CN113106082B

CN113106082B - Animal waste metagenome-derived alanine racemase and preparation and application thereof

Info

Publication number: CN113106082B
Application number: CN202110584174.7A
Authority: CN
Inventors: 许波; 杨金茹; 黄遵锡; 韩楠玉
Original assignee: Yunnan Normal University
Current assignee: Yunnan Normal University
Priority date: 2021-05-27
Filing date: 2021-05-27
Publication date: 2022-11-04
Anticipated expiration: 2041-05-27
Also published as: CN113106082A

Abstract

The invention discloses an alanine racemase from animal waste metagenome, and preparation and application thereof, wherein the amino acid sequence is shown as SEQ ID NO.2, and after the alanine racemase is treated by a buffer solution with the pH of 9.0-12.0 for 1h, the residual enzyme activity is more than 90%; the enzyme is endured for 1 hour at the temperature of 30 ℃,37 ℃, 40 ℃ and 45 ℃, and the enzyme activity of 90 percent is still kept; the optimal cofactor concentration is 10 mu mol/L, and the relative activity of 77 percent is still maintained after 10min of reaction without adding exogenous cofactor. K of enzyme under pH =12.0, 40 ℃ NaCl conditions _m And V _max Respectively 14.81mmol/L and 89.06 mu mol/L/min, has thermal stability and alkali resistance, still has higher activity without adding exogenous cofactors, and has potential application value in the aspects of enzyme synthesis of D-amino acid and targets for screening novel antibacterial drugs.

Description

Animal waste metagenome-derived alanine racemase and preparation and application thereof

Technical Field

The invention relates to alanine racemase, and in particular relates to alanine racemase from animal manure metagenome, and preparation and application thereof.

Background

Alanine racemase (alnine racemase, ALR, ec 5.1.1.1) is an enzyme which takes pyridoxal phosphate (PLP) as a cofactor and catalyzes the interconversion of L-and D-Alanine, mainly exists in prokaryotes and parts of eukaryotes, and is closely related to diseases caused by bacteria. Research has shown that ALR is present in many pathogenic bacteria, such as Mycobacterium tuberculosis (Mycobacterium tuberculosis), streptococcus mutans (Streptococcus mutans), pseudomonas pseudomonads (Bacillus pseudobacterius), and the like. The activity of ALR determines the amount of D-alanine in bacteria, namely, the synthesis of bacterial cell walls is controlled, and the survival of pathogenic bacteria is further influenced, so that ALR becomes an important target for screening novel antibacterial drugs.

In addition, ALR is one of key enzymes for synthesizing D-amino acid by a biological enzyme method, and a racemization product D-amino acid is used as a sweetening agent, a humectant and a drug synthesis intermediate, and has wide application in the fields of medicines, foods, cosmetics and the like. Researches find that the efficiency of producing D-amino acid by using the multi-enzyme coupling reaction is high, the selectivity is good, and the limitations and the defects of higher cost of a chemical resolution method and environmental pollution caused by an organic solvent can be overcome.

However, ALR has not achieved great success in the above-mentioned fields in recent years. On the one hand, many of the inhibitors currently discovered, such as O-carbamoyl-D-serine, D-cycloserine, β -trifluroalanine, etc., are structural analogs of alanine, interfere with the catalytic process of the enzyme by interacting with the enzyme cofactor PLP, lack target specificity, inactivate other unrelated PLP-dependent enzymes, cause cytotoxicity, and thus are greatly limited in clinical applications. On the other hand, the factors of low catalytic activity, poor stability and the like of microbial enzyme protein limit the large-scale popularization and application of D-amino acid synthesis by a biological enzyme method. Therefore, more ALRs are obtained from different environmental microorganisms, and the method has important significance for screening ALR inhibitors and synthesizing D-amino acid by an enzyme method.

