CN112961901A - Method for identifying action type of lactobacillus bulgaricus lactate dehydrogenase isozyme - Google Patents
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Abstract
A method for identifying the action type of lactobacillus bulgaricus lactate dehydrogenase isozyme comprises the steps of constructing recombinant escherichia coli expressing D-lactic acid and L-lactic acid lactate dehydrogenase isozyme genes, carrying out shake flask culture fermentation, detecting the generation conditions of D-lactic acid and L-lactic acid of the recombinant escherichia coli in aerobic culture and anaerobic culture by combining shake flask fermentation and lactic acid determination, and judging whether different lactate dehydrogenase isozymes have catalytic activity.
Description
Technical Field
The invention belongs to the technical field of enzyme activity identification, and particularly relates to an identification method of the action type of lactobacillus bulgaricus lactate dehydrogenase isozyme.
Background
Lactic acid is an important bio-based platform compound and is widely applied to the fields of agriculture, food, medicine, chemical industry, environmental protection and the like. They are classified into D-lactate and L-lactate according to their optical activity, and are catalyzed by D-lactate dehydrogenase (D-LDH, EC 1.1.1.28) and L-lactate dehydrogenase (L-LDH, EC 1.1.1.27), respectively.
The stereospecificity of lactic acid dehydrogenase in lactic acid bacteria cells determines the stereospecificity of lactic acid, such as Lactobacillus delbrueckii, l.bulbgaricus, l.jensenii and l.coryniforis fermentation products which are mainly D-lactic acid, and some strains contain two kinds of lactic acid dehydrogenases or have lactic acid racemase, and finally form DL-lactic acid. While some lactic acid bacteria produce L-lactic acid, when L-lactic acid accumulates, it induces the production of a lactic acid racemase, which converts L-lactic acid to D-lactic acid until equilibrium.
Studies have shown that by changing the pH or the nutrients, the proportion of D-lactic acid or L-lactic acid will change. For lactic acid bacteria synthesizing D-lactic acid, D-LDHs has higher pyruvic acid catalytic efficiency than L-LDHs. D-lactate or L-lactate with optical characteristics can be produced by biotransformation of pyruvic acid by D-LDHs or L-LDHs.
By overexpressing the ldhD or ldhL genes, high expression in a suitable host is achieved to achieve increased production of lactic acid of the corresponding configuration. Potansen peak and the like (preliminary research on genetically engineered bacteria of Escherichia coli producing high-purity L-lactic acid [ J ] modern food science and technology, 2010, 26: 126-; zhou et al (Functional replacement of the Escherichia coli D- (-) -lactate dehydrogenase gene (ldhA) with the L- (+) -lactate dehydrogenase gene (ldhL) from Pediococcus acidilactici [ J ]. Appl Environ Microbiol, 2003, 69: 2237-2244) produced the engineered bacterium SZ85 with a high chemical purity in a yield of 95% by replacing the D-LDH gene ldhA in Escherichia coli with the L-LDH gene ldhL of Pediococcus acidilactici. Okano et al (Efficient production of optional pure D-Lactic acid from raw corn by using a genetic modified L-Lactate dehydrogenase gene-specification and alpha-amylase-partitioning Lactobacillus plantarum strain [ J ]. Appl Environ Microbiol,2009,75: 462 467) knock out the ldhL.plantarum NCIMB 8826 gene, express the alpha-amylase gene from Streptococcus bovis, and the obtained engineered bacteria can effectively utilize starch to synthesize high-purity D-Lactic acid.
However, there is currently no systematic study on the identification of the role of different lactate dehydrogenase isozymes in Lactobacillus bulgaricus.
