CN116790527A - Enzyme preparation mixture, preparation method thereof and 25-hydroxycholesterol or 25-hydroxyvitamin D 3 Is prepared by the preparation method of (2) - Google Patents
Enzyme preparation mixture, preparation method thereof and 25-hydroxycholesterol or 25-hydroxyvitamin D 3 Is prepared by the preparation method of (2) Download PDFInfo
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- CN116790527A CN116790527A CN202310529525.3A CN202310529525A CN116790527A CN 116790527 A CN116790527 A CN 116790527A CN 202310529525 A CN202310529525 A CN 202310529525A CN 116790527 A CN116790527 A CN 116790527A
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Classifications
-
- 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.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
- C12N9/0077—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with a reduced iron-sulfur protein as one donor (1.14.15)
- C12N9/0081—Cholesterol monooxygenase (cytochrome P 450scc)(1.14.15.6)
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
-
- 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)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P33/00—Preparation of steroids
- C12P33/02—Dehydrogenating; Dehydroxylating
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/99—Oxidoreductases acting on the CH-OH group of donors (1.1) with other acceptors (1.1.99)
- C12Y101/9901—Glucose dehydrogenase (acceptor) (1.1.99.10)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
- C12Y114/15—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with reduced iron-sulfur protein as one donor, and incorporation of one atom of oxygen (1.14.15)
- C12Y114/15006—Cholesterol monooxygenase (side-chain-cleaving) (1.14.15.6), i.e. cytochrome P450scc
Abstract
The invention relates to the field of enzyme catalysis, and discloses an enzyme preparation mixture, a preparation method thereof and 25-hydroxycholesterol or 25-hydroxyvitamin D 3 Is prepared by the preparation method of (1). The enzyme preparation mixture packageComprising a self-consistent P450 enzyme and a glucose dehydrogenase. Co-catalyzing a substrate cholesterol or vitamin D with an enzyme preparation mixture comprising a self-consistent P450 enzyme and a glucose dehydrogenase 3 Production of 25-hydroxycholesterol or 25-hydroxyvitamin D 3 . Compared with the P450 single enzyme method, the method can use cheaper glucose as a cofactor, has higher substrate conversion rate and fewer byproducts, and is beneficial to the subsequent extraction process.
Description
Technical Field
The invention relates to the field of enzyme catalysis, in particular to an enzyme preparation mixture, a preparation method thereof and 25-hydroxycholesterol or 25-hydroxyvitamin D 3 Is prepared by the preparation method of (1).
Background
25-hydroxy vitamin D 3 Also known as calcitonin, is vitamin D 3 Has remarkable curative effects on osteoporosis, rickets, osteomalacia and other diseases, and is also used for treating hypocalcemia caused by hemodialysis. 25-hydroxy vitamin D 3 Can also be used as food and feed additive. 25-hydroxy cholesterol is produced by chemical method to produce 25-hydroxy vitamin D 3 Is an important raw material.
25-hydroxycholesterol and 25-hydroxyvitamin D 3 The synthesis can be performed by using a chemical method, but the method involves multi-step reactions such as radial ring opening, bond breaking, ring sum, reduction, coupling, oxidation, purification and the like, and has the problems of more steps, low conversion rate, more byproducts, difficult separation and purification and the like, and the large-scale production is difficult. In recent years, with the rapid development of synthetic biology and gene editing technology, a method for producing specific enzymes by fermentation of recombinant microorganisms and then producing complex compounds by catalysis of the obtained biological enzymes has been receiving attention. Biological enzyme catalyzed substrate cholesterol or vitamin D 3 To produce the corresponding 25-hydroxycholesterol or 25-hydroxyvitamin D 3 Has the advantages of low production cost, mild reaction condition, high conversion rate, easy extraction and the like, and is suitable for large-scale production. However, the existing biological enzyme method is used for preparing 25-hydroxycholesterol or 25-hydroxyvitamin D 3 The method adopts a P450 single enzyme method, requires the use of expensive enzyme reaction cofactors such as NADH or NADPH, cytochrome C and the like, has the conversion rate of 80 percent at the maximum, and has the conversion rate to be improved 。
There is therefore a need for a low cost and substrate for cholesterol or vitamin D 3 Method for preparing 25-hydroxycholesterol or 25-hydroxy vitamin D by biological enzyme with high conversion rate 3 。
Disclosure of Invention
The purpose of the invention is to overcome the defects of the prior art that 25-hydroxycholesterol or 25-hydroxyvitamin D is prepared by a single enzyme method 3 The conversion rate of (C) is low and the use of expensive enzyme reaction cofactor is required, and an enzyme preparation mixture, a preparation method thereof and 25-hydroxycholesterol or 25-hydroxyvitamin D are provided 3 The preparation method adopts a self-consistent P450 enzyme and glucose dehydrogenase double-enzyme combined catalysis method to produce 25-hydroxycholesterol or 25-hydroxyvitamin D 3 Less expensive glucose may be used as cofactor.
To achieve the above object, the present invention provides in a first aspect an enzyme preparation mixture comprising a self-consistent P450 enzyme and a glucose dehydrogenase; wherein, the liquid crystal display device comprises a liquid crystal display device,
the self-consistent P450 enzyme is the enzyme according to any one of (a) to (e):
(a) Has the sequence shown in SEQ ID NO:2 or SEQ ID NO:3, an enzyme having an amino acid sequence shown in FIG. 3;
(b) SEQ ID NO:2 or SEQ ID NO:3, and the enzyme shown in the amino acid sequence still has self-consistent P450 enzyme activity through substitution, deletion or addition of at least one amino acid residue;
(c) And SEQ ID NO:2 or SEQ ID NO:3, and has more than 80% homology, and has the enzyme shown in the amino acid sequence of self-consistent P450 enzyme activity;
(d) An enzyme represented by an amino acid sequence having a tag attached to the amino terminus and/or the carboxyl terminus of the amino acid sequence of (a), (b) or (c);
(e) An enzyme represented by an amino acid sequence wherein a signal sequence is linked to the amino terminus of the amino acid sequence of (a), (b) or (c).
The second aspect of the present invention provides a method for producing an enzyme preparation, comprising the steps of:
(1) Fermenting and culturing the genetically engineered bacterium containing the coding gene of the self-consistent P450 enzyme or the seed of the genetically engineered bacterium containing the coding gene of the glucose dehydrogenase to obtain the OD of the fermentation liquid 600 When the value is 10-15, mixing the fermentation liquor with an inducer for induction culture to obtain a bacteria-containing fermentation liquor; wherein the inducer is 5-aminolevulinic acid hydrochloride and isopropyl thiogalactoside;
(2) Breaking the wall of the bacteria-containing fermentation liquor to obtain wall-broken liquor;
(3) Separating the wall-broken liquid to obtain self-consistent P450 enzyme or glucose dehydrogenase;
(4) The self-consistent P450 enzyme and glucose dehydrogenase obtained by the above methods are mixed.
