CN116656637B - Variant of malate dehydrogenase - Google Patents
Variant of malate dehydrogenase Download PDFInfo
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- CN116656637B CN116656637B CN202310837126.3A CN202310837126A CN116656637B CN 116656637 B CN116656637 B CN 116656637B CN 202310837126 A CN202310837126 A CN 202310837126A CN 116656637 B CN116656637 B CN 116656637B
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- 102000013460 Malate Dehydrogenase Human genes 0.000 title claims abstract description 23
- 108010026217 Malate Dehydrogenase Proteins 0.000 title claims abstract description 23
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- 230000000694 effects Effects 0.000 claims abstract description 11
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 10
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- 230000003197 catalytic effect Effects 0.000 abstract description 9
- BAWFJGJZGIEFAR-NNYOXOHSSA-O NAD(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-O 0.000 abstract description 7
- BJEPYKJPYRNKOW-UHFFFAOYSA-N malic acid Chemical compound OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 abstract description 7
- 229940049920 malate Drugs 0.000 abstract description 2
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- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 9
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- 241000196324 Embryophyta Species 0.000 description 3
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 3
- 239000003242 anti bacterial agent Substances 0.000 description 3
- 229940088710 antibiotic agent Drugs 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 229940116298 l- malic acid Drugs 0.000 description 3
- 229940099690 malic acid Drugs 0.000 description 3
- 239000001630 malic acid Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- 238000009010 Bradford assay Methods 0.000 description 2
- 241000282326 Felis catus Species 0.000 description 2
- 239000012880 LB liquid culture medium Substances 0.000 description 2
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- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
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- KHPXUQMNIQBQEV-UHFFFAOYSA-N oxaloacetic acid Chemical compound OC(=O)CC(=O)C(O)=O KHPXUQMNIQBQEV-UHFFFAOYSA-N 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
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- 241001465754 Metazoa Species 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
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- 150000001413 amino acids Chemical class 0.000 description 1
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- 239000010446 mirabilite Substances 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 235000006408 oxalic acid Nutrition 0.000 description 1
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- 238000006479 redox reaction Methods 0.000 description 1
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- 238000011160 research Methods 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000021749 root development Effects 0.000 description 1
- 230000002786 root growth Effects 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
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- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01037—Malate dehydrogenase (1.1.1.37)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
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Abstract
The invention belongs to the field of biology, and discloses a variant of malate dehydrogenase, which aims to improve the catalytic efficiency of malate dehydrogenase on malate and NAD+, and specifically, the enzyme activity of the variant is at least 1-5 times that of a wild type, the amino acid sequence of the variant is shown as any one of SEQ ID NO. 1-12, or the amino acid sequence of the variant has at least 85%, 90% and more than 95% consistency with any one of SEQ ID NO. 1-12.
Description
Technical Field
The invention belongs to the field of biology, and particularly relates to a variant of malate dehydrogenase.
Background
Malate dehydrogenase (Malate dehydrogenase, abbreviated as MDH, EC 1.1.1.37) is an enzyme protein that is widely found in organisms including plants, animals, microorganisms, and the like. The function of this enzyme is to catalyze the redox reaction between malic acid and NAD+ to convert malic acid to oxalic acid, while reducing NAD+ to NADH.
Malate dehydrogenase plays an important physiological role in organisms and is involved in the regulation of many metabolic pathways, such as tricarboxylic acid cycle, photosynthesis, respiration, etc. In plants, malate dehydrogenase is also involved in regulating plant response to environmental adaptation, such as regulating root growth and development, adaptation to acidic soil, and the like.
Therefore, research on malate dehydrogenase has important significance for deeply knowing metabolic pathways and regulation mechanisms thereof in organisms and improving agricultural production efficiency.
Disclosure of Invention
The invention provides a variant of malate dehydrogenase, which aims to improve the catalytic efficiency of malate dehydrogenase on malate and NAD+.
The present invention provides a variant of malate dehydrogenase, characterized in that: the enzyme activity of the variant is at least 1-5 times that of a wild type, the amino acid sequence of the variant is shown as any one of SEQ ID NO. 1-12, or the amino acid sequence of the variant has at least 85%, 90% and 95% identity with any one of SEQ ID NO. 1-12.
