CN118516320A - D-lactate dehydrogenase mutant, preparation method, encoding DNA and D-lactate production method and application thereof - Google Patents
D-lactate dehydrogenase mutant, preparation method, encoding DNA and D-lactate production method and application thereof Download PDFInfo
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- CN118516320A CN118516320A CN202310130526.0A CN202310130526A CN118516320A CN 118516320 A CN118516320 A CN 118516320A CN 202310130526 A CN202310130526 A CN 202310130526A CN 118516320 A CN118516320 A CN 118516320A
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- lactate dehydrogenase
- amino acid
- dehydrogenase mutant
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- JZCCFEFSEZPSOG-UHFFFAOYSA-L copper(II) sulfate pentahydrate Chemical compound O.O.O.O.O.[Cu+2].[O-]S([O-])(=O)=O JZCCFEFSEZPSOG-UHFFFAOYSA-L 0.000 description 1
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- 230000037431 insertion Effects 0.000 description 1
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
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- 229910052749 magnesium Inorganic materials 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- CNFDGXZLMLFIJV-UHFFFAOYSA-L manganese(II) chloride tetrahydrate Chemical compound O.O.O.O.[Cl-].[Cl-].[Mn+2] CNFDGXZLMLFIJV-UHFFFAOYSA-L 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- RRIWRJBSCGCBID-UHFFFAOYSA-L nickel sulfate hexahydrate Chemical compound O.O.O.O.O.O.[Ni+2].[O-]S([O-])(=O)=O RRIWRJBSCGCBID-UHFFFAOYSA-L 0.000 description 1
- 229940116202 nickel sulfate hexahydrate Drugs 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
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- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 229920000747 poly(lactic acid) Polymers 0.000 description 1
- 239000004626 polylactic acid Substances 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
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- RWVGQQGBQSJDQV-UHFFFAOYSA-M sodium;3-[[4-[(e)-[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfonatophenyl)methyl]azaniumylidene]-2-methylcyclohexa-2,5-dien-1-ylidene]methyl]-n-ethyl-3-methylanilino]methyl]benzenesulfonate Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C)C=C1 RWVGQQGBQSJDQV-UHFFFAOYSA-M 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000003696 structure analysis method Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000005469 synchrotron radiation Effects 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 239000011721 thiamine Substances 0.000 description 1
- 235000019157 thiamine Nutrition 0.000 description 1
- KYMBYSLLVAOCFI-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SCN1CC1=CN=C(C)N=C1N KYMBYSLLVAOCFI-UHFFFAOYSA-N 0.000 description 1
- 229960003495 thiamine Drugs 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- RZLVQBNCHSJZPX-UHFFFAOYSA-L zinc sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Zn+2].[O-]S([O-])(=O)=O RZLVQBNCHSJZPX-UHFFFAOYSA-L 0.000 description 1
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
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Abstract
The invention provides a D-lactate dehydrogenase mutant, a preparation method, encoding DNA and a production method and application of D-lactate dehydrogenase. The amino acid sequence of the D-lactate dehydrogenase mutant is a wild-type D-lactate dehydrogenase with the following mutation on the basis of the amino acid sequence, wherein the wild-type D-lactate dehydrogenase comprises a substrate binding domain, a cofactor binding domain and a hinge region connecting the two, and the mutation is: the first amino acid of the cofactor binding domain linked to the hinge region is mutated to any one of a polar amino acid, an acidic amino acid, or a basic amino acid, and the D-lactate dehydrogenase mutant has a function of catalyzing the production of D-lactate from a substrate or a salt thereof. The D-lactate dehydrogenase mutant has catalytic activity of catalyzing the formation of D-lactate or a salt thereof from a substrate at 30 ℃ to 100 ℃. Compared with wild-type D-lactate dehydrogenase, the D-lactate dehydrogenase mutant has significantly improved catalytic performance.
Description
Technical Field
The invention relates to the fields of enzyme engineering and synthetic biology, in particular to a D-lactate dehydrogenase mutant, a preparation method, a coding DNA and a D-lactate production method and application thereof.
Background
D-lactate dehydrogenase catalyzes the production of D-lactate from pyruvate in the presence of the cofactor NAD (P) H. The optically pure D-lactic acid can be widely applied to the industries of foods, medicines, cosmetics and petrochemical industry, and is mainly used as a precursor for producing biodegradable polylactic acid. The structure of D-lactate dehydrogenase comprises a substrate binding domain, a cofactor binding domain and a hinge region connecting the two domains. At present, the catalytic performance of the D-lactic dehydrogenase has a large improvement space, and if the catalytic performance of the D-lactic dehydrogenase can be improved by modifying the D-lactic dehydrogenase, the D-lactic dehydrogenase has a large application prospect in the fields of enzyme catalytic production, biological medicine detection, sensor and microbial D-lactic acid production, and the rapid development of the related fields is greatly promoted.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a D-lactate dehydrogenase mutant, a preparation method, encoding DNA and a D-lactate production method and application thereof.
Specifically, the present invention provides:
(1) A mutant D-lactate dehydrogenase having an amino acid sequence that has a mutation based on the amino acid sequence of a wild-type D-lactate dehydrogenase, wherein the wild-type D-lactate dehydrogenase comprises a substrate binding domain, a cofactor binding domain, and a hinge region linking the two, the mutation being: the first amino acid of the cofactor binding domain linked to the hinge region is mutated to any one of a polar amino acid, an acidic amino acid, or a basic amino acid, and the D-lactate dehydrogenase mutant has a function of catalyzing the production of D-lactate from a substrate or a salt thereof.
(2) The D-lactate dehydrogenase mutant according to (1), wherein the amino acid sequence of the wild-type D-lactate dehydrogenase is as shown in SEQ ID No. 4 or has more than 30% identity with SEQ ID No. 4, and the mutation is at amino acid 101 thereof.
(3) The D-lactate dehydrogenase mutant according to (1), wherein the polar amino acids include serine, threonine, cysteine, tyrosine, glutamine and asparagine, the acidic amino acids include aspartic acid and glutamic acid, and the basic amino acids include lysine, arginine and histidine.
(4) The D-lactate dehydrogenase mutant according to (1), wherein the amino acid that has undergone mutation is mutated to glutamine or asparagine.
(5) The D-lactate dehydrogenase mutant according to (1), wherein the D-lactate dehydrogenase mutant has a tag at the N-terminus and/or the C-terminus for purification and/or detection, and the tag does not affect the catalytic performance of the D-lactate dehydrogenase mutant.
(6) The D-lactate dehydrogenase mutant according to (1), wherein the amino acid sequence of the D-lactate dehydrogenase mutant is shown as SEQ ID No.1 or SEQ ID No.2, or has identity of more than 30% to SEQ ID No.1 or SEQ ID No. 2.
(7) A DNA comprising a nucleotide sequence encoding the D-lactate dehydrogenase mutant according to any one of (1) to (6).
(8) The DNA according to (7), wherein the DNA contains a nucleotide sequence as shown in SEQ ID No. 5 or SEQ ID No. 6, or a nucleotide sequence having 30% or more identity with SEQ ID No. 5 or SEQ ID No. 6.
(9) An expression vector comprising the DNA according to (7) or (8) operably linked to a promoter.
(10) A host cell comprising the expression vector according to (9), or having incorporated into its genome a nucleotide sequence encoding the D-lactate dehydrogenase mutant according to any one of (1) to (6).
(11) The host cell of (10), wherein the host cell comprises bacillus thermocellum (Geobacillus thermoglucosidasius) and bacillus licheniformis (Bacillus licheniformis).
(12) A method for producing the D-lactate dehydrogenase mutant according to any one of (1) to (6), comprising obtaining the D-lactate dehydrogenase mutant by genetic engineering or artificial synthesis.
(13) The use of the D-lactate dehydrogenase mutant according to any one of (1) to (6), the DNA of (7) or (8), the expression vector of (9), or the host cell of (10) or (11) in the fields of enzyme-catalyzed production, biomedical detection and/or biosensors; preferably, the enzyme-catalyzed production comprises the production of D-lactic acid.
(14) A method for producing D-lactic acid or a salt thereof, characterized in that the method comprises contacting the D-lactate dehydrogenase mutant according to any one of (1) to (6) with a substrate for producing D-lactic acid and a cofactor; preferably, the substrate is pyruvic acid or a salt thereof and the cofactor is NAD (P) H.