In a third aspect, the invention provides an enzyme preparation mixture prepared by the preparation method provided by the invention.
In a fourth aspect, the present invention provides 25-hydroxycholesterol or 25-hydroxyvitamin D 3 The preparation method comprises the following steps: mixing enzyme preparation mixture, cosolvent, cofactor, substrate and solvent to obtain mixed solution, and then performing conversion reaction to obtain the 25-hydroxycholesterol or the 25-hydroxyvitamin D 3 ；
Wherein the substrate is cholesterol or vitamin D 3 The enzyme preparation mixture is provided by the invention.
Through the technical scheme, the invention has the beneficial effects that:
the invention adopts a microbial double-enzyme method to prepare the 25-hydroxy cholesterol and also can be used for preparing the 25-hydroxy vitamin D 3 The enzyme reaction condition is mild, the reaction time is short, and the method is suitable for large-scale production. The invention adopts a microbial double-enzyme method to directly convert the substrate cholesterol or vitamin D 3 Conversion to 25-hydroxycholesterol or 25-hydroxyvitamin D 3 The conversion rate is higher, the byproducts are less, and the subsequent extraction process is facilitated.
In addition, the invention adopts the self-consistent P450 enzyme and glucose dehydrogenase double-enzyme combined catalysis method to produce 25-hydroxy for the first time Basal cholesterol or 25-hydroxy vitamin D 3 The more expensive enzyme reaction cofactors such as NADH or NADPH, cytochrome C, etc. can be replaced by the cheaper cofactor glucose than by the P450 single enzyme method alone. Compared with chemical method, the method has the advantages of greatly reducing reaction steps, improving conversion rate, reducing production cost and being suitable for large-scale production.
After the cell walls are broken, flocculating settling is carried out on the wall-broken liquid, and enzyme-containing supernatant is obtained by centrifugation. Most of cell fragments and large proteins are removed by flocculation sedimentation, an enzyme reaction system is purified, and the conversion efficiency and the subsequent purification operation are improved.
In a preferred embodiment of the present invention, the substrate cholesterol or vitamin D is further increased by selecting appropriate types of self-consistent P450 enzymes and glucose dehydrogenase, the amounts of substrate, cofactor, self-consistent P450 enzymes and glucose dehydrogenase, and the conditions of the enzymatic conversion reaction 3 The conversion rate of the catalyst reaches more than 97 percent.
Drawings
FIG. 1 is a process flow diagram of the preparation of a self-consistent P450 enzyme or glucose dehydrogenase according to one embodiment of the invention.
Detailed Description
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
The first aspect of the present invention provides an enzyme preparation mixture comprising a self-consistent P450 enzyme and a glucose dehydrogenase; wherein, the liquid crystal display device comprises a liquid crystal display device,
the self-consistent P450 enzyme is the enzyme according to any one of (a) to (e):
(a) Has the sequence shown in SEQ ID NO:2 or SEQ ID NO:3, an enzyme having an amino acid sequence shown in FIG. 3;
(b) SEQ ID NO:2 or SEQ ID NO:3, and the enzyme shown in the amino acid sequence still has self-consistent P450 enzyme activity through substitution, deletion or addition of at least one amino acid residue;
(c) And SEQ ID NO:2 or SEQ ID NO:3, and has more than 80% homology, and has the enzyme shown in the amino acid sequence of self-consistent P450 enzyme activity;
(d) An enzyme represented by an amino acid sequence having a tag attached to the amino terminus and/or the carboxyl terminus of the amino acid sequence of (a), (b) or (c);
(e) An enzyme represented by an amino acid sequence wherein a signal sequence is linked to the amino terminus of the amino acid sequence of (a), (b) or (c).
In the present invention, the self-consistent (self-priming) P450 enzyme is a catalytic self-supporting P450 enzyme, i.e., comprises not only a P450 oxidation domain but also a separate reductase domain. Commercially available or self-made.
The 20 amino acid residues that make up a protein can be divided into four classes according to side chain polarity: 1. nonpolar amino acids: alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), phenylalanine (Phe), tryptophan (Trp) and proline (Pro); 2. polar uncharged amino acids: glycine (Gly), serine (Ser), threonine (Thr), cysteine (Cys), aspartic acid (Asn), glutamine (gin), and tyrosine (Tyr); 3. positively charged amino acids: arginine (Arg), lysine (Lys), and histidine (His); 4. negatively charged amino acids: aspartic acid (Asp) and glutamic acid (Glu) (see "biochemistry" (second edition) handbook, shen Tong, wang Jingyan, pages 82-83, higher education Press, 12 months 1990).
If a substitution of an amino acid residue belonging to the same class, for example Arg for Lys or Leu for Ile, occurs in a protein, the function of the residue in the protein domain (such as the function of providing a positive charge or forming a hydrophobic pocket structure) is not changed, and thus the steric structure of the protein is not affected, and thus the function of the protein can still be achieved. The substitution of the amino acid residue belonging to the same class can occur at any one of the amino acid residue positions of the self-consistent P450 enzyme.
In addition to the amino acid residue substitutions described above, the self-consistent P450 enzyme further comprises an amino acid sequence as compared to SEQ ID No.2 or SEQ ID NO:3, wherein one or more amino acid residues are deleted or added or both of the amino acid sequences shown in 3 are deleted or added at any position of the amino acid residues.
The self-consistent P450 enzyme can also be modified or mutated to obtain a derivative protein. Derived proteins are those having an amino acid sequence different from the self-consistent P450 enzyme having the above amino acid sequence, may have a modification which does not affect the sequence, or may have both. These proteins include natural or induced genetic variants. The induced variants may be obtained by various techniques such as random mutagenesis by irradiation or mutagens and the like, or by techniques such as site-directed mutagenesis or other known molecular biology techniques. Derived proteins also include analogs having residues of natural L-type amino acids (e.g., D-type amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta-amino acids, gamma-amino acids, etc.).
The modified (typically without altering the primary structure, i.e., without altering the amino acid sequence) forms include: chemically derivatized forms of proteins such as acetylated or carboxylated in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications during synthesis and processing of the protein or during further processing steps. Such modification may be accomplished by exposing the protein to an enzyme that performs glycosylation (e.g., mammalian glycosylase or deglycosylase). Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Proteins modified to increase their proteolytic resistance or to optimize their solubility properties are also included.
In the present invention, the self-consistent P450 enzyme may also be a polypeptide that hybridizes to SEQ ID NO:2 or SEQ ID NO:3, and has more than 80% homology, and has self-consistent P450 enzyme activity. Preferably, the self-consistent P450 enzyme is a polypeptide identical to SEQ ID NO:2 or SEQ ID NO:3, and more preferably has a homology of 90% or more, and more preferably 99% or more, and has an activity of a self-consistent P450 enzyme.
