Leucine dehydrogenase mutant and its preparation method and application

Abstract

The present application relates to the field of biotechnology, and specifically to a mutant of leucine dehydrogenase, a preparation method thereof, and an application thereof. For wild-type leucine dehydrogenase, the mutant has one or more of D122F and E249K, and one or more of M346A and K89R. Compared to conventional technologies, the present application provides a mutant of leucine dehydrogenase, which has a significantly improved reaction rate relative to the wild-type enzyme and can be widely used in the detection and metabolism research of branched-chain amino acids (BCAAs) and branched-chain α-keto acids.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a mutant of leucine dehydrogenase, a preparation method thereof, and an application thereof. Background Art

[0002] Leucine dehydrogenase (LeuDH) is an important enzyme widely used in the detection and metabolism of branched-chain amino acids (BCAAs) and branched-chain α-keto acids. However, the reaction rate of wild-type leucine dehydrogenase is too slow to be suitable for detection. Therefore, the development of faster-reacting leucine dehydrogenase mutants is of great significance. Summary of the Invention

[0003] Based on this, one or more embodiments of the present application provide a method including the following technical solutions:

[0004] One or more embodiments of the present application provide a mutant of leucine dehydrogenase. Compared to the wild-type leucine dehydrogenase, the mutant has one or more of D122F and E249K, and one or more of M346A and K89R.

[0005] In some embodiments of the present application, the mutant has one or more of M346A and K89R and D122F.

[0006] In some embodiments of the present application, the amino acid sequence of the wild-type leucine dehydrogenase is shown as SEQ ID NO.1.

[0007] In some embodiments of the present application, the mutant further has a functional C-terminal modification.

[0008] In some embodiments of the present application, the functionalized C-terminal modification includes two aspartic acid residues (DD).

[0009] In some embodiments of the present application, the N-terminus of the mutant is further connected to a protein tag.

[0010] In some embodiments of the present application, the protein tag satisfies one or more of the following conditions:

[0011] (1) The protein tag is connected to the N-terminus of the mutant via a flexible peptide;

[0012] (2) The protein tag includes one or more of a detection expression-promoting tag and a purification tag.

[0013] In some embodiments of the present application, the protein tag satisfies one or more of the following conditions:

[0014] (1) The detection tag includes P17-tag and / or SKIK-tag;

[0015] (2) The purification tag includes a His tag;

[0016] (3) The N-terminus of the mutant includes the expression-promoting tag and the purification tag connected in sequence.

[0017] In some embodiments of the present application, the expression-promoting tag and the purification tag are connected via an amide bond.

[0018] In some embodiments of the present application, the amino acid sequence of the mutant is shown as SEQ ID NO.4.

[0019] One or more embodiments of the present application provide a nucleic acid molecule encoding the mutant of leucine dehydrogenase.

[0020] One or more embodiments of the present application provide a vector comprising the nucleic acid molecule.

[0021] In some embodiments of the present application, the vector comprises a plasmid.

[0022] One or more embodiments of the present application provide a cell, wherein the cell expresses the mutant of leucine dehydrogenase, including the nucleic acid molecule or the vector.

[0023] In some embodiments of the present application, the cell comprises a prokaryotic cell.

[0024] One or more embodiments of the present application provide a method for preparing the mutant of leucine dehydrogenase, the method comprising the following steps:

[0025] The cells are fermented, and the mutant of leucine dehydrogenase is separated from the fermentation product.

[0026] In some embodiments of the present application, the fermentation comprises the steps of amplifying and culturing the cells and inducing the mutants.

[0027] In some embodiments of the present application, the fermentation satisfies one or more of the following conditions:

[0028] (A) The temperature of the amplification culture is 35°C-39°C;

[0029] (B) IPTG was added to induce expression when the OD600 of the culture system reached 0.6-1.2. The final concentration of IPTG in the initial induced expression system was 0.1 mmol / L-1.0 mmol / L.

[0030] (C) The induction temperature is 18-37°C, and the induction time is 6-20 hours;

[0031] (D) The fermentation medium includes 8 g / L-12 g / L tryptone, 4 g / L-6 g / L yeast extract, 8 g / L-12 g / L NaCl, 1 g / L-2 g / L glycerol, 1 mmol / L-15 mmol / L magnesium sulfate, and water.

[0032] One or more embodiments of the present application provide a detection kit for branched-chain amino acids or branched-chain α-keto acids, wherein the detection kit comprises the mutant of leucine dehydrogenase, the nucleic acid molecule, the vector or the cell.

[0033] One or more embodiments of the present application provide the use of the mutant of leucine dehydrogenase or the detection kit in detecting branched-chain amino acids or branched-chain α-keto acids in a sample to be tested.

[0034] In some embodiments of the present application, detection is performed using UV-visible spectrophotometry.

[0035] Compared with traditional technologies, the present application provides a mutant of leucine dehydrogenase, which has a significantly improved reaction rate compared with the wild-type enzyme and can be widely used in the detection and metabolism research of branched-chain amino acids (BCAAs) and branched-chain α-keto acids. DETAILED DESCRIPTION

[0036] Below in conjunction with embodiment and example, the application is described in further detail.Should be understood that these embodiment and example are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiment and example is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms, is not limited to the embodiment and example described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description hereinafter, a large amount of specific details are given in order to provide a more complete understanding of the application, and it should be understood that the application can be implemented without one or more of these details.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing embodiments and examples only and are not intended to limit this application.

