CN110438039B

CN110438039B - Strain for alanine fermentation

Info

Publication number: CN110438039B
Application number: CN201910654393.0A
Authority: CN
Inventors: 李国栋; 杨佳丽; 曹华杰; 宋书怡; 袁保梅; 邱建平
Original assignee: HENAN JULONG BIO-ENGINEERING CO LTD; Zhengzhou University
Current assignee: HENAN JULONG BIO-ENGINEERING CO LTD; Zhengzhou University
Priority date: 2019-07-19
Filing date: 2019-07-19
Publication date: 2022-01-28
Anticipated expiration: 2039-07-19
Also published as: CN110438039A

Abstract

The application belongs to the technical field of alanine fermentation, and particularly relates to a bacterial strain for alanine fermentation. The strain is Escherichia coliEscherichia coliNamed as: escherichia coli HNZZ 3728; the strain preservation number is as follows: CGMCC NO. 17166. In the application, through the primary optimization of the L-alanine producing fermentation capacity of the strain obtained by screening, a primary result shows that the L-alanine producing capacity of the strain can reach 45.8g/L, and although the actual yield is not as high as that of a part of genetically modified strain, the actual yield is higher than that of other existing strains after the strain is further modified or the fermentation process is further optimized because the strain is the originally screened strain, so that the strain has better protection and application values.

Description

Strain for alanine fermentation

Technical Field

The application belongs to the technical field of alanine fermentation, and particularly relates to a bacterial strain for alanine fermentation.

Background

Alanine (L-alanine) is not only one of 20 amino acids constituting human proteins, but also has wide applications in the industries of food, medicine, daily chemicals, and the like. For example: alanine (L-alanine) has both sweetness and freshness, is easily absorbed by human body, can play the roles of improving taste and increasing freshness only by adding trace amount in food, and also has the function of improving the nutritive value of food, so that the alanine (L-alanine) is widely used in bread, cakes, dairy products and beverages; in the pharmaceutical industry, alanine is used for the synthesis of B6 and aminopropanol; in the daily chemical industry, alanine is used for synthesizing and preparing a surfactant with high biocompatibility, and has the advantage of no stimulation to skin, so that the application is wide.

In view of the numerous effects of alanine, there is a great market demand. In the prior art, the production and preparation of alanine have various process routes such as a chemical synthesis method, a protein hydrolysis method, a biological fermentation enzyme catalysis method and the like. The chemical synthesis method has the defects of long synthesis route, low yield, high cost, serious environmental pollution and the like, so the application is less; the protein hydrolysis method has the advantages of various raw materials, low price, simple process and the like, but is also applied by teachers due to the defects of high cost, low yield, incapability of meeting the large-scale requirement and the like. The biological fermentation enzyme catalysis method is the method with the widest industrial application at present because of the advantages of mild reaction conditions, no pollution to the environment, low cost and the like.

The main technical principle of the biological fermentation enzyme catalysis method is as follows: in the microbial growth and fermentation process, the raw material L-aspartic acid can be further decarboxylated under the catalysis of L-aspartic acid alpha-decarboxylase to generate; therefore, the screening of the high-efficiency L-aspartate beta-decarboxylase strain is the basic premise for further improvement and promotion of the technology.

Disclosure of Invention

The application aims to provide a strain for alanine fermentation, thereby laying a certain technical foundation for the fermentation preparation of alanine.

The technical solution adopted in the present application is detailed as follows.

A bacterial strain for fermenting alanine is Escherichia coli (E.coli)Escherichia coli) For convenience of recording and management, the inventor names the codes as: escherichia coli HNZZ 3728; the morphological characteristics of the strain are as follows: the bacterial colony is milky white and round, the edge of the bacterial colony is neat, the surface is smooth, and the bacterial colony is wet and viscous; after the staining microscopic examination, the strain is straight rod-shaped, has no spores and periphytic flagella, and is gram-negative;

the strain is deposited by the inventor in 2019 at 11/1 month, and the preservation addresses are as follows: the microbial research institute of China academy of sciences, No. 3 of Xilu No.1 of Beijing, Chaoyang, China Committee for culture Collection of microorganisms, general microbiological center (CGMCC), the preservation number of the strain is as follows: CGMCC NO. 17166.

