CN111593038B - Glutaminase mutant with improved stability - Google Patents
Glutaminase mutant with improved stability Download PDFInfo
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Abstract
The invention discloses a glutaminase mutant with improved stability, which belongs to the technical field of enzyme engineering and microbial engineering, wherein the glutaminase mutant is obtained by mutating the 37 th tyrosine of a glutaminase parent derived from lactobacillus reuteri into cysteine, the half-life period of the glutaminase mutant at 44 ℃ reaches 12h, and the stability of the glutaminase mutant is improved by 50 percent compared with the glutaminase before mutation.
Description
Technical Field
The invention relates to a glutaminase mutant with improved stability, belonging to the technical field of enzyme engineering and microbial engineering.
Background
Glutaminase (EC 3.5.1.2) catalyzes the deamidation hydrolysis of glutamine to form glutamic acid and ammonia. When glutaminase catalyzes glutamine to hydrolyze, the method can be divided into two steps, wherein in the first step, nucleophilic groups on glutaminase and amido groups of glutamine perform nucleophilic reaction to generate covalent intermediate products-gamma-acyl-enzyme and ammonia gas; the second step is the hydrolysis of the gamma-acyl-enzyme intermediate to glutamic acid, which is a one-step reaction of nucleophilic substitution of water molecules for glutaminase. Glutamic acid plays an important role in the food industry, and not only can increase the delicate flavor of food, but also can increase the nutritional ingredients of food.
Glutamine plays a crucial role in microbial nitrogen metabolism, not only providing a nitrogen source for intermediary metabolites in the metabolic network, but also being a repressor of catabolite catabolites of the nitrogen source. The metabolic level of glutamine can also influence the balance of other amino acids and nitrogen-containing substances in a microorganism, the first step of converting glutamine into other intermediate products is to decompose glutamine into glutamic acid and ammonium ions, the most critical enzyme in the reaction is glutaminase, so the glutaminase is the critical enzyme influencing the balance of nitrogen metabolic networks of the microorganism, and in the reaction of the glutaminase, the high-temperature enzymatic degradation is very effective for improving the degradation rate of protein and can prevent the pollution of the microorganism; and the low content of glutamic acid, which is a product of glutaminase reaction, is mainly caused by the fact that glutaminase is severely inhibited by high temperature, so there is a high demand for heat-resistant glutaminase which is reliable in source, directly edible, and is in need of food production, and therefore, it is an object of the present invention to provide heat-resistant and food-grade glutaminase.
Disclosure of Invention
In order to solve the above technical problems, the present invention provides a mutant glutaminase derived from lactobacillus reuteri, wherein the tyrosine at position 37 is mutated to cysteine as compared with glutaminase having an amino acid sequence shown in SEQ ID No. 1.
In one embodiment of the invention, the nucleotide sequence encoding the glutaminase mutant is shown in SEQ ID NO. 2.
The invention also provides a gene for coding the glutaminase mutant.
The invention also provides a recombinant plasmid carrying the gene.
In one embodiment of the present invention, the vector of the recombinant plasmid is the pMA5 plasmid.
The invention also provides a host cell carrying the gene or the recombinant plasmid.
In one embodiment of the invention, the host cell is bacillus subtilis 168.
The invention also provides a preparation method of the glutaminase mutant, which comprises the steps of inoculating the host cell of any one of claims 5-7 into an LB culture medium, culturing at 35-39 ℃ and 200-220r/min for 10-12h, then inoculating into the LB culture medium according to the inoculation amount of 1%, culturing at 28-35 ℃ and 200-220r/min for 20-30h, then collecting bacterial liquid, centrifugally collecting bacterial bodies, washing the bacterial bodies by using PBS solution to break the bacterial bodies, centrifugally collecting supernatant, and separating the glutaminase mutant of claim 1 from the supernatant.
The invention also provides the application of the glutaminase mutant or the gene or the recombinant plasmid or the host cell or the preparation method in preparing food, medicines, health products and cosmetics.
In one embodiment of the invention, the use in preparing a food product comprises the use in preparing soy sauce by adding a glutaminase mutant to soy sauce to hydrolyse glutamine in soy sauce.
[ advantageous effects ]
(1) The invention provides a lactobacillus reuteri-derived glutaminase mutant which has high heat stability.
(2) The glutaminase mutant of the invention changes the 37 th tyrosine of the glutaminase parent derived from lactobacillus reuteri into cysteine, the half-life period of the glutaminase mutant reaches 12h at 44 ℃, and the stability of the glutaminase mutant is improved by 50 percent compared with the glutaminase before mutation.
(3) The invention expresses the glutaminase mutant derived from lactobacillus reuteri in a recognized food safety strain-B.subtilis 168 engineering strain, explores the enzyme activity and the thermal stability of the glutaminase mutant, widens the application range of the glutaminase mutant in the food field, and enables the glutaminase to be applied to production in a large scale.