[0038] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0039] The terms "and / or", "or / and", and "and / or" used herein include any one of two or more related listed items, and also include any and all combinations of the related listed items, wherein the arbitrary and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical and" and also undoubtedly includes technical solutions connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution of all being connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, the technical solution of all being connected by "logical AND").

[0040] In this application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0041] As used herein, "combination thereof", "any combination thereof", "any combination thereof" and the like include all suitable combinations of any two or more of the listed items.

[0042] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc. shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0043] Herein, "preferred", "better", "more preferred" and "suitable" are merely used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of this application.

[0044] In this application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of this application.

[0045] In this application, the terms "optionally," "optional," and "optional" mean optional or dispensable, i.e., they refer to either option being selected from two parallel options: "with" or "without." If a technical solution contains multiple "optional" clauses, each "optional" clause is independent unless otherwise specified and there are no contradictions or constraints.

[0046] In this application, the terms "first," "second," "third," "fourth," etc. in "the first aspect," "the second aspect," "the third aspect," "the fourth aspect," etc. are used for descriptive purposes only and should not be understood as indicating or implying relative importance or quantity, nor should they be understood as implicitly indicating the importance or quantity of the indicated technical features. Furthermore, "first," "second," "third," "fourth," etc. serve only as non-exhaustive enumeration and description, and should be understood not to constitute a closed-ended limitation on quantity.

[0047] In this application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0048] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional values ​​within the numerical interval is considered continuous and includes the two numerical endpoints of the numerical range (i.e., the minimum and maximum values), as well as every numerical value between these two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the two numerical endpoints of the numerical range, as well as every integer between the two numerical endpoints. In this document, this is equivalent to directly listing each integer. For example, "t is an integer selected from 1 to 10" means that t is any integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In addition, when multiple ranges are provided to describe a feature or characteristic, these ranges may be combined. In other words, unless otherwise specified, ranges disclosed herein should be understood to include any and all subranges subsumed therein.

[0049] Unless otherwise specified, the temperature parameters in this application allow for both constant temperature treatment and temperature fluctuations within a certain temperature range. It should be understood that the constant temperature treatment allows for temperature fluctuations within the accuracy range of instrument control. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are permitted.

[0050] In this application, %(w / w) and wt% both refer to weight percentage, %(v / v) refers to volume percentage, and %(w / v) refers to mass volume percentage.

[0051] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited in their entirety and for all purposes. When cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When cited documents are involved in this application, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be adaptively amended according to the description in this application.

[0052] In a first aspect of the embodiments of the present application, a mutant of leucine dehydrogenase is provided. Compared with the wild-type leucine dehydrogenase, the mutant has one or more of D122F and E249K, and one or more of M346A and K89R.

[0053] In the present application, the mutants have one or both of D122F and E249K, and one or both of M346A and K89R. The mutants with these mutations have significantly improved enzymatic activity relative to the wild-type enzyme. In some examples, the mutants have D122F and M346A, such as Mutant 2 in Table 4. In other examples, the mutants have D122F and K89R, such as Mutant 5 in Table 4. In yet other examples, the mutants have E249K and M346A, such as Mutant 6 in Table 4. In yet other examples, the mutants have E249K and K89R, such as Mutant 3. Alternatively, in some examples, the mutants have one or more of M346A and K89R and D122F, such as Mutants 2 and Mutant 5 in Table 4.

[0054] The present application does not impose any particular limitation on the species origin of the wild-type leucine dehydrogenase, including but not limited to that from an intermediate type of thermophilic actinomycetes.

[0055] In some examples, the wild-type leucine dehydrogenase has an amino acid sequence as shown in SEQ ID NO. 1, or has a sequence that is at least 80% (at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%) identical to SEQ ID NO. 1, or has no more than 6 amino acid substitutions relative to the wild-type leucine dehydrogenase of SEQ ID NO. 1, and has leucine dehydrogenase activity.

[0056] "Identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in the two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if every position in two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage identity between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100. For example, when the sequences are optimally aligned, if 6 out of 10 positions in the two sequences match or are homologous, then the two sequences are 60% homologous; if 95 out of 100 positions in the two sequences match or are homologous, then the two sequences are 95% homologous. Typically, when two sequences are aligned, the comparison is made to give the maximum percentage identity. For example, the comparison can be performed using the BLAST algorithm, where the parameters of the algorithm are selected to give the maximum match between each sequence over the entire length of each reference sequence. The following references relate to the BLAST algorithm commonly used for sequence analysis: BLAST ALGORITHMS: Altschul, S.F. et al., (1990) J.Mol.Biol.215:403-410; Gish, W. et al., (1993) Nature Genet.3:266-272; Madden, T.L. et al., (1996) Meth.Enzymol.266:131-141; Altschul, S.F. et al., (1997) Nucleic Acids Res.25:3389-3402; Zhang, J. et al., (1997) Genome Res.7:649-656. Other conventional BLAST algorithms, such as those provided by NCBI BLAST, are also well known to those skilled in the art.