The application of the strain for alanine fermentation in the preparation of L-alanine is used for preparing and producing the L-alanine by a biological fermentation method.

The fermentation culture medium for producing L-alanine, which is suitable for the alanine fermentation strain, is a liquid culture medium and comprises the following components: in each liter of culture medium, 20g of glucose, 1.0g of yeast powder, 1.0g of magnesium sulfate, 5.0g of monopotassium phosphate, 25.0g of corn steep liquor, 1.0g of betaine, 1.0g of sodium chloride and 200g of L-aspartic acid.

The L-alanine fermentation production method using the alanine fermentation strain comprises the following steps:

(1) preparation of seed culture solution

Inoculating the strain slant to a seed culture medium (specifically, for example, LB culture medium) and performing shake culture at constant temperature of 280r/min and 35-37 ℃ for 12-24 h (specifically, for example, 16 h);

(2) fermentation culture

Inoculating into culture solution of fermenter according to 10% (volume ratio), fermenting at 37 deg.C for 24 hr at 280 r/min.

In the prior art, in order to improve the yield of L-alanine, a plurality of aspects such as fermentation substrates, fermentation conditions and the like are researched and optimized, wherein the genome of the existing strain is typically modified so as to further improve the L-alanine production capacity by fermentation. However, strictly speaking, the screening of the original strain for obtaining high-yield L-alanine is still the basis for all strain modifications, namely: only on the basis of the level of the original strain with the capability of producing L-alanine at a high yield, the better capability of producing L-alanine can be obtained by further modification of the strain genome.

In the application, through the primary optimization of the L-alanine producing fermentation capacity of the strain obtained by screening, a primary result shows that the L-alanine producing capacity of the strain can reach 45.8g/L, and although the actual yield is not as high as that of a part of genetically modified strain, the actual yield is higher than that of other existing strains after the strain is further modified or the fermentation process is further optimized because the strain is the originally screened strain, so that the strain has better protection and application values.

Drawings

FIG. 1 shows colony morphology and under-mirror morphology of bacteria;

FIG. 2 shows the result of 16S rDNA electrophoresis, in which

lanes

1 and 2 are the strains to be identified;

figure 3 is a sequence BLAST alignment.

Detailed Description

The present application is further illustrated by the following examples. Before describing the specific embodiments, a brief description will be given of some experimental background cases in the following embodiments.

Type of medium:

the culture medium types and basic formulations referred to in the following examples are as follows (in the preparation process, reference is made to conventional preparation and sterilization operations in the prior art):

LB culture medium, tryptone 10.0 g, yeast extract 5.0g, NaCl 5.0g, distilled water 1000 mL, pH7.2;

seed culture medium: 20g of glucose, 1.0g of yeast powder, 1.0g of magnesium sulfate, 5.0g of monopotassium phosphate, 200g of L-aspartic acid and 1000 mL of distilled water, wherein the pH value is 7.2;

fermentation medium: 20g of glucose, 1.0g of yeast powder, 1.0g of magnesium sulfate, 5.0g of monopotassium phosphate, 25.0g of corn steep liquor, 1.0g of sodium chloride, 1.0g of betaine, 200g of L-aspartic acid and 1000 mL of distilled water, wherein the pH value is 7.2;

method for determining L-alanine in the experiment:

the L-alanine content of the sample is quantitatively detected by derivation of Phenylisothiocyanate (PITC), and specifically:

diluting the sample by a proper time, and filtering the sample by a 0.22 mu m filter membrane;

and (3) derivatization treatment: after filtration, the sample solution is 400 mu L + 0.5 g/L norleucine solution 20 mu L + 0.1 mol/L PITC-acetonitrile solution 200 mu L + 1mol/L triethylamine-acetonitrile solution 200 mu L, and after vortex oscillation and uniform mixing, the sample solution is placed at room temperature for 60 min;

then adding 800 mu L of n-hexane, carrying out vortex oscillation for 1min, and standing for 10 min;

