amplification Conditions:

[0180] 1) Pre-denaturation: 95°C for 5 min;

[0181] 2) Denaturation: 98°C for 10 s; Annealing: 58°C for 15 s; Extension: 72°C for 90 s; A total of 30 cycles;

[0182] 3) Post-extension: 72°C for 10 min;

[0183] 4) Store at 4°C.

[0184] 2. Template digestion:

[0185] Perform agarose gel electrophoresis on the PCR product. After recovery, digest the plasmid template in it with DpnⅠ enzyme. The digestion system is: 1 μL of DpnⅠ enzyme, 17 μL of PCR product, and 2 μL of Buffer. The digestion of the template can be completed in 2 hours at 37°C.

[0186] 3. Transformation and verification:

[0187] After verifying the digested product by nucleic acid agarose gel electrophoresis, transform Escherichia coli BL21(DE3) competent cells using the heat shock method at 42°C. The specific process is as follows:

[0188] (1) Place the competent cells on ice to thaw for 15 min;

[0189] (2) Add 10 μL of DNA to 100 μL of competent cells in a sterile environment and mix gently, then place on ice for 30 min;

[0190] (3) Place the EP tube in a 42°C metal bath for heat shock for 90 s, and then place it on ice to cool for 2 min after completion;

[0191] (4) Add 800 μL of LB medium to the EP tube and mix with a pipette tip, then incubate in a shaker at 200 rpm at 37°C for 40 - 60 min;

[0192] (5) After concentration, take an appropriate volume and spread it on the corresponding resistant plate, and colonies can appear after culturing in a 37°C incubator for 12 - 16 h.

[0193] 4. Primary screening

[0194] Add 200 μL of LB medium (containing 100 μg / mL kanamycin) to a sterilized 96-well deep plate. Use a sterilized pipette tip to pick a single colony into the 96-well deep plate. Then place the deep plate at 37°C and 220 rpm for 8 h, which is called the primary plate. Add 400 μL of LB medium (containing 100 μg / mL kanamycin) to another sterilized 96-well plate as the secondary plate. Pipette 50 μL of the bacterial liquid from the primary plate into the secondary plate, and add 20% glycerol to the primary plate and store it in a -80°C refrigerator for long-term preservation. Then place the secondary plate at 37°C for shaking culture for 2 h, add IPTG with a final concentration of 0.5 mM for induction, and then place the secondary plate at 18°C and 220 rpm for continued culture for 16 h.

[0195] The secondary plate was centrifuged at 4000 rpm and 4 °C for 20 min to collect cells, which were then placed at -80 °C for overnight freezing. The secondary plate was taken out from -80 °C and thawed at room temperature for 0.5 h. Then, 300 μL of lysing solution (10 mM phosphate buffer at pH 8.0, 750 mg / L lysozyme, 10 mg / L DNase I) was added to each well, and the cells were suspended by shaking and incubated in a 37 °C shaker at 220 rpm for 1 h. After incubation, it was centrifuged at 4000 rpm and 4 °C for 20 min, and the supernatant was taken for enzyme activity assay.

[0196] Prepare the enzyme activity assay solution (100 mL) for screening: 50 mL of chromogenic reagent (160 μg / mL 2,4,6-tribromo-3-hydroxybenzoic acid, 1 mg / mL 4-aminoantipyrine, 20 mg peroxidase), 50 mL of 50 mmol glufosinate (pH 8.0). The above substances were all prepared with 50 mM phosphate buffer at pH 8.0. Add 100 μL of the enzyme activity assay solution to each well of a new 96-well plate (reaction plate) and keep it at 30 °C for 10 min. Pipette 120 μL of enzyme solution into the reaction plate to start the reaction. After a certain time, measure the absorbance at 510 nm with an enzyme-linked immunosorbent assay reader. The higher the absorbance value, the higher its catalytic activity. Select the mutant strains with a significantly increased absorbance value compared to the control (wild type) as candidate strains for rescreening.