For ease of purification, the (a), (b) or (c) may also be additively modified with a tag common in the art, for example, by ligating the amino acid sequence of the tag (at least one of Poly-Arg, poly-His, FLAG, strep-tag II and c-myc) at the amino-and/or carboxy-terminus of (a). The tag does not influence the activity of the self-consistent P450 enzyme, and whether the tag is added or not can be selected according to the requirement in the actual application process.
In the present invention, the amino-terminal end of the self-consistent P450 enzyme may also be linked to a signal sequence. The signal sequence may be derived from bacillus licheniformis, bacillus amyloliquefaciens and bacillus subtilis, but is not limited thereto.
In the present invention, still having the enzyme activity means that the enzyme derived from (a) still has the enzyme activity at the same measurement conditions, and the percentage (relative activity) between the enzyme activity and the enzyme activity of (a) is not less than 60%, preferably not less than 70% (or 80%, or 90%, or 95%, or 96%, or 97%, or 98%, or 99%, or 100%).
According to the invention, preferably, the self-consistent P450 enzyme is a polypeptide having the sequence as set forth in SEQ ID NO: 2. SEQ ID NO:3 or SEQ ID NO:4, more preferably an enzyme having the amino acid sequence shown in SEQ ID NO:4, and an enzyme having an amino acid sequence shown in FIG. 4.
According to the invention, preferably, the self-consistent P450 enzyme is derived from Bacillus subtilis (Bacillus subtilis).
Derived from bacillus subtilis (Bacillus subtilis) and having the amino acid sequence as set forth in SEQ ID NO:1 belongs to the CYP102A1 subfamily, which is named CYP102A1-1, and NCBI accession number is CUB59589.1.
According to the invention, when CYP102A1-1 has a sequence as set forth in SEQ ID NO:1 by arginine to give a leucine having the amino acid sequence shown in SEQ ID NO:2, and the self-consistent P450 enzyme with the amino acid sequence shown in the specification is named CYP102A1-1L200R.
According to the invention, when CYP102A1-1 has a sequence as set forth in SEQ ID NO:1 by arginine to an amino acid sequence shown in SEQ ID NO:3, and the self-consistent P450 enzyme with the amino acid sequence shown in the specification is named CYP102A1-1L412R.
According to the invention, when CYP102A1-1 has a sequence as set forth in SEQ ID NO:1 and leucine at position 200 and leucine at position 412 of the amino acid sequence shown in SEQ ID NO:4, and the self-consistent P450 enzyme with the amino acid sequence shown in the formula 4 is named CYP102A1-1L200R/L412R.
Compared with CYP102A1-1, CYP102A1-1L200R, CYP A1-1L412R and CYP102A1-1L200R/L412R have higher enzymatic activities.
According to the present invention, preferably, the glucose dehydrogenase is derived from Bacillus subtilis (Bacillus subtilis 168). Glucose dehydrogenase from Bacillus subtilis (Bacillus subtilis) designated GDH with NCBI accession number NP 388275.1.
According to the present invention, preferably, the volume ratio of the self-consistent P450 enzyme to the glucose dehydrogenase is (3-5): 1.
the second aspect of the present invention provides a method for producing an enzyme preparation, comprising the steps of:
(1) Fermenting and culturing the genetically engineered bacterium containing the coding gene of the self-consistent P450 enzyme or the seed of the genetically engineered bacterium containing the coding gene of the glucose dehydrogenase to obtain the OD of the fermentation liquid 600 When the value is 10-15, mixing the fermentation liquor with an inducer for induction culture to obtain a bacteria-containing fermentation liquor; wherein the inducer is 5-aminolevulinic acid hydrochloride (5-ALA) and isopropyl thiogalactoside (IPTG);
(2) Breaking the wall of the bacteria-containing fermentation liquor to obtain wall-broken liquor;
(3) Separating the wall-broken liquid to obtain self-consistent P450 enzyme or glucose dehydrogenase;
(4) The self-consistent P450 enzyme and glucose dehydrogenase obtained by the above methods are mixed.
Namely, the self-consistent P450 enzyme can be prepared by the following method:
(1') fermenting and culturing seeds of genetically engineered bacteria containing coding genes of self-consistent P450 enzymes, and obtaining OD of fermentation liquor 600 When the value is 10-15, mixing the fermentation liquor with an inducer for induction culture to obtain a bacteria-containing fermentation liquor; wherein the inducer is 5-aminolevulinic acid hydrochloride (5-ALA) and isopropyl thiogalactoside (IPTG);
(2') breaking the wall of the bacteria-containing fermentation liquor to obtain a wall-broken liquor;
and (3') separating the wall-broken liquid to obtain the self-consistent P450 enzyme.
Glucose dehydrogenase can be prepared by the following method:
(1') fermenting and culturing seeds of genetically engineered bacteria containing genes encoding glucose dehydrogenase to obtain a fermentation broth OD 600 When the value is 10-15, mixing the fermentation liquor with an inducer for induction culture to obtain a bacteria-containing fermentation liquor; wherein the inducer is 5-aminolevulinic acid hydrochloride (5-ALA) and isopropyl thiogalactoside (IPTG);
(2') breaking the wall of the bacteria-containing fermentation broth to obtain a wall-broken liquid;
(3') separating the wall-broken liquid to obtain glucose dehydrogenase.
In the invention, the total time of the fermentation culture and the induction culture is the fermentation period.
In the invention, the inducer has the function of inducing the expression of the enzyme genes, and 5-ALA and IPTG are used together as the inducer, so that the 5-ALA and the IPTG can play a synergistic role and the expression quantity of the enzyme genes is improved.
In the method for preparing an enzyme preparation according to the second aspect of the present invention, the types and amounts of the self-consistent P450 enzyme and glucose dehydrogenase are identical to those of the self-consistent P450 enzyme and glucose dehydrogenase in the enzyme preparation mixture according to the first aspect of the present invention, and in order to avoid repetition, the present invention is not repeated in the second aspect, and those skilled in the art should not understand the limitation of the present invention.
According to the invention, the coding gene of the self-consistent P450 enzyme has a nucleotide sequence for coding the self-consistent P450 enzyme.
Accordingly, the gene may be (1) or (2) as follows:
(1) A DNA molecule having an amino acid sequence encoding the self-consistent P450 enzyme;
(2) A DNA molecule which hybridizes under stringent conditions to the DNA sequence defined in (1) and which encodes a self-consistent P450 enzyme with unchanged enzymatic activity. Wherein the stringent conditions may be: hybridization was performed in a solution of 6 XSCC, 0.5% SDS at 65℃and then washed once with 2 XSCC, 0.1% SDS and 1 XSCC, 0.1% SDS. The constant enzyme activity means that the percentage (relative activity) between the enzyme activity of the protein encoded by (2) and the enzyme activity of the protein encoded by (1) is not less than 95% (or 96%, or 97%, or 98%, or 99%, or 100%) under the same measurement conditions.
