γ-Glutamine methylamine synthetase mutant and its application

Abstract

The present invention provides a γ-glutamylmethylamine synthetase mutant and its application. The γ-glutamylmethylamine synthetase mutant or its active fragment has an amino acid sequence shown in general formula I, and compared with the amino acid sequence shown in SEQ ID NO:2, it contains at least one amino acid mutation at a corresponding position. The γ-glutamylmethylamine synthetase mutant or its active fragment of the present invention exerts a synergistic regulatory effect through a combination of different mutation sites, significantly improves the ability of γ-glutamylmethylamine synthetase to synthesize L-theanine, and has better thermal stability.

Description

Technical Field

[0001] The invention belongs to the field of enzyme engineering and genetic engineering, and relates to a gamma-glutamyl methylamine synthetase mutant or an active fragment thereof, a genetic engineering bacterium containing the mutant or the active fragment thereof, and applications thereof. Background Art

[0002] L-Theanine is a unique free amino acid in tea leaves. Its chemical name is N-ethyl-L-glutamine and its molecular formula is C7H 14 N2O3, relative molecular weight is 174.198, melting point is 207℃, soluble in water, insoluble in ethanol and ether. Its structural formula is as follows:

[0003]

[0004] L-theanine can significantly promote the release of dopamine in the brain center, increase the physiological activity of dopamine in the brain, relieve fatigue, lower blood pressure and improve learning and memory ability. As early as 1985, the U.S. Food and Drug Administration (FDA) recognized L-theanine and confirmed that synthetic L-theanine is a generally recognized safe substance (GRAS), and there is no limit on its use. Due to its good safety and stability, L-theanine is widely used. It can be used as a quality improver for tea beverages and an additive to improve the flavor of food. It plays a role in improving the flavor of products and is used in various snacks, candies, jellies and other foods; as an additive for functional foods, it can be added to functional foods used to improve sleep and relieve mental tension.

[0005] At present, there are four main methods for preparing L-theanine: extraction and separation, chemical synthesis, enzymatic synthesis and microbial fermentation. The extraction and separation method is the earliest process, which extracts and concentrates L-theanine from tea leaves, but it cannot be applied on a large scale due to raw materials and cost issues. The chemical synthesis method obtains L-theanine through chemical reactions, but the reaction process requires the use of a variety of organic solvents, which cannot be industrialized due to safety and cost issues. The microbial fermentation method uses microbial cell metabolism to produce L-theanine. The internal environment of the microorganism is relatively stable, the raw materials are simple, and the environmental pollution is small, but the subsequent extraction is more difficult. The enzymatic synthesis method refers to the conversion of L-theanine by enzymes. At present, there are four types of enzymes that can participate in the catalytic synthesis of L-theanine, including glutamine synthetase (GS), γ-glutamylmethylamine synthetase (GMAS), γ-glutamyl transpeptidase (GGT) and γ-glutaminase. However, this method has problems such as low enzyme activity and poor thermal stability.

[0006] In 2008, Yamamoto S et al. used Escherichia coli recombinant γ-glutamylmethylamine synthetase (GMAS) from Methylovorus mays NO.9, and yeast as an ATP regeneration tool to catalyze equimolar sodium glutamate and ethylamine hydrochloride, and finally obtained 110g / L of L-theanine. However, due to the low specific activity of the enzyme and the high cost, the system is difficult to be industrialized.

[0007] Patent document CN201510973289.X reports the use of γ-glutamylmethylamine synthetase (GMAS) from Methylovorus mays and a phosphokinase as catalysts, with sodium L-glutamate and ethylamine hydrochloride as substrates to synthesize L-theanine. The conversion rate of L-theanine can be as high as 80% or more, and the ee value can reach more than 99%. However, its reproducibility is low, and the enzyme activity in the reaction is low, there is obvious product inhibition, and it is difficult to achieve effective accumulation of the product, so it does not have industrial prospects.

[0008] Patent applications with publication numbers CN113151198A and CN113151199A improve the thermal stability of the enzyme by mutation, and utilize the thermal stability of the enzyme to carry out catalytic reactions at 43-46°C. The molar conversion rate is 93% at high substrate concentrations, but some substrate remains, increasing the difficulty of product extraction. At the same time, high-temperature reactions increase energy consumption and equipment investment.

[0009] In summary, the catalytic activity of the modified γ-glutamyl methylamine synthetase still needs to be improved. Rational design and modification for improving the catalytic activity of γ-glutamyl methylamine synthetase are of great significance for the industrial production of L-theanine. Summary of the invention

[0010] Therefore, the object of the present invention is to provide a γ-glutamyl methylamine synthetase mutant based on the prior art. Compared with the prior art, the γ-glutamyl methylamine synthetase mutant provided by the present invention has better catalytic activity and stability. The present invention also provides the use of the γ-glutamyl methylamine synthetase mutant for catalytic production of L-theanine.

[0011] The inventive concept of the present invention is as follows: starting from the amino acid sequence (SEQ ID NO: 1) of the wild-type γ-glutamyl methylamine synthetase, a γ-glutamyl methylamine synthetase mutant library is constructed through the rational design of the enzyme molecule, and a series of mutants with improved catalytic activity are obtained, which are significantly improved compared to the parent activity. On this basis, the beneficial mutation sites are combined, and the γ-glutamyl methylamine synthetase combination mutant of the present invention is unexpectedly obtained, which can further exert a synergistic regulatory effect on the basis of the improved γ-glutamyl methylamine synthetase, and has better enzyme specific activity and improved stability.

