CN106884001B

CN106884001B - Recombinant alkalophilic bacillus, preparation method and application thereof, and method for preparing D-lactic acid

Info

Publication number: CN106884001B
Application number: CN201510941227.0A
Authority: CN
Inventors: 薛燕芬; 马化君; 葛德永; 于波; 马延和
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2015-12-16
Filing date: 2015-12-16
Publication date: 2020-04-10
Anticipated expiration: 2035-12-16
Also published as: CN106884001A

Abstract

The invention discloses a preparation method of recombinant alkalophilic bacillus, which comprises the following steps: takes alcalophilus for producing L-lactic acid as an original strain, and the original strain does not express L-lactic dehydrogenase and expresses D-lactic dehydrogenase through genetic engineering operation. The invention also discloses the recombinant alkalophilic bacillus prepared by the method, application thereof and a method for preparing D-lactic acid. When the recombinant alkalophilic bacillus prepared by the preparation method of the recombinant alkalophilic bacillus is used for preparing D-lactic acid, the optical purity of the obtained D-lactic acid is higher than 99.8%, the conversion rate is more than 94%, and the yield can reach 142 g/L.

Description

Recombinant alkalophilic bacillus, preparation method and application thereof, and method for preparing D-lactic acid

Technical Field

The invention belongs to the field of genetic engineering, and particularly relates to a preparation method of recombinant alkalophilic bacillus, the recombinant alkalophilic bacillus prepared by the method and application thereof, and a method for preparing D-lactic acid.

Background

Lactic acid (also known as dihydroxypropionic acid) is one of three major organic acids in the world, can be used as an acidulant, an aromatic, a preservative, a plant growth regulator, a biodegradable material, a drug, a pesticide and the like, and is applied to food, pharmacy, brewing, tanning, textile, environmental protection and agriculture. Lactic acid is a chiral molecule and can be classified as L-lactic acid and D-lactic acid. D-lactic acid is an important chiral compound and is used as an intermediate for synthesizing medicines, high-efficiency low-toxicity pesticides, herbicides and other chiral compounds and a raw material for synthesizing polylactic acid. For example, Hoeehst, germany, developed a new high-efficiency herbicide weiba (Whip Super) using D-lactic acid as a raw material, BASF, germany, produced herbicide Duplosan using isopropyl D-lactic acid as a raw material, and was already put on the market on a large scale. The important application value of D-lactic acid has led to a constant search for technical processes for producing D-lactic acid with high optical purity in high yield, high substrate conversion and high production intensity.

At present, the international D-lactic acid production mainly adopts a microbial fermentation method, the production cost is low, the product safety is high, and the D-lactic acid with high optical purity can be obtained specifically. One of the key factors in the production of D-lactic acid by microbial fermentation is the performance of the strain. The fermentation strains mainly used at present are naturally isolated Lactobacillus (Lactobacillus) and Lactobacillus (Sporolactobacillus). The yield of D-lactic acid produced by fermentation of the bacillus CASD is 207g/L, and the optical purity of the product is 99.3% [ Wang et al.2011, Appl Microbiol Biotechnol.89:1009-17 ]; CN101285047B utilizes the fermentation of the bacillus Y2-8 to produce D-lactic acid, the yield reaches 165g/L, and the optical purity is 99.1%; CN102978134B utilizes lactobacillus DMDL9010 to ferment and produce D-lactic acid, the yield reaches 85g/L, and the optical purity is 90%. In addition, researchers have also adopted genetic engineering breeding strategies to improve the yield, conversion rate and optical purity of the D-lactic acid of the strain, reduce the synthesis of byproducts, and the like. The strains involved include: lactobacillus (Lactobacillus), escherichia coli (e.coli), corynebacterium glutamicum (c.glutamicum), and yeast (Saccharomyces). CN104278003A utilizes engineered recombinant Escherichia coli to ferment and produce D-lactic acid, the yield is 131g/L, and the optical purity of the product reaches more than 99.5%; CN101993850B recombinant Corynebacterium glutamicum (C.glutamicum Res 167. delta. ldh/ldhA) was fermented to produce D-lactic acid with a yield of 40g/L or more and a purity of 99% or more. However, there is still a large gap between the diversity of fermentation strains and the fermentation level in the production of D-lactic acid, and it is extremely valuable to find D-lactic acid-producing bacteria with high yield and high optical purity and production process.