Disclosure of Invention
The invention aims to provide an identification method of the action type of lactobacillus bulgaricus lactate dehydrogenase isozyme, which is characterized in that the generation conditions of D-lactic acid and L-lactic acid of recombinant bacteria during aerobic and anaerobic fermentation are judged by constructing recombinant escherichia coli expressing a lactate dehydrogenase gene, and the action of different dehydrogenase isozymes in lactobacillus bulgaricus in lactic acid synthesis is quickly and accurately judged and identified.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for identifying the action type of lactobacillus bulgaricus lactate dehydrogenase isozyme comprises the following steps:
1) culturing of bacterial strains
Inoculating the lactobacillus bulgaricus ATCC11842 bacterial liquid into 3-5 mL of MRS culture medium, and performing static culture at 37 ℃ for 48-72 hours;
inoculating escherichia coli BL21(DE3) bacterial liquid into 3-5 mL LB culture medium, and culturing at 37 ℃ and 180-220 r/min for 10-12 h to obtain E.coli BL21 competent cells;
2) construction of recombinant Escherichia coli
Respectively designing specific primers according to gene sequences ldb _ rs00400, ldb _ rs04425 and 1db _ rs03465 of Lactobacillus bulgaricus ATCC 11842D-lactate dehydrogenase in Genbank, and gene sequences ldb0120 and ldb0094 of L-lactate dehydrogenase, and carrying out PCR amplification by taking a Lactobacillus bulgaricus genome as a template to obtain corresponding target fragments;
performing double enzyme digestion on each target fragment and a vector pET28a by adopting NheI and XhoI restriction endonucleases respectively, recovering and purifying gel, and then connecting the enzyme-digested target fragment with a linearized vector pET28a by adopting T4 DNA ligase, wherein the mass concentration ratio of the target fragment to the linearized vector pET28a is 1: 3-5; thermally shocking the ligation product at 42 ℃ to transform E.coli BL21 competent cells, coating LB flat plates after recovery, and screening recombinant escherichia coli transformants;
selecting a single colony to an LB liquid culture medium with a final concentration of 30-50 mug/mL kanamycin, culturing for 10-12 h at 37 ℃, carrying out PCR verification on bacterial liquid, and sequencing to obtain recombinant escherichia coli;
3) shake flask fermentation
Using empty plasmid recombinant bacterium E.coli/pET28a as a control, and respectively carrying out aerobic culture and anaerobic culture on five recombinant escherichia coli expressing D-lactate dehydrogenase and L-lactate dehydrogenase isozyme genes;
inoculating the recombinant escherichia coli seeds into an M9 fermentation culture medium according to the inoculation amount of 0.5-2%, respectively performing aerobic fermentation and anaerobic fermentation, and adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.1-0.5 mmol/L for induction; during aerobic culture, wrapping the mouth of the culture container with gauze; during anaerobic culture, filling liquid in the bottle, and sealing the container for culture;
wherein, kanamycin with the final concentration of 30-50 mug/mL is added into the culture bacterial liquid, the temperature is 35-38 ℃, the fermentation time is 18-24 h, the thallus concentration OD600 is measured, and the concentrations of D-lactic acid and L-lactic acid are measured.
Preferably, the primer sequences involved in step 2) are as follows:
ldb _ rs00400 gene:
F：5’-CCGCGCTAGCATGACTAAAATTTTTGC-3’；
R：5’-GCTTCTCGAGTTAGCCAACCTTAACTGG-3’；
ldb _ rs04425 gene:
F：5’-GGCGGCTAGCATGACTAAAATTGCCAT-3’；
R：5’-CACGCTCGAGTTACAGGTTAACGATGCT-3’；
ldb _ rs03465 gene:
F：5’-ACTAGCTAGCATGTACCTCTTGCGC-3’；
R：5’-CGACCTCGAGTTACTGCTTTTTCGCTAA-3’；
ldb0120 gene:
F：5’-CCGAGCTAGCATGAGTAGAAAAGTCC-3’；
R：5’-CGGGCTCGAGTTATCCTAAAGAGTCC-3’；
ldb0094 gene:
F：5’-CGGAGCTAGCATGAGAAAAGTAGCAG-3’；
R：5’-CGGTCTCGAGTTATTCCAAGCTGGCC-3’。
in step 2), the PCR amplification conditions are: pre-denaturation at 94 ℃ for 10 min; denaturation at 94 ℃ for 30s, annealing at 52-55 ℃ for 30s, extension at 72 ℃ for l min, and 30 cycles; and (3) extending for 10min at 72 ℃, and detecting the PCR product by agarose gel electrophoresis.