[0197] 5. Rescreening

[0198] Use HPLC to rescreen the mutants that showed a significant increase in enzyme activity in the primary screening. After the corresponding mutant strains on the primary plate were activated by streaking on a plate, single colonies were picked and inoculated into 5 mL of LB liquid medium containing 100 μg / mL kanamycin, and cultured with shaking at 37 °C for 12 h. Transferred at an inoculation amount of 2% to 50 mL of LB liquid medium containing 100 μg / mL kanamycin as well, and cultured with shaking at 37 °C until the OD 600 reached about 0.6 - 0.8, then IPTG was added to a final concentration of 0.5 mM, and induced to culture at 18 °C for 16 h. After the culture was completed, the culture solution was centrifuged at 4000 g and 4 °C for 10 min, the supernatant was discarded, and the cells were collected. The collected cells were washed twice with 100 mM phosphate buffer at pH 8.0, then resuspended in phosphate buffer, and ultrasonically disrupted 30 times at a power of 400 W, with each ultrasonic treatment lasting for 3 s and an interval of 7 s. The cell lysate was centrifuged at 12000 g and 4 °C for 10 min to remove the precipitate, and the obtained supernatant was the crude enzyme solution.

[0199] 6. Enzyme activity assay of recombinant mutant D-amino acid oxidase

[0200] Using DL-glufosinate as a substrate, the enzyme activity of recombinant D-amino acid oxidase was detected. The assay system was as follows: The total reaction system was 2 mL, including 1 mL of 200 mM DL-glufosinate solution and 1 mL of crude enzyme solution. All of the above substances were prepared with 100 mM phosphate buffer at pH 8.0. The reaction was shaken at 30 °C for 2 h, and the reaction was terminated by adding 200 μL of 4 M HCl solution. The reaction mixture was centrifuged at 4000 rpm for 10 min to remove cells and enzyme proteins. High-performance liquid chromatography was used to determine the PPO generated in the reaction system. Enzyme activity definition: At 30 °C, the amount of enzyme that can convert the substrate to produce 1 μmol of product in 1 min is defined as 1 U. The activities of all positive mutant D-amino acid oxidases were determined.

[0201] 7. Detection of soluble expression of D-amino acid oxidase gene engineering bacteria

[0202] The cell lysates of the gene engineering bacteria after ultrasonic disruption in step 5 were unified for OD 600 Then, it was centrifuged at 12000 rpm and 4 °C for 10 min. 15 μL of the supernatant was mixed with 5 μL of 4×SDS-PAGE Loading Buffer; the supernatant was discarded, and the precipitate was resuspended with the same volume of buffer, and then 15 μL of the resuspended solution was mixed with 5 μL of 4×SDS-PAGE Loading Buffer. All protein electrophoresis samples were incubated at 99 °C for 10 min. 10 μL of protein samples were taken for protein gel electrophoresis.

[0203] All results were analyzed by independent T-test and the P value was calculated. Among them, "n.s" indicates no significant difference compared with the control group strain, "*" indicates P < 0.05 compared with the control group strain, "**" indicates P < 0.01 compared with the control group strain, and "***" indicates P < 0.001 compared with the control group strain.

[0204] According to steps 1-4, high-throughput screening of 13 sites of wild-type Wp1DAAO was carried out, and 6 positive mutants were obtained. According to steps 5 and 6, their enzyme activities were verified, and the results are shown in Table 2.

[0205] Table 2 Screening results of positive mutants in the saturation mutation library of wild-type Wp1DAAO target sites

[0206]

[0207] As can be seen from the screening results in Table 2, positive mutations occurred only at the T56 / F58 sites. Among them, the enzyme activity per unit OD of T56N / F58K was the highest, and there was a significant increase in enzyme activity compared with wild-type Wp1DAAO (P < 0.001), which was 2.1 times that of wild-type Wp1DAAO.

[0208] Example 2 Iterative saturation mutagenesis based on T56N / F58K

[0209] Using Wp1DAAO-T56N / F58K as a template, saturation mutagenesis was performed on the remaining 11 target sites, and the updated primers are shown in Table 3.

[0210] Table 3 Primers required for constructing the combined saturation mutagenesis library at G52 / N54

[0211]

[0212] The mutant library was constructed and screened according to the methods in Steps 1 to 4 of Example 1, and the expression of the positive mutant D - amino acid oxidase and the enzyme activity assay were carried out according to the methods in Steps 5 and 6 of Example 1. Finally, 24 mutants with significantly improved enzyme activity were obtained, and the results are shown in Table 4.

