CN113174378B - Glutamate dehydrogenase mutant, encoding gene thereof, genetically engineered bacterium and application thereof in preparation of L-2-aminobutyric acid - Google Patents
Glutamate dehydrogenase mutant, encoding gene thereof, genetically engineered bacterium and application thereof in preparation of L-2-aminobutyric acid Download PDFInfo
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Abstract
The invention provides a glutamate dehydrogenase mutant, a coding gene thereof, genetically engineered bacteria and application thereof in preparing L-2-aminobutyric acid, wherein the mutant is one of the following: taking wild glutamate dehydrogenase shown in SEQ ID NO.1 as a template, and mutating 76 th amino acid residue into glycine G, alanine A, valine V, leucine L or isoleucine I; mutation of amino acid residue 180 to serine S or cysteine C; the 76 th amino acid is mutated to leucine and the 180 th amino acid residue is mutated to cysteine C. According to the invention, through a molecular transformation method, the catalytic activity of AtGluDH on a non-natural substrate 2-ketobutyric acid is obviously improved, the problem of low enzyme catalytic activity in the process of preparing L-2-aminobutyric acid by a reductive amination method is solved, and when whole-cell catalysis is used, a 1M substrate can be successfully converted without adding exogenous coenzyme, so that the method has great industrial application value.
Description
Technical Field
The invention relates to the technical field of protein engineering, in particular to a glutamate dehydrogenase mutant, a coding gene thereof, genetically engineered bacteria and application thereof in preparation of L-2-aminobutyric acid.
Background
L-2-aminobutyric acid is a chiral amino acid which does not participate in protein synthesis, is present in small amounts in tissues and organs of animals and plants, and is biosynthesized by transferring oxobutyrate in isoleucine biosynthesis by non-ribosomal peptide synthetases. The L-2-aminobutyric acid can inhibit human nerve information transmission and improve the activity of glucose phosphatase so as to promote the metabolism of brain cells, and is also an important chemical raw material and chiral medicine intermediate. L-2-aminobutyric acid can obtain two important chiral prodrugs (S) -2-aminobutyric acid and (S) -2-aminobutyric acid through simple amidation and reduction, which are respectively important raw materials for synthesizing novel antiepileptic drugs, namely, buvaracetam and levetiracetam, and antituberculosis drug, namely ethambutol.
The synthesis method of the L-2-aminobutyric acid mainly comprises the following steps: chemical synthesis, biological fermentation and biocatalysis. The chemical synthesis method has the advantages of high reaction speed, but has strict conditions, low stereoselectivity, and great toxic effect of chemical reagents on the environment and human body, and is accompanied with the generation of byproducts. The biological fermentation method is more suitable for industrial production because the biological fermentation method can reach a larger scale, but the product obtained by the fermentation method is complex, and the subsequent separation and purification are difficult. The biocatalysis method for preparing the L-2-aminobutyric acid has the advantages of high stereoselectivity, mild reaction conditions and little environmental pollution, wherein the Amino Acid Dehydrogenase (AADHs) uses cheap ammonia water and equivalent cofactor NAD (P) H to catalyze the reductive amination of alpha-keto acid to generate corresponding products, has high atom economy, and is the enzyme with the most potential for synthesizing the L-2-aminobutyric acid. The existing amino acid dehydrogenases for preparing L-2-aminobutyric acid are all leucine dehydrogenases, and can catalyze a 1M substrate 2-ketobutyric acid to generate an enantiomerically pure L-type product at a conversion rate of 99%, but due to limited enzyme activity, long reaction time is required, so that the space-time yield is low, and the industrial production is not favored. In addition, the redox reaction requires the expensive cofactor NAD (P) H, and even though the efficient coenzyme circulation system is used in the reaction mediated by leucine dehydrogenase, at least 0.1mM NADH is required to be added, so that the production cost is high.
Glutamate dehydrogenase (Glutamate dehydrogenase, EC 1.4.1.2-1.4.1.4), is abundant in source and can be classified into (i) NADH dependent according to its dependence on cofactors; (ii) NADPH dependent and (iii) NADH/NADPH dependent. Wherein, the double cofactor NADH/NADPH dependent glutamate dehydrogenase can maximally utilize the cofactor endogenous in the cells in the biocatalytic reductive amination reaction of the whole cells, thereby realizing zero addition of exogenous cofactor and saving production cost. However, since glutamate dehydrogenase has strict substrate specificity for its natural substrate alpha-ketoglutarate, improving the catalytic activity of AtGluDH for its non-natural substrate 2-ketobutyrate by molecular modification is key to realizing the industrial application of the L-2-aminobutyric acid reductive amination synthesis process.
Disclosure of Invention
The invention aims to provide a glutamate dehydrogenase mutant, a coding gene thereof, a genetic engineering bacterium and application thereof in preparation of L-2-aminobutyric acid, so as to solve the problem that the low yield of L-2-aminobutyric acid cannot meet industrial requirements due to low catalytic activity of glutamate dehydrogenase on a non-natural substrate 2-ketobutyric acid in the prior art.
In order to solve the technical problems, the invention adopts the following technical scheme:
according to a first aspect of the present invention, there is provided a glutamate dehydrogenase mutant with improved activity, comprising a glutamate dehydrogenase mutant formed by mutating amino acid residue at position 76 with wild-type glutamate dehydrogenase AtGluDH as shown in SEQ ID NO.1 as template: the 76 th lysine K is mutated into a glutamic acid dehydrogenase mutant K76G of glycine G, and the amino acid sequence is shown as SEQ ID NO. 2; the 76 th lysine K is mutated into a glutamic acid dehydrogenase mutant K76A of alanine A, and the amino acid sequence is shown as SEQ ID NO. 3; the 76 th lysine K is mutated into a glutamic acid dehydrogenase mutant K76V of valine V, and the amino acid sequence is shown as SEQ ID NO. 4; a glutamate dehydrogenase mutant K76L with lysine K at position 76 mutated into leucine L, and the amino acid sequence is shown as SEQ ID NO. 5; and a glutamate dehydrogenase mutant K76I with lysine K at position 76 mutated into isoleucine I, the amino acid sequence of the glutamate dehydrogenase mutant K76I is shown as SEQ ID NO. 6.
Preferably, the invention also provides a glutamate dehydrogenase mutant with improved activity, which is formed by taking wild glutamate dehydrogenase AtGluDH shown in SEQ ID NO.1 as a template and mutating the 180 th amino acid residue as follows: a mutant T180S of glutamate dehydrogenase with threonine T at position 180 mutated into serine S, and the amino acid sequence is shown as SEQ ID NO. 7; and the 180 th threonine T is mutated into a glutamic dehydrogenase mutant T180C of cysteine C, and the amino acid sequence is shown in SEQ ID NO. 8.
Particularly preferably, the present invention provides a glutamate dehydrogenase mutant with improved activity, comprising: taking mutant glutamate dehydrogenase mutant K76L shown in SEQ ID NO.5 as a template, and carrying out threonine T mutation on the 180 th amino acid residue to obtain a glutamate dehydrogenase mutant with cysteine C, wherein the amino acid sequence is shown in SEQ ID NO. 9.
According to a second aspect of the present invention there is provided a gene encoding a glutamate dehydrogenase mutant as described above.
According to a third aspect of the present invention, there are provided a recombinant vector and a genetically engineered bacterium comprising a gene encoding the glutamate dehydrogenase mutant as described above. According to a preferred embodiment of the present invention, the host bacterium is E.coli.
According to a fourth aspect of the present invention there is provided the use of a glutamate dehydrogenase mutant as described above for catalyzing the preparation of L-2-aminobutyric acid from 2-ketobutyric acid.