It is well known in the art that of the 20 different amino acids that make up a protein, other than Met (ATG) or Trp (TGG) are each encoded by a single codon, the 18 other amino acids are each encoded by 2-6 codons (Sambrook et al, molecular cloning, cold spring harbor laboratory Press, new York, U.S. second edition, 1989, see page 950 appendix D). That is, due to the degeneracy of the genetic code, the nucleotide sequence of the gene encoding the same protein may differ, since the substitution of the third nucleotide in the triplet codon, which determines most of the codons of one amino acid, does not change the composition of the amino acid. The nucleotide sequences of the genes which can be encoded by the amino acid sequences disclosed in the present invention and the amino acid sequences which are obtained from the amino acid sequences and have unchanged activity of the self-consistent P450 enzymes can be deduced completely by the skilled person according to known codon tables, and the nucleotide sequences are obtained by biological methods (such as PCR methods, mutation methods) or chemical synthesis methods, and therefore all the partial nucleotide sequences are included in the scope of the present invention. Conversely, by using the DNA sequences disclosed herein, amino acid sequences consistent with the activity of the self-consistent P450 enzymes can also be obtained by modifying the nucleic acid sequences provided herein by methods well known in the art, such as the method of Sambrook et al (molecular cloning, cold spring harbor laboratory Press, new York, U.S. second edition, 1989).
According to the invention, preferably, the coding gene of the self-consistent P450 enzyme has a sequence encoding a polypeptide having the sequence of SEQ ID NO: 2. SEQ ID NO:3 or SEQ ID NO:4, more preferably having a nucleotide sequence encoding an enzyme having the amino acid sequence shown in SEQ ID NO:4, and a nucleotide sequence of an enzyme having an amino acid sequence shown in seq id no.
According to a preferred embodiment of the invention, there is provided a polypeptide as set forth in SEQ ID NO:1, and the nucleotide sequence of the optimized encoding gene of the CYP102A1-1 with the amino acid sequence shown in the figure 1 is shown in SEQ ID NO. 5. At least one amino acid codon of the nucleotide sequence is substituted, resulting in a nucleotide sequence having a nucleotide sequence encoding a nucleotide sequence having the amino acid sequence of SEQ ID NO: 2. SEQ ID NO:3 or SEQ ID NO:4, and a nucleotide sequence of an enzyme having an amino acid sequence shown in seq id no.
According to a preferred embodiment of the invention, there is provided a polypeptide as set forth in SEQ ID NO:2, and the nucleotide sequence of the optimized encoding gene of CYP102A1-1L200R with the amino acid sequence shown in SEQ ID NO: TG from 599 th to 600 th in 5 is replaced with GT.
According to a preferred embodiment of the invention, there is provided a polypeptide as set forth in SEQ ID NO:3, and the nucleotide sequence of the optimized encoding gene of CYP102A1-1L412R with the amino acid sequence shown in SEQ ID NO: TG from 1235 to 1236 in 5 is replaced with GT.
According to a preferred embodiment of the invention, there is provided a polypeptide as set forth in SEQ ID NO:4, the nucleotide sequence of the optimized encoding gene of CYP102A1-1L200R/L412R with the amino acid sequence shown in SEQ ID NO: the 599 th to 600 th TG and the 1235 th to 1236 th TG in 5 are simultaneously replaced with GT.
The nucleotide sequence of the coding gene may be obtained by Polymerase Chain Reaction (PCR) amplification, recombinant methods, or artificial synthesis. For example, templates and primers can be readily obtained by those skilled in the art from the nucleotide sequences provided herein, and related nucleotide sequences can be obtained by amplification using PCR. Once the relevant nucleotide sequence is obtained, the relevant amino acid sequence can be obtained in large quantities by recombinant methods. The obtained nucleotide sequence is usually cloned into a vector to obtain a recombinant vector, the recombinant vector is transferred into a host cell, and the obtained nucleotide sequence is separated from the proliferated host cell by a conventional method.
In addition, the nucleotide sequence of interest can be synthesized by well-known methods of artificial chemical synthesis.
The vectors used in the recombinant vector containing the coding gene according to the present invention may be various vectors known in the art, such as various plasmids, cosmids, phages, retroviruses and the like, which are commercially available. According to the present invention, it is preferable that the expression vector of the recombinant vector containing the gene encoding the self-consistent P450 enzyme and the expression vector of the recombinant vector containing the gene encoding the glucose dehydrogenase are both pET-series plasmids, and more preferably pET-28a (+) vectors suitable for E.coli. pET-28a (+) vectors are commercially available, for example, from biological winds. The construction of the recombinant vector can be carried out by adopting various endonucleases capable of having cleavage sites at the multiple cloning sites of the vector to carry out enzyme digestion to obtain a linear plasmid, and connecting the linear plasmid with a gene fragment cut by adopting the same endonuclease to obtain the recombinant plasmid vector.
The recombinant vector may be transformed, transduced or transfected into a host cell (strain) by methods conventional in the art, such as calcium chloride chemical transformation, high voltage shock transformation. The host cell containing the recombinant vector encoding the gene of the self-consistent P450 enzyme and the recombinant vector containing the gene encoding the glucose dehydrogenase may be a prokaryotic cell or a eukaryotic cell, preferably E.coli (Escherichia coli) or actinomycetes, more preferably E.coli (Escherichia coli), most preferably E.coli BL21 (DE 3). Coli BL21 (DE 3) is commercially available, for example from Takara Shuzo (Beijing) technology Co.
According to the present invention, preferably, the fermentation culture is performed in a fermentation medium comprising, based on 100mL of the total volume of the fermentation medium: 2-3g of glucose, 0.5-1g of peptone, 0.5-1g of yeast extract, 0.2-0.5g of monopotassium phosphate, 0.5-1g of ammonium chloride, 0.05-0.1g of magnesium sulfate heptahydrate, 0.005-0.01g of manganese chloride tetrahydrate and 0.005-0.01g of anhydrous ferric chloride.
According to the invention, the pH of the fermentation medium is between 6.5 and 7, preferably the pH of the fermentation medium is adjusted using ammonia. After the preparation of the culture medium, the culture medium was sterilized at 121℃for 30min and then used for fermentation culture.
According to the present invention, preferably, the aeration ratio of the fermentation culture is 0.8 to 1VVM and the DO value is 30% or more; the ventilation ratio of the induction culture is 0.8-1VVM, and the DO value is more than 30%. When the ventilation ratio and DO value meet the range, not only can the oxygen supply requirement of normal growth of the thalli be met, but also the oxygen supply requirement of enzyme production of the thalli can be met.
In the invention, the ventilation ratio refers to the ratio of ventilation per minute to the volume of the actual fermentation broth; DO value refers to the dissolved oxygen content of the fermentation broth measured by the dissolved oxygen electrode, and DO value 100% refers to the saturated solubility of oxygen in the fermentation broth at the current temperature, pH and tank pressure when no oxygen is consumed; DO values above 30% indicate that the dissolved oxygen in the fermentation broth is controlled to above 30% of the saturated solubility value.