[0213] Table 4 Screening results of positive mutants in the iterative saturation mutagenesis library based on T56N / F58K

[0214]

[0215]

[0216] Among the saturation mutagenesis results of the 11 target sites, there were 24 positive strains at G52 / N54, M217, and S354 / S355. After HPLC verification, as shown in Table 4, the G52G / N54I / T56N / F58K mutant had the highest enzyme activity. The crude enzyme activity was 7.83 times higher than that of the wild - type Wp1DAAO and showed a significant increase in enzyme activity per unit OD compared to the starting strain T56N / F58K (P < 0.001), which was 3.43 times its enzyme activity. In the codons of the G52G / N54I / T56N / F58K mutant, the GGC at position 52 was mutated to GGG, and the AAC at position 54 was mutated to ATT, and it was named Wp1 - 4.

[0217] Example 3 Iterative saturation mutagenesis based on G52G / N54I / T56N / F58K

[0218] Using Wp1DAAO - G52G / N54I / T56N / F58K as a template, saturation mutagenesis was performed on the remaining 9 target sites. The mutant library was constructed and screened according to the methods in Steps 1 to 4 of Example 1, and the expression of the positive mutant D - amino acid oxidase and the enzyme activity assay were carried out according to the methods in Steps 5 and 6 of Example 1. Finally, 9 mutants with significantly improved enzyme activity were obtained, and the results are shown in Table 5.

[0219] Table 5 Screening results of positive mutants in the iterative saturation mutagenesis library based on G52G / N54I / T56N / F58K

[0220]

[0221] Among the results of saturation mutagenesis at 9 target sites, there were 9 positive strains at M217 and S354 / S355. After HPLC verification, as shown in Table 5, the mutant strain G52G / N54I / T56N / F58K / S354G / S355A had the highest enzyme activity per unit OD. The crude enzyme activity was 15.58 times higher than that of the wild-type Wp1DAAO and was significantly higher than that of the starting strain G52G / N54I / T56N / F58K in terms of enzyme activity per unit OD (P<0.001), being 0.39 times its enzyme activity. Among them, the codon at position 354 of the G52G / N54I / T56N / F58K / S354G / S355A mutant strain changed from AGC to GGG, and the codon at position 355 changed from TCT to GCG, and it was named Wp1-6.

[0222] Example 4 Iterative saturation mutagenesis based on G52G / N54I / T56N / F58K / S354G / S355A

[0223] Using Wp1DAAO-G52G / N54I / T56N / F58K as a template, saturation mutagenesis was carried out on the remaining 7 target sites. The construction and screening of the mutant library were carried out according to the methods of steps 1 to 4 in Example 1, and the expression and enzyme activity determination of the positive mutant D-amino acid oxidase were carried out according to the methods of steps 5 and 6 in Example 1. Finally, 2 mutants with significantly improved enzyme activity were obtained, and the results are shown in Table 6.

[0224] Table 6 Results of screening for positive mutants in the iterative saturation mutant library based on G52G / N54I / T56N / F58K / S354G / S355A

[0225]

[0226] Among the results of saturation mutagenesis at 7 target sites, there were 2 positive strains at M217. After HPLC verification, as shown in Table 6, the mutant strain G52G / N54I / T56N / F58K / M217V / S354G / S355A had the highest enzyme activity per unit OD. The crude enzyme activity was 18.72 times higher than that of the wild-type Wp1DAAO and was 0.25 times higher than that of the starting strain G52G / N54I / T56N / F58K / S354G / S355A in terms of enzyme activity per unit OD (P<0.01). Among them, the codon at position 217 of the G52G / N54I / T56N / F58K / M217V / S354G / S355A mutant strain changed from ATG to GTT, and it was named Wp1-7.

[0227] The crude enzyme solutions of wild-type Wp1DAAO and its four-point mutant, six-point mutant, and seven-point mutant were subjected to SDS-PAGE protein electrophoresis as described in step 7 of Example 1. The results are as Figure 3 shown. The results of protein gel electrophoresis showed that the wild-type and mutant D-amino acid oxidases were present in the supernatant under shake-flask culture conditions without inclusion bodies, indicating good expression of D-amino acid oxidase and its variants in Escherichia coli.