Further, in the step 1), the MRS culture medium contains 10g/L of peptone, 8g/L of beef extract, 4g/L of yeast extract, 20g/L of glucose, 2g/L of dipotassium phosphate, 2g/L of triammonium citrate, 5g/L of sodium acetate trihydrate, 0.2g/L of magnesium sulfate heptahydrate, 0.05g/L of manganese sulfate tetrahydrate, 801 g/L of tween and pH 6.2 +/-0.2; the LB culture medium contains 10g/L of peptone, 5g/L of yeast extract and 10g/L of sodium chloride.
In the step 3), the M9 fermentation medium of the recombinant escherichia coli contains 5-9 g/L, Na of Glucose2HPO4·12H2O 10～16g/L、KH2PO42.0-3.0 g/L, NaCl 0.2.2-0.5 g/L and NH4Cl 1.0-5.0 g/L, 1M MgSO is added per liter 4 1～2mL、1M CaCl20.1-0.2 mL, 1% (w/v) vitamin B10.1-0.5 mL, and trace elements 0.1-0.5 mL.
Compared with the prior art, the invention has the following beneficial effects:
the invention judges whether different lactate dehydrogenase isoenzymes have catalytic activity or not by constructing recombinant escherichia coli expressing D-lactate dehydrogenase genes and L-lactate dehydrogenase genes of lactic acid with different configurations and combining shake flask fermentation and lactic acid determination, thereby judging the function of the isoenzymes in synthesizing lactic acid in Lactobacillus bulgaricus.
Compared with the existing method for testing the action of the isozyme of the lactate dehydrogenase, the identification method provided by the invention judges whether different lactate dehydrogenases have catalytic action or not through product synthesis, and the steps of cell disruption, enzyme activity determination and the like are omitted. The method is particularly suitable for the activity identification of enzymes with the same function of various isozymes, and can quickly screen out key enzymes from the various isozymes so as to carry out the next research on the key enzymes.
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FIG. 1 shows the results of double digestion of recombinant expression plasmids expressing D-lactate dehydrogenase gene in example 2 of the present invention, 1: pET28aldb _ rs 00400; 2: pET28aldb _ rs 04425; 3: pET28aldb _ rs 03465.
FIG. 2 shows the results of double digestion of recombinant expression plasmids expressing L-lactate dehydrogenase genes in example 2 of the present invention, wherein 1: pET28aldb 0120; 2: pET28aldb 0094.
Detailed Description
The present invention is further illustrated by the following specific examples.
EXAMPLE 1 construction of recombinant expression plasmids of D-lactate dehydrogenase Gene and L-lactate dehydrogenase Gene
Inoculating lactobacillus bulgaricus ATCC11842 bacterial liquid into 3-5 mL of MRS culture medium,
standing and culturing at 37 ℃ for 48-72 h; inoculating the Escherichia coli BL21(DE3) bacterial liquid into 3-5 mL LB culture medium, and culturing at 37 ℃ and 180-220 r/min for 10-12 h.
The MRS culture medium contains 10g/L of peptone, 8g/L of beef extract, 4g/L of yeast extract, 20g/L of glucose, 2g/L of dipotassium phosphate, 2g/L of triammonium citrate, 5g/L of sodium acetate trihydrate, 0.2g/L of magnesium sulfate heptahydrate, 0.05g/L of manganese sulfate tetrahydrate, 801 g/L of tween and pH 6.2 +/-0.2; the LB culture medium contains 10g/L of peptone, 5g/L of yeast extract and 10g/L of sodium chloride.
According to gene sequences ldb _ rs00400, ldb _ rs04425 and 1db _ rs03465 of L-lactate dehydrogenase and gene sequences ldb0120 and ldb0094 of L-lactate dehydrogenase disclosed in Genbank, specific primers are respectively designed, a Lactobacillus bulgaricus genome is used as a template, and a primer shown in Table 1 is used for PCR amplification of D-lactate dehydrogenase and L-lactate dehydrogenase isozyme genes.
TABLE 1 lactate dehydrogenase primers
Agarose gel electrophoresis all obtains a band with a specific size (figure 1-2), after sequencing, the band is compared with ldb _ rs00400(CAI96942.1, 1002bp), ldb _ rs04425(CAI97812.1, 1002bp), ldb _ rs03465(CAI98759.1, 666bp), ldb0120(CAI96942.1, 924bp) and ldb0094(CAI98759.1, 909bp) genes of L.bulbgaricus ATCC11842 of Genbank, and the sequence shows that the homology is 100 percent, thereby proving that the cloned genes are correct.