(15) The method according to (14), wherein the D-lactate dehydrogenase mutant is expressed by a microorganism and subjected to fermentation culture; preferably, the D-lactate dehydrogenase mutant is expressed in the form of a plasmid or the nucleotide sequence encoding the D-lactate dehydrogenase mutant is integrated into the genome of the microorganism to thereby effect the expression.
(16) The method according to (14), wherein the method comprises providing seeds of a microorganism expressing the D-lactate dehydrogenase mutant, and then inoculating the seeds of the microorganism into a fermentation medium at an inoculum size with an OD 620nm value of 0.2 to 0.8 for fermentation culture.
(17) The method of (15) or (16), wherein the microorganism comprises B.thermocellum and B.licheniformis.
(18) The method of (16), wherein the fermentation medium comprises one or more of yeast powder, metal ions, biotin, betaine, peanut meal, carbon source, neutral protease.
(19) The method of (18), wherein the carbon source comprises one or more of glucose, sucrose, glycerol, xylose, arabinose, and mannitol; preferably, the initial concentration of the carbon source is 80g/L to 120g/L; it is also preferable that the carbon source is replenished so that the concentration thereof is maintained in the range of 0 to 100g/L when the concentration of the carbon source is reduced to 0.0 to 40.0 g/L.
(20) The method according to (15) or (16), wherein the fermentation culture is carried out at 30℃to 70℃at a pH of 6.5 to 7.5 and a stirring rate of 50rpm to 150rpm for 40 hours to 70 hours.
Compared with the prior art, the invention has the following advantages and positive effects:
(1) The present invention proposes that the first amino acid connecting the cofactor binding domain of D-lactate dehydrogenase to the hinge region is mutated and mutated to any one of a polar amino acid, an acidic amino acid or a basic amino acid. The D-lactate dehydrogenase mutant thus modified has catalytic activity for catalyzing the formation of D-lactate or a salt thereof from a substrate at 30℃to 100 ℃. Compared with wild-type D-lactate dehydrogenase, the D-lactate dehydrogenase mutant has significantly improved catalytic performance, in particular in terms of catalytic activity (k cat), specific activity (number of enzyme activity units possessed by a unit mass of protein) and catalytic efficiency (k cat/Km).
(2) The D-lactate dehydrogenase mutant according to the present invention has good thermostability.
(3) The directional transformation method of the D-lactic dehydrogenase is simple to operate, has strong practicability, is beneficial to the rapid development of enzyme catalytic production, biological medicine detection, sensor field and microorganism D-lactic acid production field, and is more suitable for commercial and industrial application.
(4) By utilizing the D-lactate dehydrogenase mutant, the D-lactate or the salt thereof can be specifically obtained by the production method of the D-lactate or the salt thereof, and the yield can reach 93.04%, so that the utilization rate of raw materials and the post-treatment efficiency of products are improved.
Drawings
FIG. 1 shows a schematic diagram of the directed modification of D-lactate dehydrogenase and its use according to the invention, wherein a represents a wild-type D-lactate dehydrogenase, b represents a D-lactate dehydrogenase mutant, the circles in a and b show the positions of the mutated amino acids, c represents that the D-lactate dehydrogenase mutant according to the invention can be used in the fields of enzyme-catalyzed production, biomedical detection, sensor and microbial D-lactate production.
Detailed Description
Various preferred embodiments of the present invention will be described below with reference to the accompanying drawings of the specification, so that the technical contents thereof will be more clearly and easily understood. The present invention may be embodied in many different forms of embodiments and the scope of the present invention is not limited to only the embodiments described herein.
It should be understood that the above technical features of the present invention and the technical features specifically described in the following (including but not limited to the embodiments and examples) may be combined with each other in any suitable manner to form a new or preferred technical solution, so long as there is no contradiction, and the combined technical solution can be smoothly implemented and the technical problem of the present invention can be solved. The technical scheme of the invention can be realized by any suitable mode, so that the technical problem of the invention is solved, and the corresponding technical effect can be realized.
In the present invention, words "comprising," "further," "having," "further," and the like merely denote preferred or more specific embodiments or examples, and it is to be understood that they are not limiting to the scope of the invention.
In the present invention, "and/or" means any one or any combination of the listed items.
Numerical ranges in the present invention include both endpoints unless specifically stated otherwise.
Some of the English and abbreviations involved in the present invention have the following meanings:
NADH: reduced nicotinamide adenine dinucleotide; NADPH: reduced nicotinamide adenine dinucleotide phosphate; NAD +: oxidized nicotinamide adenine dinucleotide; NADP +: oxidized nicotinamide adenine dinucleotide phosphate; p: proline; q: glutamine; n: asparagine; g: glycine; D-LDH: d-lactate dehydrogenase.
The amino acid and nucleotide sequences involved in the present invention are as follows:
SEQ ID No.:1(D-LDHP101Q)：
MKVIFFSMHPYEEEFLGPILPSDWDVEMTPDFLDETTVEKAKGAQVVSLFVSDKADGPVLEALHSYGVGLLALRSAGYDHIDIETAKRLGIKVVNVPAYSQHAIADHTLAIMLALIRRLHRAHDKVRLGDFDLDGLMGFDLNGKVAGVIGLGKIGRLVATRLKAFGCKVLGYDPYIQPEIVENVDLDTLITQADIISIHCPLTRENFHMFNEETFKRMKPGAILVNTARGGLIDTKALLEALKSGKLGGAALDVYEYERGLFFKNHQKEGIKDPYLAQLLGLANVVLTGHQAFLTREAVKNIEETTVENILEWQKNPQAKLKNEI
SEQ ID No.:2(D-LDHP101N)：