[0012] The objective of the present invention is achieved through the following technical solutions:

[0013] In one aspect, the present invention provides a γ-glutamyl methylamine synthetase mutant or an active fragment thereof, which has an amino acid sequence shown in general formula I:

[0014] General formula Ⅰ:

[0015] X1KPCYDYKGITRHSPFLEKLTESLVEVGLDIYQIDHEDANGQFEINYTX2ADCLKSADDYIMFKMAASEIANELGIICSFMPKPFSNRPGNGMHMHMSIGD GX3X4

[0016] Wherein, X1 is selected from Q, S, T, L or A;

[0017] X2 is selected from Y, T, S, A or G;

[0018] X3 is selected from K, A, G, L or V;

[0019] X4 is selected from K, A, G, L or V;

[0020] Furthermore, the mutant or its active fragment comprises at least one amino acid mutation at a corresponding position compared to the amino acid sequence shown in SEQ ID NO:2.

[0021] According to the first aspect of the present invention, the amino acid sequence shown in SEQ ID NO: 2 is derived from the amino acid sequence of the wild-type γ-glutamyl methylamine synthetase of the methylotrophic beetle bacterium Methylovorus mays NO.9. Specifically, the amino acid sequence of the wild-type γ-glutamyl methylamine synthetase of the methylotrophic beetle bacterium Methylovorus mays NO.9 is shown in SEQ ID NO: 1:

[0022] MKSLEEAQKFLEDHHVKYVLAQFVDIHGVAKVKSVPASHLNDILTTGAG

[0023] FAGGAIWGTGIAPNGPDYMAIGELSTLSLIPWQPGYARLVCDGHVNGKPY

[0024] EFDTRVVLKQQIARLAEKGWTLYTGLEPEFSLLKKDEHGAVHPFDDSDTL

[0025] QKPCYDYKGITRHSPFLEKLTESLVEVGLDIYQIDHEDANGQFEINYTYAD

[0026] CLKSADDYIMFKMAASEIANELGIICSFMPKPFSNRPGNGMHMHMSIGDG

[0027] KK SLFQDDSDPSGLGLSKLAYHFLGGILAHAPALAAVCAPTVNSYKRLVV

[0028] GRSLLSGATWAPAYIAYGNNNRSTLVRIPYGRLELRLPDGSCNPYLATAAVI

[0029] AAGLDGVARELDPGTGRDDNLYDYSLEQLAEFGIGILPQNLGEALDALEADQVIMDAMGPGLSKEFVELKRMEWVDYMRHVSDWEINRYVQFY.

[0030] Based on the existing wild-type γ-glutamyl methylamine synthetase, multiple beneficial mutants were obtained through structural analysis and rational design. Comparison revealed that the mutations of these mutants are mainly concentrated in the region of the amino acid sequence shown in SEQ ID NO: 2 (the underlined region in SEQ ID NO: 1):

[0031] SEQ ID NO:2:

[0032] QKPCYDYKGITRHSPFLEKLTESLVEVGLDIYQIDHEDANGQFEINYTYAD

[0033] CLKSADDYIMFKMAASEIANELGIICSFMPKPFSNRPGNGMHMHMSIGDGKK.

[0034] Preferably, the amino acid sequence of the γ-glutamylmethylamine synthetase mutant or its active fragment according to the present invention is:

[0035] Compared with the amino acid sequence shown in SEQ ID NO: 2, it comprises at least one amino acid mutation at the corresponding position, wherein the amino acid mutation is selected from Q1S, Y49T, K102A or K103A;

[0036] Compared with the amino acid sequence shown in SEQ ID NO: 2, it comprises amino acid mutations at at least two corresponding positions, wherein the amino acid mutations are selected from Q1S / Y49T, Y49T / K102A or Y49T / K103A;

[0037] Compared with the amino acid sequence shown in SEQ ID NO: 2, it comprises amino acid mutations at at least three corresponding positions, wherein the amino acid mutations are selected from Q1S / Y49T / K102A, Q1S / Y49T / K103A or Y49T / K102A / / K103A; or

[0038] Compared with the amino acid sequence shown in SEQ ID NO: 2, it comprises amino acid mutations at at least four corresponding positions, and the amino acid mutations are Q1S / Y49T / K102A / K103A.

[0039] The γ-glutamylmethylamine synthetase mutant or its active fragment according to the present invention has or is the following amino acid sequence:

[0040] An amino acid sequence that is at least 80%, at least 85%, 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% or 100% identical to the amino acid sequence shown in any one of SEQ ID NOs: 13-23.

[0041] Preferably, the γ-glutamyl methylamine synthetase mutant or an active fragment thereof has or is an amino acid sequence shown in any one of SEQ ID NOs: 13-23.