The invention also provides a nucleic acid, characterized in that: encoding a variant of the foregoing malate dehydrogenase.
The invention also provides a carrier, which is characterized in that: contains the aforementioned nucleic acid.
The invention also provides a recombinant expression transformant, which is characterized in that: contains the aforementioned carrier.
The recombinant expression transformant provided by the invention also has the following characteristics: the recombinant expression transformant is Escherichia coli.
The variant of the malate dehydrogenase provided by the invention has an amino acid sequence shown in any one of SEQ ID NO. 1-12, or the amino acid sequence of the variant has at least 85%, 90% and 95% or more identity with any one of SEQ ID NO. 1-12, and the catalytic efficiency of the variant on substrates and coenzymes is correspondingly improved compared with that of the wild malate dehydrogenase: the enzyme activity is at least 1-fold that of the wild type.
Drawings
Fig. 1 is a graph of fit equations related to an embodiment.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings. With respect to the specific methods or materials used in the embodiments, those skilled in the art may perform conventional alternatives based on the technical idea of the present invention and are not limited to the specific descriptions of the embodiments of the present invention.
The methods used in the examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are all commercially available.
Variants herein refer to those that have amino acids that are not identical relative to the wild type, but retain the essential properties of the wild type.
The enzyme activity herein refers to a unit of measurement of the enzyme activity, that is, 1 unit of enzyme activity refers to an amount of enzyme capable of converting 1. Mu. Mole of a substrate in 1 minute under a specific condition (25 ℃ C., other is the optimum condition), or an amount of enzyme capable of converting 1. Mu. Mole of a relevant group in the substrate.
Kcat is the catalytic constant of the enzyme (CATALYTIC CONSTANT, kcat), also called turnover number or turnover number (turn number), i.e., how many substrates 1 enzyme molecule catalyzes into products per unit time. Kcat can be used to measure the catalytic efficiency of an enzyme, the greater the Kcat value, the greater the catalytic efficiency of the enzyme.
The Mirabilite constant K m is defined as the substrate concentration at which the enzyme is running at half its maximum catalytic rate; thus, it describes the affinity of an enzyme for a particular substrate. Knowledge of the K m value is crucial for quantitative understanding of enzymatic and regulatory interactions between enzymes and metabolites: it will be the intracellular concentration of the metabolite, K m can reflect the magnitude of the enzyme affinity for the substrate, i.e. the smaller the K m value, the greater the affinity of the enzyme for the substrate; conversely, the smaller the affinity.
K cat/Km combines K cat and K m together to measure the catalytic efficiency of an enzyme and to show the perfection of an enzyme.
Malate dehydrogenase Malate dehydrogenase, abbreviated as MDH, EC 1.1.1.37.
The present invention provides a variant of malate dehydrogenase having an enzymatic activity 1-5 times that of the wild-type, preferably the wild-type is from: coli, having an amino acid sequence as shown in SEQ ID NO. 13, namely:
SEQ ID NO:13：
MKVAVLGAAGGIGQALALLLKTQLPSGSELSLYDIAPVTPGVAVDLSHIPTAVKIKGFSGEDATPALEGADVVLISAGVRRKPGMDRSDLFNVNAGIVKNLVQQVAKTCPKACIGIITNPVNTTVAIAAEVLKKAGVYDKNKLFGVTTLDIIRSNTFVAELKGKQPGEVEVPVIGGHSGVTILPLLSQVPGVSFTEQEVADLTKRIQNAGTEVVEAKAGGGSATLSMGQAAARFGLSLVRALQGEQGVVECAYVEGDGQYARFFSQPLLLGKNGVEERKSIGTLSAFEQNALEGMLDTLKKDIALGQEFVNK.