MKVIFFSMHPYEEEFLGPILPSDWDVEMTPDFLDETTVEKAKGAQVVSLFVSDKADGPVLEALHSYGVGLLALRSAGYDHIDIETAKRLGIKVVNVPAYSNHAIADHTLAIMLALIRRLHRAHDKVRLGDFDLDGLMGFDLNGKVAGVIGLGKIGRLVATRLKAFGCKVLGYDPYIQPEIVENVDLDTLITQADIISIHCPLTRENFHMFNEETFKRMKPGAILVNTARGGLIDTKALLEALKSGKLGGAALDVYEYERGLFFKNHQKEGIKDPYLAQLLGLANVVLTGHQAFLTREAVKNIEETTVENILEWQKNPQAKLKNEI
SEQ ID No.:3(D-LDHP101G)：
MKVIFFSMHPYEEEFLGPILPSDWDVEMTPDFLDETTVEKAKGAQVVSLFVSDKADGPVLEALHSYGVGLLALRSAGYDHIDIETAKRLGIKVVNVPAYSGHAIADHTLAIMLALIRRLHRAHDKVRLGDFDLDGLMGFDLNGKVAGVIGLGKIGRLVATRLKAFGCKVLGYDPYIQPEIVENVDLDTLITQADIISIHCPLTRENFHMFNEETFKRMKPGAILVNTARGGLIDTKALLEALKSGKLGGAALDVYEYERGLFFKNHQKEGIKDPYLAQLLGLANVVLTGHQAFLTREAVKNIEETTVENILEWQKNPQAKLKNEI
SEQ ID No.:4(D-LDH)：
MKVIFFSMHPYEEEFLGPILPSDWDVEMTPDFLDETTVEKAKGAQVVSLFVSDKADGPVLEALHSYGVGLLALRSAGYDHIDIETAKRLGIKVVNVPAYSPHAIADHTLAIMLALIRRLHRAHDKVRLGDFDLDGLMGFDLNGKVAGVIGLGKIGRLVATRLKAFGCKVLGYDPYIQPEIVENVDLDTLITQADIISIHCPLTRENFHMFNEETFKRMKPGAILVNTARGGLIDTKALLEALKSGKLGGAALDVYEYERGLFFKNHQKEGIKDPYLAQLLGLANVVLTGHQAFLTREAVKNIEETTVENILEWQKNPQAKLKNEI
SEQ ID No. 5 (D-LDH P101Q coding sequence for codon optimised sequence for Bacillus):
ATGAAAGTAATTTTTTTTTCTATGCACCCGTATGAAGAGGAATTTCTGGGTCCGATTCTGCCGTCTGACTGGGACGTAGAAATGACCCCGGACTTTCTGGACGAAACCACCGTGGAAAAGGCTAAAGGTGCCCAGGTAGTAAGCCTGTTTGTTTCTGACAAAGCTGATGGTCCGGTACTGGAAGCGCTGCATTCTTACGGTGTGGGCCTGCTGGCCCTGCGTTCTGCTGGCTATGATCACATCGATATTGAGACCGCAAAACGCCTGGGTATCAAAGTAGTTAACGTGCCAGCCTATTCTCAGCACGCTATCGCTGACCATACTCTGGCTATCATGCTGGCTCTGATTCGTCGTCTGCACCGTGCCCATGATAAAGTGCGCCTGGGTGATTTTGATCTGGATGGTCTGATGGGCTTTGATCTGAACGGCAAAGTTGCTGGTGTAATTGGTCTGGGTAAAATCGGTCGCCTGGTAGCTACCCGCCTGAAAGCGTTTGGTTGCAAAGTTCTGGGCTATGATCCATACATTCAGCCGGAAATCGTAGAAAACGTTGATCTGGATACCCTGATCACTCAGGCTGATATCATTTCTATTCATTGTCCGCTGACCCGTGAAAACTTTCATATGTTTAACGAAGAGACTTTTAAGCGTATGAAACCGGGTGCTATTCTGGTTAACACCGCGCGTGGTGGTCTGATCGATACCAAGGCCCTGCTGGAGGCCCTGAAGTCTGGTAAACTGGGCGGCGCAGCCCTGGATGTGTATGAATATGAACGTGGCCTGTTTTTTAAAAACCACCAAAAAGAAGGTATCAAAGACCCGTATCTGGCCCAGCTGCTGGGTCTGGCCAACGTAGTGCTGACCGGTCATCAGGCCTTTCTGACCCGTGAGGCTGTAAAAAACATCGAAGAAACTACCGTAGAAAACATTCTGGAATGGCAAAAGAACCCGCAGGCAAAGCTGAAAAACGAAATCTAA
SEQ ID No.6 (D-LDH P101N coding sequence for codon optimization sequence of Bacillus):
ATGAAAGTAATTTTTTTTTCTATGCACCCGTATGAAGAGGAATTTCTGGGTCCGATTCTGCCGTCTGACTGGGACGTAGAAATGACCCCGGACTTTCTGGACGAAACCACCGTGGAAAAGGCTAAAGGTGCCCAGGTAGTAAGCCTGTTTGTTTCTGACAAAGCTGATGGTCCGGTACTGGAAGCGCTGCATTCTTACGGTGTGGGCCTGCTGGCCCTGCGTTCTGCTGGCTATGATCACATCGATATTGAGACCGCAAAACGCCTGGGTATCAAAGTAGTTAACGTGCCAGCCTATTCTAACCACGCTATCGCTGACCATACTCTGGCTATCATGCTGGCTCTGATTCGTCGTCTGCACCGTGCCCATGATAAAGTGCGCCTGGGTGATTTTGATCTGGATGGTCTGATGGGCTTTGATCTGAACGGCAAAGTTGCTGGTGTAATTGGTCTGGGTAAAATCGGTCGCCTGGTAGCTACCCGCCTGAAAGCGTTTGGTTGCAAAGTTCTGGGCTATGATCCATACATTCAGCCGGAAATCGTAGAAAACGTTGATCTGGATACCCTGATCACTCAGGCTGATATCATTTCTATTCATTGTCCGCTGACCCGTGAAAACTTTCATATGTTTAACGAAGAGACTTTTAAGCGTATGAAACCGGGTGCTATTCTGGTTAACACCGCGCGTGGTGGTCTGATCGATACCAAGGCCCTGCTGGAGGCCCTGAAGTCTGGTAAACTGGGCGGCGCAGCCCTGGATGTGTATGAATATGAACGTGGCCTGTTTTTTAAAAACCACCAAAAAGAAGGTATCAAAGACCCGTATCTGGCCCAGCTGCTGGGTCTGGCCAACGTAGTGCTGACCGGTCATCAGGCCTTTCTGACCCGTGAGGCTGTAAAAAACATCGAAGAAACTACCGTAGAAAACATTCTGGAATGGCAAAAGAACCCGCAGGCAAAGCTGAAAAACGAAATCTAA
SEQ ID No.7 (D-LDH P101G coding sequence for codon optimised sequence for Bacillus):
ATGAAAGTAATTTTTTTTTCTATGCACCCGTATGAAGAGGAATTTCTGGGTCCGATTCTGCCGTCTGACTGGGACGTAGAAATGACCCCGGACTTTCTGGACGAAACCACCGTGGAAAAGGCTAAAGGTGCCCAGGTAGTAAGCCTGTTTGTTTCTGACAAAGCTGATGGTCCGGTACTGGAAGCGCTGCATTCTTACGGTGTGGGCCTGCTGGCCCTGCGTTCTGCTGGCTATGATCACATCGATATTGAGACCGCAAAACGCCTGGGTATCAAAGTAGTTAACGTGCCAGCCTATTCTGGGCACGCTATCGCTGACCATACTCTGGCTATCATGCTGGCTCTGATTCGTCGTCTGCACCGTGCCCATGATAAAGTGCGCCTGGGTGATTTTGATCTGGATGGTCTGATGGGCTTTGATCTGAACGGCAAAGTTGCTGGTGTAATTGGTCTGGGTAAAATCGGTCGCCTGGTAGCTACCCGCCTGAAAGCGTTTGGTTGCAAAGTTCTGGGCTATGATCCATACATTCAGCCGGAAATCGTAGAAAACGTTGATCTGGATACCCTGATCACTCAGGCTGATATCATTTCTATTCATTGTCCGCTGACCCGTGAAAACTTTCATATGTTTAACGAAGAGACTTTTAAGCGTATGAAACCGGGTGCTATTCTGGTTAACACCGCGCGTGGTGGTCTGATCGATACCAAGGCCCTGCTGGAGGCCCTGAAGTCTGGTAAACTGGGCGGCGCAGCCCTGGATGTGTATGAATATGAACGTGGCCTGTTTTTTAAAAACCACCAAAAAGAAGGTATCAAAGACCCGTATCTGGCCCAGCTGCTGGGTCTGGCCAACGTAGTGCTGACCGGTCATCAGGCCTTTCTGACCCGTGAGGCTGTAAAAAACATCGAAGAAACTACCGTAGAAAACATTCTGGAATGGCAAAAGAACCCGCAGGCAAAGCTGAAAAACGAAATCTAA
SEQ ID No. 8 (D-LDH coding sequence for codon optimization sequence of Bacillus):
ATGAAAGTAATTTTTTTTTCTATGCACCCGTATGAAGAGGAATTTCTGGGTCCGATTCTGCCGTCTGACTGGGACGTAGAAATGACCCCGGACTTTCTGGACGAAACCACCGTGGAAAAGGCTAAAGGTGCCCAGGTAGTAAGCCTGTTTGTTTCTGACAAAGCTGATGGTCCGGTACTGGAAGCGCTGCATTCTTACGGTGTGGGCCTGCTGGCCCTGCGTTCTGCTGGCTATGATCACATCGATATTGAGACCGCAAAACGCCTGGGTATCAAAGTAGTTAACGTGCCAGCCTATTCTCCGCACGCTATCGCTGACCATACTCTGGCTATCATGCTGGCTCTGATTCGTCGTCTGCACCGTGCCCATGATAAAGTGCGCCTGGGTGATTTTGATCTGGATGGTCTGATGGGCTTTGATCTGAACGGCAAAGTTGCTGGTGTAATTGGTCTGGGTAAAATCGGTCGCCTGGTAGCTACCCGCCTGAAAGCGTTTGGTTGCAAAGTTCTGGGCTATGATCCATACATTCAGCCGGAAATCGTAGAAAACGTTGATCTGGATACCCTGATCACTCAGGCTGATATCATTTCTATTCATTGTCCGCTGACCCGTGAAAACTTTCATATGTTTAACGAAGAGACTTTTAAGCGTATGAAACCGGGTGCTATTCTGGTTAACACCGCGCGTGGTGGTCTGATCGATACCAAGGCCCTGCTGGAGGCCCTGAAGTCTGGTAAACTGGGCGGCGCAGCCCTGGATGTGTATGAATATGAACGTGGCCTGTTTTTTAAAAACCACCAAAAAGAAGGTATCAAAGACCCGTATCTGGCCCAGCTGCTGGGTCTGGCCAACGTAGTGCTGACCGGTCATCAGGCCTTTCTGACCCGTGAGGCTGTAAAAAACATCGAAGAAACTACCGTAGAAAACATTCTGGAATGGCAAAAGAACCCGCAGGCAAAGCTGAAAAACGAAATCTAA
The invention discovers that amino acid residues which do not directly act on the combination of a substrate and a cofactor but play a key role in enzyme catalysis exist in the D-lactate dehydrogenase, and further screens out a site and a substituted amino acid which are most suitable for point mutation, wherein the site is positioned at the first amino acid of the D-lactate dehydrogenase cofactor binding domain connected with a hinge region, and the amino acid at the site is mutated into polar amino acid, acidic amino acid or basic amino acid, so that the catalytic activity, specific enzyme activity and catalytic efficiency of the wild-type D-lactate dehydrogenase can be remarkably improved, and the D-lactate dehydrogenase can be more advantageously used for producing D-lactate.