In the lactic acid fermentation process, the acidity of the fermentation liquid is gradually increased due to continuous production of products, so that the growth of the bacteria and the acid production are severely inhibited, and therefore, a neutralizing agent such as calcium carbonate is generally added for neutralization. In addition, during the extraction and purification of lactic acid, sulfuric acid needs to be added to acidify calcium lactate, thereby generating lactic acid and insoluble calcium sulfate waste, which causes serious environmental problems and economic waste. The electrodialysis method developed in recent years is a new technology for extracting and purifying lactic acid. The process can avoid the generation of calcium carbonate and reduce environmental pollution. However, the electrodialysis membranes are paired with divalent ions such as Ca²⁺And Mg²⁺Intolerance, in order to utilize the new environment-friendly and energy-saving technology, alkaline substances of monovalent ions are required to be used as a neutralizing agent in the lactic acid fermentation process, and NaOH is an ideal neutralizing agent. However, when lactic acid fermentation is performed using NaOH as a neutralizing agent, the conventional fermentation strains have difficulty in achieving desired effects and have low yields and optical purities.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a recombinant strain which can obtain high-yield and high-optical-purity D-lactic acid and can tolerate high-concentration monovalent ion alkaline substances and a method for preparing the D-lactic acid by using the strain.

In order to achieve the above object, in a first aspect, the present invention provides a method for producing a recombinant Bacillus alkalophilus, wherein the method comprises: takes alcalophilus for producing L-lactic acid as an original strain, and the original strain does not express L-lactic dehydrogenase but expresses D-lactic dehydrogenase through genetic engineering operation.

In a second aspect, the invention also provides a recombinant alkalophilic bacillus prepared by the preparation method.

In a third aspect, the invention also provides an application of the recombinant alkalophilic bacillus in producing D-lactic acid.

In a fourth aspect, the invention also provides a method for preparing D-lactic acid, wherein the method comprises inoculating the recombinant alkalophilic bacillus of the second aspect into a fermentation medium for fermentation to obtain a fermentation liquor containing D-lactic acid.

The optical purity of the D-lactic acid prepared by the recombinant basophilic bacillus is higher than 99.8%, the conversion rate is more than 94%, and the yield can reach 142 g/L. In addition, in the preferred embodiment of the invention for preparing D-lactic acid by utilizing the recombinant basophilic bacillus, the peanut meal is used as a nitrogen source, and a strategy that fermentation equipment and/or a fermentation culture medium are not sterilized before fermentation is adopted, so that the cost is greatly reduced; meanwhile, the alkaline substance of monovalent ions can be used as a neutralizer, so that the post-treatment process is simplified and the environmental pollution is reduced.

Additional features and advantages of the invention will be set forth in the detailed description which follows.

Biological preservation

The recombinant Bacillus alcalophilus (or Bacillus sp.) provided by the preferred embodiment of the invention has a collection number of CGMCC No.11581, and has been collected in the general microbiological center of China Committee for culture Collection of microorganisms (CGMCC, address: No. 3 of Ministry of microbiology 1 of North Cheng West Lu of Ind-ward, Beijing, and postal code: 100101) 11.5 days in 2015.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:

FIG. 1 is a graph showing the change in the concentrations of glucose and D-lactic acid with fermentation time in example 2 of the present invention;

FIG. 2 is a graph showing the change in the concentrations of glucose and D-lactic acid with the fermentation time in example 3 of the present invention.

Detailed Description

The following describes in detail specific embodiments of the present invention. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.

In a first aspect, the present invention provides a method for preparing a recombinant Bacillus alcalophilus, wherein the method comprises: takes alcalophilus for producing L-lactic acid as a starting strain, and the starting strain does not express L-lactate dehydrogenase (L-LDH) but expresses D-lactate dehydrogenase (D-LDH) through genetic engineering operation.

In the present invention, the starting strain may be various alkaliphilic Bacillus species capable of producing L-lactic acid, wherein the alkaliphilic Bacillus species producing L-lactic acid may be an alkaliphilic Bacillus species expressing only the L-lactate dehydrogenase-encoding gene (L-ldh) to produce L-lactic acid, or a strain expressing both the L-ldh and D-lactate dehydrogenase-encoding genes (D-ldh) to produce L-lactic acid and D-lactic acid. The source of the L-lactic acid-producing Alkaliphilic bacillus is not particularly limited in the present invention and may be selected conventionally in the art, for example, by commercially available sources, and preferably, the L-lactic acid-producing Alkaliphilic bacillus is L-lactic acid-producing Alkaliphilic bacillus N16-5 (accession No. CGMCC NO.0369, published in CN 1266096A).