The amino acid sequence of the variant provided by the invention is shown as any one of SEQ ID NO. 1-12, or the amino acid sequence of the variant has at least 85%, 90% and 95% consistency with any one of SEQ ID NO. 1-12. SEQ ID NOS 1-12 are shown in detail as follows:
SEQ ID NO:1：
MKVAVLGAAGGIGQALALLLKTQLPAGSELSLYDIAPVTPGVALDLSHIPTNVEVKGFSGEDATPALEGADVVLISAGVARKPGMDRSDLFNINAGIVRNLVEKIAKTFPSAIIGIITNPVNTTVAIAAEVLKKAGKYDKNKLFGVTTLDIIRSETFVAELKGKDPVEVDVPVIGGHSGVTILPLLSQVPGVSFTNQEVAALTKRIQNAGTEVVEAKAGGGSATLSMGQAAVRFGLSLVRGLQGENGVVECALVEGDGKHARFCAQPLLLGKNGVEERKSYGDLSAFEQQALEGMLATLKTDITLGEEFVKK;
SEQ ID NO:2：
MKVAVLGAAGGIGQALALLLKTQLPSGSELTLYDIAPVTPGVAVDLSHIPTAVKITGFSGEDAAPALEGADIVVISAGVRRKPGMDRSDLAPVNYGIVENLTKQIAKVTPDAIVGIITNPVNATVAVAEAVLEKAGVYDPRKLFGVTTLDIIRSNTFVAELKGKQPGEVEVPVIGGHSGRTIIPLLSQVEGVTFTPEEVKALTRRIQNAGTEVVEAKAGGGSATLSMGQAAARFVLDLVAAKEGAENIVRDALVKNDGSYAHFFTRPCLLGTDGIKEVLSIGELSEFEKARLEASRPYLSAEIAKGFAYVNT;
SEQ ID NO:3：
MKVAVLGAAGGIGQALALLLKTQLPSGSTLTLYDIAPVTPGVAVDLSHIPTAVKIEGFTGEDAAPALEGADIVVISAGVRRKPGMDRSDLKPVNFGIVENLTKQIAEVTPDAIILIITNPVNTTVAIAAEVLKKAGVYDPKRLFGVTTLDIIRSNTFVAELKGKQPGEVEVPVIGGHSGKTIIPLLSKVEGLTFTDEEVEELTKRIQNAGTEVVEAKAGGGSATLSMGQAAARTVLAVARARAGAENVVLDVLVEGDGSYARFFTRPCLLGTDGVKEILSIGELSDFEKKRLEESIPYMKEEIDAGYDYVNN;
SEQ ID NO:4：
MKVAVLGAAGGIGQALALLLKTQLPAGSELSLYDIAPVTPGVAVDLSHIPTAVKVKGFSGEDHTPALEGADVVLISAGVARKPGMDRSDLFNVNAGIVKNLVEQIAKTFPKAIIGIITNPVNTTVAIAAEVLKKAGVYDKNKLFGVTTLDVIRSETFVAELKPKDPVEVDVPVIGGHSGVTILPLLSQVPGVSFTNQEVAALTKRIQNAGTEVVEAKAGGGSATLSMGQAAVRFGLSLVRGLQGENGVVECALVEGDGKHARFCAQPLLLGKNGVEERKSYGKLSAFEQQALEGMLATLKKDITLGEEFVKKGSPAATAAERILVVVITDRN;SEQ ID NO:5：
MKKKVTVVGAGNVGATAAQEIAEKESRDVVLDDGMEGLPQGKALDVLQAGPLIGQSARISGTNDSSGTAGSDVVVITAGIPRKPGMSRDDLIGTNADIVKSVTENVVKLSPKAYIIVVSNPLDAMGYTAFSATGFPIERVIGMAGALDSARFRAFIAMELNVSAGNIQAVVLGGHGDTMVPLKRRTTVAGIPITSLMSAEGIEVIVMRTRMGGAEIVILLKTGSAYAAPSASEATMVDSIVKDQKRILPCALYLEGEYGASGICVGVPVKLGANGVEEIVDIKLQEEEKLLISISAKAVREMNKVLSVL;
SEQ ID NO:6：
MKVAVLGAAGGIGQALALLLKTQLPAGSELSLYDIAPVTPGVALDLSHIPTNVEVKGFSGEDATPALEGADVVLISAGVARKPGMDRSDLFNINAGIVRNLVEQIAKTFPKAIIGIITNPVNTTVAIAAEVLKKAGKYDKNKLFGVTTLDIIRSETFVAELKGKDPVEVDVPVIGGHSGVTILPLLSQVPGVSFTNQEVAALTKRIQNAGTEVVEAKAGGGSATLSMGQAAVRFGLSLVRGLQGENGVVECALVEGDGKHARFCAQPLLLGKNGVEERKSYGDLSAFEQQALEGMLATLKKDITTGE;