[0042] SEQ ID NO:13

[0043] SKPCYDYKGITRHSPFLEKLTESLVEVGLDIYQIDHEDANGQFEINYTYAD

[0044] CLKSADDYIMFKMAASEIANELGIICSFMPKPFSNRPGNGMHMHMSIGDG

[0045] KK

[0046] SEQ ID NO:14

[0047] QKPCYDYKGITRHSPFLEKLTESLVEVGLDIYQIDHEDANGQFEINYTTAD

[0048] CLKSADDYIMFKMAASEIANELGIICSFMPKPFSNRPGNGMHMHMSIGDG

[0049] KK

[0050] SEQ ID NO:15

[0051] QKPCYDYKGITRHSPFLEKLTESLVEVGLDIYQIDHEDANGQFEINYTYAD

[0052] CLKSADDYIMFKMAASEIANELGIICSFMPKPFSNRPGNGMHMHMSIGDG

[0053] AK

[0054] SEQ ID NO:16

[0055] QKPCYDYKGITRHSPFLEKLTESLVEVGLDIYQIDHEDANGQFEINYTYAD

[0056] CLKSADDYIMFKMAASEIANELGIICSFMPKPFSNRPGNGMHMHMSIGDG

[0057] KA

[0058] SEQ ID NO:17

[0059] SKPCYDYKGITRHSPFLEKLTESLVEVGLDIYQIDHEDANGQFEINYTTAD

[0060] CLKSADDYIMFKMAASEIANELGIICSFMPKPFSNRPGNGMHMHMSIGDG

[0061] KK

[0062] SEQ ID NO:18

[0063] QKPCYDYKGITRHSPFLEKLTESLVEVGLDIYQIDHEDANGQFEINYTTAD

[0064] CLKSADDYIMFKMAASEIANELGIICSFMPKPFSNRPGNGMHMHMSIGDG

[0065] AK

[0066] SEQ ID NO:19

[0067] QKPCYDYKGITRHSPFLEKLTESLVEVGLDIYQIDHEDANGQFEINYTTAD

[0068] CLKSADDYIMFKMAASEIANELGIICSFMPKPFSNRPGNGMHMHMSIGDG

[0069] KA

[0070] SEQ ID NO:20

[0071] SKPCYDYKGITRHSPFLEKLTESLVEVGLDIYQIDHEDANGQFEINYTTAD

[0072] CLKSADDYIMFKMAASEIANELGIICSFMPKPFSNRPGNGMHMHMSIGDG

[0073] AK

[0074] SEQ ID NO:21

[0075] SKPCYDYKGITRHSPFLEKLTESLVEVGLDIYQIDHEDANGQFEINYTTAD

[0076] CLKSADDYIMFKMAASEIANELGIICSFMPKPFSNRPGNGMHMHMSIGDG

[0077] KA

[0078] SEQ ID NO:22

[0079] QKPCYDYKGITRHSPFLEKLTESLVEVGLDIYQIDHEDANGQFEINYTTAD

[0080] CLKSADDYIMFKMAASEIANELGIICSFMPKPFSNRPGNGMHMHMSIGDG

[0081] AA

[0082] SEQ ID NO:23

[0083] SKPCYDYKGITRHSPFLEKLTESLVEVGLDIYQIDHEDANGQFEINYTTAD

[0084] CLKSADDYIMFKMAASEIANELGIICSFMPKPFSNRPGNGMHMHMSIGDG

[0085] AA

[0086] The second aspect of the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the γ-glutamylmethylamine synthetase mutant or an active fragment thereof.

[0087] Preferably, the nucleotide sequence is obtained by base substitution based on the nucleotide sequence of wild-type γ-glutamylmethylamine synthetase.

[0088] The third aspect of the present invention provides an expression vector, which comprises the isolated nucleic acid molecule of the second aspect of the present invention. The expression vector selected by the present invention can stably exist in various hosts of prokaryotic or eukaryotic cells and can replicate autonomously. The expression vector of the present invention can be a plasmid vector for a prokaryotic expression system, including but not limited to the pET series, such as pET28a and pET3a; pBAD series, such as pBAD-HisA, pBAD30, pGEX-2T, pXMJ19 and pecxk99e, etc., or a plasmid vector for a fungal expression system, including but not limited to pPIC9K, pPIC9, PRS304, pUG6, pSH47, pUC110, pPZP-HYG2 and pFC330, etc. The expression vector is preferably pET28a or pPIC9K.

[0089] The fourth aspect of the present invention provides a host cell or genetically engineered bacteria, which comprises the isolated nucleic acid molecule or expression vector described in the second or third aspect of the present invention. The host cell or genetically engineered bacteria includes but is not limited to Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae or Bacillus subtilis, etc., preferably Escherichia coli or Pichia pastoris, more preferably Escherichia coli BL21 (DE3). The host cell or genetically engineered bacteria is obtained by directly inserting the isolated nucleic acid molecule of the second aspect of the present invention into the chromosome of the host cell or genetically engineered bacteria, or by using the calcium chloride method or electroporation transformation method to introduce the expression vector of the third aspect of the present invention into the host cell or genetically engineered bacteria.

[0090] The fifth aspect of the present invention provides a catalyst comprising the γ-glutamyl methylamine synthetase mutant or its active fragment of the first aspect of the present invention, or the host cell or genetically engineered bacteria of the fourth aspect of the present invention, and a preparation method thereof. The catalyst includes three forms: a whole cell catalyst, a free protein catalyst, or an immobilized enzyme catalyst. The whole cell catalyst refers to the whole cell or bacterial body obtained after enrichment culture and induced expression of the target protein by the host cell or genetically engineered bacteria of the fourth aspect of the present invention; the free protein catalyst is the whole cell or bacterial body that is ultrasonically crushed or high-pressure homogenized and crushed, and the crude enzyme liquid obtained by centrifugation, and also includes the pure enzyme obtained by protein purification. The immobilized enzyme catalyst is to select different immobilization carriers to immobilize the free protein catalyst, thereby obtaining different forms of immobilized γ-glutamyl methylamine synthetase mutants or their active fragments.

[0091] The sixth aspect of the present invention provides a method for obtaining the γ-glutamylmethylamine synthetase mutant or an active fragment thereof according to the first aspect of the present invention, which comprises the following steps:

[0092] Site-directed mutagenesis primers are designed, the nucleotide sequence of γ-glutamyl methylamine synthetase of Methylovorus mays NO.9 is used as a template for site-directed mutagenesis and a mutant expression plasmid is constructed, and a competent host cell is transformed with the mutant expression plasmid to obtain a recombinant mutant strain; the recombinant mutant strain is fermented and cultured to induce expression, thereby obtaining a γ-glutamyl methylamine synthetase mutant or an active fragment thereof.

[0093] The seventh aspect of the present invention relates to a method for preparing the γ-glutamyl methylamine synthetase mutant or its active fragment according to the first aspect of the present invention, the preparation method comprising culturing the host cell or genetically engineered bacteria according to the fourth aspect of the present invention to obtain a fermentation product, and obtaining the γ-glutamyl methylamine synthetase mutant or its active fragment from the fermentation product.

[0094] The eighth aspect of the present invention relates to a method for preparing L-theanine, which comprises using sodium glutamate and ethylamine hydrochloride as raw materials, using the γ-glutamylmethylamine synthetase mutant or its active fragment according to the first aspect of the present invention, the host cell or genetically engineered bacteria according to the fourth aspect of the present invention, or the catalyst according to the fifth aspect of the present invention to catalyze the reaction of the raw materials to produce L-theanine.