Thus, in a first aspect of the present invention, there is provided a D-lactate dehydrogenase mutant characterized by an amino acid sequence having, on the basis of the amino acid sequence of a wild-type D-lactate dehydrogenase, a mutation wherein the wild-type D-lactate dehydrogenase comprises a substrate-binding domain, a cofactor-binding domain and a hinge region joining both, said mutation being: the first amino acid of the cofactor binding domain linked to the hinge region is mutated to any one of a polar amino acid, an acidic amino acid, or a basic amino acid, and the D-lactate dehydrogenase mutant has a function of catalyzing the production of D-lactate from a substrate or a salt thereof.
The D-lactate dehydrogenase mutant of the present invention has a function of generating D-lactate and NAD (P) + using pyruvic acid (or pyruvate) and NAD (P) H (i.e., NADPH or NADH), which have a catalytic activity of catalyzing the formation of D-lactate or a salt thereof from a substrate at 30℃to 100 ℃.
The D-lactate includes, but is not limited to, sodium, magnesium, potassium and calcium salts of D-lactate, the type of D-lactate being determined by the type of substrate pyruvate.
The hinge region is the region of the protein structure of the wild-type D-lactate dehydrogenase and the D-lactate dehydrogenase mutant responsible for linking the substrate binding domain and the cofactor binding domain. For example, amino acids 95 to 100 of the amino acid sequences shown in SEQ ID No. 1 (mutant), SEQ ID No.2 (mutant), SEQ ID No. 3 (mutant) and SEQ ID No. 4 (wild type) are hinge regions.
The substrate binding domain is a domain binding to a substrate in the protein structure of wild-type D-lactate dehydrogenase and D-lactate dehydrogenase mutants. For example, amino acids 1 to 94 of the amino acid sequences shown in SEQ ID No. 1 (mutant), SEQ ID No. 2 (mutant), SEQ ID No. 3 (mutant) and SEQ ID No. 4 (wild type) are substrate binding domains.
The cofactor binding domains are domains binding to cofactors in the protein structures of wild-type D-lactate dehydrogenase and D-lactate dehydrogenase mutants. For example, amino acids 101 to 288 and 296 to 352 of the amino acid sequence shown in SEQ ID No. 1 (mutant), SEQ ID No. 2 (mutant), SEQ ID No. 3 (mutant) or SEQ ID No. 4 (wild-type) are cofactor binding domains.
The first amino acid of the cofactor binding domain is the first amino acid in the cofactor binding domain to which the hinge region in the protein structure of the wild-type D-lactate dehydrogenase and the D-lactate dehydrogenase mutant is attached.
In some embodiments of the invention, the amino acid sequence of the wild-type D-lactate dehydrogenase is as shown in SEQ ID No. 4 or has more than 30% identity to SEQ ID No. 4 and the mutation is at amino acid 101.
It will be appreciated by those skilled in the art that the amino acid sequences of wild-type D-lactate dehydrogenase in different species may be identical or may be different. Thus, the wild-type D-lactate dehydrogenase of the present invention may be other amino acid sequences as long as the mutation site is located at the first amino acid of the cofactor binding domain associated with the hinge region.
In some embodiments of the invention, the polar amino acid comprises a serine, threonine, cysteine, tyrosine, glutamine, or asparagine. Acidic amino acids include aspartic acid and glutamic acid. Basic amino acids include lysine, arginine, and histidine.
In a preferred embodiment of the invention, the mutated amino acid is mutated to glutamine or asparagine.
In some embodiments of the invention, the N-and/or C-terminus of the D-lactate dehydrogenase mutant has a tag for purification and/or detection, and the tag does not affect the catalytic performance of the D-lactate dehydrogenase mutant. The tag may be a fluorescent tag.
In a preferred embodiment of the invention, the amino acid sequence of the D-lactate dehydrogenase mutant is as shown in SEQ ID No.1 or SEQ ID No.2, or has more than 30% identity with SEQ ID No.1 or SEQ ID No. 2.
The protein having a certain sequence identity with a given protein according to the present invention should have the same or similar function as the given protein, and in particular, should be understood to have a catalytic ability to catalyze a substrate to produce D-lactic acid, more particularly, a function to produce D-lactic acid (D-lactate) and NAD (P) + using pyruvic acid (pyruvate) and NAD (P) H.
In a second aspect of the invention, there is provided a DNA comprising a nucleotide sequence encoding a D-lactate dehydrogenase mutant according to the invention.
In a preferred embodiment of the invention, the DNA contains a nucleotide sequence as shown in SEQ ID No. 5 or SEQ ID No. 6 or a nucleotide sequence having more than 30% identity to SEQ ID No. 5 or SEQ ID No. 6.
The invention also provides an expression vector comprising a DNA according to the invention operably linked to a promoter. By operably linking a promoter sequence to the DNA, the promoter sequence can direct the expression of the corresponding protein. In general, the expression vector used in the genetic engineering technique may be in the form of a plasmid, but the expression vector may be in other known forms.
The invention also provides a host cell comprising an expression vector according to the invention, or having integrated in its genome a nucleotide sequence encoding a D-lactate dehydrogenase mutant according to the invention.
In the case of an expression vector according to the invention, the host cell may have been introduced into a cell in which the expression vector according to the invention has been introduced. The cell may be, for example, a prokaryotic cell, which may be used, for example, to rapidly produce large amounts of the expression vectors of the invention. Host cells may also be transiently or stably transformed with the expression vectors of the invention. Transformation of the expression vector into a cell may be accomplished by any technique known in the art, including, but not limited to: standard bacterial transformation, calcium phosphate co-precipitation or electroporation.
In a preferred embodiment, the host cell has integrated into its genome a nucleotide sequence encoding a D-lactate dehydrogenase mutant according to the present invention. The host cells include geobacillus thermoclucoside (Geobacillus thermoglucosidasius) and bacillus licheniformis (Bacillus licheniformis).
The invention also provides a method for preparing the D-lactate dehydrogenase mutant, which comprises the step of obtaining the D-lactate dehydrogenase mutant by a genetic engineering method or an artificial synthesis method.
In some embodiments of the invention, the wild-type D-lactate dehydrogenase may be engineered in a targeted manner using genetic engineering methods to obtain the D-lactate dehydrogenase mutants according to the invention. For example, the 101 st amino acid of the amino acid sequence shown in SEQ ID No. 3 or SEQ ID No. 4 may be mutated to glutamine and asparagine, respectively, to give the amino acid sequences shown in SEQ ID No. 1 and SEQ ID No. 2, or the 101 st amino acid of the nucleotide sequence shown in SEQ ID No. 7 or SEQ ID No. 8 may be mutated to glutamine and asparagine, respectively, to give the coding sequences shown in SEQ ID No. 5 and SEQ ID No. 6. The technical method and preparation procedure used are, for example, constructing a vector plasmid containing the wild-type D-lactate dehydrogenase gene by using techniques known in the art, then selecting site-directed mutagenesis sites and mutated amino acid species, synthesizing corresponding primers, amplifying mutant DNA fragments by PCR using the vector plasmid containing the wild-type D-lactate dehydrogenase gene as a template, separating and purifying, and amplifying the obtained fragments into full-length mutant genes by PCR. Positive clones of D-lactate dehydrogenase mutants were screened by cloning the full-length mutant gene onto an appropriate vector and transforming into appropriate host cells, followed by culture. Finally, plasmid DNA is extracted from the positive clones and subjected to DNA sequencing analysis to determine the mutations introduced.
In the method for the directed modification of D-lactate dehydrogenase of the present invention, any suitable vector may be used. For example, suitable vectors include, but are not limited to, vector pETDuet-1.
In the method for the directed engineering of D-lactate dehydrogenase of the present invention, the obtained D-lactate dehydrogenase mutant gene may be expressed in a prokaryotic cell or eukaryotic cell, or may be expressed outside a prokaryotic cell or eukaryotic cell by any suitable method known in the art.