The invention firstly selects the glutamate dehydrogenase AtGluDH gene cloned from the strain Acinetobacter tandoii as a template, the amino acid sequence of the glutamate dehydrogenase AtGluDH is shown as SEQ ID NO.1, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 10. Homology modeling was performed using the crystal structure of glutamate dehydrogenase derived from Burkholderia thailandensis as a template (PDB ID:4 XGI), and 2-ketobutyrate was docked into glutamate dehydrogenase AtGluDH. The amino acid residue at position 76 and the amino acid residue at position 180, which play a key role in the substrate specificity of the glutamate dehydrogenase AtGluDH, are determined by analyzing the binding pocket of the 2-ketobutyrate and the glutamate dehydrogenase AtGluDH and simulating the binding molecular dynamics. After subjecting them to saturation mutation, the activity of each mutant in catalyzing the reductive amination reaction was measured. To analyze the synergistic effect between them, mutants with greatly improved activity were combined, and the optimal double point mutant AtGluDH-K76L/T180C, i.e., the amino acid residue at position 76 and the amino acid residue at position 180 were simultaneously mutated, was determined. By these protein engineering strategies, a number of mutants with improved glutamate dehydrogenase activity were obtained, which mutants were obtained with great advantage for industrial production.
Thus, the present invention uses the wild type amino acid dehydrogenase shown in SEQ ID NO.1 as a template, and the amino acid sites which play a key role in activity are determined through analysis, so that the following various glutamate dehydrogenase mutants are obtained, including: the mutation of K at position 76 to G, A, V, L and I forms mutants K76G, K76A, K V, K76L and K76I; the T at position 180 is mutated into S and C, forming mutants T180S and T180C; the combined mutation at position 76 and 180 forms mutant K76L/T180C.
Furthermore, the glutamate dehydrogenase mutant is applied to asymmetric reductive amination to synthesize chiral amino acid. The wild type glutamate dehydrogenase AtGluDH discovered by the invention can only convert 0.46M 2-ketobutyrate to generate L-2-aminobutyric acid in 24h, and the discovered glutamate dehydrogenase AtGluDH is modified by a saturation mutation strategy to obtain a plurality of mutants with improved catalytic performance of glutamate dehydrogenase, wherein the activities of the three mutants AtGluDH-K76L, atGluDH-T180C and AtGluDH-K76L/T180C with the greatest activity improvement range are respectively improved from 57.3U/mg to 960.6U/mg, 527.5U/mg and 985.7U/mg of the wild type AtGluDH. The two-site mutant AtGluDH-K76L/T180C of the glutamate dehydrogenase with the highest activity is used for catalyzing 1M 2-ketobutyrate, and when the addition amount of the cofactor NADH is 0.1mM, the cofactor NADH can be completely converted into L-2-aminobutyric acid in 1.5 h; when the addition amount of the cofactor NADH is 0mM, the cofactor NADH can be completely converted into L-2-aminobutyric acid in 3.5h, and the ee values reach 99.9%.
In conclusion, according to the various glutamate dehydrogenase mutants provided by the invention, the catalytic activity of 2-ketobutyric acid is obviously improved, the enantioselectivity is high, the problem that expensive cofactors are required to be additionally added in the process of synthesizing L-2-aminobutyric acid through reductive amination reaction and the activity of a biocatalyst is insufficient to provide high space-time yield is successfully solved, and the glutamate dehydrogenase mutants have great industrial application value.
Drawings
FIG. 1 is an SDS-PAGE protein gel electrophoresis of wild-type glutamate dehydrogenase AtGluDH, mutant AtGluDH-K76L and mutant AtGluDH-K76L/T180C, wherein M is a protein molecular weight standard reagent, L is a cell disruption solution supernatant, N is a cell disruption solution precipitate, and P is a fraction after nickel strain purification;
FIG. 2 is a protein gel electrophoresis diagram of enzyme co-expression in cascade reaction, wherein A is wild-type glutamate dehydrogenase AtGluDH and glucose dehydrogenase BmGDH co-expressed, and B is wild-type glutamate dehydrogenase AtGluDH, glucose dehydrogenase BmGDH and threonine deaminase EcTD three-enzyme co-expressed.
Detailed Description
The invention will be further illustrated with reference to specific examples. It should be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The technical means used in the examples are conventional in the art, unless specifically stated otherwise.
Example 1 detection of substrate, intermediate and product concentrations and biological Cascade reactions
The concentrations of L-homoalanine and L-threonine in the reaction solution were analyzed by high performance liquid chromatography: agilent C18 column (5 μm×4.6mm×250 mm), mobile phase: v (acetonitrile): v (0.02 mol/L Na) 2 HPO 4 Buffer (pH 7.2)) =15: 85, ultraviolet detection wavelength: 360nm, column temperature: 30 ℃, flow rate: 1.0mL/min. Sample derivatization conditions: 100. Mu.L of test sample diluted by a certain multiple is taken and mixed with 100. Mu.L of 0.5mol/L NaHCO 3 The solution was mixed with 50. Mu.L of a 1% solution of 2, 4-Dinitrofluorobenzonitrile (DNFB). The sample was incubated in the dark at 60℃for 1h, cooled to room temperature after the reaction, and finally 750. Mu.L NaH was added 2 PO 4 /Na 2 HPO 4 Buffer (0.2 mol/L, pH 7.0).
The concentration of 2-ketobutyric acid in the reaction solution was analyzed by high performance liquid chromatography: COSMOSIL packed PBr column (4.6 id×250 mm), mobile phase: pure water contains 0.1% H 3 PO 4 UV detection wavelength: 210nm, column temperature: 30 ℃, flow rate: 0.8mL/min.
The chiral nature of the product L-2-aminobutyric acid was analyzed by high performance liquid chromatography: SCAS SUMICHIRAL OA-5000L column (5 μm. Times.4.6 mm. Times.150 mm), mobile phase: one liter of water contains 3mL of acetonitrile and 0.5g of CuSO 4 UV detection wavelength: 254nm, column temperature: 35 ℃, flow rate: 0.8mL/min.
L-2-aminobutyric acid is prepared by biological cascade reaction, L-threonine is taken as an initial substrate, threonine deaminase EcTD derived from Escherichia coli is used for deaminizing L-threonine to generate prochiral substrate 2-ketobutyric acid, then glutamate dehydrogenase AtGluDH derived from Acinetobacter tandoii is used for carrying out reductive amination on 2-ketobutyric acid to generate target product L-2-aminobutyric acid, and glucose dehydrogenase BmGDH derived from Bacillus megaterium is coupled in a second reaction step so as to effectively recycle cofactor NAD (P) H.
Standard detection system for cascade reactions: 10mg of lyophilized powder, 1M substrate L-threonine, 1.2M glucose, and finally supplemented to 1mL with ammonium chloride buffer (200 mM, pH 9.6). The reaction was carried out for 12h at 30℃in a shaker at 200 rpm. After the reaction was completed, the reaction was terminated with 5M sodium hydroxide. And (3) putting the mixed solution into a centrifugal machine, and centrifuging at 12000rpm for 5-10min to separate the solid phase and the liquid phase in the reaction system. Sucking out the supernatant, filtering with a filter membrane, diluting by a certain multiple, derivatizing as required, and placing into a liquid phase sample bottle to detect the conversion rate of the reaction.
Example 2 docking of substrate with AtGluDH
In order to obtain structural information of glutamate dehydrogenase AtGluDH, a structural model was built for wild-type glutamate dehydrogenase AtGluDH. Homology modeling was performed using SWISS-MODEL on-line server (http:// www.swissmodel.expasy.org /), glutamate dehydrogenase from Burkholderia thailandensis was selected (PDB number: 4XGI, resolution:
) The crystal structure of (C) was used as a template, which had the highest 62% identity with glutamate dehydrogenase AtGluDH. After optimization of the model, the cofactor NADH is first docked into the model, followed by docking of the substrate 2-ketobutyrate with the complex of the enzyme molecule and NADH. Screening and scoring the docking result, and selecting the optimal docking posture, so as to carry out subsequent analysis on key amino acid residues of the enzyme molecular substrate active pocket.