[0095] Preferably, the preparation method comprises incubating the γ-glutamyl methylamine synthetase mutant or its active fragment, host cell or genetically engineered bacteria or catalyst at 40°C-45°C for 1 to 3 hours, more preferably at 45°C for 3 hours before catalyzing the raw material reaction.

[0096] The reaction in the preparation method can adopt conventional process conditions, for example, the reaction temperature is selected to be 20-40°C, such as 25-37°C.

[0097] The reaction system in the preparation method can be a buffer system, such as a phosphate buffer, pH 5.0-9.0, such as pH 6.0-8.0, preferably pH 6.5-7.5.

[0098] The concentration of the γ-glutamine methylamine synthetase mutant or its active fragment in the reaction system is 50-60 g / L, preferably 55 g / L.

[0099] The reaction system also uses MgCl2, for example, at a concentration of 28-35 mM, preferably 30 mM MgCl2, and / or MnCl2, for example, at a concentration of 4-8 mM, preferably 5 mM MnCl2.

[0100] ATP is also added to the reaction system, for example, at a concentration of 28-35 mM, preferably 30 mM.

[0101] Compared with the γ-glutamylmethylamine synthetase SEQ ID NO: 1 derived from the microorganism Methylovorus mays NO.9, the γ-glutamylmethylamine synthetase mutant or its active fragment constructed by the present invention has significantly improved enzyme activity in the catalytic reaction and better stability. The γ-glutamylmethylamine synthetase mutant or its active fragment can be heat-treated at a higher temperature, such as 45° C., before being used in the reaction. The heat-treatment is beneficial to removing miscellaneous enzymes such as amino acid hydrolases and racemases of the host cell itself, thereby reducing the consumption of substrates.

[0102] The ninth aspect of the present invention relates to the use of the mutant of γ-glutamylmethylamine synthetase or its active fragment, the host cell or genetically engineered bacteria, or the catalyst in the preparation of L-theanine.

[0103] Specifically, theanine is preferably produced by the following preparation method:

[0104] Sodium glutamate and ethylamine hydrochloride are used as raw materials, and the mutant or active fragment thereof according to the first aspect of the present invention, the host cell or genetically engineered bacteria according to the fourth aspect of the present invention, or the catalyst according to the fifth aspect of the present invention is used to catalyze the reaction of the raw materials to obtain the product L-theanine.

[0105] Preferably, the method comprises, before the catalytic reaction, incubating the γ-glutamylmethylamine synthetase mutant or its active fragment, the host cell or genetically engineered bacteria or the catalyst at 40°C-45°C for 1 to 3 hours, more preferably at 45°C for 3 hours.

[0106] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0107] The beneficial mutation sites discovered by the inventors significantly improve the activity of γ-glutamylmethylamine synthetase. By combining these beneficial mutation sites, the γ-glutamylmethylamine synthetase mutant of the present invention or its active fragment is unexpectedly obtained, which can play a synergistic regulatory effect on the basis of the improved γ-glutamylmethylamine synthetase, and further significantly improve the activity while having better thermal stability. The γ-glutamylmethylamine synthetase mutant of the present invention or its active fragment can be heat-treated at a higher temperature, such as 45°C, before being used for the reaction. The heat-treatment is beneficial to remove the host cell's own amino acid hydrolase, racemase and other miscellaneous enzymes, and reduce the consumption of substrates. Therefore, the γ-glutamylmethylamine synthetase mutant of the present invention or its active fragment has an industrial development and application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0108] In order to more clearly illustrate the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to the accompanying drawings:

[0109] Figure 1 The enzyme activity is measured by the enzyme activity detection method in Example 4 of the present invention, and the maximum enzyme activity is taken as 100%, and the relative enzyme activity at other temperatures is calculated;

[0110] Figure 2 As shown in Example 4 of the present invention, the thermal stability of each GMAS was compared according to its relative enzyme activity. DETAILED DESCRIPTION

[0111] The features and exemplary embodiments of various aspects of the present invention will be described in detail below. The specific embodiments listed in the present invention are only examples of the present invention, and the present invention is not limited to the specific embodiments described below.

[0112] For those skilled in the art, any equivalent modifications and substitutions to the embodiments described below are also within the scope of the present invention. Therefore, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention. The specific conditions not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. All reagents or instruments that do not specify the manufacturer are conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are given in the specific embodiments below. It should be understood by those skilled in the art that the present invention can also be implemented without certain specific details. In other embodiments, methods, means, equipment and steps well known to those skilled in the art are not described in detail to highlight the purport of the present invention.

[0113] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Unless otherwise specified, the units used in this specification are all international standard units, and the values ​​and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0114] definition

[0115] Throughout this discussion, the standard single-letter codes for amino acids are used, as are standard substitution notations.

[0116] 1) Amino acid residues are represented by single-letter symbols using the generally accepted IUPAC nomenclature. DNA sequences are also represented by the generally accepted IUPAC nomenclature.

[0117] 2) Mutant identification: The mutated amino acid in the GMAS mutant is represented by "amino acid replaced by the original amino acid position". For example, Q1S means that the amino acid at position 1 is replaced by serine Ser from glutamine Gln in the original γ-glutamylmethylamine synthetase, and the position number corresponds to the amino acid sequence number in SEQ ID NO:2.

[0118] In the expressions separated by the " / " symbol at different positions, the " / " symbol means "and". For example, Q1S / Y49T means that the amino acid at position 1 is replaced by glutamine Gln in the original γ-glutamylmethylamine synthetase to serine Ser, and the amino acid at position 49 is replaced by tyrosine Tyr in the original γ-glutamylmethylamine synthetase to threonine Thr.