SEQ ID NO:7：
MKVAVLGAAGGIGQALALLLKTQLPAGSELSLYDIAPVTPGVAADLSHIPTNVFVKGFSGEDATPALEGADVVLISAGVARKPGMDRSDLFNVNAGIVKNLVEQIAKTFPKAIIGIITNPVNTTVAIAAEVLKKAGKYDKNKLFGVTTLDVIRSETFVAELKPKDPVEVDVPVIGGHSGVTILPLLSQVPGVSFTNQEVADLTKRIQNAGTEVVEAKAGGGSATLSMGQAAVRFGLSLVRALQGENGVVECALVEGDGKHARFCAQPLLLGKNGVEERKSYGDLSAFEQQALDGMLATLKKDITTME;
SEQ ID NO:8：
MKVAVLGAAGGIGQALALLLKTQLPSGSELTLYDIAPVTPGVAVDLSHIPTAVKIKGFSGEDASPALEGADVVVISAGVRRKPGMDRSDLAPVNFGIVENLTRQIAKVTPNAIVGIITNPVNSTVAVAAEVLKKEGVYDPKRLFGVTTLDIIRSNTFVAELKGKQPGEVEVPVIGGHSGETIIPLLSQVKGLTFSDEEIRDLTARIQNAGTEVVEAKAGGGSATLSMGQAAARFVLDVVAALEGEKNIIRDALVENDGSYARFFTAPCLLGTDGIEKVLSIGTLSAFEKAQLAASRPIMNAEIDKGFDYVNK;
SEQ ID NO:9：
MKVTVVGAGAVGATCAENIANKQIASEVVLLDIKEGFAEGKALDIMQTASLNGFDTKITGVTNDYSKTAGSDVVVITSGIPRKPGMTREELIGINAGIVKSVTENLLKLSPDRIIIVVSNPMDTMTYLAFKATGLPKNRIIGMGGALDSVRFRYFLSLALNVSASDLQAMVIGGHGDTTMIPLIRLATLNSIPVSKMLAGEELDEVAQDTMVGGATLTKLIGTSAWYAPGAAVATLVDSIVKDQKKIFPCSVYLEGEYGQKDICIGVPVILGANGVEKIVDIDLQDAEKAKLSKSADAVREMNKVLSV;
SEQ ID NO:10：
MVLKKILVGGAGNVGHTAANRAADERIGVVVLFDIVAGVPQGKELDIAESGPNEGFDRKTKGTNDYAGIAGSDVVIITAGIPRKPGMSRDDLLEINAKIVKSVVEGILKYSPDAIVIVVSNPLDVMVWVAQKFSGFPKNRVLGMAGVLDSSRFKYFEAEYLEVSMEDVLAFVLGGHGDTMVPLVRYDTVAGIPVTELLDSPEIAAIVERTRGGGAEIVTLLKTGSAYYAPSAAVAELVEAILPDTKKILPVAAHLAGEYGVSDMFVGVPVKLGSHGVEGIIEGKLTEAEDAAFQSSAESVDEGLAVLAAL;
SEQ ID NO:11：
MKVAVLGAAGGIGQALALLLKTRLPAGSELSLYDIAPVTPGVAVDLSHIPTAVKIKGFAGEDPTPALEGADVVLISAGVARKPGMDRSDLFNINAGIVKNLVEQNAKIFPKAIIGIITNPVNTTVAIAAEVLKKAGVYDKNKLFIVTTLDVIRSETFVAELKGLDPAEVDVPVIGGHSGVTILPLLSQVPGVSFTNQEVAALTKRIQNAGTEVVEAKAGGGSATLSMGQAAARFGLSLVRALQGENGVVECALVEGDGKHARFGAQPLLLGKNGVEAVKSYGKLSAFEQQALEGMLATLKADIVLGEEFVKK;
SEQ ID NO:12：
MKVAVLGAAGGIGQALALLLKTQLPSGSELKLYDIAPVTPGVAVDLSHIPTAVRIEGFTGEDATPALEGADVVVISAGVRRKPGMDRSDLIPVNFGIVENLIKQIAETTPDAVILIITNPVNSTVAVAAEVLEKAGVYDPKRLFGVTTLDIIRSNTFVAELKGKQPGEVEVRVIGGHSGETIIPLLSQVEGVTFTEEEKKELTDRIQNAGTEVVEAKAGGGSATLSMGQAAARTVLAVVRALRGEKDVVLDLLVKGDGSYSEFFTAPCLLGKDGVEEILSIGELDEYEKELLESSLPYLNRLIAIGKDYVNN.