EXAMPLE 3 construction and screening of AtGluDH mutant
E.coli engineering bacteria with pET-28a (+) -AtGluDH recombinant plasmid are cultivated in LB culture medium for 10-12h, and the plasmid is extracted as a template for constructing subsequent mutants. The mutant library was constructed using KOD-One point mutation kit from Toyobo of Japan. The specific operation is as follows:
the primer design was performed using the above-mentioned extracted plasmid as a template and the mutation residues introduced as needed as shown in Table 1.
TABLE 1 primers for mutation
Point mutations were introduced by inverse PCR using high fidelity KOD-One-enzyme, and the PCR reaction system is shown in Table 2.
TABLE 2 PCR reaction System
| Component (A) | Volume (mu L) |
| KOD-One-enzyme | 10 |
| Primer F | 1 |
| Primer R | 1 |
| Template | 1 |
| ddH 2 O | 7 |
PCR reaction conditions: pre-denaturation at 94℃for 2min; denaturation at 98℃for 10s, annealing at 58℃for 5s, extension at 68℃for 2min, cycle 12 times at this stage; then extending for 2min at 68 ℃; preserving at 4 ℃.
After the PCR amplification, the template plasmid DNA is digested by Dpn I at 37 ℃ for 2 hours;
the PCR products were subjected to self-cyclization at 16℃for 3h using T4 Polynucleotide Kinase and Ligation high in the kit, and the self-cyclization system is shown in Table 3.
TABLE 3 self-cyclization of digestion products
| Component (A) | Volume (mu L) |
| Digestion products | 2 |
| Connecting liquid | 5 |
| T4 polynucleotide kinase | 1 |
| ddH 2 O | 7 |
Transforming, and introducing the obtained cyclization product into competent cells of escherichia coli BL21 (DE 3);
picking single colony and inoculating to 5mL LB culture medium, culturing overnight at 37 ℃ and sequencing the strain;
after ensuring the correct sequence, carrying out induced expression on each mutant, and preparing crude enzyme solution or freeze-drying by using ultrasonic crushing after harvesting thalli; among them, SDS-PAGE protein gel electrophoresis of wild-type glutamate dehydrogenase AtGluDH, mutant AtGluDH-K76L and mutant AtGluDH-K76L/T180C is shown in FIG. 1.
The activity of the wild-type glutamate dehydrogenase AtGluDH and mutants thereof was determined at 30℃and pH 9.6. By measurement at 340nm (epsilon=6220m -1 cm -1 ) Absorbance at which the concentration of NADH consumed in the reductive amination reaction was obtained to calculate the amount of the corresponding substrate consumed. The specific operation is as follows: first, a reaction mixture consisting of 2-ketobutyric acid (50 mM), NADH (0.1 mM), NH was added to the ELISA plate 4 Cl-NH 3 OH (200 mM, pH 9.6) buffer, and adding a certain amount of crude cell extract or purified enzyme solution to start the reaction; secondly, a measurement program is set for the enzyme labeling instrument, the system is firstly oscillated for 5s, then readings are taken at intervals of 10s under the ultraviolet band of 340nm, the total measurement is carried out for 2min, and 3 groups are measured in parallel to calculate the average value and the error. One unit of AtGluDH activity (U) is defined as the amount of enzyme required to catalyze the reduction of 2-ketobutyrate and then produce 1. Mu. Mol L-homoalanine per minute. The concentration of protein contained in the pure enzyme was measured using the Bradford method to calculate specific activity (U/mg).
EXAMPLE 4 expression and purification of wild-type AtGluDH and mutants thereof
Recombinant cells were plated on agar plates, incubated at 37℃for 12 hours, and then individual colonies were picked into tubes containing 5ml of LB medium and 50. Mu.g/ml kanamycin. Thereafter, the cells were cultured at 200rpm for 10-12 hours at 37 ℃. Then, they were transferred to a flask containing 200ml of LB medium and 50. Mu.g/ml of kanamycin, and cultured at 200rpm for about 3.5 hours at 37 ℃. When the OD value (λ=600 nm) reached 0.6 to 0.8, 0.1mM IPTG was added for induction and the cells were incubated at 200rpm for 18 hours at 20 ℃. The recombinant cells were then centrifuged at 8000rpm for 10 minutes at 4℃and washed 3 times with 0.5% NaCl solution, resuspended in 20mM PB buffer (pH 8.0) placed on ice, and then the recombinant cells were disrupted by sonication and centrifuged at 8000rpm for 30 minutes at 4℃to remove unbroken cells and cell debris. The supernatant was loaded onto a Ni-NTA column, and the 6 XHis-tagged protein was bound to the Ni-NTA column at a flow rate of 1ml/min, followed by elution with 20mM,50mM,100mM,250mM and 500mM imidazole at the same flow rate. The purity of the collected proteins was checked by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Fractions containing the target protein were collected and dialyzed against 20mM PB buffer (pH 8.0) for desalting, followed by concentration of the enzyme solution, addition of 20% (v/v) glycerol, and storage at-80℃for later use.
Example 5 kinetic parameters of wild-type AtGluDH and mutants thereof
Kinetic analysis was performed at 30℃and pH 9.6. Enzyme activities were measured at different substrate concentrations (0.1-20 mM) using purified wild-type glutamate dehydrogenase AtGluDH and mutants thereof at a fixed concentration of NADH (0.1 mM). The maximum reaction rate (v) was plotted using the obtained data max ) Correlation with substrate concentration and fitting using Michelis-Menten equation to obtain v max And K m Values. By v max And calculating k from the concentration of the enzyme cat Values. The results showed that the wild type AtGluDH, K76L mutant, T180C mutant and K76L/T180C mutant were K m The values were 4.12, 2.96, 2.88 and 2.52mM, respectively. K of wild type AtGluDH, K76L mutant, T180C mutant and K76L/T180C mutant cat Values of 0.53, 3.03, 2.81 and 3.24s, respectively -1 . K of wild type AtGluDH, K76L mutant, T180C mutant and K76L/T180C mutant cat /K m Values of 0.13, 1.02, 0.98 and 1.29mM, respectively -1 s -1 。
TABLE 4 kinetic parameters
EXAMPLE 6 Co-expression of enzymes in a Cascade reaction
The plasmid pET28a-BmGDH was linearized while amplifying the AtGluDH gene, and then the Open Reading Frame (ORF) of the AtGluDH was cloned into the backbone of pET28a-BmGDH and placed in front of BmGDH to obtain recombinant plasmid pET28a-AtGluDH-BmGDH expressed in tandem with glutamate dehydrogenase. The pACYC184 plasmid was cut singly using the restriction enzyme HindIII to obtain a linearized plasmid, and then the ORF of EcTD was inserted into the pACYC184 plasmid from pET28a-EcTD to construct pACYC184-EcTD. Finally, pET28a-AtGluDH-BmGDH and pACYC184-EcTD are co-transformed into competent cells of the escherichia coli BL21 (DE 3) to obtain recombinant escherichia coli co-expressing AtGluDH, bmGDH and EcTD. The results of the protein electrophoresis of tandem expression of AtGluDH and BmGDH and the protein electrophoresis of co-expression of AtGluDH, bmGDH and EcTD three enzymes are shown in FIG. 2.
The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, and various modifications can be made to the above-described embodiment of the present invention. All simple, equivalent changes and modifications made in accordance with the claims and the specification of the present application fall within the scope of the patent claims. The present invention is not described in detail in the conventional art.