[0119] The term "wild type" refers to a gene or gene product that is separated from a naturally occurring source. A wild-type gene is the most commonly observed gene in a population and is therefore arbitrarily designed to be the "normal" or "wild-type" form of a gene. In contrast, the term "modified", "mutant" or "variant" refers to a gene or gene product that displays modification (e.g., substitution, truncation or insertion) of a nucleotide sequence, post-translational modification and / or functional properties (e.g., altered properties) compared to a wild-type gene or gene product. Note that naturally occurring mutants can be isolated and identified by the fact that they have altered properties compared to a wild-type gene or gene product. Methods for introducing or replacing naturally occurring amino acids are well known in the art. For example, methionine (M) can be replaced by arginine (R) by replacing the codon (ATG) of methionine at the relevant position in the polynucleotide encoding the mutant. Methods for introducing or replacing non-naturally occurring amino acids are also well known in the art.

[0120] Wild-type γ-glutamyl methylamine synthetase

[0121] γ-glutamylmethylamide synthetase (GMAS) is a catalytic enzyme that can catalyze the synthesis of N-methyl-L-glutamine from L-glutamate and methylamine, and the reaction requires consumption of ATP. In this article, the terms "wild (type) enzyme" and "wild γ-glutamylmethylamine synthetase" have the same meaning, both referring to the γ-glutamylmethylamine synthetase derived from Methylovorus mays NO.9, and its amino acid sequence is SEQ ID NO: 1.

[0122] Correspondingly, the terms "γ-glutamylmethylamine synthetase mutant", "mutant γ-glutamylmethylamine synthetase", "mutant GMAS" and "mutant enzyme" have the same meaning, and all refer to mutants formed by changing amino acids based on the wild-type enzyme.

[0123] Determination of the catalytic activity of γ-glutamylmethylamine synthetase

[0124] 1. Determination of enzyme properties

[0125] Enzyme activity definition: 1U enzyme activity is defined as the amount of enzyme required to generate 1μM L-theanine per minute at 35°C. The reaction system contains: 100mM sodium glutamate, 150mM ethylamine hydrochloride, 150mM ATP, 100mM potassium phosphate buffer (pH7.0), 0.5% wet bacteria. The reaction is carried out at 37°C for 2h, and the L-theanine content is determined by HPLC.

[0126] The enzymatic properties of GMAS may change after mutation. In order to explore the optimal enzyme activity conditions, the enzymatic properties of the unmutated WT (original bacteria) and the mutant under the same conditions were analyzed and compared.

[0127] 2. Thermal stability

[0128] Determination of temperature stability: Keep the sample at 25°C to 60°C for 30 minutes, then determine the residual enzyme activity in the sample according to the enzyme activity detection method, define the enzyme activity of the sample without heat treatment as 100% to calculate the relative enzyme activity, and draw a temperature-relative enzyme activity curve.

[0129] 3. Recombinant vector and transformant of γ-glutamyl methylamine synthetase mutant

[0130] Depending on the host cell, the nucleotide sequence encoding the γ-glutamyl methylamine synthetase mutant can be constructed into different types of recombinant vectors, or directly integrated into the chromosome of the host bacteria. As usable vectors, plasmid DNA, phage DNA, retrotransposon DNA, artificial chromosome DNA, etc. can be listed.

[0131] The host used for the transformant of the present invention is not particularly limited as long as it can express the target γ-glutamylmethylamine synthetase after the introduction of the above-mentioned recombinant vector or the nucleotide sequence encoding the γ-glutamylmethylamine synthetase mutant, and for example, bacteria such as Escherichia coli, yeast, animal cells, insect cells, plant cells, etc. can be used.

[0132] The method for introducing a recombinant vector into bacteria is not particularly limited as long as it is a method for introducing DNA into bacteria, and examples thereof include a method using calcium ions and an electroporation method.

[0133] The method for integrating the nucleotide sequence encoding the γ-glutamylmethylamine synthetase mutant into bacteria is not particularly limited as long as it is a method for introducing DNA into bacteria, and examples thereof include homologous recombination and gene editing.

[0134] Example

[0135] The experimental methods without specific conditions in the following examples were carried out according to conventional methods and conditions, or according to the product instructions.

[0136] Plasmid pET28a was purchased from Wuhan Miaoling Biotechnology Co., Ltd.;

[0137] EcoRI, HindIII and other restriction enzymes, Escherichia coli DH5α, BL21 (DE3) competent cells, DNA marker, plasmid extraction kit, DNA gel recovery and purification kit, and T4 ligase were purchased from Takara Biotechnology (Dalian) Co., Ltd.;

[0138] DNA polymerase (Q5 High-Fidelity DNA Polymerase) was purchased from Gene Co., Ltd.;

[0139] Kanamycin sulfate and BCA protein content determination kits were purchased from Biosharp; Ni-NTA protein purification kit was purchased from Sangon Biotechnology;

[0140] All chemical reagents were of analytical grade from Chinese medicine. The plasmid extraction procedure was based on the instructions of the plasmid extraction kit.

[0141] The DNA gel recovery operation steps refer to the DNA gel recovery kit instructions; the DNA fragment ligation operation steps refer to the T4 ligase instructions;

[0142] The protein purification steps refer to the instructions of the Ni-NTA protein purification kit;

[0143] The protein content determination procedure refers to the BCA protein content determination kit instructions.

[0144] PCR amplification experiments were performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. If necessary, adjustments could be made through simple experiments.

[0145] LB medium (g / L): tryptone 10, yeast extract 5, sodium chloride 10, natural pH (LB solid medium plus 20g / L agar powder), sterilized at 121°C for 20min;

[0146] Slant medium (g / L): glucose 1, tryptone 10, yeast extract 5, potassium dihydrogen phosphate 1, magnesium sulfate heptahydrate 0.2, agar powder 20, pH 7.20, sterilized at 121°C for 20 min;

[0147] Fermentation medium (g / L): glucose 20, yeast peptone 5, yeast extract powder 5, citric acid 2, potassium dihydrogen phosphate 5.2, magnesium sulfate heptahydrate 2, ferrous sulfate heptahydrate 10 mg, manganese sulfate monohydrate 10 mg, biotin 0.1 mg, trace elements 1 mL (zinc sulfate 6.4, cobalt nitrate hexahydrate 4.9, copper sulfate pentahydrate 0.6), defoaming agent 0.3 ml / L, sterilize at 121°C for 20 min.