And (3) performing double enzyme digestion on the target gene fragment and the plasmid pET28a according to primers shown in a primer design table, performing agarose gel electrophoresis, recovering and purifying by using a kit, further connecting the recovered fragment with a linearized pET28a vector, transforming E.coli BL21 competent cells by using a connection product, and screening recombinant escherichia coli by using kanamycin resistance.
And extracting the recombinant expression plasmids for double enzyme digestion verification, wherein each recombinant expression plasmid is cut into two strips as shown in figure 2, the larger strip is a pET28a linearized vector with about 5.3kb, the smaller strip is each target gene and is consistent with the expected size, and the construction of the recombinant expression plasmids is completed.
EXAMPLE 2 Shake flask fermentation of recombinant Strain overexpressing D-lactate dehydrogenase Gene
Taking recombinant strains BL21/pET28a for expressing empty plasmids as a control, respectively inoculating three kinds of fresh-cultured recombinant Escherichia coli BL21/pET28aldb _ rs00400, BL21/pET28aldb _ rs04425 and BL21/pET28aldb _ rs03465 seeds for expressing D-lactate dehydrogenase genes into a triangular flask containing 30mL of 250mL and a blue flask containing 40mL of fermentation medium, and respectively carrying out aerobic fermentation and anaerobic fermentation on the recombinant Escherichia coli expressing the D-lactate dehydrogenase isozyme genes, wherein the fermentation medium adopts M21/pET 28a9 Medium (g/L): glucose 5-9, Na2HPO4·12H2O 10～16，KH2PO42.0～3.0，NaCl 0.2～0.5，NH4Cl 1.0-5.0, adding 1M MgSO (MgSO) per liter 4 1～2mL，1M CaCl20.1-0.2 mL, 1% (w/v) vitamin B10.1-0.5 mL, trace elements 0.1-0.5 mL; culturing at 37 deg.C and 220r/min for 24h, and determining thallus concentration OD 600; the fermentation samples were collected by centrifugation at 12000r/min at 4 ℃ and the D-lactic acid content was determined using a D-lactic acid kit, and the results of shake flask fermentation are shown in Table 2.
TABLE 2 comparison of the Performance of the recombinant E.coli in shake flask for the fermentation synthesis of D-lactic acid
The results of aerobic and anaerobic fermentations, respectively, on recombinant E.coli expressing D-lactate dehydrogenase isozyme genes showed that the growth of each strain was inhibited to a different extent under anaerobic conditions compared to aerobic fermentation conditions; the strains BL21/pET28aldb _ rs00400 and BL21/pET28aldb _ rs04425 show better performance of synthesizing D-lactic acid, wherein the content of the D-lactic acid under the anaerobic condition is 1722.4mg/L and 1524.7mg/L respectively; under aerobic conditions, the synthesis of D-lactic acid of the three recombinant strains is increased compared with that of an empty plasmid recombinant strain, wherein the strain BL21/pET28aldb _ rs04425 can synthesize D-lactic acid with higher concentration, and the concentration is 643.5mg/L, which shows that the enzyme LDB _ rs00400 has higher enzyme activity under aerobic conditions and can catalyze substrates to synthesize the D-lactic acid.
From the shake flask fermentation data of recombinant escherichia coli, it can be speculated that lactate dehydrogenases LDB _ rs00400 and LDB _ rs04425 are key enzymes for synthesizing D-lactic acid by lactobacillus bulgaricus and are regulated and controlled by oxygen, the lactate dehydrogenases LDB _ rs00400 and LDB _ rs04425 are key enzymes for catalyzing pyruvate to synthesize D-lactic acid under anaerobic conditions, and the lactate dehydrogenase LDB _ rs04425 is key enzymes for catalyzing D-lactic acid under aerobic conditions.