SEQUENCE LISTING
<110> university of Industy of Huadong
<120> glutamic acid dehydrogenase mutant and encoding gene, genetically engineered bacterium and preparation of L-2-aminobutyric acid
Use in acids
<160> 24
<170> PatentIn version 3.5
<210> 1
<211> 423
<212> PRT
<213> Acinetobacter tandoii
<400> 1
Met Ser Leu Ser Tyr Glu Met Glu Asn Ser Gly Ala Trp Gln Thr Tyr
1 5 10 15
Leu Ala Gln Ile Asn Arg Val Ala Pro Tyr Leu Glu Glu Asp Leu Ile
20 25 30
Pro Phe Ile Asn Thr Leu Lys Arg Pro Lys Arg Ala Leu Ile Val Asp
35 40 45
Val Pro Ile Val Met Asp Asp Gly Ser Ile Gln His Phe Glu Gly Tyr
50 55 60
Arg Val Gln His Asn Leu Ser Arg Gly Pro Gly Lys Gly Gly Ile Arg
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Tyr His Pro Asp Val Glu Leu Asn Glu Val Met Ala Leu Ser Ala Trp
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Met Thr Ile Lys Thr Ala Val Leu Asn Leu Pro Tyr Gly Gly Ala Lys
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Gly Gly Ile Arg Val Asp Pro Arg Lys Leu Ser Pro Arg Glu Leu Glu
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Arg Leu Thr Arg Arg Phe Thr Thr Glu Ile Ser Pro Ile Ile Gly Pro
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Gln Ile Asp Ile Pro Ala Pro Asp Val Gly Thr Asn Ala Asp Ile Met
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Gly Trp Met Met Asp Thr Tyr Ser Thr Ile Lys Gly His Thr Val Thr
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Gly Val Val Thr Gly Lys Pro Val His Leu Gly Gly Ser Leu Gly Arg
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Val Arg Ala Thr Gly Arg Gly Val Phe Val Thr Gly Leu Glu Val Ala
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Lys Lys Ile Asn Leu Ala Leu Glu Gly Ser Arg Ile Ala Val Gln Gly
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Phe Gly Asn Val Gly Ser Glu Ala Ala Tyr Leu Phe His Lys Ala Asn
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Ala Lys Val Val Cys Val Gln Asp His Thr Gly Thr Ile Phe Asn Ala
245 250 255
Asp Gly Phe Asp Val Lys Gln Leu Gln Asp Tyr Val Ala Ile His Lys
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Gly Val Ala Gly Phe Pro Asn Ala Thr Val Ile Glu Asp Glu Ala Phe
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Trp Thr Val Glu Met Asp Ile Leu Ile Pro Ala Ala Leu Glu Gly Gln
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Ile Thr Ala Glu Arg Ala Gln Lys Leu Thr Ala Lys Leu Val Leu Glu
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Gly Ala Asn Gly Pro Thr Tyr Pro Glu Ala Glu Asp Ile Leu Leu Gln
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Arg Gln Ile Met Ile Val Pro Asp Val Leu Cys Asn Ala Gly Gly Val
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Thr Val Ser Tyr Phe Glu Trp Val Gln Asp Met Ala Ser Tyr Phe Trp
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Thr Glu Glu Glu Ile Asn Glu Arg Leu Asp Lys Leu Met Ile Lys Ala
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Val Glu Asp Val Trp His Thr Ala Asp Asp Lys Asp Cys Ser Leu Arg
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Thr Ala Ala Tyr Ile Leu Ala Cys Glu Arg Ile Leu Lys Ala Arg Lys
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Glu Arg Gly Ile Phe Pro Gly
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Met Ser Leu Ser Tyr Glu Met Glu Asn Ser Gly Ala Trp Gln Thr Tyr
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Leu Ala Gln Ile Asn Arg Val Ala Pro Tyr Leu Glu Glu Asp Leu Ile
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Pro Phe Ile Asn Thr Leu Lys Arg Pro Lys Arg Ala Leu Ile Val Asp
35 40 45
Val Pro Ile Val Met Asp Asp Gly Ser Ile Gln His Phe Glu Gly Tyr
50 55 60
Arg Val Gln His Asn Leu Ser Arg Gly Pro Gly Gly Gly Gly Ile Arg
65 70 75 80
Tyr His Pro Asp Val Glu Leu Asn Glu Val Met Ala Leu Ser Ala Trp
85 90 95
Met Thr Ile Lys Thr Ala Val Leu Asn Leu Pro Tyr Gly Gly Ala Lys
100 105 110
Gly Gly Ile Arg Val Asp Pro Arg Lys Leu Ser Pro Arg Glu Leu Glu
115 120 125
Arg Leu Thr Arg Arg Phe Thr Thr Glu Ile Ser Pro Ile Ile Gly Pro
130 135 140
Gln Ile Asp Ile Pro Ala Pro Asp Val Gly Thr Asn Ala Asp Ile Met
145 150 155 160
Gly Trp Met Met Asp Thr Tyr Ser Thr Ile Lys Gly His Thr Val Thr
165 170 175
Gly Val Val Thr Gly Lys Pro Val His Leu Gly Gly Ser Leu Gly Arg
180 185 190
Val Arg Ala Thr Gly Arg Gly Val Phe Val Thr Gly Leu Glu Val Ala
195 200 205
Lys Lys Ile Asn Leu Ala Leu Glu Gly Ser Arg Ile Ala Val Gln Gly
210 215 220
Phe Gly Asn Val Gly Ser Glu Ala Ala Tyr Leu Phe His Lys Ala Asn
225 230 235 240
Ala Lys Val Val Cys Val Gln Asp His Thr Gly Thr Ile Phe Asn Ala
245 250 255
Asp Gly Phe Asp Val Lys Gln Leu Gln Asp Tyr Val Ala Ile His Lys
260 265 270
Gly Val Ala Gly Phe Pro Asn Ala Thr Val Ile Glu Asp Glu Ala Phe
275 280 285
Trp Thr Val Glu Met Asp Ile Leu Ile Pro Ala Ala Leu Glu Gly Gln
290 295 300
Ile Thr Ala Glu Arg Ala Gln Lys Leu Thr Ala Lys Leu Val Leu Glu
305 310 315 320
Gly Ala Asn Gly Pro Thr Tyr Pro Glu Ala Glu Asp Ile Leu Leu Gln
325 330 335
Arg Gln Ile Met Ile Val Pro Asp Val Leu Cys Asn Ala Gly Gly Val
340 345 350
Thr Val Ser Tyr Phe Glu Trp Val Gln Asp Met Ala Ser Tyr Phe Trp
355 360 365
Thr Glu Glu Glu Ile Asn Glu Arg Leu Asp Lys Leu Met Ile Lys Ala
370 375 380
Val Glu Asp Val Trp His Thr Ala Asp Asp Lys Asp Cys Ser Leu Arg
385 390 395 400
Thr Ala Ala Tyr Ile Leu Ala Cys Glu Arg Ile Leu Lys Ala Arg Lys
405 410 415