[0057] The biological activity of leucine dehydrogenase is retained by amino acid substitutions ("conservative modifications" or "conservative replacements"). For example, one amino acid may be replaced by another amino acid, or one amino acid may be replaced by multiple (e.g., 2, 3, 4, 5, or 6) amino acids. Amino acid substitution refers to the replacement of an amino acid with an amino acid having similar or similar properties. For example, amino acid substitutions are made with reference to Table 1.

[0058] Table 1

[0059]

[0060] "Conservative modification" or "conservative substitution or replacement" refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation and rigidity, etc.), so that changes can be made frequently without changing the biological activity of the protein. It is known to those skilled in the art that, in general, single amino acid replacements in non-essential regions of a polypeptide do not substantially change the biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th ed.)). In addition, replacement of amino acids with similar structure or function is unlikely to destroy biological activity.

[0061] This application does not impose any particular restrictions on the method for synthesizing the mutants. The mutants may be prepared by bioengineering methods or by chemical synthesis (e.g., solid-phase synthesis). It is understood that during the bioengineering process for synthesizing the mutants, functional modifications may be made to the mutants for reasons of stability, purification, yield, and the like.

[0062] In some examples, the mutants have a functionalized C-terminal modification. The purpose of the functionalized C-terminal modification includes, but is not limited to, adding two aspartic acid residues (DD) to the C-terminus, such as mutants 7 and 8 in Tables 8 to 11.

[0063] In some examples, the N-terminus of the mutant is also connected to a protein tag. Furthermore, the protein tag satisfies one or more of the following conditions: (1) the protein tag is connected to the N-terminus of the mutant through a flexible peptide; (2) the protein tag includes one or more of a detection tag, a degradation tag, and a purification tag. Furthermore, the protein tag satisfies one or more of the following conditions: (1) the expression-promoting tag includes a P17-tag and / or a SKIK-tag; (2) the purification tag includes a His tag; (3) the N-terminus of the mutant includes the expression-promoting tag and the purification tag connected in sequence. Furthermore, the expression-promoting tag and the purification tag are connected through an amide bond.

[0064] In some examples, the amino acid sequence of the mutant is as shown in SEQ ID NO. 4, or has a sequence that is at least 80% (at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%) identical to SEQ ID NO. 4, or has no more than 6 positions (e.g., 1, 2, 3, 4, 5, 6) of amino acid substitutions relative to the wild-type leucine dehydrogenase described in SEQ ID NO. 4 (refer to Table 1 for substitutions), and has leucine dehydrogenase activity.

[0065] In a second aspect of the embodiments of the present application, a nucleic acid molecule is provided, which encodes the mutant of the leucine dehydrogenase.

[0066] The nucleic acid molecules of the present application primarily refer to isolated nucleic acid molecules. "Isolated" refers to molecules that are substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cell debris and growth medium. Generally, the term "isolated" is not intended to refer to the complete absence of such materials or the absence of water, buffers, or salts, unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the compounds as described herein.

[0067] According to a third aspect of the embodiments of the present application, a vector is provided, wherein the vector comprises the nucleic acid molecule.

[0068] Optionally, the vector is selected from mammalian cell virus, bacterial plasmid, phage, yeast plasmid or a combination thereof.

[0069] The term "vector" is also "expression vector" and refers to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. In one embodiment, the vector is a "plasmid", which refers to a circular double-stranded DNA loop into which another DNA segment can be connected. In another embodiment, the vector is a viral vector, in which another DNA segment can be connected to a viral genome. The vector disclosed herein can be autonomously replicated in the host cell into which they have been introduced (for example, bacterial vectors and additional mammalian vectors with a bacterial origin of replication) or can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating together with the host genome (for example, non-additional mammalian vectors). On the whole, the vector can be selected from, but is not limited to, mammalian cell viruses, bacterial plasmids, bacteriophages, yeast plasmids, or a combination thereof.

[0070] A fourth aspect of the embodiments of the present application provides a cell, wherein the cell expresses the mutant of leucine dehydrogenase, including the nucleic acid molecule or the vector.

[0071] The term "cell," also known as "host cell," refers to a cell into which an expression vector has been introduced. Host cells can include bacteria, microorganisms, and animal cells. Easily transformed bacteria include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus; and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, or HEK293 cells.

[0072] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, without regard to the number of passages. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or unintentional mutations. Mutant progeny that possess the same function or biological activity as screened for in the originally transformed cell are included.

[0073] In a fifth aspect of the embodiments of the present application, a method for preparing the mutant of leucine dehydrogenase is provided, the method comprising the following steps:

[0074] The cells are fermented, and the mutant of leucine dehydrogenase is separated from the fermentation product.

[0075] Optionally, the fermentation comprises the steps of amplifying and culturing the cells and inducing the mutants.