The concentrations of recombinant bacteria BL21/pET28aldb _ rs 03465D-lactic acid under anaerobic and aerobic conditions are 894.8 and 103.1mg/L respectively, and the concentrations of the recombinant bacteria BL21/pET28a under anaerobic and aerobic conditions are 684.8 and 3.5mg/L respectively, which shows that the LDB _ rs03465 enzyme also has catalytic activity, but the enzyme activity is lower, and the activity is not controlled by oxygen.
EXAMPLE 3 Shake flask fermentation of recombinant Strain overexpressing L-lactate dehydrogenase Gene
Respectively carrying out aerobic fermentation and anaerobic fermentation on recombinant escherichia coli BL21/pET28aldb0120 and BL21/pET28aldb0094 for expressing L-lactate dehydrogenase by taking an empty plasmid recombinant bacterium BL21/pET28a as a control, wherein the fermentation medium adopts an M9 medium (g/L): glucose 5-9, Na2HPO4·12H2O 10～16，KH2PO4 2.0～3.0，NaCl 0.2～0.5，NH4Cl 1.0-5.0, adding 1M MgSO (MgSO) per liter 4 1～2mL，1M CaCl20.1-0.2 mL, 1% (w/v) vitamin B10.1-0.5 mL, culturing at 37 ℃ and 220r/min for 24h, measuring the thallus concentration OD600, centrifuging at 4 ℃ and 12000r/min, collecting fermentation samples, measuring the content of L-lactic acid by using a lactic acid kit, and the shake flask fermentation result is shown in Table 3.
TABLE 3 Shake flask fermentation results of recombinant bacteria expressing L-lactate dehydrogenase gene
It can be seen that compared with a control BL21/pET28a, the recombinant bacterium BL21/pET28aldb0120 is not different from the control BL21/pET28a, the concentration of L-lactic acid is very low and is only 3-6 mg/L, the strain BL21/pET28aldb0094 can synthesize L-lactic acid under an anaerobic condition, the concentration is 227.9mg/L, the concentration of L-lactic acid is 41.9mg/L under an aerobic condition, and it is presumed that LDB0120 may be inactive, so that the L-lactic acid cannot be synthesized from a substrate pyruvic acid, while LDB0094 has catalytic activity and is negatively regulated by oxygen, L-lactic acid can be synthesized under an anaerobic condition, and the enzyme activity is reduced under an aerobic condition, so that the synthesis of L-lactic acid is influenced.
Claims (5)
1. A method for identifying the action type of lactobacillus bulgaricus lactate dehydrogenase isozyme comprises the following steps:
1) culturing of bacterial strains
Inoculating the lactobacillus bulgaricus ATCC11842 bacterial liquid into 3-5 mL of MRS culture medium, and performing static culture at 37 ℃ for 48-72 hours;
inoculating escherichia coli BL21(DE3) bacterial liquid into 3-5 mL LB culture medium, culturing at 37 ℃ and 180-220 r/min for 10-12 h to obtain E.coli BL21 competent cells;
2) construction of recombinant Escherichia coli
Respectively designing specific primers according to gene sequences ldb _ rs00400, ldb _ rs04425 and 1db _ rs03465 of Lactobacillus bulgaricus ATCC 11842D-lactate dehydrogenase in Genbank, and gene sequences ldb0120 and ldb0094 of L-lactate dehydrogenase, and carrying out PCR amplification by taking a Lactobacillus bulgaricus genome as a template to obtain corresponding target fragments;
performing double enzyme digestion on each target fragment and a vector pET28a by adopting NheI and XhoI restriction endonucleases respectively, recovering and purifying gel, and then connecting the enzyme-digested target fragment with a linearized vector pET28a by adopting T4 DNA ligase, wherein the mass concentration ratio of the target fragment to the linearized vector pET28a is 1: 3-5; thermally shocking the ligation product at 42 ℃ to transform E.coliBL21 competent cells, coating an LB flat plate after recovery, and screening recombinant escherichia coli transformants;
selecting a single colony to an LB liquid culture medium with a final concentration of 30-50 mug/mL kanamycin, culturing for 10-12 h at 37 ℃, carrying out PCR verification on bacterial liquid, and sequencing to obtain recombinant escherichia coli;
3) shake flask fermentation
Using empty plasmid recombinant strain E.coli/pET28a as a control, and respectively carrying out aerobic culture and anaerobic culture on five recombinant escherichia coli expressing D-lactate dehydrogenase and L-lactate dehydrogenase isozyme genes;
inoculating the recombinant escherichia coli seeds into an M9 fermentation culture medium according to the inoculation amount of 0.5-2%, respectively performing aerobic fermentation and anaerobic fermentation, and adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.1-0.5 mmol/L for induction; during aerobic culture, wrapping the mouth of the culture container with gauze; during anaerobic culture, the liquid loading amount is close to the full bottle, and the container is sealed for culture;
wherein, kanamycin with the final concentration of 30-50 mug/mL is added into the culture bacterial liquid, the temperature is 35-38 ℃, the fermentation time is 18-24 h, the thallus concentration OD600 is measured, and the concentrations of D-lactic acid and L-lactic acid are measured.