sucking the lower layer solution by using an injector, filtering by using a filter membrane of 0.22 m, and then carrying out HPLC detection;

during HPLC detection, specific parameters are set as follows:

the column was ZORBAX SB-C18 (5 μm, 4.6 mm. times.150 mm);

setting the wavelength of the ultraviolet detector to be 254 nm;

the column temperature is 38 ℃, the flow rate is set to be 0.6 mL/min, and the sample injection amount is 10 mu L;

the mobile phase A is 0.1 mol/L ammonium acetate-acetonitrile (volume ratio 97: 3) solution (pH 6.5), and the mobile phase B is acetonitrile solution;

the elution procedure was: the proportion of the mobile phase B is 18% in 0-8 min, and the proportion of the mobile phase B is increased to 80% from 18% in 8-16 min in a gradient manner.

Example 1

It should be noted that the strain obtained in this example is obtained by screening from natural soil by a conventional screening method, and therefore the screening process of this strain is briefly described below in this example.

Sample source: the soil sample is collected from surface layer (0-20 cm) fresh soil rich in neutral and slightly alkaline protein waste in Zhengzhou college, and after the collection is finished, the collected soil sample is placed in a closed sterile plastic bag to be stored at 4 ℃, and flora separation is carried out within 1 week.

(I) preliminary screening of strains

Suspending 1g of soil sample in sterile distilled water, sterilizing ddH₂O is prepared into 1% (W/V) suspension; coating the diluent on a culture medium flat plate by using a coating rod for culturing, and selecting and separating after independent single bacteria grow on the flat plate; specific examples thereof include:

get it 10^-3Inoculating 100 μ L of the diluted solution into 100 mL of beef extract peptone culture solution for enrichment culture for 24h, and then taking 10% of the extract after enrichment culture^-4The diluted solution (100 μ L) was applied to beef extract peptone plate at 30 deg.CAfter the culture and separation of single bacterium, streaking, separating and purifying are carried out, and the culture condition is observed and determined.

And finally, selecting a required bacterial colony according to the bacterial colony morphology, inoculating the bacterial colony in a 10ml LB liquid culture tube, culturing for 24 hours by shaking, taking a supernatant after centrifugal precipitation, detecting the content of L-alanine by using a high performance liquid chromatograph, and primarily obtaining 46 wild strains with higher content of L-alanine, wherein partial results are shown in Table 1.

TABLE 1 amount of alanine produced by part of the prescreened strains (g. L)^-1）

。

Second, re-screening of bacterial strains

Performing multi-round re-screening on 46 single colonies obtained by primary screening, and taking the strain with the highest alanine yield capability as a final target strain; the specific re-screening process comprises the following steps:

respectively inoculating the wild type strains obtained by primary screening into 50ml of LB liquid culture medium for amplification culture for 12 h;

after the culture is finished, inoculating the seed solution into 50mL of fermentation liquor (20 g of glucose, 1.0g of yeast powder, 1.0g of magnesium sulfate, 5.0g of monopotassium phosphate, 25.0g of corn steep liquor, 1.0g of betaine, 1.0g of sodium chloride and 200g of L-aspartic acid) according to the amount of 10%, shaking the flask for 24 hours at 37 ℃, centrifuging and taking the supernatant to determine the content of the L-alanine.

TABLE 2 alanine production (g. L) by partial rescreening of the strains^-1）

。

From the rescreening results above, it can be seen that alanine production was still highest for strain No. 20, and strain No. 20 was therefore selected for further strain identification and subsequent optimization.

(III) morphological and physiological and biochemical characterization

Streaking the final screened and determined strain No. 20 on a blood plate cultureCulturing in culture medium at 37 deg.C for 1-2 days, observing single colony morphology, observing cell morphology of strain under electron microscope, and performing physiological and biochemical tests (mainly including acid production and gas production by glucose fermentation, lactose fermentation test, methyl red test, V-P test, indole production, puncture culture, and H production) according to literature methods such as Bergey's Manual of bacteria identification (eighth edition) and Industrial microorganism experiment technical Manual₂S experiment, and the like, and the measurement methods thereof can be determined according to the conventional methods shown in the prior documents).