The invention also provides a nucleic acid encoding a variant of the aforementioned malate dehydrogenase.
The invention also provides a vector containing the nucleic acid.
The invention also provides a recombinant expression transformant containing the vector, preferably, the recombinant expression transformant is escherichia coli.
Examples
Plasmids containing variants were constructed and synthesized by Beijing qing Biotechnology Co.
1. Recombinant escherichia coli culture and crude enzyme preparation experiment
1. Transformation of the plasmid into E.coli BL21 (DE 3): on a super clean bench, 2. Mu.L of plasmid at a concentration of 50mg/L was added to 100. Mu.L of BL21 (DE 3) competent cell suspension. Flick by hand or mix with gun, place on ice for 30min; heat shock is carried out for 90s in a water bath at the temperature of 42 ℃, and the heat shock is quickly carried out on ice for cooling for 5min, so that shaking is avoided. On an ultra-clean bench, 0.9mL of LB liquid medium is heated into the cell suspension, and after uniform mixing, the cells are cultured for 45min at 37 ℃ in a shaking way, and the rotating speed is 150-225rpm, so that the cells are recovered to a normal growth state. Centrifuging at 4000rpm for 1min, sucking the supernatant on an ultra-clean bench until 200uL of bacterial liquid remains, and blowing and sucking uniformly;
2. Coating: 200. Mu.L of the mixture was plated on LB medium plates containing kana antibiotics, and the mixture was spread with a disposable spreading bar. Sealing the flat plate with sealing film, and standing until bacteria liquid is fully absorbed. Inverting the plate, and culturing at 37 ℃ for 12-24 hours until the transformant appears;
3. Culturing primary seed liquid: 10mL of LB liquid culture medium and final concentration of kana antibiotics of 50 mug/mL are used for picking 1 monoclonal and culturing first-stage seed liquid. Culture conditions: 37 ℃,200rpm,12-18h;
4. And (5) gene delivery sequencing: 2mL of the cultured primary seed solution is taken to send gene sequencing, and whether the target gene fragment sequence of the MDH variant on the plasmid is correct or not is checked;
5. Glycerol-retaining bacteria: on an ultra clean bench, a sterile bacteria-preserving pipe is opened, 0.5mL of first-stage seed liquid is added by a liquid-transferring gun, then 0.5mL of sterilized 50% glycerol is added, the mixture is uniformly mixed by the liquid-transferring gun, and a cover is covered. Placing into a refrigerator at-80deg.C for preservation;
6. Culturing first-stage seed liquid by glycerol bacteria: and (3) verifying the sequence to be tested, and culturing the first-stage seed solution by using 10mL of LB liquid culture medium and 10 mu L of MDH variant glycerol bacteria with the final concentration of kana antibiotics of 50 mu g/mL. Culture conditions: 37 ℃,200rpm,12-18h;
7. culturing a secondary seed solution: the second seed solution was cultured with 200mL of LB medium, final kana antibiotic concentration of 50. Mu.g/mL, 10mL of MDH variant first seed solution. Culture conditions: sampling and measuring OD600nm to OD value of 0.6-0.8 in the culture process at 37 ℃ and 180 rpm;
8. Induction: and after the secondary seed solution is cultured, adding an IPTG aqueous solution into the secondary seed solution on an ultra-clean bench to ensure that the final concentration of the IPTG in the secondary seed solution reaches 0.5mM. Culture conditions: 25 ℃,180rpm,16-24 hours;
9. And (3) centrifugally collecting thalli at a low temperature: for the induced bacterial liquid, 40mL of liquid is filled in a 50mL centrifuge tube, the liquid is centrifuged for 5min at 4 ℃ and 8000rpm, the supernatant is removed, and bacterial mud is reserved;
