CN103074309A - L-alanine dehydrogenase mutant zymoprotein and preparation method thereof - Google Patents

Abstract

The present invention discloses L-alanine dehydrogenase mutant zymoprotein and a preparation method thereof. According to a homologous sequence secondary structure comparison result, site-specific mutagenesis of 73th lysine near an L-alanine dehydrogenase activity center of Bacilluspseudofirmus into alanine through PCR is performed to construct a mutant expression vector, and prokaryotic expression and Ni-NTA affinity chromatography purification are performed to obtain a mutant enzyme K73A, wherein specific activity of the obtained mutant enzyme K73A is 202% of specific activity the wild type, and other enzymatic properties are not changed. In addition, a turnover number Kcat on a substrate L-alanine by the mutant zymoprotein is 376.7 min<-1> and is 2.0 times the turnover number Kcat of the wild type, a turnover number Kcat on beta-NAD<+> by the mutant zymoprotein is 290.1 min<-1> and is 2.1 times the turnover number Kcat of the wild type, and the mutant zymoprotein can be applicable for production processes of production of pyruvic acid, L/D-alanine, and the like through improvement biology methods.

Description

Mutant enzyme albumen of a kind of ALANINE desaturase and preparation method thereof

Technical field

The present invention relates to a kind of mutant enzyme albumen of ALANINE desaturase, belong to genetically engineered and technical field of enzyme engineering.

Background technology

ALANINE desaturase (L-alanine dehydrogenase, Ald, EC 1.4.1.1) is a kind of amino acid dehydrogenase take nicotinamide-adenine (NAD) as coenzyme.The reversible catalysis ALANINE of ALANINE desaturase oxidative deamination generates pyruvic acid, ammonia and NADH.React as follows:

L-alanine + NAD ⁺ + H ₂O <->NH ₄ ⁺ + pyruvate + NADH + H ⁺

The ALANINE desaturase is to participate in regulating amino acid and glycometabolic important enzyme, and product pyruvic acid or the ALANINE of catalyzed reaction are widely used in the fields such as medicine, agricultural chemicals, food, have good development prospect.As with this enzyme and alanine racemase acting in conjunction, then pyruvic acid, ammonia etc. finally can be generated D-alanine.Therefore, the ALANINE desaturase has important using value in fields such as medicine, agricultural chemicals and food.The ALANINE desaturase extensively is present among the microorganism, is to be found in nineteen fifty-five by people such as Wiame at first, but until nineteen ninety its encoding gene just be cloned by people such as Kuroda.From super hyperthermophilic archaeon strain Archaeoglobus fulgidus, elongated synechococcus Synechococcus elongatus PCC 7942 has found the existence of alanine dehydrogenase in a plurality of organisms such as Bacillus natto Bacillus subtilis at present.Since the people such as Patrick in 1998 have resolved the space structure that comes from alanine dehydrogenase among the Phormidium lapideum for the first time, at present the crystalline structure of total alanine dehydrogenase from 6 bacterial strains is resolved, and the catalytic mechanism that resolves to the in-depth explanation alanine dehydrogenase of space structure provides more easily condition.

The false bacillus firmus (Bacillus pseudofirmus OF4) of extreme basophilic, to equal at first discovery in 1986 by Guffanti, Takami etc. have carried out the analysis on the taxonomy, this bacterium is strict aerobic-type, product spore, Gram-positive, sports type rod-shaped bacterium, optimum growth temperature is 30 ℃, at pH 7.5-11.5

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Condition under grow optimal pH 10.5.Genomic sequence analysis is found, operon of the adjacent formation of ALANINE dehydrogenase gene and alanine racemase, and the two acting in conjunction can be synthesized D-alanine.But because these enzymes content in microorganism is few, activity is low, purposes has been subject to certain limitation.

Summary of the invention

One of purpose of the present invention provides a kind of mutant enzyme albumen of ALANINE desaturase of enzyme activity raising.

Two of purpose of the present invention provides a kind of preparation method of mutant enzyme albumen of ALANINE desaturase.

The object of the present invention is achieved like this.The mutant enzyme albumen of a kind of ALANINE desaturase provided by the invention, its aminoacid sequence is shown in SEQ ID No.1.

The present invention also provides the gene of coding said mutation body zymoprotein, and its nucleotide sequence is shown in SEQ ID No.2.

The present invention also provides carrier and the host cell that contains said gene.

The present invention also provides the engineering bacteria that contains said gene.