In the method for the directed engineering of D-lactate dehydrogenase of the present invention, the microbial host cell of the vector may be a prokaryotic cell or a eukaryotic cell. Such prokaryotic microorganisms include, but are not limited to, E.coli (ESCHERICHIA COLI), bacillus coagulans (Bacillus coagulans), bacillus subtilis (Bacillus subtilis), geobacillus (Geobacillus), bacillus smithii (Bacillus smithii), bacillus megaterium (Bacillus magaterium), and Streptomyces (Streptomyces). Such eukaryotic microorganisms include, but are not limited to, saccharomyces cerevisiae (Saccharomyces cerevisiae) and Pichia pastoris (Pichia pastoris).
The invention also provides the use of the D-lactate dehydrogenase mutants, DNA, expression vectors or host cells according to the invention in enzyme-catalyzed production, biomedical detection, biosensor and microbial D-lactate production.
In some embodiments of the invention, the catalytic performance of the D-lactate dehydrogenase mutants according to the invention is significantly improved compared to the wild-type D-lactate dehydrogenase, which benefits from the smart design and selection of mutation sites and mutant amino acids. In particular, the D-lactate dehydrogenase mutant according to the present invention has one or more properties of catalytic activity (k cat), specific activity (number of units of enzyme activity per unit mass of protein) and catalytic efficiency (k cat/Km) that are significantly higher than those of the wild-type D-lactate dehydrogenase.
In some embodiments of the invention, the catalytic activity (k cat) of the D-lactate dehydrogenase mutant according to the invention is more than 3 times the catalytic activity (k cat) of the wild-type D-lactate dehydrogenase, in particular the catalytic activity (k cat) of the D-lactate dehydrogenase mutant according to the invention is 3 to 30 times, e.g. 5 to 25 times, the catalytic activity (k cat) of the wild-type D-lactate dehydrogenase.
In some embodiments of the invention, the specific activity of the D-lactate dehydrogenase mutant according to the invention is more than 3 times the specific activity of the wild-type D-lactate dehydrogenase, in particular, the specific activity of the D-lactate dehydrogenase mutant according to the invention is 3 to 30 times, e.g., 5 to 25 times the specific activity of the wild-type D-lactate dehydrogenase.
In some embodiments of the invention, the catalytic efficiency (k cat/Km) of the D-lactate dehydrogenase mutant according to the invention is more than 2 times the catalytic efficiency (k cat/Km) of the wild-type D-lactate dehydrogenase, in particular the catalytic efficiency (k cat/Km) of the D-lactate dehydrogenase mutant according to the invention is 2 to 15 times, e.g. 3 to 10 times, the catalytic efficiency (k cat/Km) of the wild-type D-lactate dehydrogenase.
In some embodiments of the invention, the D-lactate dehydrogenase mutants according to the invention have good thermostability. In particular, the D-lactate dehydrogenase mutants according to the present invention are maintained at higher temperatures for longer periods of time while still maintaining higher levels of enzyme activity. For example, the D-lactate dehydrogenase mutant according to the present invention is capable of maintaining at least 60% to 100% of the initial enzyme activity after being maintained at 50℃for 0 to 70 hours (h).
In some embodiments of the invention, the D-lactate dehydrogenase mutants according to the invention are highly symmetrical tetrameric structures.
The present invention also provides a method for producing D-lactic acid, comprising contacting the D-lactate dehydrogenase mutant according to the present invention with a substrate and a cofactor for the production of D-lactic acid.
Preferably, the substrate is pyruvic acid or a salt thereof and the cofactor is NAD (P) H.
The method for producing D-lactic acid may be performed in vitro. In this case, the D-lactate dehydrogenase mutant is used as a substrate for pyruvic acid or a salt thereof at 30℃to 100℃and reacted in the presence of NAD (P) H for 0 to 70 hours, thereby producing D-lactate.
The method for producing D-lactic acid may also be carried out by a microbial fermentation method. In this case, the coding sequence of the D-lactate dehydrogenase mutant is introduced into a microorganism for expression and fermentation culture, thereby producing D-lactic acid. The D-lactate dehydrogenase mutant may be expressed in the form of a plasmid, or the expression may be performed by integrating a nucleotide sequence encoding the D-lactate dehydrogenase mutant into the genome of the microorganism.
In some embodiments of the invention, a method of producing D-lactic acid comprises providing seeds of a microorganism expressing the D-lactate dehydrogenase mutant at 30℃to 70℃and then inoculating the seeds of the microorganism into a fermentation medium for fermentation culture at an inoculum size with an OD 620nm value of 0.2 to 0.8. OD 620nm is the absorbance of the sample at 620nm, and can be used as an index of the cell density in the sample.
The microorganism is preferably geobacillus thermoclucoside (Geobacillus thermoglucosidasius) or bacillus licheniformis (Bacillus licheniformis).
The fermentation medium may comprise one or more of yeast powder, metal ions, biotin, betaine, peanut meal, a carbon source, and a neutral protease.
The carbon source may include one or more of glucose, sucrose, glycerol, xylose, arabinose, and mannitol. The initial concentration of the carbon source may be 80g/L to 120g/L.
In some embodiments, fed-batch fermentation strategies are used. When the concentration of the carbon source is reduced to 0.0g/L to 40.0g/L, the carbon source is replenished so that the concentration thereof is maintained in the range of 0 to 100 g/L.
In some embodiments of the invention, the temperature of fermentation may be from 30 ℃ to 70 ℃, and the skilled person can adjust the fermentation temperature according to the specific fermentation strain used. The fermentation time may be 40 hours to 70 hours.
In a preferred embodiment of the present invention, the time of fermentation may be 45 hours to 60 hours.
The pH of the fermentation culture may be 6.5 to 7.5 and the stirring rate may be 50rpm to 150rpm.
In some embodiments of the invention, the substrate may be fermented using fermentation vessels (e.g., fermenters) known in the art.
In some embodiments of the invention, the fermentation product may be isolated and purified. Isolation and purification of the fermentation product may be performed using methods known in the art.
The present invention will be described in further detail with reference to examples of the present invention. The following examples are illustrative, not limiting, and should not be taken as limiting the scope of the invention. The examples of the present invention are implemented on the premise of the technical scheme of the present invention, and detailed embodiments and specific operation procedures are given, but the scope of the present invention is not limited to the following examples.
Examples
Experimental methods (such as PCR amplification, transformation, gene insertion, protein purification, etc.) without specifying specific conditions in the examples are generally performed according to conventional methods and conditions, for example, molecular cloning of Sambrook et al: the methods and conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) are carried out or are carried out according to the conditions recommended by the manufacturer.
The detection conditions and the method of the high performance liquid chromatography system are as follows: a high performance liquid chromatography system (Agilent 1260 series, hewlett-Packard, USA) was equipped with a Bio-Rad Aminex HPX-87H chromatography column (300X 7.8 mM) and a differential refractive detector (DIFFERENTIAL REFRACTIVE index detector, RID), column temperature 55 ℃, flow rate 0.5mL/min, mobile phase 5mM sulfuric acid, sample loading 10. Mu.L.
The pETDuet-1 vector (from Novagen) is commercially available.
Materials, reagents and the like used in the examples are commercially available unless otherwise specified.
EXAMPLE 1 directed engineering, expression and purification of D-lactate dehydrogenase
In example 1, wild-type D-lactate dehydrogenase D-LDH, D-lactate dehydrogenase mutant D-LDH P101Q、D-LDHP101N according to the invention and comparative D-lactate dehydrogenase mutant D-LDH P101G were prepared. Wherein the amino acid sequence of the D-LDH is shown as SEQ ID No. 4; D-LDH P101Q is obtained by mutating proline at position 101 of D-LDH into glutamine, and the amino acid sequence is shown as SEQ ID No. 1; D-LDH P101N is obtained by mutating proline at position 101 of D-LDH into asparagine, and the amino acid sequence is shown as SEQ ID No. 2; D-LDH P101G is obtained by mutating proline at position 101 of D-LDH into glycine, and the amino acid sequence is shown as SEQ ID No. 3.