[0148] Example 1 Construction of GMAS genetically engineered bacteria

[0149] According to the NCBI database and patent disclosure information, the sequence information of the γ-glutamylmethylamine synthetase (GMAS) gene from Methylovorus mays NO.9 was consulted (SEQ ID No: 1), the gene sequence was codon optimized according to the codon preference rule of Escherichia coli, and EcoRI and HindIII restriction sites were designed and added at both ends of the gene. Then, it was sent to Jinkairui Biotechnology Co., Ltd. for artificial synthesis.

[0150] The plasmid pET28a and the synthesized GMAS gene fragment were double-digested with EcoRI / HindIII, respectively. The reaction system was: 1 μg of plasmid or GMAS gene fragment, 5 μl of 10× buffer, 1 μl of EcoRI, 1 μl of HindIII, and water was added to 50 μl. The enzyme digestion was carried out at 37℃ for 5 h. The 5.5 kb nucleotide fragment was recovered by 1% agarose gel electrophoresis and DNA gel recovery and purification kit;

[0151] The GMAS gene DNA fragment was connected to the pET28a vector using T4 DNA ligase. The connection system was: pET28a 0.5 μl, Solution I 5 μl, GMAS gene DNA fragment 4.5 μl. The connection was carried out at 16°C overnight.

[0152] The ligation product was transformed into Escherichia coli DH5α competent cells by heat shock transformation method, and the transformation product was spread on LB plate containing 100 μg / ml kanamycin sulfate. After overnight culture at 37°C, positive transformants were selected and sent to Jinkairui Biotechnology Co., Ltd. for sequencing. The transformants with correct sequencing were the plasmid vector GMAS-pET28a containing the γ-glutamylmethylamine synthetase gene.

[0153] The correctly sequenced transformants were transferred to 5 ml LB medium, cultured at 37°C and 200 rpm for 16 hours, and 2 ml of bacterial solution was taken and centrifuged at 10,000 rpm for 2 minutes, and the supernatant was discarded. The plasmid was extracted from the wet bacteria using a plasmid mini-extraction kit. The plasmid was transformed into Escherichia coli BL21 (DE3) by heat shock method, and after overnight culture at 37°C, the positive transformants were selected for resequencing. The correctly sequenced transformants were GMAS genetically engineered bacteria W1.

[0154] Example 2 Construction of GMAS mutant genetically engineered bacteria

[0155] Based on the GMAS genetically engineered bacteria W1, the inventors designed a list of single mutations and combined mutations, as shown in Table 1:

[0156] Table 1

[0157] Mutant number Mutation site W2 Q1S W3 K102A W4 K103A W5 Y49T W6 Q1S / Y49T W7 K102A / Y49T W8 K103A / Y49T W9 Q1S / K102A / Y49T W10 Q1S / K103A / Y49T W11 K102A / K103A / Y49T W12 Q1S / K102A / K103A / Y49T

[0158] Design mutation primers for the mutation sites, and the primer sequences are shown in Table 2:

[0159] Table 2

[0160]

[0161] Single point mutation: Using GMAS FP / Q1S RP, GMAS FP / K102A RP, GMAS FP / K103A RP, and GMASFP / Y49T RP as primers, and the GMAS-pET28a plasmid in Example 1 as a template, high-fidelity Q5 High-Fidelity DNA Polymerase polymerase was used to amplify the left fragment of each mutation site by PCR; using Q1S FP / GMAS RP, K102A FP / GMASRP, K103AFP / GMAS RP, and Y49T FP / GMAS RP as primers, and GMAS-pET28a was also used as a template to amplify the right fragment of the mutation site; then using GMAS FP / GMAS RP as primers, overlap extension PCR was performed using the left and right fragments corresponding to the amplified mutation as templates, respectively, to amplify the full-length GMAS single mutation fragments W2 to W5.

[0162] Combined mutation: Using the amplified single mutation DNA gene fragment as a template, referring to the above-mentioned primer combination method, first amplify the left fragment of the mutation site, then amplify the right fragment of the mutation site, and then use GMAS FP / GMAS RP as primers, and use the left and right fragments corresponding to the amplified mutations as templates to perform overlapping extension PCR to amplify the full-length GMAS double mutation fragments W6~W8; similarly, using the amplified double mutation DNA gene fragment as a template, amplify the full-length GMAS triple mutation fragments W9~W11; similarly, using the amplified triple mutation DNA gene fragment as a template, amplify the full-length GMAS quadruple mutation fragment W12.

[0163] The PCR reaction system (50 μl) is: 10 μl of 5×Q5 reaction buffer, 1 μl of 10 mM dNTP, 2.5 μl of each primer, 0.5 μl of Q5 enzyme, the template concentration depends on the sample concentration, and water is added to 50 μl.

[0164] The final PCR product was detected by 1% agarose gel electrophoresis and purified using a DNA gel recovery kit.

[0165] The plasmid pET28a was double-digested with EcoRI / HindIII, and the reaction system was: 1μg of plasmid, 5μl of 10× buffer, 1μl of EcoRI, 1μl of HindIII, and water was added to 50μl. The enzyme digestion was carried out at 37℃ for 5h. The linear fragment of 5.5kb was recovered by 1% agarose gel electrophoresis and DNA gel recovery and purification kit.

[0166] The Onestep clonning cloning recombination kit was used to recombinant the above-mentioned double enzyme linear fragments and the mutant fragments recovered by amplification, and the recombinant products were used to transform Escherichia coli DH5α competent cells. The transformation products were spread on LB plates containing 100 μg / ml kanamycin sulfate. After overnight culture at 37°C, the positive transformants were selected and sent to Jinkairui Biotechnology Co., Ltd. for sequencing, and the correctly sequenced transformant W2~W12 mutant plasmid vectors were obtained.