According to the present invention, in the above preparation method, the manner in which the starting strain does not express L-LDH may be: the L-ldh in the starting strain is mutated or knocked out. Preferably, L-ldh is not expressed at all by knocking out the L-ldh in the starting strain. In a preferred aspect, the sequence of L-ldh is shown as SEQ ID NO 1.

According to the present invention, in the above production method, the manner of allowing D-LDH to be expressed is: the expression vector containing D-LDH was introduced into Bacillus alcalophilus not expressing L-LDH. Preferably, the sequence of D-ldh is shown in GenBank number AAA 25246.

In a preferred embodiment, the method for preparing recombinant basophilic bacillus provided by the invention can specifically comprise the following steps:

(1) constructing L-ldh-deficient alkalophilic Bacillus (Bacillus sp. N16-5 delta ldh);

(2) constructing an expression vector containing D-ldh;

(3) transforming L-ldh-deleted Bacillus alcalophilus (Bacillus sp. N16-5. delta. ldh) with the constructed expression vector containing D-ldh;

(4) positive clones were screened.

Among them, in step (1), L-ldh-deleted Alcaliphilus sp.N16-5. delta. ldh can be constructed by a method conventionally used in the art, for example, by a method of homologous recombination or gene mutation, preferably by a method of homologous recombination.

In step (2), the expression vector may be at least one of a plasmid, a virus, a cosmid, and a phage, and is preferably a plasmid.

In addition, in step (2), a specific manner of constructing the expression vector containing D-ldh may be: designing a primer according to the gene sequence of the D-ldh, and amplifying the D-ldh; in a preferred case, the primer can be designed based on the gene sequence of D-ldh in Lactobacillus delbrueckii or Lactobacillus bulgaricus.

In step (3), the transformation may be carried out by protoplast method, chemical transformation method or electroporation method.

In the step (4), preferably, the step (4) can also comprise verifying the obtained recombinant alkalophilic Bacillus (Bacillus sp.N16-5 delta ldh-pDalac) carrying the exogenous D-ldh by a PCR method.

In a second aspect, the invention also provides a recombinant alkalophilic bacillus prepared by the preparation method. According to a preferred embodiment of the invention, the recombinant Bacillus alcalophilus is a recombinant strain with the preservation number CGMCC No. 11581.

In a fourth aspect, the invention also provides a method for preparing D-lactic acid, wherein the method comprises inoculating the recombinant alkalophilic bacillus into a fermentation medium for fermentation to obtain a fermentation liquid containing the D-lactic acid.

According to the invention, the fermentation medium may contain a carbon source, a nitrogen source and optionally inorganic salts. Preferably, the carbon source is present in the fermentation medium in an amount of 60 to 150 g/l, preferably 70 to 100 g/l. The content of the nitrogen source is preferably 10 to 50 g/l, more preferably 20 to 40 g/l. The content of the inorganic salt is preferably 0 to 5g/l, more preferably 1 to 4 g/l.

Preferably, the fermentation medium may also contain an antibiotic, preferably chloramphenicol, and water. The concentration of antibiotic in the fermentation medium may be 2-4. mu.g/mL.

According to the present invention, the carbon source is not particularly limited and may be conventionally selected in the art, and for example, may be a sugar, preferably a monosaccharide and/or an oligosaccharide, more preferably at least one of glucose, sucrose, fructose, maltose, mannose and galactose, and further preferably glucose and/or fructose.

In the present invention, the nitrogen source is not particularly limited and may be conventionally selected in the art, and for example, may be an inorganic nitrogen source and/or an organic nitrogen source, preferably an organic nitrogen source, and more preferably provided by at least one of peanut meal, peptone, yeast powder and soybean meal. In addition, when an organic nitrogen source is used, in order to hydrolyze large molecular proteins therein into small molecular peptides or amino acids for efficient absorption and utilization by the strain, it is preferable that the organic nitrogen source is subjected to a pretreatment before fermentation, which may include: sterilization pretreatment and enzymatic pretreatment. The mode of the sterilization pretreatment in the present invention is not particularly limited, and may be a conventional one in the art, and for example, a high-temperature sterilization mode may be employed. The enzyme pretreatment may be performed using a neutral protease, and preferably, the conditions of the enzyme pretreatment may include: the pH value is 6.8-7.2, the pretreatment temperature is 40-50 ℃, the pretreatment time is 6-10 hours, and the concentration of neutral protease is 10-50 g/L.