According to the invention, preferably, the temperature of the fermentation culture is 35-37 ℃. When the temperature of the fermentation culture is within this range, the bacterial cells can be grown rapidly.
According to the present invention, preferably, the temperature of the induction culture is 22-25 ℃. When the temperature of the induction culture is within the range, the growth and metabolism rate of the thalli can be reduced, and the synthesis of the product enzyme is facilitated.
According to the invention, preferably, the pH of the fermentation culture is between 6.5 and 7; the pH value of the induction culture is 6.5-7. When the pH of the fermentation culture and the induction culture is within this range, the cells can grow and produce enzymes relatively rapidly.
According to the present invention, preferably, the total time of the fermentation culture and the induction culture is 22 to 26 hours. When the total time of fermentation culture and induction culture is within this range, the enzyme yield of the cells can be increased.
According to the invention, preferably, 5-ALA is used in an amount of 100 to 200mg and IPTG is used in an amount of 0.1 to 0.15mmol, based on 1L of the total volume of the fermentation broth. When the induction concentration of 5-ALA and IPTG is within this range, the expression level of the bacterial enzymes can be increased.
According to the present invention, preferably, the preparation method further comprises: in the step (1), when the fermentation culture is carried out for 4-6 hours, continuously feeding a feed medium into the fermentation broth until the fermentation culture and the induction culture are finished, so as to obtain the bacteria-containing fermentation broth. When the culture is carried out for 4-6 hours, the glucose in the bottom material in the initial culture medium is consumed more, and the culture medium is fed with the feed in time, so that the thalli can continue to grow.
According to the present invention, preferably, the feed medium comprises, based on 100mL of total volume of the feed medium: glucose 40-60g and magnesium sulfate heptahydrate 0.5-1g. Sterilizing at 121deg.C for 30min. When the feed medium is in the concentration range, the thalli can continue to grow and produce enzyme faster.
According to the invention, the feed medium is preferably fed at a rate of 5-10mL/L/h, i.e. 5-10mL of feed medium per hour per liter of fermentation broth. When the feeding speed is controlled within the range, the nutrient requirements of normal growth and enzyme production of thalli can be met.
According to the present invention, preferably, in step (1), the seed preparation method includes: culturing the genetically engineered bacterium containing the coding gene of the self-consistent P450 enzyme or the genetically engineered bacterium containing the coding gene of the glucose dehydrogenase in a seed culture medium at the temperature of 30-35 ℃ for 3-5h to obtain the seeds.
According to the present invention, preferably, the seed medium is LB medium, and the cultivation is performed under the condition that the rotation speed of the shaking table is 220 rpm.
According to the invention, in the step (2), the bacteria-containing fermentation broth is subjected to solid-liquid separation by a first centrifugation, the supernatant is removed, and the bacteria are collected, and then the bacteria are broken. Preferably, the wall breaking method comprises the following steps: suspending the thalli by using phosphate buffer solution with pH value of 7.2-7.5 to obtain suspension, and breaking the wall of the suspension to obtain the wall breaking solution.
According to a specific embodiment of the invention, the first centrifugation is performed by using a centrifuge, preferably the rotation speed of the centrifuge is 5000-10000rpm, and the time of the first centrifugation is 20-30min; the wall breaking is carried out by adopting a high-pressure pulp homogenizing machine, preferably, the wall breaking condition of the high-pressure pulp homogenizing machine is that the wall breaking is carried out twice under the pressure condition of 80-100 MPa.
According to the present invention, preferably, in the step (2), the phosphate buffer is used in an amount of 3 to 6 times by weight of the cells.
According to the present invention, preferably, in the step (2), the phosphate buffer contains phosphate groups in an amount of 0.1mol based on 1L of the volume of the phosphate buffer.
According to the present invention, preferably, in step (3), the separation method comprises: mixing the wall breaking liquid with a Polyethyleneimine (PEI) solution with the concentration of 10-15wt% for flocculation precipitation to remove cell fragments and large protein particles, and then performing second centrifugation and collecting supernatant to obtain the self-consistent P450 enzyme or the glucose dehydrogenase.
According to a specific embodiment of the invention, the second centrifugation is performed with a centrifuge, preferably with a rotational speed of 5000-10000rpm, and the second centrifugation is performed for 20-30min.
According to the invention, a solution of Polyethylenimine (PEI) having a concentration of 10-15 wt.% can be formulated by the following method: PEI stock solution with concentration of 30wt% is diluted 2-3 times with deionized water, and then pH is adjusted to 7.2-7.5 with 30wt% sulfuric acid.
According to the present invention, preferably, in the step (3), the PEI solution is used in an amount of 0.25 to 0.45 times the weight of the cells in the step (2).
The method for producing a self-consistent P450 enzyme or glucose dehydrogenase according to an embodiment of the present invention will be described with reference to FIG. 1. Firstly, culturing seeds of genetically engineered bacteria containing coding genes of self-consistent P450 enzymes or genetically engineered bacteria containing coding genes of glucose dehydrogenase to obtain bacteria-containing fermentation liquor; centrifuging the bacteria-containing fermentation liquor to collect thalli, suspending the thalli by using a phosphate buffer solution to obtain a suspension, and breaking the wall of the suspension to obtain a wall-broken liquid; mixing the wall-broken liquid with PEI solution for flocculation precipitation to remove cell fragments and large protein particles, centrifuging and collecting enzyme-containing supernatant to obtain the self-consistent P450 enzyme or the glucose dehydrogenase.
In a third aspect, the invention provides an enzyme preparation mixture prepared by the preparation method provided by the invention.
In a fourth aspect, the present invention provides 25-hydroxycholesterol or 25-hydroxyvitamin D 3 The preparation method comprises the following steps: mixing enzyme preparation mixture, cosolvent, cofactor, substrate and solvent to obtain mixed solution, and then performing conversion reaction to obtain the 25-hydroxycholesterol or the 25-hydroxyvitamin D 3 ；
Wherein the substrate is cholesterol or vitamin D 3 The enzyme preparation mixture is provided by the invention.
According to the present invention, preferably, the temperature of the enzymatic conversion reaction is 25 to 35℃for 2 to 5 hours.
According to the present invention, the cofactor functions to promote binding of a substrate to an enzyme active site, thereby improving the reaction efficiency of an enzyme. Preferably, the cofactor is glucose.
According to the invention, the co-solvent acts to increase the substrate vitamin D 3 Or the solubility of cholesterol in an enzyme reaction system, so that a substrate is better contacted with an enzyme active site, and the enzyme reaction efficiency is improved. Preferably, the cosolvent is cyclodextrin.