The present invention also provides the preparation method of said mutation body zymoprotein, may further comprise the steps:

(1) according to the aminoacid sequence of disclosed ALANINE desaturase from false bacillus firmus in the ncbi database, determines the mutational site having resolved the basis that the secondary structure sequence of homologous protein compares with structure; The mutant primer pair of design rite-directed mutagenesis; Described mutant primer to be 5 '-TCTTTAACTGCCATCACCAT-3 ' and 5 '-ATGGTGATGGCAGTTAAAGA-3 ';

(2) take the carrier that carries the ALANINE dehydrogenase gene as template, use the primer of above-mentioned design to carry out pcr amplification, the amplified production that will contain the mutational site is connected in the expression vector, makes up mutant plasmid;

(3) the said mutation Plasmid Transformation can be expressed in the engineering bacteria of goal gene, express this mutant enzyme albumen with copying of engineering bacteria.

Preparation method's optimum condition: expression vector described in the step (2) is pET series, pUC series, pT7-7, or among the pGEX any one;

Engineering bacteria described in the step (3) is BL21 (DE3).

The present invention further provides the application of said mutation body zymoprotein, specifically mutant enzyme albumen is used

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Produce pyruvic acid or ALANINE and D-alanine in biological process.

Particularly, the present invention obtains ALANINE dehydrogenase gene (NCBI numbering: YP_003426902) from false bacillus firmus (Bacillus Pseudofirmus), secondary structure comparison result according to homologous protein is determined the mutational site, replace near the amino acid sites in active centre by site-directed mutagenesis technique (site-directed mutagenesis), the mutant gene fragment is connected with pET22b (+) plasmid, successfully makes up recombinant expression plasmid pET22b-K73A; This recombinant plasmid transformed competent escherichia coli cell, the genetic engineering bacterium BL21 (DE3) that structure is expressed for mutant enzyme/pET22b-K73A; Under 37 ℃ and 1.0mM IPTG condition, obtained to express preferably.With turnover number K _CatWith apparent secondary rate constant K _Cat/ K _mExpression, the enzyme work of mutant K73A is improved than wild-type ALANINE desaturase; The multiple that the Kcat of mutant and Kcat/Km improve:

False bacillus firmus ALANINE dehydrogenase mutant nucleotides sequence is classified as: SEQ ID NO:2, aminoacid sequence is: SEQ ID NO:1.

The beneficial effect that the present invention obtains: the present invention determines that by homologous sequence secondary structure comparison result amino acid is the mutational site near the false bacillus firmus ALANINE dehydrogenase activity center, replace by site-directed mutagenesis technique (site-directed mutagenesis), obtain ALANINE dehydrogenase mutant K73A, with turnover number K _CatWith apparent secondary rate constant K _Cat/ K _mExpression, the enzyme work of ALANINE desaturase is improved.

Description of drawings

Fig. 1 is the electrophoretogram of pcr amplification product of the present invention.

M:Marker among Fig. 1; The pcr amplification product of 1:ald gene.

Fig. 2 is the electrophoretogram of rite-directed mutagenesis pcr amplification product.

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M:Marker among Fig. 2; 1: take the pET22b-Ald plasmid as template rite-directed mutagenesis pcr amplification product.

Fig. 3 is the electrophoretogram of mutant plasmid pET22b-K73A double digestion checking.

M:Marker among Fig. 3; 1: through the plasmid pET22b-K73A of double digestion processing.

Fig. 4 is the wild-type protein of purifying of the present invention and the electrophoretogram of mutain.

Among Fig. 41: wild-type Ald, 2: mutain K73A, M: albumen marker.

Embodiment

Following examples are used for explanation the present invention.Need explanation, employed experimental technique is ordinary method if no special instructions among the following embodiment.

Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.

Embodiment 1 ALANINE dehydrogenase gene ald obtains and expression vector establishment

(1) the ald gene obtains

According to false bacillus firmus OF4(Bacillus pseudofirmus) known ALANINE dehydrogenase gene (NCBI numbering: YP_003426902) design primer, take B. pseudofirmus genomic dna as template, pcr amplification, the result obtains 1 specific fragment (Fig. 1).Ald amplimer and PCR condition are respectively:

Sense:5 '-G CATATGATTATCGGTATTCCA-3 ' (underscore is restriction enzyme site);

Anti-sense:5 '-C CTCGAGTGCTTGAACAGGTGTTTTC-3 ' (underscore is restriction enzyme site);

94 ℃ of denaturation 4min; 94 ℃ of sex change 45sec, 55 ℃ of annealing 1min, 72 ℃ are extended 2min, circulate 25 times; 72 ℃ are fully extended 10min.The PCR fragment that obtains of amplification, after glue reclaimed, pMD18-T was connected with carrier, and will connect product and be converted into intestinal bacteria E. coli DH5 α, through blue hickie screening, selected the white clone and extracted plasmid pMD18-Ald, sent the greatly gene sequencing evaluation of Beijing China.Sequence alignment finds that sequenced genes and Genbank(NCBI number: in full accord YP_003426902);