The primer sequences used in this example are as follows:
ldh-F：CGCGGATCCGATGAAAGTAATTTTTTTTTCTATGCAC(SEQ ID
No.:9)
ldh-R：CCCAAGCTTTTAGATTTCGTTTTTCAGCTTTG(SEQ ID No.:10)
ldh1-R：TAGCGTGCTGAGAATAGG(SEQ ID No.:11)
ldh1-F：CCTATTCTCAGCACGCTA(SEQ ID No.:12)
ldh2-R：TAGCGTGCCCAGAATAGG(SEQ ID No.:13)
ldh2-F：CCTATTCTGGGCACGCTA(SEQ ID No.:14)
ldh3-R：TAGCGTGGTTAGAATAGGCTG(SEQ ID No.:15)
ldh3-F：CAGCCTATTCTAACCACGCTA(SEQ ID No.:16)
The preparation process comprises the following steps:
The original D-ldh gene was amplified by Polymerase Chain Reaction (PCR) using the genome of Bacillus licheniformis BJQ using primer pairs ldh-F (BamHI) and ldh-R (HindIII) and BamHI and HindIII restriction sites were introduced; the vector pETDuet-1 was digested with BamHI and HindIII, and then the D-LDH gene was cloned into the BamHI-HindIII site of the Multiple Cloning Site (MCS) I of pETDuet-1 to generate pETDuet-LDH for expression and purification of D-LDH.
The mutant gene D-LDH1 (CCG/CAG, encoding protein D-LDH P101Q) was amplified using the primer pairs LDH-F, LDH-R and LDH1F, LDH-R using the D-LDH gene as template using the scissoring overlap extension polymerase chain reaction technique (SOE-PCR), and BamHI and HindIII restriction sites were introduced; the vector pETDuet-1 was digested with BamHI and HindIII, and then the D-LDH1 gene was cloned into the BamHI-HindIII site of pETDuet-1's Multiple Cloning Site (MCS) I to generate pETDuet-LDH1 for expression and purification of D-LDH P101Q.
The mutant gene D-LDH2 (CCG/GGG, encoding protein D-LDH P101G) was amplified using the primer pairs LDH-F, LDH2-R and LDH2-F, LDH-R using the D-LDH gene as a template using the scissoring overlap extension polymerase chain reaction technique (SOE-PCR), and BamHI and HindIII restriction sites were introduced; the vector pETDuet-1 was digested with BamHI and HindIII, and then the D-LDH2 gene was cloned into the BamHI-HindIII site of the Multiple Cloning Site (MCS) I of pETDuet-1 to generate pETDuet-LDH2 for expression and purification of D-LDH P101G.
The mutant gene D-LDH3 (CCG/AAC, encoding protein D-LDH P101N) was amplified using the primer pairs LDH-F, LDH3-R and LDH3-F, LDH-R using the D-LDH gene as a template using the scissoring overlap extension polymerase chain reaction technique (SOE-PCR), and BamHI and HindIII restriction sites were introduced; the vector pETDuet-1 was digested with BamHI and HindIII, and then the D-LDH3 gene was cloned into the BamHI-HindIII site of the Multiple Cloning Site (MCS) I of pETDuet-1 to generate pETDuet-LDH3 for expression and purification of D-LDH P101N.
Respectively introducing pETDuet-ldh, pETDuet-ldh1, pETDuet-ldh2 and pETDuet-ldh3 into an escherichia coli BL21 (DE 3) strain (purchased from Novagen company) to obtain four escherichia coli recombinant strains; four E.coli recombinant strains were cultured in seed medium (composition: 10g L -1 sodium chloride, 5g L -1 yeast powder, 10g L -1 peptone) containing ampicillin (100. Mu.g mL -1) at 37℃and 200 rpm. When the cell density OD 620nm reached 0.6 to 0.8, expression of recombinant D-LDH was induced by addition of 0.4mM isopropyl- β -D-thiogalactoside, which was incubated at 16 ℃ for about 20 hours; the collected cells were washed twice with 10mM phosphate buffer, and then the cell pellet was resuspended in equilibration buffer (25 mM tris hydrochloride, 500mM sodium chloride, 25mM imidazole, pH 8.0) and disrupted by high pressure treatment at 4 ℃; the lysed cells were centrifuged at 12,000rpm for 40 minutes at 4℃and the supernatant was then used for subsequent purification.
The D-lactate dehydrogenase and each mutant were purified by using a nickel column purification method. Specifically, D-lactate dehydrogenase and each mutant were purified from the above crude extract by affinity chromatography using a Ni 2+ -NTA column, which had been equilibrated with 30mL of equilibration buffer; D-LDH was washed and eluted by using wash buffer (25 mM tris hydrochloride, 500mM sodium chloride, 80mM imidazole, pH 8.0) and elution dissolution buffer (25 mM tris hydrochloride, 500mM sodium chloride, 300mM imidazole, pH 8.0); the protein solution of interest was then transferred to an Amicon Ultra-15 30K centrifugal ultrafiltration tube for further protein concentration and finally passed through a column packed with Superdex 200 10/300GLPurifier gel filtration desalting. The gel filtration buffer was 10mM phosphate buffer (pH 7.0); expressed and purified enzymes were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), all purification procedures were performed at 4 ℃ to ensure enzyme activity, and the purified enzyme was snap frozen in liquid nitrogen and then stored at-80 ℃ for further study experiments.
Based on SDS-PAGE test resultsPurifier gel filtration chromatography gave D-LDH, D-LDH P101Q、D-LDHP101N and D-LDH P101G each having a molecular weight of about 35kD and a tetrameric structure.
Example 2.D-enzymatic kinetic Properties of lactate dehydrogenase and mutants
The method for detecting the enzyme catalytic performance of the D-lactate dehydrogenase and the mutant comprises the following steps: the total volume of the reaction mixture was 0.8mL, which contained: 10mM phosphate buffer (pH 7.0), an appropriate amount (0.0001 mg/L to 0.0035 mg/L) of purified D-lactate dehydrogenase or mutant, 0.25mM NADH, sodium pyruvate at a concentration of 0.01mM to 0.4mM or sodium pyruvate at a concentration of 0.625mM, NADH at a concentration of 0.005mM to 0.2 mM. In this detection method, the reaction conditions used for the wild-type D-lactate dehydrogenase and the mutant are the same. The measurement temperature was 50 ℃. The catalytic performance of the D-lactate dehydrogenase and mutants was determined based on measuring the change in the initial rate of absorbance at 340nm, which corresponds to the oxidation of NADH (ε340=6220M -1cm-1).
According to the results of the above detection method, the catalytic activity (k cat), specific enzyme activity and catalytic efficiency were calculated, and the calculation method was the same as that conventionally used in the art.
Through the above detection, the enzymatic kinetic properties of D-lactate dehydrogenase and mutants were characterized, and the data are summarized in Table 1 below.
[ Table 1]D-lactic dehydrogenase and enzyme kinetic Properties of mutants ]
From the results in Table 1, it can be seen that the catalytic performance is significantly improved when the D-LDH is mutated from proline to glutamine at position 101 to obtain mutant D-LDH P101Q. Specifically, in the case where the substrate is sodium pyruvate, the catalytic activity (k cat) of D-LDH P101Q is about 22 times that of D-LDH, the specific enzyme activity is about 22 times that of D-LDH, and the catalytic efficiency (k cat/Km) is about 5 times that of D-LDH; in the case of NADH as substrate, the catalytic activity (k cat) of D-LDH P101Q is about 11 times that of D-LDH, the specific enzyme activity is about 11 times that of D-LDH, and the catalytic efficiency (k cat/Km) is about 6 times that of D-LDH.
Similarly, when proline at position 101 of D-LDH is mutated to asparagine, the catalytic performance is significantly improved when the mutant D-LDH P101N is obtained. Specifically, in the case where the substrate is sodium pyruvate, the catalytic activity (k cat) of D-LDH P101N is about 16 times that of D-LDH, the specific enzyme activity is about 16 times that of D-LDH, and the catalytic efficiency (k cat/Km) is about 3.5 times that of D-LDH; in the case of NADH as substrate, the catalytic activity (k cat) of D-LDH P101N is about 8 times that of D-LDH, the specific enzyme activity is about 8 times that of D-LDH, and the catalytic efficiency (k cat/Km) is about 8 times that of D-LDH.
However, when proline at position 101 of D-LDH was mutated to glycine to obtain mutant D-LDH P101G, the catalytic activity (k cat) and specific enzyme activity were only slightly increased (less than 2-fold) compared to D-LDH, whereas the catalytic efficiency (k cat/Km) was even lower than that of D-LDH.
This shows that the mutation sites and the kind of mutation target amino acid selected in the present invention can significantly improve the catalytic performance of the resulting D-lactate dehydrogenase mutant, which is the result of creative efforts. When the amino acid is mutated into glycine, the desired effect of improving the catalytic performance is not obtained.
Example 3 thermostability of D-lactate dehydrogenase and mutant
The method for detecting the thermal stability of the D-lactate dehydrogenase and the mutant comprises the following steps: the D-lactate dehydrogenase or a mutant thereof was subjected to a water bath at a temperature of 50℃and sampled at various time points over a period of 0 to 70 hours for measurement of enzyme activity.