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FIG. 1: nucleic acid gel electrophoresis images of wild type and mutant recombinant plasmid verification; wherein M is marker; 1 is wild type; 2 is mutant Y37C; 3 is mutant T85C/G244C; 4 is mutant D144C/S200C; mutant A290C was designated 5.
FIG. 2: SDS-PAGE analysis of wild-type glutaminase and glutaminase mutant expression; wherein, M: protein marker, 1: bs168/pMA5-lglsA extracellular, 2: bs168/pMA5-lglsA intracellularly, 3: bs168/pMA5-lglsA1 extracellular, 4: bs168/pMA5-lglsA1 intracellular, 5: bs168/pMA5-lglsA2 extracellular, 6: bs168/pMA 5-lglysA 2 intracellular, 7: bs168/pMA5-lglsA3 extracellular, 8: bs168/pMA5-lglsA3 intracellular, 9: bs168/pMA 5-lglysA 4 extracellular 10: bs168/pMA 5-lglysA 4.
FIG. 3: the thermal stability of wild type, mutant Y37C, T85C/G244C, D144C/S200C and A290C at 44 ℃.
Detailed Description
The invention will be further illustrated with reference to specific examples.
The pMA5 plasmid vector and Escherichia coli JM109 referred to in the following examples were purchased from invitrogen.
The media involved in the following examples are as follows:
LB culture medium: 10g/L of Tryptone (Tryptone), 5g/L of Yeast extract (Yeast extract) and 10g/L of sodium chloride (NaCl).
The detection methods referred to in the following examples are as follows:
the method for measuring the enzyme activity of the glutaminase comprises the following steps:
the content of glutamic acid in the enzyme product is directly measured by adopting an SBA biosensing analyzer: glutaminase enzyme activity measurement reaction system (total system 1 mL): mu.L of 50 mmol.L-1 Tris-HCl buffer solution and 440 mu.L of 50 mmol.L-1L-glutamine mixed solution are placed in a 37 ℃ water bath to be preheated for 5min, then 20 mu.L of enzyme solution is added, 100 mu.L of 15% trichloroacetic acid is directly added in a contrast reaction, the reaction is carried out in 37 ℃ constant temperature water bath for 5min, 100 mu.L of 15% trichloroacetic acid is rapidly added, the mixture is shaken up and down and mixed evenly, and the reaction is terminated. Placing the enzyme reaction solution into a centrifuge at 10000 r.min-1Centrifuging for 10min under the condition of (1), removing protein, collecting supernatant, and diluting the product concentration of the reaction solution to 0.3-1.0 g.L-1Then, 25. mu.L of the reaction solution was subjected to glutamic acid assay.
Enzyme activity determination related reagent of glutaminase:
a)50mM Tris-HCl (pH 7.5) buffer: 6.05g of Tris was weighed and dissolved in distilled water, pH was adjusted to 7.5 with HCl, and a volume of 1L was determined.
b) Reaction substrate solution (50 mM): 0.73g of glutamine was weighed and dissolved in 50mM Tris-HCl buffer solution pH7.5 to 100mL, and stored in a refrigerator at 4 ℃.
c) 15% (w/v) trichloroacetic acid solution: 15g of trichloroacetic acid was weighed into 100mL of distilled water, and the solution was dissolved and mixed well for use as a solution for terminating the enzymatic reaction.
Definition of enzyme activity: the amount of enzyme required to catalyze the formation of 1. mu. mol of glutamic acid within 1min is defined as one unit of enzyme activity.
Example 1: expression of the wild enzyme glutaminase
(1) A gene sequence of glutaminase (GenBank accession number: WP-003668502.1) derived from Lactobacillus reuteri is obtained from NCBI, and the gene sequence is optimized according to the preference of Bacillus subtilis codons and then sent to Jinzhi Biotech limited company to be synthesized to obtain the glutaminase with a coding nucleotide sequence shown as SEQ ID NO. 3.
(2) Construction and transformation of Gene expression vectors
Carrying out double enzyme digestion on glutaminase lgsA and pMA5 vectors with coding nucleotide sequences shown as SEQ ID NO.3 by using enzymes MluI and NdeI, and connecting products obtained after enzyme digestion by using homologous recombinase to obtain a recombinant vector pMA 5-lgsA; the recombinant plasmid pMA 5-lglysA was introduced into Escherichia coli JM109 by chemical transformation for plasmid amplification, and the plasmid was inoculated into 10ml of LB medium at 37 ℃ and 180 r.min after successful confirmation of colony PCR-1Culturing for 10h, and extracting plasmids according to a bacterial plasmid extraction kit; introducing the amplified plasmid into Bacillus subtilis168 by chemical transformation, verifying by colony PCR, inoculating into 10ml LB culture medium at 37 deg.C for 180r min-1The recombinant strain B.subtiliss 168/pMA 5-lgsA was obtained after 10h of culture.