(2) plasmid pET22b-Ald makes up

Plasmid pMD18-Ald and pET22b(+) through Nde I, Xho I double digestion, enzyme is cut product after glue reclaims, and with the connection of spending the night of 16 ℃ of T4 ligase enzymes, the connection product is converted into intestinal bacteria E. coli DH5 α,

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Screening positive clone obtains expression plasmid pET22b-Ald.By the dna fragmentation of agarose gel electrophoresis Separation and Recovery gene ald, and is connected acquisition expression plasmid pET22b-Ald with the carrier pET-22b that processes through same double digestion.

The selection in embodiment 2 mutational sites

Utilize ClustalX software, the aminoacid sequence of false bacillus firmus ALANINE desaturase and ALANINE desaturase (PDB ID:2VOE) from mycobacterium tuberculosis (Mycobacterium tuberculosis) are carried out sequence alignment, with ESPript(http: //espript.ibcp.fr/ESPript/ESPript/index.php; Gouet P, Courcelle E, Stuart DI, Metoz F. ESPript:multiple sequence alignments in PostScript. Bioinformatics. 1999,15,305-308) the secondary structure of predicted protein matter, the site of determining rite-directed mutagenesis are 73 Methionin K, and the sudden change direction is that 73 Methionin K is substituted by L-Ala A.

The structure of embodiment 3 mutant plasmid pET22b-K73A

According to the gene order of the strong gemma ALANINE of vacation desaturase (NCBI numbering: YP_003426902), and selected mutational site 73K, design following 2 rite-directed mutagenesis primers:

Sense-K73A:5 '-ATGGTGATG GCAGTTAAAGA-3 ' (underscore is mutating alkali yl);

Anti-sense-K73A:5 '-TCTTTAAC TGCCATCACCAT-3 ' (underscore is mutating alkali yl);

With reference to fast PCR mutating technology (QuickChange Site-Directed Mutagenesis), carry out PCR take recombinant expression vector pET22b-Ald as template, the PCR reaction conditions is: 94 ℃ of denaturation 4min; 94 ℃ of sex change 35sec, 55 ℃ of annealing 1min, 72 ℃ are extended 7min, circulate 16 times; 72 ℃ are fully extended 10min.

PCR product (Fig. 2) digests through the Dpn I, be converted into intestinal bacteria E. coli DH5 α, after cultivating, extracts the picking mono-clonal plasmid, by containing target DNA fragment (Fig. 3) in restriction restriction endonuclease Nde I, the Xho I double digestion checking plasmid, determine to suddenly change successfully through checking order, obtain mutant plasmid pET22b-K73A.

Embodiment 4 mutant enzymes and wild-type enzyme protein expression and and purifying

Plasmid pET22b-K73A is converted into e. coli bl21 (DE3) competent cell, selects transformant in 37 ℃ of incubated overnight of LB substratum (containing 100 μ g/mL penbritins); Nutrient solution is inoculated in 1:100

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100mLLB liquid nutrient medium (containing 100 μ g/mL penbritins), 37 ℃, 180rpm, shaking culture is to OD ₆₀₀Be 0.5; Add 1mM isopropylthiogalactoside (isopropyl-β-D-thiogalactopyranoside, IPTG), 37 ℃ of abduction delivering 6h.In 4 ℃, the centrifugal 10min of 8000rpm collects thalline, ultrasonic disruption.With Ni-NTA affinity column (Ni-NTA affinity chromatography) purifying, imidazole concentration is 250mM in the elutriant, obtains the mutant protein K73A of purifying.

Adopting uses the same method changes carrier pET22b-Ald over to BL21(DE3) the middle recombinant bacterium BL21(DE3 that obtains)/pET22b-Ald, this recombinant bacterium is cultivated and purifying with identical step, preferment albumin A ld obtains suddenling change;

Zymoprotein Ald and K73A behind the purifying identify at molecular weight 40kDA place the very high band of one specificity (Fig. 4) is arranged all through 12.5%SDS-PAGE, illustrate that the purity of protein behind the purifying is higher.SDS-PAGE shows that the molecular size range of protein is about 40kDa, and is consistent with the molecular size range that calculates by aminoacid sequence.