At present, the research on alanine racemase mainly comprises the steps of directly screening strains for producing the alanine racemase from environmental samples, extracting single-strain genome DNA, designing degenerate primers to clone alanine racemase genes, but most microorganisms in the environment are non-culturable microorganisms, so that the screening universality, effectiveness and safety of the novel ALR by the traditional separation and culture method are greatly limited.

Disclosure of Invention

The alanine racemase is obtained by using a metagenome technology, has good thermal stability and alkali resistance, and has potential application value in the aspects of enzymatic synthesis of D-amino acid, screening targets of novel antibacterial drugs and the like.

In order to achieve the aim, the invention provides the alanine racemase from animal manure metagenome, and the amino acid sequence of the alanine racemase is shown as SEQ ID NO. 2.

Another purpose of the invention is to provide the coding gene of the alanine racemase, and the nucleotide sequence of the coding gene of the alanine racemase is shown as SEQ ID NO. 1.

Another object of the present invention is to provide a recombinant vector comprising said encoding gene.

Preferably, the vector used is plasmid pEASY-E2.

Another object of the present invention is to provide a recombinant bacterium comprising the encoding gene.

Preferably, the bacterium used is Escherichia coli BL21 (DE 3).

Another object of the present invention is to provide a method for preparing the alanine racemase, the method comprising:

transforming a recombinant expression vector containing the coding gene of the alanine racemase into a host cell to obtain a recombinant strain, culturing the recombinant strain, and inducing the expression of the recombinant alanine racemase; recovering and purifying the expressed alanine racemase to obtain the alanine racemase.

Preferably, the vector used is plasmid pEASY-E2; the strain used is Escherichia coli BL21 (DE 3).

Preferably, the coding gene of the alanine racemase is amplified by PCR by using metagenome DNA as a template and primers with nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO. 4.

The invention also aims to provide the application of the alanine racemase in preparing D-alanine or in antibacterial drug targets.

The alanine racemase from the animal manure metagenome source, and the preparation and the application thereof have the following advantages:

the invention utilizes the metagenome technology to directly screen the ALR gene from the Western black-crown ape excrement microorganism metagenome, designs a primer to amplify the ALR gene segment, and performs heterologous expression in escherichia coli. Metagenome avoids the problem of microorganism separation culture, directly sequences all microorganisms in an environmental sample, and greatly expands the utilization space of microorganism resources.

The optimum pH of the alanine racemase is 12.0; after being treated by buffer solution with pH of 9.0-12.0 for 1 hour, the residual enzyme activity is more than 90 percent; the optimal temperature is 40 ℃; the enzyme is endured for 1 hour at the temperature of 30 ℃,37 ℃, 40 ℃ and 45 ℃, and the enzyme activity of 90 percent is still kept; the optimal PLP concentration is 10 mu mol/L, and 77 percent of relative enzyme activity can be exerted after reaction for 10min without adding exogenous PLP; k of the enzyme at pH =12.0, temperature 40 ℃ NaCl _m And V _max Respectively 14.81mmol/L and 89.06 mu mol/L/min; hg is a mercury vapor ²⁺ 、Ag ⁺ 、Zn ²⁺ 、Ni ²⁺ 、Fe ²⁺ 、Pb ²⁺ 、Mg ²⁺ 、Mn ²⁺ And SDS almost or completely inhibit the activity of the recombinase, fe ³⁺ 、Cu ²⁺ Has activating effect on recombinant enzyme, and can increase activity by 0.5-2.6 times. The enzyme activity is reduced along with the increase of NaCl concentration, when the NaCl concentration is 2-4M, the relative enzyme activity is lower than 10%, and the enzyme activity is completely inhibited after the enzyme activity is treated in 0.5-5M NaCl for 1 hour. The properties show that the alanine racemase prepared by the invention has thermal stability and alkali resistance, and has potential application value in the aspects of enzymatic synthesis of D-amino acid, novel antibacterial drug screening targets and the like.

Drawings

FIG. 1 is a SDS-PAGE analysis of recombinant alanine racemase NC ALR1 expressed in E.coli as provided in Experimental example 2 of the present invention.