[0167] Transfer the correctly sequenced transformants to 5 ml LB medium, shake and culture at 37°C and 200 rpm for 16 hours, take 2 ml of bacterial solution, centrifuge at 10,000 rpm for 2 minutes, and discard the supernatant. Extract the plasmid from the wet bacteria using a plasmid mini-extraction kit. Transform the plasmid into Escherichia coli BL21 (DE3) by heat shock method, culture at 37°C overnight, select the positive transformants and sequence them again. The correctly sequenced transformants are the W2-W12 mutant genetic engineering bacteria.

[0168] Example 3 Expression and purification of original GMAS and mutant GMAS

[0169] The original GMAS genetically engineered bacteria W1 and mutant genetically engineered bacteria W2-W12 were inoculated into 5 ml of LB liquid medium containing 100 μg / ml kanamycin sulfate, and cultured overnight at 37°C and 200 rpm. 2 ml of the culture was transferred to 150 ml of fermentation medium containing 100 μg / ml kanamycin sulfate, and cultured at 37°C and 220 rpm until OD600 reached 0.8-1.0, then 0.08 mM IPTG was added and cultured overnight at 25°C to express the recombinant protein.

[0170] The fermentation broth was centrifuged at 4°C and 10,000 r / min for 15 min, the bacterial precipitate was collected, and the bacterial cells were washed and resuspended with buffer (20 mM potassium phosphate buffer, pH 7.0), and ultrasonically disrupted in an ice-water bath (the disruption conditions were 100 W, 3s / 5s, 30min), then centrifuged at 4°C and 12,000 r / min for 20 min, and the supernatant was collected to obtain the crude enzyme solution.

[0171] The obtained crude enzyme solution was purified using a Ni-NTA protein purification kit, the purification effect was verified by SDS-PAGE, and the protein concentration was determined by the BCA method.

[0172] Example 4 GMAS enzyme activity assay

[0173] Reaction system: 100 mM sodium glutamate, 150 mM ethylamine hydrochloride, 150 mM ATP, 100 mM potassium phosphate buffer (pH 7.0), 0.5% wet bacteria, react at 37° C. for 2 h, and determine the content of L-theanine by HPLC.

[0174] 50 μl of the purified W1 and W2-W12 pure proteases in Example 3 were added to a 1.5 ml EP tube, substrate was added, and the GMAS enzyme activity was calculated. The results are shown in Table 3.

[0175] HPLC analysis method: XDB-C18 250mm*4.6, 5μm column, wavelength 338nm, mobile phase 40mM sodium dihydrogen phosphate (pH3.0): acetonitrile = 80:20, flow rate 1ml / min, temperature 35℃.

[0176] Table 3 Comparison of γ-glutamyl methylamine synthetase activity between wild strain and mutant strain

[0177] Strain name L-Theanine(mM) W1 100％ W2 152.3％ W3 156.2％ W4 157.3％ W5 222.4％ W6 244.4％ W7 246.5％ W8 247.2％ W9 270.5％ W10 272.6％ W11 274.4％ W12 290.3％

[0178] The test results showed that the enzyme activity of the mutant was improved, reaching up to 2.903 times the enzyme activity of the original W1, indicating that the enzyme's catalytic activity was improved by modifying the enzyme through site-directed mutagenesis.

[0179] Example 5 GMAS optimum temperature and thermal stability determination

[0180] The purified W1-W12 pure proteases in Example 3 were reacted with the substrate reaction solution at different temperatures (25-60°C), and then the enzyme activity was determined according to the enzyme activity detection method in Example 4. The maximum enzyme activity was taken as 100%, and the relative enzyme activity at other temperatures was calculated. The results are shown in FIG. Figure 1 shown.

[0181] Keep the sample at 25°C to 60°C for 30 minutes, and then measure the residual enzyme activity in the sample according to the enzyme activity detection method of Example 4. The enzyme activity of the sample without heat treatment is defined as 100% to calculate the relative enzyme activity. A temperature-relative enzyme activity curve is drawn. When the residual enzyme activity reaches more than 85%, it is defined as good thermal stability. The relative enzyme activity results are shown in Figure 4. Figure 1 The optimum temperature of the GMAS mutant is the same as that of the wild type, which is 37°C. When the temperature is lower or higher than 37°C, the catalytic activity of the GMAS mutant changes less than that of the wild type, indicating that its temperature range is wider than that of the wild type.

[0182] The enzyme was placed in a 20 mM potassium phosphate buffer at pH 7.0, and kept at 37°C, 45°C, 50°C, 55°C, 60°C, 65°C, and 70°C for 3 h, respectively, and then cooled in an ice water bath. The residual enzyme activity was then determined at 37°C with reference to the enzyme activity detection method in Example 4. The highest enzyme activity was defined as 100%, and the enzyme activities measured under different conditions were compared with it to calculate the relative enzyme activity. The thermal stability of each GMAS was compared according to its relative enzyme activity. The results are as follows: Figure 2 As shown in the figure, the thermal stability of GMAS mutants is significantly higher than that of the original enzyme, especially the W12 mutant, which has no significant change in enzyme activity after being incubated at 45°C for 3h. Moreover, when the temperature is higher than 55°C, the relative enzyme activity of GMAS of the W12 mutant is maintained at more than 85%, and the relative enzyme activity of the other mutants is also maintained at more than 75%, while the relative enzyme activity of the wild type is already less than 70%.