10. washing: for bacterial mud in each centrifuge tube, adding 5mL of Tris-HCl buffer (50 mM, pH 6.8-7.2) into each tube, blowing and sucking uniformly, combining a plurality of tubes into one tube, swirling, centrifuging at 8000rpm for 5min, removing supernatant, and reserving bacterial mud;
11. Ultrasonic crushing: for the washed bacterial mud, adding 15mL of Tris-HCl buffer (50 mM, pH 6.8-7.2) into a 50mL centrifuge tube, blowing, sucking and suspending, vortex mixing uniformly, setting the working power of an ultrasonic breaker to 230W, carrying out ultrasonic treatment for 3s, stopping for 7s, and carrying out total working time of 40min. The centrifuge tube is in ice water bath in the ultrasonic breaking stage, and the thalli starts to be broken;
12. centrifuging to remove sediment: and (3) centrifuging the bacterial liquid after ultrasonic disruption at 4 ℃ and 10000rpm for 40min. Removing sediment, and reserving supernatant to obtain crude enzyme liquid of MDH variant enzyme.
2. Enzyme activity assay:
1. Preheating an ultraviolet spectrophotometer for 30min in advance, adjusting the wavelength to 340nm, and setting background absorption to 0 by distilled water;
2. keeping the temperature in a water bath with the temperature of 37 ℃ of standby distilled water;
Sequentially adding 760 mu L of distilled water, 10 mu L of 0.8mM NADH aqueous solution, 10 mu L of 1.6 mM-malic acid aqueous solution and 20 mu L of crude enzyme solution to be detected into A1 mL quartz cuvette, fully blowing, sucking and uniformly mixing, immediately recording initial absorbance A1 and absorbance A2 after 1min of reaction at a wavelength of 340nm, and keeping the temperature of the reaction solution at 37 ℃ in the reaction process;
3. Enzyme activity calculation: under optimal conditions, the amount of enzyme that converts 1. Mu. Mol of substrate in 1min is 1U. Here, the enzyme activity calculation formula of the crude enzyme solution (U/mL) =Δa×v reaction solution/(ε×l×t×v enzyme solution). The notes for each term in the formula are as in Table 1:
both wild-type MDH and MDH variants were tested and the enzyme activity was calculated as described above, and the results are shown in Table 2:
conclusion: as can be seen from the enzyme activity measurement Table 2, the enzyme activities of the 12 groups of MDH variants are significantly better than those of the MDH wild type.
3. Calculation of Kcat value, K M value for wild type MDH and 12 MDH variants:
The calculation methods of the Kcat value and the K M value of MDH take wild type MDH as an example:
after the wild-type MDH crude enzyme solution was purified, the concentration of the purified enzyme was measured by the Bradford method.
The reaction was designed to catalyze the conversion of the substrates L-malate and NAD+ to oxaloacetate and NADH with wild-type MDH, and the concentration of NADH generated by the reaction was measured by High Performance Liquid Chromatography (HPLC).
The reaction system: the total volume of the reaction was 10mL, and the addition amount of the wild-type MDH-purified enzyme solution was 1mL. L-malic acid concentration 9 gradients [ S ] were set: 5. Mu.M, 10. Mu.M, 20. Mu.M, 40. Mu.M, 80. Mu.M, 160. Mu.M, 320. Mu.M, 640. Mu.M, 1280. Mu.M, L-malic acid was prepared as a 10mM stock solution at the time of use, and the loading volume was calculated from the desired concentration. NAD+ was set at 2mM, and NAD+ was prepared as a 10mM stock solution at the time of use, and the loading volume was calculated from the desired concentration. The whole reaction volume was made up to 10mL with ultrapure water.