In a preferred aspect, the inorganic salt of the present invention is sodium acetate.

According to the invention, the conditions of the fermentation may include: the fermentation temperature is 35-45 deg.C, the fermentation time is 90-100 hr, the fermentation pH is 9-10, and the carbon source content is controlled at 20-50 g/L during fermentation. Preferably, the fermentation is carried out under stirring conditions, with a stirring speed of 80-120 rpm. Further preferably, aeration treatment is performed before fermentation in such a manner that air is introduced at a flow rate of 0.8 to 1.5 liters/minute 10 to 14 hours before fermentation.

According to the present invention, the pH value of the fermentation may be controlled by adding a neutralizer to the fermentation system, and the neutralizer may include NaOH, ammonia and Na₂CO₃Preferably NaOH and/or Na₂CO₃。

In the present invention, since the carbon source is continuously consumed during the fermentation process to continuously decrease the content thereof, the content of the carbon source during the fermentation process needs to be controlled in order to better ensure the normal growth of the strain. The method for controlling the content of the carbon source in the fermentation process is not particularly limited in the present invention, and can be selected conventionally in the art, for example, a method of supplementing an aqueous solution of a carbon source (such as glucose) can be adopted, and preferably, the concentration of the aqueous solution of the carbon source can be 800-1000 g/L. The number of additions is usually 3 to 5.

According to the invention, the fermentation apparatus and/or the fermentation medium may or may not be sterilized, preferably not, prior to fermentation. The mode of sterilization in the present invention is not particularly limited, and may be selected conventionally in the art, and for example, high temperature sterilization may be used. In a preferred case, the conditions of the high temperature sterilization may include: the sterilization temperature is 110-120 ℃, and the sterilization time is 15-25 minutes. The recombinant basophilic bacillus can prepare D-lactic acid by fermentation without sterilization, and is particularly beneficial to energy consumption saving.

The present invention will be described in detail below by way of examples.

The media formulations used in the following examples are as follows:

SA5 medium (1L): 81g of sodium succinate, 1.21g of Tris Base, 5g of yeast extract, 5g of Casamino acid5g, 5g of glucose and MgCl₂·6H₂O 4.06g，CaCl₂1.39g, NaCl 10g and agar 5%. Preparation of 200mL solid MediumThe method comprises dissolving the components in 190mL, adding 10g agar, sterilizing, adding 10mL 10% glucose solution, cooling at 70 deg.C, and pouring into plate.

NCM medium (1L): 5g of peptone, 2g of yeast extract, 0.34g of citric acid, 5g of glucose and MgSO₄·7H₂O0.05g and NaCl 11.7 g. After sterilization 1/10 final volume of separately sterilized 22.8% K was added₂HPO₄·3H₂And O, and mixing uniformly. The pH value of the culture medium is 7.6-7.8. Solid medium was added with 3% agar.

Seed medium (g/l): glucose 10, yeast powder 5, polypeptone 5, K₂HPO₄·3H₂O1，MgSO₄·7H₂0.2O, 50 NaCl and 900mL distilled water, sterilizing at 115 ℃ for 20 minutes, and adding 10% (w/v) Na sterilized at high temperature₂CO₃100mL。

Fermentation medium (g/l): glucose 80, peanut meal (Beijing Congmingwei culture medium technology, Inc.) 30 and sodium acetate 2.

Example 1

This example illustrates the construction of recombinant Bacillus alcalophilus sp.N16-5 Δ ldh-pDlac provided by the present invention.