[0183] Example 6 Fermentation of original GMAS and mutants

[0184] Take 1 ml of glycerol strains containing original W1 and W9-W12 mutants respectively and activate them on slant medium, and culture them at 37°C for 16 hours; pick an inoculated loop of bacteria from the slant medium and transfer it to 100 ml of LB seed medium, and culture it at 37°C and 200 rpm for 10-16 hours. Inoculate seeds into a 15L fermentation tank containing 8L fermentation medium at a volume ratio of 5%, control the temperature at 37°C±1°C, supplement ammonia water during the fermentation process to control the pH to 7.0±0.1, control the ventilation volume to 0.5vvm-3.0vvm, control the speed to 200rpm-800rpm, maintain the dissolved oxygen at 30% by adjusting the speed and ventilation volume, and after fermentation for 7-8 hours, cool down to 25-30°C, add 0.08mM IPTG, induce for about 16 hours, and end the fermentation when the enzyme activity no longer increases.

[0185] The fermentation liquid was centrifuged at 6000rpm for 20min, the supernatant was discarded to obtain the bacterial cells, and the bacterial cells were washed and resuspended with a buffer solution (20mM potassium phosphate buffer solution, pH 7.0), and the bacterial cells were crushed by a high-pressure homogenizer (crushing conditions were 700-800bar high-pressure homogenization treatment twice), and then centrifuged at 4°C and 12000r / min for 20min, and the supernatant was collected to obtain a crude enzyme solution. The crude enzyme solution was kept at 45°C for 3h to remove the host cell's own amino acid hydrolase, racemase and other miscellaneous enzymes and reduce the consumption of substrates.

[0186] Example 7 GMAS conversion to produce theanine

[0187] The reaction system was 1 L, containing 400 mM sodium glutamate, 450 mM ethylamine hydrochloride, 30 mM MgCl2, 5 mM MnCl2, 30 mM ATP, and 55 g / L of the crude enzyme solution obtained in Example 6. The reaction was carried out at 37°C and 200 rpm for 16 h. The pH was controlled at 7.0 during the reaction. The concentration of theanine was detected by sampling, and the conversion rate was calculated. The results are shown in Table 4.

[0188] Table 4

[0189]

[0190] The concentration of theanine in the transformation liquid of the original GMAS was 28.5 g / L, while the product concentration of theanine in the transformation liquid of the mutant GMAS was above 64.0 g / L, and the product generation rate exceeded 90%, indicating that the site-directed mutagenesis of GMAS improved the catalytic activity and product generation rate of the enzyme. After the reaction of the W12 mutant strain was completed, the substrate could no longer be detected, and the product generation rate reached more than 99.7%, which was convenient for subsequent product extraction.

[0191] In summary, the activity of the γ-glutamyl methylamine synthetase mutant constructed by the present invention is significantly higher than that of the original γ-glutamyl methylamine synthetase SEQ ID NO: 1. The enzyme mutant is heat-treated at 45° C. for 3 h and then catalyzes sodium glutamate and ethylamine hydrochloride to react for 16 h under the action of ATP to generate 67.4 g / L of L-theanine without any substrate residue, and has industrial development and application prospects.

[0192] The above embodiments are exemplary and should not be construed as limiting the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A γ-glutamyl methylamine synthetase mutant, whose amino acid sequence is the amino acid sequence shown in any one of SEQ ID NOs: 13-23.

2. An isolated nucleic acid molecule, the nucleotide sequence of which encodes the γ-glutamylmethylamine synthetase mutant according to claim 1.

3. An expression vector comprising the isolated nucleic acid molecule according to claim 2. The expression vector according to claim 3 , which is a plasmid vector. The expression vector according to claim 4 , which is a plasmid vector for a prokaryotic expression system or a plasmid vector for a fungal expression system. The expression vector according to claim 5 , wherein the plasmid vector for prokaryotic expression system is selected from the group consisting of pET series and pBAD series.

7. The expression vector according to claim 6, wherein the plasmid vector for prokaryotic expression system is pET28a or pET3a.

8. The expression vector according to claim 6, wherein the plasmid vector for prokaryotic expression system is pBADHisA, pBAD30, pGEX 2T, pXMJ19 or pecxk99e.

9. The expression vector according to claim 5, wherein the plasmid vector for fungal expression system is selected from pPIC9K, pPIC9, PRS304, pUG6, pSH47, pUC110, pPZP HYG2 or pFC330. 10 . The expression vector according to claim 5 , which is pET28a or pPIC9K.

11. A host cell or genetically engineered bacterium comprising the isolated nucleic acid molecule according to claim 2 or the expression vector according to any one of claims 3 to 10.

12. The host cell or genetically engineered bacterium according to claim 11, which is selected from Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae or Bacillus subtilis. The host cell or genetically engineered bacterium according to claim 12 , which is Escherichia coli or Pichia pastoris. The host cell or genetically engineered bacterium according to claim 13 , which is Escherichia coli BL21 (DE3). 15 . A catalyst comprising the γ-glutamylmethylamine synthetase mutant according to claim 1 , or the host cell or genetically engineered bacterium according to any one of claims 11 to 14 .

16. Use of the γ-glutamylmethylamine synthetase mutant according to claim 1, the host cell or genetically engineered bacterium according to any one of claims 11 to 14, or the catalyst according to claim 15 in preparing L-theanine.

17. A method for preparing L-theanine, comprising using the γ-glutamylmethylamine synthetase mutant according to claim 1, the host cell or genetically engineered bacteria according to any one of claims 11 to 14, or the catalyst according to claim 15 to catalyze a raw material reaction to produce L-theanine.

18. The preparation method according to claim 17, wherein the raw materials include sodium glutamate and ethylamine hydrochloride.

19. The preparation method according to claim 17 or 18, comprising keeping the γ-glutamylmethylamine synthetase mutant according to claim 1, the host cell or genetically engineered bacterium according to any one of claims 11 to 14, or the catalyst according to claim 15 at 40°C-45°C for 1 to 3 hours before catalyzing the reaction of the raw materials.

20. The preparation method according to claim 19, comprising keeping the γ-glutamylmethylamine synthetase mutant according to claim 1, the host cell or genetically engineered bacterium according to any one of claims 11 to 14, or the catalyst according to claim 15 at 45°C for 3 hours before catalyzing the reaction of the raw materials.

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