2. The method for identifying the action type of the lactobacillus bulgaricus lactate dehydrogenase isozyme according to claim 1, wherein the primer sequences involved in the step 2) are as follows:
ldb _ rs00400 gene:
F：5’-CCGCGCTAGCATGACTAAAATTTTTGC-3’；
R：5’-GCTTCTCGAGTTAGCCAACCTTAACTGG-3’；
ldb _ rs04425 gene:
F：5’-GGCGGCTAGCATGACTAAAATTGCCAT-3’；
R：5’-CACGCTCGAGTTACAGGTTAACGATGCT-3’；
ldb _ rs03465 gene:
F：5’-ACTAGCTAGCATGTACCTCTTGCGC-3’；
R：5’-CGACCTCGAGTTACTGCTTTTTCGCTAA-3’；
ldb0120 gene:
F：5’-CCGAGCTAGCATGAGTAGAAAAGTCC-3’；
R：5’-CGGGCTCGAGTTATCCTAAAGAGTCC-3’；
ldb0094 gene:
F：5’-CGGAGCTAGCATGAGAAAAGTAGCAG-3’；
R：5’-CGGTCTCGAGTTATTCCAAGCTGGCC-3’。
3. the method for identifying the action type of the lactobacillus bulgaricus lactate dehydrogenase isozyme according to claim 1, wherein in the step 2), the PCR amplification conditions are as follows: pre-denaturation at 94 ℃ for 10 min; denaturation at 94 ℃ for 30s, annealing at 52-55 ℃ for 30s, extension at 72 ℃ for 1min, and 30 cycles; and (3) extending for 10min at 72 ℃, and detecting the PCR product by agarose gel electrophoresis.
4. The method for identifying the action pattern of the lactobacillus bulgaricus lactate dehydrogenase isozyme according to claim 1, wherein in the step 1), the MRS medium contains 10g/L of peptone, 8g/L of beef extract, 4g/L of yeast extract, 20g/L of glucose, 2g/L of dipotassium hydrogen phosphate, 2g/L of triammonium citrate, 5g/L of sodium acetate trihydrate, 0.2g/L of magnesium sulfate heptahydrate, 0.05g/L of manganese sulfate tetrahydrate, 801 g/L of Tween, and pH 6.2 +/-0.2; the LB culture medium contains 10g/L of peptone, 5g/L of yeast extract and 10g/L of sodium chloride.
5. The method for identifying the action type of the lactic dehydrogenase isozyme of Lactobacillus bulgaricus according to claim 1, wherein in the step 3), the M9 fermentation medium of the recombinant Escherichia coli contains 5-9 g/L, Na of Glucose2HPO4·12H2O 10～16g/L、KH2PO42.0-3.0 g/L, NaCl 0.2.2-0.5 g/L and NH4Cl 1.0-5.0 g/L, 1M MgSO is added per liter4 1～2mL、1M CaCl20.1-0.2 mL, 1% (w/v) vitamin B10.1-0.5 mL, and trace elements 0.1-0.5 mL.
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| CN115786228A (en) * | 2022-12-27 | 2023-03-14 | 合肥迈可罗生物工程有限公司 | Lactobacillus genetically engineered bacterium and application thereof |
| CN115786228B (en) * | 2022-12-27 | 2024-01-09 | 合肥迈可罗生物工程有限公司 | Lactobacillus genetically engineered bacterium and application thereof |
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