The specific method comprises the following steps: 0.004mg/L of D-lactate dehydrogenase or mutant (ensuring that the final concentrations of the different D-lactate dehydrogenases or mutants in the reaction system are consistent) was added to the reaction system, and the total volume of the reaction mixture was 0.8mL containing 10mM PBS (pH 7.0), 0.25mM NADH and 0.625mM sodium pyruvate, measured at 50 ℃. The enzyme activity measured when the D-lactate dehydrogenase or mutant was incubated for 0 hour was defined as 100% (initial enzyme activity), and the enzyme activities measured by sampling at other time points were expressed as percentages relative to the initial enzyme activity.
The thermostability data of the D-lactate dehydrogenase and mutants are summarized in Table 2 below.
[ Table 2]D-lactate dehydrogenase and thermal stability of mutant at 50 ]
From the results in Table 2, it was found that, for the D-lactate dehydrogenase mutant D-LDH P101Q according to the present invention, the thermostability at 50℃was very good, the enzyme activity of the D-LDH P101Q protein was relatively slightly increased from the initial stage over the incubation period of 0 to 9 hours, and gradually decreased from the initial stage over the period of 9 to 67 hours, and finally 60.53% of the initial enzyme activity was maintained at a relatively high enzyme activity level. This indicates that D-LDH P101Q has good thermostability.
For the D-lactate dehydrogenase mutant D-LDH P101N according to the invention, the heat stability at 50℃is very good, and the enzyme activity is maintained at a relatively high level of enzyme activity throughout the heating period of 0 to 67 hours, and the final enzyme activity is maintained at 100.73% of the initial enzyme activity. This indicates that D-LDH P101N has excellent thermostability.
It will be appreciated that in performing the comparative analysis of the thermostability of the enzymes, the comparison of each enzyme itself over a respective different time period is of biological value, whereas the comparison between the enzymes is of no biological value, since the initial enzyme activity of each enzyme is different, and the remaining time points are all relative enzyme activities calculated from the initial enzyme activities of the respective enzymes.
Example 4.D Single Crystal Structure of lactate dehydrogenase mutant D-LDH P101Q
The crystallization, data collection, processing and structure analysis methods of the D-lactate dehydrogenase and the mutant are as follows: crystals of D-LDH P101Q were grown by using hanging drop vapor diffusion from a 1:1 (v/v) mixture of protein solution of D-LDH P101Q (10 mM phosphate buffer, pH 7.0) and storage solution (0.1M Bis-Tris (pH 5.7), 0.2M ammonium acetate and 47% (v/v) (+/-) -2-methyl-2, 4-pentanediol) at 20℃and after growing the crystals, they were quickly fished out using a loop and placed in a cryoprotectant, wherein the cryoprotectant was 25% (v/v) glycerol was added to the above crystallization solution, and then placed in a Puck freezer in liquid nitrogen for quick freezing.
Collecting all at 100K at Shanghai Synchrotron Radiation Facility (SSRF) BL19U1 line station using Dectris PILATUS3 6M detectorWavelength data is processed and scaled by HKL3000 software, a molecular replacement method is adopted, a D-LDH structure of pseudomonas aeruginosa (Pseudomonas aeruginosa) PAO1 is taken as a search model, a program Phaser is used for analyzing the crystal structure of D-lactate dehydrogenase, and the structure optimization is carried out by Coot and Refmac5 programs.
By the method, the resolution is obtainedD-LDH P101Q crystal structure of (a). The results show that D-LDH P101Q is a tetrameric structure with high symmetry.
Example 5 fed-batch fermentation production of D-lactic acid Using recombinant Geobacillus thermoglucosides (Geobacillus thermoglucosidasius) containing the D-LDH P101Q Gene
The nucleotide sequence (SEQ ID No. 5) encoding the D-LDH P101Q mutant protein was integrated into Geobacillus thermoglucosides, thus obtaining recombinant Geobacillus thermoglucosides. Recombinant Geobacillus thermocellum was cultured in fermentation seed medium A (ingredients: 5g L -1 soytone, 15g L -1 peptone, 5g L -1 sodium chloride) at 60℃overnight at 200rpm to achieve an OD 620nm value of 2.0 to 8.0.
The fermentation seed inoculum size may be 0.3% (v/v) to 10% (v/v). In this example, fermentation seeds were inoculated at 10% (v/v) into fermentation medium A (composition: 5g L -1 yeast powder, 3g L -1 disodium hydrogen phosphate, 3g L -1 potassium dihydrogen phosphate, 1g L -1 ammonium chloride, 0.48g L -1 magnesium sulfate, 0.5g L -1 sodium chloride, 0.42g L -1 citric acid, 0.028g L -1 ferrous sulfate heptahydrate, 0.01g L -1 thiamine, metal ion mixtures (4.4 mg L -1 nickel sulfate hexahydrate, 2.86mg L -1 boric acid, 1.81mg L -1 manganese chloride tetrahydrate, 0.39mg of L -1 sodium molybdate dihydrate, 0.222mg of L -1 zinc sulfate heptahydrate, 0.079mg of L -1 copper sulfate pentahydrate, 0.049mg of L -1 cobalt nitrate hexahydrate), 3.1mg L -1 biotin, 0.1g L -1 betaine). The fermentation conditions are as follows: A5L fermenter was used, the pH was maintained at 7.0 using 25% (w/v) calcium hydroxide, the stirring speed was 80rpm, and the incubation temperature was 60 ℃. The initial glucose concentration was 100g L -1, and glucose was added to the fermenter to maintain the glucose concentration in the range of 0 to 70g/L for 48 hours when the glucose concentration was 0.0g/L to 40.0g/L during fermentation.
The fermentation broth was boiled at 100℃for 10 minutes, then 2mL was taken and placed in a 100mL volumetric flask, acidolysis was performed with 2mL of 2M sulfuric acid for 10 minutes, then the volume was set to 100mL, and finally the above solution was centrifuged at 8,000rpm for 10 minutes and filtered with a 0.22 μm syringe aqueous filter, and then D-lactic acid was detected by high performance liquid chromatography.
According to the detection result of high performance liquid chromatography, D-lactic acid titer in the fermentation broth obtained after 48 hours of fermentation was 153.07g L -1, D-lactic acid yield was 93.04%, and D-lactic acid productivity was 3.189g L -1h-1.
Example 6 production of D-lactic acid by fed-batch fermentation with recombinant Bacillus licheniformis (Bacillus licheniformis) comprising the D-LDH P101Q Gene
The nucleotide sequence (SEQ ID No. 5) encoding the D-LDH P101Q mutant protein was integrated into B.licheniformis to obtain recombinant B.licheniformis. The recombinant Bacillus licheniformis was cultivated in fermentation seed medium B (composition: 100g L -1 glucose, 10g L -1 yeast powder, 5g L -1 peptone, 50g L -1 calcium carbonate) at 50℃overnight at rest to an OD 620nm value of 2.0 to 8.0.
The fermentation seeds were inoculated into fermentation medium B (composition: 40g L -1 peanut meal, 100g L -1 glucose, 0.3g L -1 neutral protease) at 10% (v/v) for fermentation culture. The fermentation conditions are as follows: A5L fermenter was used, the pH was maintained at 7.0 using 25% (w/v) calcium hydroxide, the stirring speed was 80rpm, and the incubation temperature was 50 ℃. The initial glucose concentration was 100g/L, and glucose was added to the fermenter to maintain the glucose concentration in the range of 0 to 100g/L for 60 hours when the glucose concentration was 0.0g/L to 40.0g/L during fermentation.
The fermentation broth was boiled at 100℃for 10 minutes, then 2mL was taken and placed in a 100mL volumetric flask, acidolysis was performed with 2mL of 2M sulfuric acid for 10 minutes, then the volume was set to 100mL, and finally the above solution was centrifuged at 8,000rpm for 10 minutes and filtered with a 0.22 μm syringe aqueous filter, and then D-lactic acid was detected by high performance liquid chromatography.
According to the detection result of high performance liquid chromatography, D-lactic acid titer in the fermentation broth obtained by fermenting for 60 hours was 145.91g L -1, D-lactic acid yield was 86.00%, and D-lactic acid productivity was 2.43g L -1h-1.