The bacterial colony morphology and the under-lens morphology are shown in FIG. 1. Generally, the colony of the separated strain on a culture medium is milky white and round, the edge of the colony is neat, the surface is smooth, and the colony is wet and viscous; after the staining microscopy, the strain is straight rod-shaped, has no spores, is periphytic flagellum and is gram-negative.

The specific physiological and biochemical characteristics are identified as shown in the following table 3:

TABLE 3 physiological and biochemical identification of bacteria

。

(IV) 16S rDNA identification

The 16S rDNA identification refers to species identification of bacteria by a method of sequencing a 16S rDNA sequence of the bacteria; the specific identification process comprises the steps of bacterial genome DNA extraction, 16S rDNA specific primer PCR amplification, amplification product purification, DNA sequencing, sequence comparison and the like. The specific identification process is described below.

(1) Bacterial colony culture

3ml of LB medium was added to a sterile tube, and a single colony was picked up and added to the medium, and shaken overnight at 37 ℃ and 200 rpm.

(2) Genomic DNA extraction

Taking 1ml of the bacterial liquid obtained in the step (1) to a centrifugal tube, centrifuging for 2min at 12000g, and removing the supernatant;

adding 500 mu L of SLA, and fully and uniformly mixing; sucking the mixed solution into a Spin column tube, centrifuging at 12000g for 1min, and removing the filtrate;

adding 600 mu L of Wash Buffer, centrifuging for 1min at 12000g, and removing the filtrate;

taking out Spin column, and putting the Spin column into a new 1.5ml centrifuge tube; 50. mu.L of sterile water was aspirated, and the mixture was centrifuged at 12000g for 1min, and the filtrate was collected to obtain genomic DNA.

(3) PCR amplification

During PCR amplification, the primer sequence design is shown as SEQ ID NO. 1-2, and specifically comprises the following steps:

27F：5’-AGAGTTTGATCCTGGCTCAG-3’，

1492R：5’-GGTTACCTTGTTACGACTT-3’；

during PCR amplification, a 40 mu L amplification system is adopted;

the PCR amplification procedure was: 94 ℃ for 5 min; 94 ℃, 30s, 51 ℃, 30s, 72 ℃, 60s, 20 cycles; 72 ℃ for 10 min;

the PCR amplification products were subjected to 1% agarose gel detection (MarkerDL 2000 as molecular weight standard).

(4) Sequencing alignment

And (4) recovering the electrophoresis band in the step (3), sequencing, and performing Blast comparison on NCBI according to a sequencing result.

The electrophoresis results are shown in FIG. 2. About 1 specific band of 1500 bp can be seen. Based on the sequencing and alignment results (sequence BLAST alignment shown in FIG. 3), the homology between the obtained sequence and the 16S rDNA gene of 11 bacteria was as high as 99%. It was preliminarily determined that the strain to which the sequence belongs may be a member of the above genus. Further, the results of the physiological and biochemical identification were combined, and the strain to be identified was determined to be of the e.coli species (further analysis revealed that homology with the 16S rRNA gene sequence of known e.coli was 99.99%).

The specific sequencing results are illustrated below.

The sequence is as follows:

GGCCGTGCGGCAGCTACACATGCAGTCGAACGGTAACAGGAAGCAGCTTGCTTCTTTGCTGACGAGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCTCTTGCCATCGGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGGAGTAAAGTTAATACCTTTGCTCATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTTGTTAAGTCAGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCTGATACTGGCAAGCTTGAGTCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTTGGAGGTTGTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCTTGACATCCACGGAGTTTTCAGAGATGAGATGTGCTTCGGACGTGAGAACAGGTGCTGCATGCTGTCGTCAGCTCGTGTGTGAAATGTGGGTAGTCCGCACCGAGCGCACCTATCTTGTGCCAGCGTCCGCGAACTCAGGAACTGCATGATACTGAGAGGTGGGGATGACGTCAGTCACCCGGAAAAAAGTGGTAGCGCCCTCCCGAAGGTTAAGCTACCTACTTCTTTTGCAACCCACTCCC。

the sequence is as follows:

ATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGGCATTCTGATCCACGATTACTAGCGATTCCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTACGACGCACTTTATGAGGTCCGCTTGCTCTCGCGAGGTCGCTTCTCTTTGTATGCGCCATTGTAGCACGTGTGTAGCCCTGGTCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCAGTTTATCACTGGCAGTCTCCTTTGAGTTCCCGGCCGGACCGCTGGCAACAAAGGATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATTTCACAACACGAGCTGACGACAGCCATGCAGCACCTGTCTCACGGTTCCCGAAGGCACATTCTCATCTCTGAAAACTTCCGTGGATGTCAAGACCAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGACTTAACGCGTTAGCTCCGGAAGCCACGCCTCAAGGGCACAACCTCCAAGTCGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTGAGCGTCAGTCTTCGTCCAGGGGGCCGCCTTCGCCACCGGTATTCCTCCAGATCTCTACGCATTTCACCGCTACACCTGGAATTCTACCCCCCTCTACGAGACTCAAGCTTGCCAGTATCAGATGCAGTTCCCAGGTTGAGCCCGGGGATTTCACATCTGACTTAACAAACCGCCTGCGTGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCCGTATTACGCGGCTGCTGGCACGGAGTTAGCCGTGCTTCTTCTGCGGTAACGTCATGAGCAAAGTATTACTTACTCCTTCTCCCGCTGAAAGTACTTACACCCGAGCTCTCATACACGCGCATGCTGCATCAAGCTTGCGCCCATTGGTGGCA。

example 2

On the basis of example 1, the inventors have optimized specific fermentation conditions of the strain and examined the application conditions such as mass production ability and genetic stability. The specific process is briefly described as follows.

Optimization of fermentation process

On the basis of the liquid medium used in example 1 (1L of the medium, 20g of glucose, 1.0g of yeast powder, 1.0g of magnesium sulfate, 5.0g of monopotassium phosphate, 25.0g of corn steep liquor, 1.0g of betaine, 1.0g of sodium chloride, and 200g of L-aspartic acid), the inventors selected optimum production conditions for the fermentation temperature and the pH of the medium, respectively.

(1) Optimal fermentation temperature determination

The culture solution of the strain No. 20 after amplification culture is inoculated into 3L fermentation culture solution (5L fermentation tank, initial ph of fermentation liquid = 6.5) according to the proportion of 10%, five different fermentation temperatures of 33 ℃, 35 ℃, 37 ℃, 39 ℃ and 41 ℃ are respectively set, and the influence of the temperature on the growth of microorganisms and the alanine production amount is researched.

The alanine yield under different fermentation temperature conditions was determined using the alanine content in the fermentation broth after 24h fermentation at 250 r/min as an evaluation index, and the results are shown in table 4 below.

TABLE 4 influence of different temperatures on alanine production (pH 6.5 of the initial fermentation broth)

。

As can be seen from the results in the above table, alanine is produced by transformation at 37 ℃ which is more suitable for the metabolism of the strain.

(2) Initial optimal pH determination of the culture Medium

Based on the determination of the optimal fermentation temperature, the pH value of the initial fermentation broth of the culture medium is respectively adjusted to 5.5, 6.0, 6.5, 7.0, 7.5, 8.0 and 8.5 by using dilute hydrochloric acid and sodium hydroxide at 37 ℃ as the fermentation temperature.

The yield of alanine in the fermentation broth after 24h of shake flask (250 r/min) fermentation culture was used as an evaluation index to determine the yield of alanine in the initial fermentation broth under different pH conditions, and the results are shown in Table 5 below.

TABLE 5 influence of different pH on alanine production (fermentation temperature 37 ℃ C.)

。

From the above results, it can be seen that the optimal fermentation temperature of 37 ℃ is most favorable for the fermentative conversion of the strain to produce alanine when the initial fermentation broth ph = 6.5.

(3) Genetic stability

To determine the genetic stability of the strain, the inventors further performed continuous 10 subcultures of the strain on a slant culture medium, followed by shake flask fermentation culture (at 37 ℃, pH6.5, and 250 r/min for 24 h) for each generation, and determined the alanine production and evaluated the genetic stability of the strain. The results of alanine fermentation production of the partial generation lines are shown in Table 6 below.