1. Knock-out of L-ldh gene in Bacillus alcalophilus sp.N16-5

Designing an upstream primer and a downstream primer of an L-ldh gene (SEQ ID NO:1) according to genome information of Bacillus alcalophilus sp.N16-5, wherein the upstream primer is 5'-TATATAGAAAGGACGATGTAAATGAGTG-3' (SEQ ID NO:2), and the downstream primer is 5'-TCTTATCTTATTTGCCTGATCAAATGCC-3' (SEQ ID NO: 3); the L-ldh gene was amplified by PCR using Bacillus alcalophilus sp.N16-5 genome as a template by high fidelity nucleic acid polymerase Pyrobest (Takara Co.), under the conditions: pre-denaturation at 95 ℃ for 5min, then at 95 ℃ for 30s, 55 ℃ for 30s, and 72 ℃ for 90s for 35 cycles; finally, extension was carried out at 72 ℃ for 7 min. Detecting the size of the target gene by 1% agarose gel electrophoresis, and sending the PCR product to Beijing Optimalaceae gene company for sequencing verification; the purified PCR product was ligated with pMD18T vector using Fast clone Kit (PUEX Co.), the constructed recombinant vector pMD18(Bspn165-ldh) was introduced into E.coli DH5a by chemical transformation, positive clones were obtained by screening of LB plate containing 100. mu.g/mL ampicillin and PCR verification, plasmids were extracted, BamHI and SalI (New England Biolabs) were digested and ligated to shuttle vector pNNB194 to obtain suicide plasmid pNNB-. DELTA.ldh; the suicide plasmid pNNB-delta ldh is transferred into Alkaliphilic Bacillus sp.N16-5 (the preservation number is CGMCC NO.0369 and published in CN 1266096A) [ Gao et al 2011PLoS ONE.6(11): e28148], transformants are screened on an SA5 solid medium plate containing 0.5 mu g/mL erythromycin, the grown colonies are verified to be positive clones by a colony PCR method, the obtained positive clones are subjected to homologous exchange by a temperature shift method [ Connelly et al 2004, JBact 186: 4159-67 ], a knockout strain is screened by an NCM plate, and the L-ldh gene knockout strain is detected by PCR amplification and sequencing to obtain the Alkaliphilic Bacillus N16-5L-ldh gene (Bstpn. delta. 165-ldh) completely deleted mutant Bacillus sp.N16-5 delta.

2. Construction of recombinant Bacillus alcalophilus for production of D-lactic acid

Pyrobest DNA polymerase was used, and Bacillus alkalophilus N16-5 genome was used as a template, and primer 165PRF (5' -GGAATTC)CATATGCTGATGGTAGGACGCTTGTAC-3 ', S EQ ID NO:4) and PR-LDH (5'-CGTAAGCAAAAATTTTAGTCATGTTTA AACATCTACCTTTCC-3', SEQ ID NO:5) were PCR amplified with the L-LDH promoter under the following conditions: pre-denaturation at 94 ℃ for 10min, then 30 cycles of 94 ℃ for 30s, 60 ℃ for 30s and 72 ℃ for 30 s; finally, extension was carried out at 72 ℃ for 7 min. With Lactobacillus delbrueckii genome as template, and with primers LDH-PR (5'-GGAAAGGTAGATGTTTAAACATGACTAAAATTTTTGCTTACG-3', SEQ ID NO:6) and 165LDHR (5 ' -CGC)GGATCCTTAGCCAACCTTAACTGGAG-3', SEQ ID NO:7) was amplified by PCR of the D-ldh gene (GenBank: AAA25246), PCR amplification conditions were: pre-denaturation at 94 ℃ for 10min, then 30 cycles of 94 ℃ for 30s, 60 ℃ for 30s and 72 ℃ for 90 s; finally, extension was carried out at 72 ℃ for 7 min. Fusion of L-ldh promoter and D-ldh gene by overlap PCR and ligation to pMK4 vector to obtain expression plasmid pMK4-ldh, transformation of Bacillus sp.N16-5. delta. ldh protoplast competence, and bacterial growth by using SA5 medium containing 2.5. mu.g/mL chloramphenicol and using primer Cm-F/Cm-RAnd selecting positive clones by colony PCR, inoculating the positive clones into an NCM culture medium containing 2.5 mu g/mL chloramphenicol for culture, and obtaining the recombinant Bacillus alcalophilus sp.N16-5 delta ldh-pDalac for producing the D-lactic acid.

Example 2

This example illustrates the use of recombinant Bacillus alcalophilus provided by the present invention for the production of D-lactic acid.