According to the present invention, preferably, the solvent is selected from at least one of absolute ethanol, ethyl acetate, and diethyl ether.
According to the present invention, preferably, the substrate is used in an amount of 0.1 to 0.5g, the cosolvent is used in an amount of 10 to 15g, and the cofactor is used in an amount of 2 to 5g, based on 100mL of the total volume of the mixed solution.
According to the invention, the amount of the enzyme preparation mixture may be determined according to the actual requirements of the conversion reaction. Preferably, the enzyme preparation mixture is used in an amount of 85-95mL, based on 100mL of the total volume of the mixture.
According to one embodiment of the invention, the preparation method comprises the following steps: mixing the enzyme preparation mixture, cosolvent and cofactor to obtain a mixtureLiquid-1; mixing a substrate and a solvent to obtain a mixed solution-2; mixing the mixed solution-1 and the mixed solution-2 to obtain a mixed solution-3, and then performing an enzymatic conversion reaction to obtain the 25-hydroxycholesterol or the 25-hydroxyvitamin D 3 。
The present invention will be described in detail below by way of examples and comparative examples. In the following examples and comparative examples, conventional methods are employed unless otherwise specified; the reagents and materials used, unless otherwise indicated, are all those commercially available.
The materials and measurement methods involved in the examples and comparative examples of the present invention are as follows:
host cell escherichia coli BL21 (DE 3), purchased from bao bio (beijing) technologies limited;
plasmid vector pET-28a (+), purchased from biological wind;
the gene encoding the amino acid sequence of CYP102A1-1 (shown as SEQ ID NO: 1) (the optimized nucleic acid sequence of the encoding gene is shown as SEQ ID NO: 5) is synthesized by optimizing codons by Suzhou gold intelligent biotechnology Co., ltd.
Detecting 25-hydroxycholesterol or 25-hydroxyvitamin D in reaction system by HPLC 3 The content and the conversion rate of (3);
percent conversion = (product 25-hydroxycholesterol or 25-hydroxyvitamin D) 3 Is the amount of substance/substrate cholesterol or vitamin D 3 The amount of substance) x 100%.
Preparation example 1
The genetic engineering bacteria containing self-consistent P450 enzyme CYP102A1-1 gene are prepared according to the following method:
(a) The synthetic coding gene of CYP102A1-1 is used as a template, ndeI-CYP102-A1-1F (SEQ ID NO: 6) and HindIII-CYP102A1-1R (SEQ ID NO: 7) are used as primers for PCR amplification, and after gel cutting and recovery, a gene fragment with enzyme cutting sites and carrier homologous sequences at two ends is obtained.
(b) The pET-28a (+) plasmid was digested with the restriction enzyme NdeI/HindIII and cleaned using a PCR cleaning kit to give the vector backbone tangential to the NdeI/HindIII double enzyme, using ClonExpress II One Step Cloning Kit (purchasedOne-step ligation kit from Nanjinouzan Biotechnology Co., ltd.) links the NdeI/HindIII double-enzyme tangential vector backbone and CYP102A1-1 gene fragment, and then transforms the vector into competent cells of Escherichia coli BL21 (DE 3) and coats the competent cells to LB+Kn r Is incubated overnight at 37℃and the grown transformants are subjected to PCR verification and positive transformants are grown on LB+Kn r The plasmid was extracted by NdeI and HindIII double digestion and sequencing to obtain pET28a (+) -CYP102A1-1 plasmid.
(c) Transforming the recombinant plasmid obtained by construction into expression host escherichia coli BL21 (DE 3), and coating the recombinant plasmid on LB+Kn r Is incubated overnight at 37℃to give a monoclonal carrying the recombinant plasmid.
The self-consistent P450 enzyme CYP102A1-1 is prepared according to the following method:
(1) Fermentation culture: culturing the prepared genetically engineered bacteria in a shake flask filled with LB culture medium at a rotation speed of 220rpm and a temperature of 32 ℃ for 3 hours to obtain seed liquid, and then inoculating the seed liquid into a fermentation tank filled with fermentation culture medium. Culturing at 35deg.C, pH 7 and ventilation ratio of 1VVM, controlling DO value to 30% by stirring rotation speed of fermenter, continuously feeding culture medium after culturing for 4 hr, maintaining feeding speed at 6 ml/L/hr, adding 5-ALA and IPTG inducer (the dosage of 5-ALA and IPTG is 150mg and 0.12mmol respectively based on total volume of fermentation broth of 1L) when OD (600 nm) value of thallus in fermentation broth reaches 10, and simultaneously reducing culture temperature to 25deg.C for induction culture to obtain bacteria-containing fermentation broth when fermentation period is 25 hr; wherein, the liquid crystal display device comprises a liquid crystal display device,
The formula of the LB culture medium is as follows: based on 100mL of total volume of LB culture medium, 0.5g of yeast extract powder, 1g of sodium chloride and 1g of tryptone;
the formula of the fermentation medium is as follows: the total volume of the fermentation medium is 100mL, 2g of glucose, 0.8g of peptone, 0.5g of yeast extract, 0.4g of monopotassium phosphate, 0.7g of ammonium chloride, 0.06g of magnesium sulfate heptahydrate, 0.006g of manganese chloride tetrahydrate and 0.006g of anhydrous ferric chloride. Adjusting pH to 7 with ammonia water, and sterilizing at 121deg.C for 30min;
the formula of the feed medium is as follows: glucose 50g and magnesium sulfate heptahydrate 0.8g were calculated to make up 100mL of the total volume of the feed medium. Sterilizing at 121deg.C for 30min;
(2) Breaking the wall: centrifuging the bacteria-containing fermentation liquor for 20min at 5000rpm of a centrifuge, removing the supernatant, collecting wet bacterial sludge, re-suspending the bacterial sludge by using phosphate buffer solution with pH of 7.5 and phosphate content of 0.1mol/L, wherein the dosage of the phosphate buffer solution is 4 times of the weight of the bacterial sludge, and breaking the wall of the bacterial suspension by using a high-pressure homogenizer for two times under the condition of 95MPa to obtain wall-broken liquor;
(3) Flocculation sedimentation centrifugation: slowly adding the prepared PEI solution with the pH of 7.5 and the concentration of 10wt% into the wall-broken solution to perform full flocculation precipitation, wherein the dosage of the PEI solution is 0.3 time of the weight of the wet bacterial sludge, centrifuging for 30min by using a centrifugal machine at 10000rpm, and collecting the supernatant to obtain the enzyme-containing supernatant of the self-consistent P450 enzyme CYP102A 1-1.