FIG. 2 is the optimum pH of the recombinant alanine racemase in Experimental example 3 of the present invention.

FIG. 3 is a graph showing the pH stability of recombinant alanine racemase in Experimental example 3 of the present invention.

FIG. 4 is a graph showing the optimum temperature of the recombinant alanine racemase in Experimental example 3 of the present invention.

FIG. 5 is a graph showing the temperature stability of recombinant alanine racemase in Experimental example 3 of the present invention.

FIG. 6 shows the effect of recombinant alanine racemase cofactor in Experimental example 3 of the present invention.

FIG. 7 shows the effect of recombinant alanine racemase NaCl in Experimental example 3 of the present invention.

FIG. 8 shows the NaCl tolerance of the recombinant alanine racemase in Experimental example 3 of the present invention.

FIG. 9 shows the catalytic activity of the recombinant alanine racemase in Experimental example 3 of the present invention on 8L-amino acids.

Note: in fig. 1, M: low molecular weight protein Marker;1: primary enzyme after induction of Escherichia coli containing only pEASY-E2 vector; 2: unpurified recombinant alanine racemase; 3: purified recombinant alanine racemase.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.

Test materials and reagents

1. Sample, strain and carrier: western black-crown ape feces microorganism metagenome DNA and an expression vector pEASY-E2; BL21 (DE 3) was purchased from Kyork Hippon Biotechnology Ltd.

2. Genetically engineered operating enzymes, kits and other biochemical reagents: restriction enzyme, DNA polymerase and ligase were purchased from TaKaRa company, and plasmid extraction kit and gel recovery and purification kit were from Omega company; other reagents are analytically pure.

3. LB medium: 10g of Peptone, 5g of Yeast extract, 10g of NaCl, distilled water was added to 1000mL, and the pH was adjusted to about 7. Solid media 2.0% (w/v) agar was added on the above basis.

Description of the invention: the molecular biological experiments which are not specifically described in the following experimental examples are carried out by referring to the specific methods listed in molecular cloning, a laboratory manual (third edition) J. SammBruke, or according to kits and product instructions.

Experimental example 1 obtaining of alanine racemase Gene NC Alr1

(1) Screening of gibbon ape fecal microorganism metagenome alanine racemase gene

And screening the gene of which the annotation result is alanine racemase from the constructed gibbon ape stool microorganism library according to the gene prediction, functional annotation and secretory protein prediction analysis results, thereby obtaining the alanine racemase gene NC Alr1, wherein the gene sequence is shown as SEQ ID NO. 1.

(2) Cloning of alanine racemase Gene NC Alr1

With NC Alr1-F ₁ :5 'TAAGAAGGAGATATACATGGAATTGATGGATTCAACATTAAAAGCGG-containing 3' (SEQ ID NO. 3) and NC Alr 1-R ₁ :5 'GTGGTGGTGGTG GTGCTCGAGCCCCCTTAAAAAGAAGCTC-3' (SEQ ID NO. 4) is used as a primer pair, and the PCR amplification is carried out by using the western black-crown ape fecal microorganism metagenome DNA as a template.

PCR reaction (20.0. Mu.L): metagenomic library DNA 0.5. Mu.L, primeSTAR Max 10. Mu.L, NCAlr1-F ₁ 0.5μL，NCAlr 1-R ₁ 0.5μL，ddH ₂ O make up to 20.0. Mu.L.

The PCR reaction parameters are as follows: 10s at 98 ℃; 15s at 55 ℃;72 ℃,1min,30s;30 cycles; 72 ℃ for 10min;4 ℃ for 10min.

The objective gene NCAlr1 was obtained as a result of PCR.