Example 7 production of D-lactic acid by fed-batch fermentation with recombinant Bacillus licheniformis (Bacillus licheniformis) containing the D-LDH Gene
The nucleotide sequence encoding the D-LDH protein (SEQ ID No. 8) was integrated into Bacillus licheniformis to thereby obtain recombinant Bacillus licheniformis. The recombinant Bacillus licheniformis was cultivated in fermentation seed medium B (composition: 100g L -1 glucose, 10g L -1 yeast powder, 5g L -1 peptone, 50g L -1 calcium carbonate) at 50℃overnight at rest to an OD 620nm value of 2.0 to 8.0.
The fermentation seeds were inoculated into fermentation medium B (composition: 40g L -1 peanut meal, 100g L -1 glucose, 0.3g L -1 neutral protease) at 10% (v/v) for fermentation culture. The fermentation conditions are as follows: A5L fermenter was used, the pH was maintained at 7.0 using 25% (w/v) calcium hydroxide, the stirring speed was 80rpm, and the incubation temperature was 50 ℃. The initial glucose concentration was 100g/L, and glucose was added to the fermenter to maintain the glucose concentration in the range of 0 to 100g/L for 60 hours when the glucose concentration was 0.0g/L to 40.0g/L during fermentation.
The fermentation broth was boiled at 100℃for 10 minutes, then 2mL was taken and placed in a 100mL volumetric flask, acidolysis was performed with 2mL of 2M sulfuric acid for 10 minutes, then the volume was set to 100mL, and finally the above solution was centrifuged at 8,000rpm for 10 minutes and filtered with a 0.22 μm syringe aqueous filter, and then D-lactic acid was detected by high performance liquid chromatography.
According to the detection result of high performance liquid chromatography, D-lactic acid titer in the fermentation broth obtained by fermenting for 60 hours is 48.05g L -1, D-lactic acid yield is 56.00%, and D-lactic acid productivity is 0.80g L -1h-1.
The results of the fed-batch fermentation production of D-lactic acid using the recombinant microorganisms in examples 5 to 7 are summarized in Table 3 below.
TABLE 3 results of the fed-batch fermentation of D-lactic acid by recombinant microorganisms
As can be seen from the results of Table 3, when D-lactic acid is produced by fed-batch fermentation using the recombinant B.thermoglucosidica containing the coding sequence of D-LDH P101Q or the recombinant B.licheniformis containing the coding sequence of D-LDH P101Q according to the present invention, the D-lactic acid titer obtained can be 145 or more g L -1, the D-lactic acid yield can be 86% or more, and the D-lactic acid productivity can be as high as 3.189g L -1h-1.
In contrast, in example 7, when D-lactic acid was produced by fed-batch fermentation using recombinant Bacillus licheniformis containing the unmutated D-LDH gene, the D-lactic acid titer was only 48.05g L -1, much lower than the D-lactic acid titer 145.91g L -1 of example 6, under the same fermentation conditions as in example 6 using recombinant Bacillus licheniformis containing the mutated D-LDH P101Q gene; meanwhile, the D-lactic acid yield of example 7 was only 56%, and the D-lactic acid productivity was only 0.80g L -1h-1, which were significantly lower than the corresponding values in example 6. This shows that the D-lactate dehydrogenase mutant of the invention significantly improves the titer, yield and productivity of D-lactate produced by fed-batch fermentation, and shows significantly improved catalytic performance.
The foregoing describes in detail preferred embodiments of the present invention. It should be understood that numerous modifications and variations can be made in accordance with the concepts of the invention without requiring creative effort by one of ordinary skill in the art. Therefore, all technical solutions which can be obtained by logic analysis, reasoning or limited experiments based on the prior art by the person skilled in the art according to the inventive concept shall be within the scope of protection defined by the claims.
Claims (20)
1. A mutant D-lactate dehydrogenase, characterized by an amino acid sequence that has a mutation based on the amino acid sequence of a wild-type D-lactate dehydrogenase, wherein the wild-type D-lactate dehydrogenase comprises a substrate binding domain, a cofactor binding domain, and a hinge region connecting the two, wherein the mutation is: the first amino acid of the cofactor binding domain linked to the hinge region is mutated to any one of a polar amino acid, an acidic amino acid, or a basic amino acid, and the D-lactate dehydrogenase mutant has a function of catalyzing the production of D-lactate from a substrate or a salt thereof.
2. The D-lactate dehydrogenase mutant according to claim 1, wherein the amino acid sequence of the wild-type D-lactate dehydrogenase is as shown in SEQ ID No. 4 or has more than 30% identity to SEQ ID No. 4 and the mutation is at amino acid 101 thereof.
3. The D-lactate dehydrogenase mutant according to claim 1, wherein the polar amino acids include serine, threonine, cysteine, tyrosine, glutamine, and asparagine, the acidic amino acids include aspartic acid and glutamic acid, and the basic amino acids include lysine, arginine, and histidine.
4. The D-lactate dehydrogenase mutant according to claim 1, wherein the mutated amino acid is mutated to glutamine or asparagine.
5. The D-lactate dehydrogenase mutant according to claim 1, wherein the D-lactate dehydrogenase mutant has a tag at the N-terminus and/or the C-terminus for purification and/or detection and the tag does not affect the catalytic performance of the D-lactate dehydrogenase mutant.
6. The D-lactate dehydrogenase mutant according to claim 1, wherein the amino acid sequence of the D-lactate dehydrogenase mutant is as shown in SEQ ID No.1 or SEQ ID No.2, or has more than 30% identity with SEQ ID No.1 or SEQ ID No. 2.
7. A DNA comprising a nucleotide sequence encoding the D-lactate dehydrogenase mutant according to any one of claims 1-6.
8. The DNA of claim 7, wherein the DNA contains a nucleotide sequence as set forth in SEQ ID No. 5 or SEQ ID No. 6, or a nucleotide sequence having greater than 30% identity to SEQ ID No. 5 or SEQ ID No. 6.
9. An expression vector comprising the DNA according to claim 7 or 8 operably linked to a promoter.
10. A host cell comprising the expression vector of claim 9, or having incorporated into its genome a nucleotide sequence encoding the D-lactate dehydrogenase mutant of any one of claims 1-6.
11. The host cell of claim 10, wherein the host cell comprises bacillus thermocellus (Geobacillus thermoglucosidasius) and bacillus licheniformis (Bacillus licheniformis).
12. A method for preparing a D-lactate dehydrogenase mutant according to any one of claims 1-6, comprising obtaining the D-lactate dehydrogenase mutant by genetic engineering or artificial synthesis.
13. Use of the D-lactate dehydrogenase mutant according to any one of claims 1-6, the DNA of claim 7 or 8, the expression vector of claim 9, or the host cell of claim 10 or 11 in the fields of enzyme-catalyzed production, biomedical detection and/or biosensors; preferably, the enzyme-catalyzed production comprises the production of D-lactic acid.
14. A method for producing D-lactic acid or a salt thereof, characterized in that the method comprises contacting the D-lactate dehydrogenase mutant according to any one of claims 1-6 with a substrate for producing D-lactic acid and a cofactor; preferably, the substrate is pyruvic acid or a salt thereof and the cofactor is NAD (P) H.
15. The method of claim 14, wherein the D-lactate dehydrogenase mutant is expressed by a microorganism and subjected to fermentation culture; preferably, the D-lactate dehydrogenase mutant is expressed in the form of a plasmid or the nucleotide sequence encoding the D-lactate dehydrogenase mutant is integrated into the genome of the microorganism to thereby effect the expression.
16. The method according to claim 14, wherein the method comprises providing seeds of a microorganism expressing the D-lactate dehydrogenase mutant, and then inoculating the seeds of the microorganism into a fermentation medium for fermentation culture at an inoculum size with an OD 620nm value of 0.2 to 0.8.
17. The method of claim 15 or 16, wherein the microorganism comprises bacillus thermocellum and bacillus licheniformis.
18. The method of claim 16, wherein the fermentation medium comprises one or more of yeast powder, metal ions, biotin, betaine, peanut meal, carbon source, neutral protease.
19. The method of claim 18, wherein the carbon source comprises one or more of glucose, sucrose, glycerol, xylose, arabinose, and mannitol; preferably, the initial concentration of the carbon source is 80g/L to 120g/L; it is also preferable that the carbon source is replenished so that the concentration thereof is maintained in the range of 0 to 100g/L when the concentration of the carbon source is reduced to 0.0 to 40.0 g/L.
20. The method of claim 15 or 16, wherein the fermentation culture is performed at 30 ℃ to 70 ℃, pH 6.5 to 7.5, stirring rate 50rpm to 150rpm for 40 hours to 70 hours.
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