TABLE 6 variation in the amount of alanine produced by strains of different passage numbers (37 ℃ C., pH 6.5)

。

From the results of the above table, it can be seen that: the strain can keep very stable production performance, and is a wild high-yield strain meeting the industrial production requirements. Based on the characteristics, the strain can be further used as a template for engineering so as to further obtain an engineered strain with high L-alanine yield.

(4) Large-scale fermentation verification

In the experimental process, as small-scale verification experiments are carried out, in order to further determine the industrial production capacity of the alanine, the inventor further carries out 30L large-scale fermentation verification to investigate the actual industrial yield performance of the alanine, and the specific process is as follows:

inoculating the strain slant to a seed culture medium (20 g of glucose, 1.0g of yeast powder, 1.0g of magnesium sulfate, 5.0g of monopotassium phosphate, 200g of L-aspartic acid, 1000 mL of distilled water and pH 7.2), and carrying out shake culture at a constant temperature of 280r/min for 16 hours;

then inoculating the strain into 30L of culture solution (namely, fermentation medium) according to the inoculation amount (volume ratio) of 10% (50L fermentation tank, liquid filling 30L), at 37 ℃, 280r/min, and controlling the tank pressure at 0.04 +/-0.01 MPa; the air flow is controlled at 80 m³And h, controlling the whole dissolved oxygen to be more than 30%, fermenting for 24h, putting the fermented product into a tank, and sampling to determine the alanine content.

The determination result shows that the amount of the L-alanine is 45.8g/L, which is equivalent to the yield under the condition of small-scale experimental verification, and further shows that the strain is a wild strain with higher yield and better industrial application prospect.

SEQUENCE LISTING

<110> Zhengzhou university

<120> a strain for alanine fermentation

<130> none

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 20

<212> DNA

<213> Artificial design

<400> 1

agagtttgat cctggctcag 20

<210> 2

<211> 19

<212> DNA

<213> Artificial design

<400> 2

ggttaccttg ttacgactt 19

Claims

1. A bacterial strain for alanine fermentation is characterized in that the bacterial strain is Escherichia coli (E.coli)Escherichia coli) The inventors coded itIs named as: escherichia coli HNZZ 3728; the strain is deposited by the inventor in 2019 at 11/1 month, and the preservation addresses are as follows: the microbial research institute of China academy of sciences, No. 3 of Xilu No.1 of Beijing, Chaoyang, China Committee for culture Collection of microorganisms, CGMCC, the strain preservation number is: CGMCC No. 17166.

2. The use of the alanine fermentation strain of claim 1 for the production of L-alanine by the biological fermentation method.

3. A process for fermentative production of L-alanine using the alanine fermentation strain according to claim 1, comprising the steps of:

(1) preparation of seed culture solution

Activating an Escherichia coli HNZZ3728 strain, inoculating the activated Escherichia coli HNZZ3728 strain in a seed culture medium, and performing shake culture at 35-37 ℃ for 12-24 h to prepare a seed solution;

the Escherichia coli HNZZ3728 has the strain preservation number as follows: CGMCC No. 17166;

(2) fermentation culture

Inoculating the seed solution in the step (1) into a fermentation culture medium, and performing shake fermentation culture for 16-48 h at 33-41 ℃.

4. The process for the fermentative production of L-alanine according to claim 3, wherein in step (2), the fermentation medium is a liquid medium comprising: in each liter of culture medium, 20g of glucose, 1.0g of yeast powder, 1.0g of magnesium sulfate, 5.0g of monopotassium phosphate, 25.0g of corn steep liquor, 1.0g of betaine, 1.0g of sodium chloride and 200g of L-aspartic acid;

the ph of the fermentation medium is = 5.5-7.5.

5. The process for the fermentative production of L-alanine according to claim 4, wherein the fermentation medium has a ph of = 6.5.

6. The fermentation production method of L-alanine as claimed in claim 3, wherein in the step (2), the fermentation temperature is 37 ℃, and the fermentation culture is carried out for 24h at 280 r/min.