Glu Arg Gly Ile Phe Pro Gly
420
<210> 3
<211> 423
<212> PRT
<213> Acinetobacter tandoii
<400> 3
Met Ser Leu Ser Tyr Glu Met Glu Asn Ser Gly Ala Trp Gln Thr Tyr
1 5 10 15
Leu Ala Gln Ile Asn Arg Val Ala Pro Tyr Leu Glu Glu Asp Leu Ile
20 25 30
Pro Phe Ile Asn Thr Leu Lys Arg Pro Lys Arg Ala Leu Ile Val Asp
35 40 45
Val Pro Ile Val Met Asp Asp Gly Ser Ile Gln His Phe Glu Gly Tyr
50 55 60
Arg Val Gln His Asn Leu Ser Arg Gly Pro Gly Ala Gly Gly Ile Arg
65 70 75 80
Tyr His Pro Asp Val Glu Leu Asn Glu Val Met Ala Leu Ser Ala Trp
85 90 95
Met Thr Ile Lys Thr Ala Val Leu Asn Leu Pro Tyr Gly Gly Ala Lys
100 105 110
Gly Gly Ile Arg Val Asp Pro Arg Lys Leu Ser Pro Arg Glu Leu Glu
115 120 125
Arg Leu Thr Arg Arg Phe Thr Thr Glu Ile Ser Pro Ile Ile Gly Pro
130 135 140
Gln Ile Asp Ile Pro Ala Pro Asp Val Gly Thr Asn Ala Asp Ile Met
145 150 155 160
Gly Trp Met Met Asp Thr Tyr Ser Thr Ile Lys Gly His Thr Val Thr
165 170 175
Gly Val Val Thr Gly Lys Pro Val His Leu Gly Gly Ser Leu Gly Arg
180 185 190
Val Arg Ala Thr Gly Arg Gly Val Phe Val Thr Gly Leu Glu Val Ala
195 200 205
Lys Lys Ile Asn Leu Ala Leu Glu Gly Ser Arg Ile Ala Val Gln Gly
210 215 220
Phe Gly Asn Val Gly Ser Glu Ala Ala Tyr Leu Phe His Lys Ala Asn
225 230 235 240
Ala Lys Val Val Cys Val Gln Asp His Thr Gly Thr Ile Phe Asn Ala
245 250 255
Asp Gly Phe Asp Val Lys Gln Leu Gln Asp Tyr Val Ala Ile His Lys
260 265 270
Gly Val Ala Gly Phe Pro Asn Ala Thr Val Ile Glu Asp Glu Ala Phe
275 280 285
Trp Thr Val Glu Met Asp Ile Leu Ile Pro Ala Ala Leu Glu Gly Gln
290 295 300
Ile Thr Ala Glu Arg Ala Gln Lys Leu Thr Ala Lys Leu Val Leu Glu
305 310 315 320
Gly Ala Asn Gly Pro Thr Tyr Pro Glu Ala Glu Asp Ile Leu Leu Gln
325 330 335
Arg Gln Ile Met Ile Val Pro Asp Val Leu Cys Asn Ala Gly Gly Val
340 345 350
Thr Val Ser Tyr Phe Glu Trp Val Gln Asp Met Ala Ser Tyr Phe Trp
355 360 365
Thr Glu Glu Glu Ile Asn Glu Arg Leu Asp Lys Leu Met Ile Lys Ala
370 375 380
Val Glu Asp Val Trp His Thr Ala Asp Asp Lys Asp Cys Ser Leu Arg
385 390 395 400
Thr Ala Ala Tyr Ile Leu Ala Cys Glu Arg Ile Leu Lys Ala Arg Lys
405 410 415
Glu Arg Gly Ile Phe Pro Gly
420
<210> 4
<211> 423
<212> PRT
<213> Acinetobacter tandoii
<400> 4
Met Ser Leu Ser Tyr Glu Met Glu Asn Ser Gly Ala Trp Gln Thr Tyr
1 5 10 15
Leu Ala Gln Ile Asn Arg Val Ala Pro Tyr Leu Glu Glu Asp Leu Ile
20 25 30
Pro Phe Ile Asn Thr Leu Lys Arg Pro Lys Arg Ala Leu Ile Val Asp
35 40 45
Val Pro Ile Val Met Asp Asp Gly Ser Ile Gln His Phe Glu Gly Tyr
50 55 60
Arg Val Gln His Asn Leu Ser Arg Gly Pro Gly Val Gly Gly Ile Arg
65 70 75 80
Tyr His Pro Asp Val Glu Leu Asn Glu Val Met Ala Leu Ser Ala Trp
85 90 95
Met Thr Ile Lys Thr Ala Val Leu Asn Leu Pro Tyr Gly Gly Ala Lys
100 105 110
Gly Gly Ile Arg Val Asp Pro Arg Lys Leu Ser Pro Arg Glu Leu Glu
115 120 125
Arg Leu Thr Arg Arg Phe Thr Thr Glu Ile Ser Pro Ile Ile Gly Pro
130 135 140
Gln Ile Asp Ile Pro Ala Pro Asp Val Gly Thr Asn Ala Asp Ile Met
145 150 155 160
Gly Trp Met Met Asp Thr Tyr Ser Thr Ile Lys Gly His Thr Val Thr
165 170 175
Gly Val Val Thr Gly Lys Pro Val His Leu Gly Gly Ser Leu Gly Arg
180 185 190
Val Arg Ala Thr Gly Arg Gly Val Phe Val Thr Gly Leu Glu Val Ala
195 200 205
Lys Lys Ile Asn Leu Ala Leu Glu Gly Ser Arg Ile Ala Val Gln Gly
210 215 220
Phe Gly Asn Val Gly Ser Glu Ala Ala Tyr Leu Phe His Lys Ala Asn
225 230 235 240
Ala Lys Val Val Cys Val Gln Asp His Thr Gly Thr Ile Phe Asn Ala
245 250 255
Asp Gly Phe Asp Val Lys Gln Leu Gln Asp Tyr Val Ala Ile His Lys
260 265 270
Gly Val Ala Gly Phe Pro Asn Ala Thr Val Ile Glu Asp Glu Ala Phe
275 280 285
Trp Thr Val Glu Met Asp Ile Leu Ile Pro Ala Ala Leu Glu Gly Gln
290 295 300
Ile Thr Ala Glu Arg Ala Gln Lys Leu Thr Ala Lys Leu Val Leu Glu
305 310 315 320
Gly Ala Asn Gly Pro Thr Tyr Pro Glu Ala Glu Asp Ile Leu Leu Gln
325 330 335
Arg Gln Ile Met Ile Val Pro Asp Val Leu Cys Asn Ala Gly Gly Val
340 345 350
Thr Val Ser Tyr Phe Glu Trp Val Gln Asp Met Ala Ser Tyr Phe Trp
355 360 365
Thr Glu Glu Glu Ile Asn Glu Arg Leu Asp Lys Leu Met Ile Lys Ala
370 375 380
Val Glu Asp Val Trp His Thr Ala Asp Asp Lys Asp Cys Ser Leu Arg
385 390 395 400
Thr Ala Ala Tyr Ile Leu Ala Cys Glu Arg Ile Leu Lys Ala Arg Lys
405 410 415
Glu Arg Gly Ile Phe Pro Gly
420
<210> 5
<211> 423
<212> PRT
<213> Acinetobacter tandoii
<400> 5
Met Ser Leu Ser Tyr Glu Met Glu Asn Ser Gly Ala Trp Gln Thr Tyr
1 5 10 15
Leu Ala Gln Ile Asn Arg Val Ala Pro Tyr Leu Glu Glu Asp Leu Ile
20 25 30
Pro Phe Ile Asn Thr Leu Lys Arg Pro Lys Arg Ala Leu Ile Val Asp
35 40 45
Val Pro Ile Val Met Asp Asp Gly Ser Ile Gln His Phe Glu Gly Tyr
50 55 60
Arg Val Gln His Asn Leu Ser Arg Gly Pro Gly Leu Gly Gly Ile Arg
65 70 75 80
Tyr His Pro Asp Val Glu Leu Asn Glu Val Met Ala Leu Ser Ala Trp
85 90 95
Met Thr Ile Lys Thr Ala Val Leu Asn Leu Pro Tyr Gly Gly Ala Lys
100 105 110
Gly Gly Ile Arg Val Asp Pro Arg Lys Leu Ser Pro Arg Glu Leu Glu
115 120 125
Arg Leu Thr Arg Arg Phe Thr Thr Glu Ile Ser Pro Ile Ile Gly Pro
130 135 140
Gln Ile Asp Ile Pro Ala Pro Asp Val Gly Thr Asn Ala Asp Ile Met
145 150 155 160
Gly Trp Met Met Asp Thr Tyr Ser Thr Ile Lys Gly His Thr Val Thr
165 170 175
Gly Val Val Thr Gly Lys Pro Val His Leu Gly Gly Ser Leu Gly Arg
180 185 190
Val Arg Ala Thr Gly Arg Gly Val Phe Val Thr Gly Leu Glu Val Ala
195 200 205
Lys Lys Ile Asn Leu Ala Leu Glu Gly Ser Arg Ile Ala Val Gln Gly
210 215 220