Experimental example 2 preparation of alanine racemase NC ALR1

The alanine racemase gene NCAlr1 prepared in example 1 was ligated with plasmid pEASY-E2 to obtain recombinant expression vector pEASY-E2-NCAlr1, thenThen transforming the Escherichia coli BL21 (DE 3) to obtain a recombinant Escherichia coli strain BL21 (DE 3)/NCAlr 1. Escherichia coli strain BL21 (DE 3)/NC Alr1 containing recombinant expression vector pEasy-E2-NCAlr1 was inoculated in LB (100. Mu.g/mLAmp) culture medium at 0.1% and cultured overnight at 37 ℃ and 180 rpm. Then, the activated bacterial suspension was inoculated into a fresh LB (100. Mu.g/ml Amp) -containing culture medium at an inoculum size of 1%, and cultured at 37 ℃ and 180rpm for about 5 to 6 hours (OD) ₆₀₀ 0.8-1.0), adding IPTG with the final concentration of 0.7mmol/L for induction, and culturing at 16 ℃ and 180rpm for about 16h. Centrifuging at 5000rpm for 10min, and collecting thallus. After suspending the cells in an appropriate amount of Tris-HCl buffer solution with pH =7.0, the cells were sonicated in an ice bath. The above-mentioned concentrated initial enzyme solution in the cells was centrifuged at 12,000rpm at 4 ℃ for 10min, and then the supernatant was aspirated and the target protein was purified by Nickel-NTA Agarose to obtain alanine racemase NCALR1.

The purified target protein was subjected to SDS-PAGE, and the results are shown in FIG. 1, FIG. 1 is a SDS-PAGE analysis of recombinant alanine racemase expressed in E.coli provided by the embodiment of the present invention, wherein, M: a protein Marker;1: primary enzyme after induction of Escherichia coli containing only pEASY-E2 vector; 2: unpurified recombinant alanine racemase 3: purified recombinant alanine racemase. As can be seen from FIG. 1, the recombinant alanine racemase was expressed in E.coli and was a single band after purification with Nickel-NTAAgarose.

Experimental example 3 determination of Properties of alanine racemase NC ALR1

Methods for measuring enzyme activity are referred to Soda K (Determination of D-amino acids and D-amino acid oxidase activity with 3, methyl-2-benzothiazole hydroxide, 1968) and Lida F et al (Electrochemical Study of iodine in the Presence of Phenol and o-Cresol: application to the Catalytic Determination of Phenol and o-Cresol, 2004): the activity of ALR is determined by adopting a two-step method of racemization reaction and oxidation reaction.

Racemization reaction: the reaction system contained 20mmol/L birutan-robinson buffer, 50mmol/L L alanine, and 0-100. Mu. Mol/LPLP (pyridoxal phosphate) in 200. Mu.L. Preheating at optimum temperature for 5min, adding appropriate amount of crude enzyme solution or purified enzyme protein (boiling inactivated enzyme solution as blank control), reacting for 10min, immediately adding 25 μ L of 2mol/L HCl to terminate the reaction, standing on ice for 2min, adding 25 μ L of 2mol/L NaOH solution to neutralize excessive acid, centrifuging at 12000r/min at 4 deg.C for 10min, and transferring the supernatant to new test tube.

And (3) oxidation reaction: 200 μ L of the reaction mixture contained 200mmol/L Tris-HCl (pH = 8.0), 0.1mg/mL 4-aminoantipyrine, 0.1mg/mL sodium salt of ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline (TOOS), 0.1U D-amino acid oxidase, 2U (horseradish) peroxidase and 200 μ L of the racemized reaction product, and was reacted at 37 ℃ for 20min to determine the absorbance at 550 nm.

One enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the formation of 1. Mu. Mol D-Ala in 1 min.

(1) Determination of the optimum pH and pH stability of the alanine racemase NCALR1

Determination of the optimum pH of the enzyme: the alanine racemase NCALR1 purified in example 2 was subjected to an enzymatic reaction at 30 ℃ in a buffer solution of pH 3 to pH 13.

Determination of the pH stability of the enzyme: the purified enzyme solution was placed in a buffer solution with pH =3 to 13, treated at 30 ℃ for 1 hour, and then subjected to an enzymatic reaction, with the untreated enzyme solution as a control.