1. Fermentation of

1) Activation culture: inoculating the preserved recombinant Bacillus alcalophilus sp.N16-5 delta ldh-pDalac glycerol bacterial liquid into a test tube reciprocating type shaking table filled with 4mL of seed culture medium according to the inoculum size of 4% by volume ratio, and culturing for 12 hours at the temperature of 37 ℃ at 200 rpm;

2) seed culture: inoculating the liquid culture obtained in the step 1) into a 250mL triangular flask filled with 100mL of seed culture medium under an aseptic condition, and performing static culture at 40 ℃ for 8 hours to obtain a seed culture solution;

3) fermentation culture: 700mL of fermentation medium was added to a 1.4 liter fermentor of multiforms manufactured by Infors HT of Switzerland, and sterilized at 115 ℃ for 20 minutes. The peanut meal was treated with filter-sterilized neutral protease (EC3.4.24.28) at a final concentration of 0.15 g/l for 8h at 45 ℃ and pH 7. Inoculating the seed solution with 10% (volume ratio), adding filter-sterilized chloramphenicol at a final concentration of 2.5. mu.g/mL, and supplementing 45mL of 750 g/L glucose aqueous solution when the glucose concentration of the fermentation solution is lower than 20 g/L during the fermentation process (3 times in total). Aeration is carried out for 1.0 liter/min 12 hours before fermentation, and then aeration is stopped until the fermentation is finished, the rotating speed is 100 revolutions per minute, and the rotating radius is 48 mm. The pH was kept constant at 9 with 10mol/L NaOH, and the fermentation was terminated by culturing at 45 ℃ for 96 hours.

Taking the fermentation liquor once every 4 hours at 6000 rpm, centrifuging for 5 minutes with the centrifugal radius of 36mm, and taking the supernatant.

2. Detection of

The concentration of glucose was measured by Agilent 1100 liquid chromatography (Agilent technologies, Ltd.) using an Aminex HPX-87H analytical column (300X 7.8mm) from Bio-Rad, a diode array detector, a detection wavelength of 215nm, and a flow ratePhase 18mmol/L H₂SO₄The flow rate was 0.5 mL/min, the column temperature was 65 ℃ and the amount of sample was 10. mu.L.

The concentration of D-lactic acid was measured by Agilent 1100 liquid chromatography (Agilent technologies, Ltd.), MCIGEL CRS10W chiral analytical column, diode array detector, detection wavelength of 254nm, mobile phase of 2mM CuSO₄The flow rate was 0.5 mL/min, the column temperature was 25 ℃ and the amount of sample was 10. mu.L. The L-lactic acid standard is a product of Sigma-Aldrich company with the product number of L1750. The standard product of the D-lactic acid is a product of Sigma-Aldrich company, and the product number is L0625.

The method for calculating the optical purity of the D-lactic acid comprises the following steps:

optical purity (%) of D-lactic acid ═ D-lactic acid concentration (g/L)/[ L-lactic acid concentration (g/L) + D-lactic acid concentration (g/L) ] × 100%

Calculation method of conversion:

conversion (%) ═ D-lactic acid concentration (g/l)/glucose consumption (g/l) × 100%

The experiment was repeated 3 times in total and the results averaged. As shown in FIG. 1, the concentration of glucose in the final fermentation broth was 0g/L, the concentration of D-lactic acid was 142 g/L, the conversion was 94%, and the optical purity of D-lactic acid was 99.85%.

Example 3

This example illustrates the use of recombinant Bacillus alcalophilus provided by the present invention for the production of D-lactic acid without sterilization.

1. Fermentation of

1) Activation culture: the procedure is as in example 1;

2) seed culture: the procedure is as in example 1;

3) fermentation culture: 700mL of fermentation medium was added to a 1.4 liter fermentor of multiforms manufactured by Infors HT of Switzerland, to a final concentration of 0.15 g/L, filter-sterilized neutral protease (EC3.4.24.28) was added, and the peanut meal was treated at pH7 and 45 ℃ for 8 hours. Inoculating the seed solution with 10% (volume ratio), adding filter-sterilized chloramphenicol at final concentration of 2.5 μ g/mL, and supplementing 45mL 750 g/L glucose aqueous solution once during fermentation process when the glucose concentration of the fermentation solution is lower than 20 g/L, wherein the whole process is supplemented for 3 times. Aeration is carried out for 1.0 liter/min 12 hours before fermentation, and then aeration is stopped until the fermentation is finished, the rotating speed is 100 revolutions per minute, and the rotating radius is 48 mm. The fermentation was terminated by culturing at 45 ℃ for 96 hours while maintaining the pH constant at 9 with 10M NaOH.