Preparation example 2
The genetic engineering bacteria containing self-consistent P450 enzyme CYP102A1-1L200R genes are prepared according to the following method:
the constructed pET28a (+) -CYP102A1-1 plasmid is used as a template, primers L200R-F (SEQ ID NO: 8) and L200R-R (SEQ ID NO: 9) are used for amplification, PCR products are cleaned and recovered, dpnI single enzyme digestion is used for converting the digested PCR products into competent cells of escherichia coli BL21 (DE 3), and the competent cells are coated on LB+Kn r On the plate, the transformant is cultured upside down at 37 ℃ overnight, and sequencing verification is carried out on the grown transformant, thus obtaining the pET28a (+) -CYP102A1-1L200R plasmid which mutates leucine at 200 th position of CYP102A1-1 amino acid sequence into arginine. Transforming the recombinant plasmid obtained by construction into expression host escherichia coli BL21 (DE 3), and coating the recombinant plasmid on LB+Kn r Is incubated overnight at 37℃to give a monoclonal carrying the recombinant plasmid.
According to the method of preparation example 1, a self-consistent P450 enzyme CYP102A1-1L200R enzyme-containing supernatant was prepared by using a genetically engineered bacterium containing a self-consistent P450 enzyme CYP102A1-1L200R gene.
Preparation example 3
The genetic engineering bacteria containing self-consistent P450 enzyme CYP102A1-1L200R/L412R genes are prepared according to the following method:
to construct pET28a (+) The CYP102A1-1L200R plasmid is used as a template, the primers L412R-F (SEQ ID NO: 10) and L412R-R (SEQ ID NO: 11) are used for amplification, the PCR product is cleaned and recovered, the DpnI single enzyme digestion is used for converting the PCR product after enzyme digestion into competent cells of escherichia coli BL21 (DE 3), and the competent cells are coated on LB+Kn r On the plate, the transformant is cultured upside down at 37 ℃ overnight, and sequencing verification is carried out on the grown transformant, thus obtaining the pET28a (+) -CYP102A1-1L200R/L412R plasmid which simultaneously mutates leucine at 200 th and 412 th positions of the CYP102A1-1 amino acid sequence into arginine. Transforming the recombinant plasmid obtained by construction into expression host escherichia coli BL21 (DE 3), and coating the recombinant plasmid on LB+Kn r Is incubated overnight at 37℃to give a monoclonal carrying the recombinant plasmid.
According to the method of preparation example 1, a self-consistent P450 enzyme CYP102A1-1L200R/L412R enzyme-containing supernatant was prepared by using a genetically engineered bacterium containing a self-consistent P450 enzyme CYP102A1-1L200R/L412R gene.
Preparation example 4
Glucose dehydrogenase from Bacillus subtilis (Bacillus subtilis) designated GDH with NCBI accession number NP 388275.1.
The preparation method comprises the following steps of:
the cells of bacillus subtilis 168 are used as templates, ndeI-GDH-F (SEQ ID NO: 12) and HindIII-GDH-R (SEQ ID NO: 13) are used as primers for PCR amplification, and after gel cutting and recovery, the gene fragment with enzyme cutting sites and carrier homologous sequences at two ends is obtained. The NdeI/HindIII double enzyme tangential vector backbone and GDH fragment were ligated using ClonExpress II One Step Cloning Kit (purchased from Nanjinouzan Biotechnology Co., ltd.) one-step ligation kit, transformed into E.coli BL21 (DE 3) competent cells, and then coated to LB+Kn r Is incubated overnight at 37℃and the grown transformants are subjected to PCR verification and positive transformants are grown on LB+Kn r The plasmid was extracted overnight by shaking in a tube of (1) and verified by NdeI and HindIII double digestion and sequencing to give the pET28a (+) -GDH plasmid. Transforming the recombinant plasmid obtained by construction into expression host escherichia coli BL21 (DE 3), and coating the recombinant plasmid on LB+Kn r Is incubated overnight at 37℃to give a monoclonal carrying the recombinant plasmid.
Glucose dehydrogenase was prepared as follows:
(1) Fermentation culture: culturing the prepared genetically engineered bacteria in a shake flask filled with LB culture medium at a rotation speed of 220rpm and a temperature of 30 ℃ for 5 hours to obtain seed liquid, and then inoculating the seed liquid into a fermentation tank filled with fermentation culture medium. Culturing at 37deg.C, pH 6.8 and ventilation ratio of 1VVM, controlling DO value to 30% by stirring rotation speed of fermenter, continuously feeding culture medium after culturing for 6 hr, maintaining feeding speed at 10 ml/L/hr, adding 5-ALA and IPTG inducer (the dosage of 5-ALA and IPTG is 200mg and 0.15mmol respectively based on total volume of fermentation broth 1L) when thallus OD (600 nm) in fermentation broth reaches 15, and simultaneously reducing culture temperature to 22deg.C for induction culture until fermentation period is 24 hr to obtain bacteria-containing fermentation broth; wherein, the liquid crystal display device comprises a liquid crystal display device,
The formula of LB culture medium is the same as that of preparation example 1;
the formula of the fermentation medium is as follows: the total volume of the fermentation medium is 100mL, glucose 3g, peptone 1g, yeast extract 0.8g, monopotassium phosphate 0.5g, ammonium chloride 0.5g, magnesium sulfate heptahydrate 0.08g, manganese chloride tetrahydrate 0.01g and anhydrous ferric chloride 0.008g. Ammonia water to adjust pH value to 6.8, sterilizing at 121deg.C for 30min.
The formula of the feed medium is as follows: glucose 60g and magnesium sulfate heptahydrate 0.5g were added to the total volume of the feed medium of 100 mL. Sterilizing at 121deg.C for 30min;
(2) Breaking the wall: centrifuging the bacteria-containing fermentation liquor for 30min at 8000rpm by using a centrifuge, removing the supernatant, collecting wet bacterial sludge, re-suspending by using a phosphate buffer solution with the pH of 7.2 and the phosphate content of 0.1mol/L, wherein the dosage of the phosphate buffer solution is 6 times of the weight of the bacterial sludge, and breaking the wall of the bacterial suspension by using a high-pressure homogenizer for two times under the condition of the pressure of 90MPa to obtain wall-broken liquor;
(3) Flocculation sedimentation centrifugation: slowly adding the prepared PEI solution with the pH of 7.2 and the concentration of 10wt% into the wall-broken solution to perform full flocculation precipitation, wherein the dosage of the PEI solution is 0.4 time of the weight of the wet bacterial sludge, centrifuging for 30min by using a centrifugal machine at 8000rpm, and collecting the supernatant to obtain the glucose dehydrogenase GDH enzyme-containing supernatant.
Preparation example 5
The self-consistent P450 enzyme CYP102A1-1L200R/L412R was prepared as described in preparation example 3, except that in step (3) flocculation precipitation was performed using PEI at a concentration of 15% wt. The enzyme-containing supernatant of the self-consistent P450 enzyme CYP102A1-1L200R/L412R is obtained.
Preparation example 6
The self-consistent P450 enzyme CYP102A1-1L200R/L412R was prepared as in preparation example 3, except that only IPTG inducer was used, specifically, the amount of IPTG was 0.15mmol based on 1L total volume of fermentation broth. The enzyme-containing supernatant of the self-consistent P450 enzyme CYP102A1-1L200R/L412R is obtained.