Phe Gly Asn Val Gly Ser Glu Ala Ala Tyr Leu Phe His Lys Ala Asn
225 230 235 240
Ala Lys Val Val Cys Val Gln Asp His Thr Gly Thr Ile Phe Asn Ala
245 250 255
Asp Gly Phe Asp Val Lys Gln Leu Gln Asp Tyr Val Ala Ile His Lys
260 265 270
Gly Val Ala Gly Phe Pro Asn Ala Thr Val Ile Glu Asp Glu Ala Phe
275 280 285
Trp Thr Val Glu Met Asp Ile Leu Ile Pro Ala Ala Leu Glu Gly Gln
290 295 300
Ile Thr Ala Glu Arg Ala Gln Lys Leu Thr Ala Lys Leu Val Leu Glu
305 310 315 320
Gly Ala Asn Gly Pro Thr Tyr Pro Glu Ala Glu Asp Ile Leu Leu Gln
325 330 335
Arg Gln Ile Met Ile Val Pro Asp Val Leu Cys Asn Ala Gly Gly Val
340 345 350
Thr Val Ser Tyr Phe Glu Trp Val Gln Asp Met Ala Ser Tyr Phe Trp
355 360 365
Thr Glu Glu Glu Ile Asn Glu Arg Leu Asp Lys Leu Met Ile Lys Ala
370 375 380
Val Glu Asp Val Trp His Thr Ala Asp Asp Lys Asp Cys Ser Leu Arg
385 390 395 400
Thr Ala Ala Tyr Ile Leu Ala Cys Glu Arg Ile Leu Lys Ala Arg Lys
405 410 415
Glu Arg Gly Ile Phe Pro Gly
420
<210> 6
<211> 423
<212> PRT
<213> Acinetobacter tandoii
<400> 6
Met Ser Leu Ser Tyr Glu Met Glu Asn Ser Gly Ala Trp Gln Thr Tyr
1 5 10 15
Leu Ala Gln Ile Asn Arg Val Ala Pro Tyr Leu Glu Glu Asp Leu Ile
20 25 30
Pro Phe Ile Asn Thr Leu Lys Arg Pro Lys Arg Ala Leu Ile Val Asp
35 40 45
Val Pro Ile Val Met Asp Asp Gly Ser Ile Gln His Phe Glu Gly Tyr
50 55 60
Arg Val Gln His Asn Leu Ser Arg Gly Pro Gly Ile Gly Gly Ile Arg
65 70 75 80
Tyr His Pro Asp Val Glu Leu Asn Glu Val Met Ala Leu Ser Ala Trp
85 90 95
Met Thr Ile Lys Thr Ala Val Leu Asn Leu Pro Tyr Gly Gly Ala Lys
100 105 110
Gly Gly Ile Arg Val Asp Pro Arg Lys Leu Ser Pro Arg Glu Leu Glu
115 120 125
Arg Leu Thr Arg Arg Phe Thr Thr Glu Ile Ser Pro Ile Ile Gly Pro
130 135 140
Gln Ile Asp Ile Pro Ala Pro Asp Val Gly Thr Asn Ala Asp Ile Met
145 150 155 160
Gly Trp Met Met Asp Thr Tyr Ser Thr Ile Lys Gly His Thr Val Thr
165 170 175
Gly Val Val Thr Gly Lys Pro Val His Leu Gly Gly Ser Leu Gly Arg
180 185 190
Val Arg Ala Thr Gly Arg Gly Val Phe Val Thr Gly Leu Glu Val Ala
195 200 205
Lys Lys Ile Asn Leu Ala Leu Glu Gly Ser Arg Ile Ala Val Gln Gly
210 215 220
Phe Gly Asn Val Gly Ser Glu Ala Ala Tyr Leu Phe His Lys Ala Asn
225 230 235 240
Ala Lys Val Val Cys Val Gln Asp His Thr Gly Thr Ile Phe Asn Ala
245 250 255
Asp Gly Phe Asp Val Lys Gln Leu Gln Asp Tyr Val Ala Ile His Lys
260 265 270
Gly Val Ala Gly Phe Pro Asn Ala Thr Val Ile Glu Asp Glu Ala Phe
275 280 285
Trp Thr Val Glu Met Asp Ile Leu Ile Pro Ala Ala Leu Glu Gly Gln
290 295 300
Ile Thr Ala Glu Arg Ala Gln Lys Leu Thr Ala Lys Leu Val Leu Glu
305 310 315 320
Gly Ala Asn Gly Pro Thr Tyr Pro Glu Ala Glu Asp Ile Leu Leu Gln
325 330 335
Arg Gln Ile Met Ile Val Pro Asp Val Leu Cys Asn Ala Gly Gly Val
340 345 350
Thr Val Ser Tyr Phe Glu Trp Val Gln Asp Met Ala Ser Tyr Phe Trp
355 360 365
Thr Glu Glu Glu Ile Asn Glu Arg Leu Asp Lys Leu Met Ile Lys Ala
370 375 380
Val Glu Asp Val Trp His Thr Ala Asp Asp Lys Asp Cys Ser Leu Arg
385 390 395 400
Thr Ala Ala Tyr Ile Leu Ala Cys Glu Arg Ile Leu Lys Ala Arg Lys
405 410 415
Glu Arg Gly Ile Phe Pro Gly
420
<210> 7
<211> 423
<212> PRT
<213> Acinetobacter tandoii
<400> 7
Met Ser Leu Ser Tyr Glu Met Glu Asn Ser Gly Ala Trp Gln Thr Tyr
1 5 10 15
Leu Ala Gln Ile Asn Arg Val Ala Pro Tyr Leu Glu Glu Asp Leu Ile
20 25 30
Pro Phe Ile Asn Thr Leu Lys Arg Pro Lys Arg Ala Leu Ile Val Asp
35 40 45
Val Pro Ile Val Met Asp Asp Gly Ser Ile Gln His Phe Glu Gly Tyr
50 55 60
Arg Val Gln His Asn Leu Ser Arg Gly Pro Gly Lys Gly Gly Ile Arg
65 70 75 80
Tyr His Pro Asp Val Glu Leu Asn Glu Val Met Ala Leu Ser Ala Trp
85 90 95
Met Thr Ile Lys Thr Ala Val Leu Asn Leu Pro Tyr Gly Gly Ala Lys
100 105 110
Gly Gly Ile Arg Val Asp Pro Arg Lys Leu Ser Pro Arg Glu Leu Glu
115 120 125
Arg Leu Thr Arg Arg Phe Thr Thr Glu Ile Ser Pro Ile Ile Gly Pro
130 135 140
Gln Ile Asp Ile Pro Ala Pro Asp Val Gly Thr Asn Ala Asp Ile Met
145 150 155 160
Gly Trp Met Met Asp Thr Tyr Ser Thr Ile Lys Gly His Thr Val Thr
165 170 175
Gly Val Val Ser Gly Lys Pro Val His Leu Gly Gly Ser Leu Gly Arg
180 185 190
Val Arg Ala Thr Gly Arg Gly Val Phe Val Thr Gly Leu Glu Val Ala
195 200 205
Lys Lys Ile Asn Leu Ala Leu Glu Gly Ser Arg Ile Ala Val Gln Gly
210 215 220
Phe Gly Asn Val Gly Ser Glu Ala Ala Tyr Leu Phe His Lys Ala Asn
225 230 235 240
Ala Lys Val Val Cys Val Gln Asp His Thr Gly Thr Ile Phe Asn Ala
245 250 255
Asp Gly Phe Asp Val Lys Gln Leu Gln Asp Tyr Val Ala Ile His Lys
260 265 270
Gly Val Ala Gly Phe Pro Asn Ala Thr Val Ile Glu Asp Glu Ala Phe
275 280 285
Trp Thr Val Glu Met Asp Ile Leu Ile Pro Ala Ala Leu Glu Gly Gln
290 295 300
Ile Thr Ala Glu Arg Ala Gln Lys Leu Thr Ala Lys Leu Val Leu Glu
305 310 315 320
Gly Ala Asn Gly Pro Thr Tyr Pro Glu Ala Glu Asp Ile Leu Leu Gln
325 330 335
Arg Gln Ile Met Ile Val Pro Asp Val Leu Cys Asn Ala Gly Gly Val
340 345 350
Thr Val Ser Tyr Phe Glu Trp Val Gln Asp Met Ala Ser Tyr Phe Trp
355 360 365
Thr Glu Glu Glu Ile Asn Glu Arg Leu Asp Lys Leu Met Ile Lys Ala
370 375 380
Val Glu Asp Val Trp His Thr Ala Asp Asp Lys Asp Cys Ser Leu Arg
385 390 395 400
Thr Ala Ala Tyr Ile Leu Ala Cys Glu Arg Ile Leu Lys Ala Arg Lys
405 410 415
Glu Arg Gly Ile Phe Pro Gly
420
<210> 8
<211> 423
<212> PRT
<213> Acinetobacter tandoii
<400> 8
Met Ser Leu Ser Tyr Glu Met Glu Asn Ser Gly Ala Trp Gln Thr Tyr
1 5 10 15
Leu Ala Gln Ile Asn Arg Val Ala Pro Tyr Leu Glu Glu Asp Leu Ile