2. Detection of

The detection method was the same as described in example 1. The experiment was repeated 3 times in total and the results averaged. As shown in FIG. 2, the concentration of glucose in the final fermentation broth was 0g/L, the concentration of D-lactic acid was 144 g/L, the conversion was 96%, and the optical purity of D-lactic acid was 99.85%

From the above examples, it can be seen that the recombinant basophilic Bacillus prepared by the preparation method of the recombinant basophilic Bacillus provided by the invention can be used for preparing D-lactic acid, wherein the optical purity of the obtained D-lactic acid is higher than 99.8%, the conversion rate is as high as 94% or more, and the yield is as high as 142 g/L.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various features described in the above embodiments may be combined in any suitable manner without departing from the scope of the invention. The invention is not described in detail in order to avoid unnecessary repetition.

In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.

Claims

1. A method for producing a recombinant Bacillus alcalophilus, comprising: takes alcalophilus for producing L-lactic acid as an original strain, and the original strain does not express L-lactic dehydrogenase but expresses D-lactic dehydrogenase through genetic engineering operation.

2. The method according to claim 1, wherein the starting strain is allowed to express no L-lactate dehydrogenase in such a manner that: mutating or knocking out the encoding gene of the L-lactate dehydrogenase in the original strain.

3. The preparation method according to claim 1, wherein the starting strain is Bacillus alcalophilus with the preservation number of CGMCC NO. 0369.

4. The production method according to any one of claims 1 to 3, wherein the D-lactate dehydrogenase is expressed in such a manner that: an expression vector containing a gene encoding D-lactate dehydrogenase is introduced into Bacillus alcalophilus which does not express L-lactate dehydrogenase.

5. A recombinant Bacillus alkalophilus produced by the production method according to any one of claims 1 to 4.

6. The recombinant Bacillus alkalophilus of claim 5, wherein the preservation number of the recombinant Bacillus alkalophilus is CGMCC No. 11581.

7. Use of the recombinant Bacillus alkalophilus of claim 5 or 6 for the preparation of D-lactic acid.

8. A method for producing D-lactic acid, which comprises inoculating the recombinant Bacillus alkalophilus of claim 5 or 6 into a fermentation medium and fermenting to obtain a fermentation broth containing D-lactic acid.

9. The process according to claim 8, wherein the fermentation medium contains a carbon source, a nitrogen source and optionally inorganic salts.

10. The method according to claim 9, wherein the carbon source is contained in an amount of 60 to 150 g/l, the nitrogen source is contained in an amount of 10 to 50 g/l, and the inorganic salt is contained in an amount of 0 to 5g/l in the fermentation medium.

11. The method of claim 9, wherein the carbon source is a sugar; the nitrogen source is an inorganic nitrogen source and/or an organic nitrogen source; the inorganic salt is sodium acetate.

12. The method of claim 11, wherein the carbon source is at least one of glucose, sucrose, fructose, maltose, mannose, and galactose.

13. The method of claim 12, wherein the carbon source is glucose and/or fructose.

14. The method of claim 11, wherein the nitrogen source is an organic nitrogen source.

15. The method of claim 14, wherein the nitrogen source is provided by at least one of peanut meal, peptone, yeast powder, and soybean meal.

16. The method of claim 8, wherein the conditions of the fermentation comprise: the fermentation temperature is 35-45 deg.C, the fermentation time is 90-100 hr, the fermentation pH is 9-10, and the carbon source content is controlled at 20-50 g/L during fermentation.

17. The method of claim 16, wherein the pH of the fermentation is controlled by adding neutralizing agents to the fermentation system, the neutralizing agents comprising NaOH, ammonia, and Na₂CO₃At least one of (1).

18. The method of claim 17, wherein the neutralizing agent is NaOH and/or Na₂CO₃。

19. The method according to any one of claims 8-18, wherein the fermentation equipment and/or the fermentation medium is sterilized or not sterilized prior to the fermentation.

20. The method of claim 19, wherein fermentation equipment and/or fermentation media are not sterilized prior to the fermentation.