[0076] Optionally, the fermentation satisfies one or more of the following conditions:

[0077] (A) The temperature of the expansion culture is 35°C-39°C (e.g., 35°C, 36°C, 37°C, 38°C, 39°C);

[0078] (B) amplifying and culturing the culture until the OD600 of the culture system is 0.6-1.2 (e.g., 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2), then inducing expression by adding IPTG, wherein the final concentration of IPTG in the initial induced expression system is 0.1 mmol / L-1.0 mmol / L (e.g., 0.1 mmol / L, 0.2 mmol / L, 0.3 mmol / L, 0.4 mmol / L, 0.5 mmol / L, 0.6 mmol / L, 0.7 mmol / L, 0.8 mmol / L, 0.9 mmol / L, 1.0 mmol / L);

[0079] (C) the temperature for inducing expression is 18°C-37°C (for example, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C), and the time for inducing expression is 6h-20h (for example, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h);

[0080] (D) The culture medium used for fermentation includes 8 g / L-12 g / L (e.g., 8 g / L, 8.5 g / L, 9 g / L, 9.5 g / L, 10 g / L, 10.5 g / L, 11 g / L, 11.5 g / L, 12 g / L) of tryptone, 4 g / L-6 g / L (e.g., 4 g / L, 4.5 g / L, 5 g / L, 5.5 g / L, 6 g / L) of yeast extract, 8 g / L-12 g / L (e.g., 8 g / L, 8.5 g / L, 9 g / L, 9.5 g / L, 10 g / L, 10.5 g / L, 11 g / L, 11.5 g / L, 12 g / L) of N aCl, 1 g / L-2 g / L (for example, 1 g / L, 1.2 g / L, 1.4 g / L, 1.6 g / L, 1.8 g / L, 2 g / L) of glycerol, 1 mmol / L-15 mmol / L (for example, 1 mmol / L, 2 mmol / L, 3 mmol / L, 4 mmol / L, 5 mmol / L, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, 11 mmol / L, 12 mmol / L, 13 mmol / L, 14 mmol / L, 15 mmol / L) of magnesium sulfate, and water.

[0081] In a sixth aspect of the embodiments of the present application, a detection kit for branched-chain amino acids or branched-chain α-keto acids is provided, wherein the detection kit comprises the mutant of leucine dehydrogenase, the nucleic acid molecule, the vector or the cell.

[0082] In a seventh aspect of the embodiments of the present application, there is provided a use of the mutant of leucine dehydrogenase or the detection kit in detecting branched-chain amino acids or branched-chain α-keto acids in a sample to be tested.

[0083] In some of these examples, detection is performed using UV-Vis spectrophotometry.

[0084] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manuals or conventional conditions in this area, or according to the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0085] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0086] 1. Preparation of various mutants and wild-type enzymes

[0087] (1) Construction of recombinant plasmid

[0088] Based on the amino acid sequence characteristics, three-dimensional structure, and active site of the wild-type leucine dehydrogenase from Thermoactinomyces intermedius (intermediate thermophilic actinomycete), three mutation sites (D122F, D122S, and E249K) that may affect the reaction activity and two mutation sites (K89R and M346A) that may affect the three-dimensional structural stability were preliminarily screened. Pairwise combinations of these two types of mutation sites resulted in a total of six mutant combination proteins (referred to as Mutants 1 to 6, respectively). Together with the wild-type protein, a total of seven proteins were obtained. The amino acid sequence of the wild-type enzyme is shown in SEQ ID NO. 1. Relative to the wild-type leucine dehydrogenase, the mutation site combinations carried by Mutants 1 to 6 are shown in Table 2 below.

[0089] Table 2

[0090]

[0091] SEQ ID NO.1:

[0092] MKIFDYMEKYDYEQLVMCQDKESGLKAIICIHVTTLGPALGGMRMWTYASEEEAIEDALRLGRGMTYKNAAAGLNLGGGKTVIIGDPRKDKNEAMFRALGRFIQGLNGRYITAEDVGTTVEDMDIIHEETRYVTGVSPAFGSSGNPSPVTAYGVYRGMKAAAKEAFGDDSLEGKVVAVQGVGH VAYELCKHLHNEGAKLIVTDINKENADRAVQEFGAEFVHPDKIYDVECDIFAPCALGAIINDETIERLKCKVVAGSANNQLKEERHGKMLEEKGIVYAPDYVINAGGVINVADELLGYNRERAMKKVEGIYDKILKVFEIAKRDGIPSYLAADRMAEERIEMMRKTRSTFLQDQRNLINFNNK.

[0093] Each protein was cloned into the NdeI and XhoI restriction sites of pET28a, each with a 6×His tag at the N-terminus. The recombinant plasmid pET28a-LeuDH-m was then constructed and transformed into the host E. coli BL21 (DE3). Single colonies were selected and transferred to culture medium. Shake overnight at 30°C-37°C to obtain the seed culture solution, which became the expression strain.

[0094] (2) Inducible expression

[0095] The seed bacterial liquid was inoculated into the culture medium at a 2% inoculum volume, cultured in a shaking incubator at 37°C until the OD600 reached 0.8, IPTG was added to a final concentration of 1.0 mmol / L, and expression was induced at 20°C for 16 h to prepare a recombinant cell culture medium containing the leucine dehydrogenase mutant.

[0096] (3) Affinity chromatography purification

[0097] The recombinant cells were disrupted and centrifuged at 10,000 rpm for 15 minutes to separate the supernatant and obtain a crude enzyme solution. The crude enzyme solution was added to an affinity chromatography column (Ni-NTA 6FF), and the impurities were washed with Tris / HCl (pH 8.0, 30 mmol / L imidazole). The target protein was eluted with Tris / HCl (pH 8.0, 250 mmol / L imidazole). The imidazole was removed using a tangential flow ultrafiltration system, and the protein was concentrated to obtain the purified recombinant leucine dehydrogenase mutant.

[0098] Subsequently, the recombinant protein was expressed and purified on a 100 mL culture medium scale according to the above preparation method.