[0228] Example 5 Determination of Specific Enzyme Activity of Wp1DAAO and Its Mutants

[0229] 1. Shake-Flask Fermentation of Mutant D-Amino Acid Oxidase

[0230] After activating the mutant strains Wp1-4, Wp1-6, Wp1-7 and the wild-type Wp1DAAO by streaking on a plate, a single colony was picked and inoculated into 5 mL of LB liquid medium containing 100 μg / mL kanamycin, and cultured with shaking at 37 °C for 12 h. Transferred at an inoculation amount of 2% to 200 mL of TB liquid medium containing 100 μg / mL kanamycin as well, and cultured with shaking at 37 °C until the OD 600 reached about 0.6 - 0.8, then IPTG was added to a final concentration of 0.5 mM, and induced to culture at 18 °C for 24 h. After the culture was completed, the culture solution was centrifuged at 4000 g and 4 °C for 10 min, the supernatant was discarded, and the cells were collected. The collected cells were washed twice with 100 mM phosphate buffer at pH 8.0, then resuspended in phosphate buffer, and ultrasonically disrupted on ice bath until the solution became clear. The cell lysate was centrifuged at 12000 g and 4 °C for 30 min to remove the precipitate, and the supernatant was filtered through a 0.22 μm filter membrane and reserved for use.

[0231] 2. Protein Purification by Gravity Column

[0232] (1) Loading: After the preservation solution in the gravity column drained completely, the nickel column was rinsed with 50 mL of Ni-0-native Buffer (10 column volumes), then the prepared sample was slowly loaded onto the column, repeated three times to make the protein fully bind to the nickel column;

[0233] (2) Impurity Removal and Elution: The nickel column was rinsed successively with 50 mL (10 column volumes) of Ni-50-native Buffer, Ni-100-native Buffer, Ni-150-native Buffer, Ni-200-native Buffer, Ni-250-native Buffer, and Ni-500-native Buffer solutions respectively, and the eluate of each time was collected, labeled and recorded;

[0234] (3) Nickel column cleaning and preservation: Wash the nickel column after eluting the protein with 50 mL of deionized water, add 20% alcohol, and store it in a 4°C refrigerator for later use.

[0235] (4) Ultrafiltration and preservation: After confirming the collection tube corresponding to the target protein, transfer the sample to an ultrafiltration tube for centrifugal ultrafiltration. Centrifuge at 4000 rpm for 20 min at 4°C each time, pour off the ultrafiltered filtrate, and repeat several times until all the samples are ultrafiltered thoroughly to complete the concentration of the target protein. Then ultrafilter three times with 0.25 M phosphate buffer at pH 7.5 to desalt the protein. After ultrafiltration is completed, add 10% glycerol, aliquot, and store in a -80°C refrigerator for later use.

[0236] 3. Protein concentration determination

[0237] The protein concentration in the solution was determined using a Bradford protein concentration assay kit. The specific operation steps refer to the kit instruction manual.

[0238] 4. Specific enzyme activity determination

[0239] Using DL-glufosinate as the substrate, the specific enzyme activity of recombinant D-amino acid oxidase was detected. The assay system was as follows: The total reaction system was 400 μL, including 200 μL of 200 mM DL-glufosinate solution and 200 μL of pure enzyme solution. The above substances were all prepared with 100 mM phosphate buffer at pH 8.0. React with shaking at 30°C for 1 h, and add 40 μL of 4 M HCl solution to terminate the reaction. Centrifuge the reaction mixture at 12000 rpm for 10 min. High-performance liquid chromatography was used to determine the PPO generated in the reaction system, and the specific activities of wild-type and mutant D-amino acid oxidase pure enzyme solutions were determined.

[0240] Definition of specific enzyme activity: The enzyme activity unit per unit weight of protein (U / g).

[0241] Table 7 Specific enzyme activities of Wp1DAAO and its mutants

[0242]

[0243] It can be seen from Figure 4 that the purified Wp1DAAO and mutant protein bands are clear without impurity bands. From the results in Table 7, it can be seen that the specific enzyme activity of wild-type Wp1DAAO for catalyzing DL-glufosinate is relatively low, only 0.71 U / g. The specific enzyme activities of the D-amino acid oxidase after semi-rational mutagenesis modification are all significantly improved (P < 0.001). The enzyme activity of the seven-point mutant D-amino acid oxidase reaches the highest 43.23 U / g, which is 59.89 times higher than that of wild-type Wp1DAAO.