The activity of embodiment 5 ALANINE dehydrogenase mutants detects

(1) relative enzyme activity determination

To get respectively 5 μ L by purifying enzyme liquid Ald and the K73A that embodiment 5 obtains, with contain 50mM Sodium Carbonate Buffer (pH 10.5), 50mM L-alanine, 0.6mM β-Nicotinamide Adenine Dinucleotide (NAD ⁺, Roche company, production code member is 004626) damping fluid 1000 μ L(40 ℃ preheating 30min) mix rapidly, with in the ultraviolet spectrophotometer, detect A ₃₄₀Variation, average and calculate enzyme and live.Ld compares with the wild-type enzyme albumin A, and the relative enzyme work of mutant K73A is 202% of wild-type.

(2) the enzyme kinetics parametric measurement reaches with the parameter of wild-type Ald and haggles over

1 enzyme unit alive (U) is defined as: it is a unit that per minute produces the needed enzyme amount of 1 μ mol NADH.

At the 50mM of 40 ℃ of preheatings Sodium Carbonate Buffer (pH 10.5), 6mM β-NAD ⁺The fixation reaction system in, change the final concentration of L-alanine, mix rapidly after adding zymoprotein, detect A with spectrophotometer ₃₄₀Variation, parallel 3 ~ 4 secondary responses of doing of identical L-alanine concentration are averaged and are done curve, the method for the nonlinear fitting that provides according to Graphpad software is calculated the kinetics-6-of each zymoprotein

Constant K _mAnd V _Max, K _CatBy V _MaxGet with the ratio calculation of zymoprotein concentration.Use the same method and calculate each enzyme to NAD ⁺K _m, V _MaxAnd K _CatValue.With turnover number K _CatExpression, L-alanine is the reaction substrate variable, the enzyme work of mutant K73A is 2.0 times of wild-type Ald; β-NAD ⁺Be the reaction substrate variable, the enzyme work of mutant K73A is 2.1 times of wild-type Ald.With apparent secondary rate constant K _Cat/ K _mExpression, L-alanine is the substrate variable, the enzyme work of mutant K73A is 5.9 times of wild-type Ald; β-NAD ⁺Be the reaction substrate variable, the enzyme work of mutant K73A is 1.7 times of wild-type Ald.Concrete numerical value sees Table 1.

Table 1

Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.

SEQUENCE LISTING

＜110〉Hebei Normal University

＜120〉mutant enzyme albumen of a kind of ALANINE desaturase and preparation method thereof

<130>

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 385

<212> PRT

＜213〉false bacillus firmus (Bacillus pseudofirmus)