Example 2: preparation and expression of glutaminase mutant
The method comprises the following specific steps:
(1) a glutaminase mutant Y37C with a coding nucleotide sequence shown as SEQ ID NO.2, a glutaminase mutant T85C/G244C with a coding nucleotide sequence shown as SEQ ID NO.4, a glutaminase mutant D144C/S200C with a coding nucleotide sequence shown as SEQ ID NO.5 and a glutaminase mutant A290C with a coding nucleotide sequence shown as SEQ ID NO.6 are chemically synthesized.
(2) Construction and transformation of Gene expression vectors
Carrying out double enzyme digestion on gene lgsA 1 of glutaminase mutant Y37C with a nucleotide sequence shown as SEQ ID NO.2, gene lgsA 2 of glutaminase mutant T85C/G244C with a nucleotide sequence shown as SEQ ID NO.4, gene lgsA 3 of glutaminase mutant D144C/S200C with a nucleotide sequence shown as SEQ ID NO.5, gene lgsA 4 of glutaminase mutant A290C with a nucleotide sequence shown as SEQ ID NO.6 and pMA5 carrier by using enzymes MluI and NdeI respectively, and connecting the enzyme digestion products by using homologous recombinase to obtain recombinant plasmids pMA 5-lgsA 1-pMA 5-lgsA 4;
the recombinant plasmid pMA5-lgls was chemically transformedA1-pMA 5-lglysA 4 were introduced into Escherichia coli JM109, amplified, subjected to nucleic acid gel electrophoresis as shown in FIG. 1, and subjected to colony PCR verification, then inoculated into 10ml of LB medium at 37 ℃ and 180 r.min-1Culturing for 10h, and extracting plasmids according to a bacterial plasmid extraction kit; introducing the amplified plasmid into Bacillus subtilis168 by chemical transformation, verifying by colony PCR, inoculating into 10ml LB culture medium at 37 deg.C for 180r min-1Culturing for 10h to obtain recombinant strain B.subtiliss 168/pMA 5-lglysA 1-B.subtiliss 168/pMA 5-lglysA 4.
The recombinant Bacillus subtilis B.subtiliss 168/pMA 5-lgsA obtained in example 1 and the recombinant Bacillus subtilis B.subtiliss 168/pMA 5-lgsA 1-B.subtiliss 168/pMA 5-lgsA 4 were respectively cultured in 10mL of LB medium at 37 ℃ and 180 r.min-1After 10h of culture, the cells were transferred to 50ml of LB medium at a inoculum size of 1% and incubated at 37 ℃ for 180 r.min-1Culturing for 30h for glutaminase expression, collecting bacterial liquid, washing cells with PBS solution, centrifuging, collecting cells, adding 30 μ L lysozyme (200mg/mL) into cells, and carrying out ultrasonication: breaking for 2s at 400w for 5s, centrifuging at 12000rpm for 20min at 4 deg.C after breaking for 30min, separating wall-broken supernatant and wall-broken precipitate, and filtering the supernatant with 0.45 μm filter membrane to obtain wall-broken supernatant.
Enzyme activity determination is carried out on the fermentation supernatant, the wall-breaking supernatant and the wall-breaking sediment respectively, and the enzyme activity is found to be only detected in the wall-breaking supernatant, the enzyme activity is not detected in the other 2 parts, and the enzyme activity results of the wall-breaking supernatant are shown in table 1. Glutaminase mutants Y37C, T85C/G244C, D144C/S200C and A290C are proved to be intracellular enzymes (shown in figure 2). SDS-PAGE analysis of the wall-broken supernatant shows that the mutants have obvious bands near 30kDa, which indicates that the proteins of the mutants are normally expressed.
TABLE 1 Glutamine mutant enzyme Activity
Example 3: thermostability of different glutaminase mutants
The method comprises the following specific steps:
the enzyme solutions containing the mutants Y37C, T85C/G244C, D144C/S200C and A290C obtained in example 2 were respectively placed at 44 ℃ and kept warm for 14h, samples were taken every 1h and glutaminase activity was measured, and glutaminase activity measured in the initial enzyme solution was defined as 100%; the half-life of each mutant at 44 ℃ was calculated using an exponential function fit.
As can be seen from FIG. 3 and Table 2, the results show that the stability of wild-type glutaminase reached a half-life of 8h at 44 ℃. Compared with the wild type, the half-life of the mutants T85C/G244C, D144C/S200C and A290C is reduced, only the mutant Y37C obtained by mutating the 37 th tyrosine of the parent enzyme into cysteine has the half-life of 12h at 44 ℃, and the stability is improved by about 50%.