[0244] Example 6 Preparation of L-glufosinate by multi-enzyme cascade reaction

[0245] After screening and engineering Wp1DAAO, we obtained a strain Wp1-7 with the highest enzyme activity and constructed a multi-enzyme cascade system. Starting from DL-glufosinate, D-amino acid oxidase was used to catalyze the oxidation of D-amino acids to generate keto acids, while L-amino acids were completely retained. Then, glutamate dehydrogenase was used to in-situ reduce the keto acids to L-amino acids, achieving the racemization of D,L-amino acids. Under the conditions of 40 °C, pH 8.0 and oxygen supply, L-glufosinate was prepared by a thermostatic magnetic stirrer. The total reaction volume was 100 mL, and the concentrations of the components were as follows: 200 mM DL-glufosinate solution, 300 mM isopropanol, 0.2 mM NAPD+, 0.1 g / L (dry cell weight) PpGluDH, 0.2 g / L (dry cell weight) alcohol dehydrogenase (BsADH), and 10 g / L (dry cell weight) Wp1-7 D-amino acid oxidase (DAAO) and catalase (MaCAT). The reaction technical route is as Figure 4 shown. The specific operation steps are as follows: Prepare an aqueous solution of 0.2 M (NH4)2SO4 and adjust its pH to 8.0 with 25% ammonium hydroxide to obtain an NH3·(NH3)2SO4 buffer solution. Dissolve DL-glufosinate and isopropanol in the buffer solution and adjust the pH to 8.0 with 25% ammonium hydroxide to obtain a substrate solution (80 mL). Resuspend the collected PpGluDH, BsADH, DAAO, and MaCAT cells in the buffer solution, disrupt them by sonication, and then add NAPD+ to obtain an enzyme solution (20 mL). Mix the substrate solution and the enzyme solution and add them to a three-necked flask for reaction. During the reaction, maintain the reaction solution at pH 8.0 by adding 10% ammonium hydroxide solution.

[0246] The catalytic effect of the multi-enzyme catalytic system was investigated by substrate conversion rate, conversion efficiency, and enantiomeric excess (ee value). The ee value represents the excess of one enantiomer over the other and is usually expressed as a percentage: ee = ([R]-[S] / [R]+[S])*100%.

[0247] The highest enzyme activity of Wp1-7 D-amino acid oxidase was used for the multi-enzyme cascade reaction to prepare L-glufosinate. During the whole reaction, the contents of L- and D-glufosinate were measured simultaneously, and the ee value was calculated. The results are as Figure 6 shown. As Figure 6 can be seen, after 400 min of reaction, all D-glufosinate was converted to L-glufosinate, and the conversion rate reached 100%. Finally, about 185.69 mM L-glufosinate was generated in total, and the measured ee value was greater than 99%.

[0248] Comparative Example

[0249] The enzyme activity was compared with the optimal mutant M213S / F58H / N54V / L239G of D - amino acid oxidase (NCBI accession number: POY70719.1) from Rhodotorula taiwanensis in the prior art. The production amount of PPO was measured by HPLC method to compare the catalytic efficiency of DAAO and its mutant. The 1 ml reaction system included: 50 mM racemic PPT ammonium salt, 50 mM phosphate buffer at pH 8.0, 8000 U / L catalase, and 50 g / L frozen dry cells of DAAO mutant. After reacting for 2 h, the reaction solution sample was taken for treatment, the concentration of PPO was measured, and the conversion rate was calculated (product PPO concentration / initial substrate D,L - PPT concentration × 100%), and the results are shown in Table 8.

[0250] Table 8 Determination of PPO concentration and conversion rate of the comparison strains and Wp1 - 7 product

[0251]

[0252] The results showed that the conversion rate of the Wp1 - 7 strain was higher under the same conditions, and there was a significant improvement compared with the comparison strains (P < 0.001), having better technical effects and application prospects.

[0253] The above - mentioned is only the preferred embodiment of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Use of a mutation site as a target in the preparation of α-keto acid and / or L-glufosinate; The mutation sites include: One or more of positions 52, 54, 56, 58, 217, 354, and 355 of D-amino acid oxidase.