[0099] The purified protein was assessed for purity by reducing SDS-PAGE electrophoresis (≥90% was acceptable, as shown in Table 3 below) and dialyzed into storage buffer (10 mM Tris, 0.5 M NaCl, 0.1% Triton X-100, 1 mM EDTA, 0.01% DTT, 30% glycerol, pH 8.0). The protein was aliquoted into 1 mL / tubes and stored frozen at -20°C ± 5°C until use.

[0100] Table 3

[0101]

[0102] The culture medium used in this part is modified LB medium: 10 g / L tryptone, 5 g / L yeast extract powder, 10 g / L NaCl, 2.0 g / L glycerol, 2 mmol / L magnesium sulfate and water.

[0103] 2. Characterization of various mutants and wild-type enzymes

[0104] (1) Enzyme activity, concentration, yield, specific activity and reaction rate of various mutants and wild-type enzymes

[0105] 1) Enzyme activity detection method

[0106] Reaction principle: Under the action of leucine dehydrogenase, L-leucine + oxidized coenzyme I + water → 4-methyl-2-oxopentanoic acid + ammonium ions + reduced coenzyme I.

[0107] Reaction system: 0.15 mol / L glycine / KCl / KOH Buffer (pH 10.5), 3 mM NAD + , 20 mM L-leucine.

[0108] Reaction temperature: 37℃±1℃.

[0109] Reaction time: 10min.

[0110] Detection instrument: iBC900 fully automatic biochemical analyzer.

[0111] Detection parameters: wavelength 340 nm, sample volume 2 μL / reagent volume 120 μL.

[0112] Method: RateA metering interval 4-11.

[0113] The enzyme solution with different dilutions was used as the sample to measure the reaction rate ΔAbs. The enzyme activity (U / mL) was calculated by the following formula:

[0114] Enzyme activity calculation method: Enzyme activity per unit volume (U / mL) = [△Abs 340 × (3.00 + 0.001)] ÷ [6.22 × 0.01] × dilution factor of the enzyme solution to be tested;

[0115] The above formula can be simplified as follows: Volume enzyme activity (U / mL) = 48.392 × ΔAbs × dilution factor of the enzyme solution to be tested;

[0116] Specific activity calculation formula: specific activity value (U / mg) = volumetric enzyme activity (U / mL) / enzyme solution protein concentration (mg / mL).

[0117] 2) The protein concentration of the enzyme solution was measured using a nucleic acid protein analyzer.

[0118] 3) Enzyme reaction rate detection method

[0119] Reaction principle: Under the action of leucine dehydrogenase, L-leucine + oxidized coenzyme I + water → α-ketoisocaproic acid sodium salt + ammonium ions + reduced coenzyme I.

[0120] Reaction system: 0.15 mol / L glycine / KCl / KOH Buffer (pH 10.5), 3 mM NAD + , 0.1% BSA, 10 U / mL leucine dehydrogenase (if the volume enzyme activity is insufficient, prepare in large quantities and then freeze-dry and prepare).

[0121] Reaction temperature: 37℃±1℃.

[0122] Reaction time: 10 minutes.

[0123] Detection instrument: iBC900 fully automatic biochemical analyzer.

[0124] Detection parameters: wavelength 340 nm, sample volume 2 μL / reagent volume 120 μL.

[0125] Method: RateA metering interval 1-6.

[0126] Using the substrate L-leucine as the sample, a concentration of 500 μmol / L was selected, and the reaction rate ΔAbs (unit ΔA / min) was directly measured on the instrument.

[0127] The protein concentration and enzyme activity were measured using the storage buffer as a blank. The results are shown in Table 4 below:

[0128] Table 4

[0129]

[0130] According to Table 4 above, calculate the enzyme yield (mg protein / L culture medium) and specific activity (U / mg) and record the reaction rate measurements, as shown in Table 5 below.

[0131] Table 5

[0132]

[0133] It can be seen from Table 5 that:

[0134] In terms of yield, mutants 1 to 6 were relatively close to the wild type, indicating that these mutation sites had no significant effect on protein expression.

[0135] In terms of specific activity, mutants 2 and 5, carrying the D122F mutation, showed significantly higher specific activities than the wild type and other mutants, with specific activities approximately 4.5 times that of the wild type. Mutants 1 and 4, carrying the D122S mutation, showed significantly lower enzyme activities than the wild type. Mutants 3 and 6, carrying the E249K mutation, showed approximately twice the enzyme activity of the wild type.

[0136] In terms of reaction rate, Mutants 2 and 5 had the highest reaction rates, 8 times that of the wild type. Mutants 1 and 4 had the lowest reaction rates, even lower than the wild type. Mutants 3 and 6 had reaction rates 3.7 times that of the wild type, lower than those of Mutants 2 and 5. The distribution trend of reaction rates for mutants carrying different mutation combinations was consistent with the specific activity, indicating that mutations affecting enzyme activity also affect reaction rate.

[0137] (2) Enzyme stability test The enzyme solutions of mutants 2 and 5 with the highest specific activity and reaction rate, as well as the wild-type enzyme, were selected from the above six mutants for stability testing.