<400> 1

Met Ile Ile Gly Ile Pro Lys Glu Ile Lys Asn Asn Glu Asn Arg Val

1 5 10 15

Ala Ile Thr Pro Ala Gly Val Val Ala Leu Thr Lys Ala Gly His Gln

20 25 30

Ile Leu Ile Glu Gln Gly Ala Gly Ile Gly Ser Gly Phe Glu Asp Val

35 40 45

Asp Tyr Thr Ala Ala Gly Ala Thr Ile Ile Pro Glu Ala Lys Asp Val

50 55 60

Trp Ala Lys Ala Glu Met Val Met Ala Val Lys Glu Pro Leu Ser Ser

65 70 75 80

Glu Tyr Gly Tyr Phe Arg Lys Gly Leu Ile Leu Phe Thr Tyr Leu His

85 90 95

Leu Ala Ala Glu Pro Glu Leu Ala Lys Ala Leu Val Asp Ser Gly Val

100 105 110

Ile Ala Ile Ala Tyr Glu Thr Val Glu Val Asn Arg Thr Leu Pro Leu

115 120 125

Leu Thr Pro Met Ser Glu Val Ala Gly Arg Met Ala Ser Gln Ile Gly

130 135 140

Ala Gln Phe Leu Glu Lys Ser Lys Gly Gly Lys Gly Ile Leu Leu Ser

145 150 155 160

Gly Val Pro Gly Val Lys Arg Gly Lys Val Thr Ile Ile Gly Gly Gly

165 170 175

Val Val Gly Thr Asn Ala Ala Lys Ile Ala Val Gly Leu Gly Ala Asp

180 185 190

Val Thr Leu Ile Asp Leu Ser Ala Asp Arg Leu Arg Gln Leu Asp Asp

195 200 205

Gln Phe Gly Asn Asp Ile Gln Thr Leu Met Ser Asn Pro Leu Asn Ile

210 215 220

Ala Glu Ala Val Lys Glu Ser Asp Leu Val Ile Gly Ala Val Leu Ile

225 230 235 240

Pro Gly Ala Lys Ala Pro Lys Leu Val Thr Glu Glu Met Ile Lys Ser

245 250 255

Met Thr Pro Gly Ser Val Val Val Asp Val Ala Ile Asp Gln Gly Gly

260 265 270

Ile Ile Glu Thr Val Asp Gln Ile Thr Thr His Asp Asn Pro Thr Tyr

275 280 285

Thr Lys His Gly Val Val His Tyr Ala Val Ala Asn Met Pro Gly Ala

290 295 300

Val Pro Arg Thr Ser Thr Ile Gly Leu Thr Asn Val Thr Ile Pro Tyr

305 310 315 320

Ala Met Gln Ile Ala Asn Lys Gly Val Glu Lys Ala Val Ala Glu Asn

325 330 335

Pro Ala Leu Ala Leu Gly Val Asn Val Ala Asn Gly Asp Val Thr Tyr

340 345 350

Asn Ala Val Ala Arg Asp Leu Gly Tyr Glu Leu Val Ser Val Glu Asp

355 360 365

Ala Leu Lys Lys Thr Pro Val Gln Ala Leu Glu His His His His His

370 375 380

His

385

<210> 2

<211> 1155

<212> DNA

＜213〉false bacillus firmus (Bacillus pseudofirmus)

<400> 2

atgattatcg gtattccaaa ggaaattaaa aataatgaaa accgcgtagc aattacacca 60

gcaggagttg ttgctttaac aaaagcaggc caccaaattc taatcgaaca aggcgctgga 120

attggcagcg gatttgaaga tgtagattac acagctgctg gagcaacaat tattccagaa 180

gcgaaagatg tatgggctaa agctgaaatg gtgatggcag ttaaagaacc attaagctct 240

gagtacggct acttccgcaa aggattaatc ctattcacat accttcacct agctgctgag 300

cctgaacttg caaaagcact agtagacagc ggcgttattg cgatcgctta tgaaacagtt 360

gaagtaaacc gcactcttcc tcttttaact cctatgagtg aagtggctgg acgcatggca 420

tcacaaattg gtgctcaatt cctagagaag tctaaaggcg gaaaaggaat tctattatca 480

ggtgttcctg gagttaaacg tggtaaagta acaatcatcg gcggcggtgt tgttggtaca 540

aacgcagcta aaattgctgt tggccttggt gctgatgtaa cacttatcga cttaagtgca 600

gatcgtcttc gccagcttga tgatcaattt ggaaacgata ttcaaacact tatgtctaac 660

ccgcttaaca ttgctgaggc agtaaaagaa tctgacttag taatcggtgc tgtattaatt 720

cctggtgcaa aagctcctaa gcttgtaaca gaggaaatga tcaaatctat gactcctgga 780

tcggttgttg ttgacgtagc gattgaccaa ggcggtatca ttgaaacagt tgatcaaatt 840

acaacacatg ataacccaac gtatacaaaa cacggtgttg ttcactatgc agttgctaac 900

atgcctggag ctgttccgcg cacatcaaca atcggcttaa caaacgtaac aattccttac 960

gctatgcaga ttgctaacaa aggcgtagaa aaagctgttg ctgagaaccc tgcacttgct 1020

cttggtgtaa acgttgcaaa cggtgatgta acatacaacg ctgtagcacg tgatcttgga 1080

tatgagttag tatctgtaga agatgcatta aagaaaacac ctgttcaagc actcgagcac 1140

caccaccacc accac 1155

Claims (5)

1. the mutant enzyme albumen of an ALANINE desaturase is characterized in that its aminoacid sequence is shown in SEQ ID No.1.

2. the gene of the described mutant enzyme albumen of the claim 1 of encoding is characterized in that its nucleotide sequence is shown in SEQ ID No.2.

3. method for preparing the described mutant enzyme albumen of claim 1 is characterized in that may further comprise the steps:

(1) according to the aminoacid sequence of disclosed ALANINE desaturase from false bacillus firmus in the ncbi database, determines the mutational site having resolved the basis that the secondary structure sequence of homologous protein compares with structure; The mutant primer pair of design rite-directed mutagenesis; Described mutant primer to be 5 '-TCTTTAACTGCCATCACCAT-3 ' and 5 '-ATGGTGATGGCAGTTAAAGA-3 ';

(2) take the carrier that carries the ALANINE dehydrogenase gene as template, use the primer of above-mentioned design, utilize the fast PCR mutating technology, make up the mutant plasmid of 73 amino acids sudden change;

(3) the said mutation Plasmid Transformation can be expressed in the engineering bacteria of goal gene, express this mutant enzyme albumen with copying of engineering bacteria.

4. method according to claim 3 is characterized in that, carrier is any one in the pET series in the step (2).

5. method according to claim 3 is characterized in that, engineering bacteria is BL21 (DE3) in the step (3).

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