2. Mutants of D-amino acid oxidase, characterized in that, The mutation includes: amino acid mutation and / or codon mutation; The amino acid mutation is obtained by single or multiple point mutations at positions 52, 54, 56, 58, 217, 354, and 355 of D-amino acid oxidase; The codon mutation is obtained by mutation of one or more of positions 154-156, 160-162, 166-168, 172-174, 649-651, and 1060-1065 of the nucleic acid molecule encoding the D-amino acid oxidase.

3. The mutant according to claim 2, wherein Including: The G at position 52 of the D-amino acid oxidase is mutated to R; and / or The G at position 52 of the D-amino acid oxidase is mutated to V; and / or The N at position 54 of the D-amino acid oxidase is mutated to I; and / or The N at position 54 of the D-amino acid oxidase is mutated to S; and / or The N at position 54 of the D-amino acid oxidase is mutated to L; and / or The N at position 54 of the D-amino acid oxidase is mutated to G; and / or The N at position 54 of the D-amino acid oxidase is mutated to V; and / or The N at position 54 of the D-amino acid oxidase is mutated to Q; and / or The N at position 54 of the D-amino acid oxidase is mutated to R; and / or The T at position 56 of the D-amino acid oxidase is mutated to N; and / or The T at position 56 of the D-amino acid oxidase is mutated to S; and / or The T at position 56 of the D-amino acid oxidase is mutated to Q; and / or The F at position 58 of the D-amino acid oxidase is mutated to K; and / or The F at position 58 of the D-amino acid oxidase is mutated to H; and / or The F at position 58 of the D-amino acid oxidase is mutated to R; and / or The M at position 217 of the D-amino acid oxidase is mutated to I; and / or The M at position 217 of the D-amino acid oxidase is mutated to T; and / or The M at position 217 of the D-amino acid oxidase is mutated to V; and / or The S at position 354 of the D-amino acid oxidase is mutated to G; and / or The S at position 354 of the D-amino acid oxidase is mutated to P; and / or The S at position 354 of the D-amino acid oxidase is mutated to A; and / or The S at position 355 of the D-amino acid oxidase is mutated to G; and / or The S at position 355 of the D-amino acid oxidase is mutated to A; and / or The S at position 355 of the D-amino acid oxidase is mutated to P.

4. The mutant according to claim 2 or 3, characterized in that, Including: The C at position 156 of the gene encoding the D-amino acid oxidase is mutated to G; and / or The C at position 156 of the gene encoding the D-amino acid oxidase is mutated to T; and / or The C at position 1062 of the gene encoding the D-amino acid oxidase is mutated to T.

5. The mutant according to any one of claims 2 to 4, characterized in that, The mutant has: (1) An amino acid sequence as shown in any one of SEQ ID NO:1 to SEQ ID NO:28; or (2) An amino acid sequence obtained by substituting, deleting or adding one or more amino groups to the amino acid sequence as shown in (1), and having the same or similar function as the amino acid sequence as shown in (1); or (3) An amino acid sequence having at least 80% identity with the amino acid sequence as shown in (1) or (2).

6. A nucleic acid molecule encoding a mutant according to any one of claims 2 to 5, characterized in that, The nucleic acid molecule has: (4) A nucleotide sequence as shown in any one of SEQ ID NO:29 to SEQ ID NO:56; or (5) A nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence as shown in (4), and having the same or similar function as the nucleotide sequence as shown in (4); or (6) A nucleotide sequence having at least 80% identity with the nucleotide sequence as shown in (4) or (5).

7. Expression vector, characterized in that, Comprising: The nucleic acid molecule as claimed in claim 6 and an acceptable element.

8. A host, characterized in that, Transforming and / or transfecting the expression vector as claimed in claim 7.

9. Use of the mutant as claimed in any one of claims 2 to 5, the nucleic acid molecule as claimed in claim 6, the expression vector as claimed in claim 7 and / or the host as claimed in claim 8 in the preparation of α-keto acid and / or L-glufosinate.

10. A method for preparing α-keto acid and / or L-glufosinate, characterized in that, Using the mutant as claimed in any one of claims 2 to 5 or the host as claimed in claim 8 to carry out an oxidation reaction with D-amino acid or racemic amino acid as the substrate to obtain the α-keto acid and / or the L-glufosinate.

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