[0138] The enzyme solution to be tested was divided into small portions and kept at 40℃±1℃ for 6h and 55℃±1℃ for 1h respectively. Based on the freezing at -20℃±5℃, the percentage of enzyme activity after the other two high temperature conditions was calculated. The higher the percentage of residual enzyme activity, the more stable the enzyme.

[0139] The results are shown in Table 6:

[0140] Table 6 (enzyme activity unit: U / mL)

[0141]

[0142] According to Table 6 above, the percentage of residual enzyme activity at each storage temperature was calculated as shown in Table 7 below:

[0143] Table 7

[0144]

[0145] As can be seen from Table 7 above, the residual enzyme activity of mutant 5 is significantly higher than that of the wild type and mutant 2, and that of mutant 2 is higher than that of the wild type, indicating that both K89R and M346A mutations can improve the stability of the enzyme, among which the improvement effect of K89R mutation is significantly better than that of M346A mutation.

[0146] Thus, the present application has obtained two mutants, which are significantly better than the wild type in terms of activity and stability, and have an expression yield comparable to that of the wild type enzyme.

[0147] 3. Optimize the carbon end (C end) to further improve stability

[0148] Based on mutant 2 (D122F+M346A) and mutant 5 (D122F+K89R) obtained in the second screening, two hydrophilic and strongly negatively charged aspartic acid residues (DD) were added to the C-terminus. The corresponding mutants were recorded as mutant 7 and mutant 8, respectively. It is expected that increasing the hydrophilicity and negative charge of the mutants will help resist the tendency of molecules to aggregate, and thus help increase the tendency of the mutants to aggregate at high temperatures, thereby improving stability.

[0149] As described in item 1, for both mutants 7 and 8, optimize the DNA according to the codon bias of E. coli. Express and purify the recombinant protein at a 100 mL culture scale. The purified protein was assessed for purity by reducing SDS-PAGE electrophoresis (≥90% acceptable). The protein was dialyzed into storage buffer (10 mM Tris, 0.5 M NaCl, 0.1% Triton X-100, 1 mM EDTA, 0.01% DTT, 30% glycerol, pH 8.0), aliquoted into 1 mL / tube, and stored frozen at -20°C ± 5°C until use.

[0150] Referring to the description under item 2, the protein concentration and enzyme activity were measured using the storage buffer as a blank. The results are shown in Table 8 below:

[0151] Table 8

[0152]

[0153] According to Table 8 above, calculate the enzyme yield (mg protein / L culture medium), specific activity (U / mg) and record the reaction rate value, as shown in Table 9 below:

[0154] Table 9

[0155]

[0156] As shown in Table 9 above, there was no significant change in the expression yield, specific activity and reaction rate of mutants 7 and 8 compared with the corresponding mutants 2 and 5, indicating that the addition of two aspartic acid residues (DD) at the C-terminus had no effect on the yield and activity of the mutants.

[0157] Referring to the record under item 2, the above enzyme solution was tested for stability. The results are shown in Table 10 below:

[0158] Table 10 (enzyme activity unit: U / mL)

[0159]

[0160] According to Table 10 above, the percentage of residual enzyme activity at each storage temperature was calculated as shown in Table 11 below:

[0161] Table 11

[0162]

[0163] As can be seen from Table 11 above, the residual enzyme activities of mutants 7 and 8 were significantly improved under high temperature conditions compared with the corresponding mutants 2 and 5, indicating that adding two aspartic acid residues (DD) at the C-terminus can significantly improve thermal stability.

[0164] Combined with the above data, it can be seen that the enzyme activity and thermal stability of mutant 8 are significantly improved compared with the wild type and other mutants, but the expression yield is not significantly improved compared with the wild type and other mutants. The yield is low and needs further optimization.

[0165] 4. N-terminal optimization to improve expression yield

[0166] Based on the mutant 8 obtained by screening under item 3, this application further set up the following mutants:

[0167] Mutant 9: N-terminal P17-tag + His tag + (D122F + K89R) + two aspartic acid residues at the C-terminus (DD);

[0168] Mutant 10: N-terminal SKIK-tag + His tag + (D122F + K89R) + two aspartic acid residues at the C-terminus (DD);

[0169] Mutant 9 and mutant 10 differ only in the N-terminal tag, and the rest of the sequences are identical.

[0170] The amino acid sequence of P17-tag is KNESSTNATNTKQWRDETKGFRDEAKRFKNTAG (SEQ ID NO. 2); the amino acid sequence of SKIK-tag is SKIK; the amino acid sequence of His tag is HHHHHHHH (SEQ ID NO. 3); adjacent tags are directly connected by amide bonds, and the His tag and the N segment of mutant 8 are connected by a flexible short peptide gsg.

[0171] Refer to the instructions under item 1. For the two mutants described above, optimize the DNA according to the codon preference of E. coli and clone it into the Nde I and Xho I restriction sites of pET28a. Express and purify the recombinant protein at a 100 mL culture scale. Verify the purity of the purified protein by reducing SDS-PAGE electrophoresis (≥90% purity is acceptable) and dialyze it into storage buffer (10 mM Tris, 0.5 M NaCl, 0.1% Triton X-100, 1 mM EDTA, 0.01% DTT, 30% glycerol, pH 8.0). Aliquot into 1 mL / tube and freeze at -20°C ± 5°C until use.