20 25 30
Pro Phe Ile Asn Thr Leu Lys Arg Pro Lys Arg Ala Leu Ile Val Asp
35 40 45
Val Pro Ile Val Met Asp Asp Gly Ser Ile Gln His Phe Glu Gly Tyr
50 55 60
Arg Val Gln His Asn Leu Ser Arg Gly Pro Gly Lys Gly Gly Ile Arg
65 70 75 80
Tyr His Pro Asp Val Glu Leu Asn Glu Val Met Ala Leu Ser Ala Trp
85 90 95
Met Thr Ile Lys Thr Ala Val Leu Asn Leu Pro Tyr Gly Gly Ala Lys
100 105 110
Gly Gly Ile Arg Val Asp Pro Arg Lys Leu Ser Pro Arg Glu Leu Glu
115 120 125
Arg Leu Thr Arg Arg Phe Thr Thr Glu Ile Ser Pro Ile Ile Gly Pro
130 135 140
Gln Ile Asp Ile Pro Ala Pro Asp Val Gly Thr Asn Ala Asp Ile Met
145 150 155 160
Gly Trp Met Met Asp Thr Tyr Ser Thr Ile Lys Gly His Thr Val Thr
165 170 175
Gly Val Val Cys Gly Lys Pro Val His Leu Gly Gly Ser Leu Gly Arg
180 185 190
Val Arg Ala Thr Gly Arg Gly Val Phe Val Thr Gly Leu Glu Val Ala
195 200 205
Lys Lys Ile Asn Leu Ala Leu Glu Gly Ser Arg Ile Ala Val Gln Gly
210 215 220
Phe Gly Asn Val Gly Ser Glu Ala Ala Tyr Leu Phe His Lys Ala Asn
225 230 235 240
Ala Lys Val Val Cys Val Gln Asp His Thr Gly Thr Ile Phe Asn Ala
245 250 255
Asp Gly Phe Asp Val Lys Gln Leu Gln Asp Tyr Val Ala Ile His Lys
260 265 270
Gly Val Ala Gly Phe Pro Asn Ala Thr Val Ile Glu Asp Glu Ala Phe
275 280 285
Trp Thr Val Glu Met Asp Ile Leu Ile Pro Ala Ala Leu Glu Gly Gln
290 295 300
Ile Thr Ala Glu Arg Ala Gln Lys Leu Thr Ala Lys Leu Val Leu Glu
305 310 315 320
Gly Ala Asn Gly Pro Thr Tyr Pro Glu Ala Glu Asp Ile Leu Leu Gln
325 330 335
Arg Gln Ile Met Ile Val Pro Asp Val Leu Cys Asn Ala Gly Gly Val
340 345 350
Thr Val Ser Tyr Phe Glu Trp Val Gln Asp Met Ala Ser Tyr Phe Trp
355 360 365
Thr Glu Glu Glu Ile Asn Glu Arg Leu Asp Lys Leu Met Ile Lys Ala
370 375 380
Val Glu Asp Val Trp His Thr Ala Asp Asp Lys Asp Cys Ser Leu Arg
385 390 395 400
Thr Ala Ala Tyr Ile Leu Ala Cys Glu Arg Ile Leu Lys Ala Arg Lys
405 410 415
Glu Arg Gly Ile Phe Pro Gly
420
<210> 9
<211> 423
<212> PRT
<213> Acinetobacter tandoii
<400> 9
Met Ser Leu Ser Tyr Glu Met Glu Asn Ser Gly Ala Trp Gln Thr Tyr
1 5 10 15
Leu Ala Gln Ile Asn Arg Val Ala Pro Tyr Leu Glu Glu Asp Leu Ile
20 25 30
Pro Phe Ile Asn Thr Leu Lys Arg Pro Lys Arg Ala Leu Ile Val Asp
35 40 45
Val Pro Ile Val Met Asp Asp Gly Ser Ile Gln His Phe Glu Gly Tyr
50 55 60
Arg Val Gln His Asn Leu Ser Arg Gly Pro Gly Leu Gly Gly Ile Arg
65 70 75 80
Tyr His Pro Asp Val Glu Leu Asn Glu Val Met Ala Leu Ser Ala Trp
85 90 95
Met Thr Ile Lys Thr Ala Val Leu Asn Leu Pro Tyr Gly Gly Ala Lys
100 105 110
Gly Gly Ile Arg Val Asp Pro Arg Lys Leu Ser Pro Arg Glu Leu Glu
115 120 125
Arg Leu Thr Arg Arg Phe Thr Thr Glu Ile Ser Pro Ile Ile Gly Pro
130 135 140
Gln Ile Asp Ile Pro Ala Pro Asp Val Gly Thr Asn Ala Asp Ile Met
145 150 155 160
Gly Trp Met Met Asp Thr Tyr Ser Thr Ile Lys Gly His Thr Val Thr
165 170 175
Gly Val Val Cys Gly Lys Pro Val His Leu Gly Gly Ser Leu Gly Arg
180 185 190
Val Arg Ala Thr Gly Arg Gly Val Phe Val Thr Gly Leu Glu Val Ala
195 200 205
Lys Lys Ile Asn Leu Ala Leu Glu Gly Ser Arg Ile Ala Val Gln Gly
210 215 220
Phe Gly Asn Val Gly Ser Glu Ala Ala Tyr Leu Phe His Lys Ala Asn
225 230 235 240
Ala Lys Val Val Cys Val Gln Asp His Thr Gly Thr Ile Phe Asn Ala
245 250 255
Asp Gly Phe Asp Val Lys Gln Leu Gln Asp Tyr Val Ala Ile His Lys
260 265 270
Gly Val Ala Gly Phe Pro Asn Ala Thr Val Ile Glu Asp Glu Ala Phe
275 280 285
Trp Thr Val Glu Met Asp Ile Leu Ile Pro Ala Ala Leu Glu Gly Gln
290 295 300
Ile Thr Ala Glu Arg Ala Gln Lys Leu Thr Ala Lys Leu Val Leu Glu
305 310 315 320
Gly Ala Asn Gly Pro Thr Tyr Pro Glu Ala Glu Asp Ile Leu Leu Gln
325 330 335
Arg Gln Ile Met Ile Val Pro Asp Val Leu Cys Asn Ala Gly Gly Val
340 345 350
Thr Val Ser Tyr Phe Glu Trp Val Gln Asp Met Ala Ser Tyr Phe Trp
355 360 365
Thr Glu Glu Glu Ile Asn Glu Arg Leu Asp Lys Leu Met Ile Lys Ala
370 375 380
Val Glu Asp Val Trp His Thr Ala Asp Asp Lys Asp Cys Ser Leu Arg
385 390 395 400
Thr Ala Ala Tyr Ile Leu Ala Cys Glu Arg Ile Leu Lys Ala Arg Lys
405 410 415
Glu Arg Gly Ile Phe Pro Gly
420
<210> 10
<211> 1272
<212> DNA
<213> Acinetobacter tandoii
<400> 10
atgtctttgt catatgaaat ggaaaatagt ggtgcatggc aaacctacct tgctcagatt 60
aaccgtgtag ccccttatct tgaagaagac ttaatcccat ttatcaatac tttaaagcgt 120
ccaaagcgtg cgctcattgt tgatgtgcct attgtaatgg acgacggttc tattcagcat 180
ttcgaaggtt accgtgtaca acacaatttg tcgcgtggac caggtaaggg tggtattcgt 240
taccatccag atgtagaact gaatgaggtc atggccctct cagcatggat gaccattaaa 300
actgcggtat tgaacttacc gtatggtggt gcaaaaggcg gtattcgagt agacccgcgt 360
aaattgtcac cacgtgaact tgaacgctta acgcgtcgtt ttaccactga aattagccca 420
atcattggtc cacaaattga tattccagcg ccagatgtgg gtactaatgc agacattatg 480
ggttggatga tggataccta ttccacaatt aagggtcata ctgtcacagg tgtagtgacg 540
ggtaaacctg tacatttagg tggttcatta ggtcgtgtcc gtgcaacagg tcgtggtgta 600
ttcgttacag gtttagaagt tgccaaaaaa attaacttag cacttgaagg cagccgtatt 660
gcagttcagg gctttggtaa cgtaggtagc gaagctgcat atttattcca taaagccaac 720
gcaaaagtag tatgtgtaca agaccacaca ggcacaattt tcaacgcaga tggttttgac 780
gttaagcaat tacaagacta cgttgcgatt cataaaggtg ttgcaggctt ccctaatgca 840
accgtaattg aagatgaagc gttctggaca gtagagatgg atattttaat ccctgctgct 900
ttagaaggtc agattactgc tgagcgtgcg caaaaattga ctgcgaaatt ggtcttggaa 960
ggtgcgaacg gcccaactta tccagaagca gaagatattc ttttacaacg tcaaatcatg 1020
attgttccag atgtgctctg taatgctggc ggtgtaaccg tgagttactt cgagtgggtt 1080