The buffer solution is as follows: 20mmol/L birutan-robinson buffer (pH = 3-13). Taking L-alanine as a substrate, reacting for 10min, and determining the enzymatic properties of the purified alanine racemase.

Results referring to fig. 2 and 3, fig. 2 is the optimum pH of the alanine racemase provided by the embodiments of the present invention, and fig. 3 is the pH stability of the alanine racemase provided by the embodiments of the present invention. As can be seen from FIGS. 2 and 3, the optimum pH of the alanine racemase provided by the present invention is 12.0; after being treated by buffer solution with pH of 9.0-12.0 for 1 hour, more than 90 percent of enzyme activity remains.

(2) Determination of optimum temperature and temperature stability of alanine racemase

Determination of optimum temperature of enzyme: the enzymatic reaction is carried out at pH =12.0 at 0-70 ℃.

Temperature stability assay of enzymes: the enzyme solution was treated at a predetermined temperature (30, 37, 40, 45 ℃) for 1 hour for the same amount of enzyme, and the enzyme reaction was carried out at pH =12.0 and 40 ℃ every 10 minutes, with the untreated enzyme solution being used as a control.

Referring to fig. 4 and 5, fig. 4 shows the optimal temperature of the alanine racemase according to the embodiment of the present invention, and fig. 5 shows the temperature stability of the alanine racemase according to the embodiment of the present invention. The results show that: the optimal temperature of the alanine racemase is 40 ℃, and the alanine racemase is stable under the conditions of 30 ℃,37 ℃, 40 ℃ and 45 ℃.

(3) Determination of the Effect of the cofactor concentration of alanine racemase

Determination of cofactor concentration of the enzyme: the enzymatic reaction was carried out at 40 ℃, pH =12.0, 0-100 μmol/L PLP.

See figure 6 for results. FIG. 6 is a graph showing the effect of cofactor concentration for alanine racemase provided in the present examples. The results show that: the optimal PLP concentration of the alanine racemase is 10 mu mol/L, and the enzyme activity is kept above 77 percent under the condition of not adding exogenous PLP.

(4) Determination of NaCl Effect and NaCl tolerance of alanine racemase

NaCl effect assay of the enzyme: the enzymatic reaction was carried out at 40 ℃, pH =12.0, 0.5-5 m nacl reaction conditions.

NaCl stability assay of the enzyme: the enzyme solution of the same amount of enzyme was treated for 1 hour under the reaction condition of 0.5 to 5M NaCl, and the enzymatic reaction was carried out at pH =12 and 40 ℃ with the untreated enzyme solution as a control.

Referring to fig. 7 and 8, fig. 7 shows NaCl effect of alanine racemase provided by the embodiment of the present invention, and fig. 8 shows NaCl tolerance of alanine racemase provided by the embodiment of the present invention. The results show that: the enzyme activity is reduced along with the increase of NaCl concentration, and the enzyme activity is lower than 10% when the NaCl concentration is 2-5M. There was little enzymatic activity after 1h of treatment under 0.5-5M NaCl conditions.

(5) Determination of kinetic parameters of recombinant alanine racemase

Kinetic parameters were determined at pH =12.0, temperature 40 ℃ and first order reaction time with different concentrations of L-alanine as substrate (0-50 mM), K was calculated according to the Lineweaver-Burk method _m And V _max The value is obtained. Measured byDetermining the K of the enzyme at pH 12.0 and temperature 40 ℃ _m And V _max Respectively 14.81mmol/L and 89.06. Mu. Mol/L/min.

(6) Determination of influence of different metal ions and chemical reagents on activity of recombinant alanine racemase

A chemical reagent (10 mM final concentration) was added to the enzymatic reaction system, and the effect on the enzyme activity was investigated. The enzyme activity was measured at 40 ℃ and pH =12.0 (enzymatic reaction without addition of metal ions and chemicals was used as a control under the same conditions), and the results are shown in table 1.