[0172] Referring to the description under item 2, the protein concentration and enzyme activity were measured using the storage buffer as a blank. The results are shown in Table 12 below:

[0173] Table 12

[0174]

[0175] According to Table 12 above, the enzyme yield (mg protein / L culture medium) and specific activity (U / mg) were calculated and the reaction rate values ​​were recorded. The results are shown in Table 13 below:

[0176] Table 13

[0177]

[0178] As can be seen from Table 13 above: in terms of expression yield, mutant 9 has a significant increase of about 8 times compared with the wild-type enzyme; the expression yield of mutant 10 is also improved compared with the wild type. In terms of specific activity and reaction rate, there is no significant difference between mutant 8, mutant 9 and mutant 10, which is about 4.5 times higher than the wild type. Comparison between mutant 9 and mutant 10 shows that there is no difference in specific activity, but mutant 9 is significantly higher than mutant 10 in terms of yield. In summary, the two N-terminal expression-promoting tags have a significant effect on the expression yield of the leucine dehydrogenase of the present application, among which the P17-Tag carried by mutant 9 can greatly increase the expression yield, and the SKIK-Tag has only a weak expression-promoting effect. At the same time, these two tags have no effect on the activity and reaction rate of the enzyme.

[0179] Referring to the description under item 2, the stability test of the above two mutants was carried out, and the results are shown in Table 14 below:

[0180] Table 14 (enzyme activity unit: U / mL)

[0181]

[0182] According to Table 14 above, the percentage of residual enzyme activity at each storage temperature was calculated and the results are shown in Table 15 below:

[0183] Table 15

[0184]

[0185] As can be seen from Table 15 above, compared with mutant 8 under item 3, the residual enzyme activity of mutant 9 was significantly improved under high temperature conditions, indicating that the addition of P17-Tag to the N-terminus not only increased the expression yield but also improved the stability of the enzyme to a certain extent.

[0186] As a result, the present application screened out a mutant that showed significant improvements in yield, activity, and stability compared to the wild-type enzyme, namely mutant 9, which has an N-terminal P17-tag + (D122F + K89R) + two aspartic acid residues (DD) at the C-terminus, and the sequence is shown in SEQ ID NO. 4:

[0187] MGKNESSTNATNTKQWRDETKGFRDEAKRFKNTAGHHHHHHHHGSG MKIFDYMEKYDYEQLVMCQDKESGLKAIICIHVTTLGPALGGMRMWTYASEEEAIEDALRLGRGMTYKNAAAGLNLGGGKTVIIGDPRRDKNEAMFRALGRFIQGLNGRYITAEDVGTTVEFMDIIHEETRYVTGVSPAFGSSGNPSPVTAYGVYRGMKAAAKEAFGDDSLEGKVVAVQGVGH VAYELCKHLHNEGAKLIVTDINKENADRAVQEFGAEFVHPDKIYDVECDIFAPCALGAIINDETIERLKCKVVAGSANNQLKEERHGKMLEEKGIVYAPDYVINAGGVINVADELLGYNRERAMKKVEGIYDKILKVFEIAKRDGIPSYLAADRMAEERIEMMRKTRSTFLQDQRNLINFNNK DD .

[0188] 5. Comparison of sensitivity between mutant 9 and wild-type enzyme

[0189] Leucine detection reagents were prepared using wild-type and mutant leucine dehydrogenases, respectively. The blank limit of the detection reagent was used to characterize the sensitivity of the corresponding enzyme. The lower the blank limit, the better the sensitivity.

[0190] Reagent 1 was prepared for the wild type and mutant 9 according to the following formula:

[0191] Reagent 1 - Wild-type enzyme formula: 20 mM Tris-HCl buffer (pH 9.0), trehalose 50 g / L, EDTA 2Na 0.2 g / L, Triton X-100 0.2 g / L, bovine serum albumin 1 g / L, ascorbate oxidase 2 kU / L, leucine dehydrogenase (wild-type) 10 kU / L, Proclin 300 1 mL / L.

[0192] Reagent 1-Mutant 9 formula: 20 mM Tris-HCl buffer (pH 9.0), trehalose 50 g / L, EDTA 2Na 0.2 g / L, Triton X-100 0.2 g / L, bovine serum albumin 1 g / L, ascorbate oxidase 2 kU / L, leucine dehydrogenase (Mutant 9) 10 kU / L, Proclin 300 1 mL / L.

[0193] The difference between the two reagents 1 is only the type of leucine dehydrogenase, and the amount of enzyme used and other components and contents are the same.

[0194] The formula of reagent 2 is as follows: 50mM pH=4.0 glycine buffer, EDTA 2Na 0.5g / L, oxidized coenzyme NAD + 25g / L, Proclin 300 1mL / L. This reagent 2 is universal and can be used with the above two reagents 1 to form two different leucine dehydrogenase detection reagents.

[0195] Calibration curves were established on the iBC900 fully automated biochemical analyzer using leucine calibrators at varying concentrations (test parameters: sample volume 15 μL, reagent 1 90 μL, reagent 2 30 μL, detection wavelength 340 nm, photometric points 15–36). A blank sample (normal saline) was then tested 25 times. Outliers were removed using the Grubbs test (for detailed methods, see GB / T 4883-2008). The mean and standard deviation were then calculated, and the sensitivity index was approximated using (mean + 1.96 times the standard deviation). The test results are shown in Tables 16 and 17 below:

[0196] The test results of the detection reagent corresponding to the wild-type enzyme are shown in Table 16:

[0197] Table 16

[0198]

[0199] The test results of the detection reagent corresponding to mutant 9 are shown in Table 17:

[0200] Table 17

[0201]

[0202] Comparison of the two blank limits above shows that the blank limit of the wild-type enzyme is 15.5, and the blank limit of mutant 9 is 1.21, indicating that the sensitivity of mutant 9 is greatly improved compared with the wild-type.