Reaction sampling and detection: the reaction temperature is 37 ℃ and the reaction time is 1min, 1mL of reaction solution is taken, and the reaction solution is inactivated for 1min at a high temperature in a water bath with the temperature of 80 ℃. The obtained inactivated sample is diluted to a proper concentration, the absorption peak value of NADH at the wavelength of 340nm is detected by HPLC, and the actual NADH concentration in the reaction liquid is calculated according to the standard concentration curve of NADH standard substance. The reaction rate v (NADH is formed in equivalent to oxaloacetate) was calculated from the concentration of NADH formed.
As shown in FIG. 1, the Lineweaver-Burk equation for wild-type MDH under the experimental reaction conditions was linearly fitted using a double reciprocal mapping method: taking the reciprocal 1/[ S ] of the initial concentration of L-malic acid as an abscissa and taking the reciprocal 1/v of the reaction rate measured at each concentration as an ordinate, making a scatter diagram in Excel, and calculating a corresponding linear equation y=kx+b, wherein k in the equation is KM/Vmax in a Lineweaver-Burk equation, and b is 1/Vmax in the Lineweaver-Burk equation. The values of the variables are shown in Table 3.
Vmax=1/105.3=9.50×10 -3(mol/min),KM＝0.0126*Vmax＝1.20*10-4 (mol/L) can be calculated from the fitted equation.
Kcat=vmax per the molar amount of enzyme in the reaction is known from the definition. Vmax=9.50×10 -3mol/min＝1.58*10-4 mol/s. The concentration of the mother liquor of the wild-type MDH pure enzyme was 1.01mg/mL as measured by the Bradford method, and 1mL was used in the reaction, and the molecular weight of the wild-type MDH was 34458.63Da, so that kcat=Vmax/(1.01 mg/34458.63 g.mol -1)＝5387s-1) could be calculated.
The Kcat value and K M value calculation methods of other mutants of MDH are consistent with those of wild MDH.
The measurement results are shown in Table 4:
Claims (5)
1. A variant of malate dehydrogenase, characterized by:
The enzyme activity of the variant is at least 1-5 times that of the wild type,
The amino acid sequence of the variant is shown in any one of SEQ ID NO 5 and SEQ ID NO 12.
2. A nucleic acid, characterized in that:
encoding the variant of malate dehydrogenase of claim 1.
3. A carrier, characterized in that:
comprising the nucleic acid of claim 2.
4. A recombinant expression transformant characterized in that:
a vector comprising the vector of claim 3.
5. The recombinant expression transformant according to claim 4, wherein:
Is Escherichia coli.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JPH09107959A (en) * | 1995-10-17 | 1997-04-28 | Unitika Ltd | Malate dehydrogenase and its production |
| CN104854244A (en) * | 2012-10-02 | 2015-08-19 | 密歇根生物技术研究所 | Recombinant microorganisms for producing organic acids |
| CN105505971A (en) * | 2016-01-19 | 2016-04-20 | 中国科学院微生物研究所 | Method for improving yield of butanol produced by escherichia coli |
| CN109306355A (en) * | 2018-11-23 | 2019-02-05 | 安徽师范大学 | A kind of malate dehydrogenase enzyme mutant gene, construction method and its application |
| CN112592940A (en) * | 2020-12-16 | 2021-04-02 | 江西邦泰绿色生物合成生态产业园发展有限公司 | Biological enzyme synthesis method of N-fluorenyl methoxycarbonyl-decyl acetaldehyde |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JPH09107959A (en) * | 1995-10-17 | 1997-04-28 | Unitika Ltd | Malate dehydrogenase and its production |
| CN104854244A (en) * | 2012-10-02 | 2015-08-19 | 密歇根生物技术研究所 | Recombinant microorganisms for producing organic acids |
| CN105505971A (en) * | 2016-01-19 | 2016-04-20 | 中国科学院微生物研究所 | Method for improving yield of butanol produced by escherichia coli |
| CN109306355A (en) * | 2018-11-23 | 2019-02-05 | 安徽师范大学 | A kind of malate dehydrogenase enzyme mutant gene, construction method and its application |
| CN112592940A (en) * | 2020-12-16 | 2021-04-02 | 江西邦泰绿色生物合成生态产业园发展有限公司 | Biological enzyme synthesis method of N-fluorenyl methoxycarbonyl-decyl acetaldehyde |
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