Table 1 shows Hg ²⁺ 、Ag ⁺ 、Zn ²⁺ 、Ni ²⁺ 、Fe ²⁺ 、Pb ²⁺ 、Mg ²⁺ 、Mn ²⁺ And SDS almost or completely inhibits the activity of the recombinase, fe ³⁺ 、Cu ²⁺ Activating the recombinant enzyme to increase its activity by 0.5-2.6 times.

TABLE 1 Effect of Metal ions and chemical reagents on the Activity of the recombinase NCALR1

(7) Determination of degradation of different substrates by recombinant alanine racemase

The enzyme activity assay system described above was added with the same concentrations of different substrates at 40 ℃, pH = 12.0: alanine (Ala), serine (Ser), cysteine (Cys), proline (Pro), tyrosine (Tyr), leucine (Leu), glutamic acid (Glu) and aspartic acid (Asp), and the enzyme activity is measured (the enzyme activity taking L-alanine as a substrate is used as a reference), and the result shows that the recombinant alanine racemase does not decompose other substrates.

The results are shown in FIG. 9, the recombinant alanine racemase substrate has stronger specificity, and the catalytic activity to other L-amino acids is lower than 10%.

While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the scope of the invention should be determined from the following claims.

Sequence listing

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<120> animal waste metagenome-derived alanine racemase and preparation and application thereof

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acaacccgga tgggcgtcat tccttacggc tatgcagacg gttttttcag atgcctttcc 900

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gtggaaaagg agcttctttt aagggggtaa 1170

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Leu Ala His Asn Tyr Thr Ile Leu Arg Lys Arg Ile Ser Ala Asp Val

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Glu Tyr Asn Glu Glu Ala Val Arg Cys Gln Gly Thr Leu Lys Val His

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Ile Lys Val Asp Thr Gly Met Ser Arg Leu Gly Phe Leu Cys Asp Gly

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Asp Tyr Phe Glu Asn Gly Val Glu Gly Ile Cys Glu Ala Cys Leu Leu

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Pro Gly Leu Ser Ala Glu Gly Ile Phe Thr His Phe Ala Val Ser Asp

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Glu Phe Ala Arg Glu Leu Asn Leu Gln Pro Val Met Ser Leu Lys Thr

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Thr Val Ser Thr Ile Lys Thr Tyr Pro Ala Gly Thr Ala Val Ser Tyr

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Gly Gly Ile Phe Val Thr Pro Gln Thr Thr Arg Met Gly Val Ile Pro

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Tyr Gly Tyr Ala Asp Gly Phe Phe Arg Cys Leu Ser Asn Lys Cys Ser

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Ala Leu Ala Gly Thr Ile Pro Tyr Glu Leu Thr Cys Ala Val Ser Lys

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gtggtggtgg tggtgctcga gcccccttaa aagaagctc 39

Claims

1. An alanine racemase from animal waste metagenome, which is characterized in that the amino acid sequence of the alanine racemase is shown in SEQ ID NO. 2.

2. The gene encoding alanine racemase as claimed in claim 1, wherein the nucleotide sequence of the gene encoding alanine racemase is shown in SEQ ID No. 1.

3. A recombinant vector comprising the encoding gene of claim 2.

4. The recombinant vector according to claim 3, wherein the vector used is plasmid pEASY-E2.

5. A recombinant bacterium comprising the coding gene according to claim 2.

6. The recombinant bacterium according to claim 5, wherein the bacterium used is Escherichia coli BL21 (DE 3).

7. The method of claim 1, comprising:

transforming a recombinant expression vector containing the gene encoding alanine racemase according to claim 2 into a host cell to obtain a recombinant strain, culturing the recombinant strain, and inducing expression of the recombinant alanine racemase;

recovering and purifying the expressed alanine racemase to obtain the alanine racemase as recited in claim 1.

8. The method according to claim 7, wherein the vector used is plasmid pEASY-E2; the strain used is Escherichia coli BL21 (DE 3).

9. Use of the alanine racemase as claimed in claim 1 in the preparation of D-alanine or in an antibacterial drug target.