[0203] Overall, the mutant 9 of the present embodiment has the following advantages:

[0204] (1) Stability: Compared with the wild-type enzyme, the thermal stability of the leucine dehydrogenase mutant 9 of the present application is significantly improved. Mutant 9 has a residual enzyme activity of 86% after being incubated at 40°C for 6 hours and 66% after being incubated at 55°C for 1 hour, while the wild-type enzyme only has 38% and 13% residual enzyme activity under the same two conditions, respectively.

[0205] (2) Reaction rate: Compared with the wild-type enzyme, the reaction rate of mutant 9 of the present application is significantly improved, reaching 0.28ΔA / min, while the reaction rate of the wild-type is only 0.0352. Mutant 9 of the present application has a greater speed advantage in clinical testing.

[0206] (3) Yield: Compared with the wild-type enzyme, mutant 9 introduced an expression-promoting tag at its N-terminus and was recombinantly expressed in E. coli. The yield in shake flask fermentation exceeded 100 mg / L, while the yield of the wild-type was only about 14 mg / L. This makes industrial large-scale production possible.

[0207] (4) In terms of sensitivity, the sensitivity of mutant 9 was significantly improved compared with the wild-type enzyme.

[0208] The various technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. In order to make the description concise, not all possible combinations of the various technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of the description in this specification.

[0209] The embodiments described above only express several implementation methods of the present application, which are convenient for understanding the technical solutions of the present application in a specific and detailed manner, but they cannot be understood as limiting the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several variations and improvements can be made, which all fall within the scope of protection of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the scope of protection of the present application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the scope of protection of the claims attached to the present application. Therefore, the scope of protection of the patent application of this application shall be based on the content of the attached claims, and the description can be used to interpret the content of the claims.

Claims

1. A mutant of leucine dehydrogenase, characterized in that Relative to the wild-type leucine dehydrogenase, the mutations of the mutant are M346A and D122F, or K89R and D122F; The amino acid sequence of the wild-type leucine dehydrogenase is shown in SEQ ID NO.1; The mutant also has a functional C-terminal modification, wherein the functional C-terminal modification is two aspartic acid residues; The N-terminus of the mutant is further connected to a protein tag, wherein the protein tag includes an expression-promoting tag and a purification tag, wherein the expression-promoting tag includes a P17-tag and / or a SKIK-tag, and the purification tag includes a His tag; The N-terminus of the mutant includes the expression-promoting tag and the purification tag connected in sequence.

2. The mutant of leucine dehydrogenase according to claim 1, characterized in that The protein tag is connected to the N-terminus of the mutant via a flexible peptide.

3. The mutant of leucine dehydrogenase according to claim 2, characterized in that The expression-promoting tag and the purification tag are connected via an amide bond.

4. The mutant of leucine dehydrogenase according to any one of claims 1 to 3, characterized in that The amino acid sequence of the mutant is shown in SEQ ID NO.

4.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the mutant of leucine dehydrogenase according to any one of claims 1 to 4.

6. A carrier, characterized in that The vector comprises the nucleic acid molecule of claim 5.

7. A cell, characterized in that The cell expresses the mutant of leucine dehydrogenase according to any one of claims 1 to 4, comprising the nucleic acid molecule according to claim 5 or the vector according to claim 6; The cells are microorganisms or animal cells.

8. The method for preparing a mutant of leucine dehydrogenase according to any one of claims 1 to 4, characterized in that: The preparation method comprises the following steps: The cell according to claim 7 is fermented, and the mutant of leucine dehydrogenase is isolated from the fermentation product.

9. The method for preparing a mutant of leucine dehydrogenase according to claim 8, characterized in that: The fermentation comprises the steps of amplifying and culturing the cells and inducing the mutants.

10. The method for preparing a mutant of leucine dehydrogenase according to claim 9, characterized in that: Fermentation meets one or more of the following conditions: (A) The temperature of the amplification culture is 35°C-39°C; (B) IPTG was added to induce expression when the OD600 of the culture system reached 0.6-1.

2. The final concentration of IPTG in the initial induced expression system was 0.1-1.0 mmol / L. (C) The induction temperature is 18-37°C, and the induction time is 6-20 hours; (D) The fermentation medium includes 8 g / L-12 g / L tryptone, 4 g / L-6 g / L yeast extract, 8 g / L-12 g / L NaCl, 1 g / L-2 g / L glycerol, 1 mmol / L-15 mmol / L magnesium sulfate, and water.

11. A detection kit for branched-chain amino acids or branched-chain α-keto acids, characterized in that: The detection kit comprises the mutant of leucine dehydrogenase according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, the vector according to claim 6 or the cell according to claim 7.

12. Use of the mutant of leucine dehydrogenase according to any one of claims 1 to 4 or the detection kit according to claim 11 in detecting branched-chain amino acids or branched-chain α-keto acids in a sample to be tested.

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