TABLE 2 half-lives of glutaminase wild-type and mutant at 44 ℃
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> glutaminase mutant having improved stability
<130> BAA200564A
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<170> PatentIn version 3.3
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atctgcaatt gcccagcgat cacgctgaac gaggagatct accagagcga actgcgcacc 480
ggcgatatga accgcagcct cgcgtactat ctgaaagcca aggaagtgct gacgaacgac 540
gtgacgctca gtctggatac gtacttcaag cagtgcagca tgatggtgac gtgccagtgc 600
ctggcgaatc tgggcgccgt tctggccaac gatggcattg ccccgtggaa caatgagcgc 660
atcatcagca gcgaggccgc cacctacacc aaaagcgtga tgatgaccac cggtctgtac 720
aacgaaagcg gcacctacag cgtgcgcatc ggcgttccga ccaaaagcgg tgttggcggc 780
ggtctggtta gtgcgagccc gaatcactat ggcatcggca tcttcagccc agcgctggac 840
cacgcgggca atagtgttgc cggtctggcg ctgctggagc tgatcagcaa gaagctgaag 900
ctggacgtgt tccgctacta a 921
<210> 6
<211> 921
<212> DNA
<213> Artificial sequence
<400> 6
atgaatctga acgatgccat ccaagaatgc tggagtaaga tcgacgaagg ccaagttgcg 60
acctacattc cggcgctggc caaagttgat ccgtaccagc tgggcgtgta tctgttcgac 120
gtgaccaacg acaagaaagc cgaagcgggt gcgagtcaag ttcgcttcgc gatcgaaagc 180
gtgagcaaag tgatcacgct gctgtacgcc atcgaacgtc tgggtctgag cgccgtggaa 240
gaacaagttg gcacccgcca aaccggcttc ccattcgaca ccattctgaa catggagatc 300
accaaggaaa cgcacccgct gaacgcgttc atcaatagcg gcgccattct gatcagtagc 360
ctcatcgagg agcaagatgg tctgagcccg ttcgatcaga tcctcgagtt cagccgcaag 420
atctgcaatg atccagcgat cacgctgaac gaggagatct accagagcga actgcgcacc 480
ggcgatatga accgcagcct cgcgtactat ctgaaagcca aggaagtgct gacgaacgac 540
gtgacgctca gtctggatac gtacttcaag cagtgcagca tgatggtgac gtgccagagt 600
ctggcgaatc tgggcgccgt tctggccaac gatggcattg ccccgtggaa caatgagcgc 660
atcatcagca gcgaggccgc cacctacacc aaaagcgtga tgatgaccac cggtctgtac 720
aacgaaagcg gcacctacag cgtgcgcatc ggcgttccga ccaaaagcgg tgttggcggc 780
ggtctggtta gtgcgagccc gaatcactat ggcatcggca tcttcagccc agcgctggac 840
cacgcgggca atagtgttgc cggtctgtgc ctgctggagc tgatcagcaa gaagctgaag 900
ctggacgtgt tccgctacta a 921
Claims (9)
1. A glutaminase mutant, characterized in that the tyrosine at position 37 of the glutaminase mutant is mutated to cysteine compared with glutaminase having an amino acid sequence shown in SEQ ID NO. 1.
2. A gene encoding the glutaminase mutant according to claim 1.
3. A recombinant plasmid carrying the gene of claim 2.
4. The recombinant plasmid of claim 3 wherein the vector of the recombinant plasmid is the pMA5 plasmid.
5. A host cell carrying the gene of claim 2 or the recombinant plasmid of claim 3 or 4.
6. The host cell of claim 5, wherein the host cell is Bacillus subtilisB. subtilis 168。
7. The method for preparing the glutaminase mutant according to claim 1, wherein the method comprises inoculating the host cell according to claim 5 or 6 into LB medium, culturing at 35-39 ℃ and 200-220r/min for 10-12h, inoculating to LB medium in an amount of 1%, culturing at 28-35 ℃ and 200-220r/min for 20-30h, collecting the bacterial liquid, centrifuging to collect the bacterial liquid, washing the cell with PBS solution to break the bacterial liquid, centrifuging to collect the supernatant, and separating the glutaminase mutant according to claim 1 from the supernatant.
8. Use of the glutaminase mutant according to claim 1, or the gene according to claim 2, or the recombinant plasmid according to claim 3 or 4, or the host cell according to claim 5 or 6, or the method according to claim 7 for the preparation of a food, a pharmaceutical, or a cosmetic product.
9. The use according to claim 8, wherein the use in the preparation of a food product comprises use in the preparation of soy sauce, wherein the use is the addition of a glutaminase mutant to soy sauce, hydrolysing glutamine in soy sauce.
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