caagacatgg caagttactt ctggaccgaa gaagaaatta atgaacgctt agataagctc 1140
atgattaaag cggttgaaga tgtttggcat acagcagatg acaaagattg tagcttgcgt 1200
accgcagcgt acattttggc ttgtgagcgc attttgaaag cacgtaaaga acgaggaatt 1260
ttcccaggtt aa 1272
<210> 11
<211> 24
<212> DNA
<213> artificial sequence
<400> 11
ggtggcggcg gtattcgtta ccat 24
<210> 12
<211> 24
<212> DNA
<213> artificial sequence
<400> 12
tggtccacgc gacaaattat gttg 24
<210> 13
<211> 24
<212> DNA
<213> artificial sequence
<400> 13
ggtgcgggcg gtattcgtta ccat 24
<210> 14
<211> 24
<212> DNA
<213> artificial sequence
<400> 14
tggtccacgc gacaaattat gttg 24
<210> 15
<211> 24
<212> DNA
<213> artificial sequence
<400> 15
ggtgtgggcg gtattcgtta ccat 24
<210> 16
<211> 24
<212> DNA
<213> artificial sequence
<400> 16
tggtccacgc gacaaattat gttg 24
<210> 17
<211> 24
<212> DNA
<213> artificial sequence
<400> 17
ggtctgggcg gtattcgtta ccat 24
<210> 18
<211> 24
<212> DNA
<213> artificial sequence
<400> 18
tggtccacgc gacaaattat gttg 24
<210> 19
<211> 24
<212> DNA
<213> artificial sequence
<400> 19
ggtatcggcg gtattcgtta ccat 24
<210> 20
<211> 24
<212> DNA
<213> artificial sequence
<400> 20
tggtccacgc gacaaattat gttg 24
<210> 21
<211> 27
<212> DNA
<213> artificial sequence
<400> 21
gtgtctggta aacctgtaca tttaggt 27
<210> 22
<211> 24
<212> DNA
<213> artificial sequence
<400> 22
tacacctgtg acagtatgac cctt 24
<210> 23
<211> 27
<212> DNA
<213> artificial sequence
<400> 23
gtgtgcggta aacctgtaca tttaggt 27
<210> 24
<211> 24
<212> DNA
<213> artificial sequence
<400> 24
tacacctgtg acagtatgac cctt 24
Claims (7)
1. A glutamate dehydrogenase mutant having improved activity, comprising:
taking wild glutamate dehydrogenase AtGluDH shown in SEQ ID NO.1 as a template, and mutating lysine K at position 76 into a glutamate dehydrogenase mutant K76L of leucine L, wherein the amino acid sequence is shown in SEQ ID NO. 5;
taking wild glutamate dehydrogenase AtGluDH shown in SEQ ID NO.1 as a template, mutating threonine T at position 180 into glutamate dehydrogenase mutant T180C of cysteine C, wherein the amino acid sequence is shown in SEQ ID NO. 8; and
taking mutant glutamate dehydrogenase mutant K76L shown in SEQ ID NO.5 as a template, and carrying out threonine T mutation on the 180 th amino acid residue to obtain a glutamate dehydrogenase mutant with cysteine C, wherein the amino acid sequence is shown in SEQ ID NO. 9.
2. The glutamate dehydrogenase mutant encoding gene according to claim 1.
3. A recombinant vector comprising a gene encoding the glutamate dehydrogenase mutant according to claim 2.
4. A genetically engineered bacterium comprising a gene encoding the glutamate dehydrogenase mutant of claim 2.
5. Use of a glutamate dehydrogenase mutant according to claim 1 for catalyzing the preparation of L-2-aminobutyric acid from 2-ketobutyric acid.
6. Use of a recombinant vector comprising a gene encoding a glutamate dehydrogenase mutant according to claim 3 for catalyzing 2-ketobutyrate production of L-2-aminobutyric acid.
7. The use of a genetically engineered bacterium comprising a gene encoding a glutamate dehydrogenase mutant according to claim 4 for catalyzing 2-ketobutyrate to produce L-2-aminobutyric acid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110334184.5A CN113174378B (en) | 2021-03-29 | 2021-03-29 | Glutamate dehydrogenase mutant, encoding gene thereof, genetically engineered bacterium and application thereof in preparation of L-2-aminobutyric acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110334184.5A CN113174378B (en) | 2021-03-29 | 2021-03-29 | Glutamate dehydrogenase mutant, encoding gene thereof, genetically engineered bacterium and application thereof in preparation of L-2-aminobutyric acid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113174378A CN113174378A (en) | 2021-07-27 |
| CN113174378B true CN113174378B (en) | 2023-07-07 |
Family
ID=76922538
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| WO1995034672A1 (en) * | 1994-06-14 | 1995-12-21 | Ajinomoto Co., Inc. | α-KETOGLUTARIC DEHYDROGENASE GENE |
| CN108588045A (en) * | 2018-03-09 | 2018-09-28 | 浙江大学 | Glutamate dehydrogenase enzyme mutant and its application in preparing L-glufosinate-ammonium |
| CN110184246A (en) * | 2019-05-15 | 2019-08-30 | 浙江大学 | Glutamate dehydrogenase enzyme mutant and its application |
| WO2020034209A1 (en) * | 2018-08-17 | 2020-02-20 | 凯莱英生命科学技术(天津)有限公司 | Amino acid dehydrogenase mutant and application thereof |
| CN111676203A (en) * | 2020-06-28 | 2020-09-18 | 江南大学 | Leucine dehydrogenase mutant and application thereof |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1995034672A1 (en) * | 1994-06-14 | 1995-12-21 | Ajinomoto Co., Inc. | α-KETOGLUTARIC DEHYDROGENASE GENE |
| CN108588045A (en) * | 2018-03-09 | 2018-09-28 | 浙江大学 | Glutamate dehydrogenase enzyme mutant and its application in preparing L-glufosinate-ammonium |
| WO2020034209A1 (en) * | 2018-08-17 | 2020-02-20 | 凯莱英生命科学技术(天津)有限公司 | Amino acid dehydrogenase mutant and application thereof |
| CN110184246A (en) * | 2019-05-15 | 2019-08-30 | 浙江大学 | Glutamate dehydrogenase enzyme mutant and its application |
| CN111676203A (en) * | 2020-06-28 | 2020-09-18 | 江南大学 | Leucine dehydrogenase mutant and application thereof |
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