Preparation example 7
Glucose dehydrogenase GDH was prepared as in preparation example 4, except that 5-ALA and IPTG were added to induce culture when the OD (600 nm) of the cells in the broth to be fermented reached 25. The glucose dehydrogenase GDH enzyme-containing supernatant was obtained.
The sequences of the primers used in preparation examples 1 to 7 are shown in Table 1.
TABLE 1
Example 1
25-hydroxycholesterol was prepared as follows:
mixing the self-consistent P450 enzyme CYP102A1-1L200R/L412R enzyme-containing supernatant prepared in preparation example 3 and the glucose dehydrogenase GDH enzyme-containing supernatant prepared in preparation example 4, adding cyclodextrin and enzyme reaction cofactor glucose, and fully stirring and dissolving to obtain a mixed solution-1; dissolving cholesterol in absolute ethyl alcohol to obtain a mixed solution-2; and then mixing the mixed solution-1 and the mixed solution-2 to obtain a mixed solution-3, and stirring the mixed solution at 28 ℃ for enzymatic conversion reaction for 3 hours to obtain the 25-hydroxycholesterol. Detecting the content of 25-hydroxycholesterol in the reaction system to be 3.06g/L by HPLC, wherein the conversion rate is 98.08%; wherein, the liquid crystal display device comprises a liquid crystal display device,
The total volume of the mixed solution-3 is 100mL, the dosage of cyclodextrin is 10g, the dosage of cofactor glucose is 2g, the dosage of cholesterol is 0.3g, the dosage of self-consistent P450 enzyme containing enzyme supernatant is 67.5mL, and the dosage of glucose dehydrogenase enzyme containing supernatant is 22.5mL.
Example 2
The 25-hydroxy vitamin D is prepared as follows 3 ：
Mixing the self-consistent P450 enzyme CYP102A1-1L200R/L412R enzyme-containing supernatant prepared in preparation example 3 and the glucose dehydrogenase GDH enzyme-containing supernatant prepared in preparation example 4, adding cyclodextrin and enzyme reaction cofactor glucose, and fully stirring and dissolving to obtain a mixed solution-1; vitamin D 3 Dissolving in anhydrous diethyl ether to obtain mixed solution-2; then mixing the mixed solution-1 and the mixed solution-2 to obtain a mixed solution-3, and stirring the mixed solution at 30 ℃ for enzyme conversion reaction for 5 hours to obtain 25-hydroxy vitamin D 3 . Detection of 25-hydroxyvitamin D in reaction System Using HPLC 3 The content of (2) is 5.09g/L, and the conversion rate is 97.88%; wherein, the liquid crystal display device comprises a liquid crystal display device,
the total volume of the mixed solution-3 is 100mL, the dosage of cyclodextrin is 15g, the dosage of the cofactor glucose is 5g, and the vitamin D 3 The amount of the enzyme-containing supernatant of the self-consistent P450 enzyme was 0.5g, the amount of the enzyme-containing supernatant of the self-consistent P450 enzyme was 72mL, and the amount of the enzyme-containing supernatant of the glucose dehydrogenase was 18mL.
Example 3
25-hydroxycholesterol was prepared as in example 1, except that the self-consistent P450 enzyme used was varied and the self-consistent P450 enzyme CYP102A1-1L200R/L412R containing enzyme supernatant prepared in preparation 5 was used instead of the equivalent volume of the self-consistent P450 enzyme CYP102A1-1L200R/L412R containing enzyme supernatant prepared in preparation 3. The content of 25-hydroxycholesterol in the reaction system was 3.13g/L as determined by HPLC, and the conversion was 97.08%.
Example 4
25-hydroxycholesterol was prepared as in example 1, except that the amount of cofactor glucose was varied. Specifically, the amount of cofactor glucose used was 8g, based on 100mL of the total volume of the mixture-3. Obtaining 25-hydroxycholesterol. The content of 25-hydroxycholesterol in the reaction system was 2.6g/L as determined by HPLC, and the conversion was 80.61%.
Example 5
25-hydroxycholesterol was prepared as in example 1, except that the amount of substrate cholesterol was varied. Specifically, the amount of cholesterol was 0.08g based on 100mL of the total volume of the mixed solution-3. Obtaining 25-hydroxycholesterol. The content of 25-hydroxycholesterol in the reaction system was 0.78g/L as determined by HPLC, and the conversion was 90.68%.
Example 6
Preparation of 25-hydroxyvitamin D by the method of example 2 3 The enzyme conversion reaction conditions are different. Specifically, the temperature of the enzymatic conversion reaction was 37℃for 1.5 hours. Obtaining 25-hydroxy vitamin D 3 . Detection of 25-hydroxyvitamin D in reaction System Using HPLC 3 The content of (C) was 2.2g/L, and the conversion was 78.36%.
Example 7
Preparation of 25-hydroxyvitamin D by the method of example 2 3 The difference is that the ratio of the amounts of self-consistent P450 enzyme and glucose dehydrogenase is different. Specifically, the amount of the enzyme-containing supernatant of the self-consistent P450 enzyme was 45mL and the amount of the enzyme-containing supernatant of the glucose dehydrogenase was 45mL, based on 100mL of the total volume of the mixed solution-3. Obtaining 25-hydroxy vitamin D 3 . Detection of 25-hydroxyvitamin D in reaction System Using HPLC 3 The content of (C) was 3.5g/L, and the conversion was 73.3%.
Example 8
25-hydroxycholesterol was prepared as in example 1, except that the self-consistent P450 enzyme CYP102A1-1 enzyme-containing supernatant prepared in preparation example 1 was used instead of the equivalent volume of the self-consistent P450 enzyme CYP102A1-1L200R/L412R enzyme-containing supernatant prepared in preparation example 3. Obtaining 25-hydroxycholesterol. The content of 25-hydroxycholesterol in the reaction system was 2.75g/L as determined by HPLC, and the conversion was 88.14%.
Example 9
25-hydroxycholesterol was prepared as in example 1, except that the self-consistent P450 enzyme CYP102A1-1L200R/L412R enzyme-containing supernatant prepared in preparation example 2 was used instead of the equivalent volume of the self-consistent P450 enzyme CYP102A1-1L200R/L412R enzyme-containing supernatant prepared in preparation example 3. Obtaining 25-hydroxycholesterol. The content of 25-hydroxycholesterol in the reaction system was 2.88g/L as determined by HPLC, and the conversion was 92.31%.
Comparative example 1
Preparation of 25-hydroxyvitamin D by the method of example 2 3 The difference is that the self-consistent P450 enzyme used was different, and the self-consistent P450 enzyme CYP102A1-1L200R/L412R enzyme-containing supernatant prepared in preparation example 6 was used instead of the self-consistent P450 enzyme CYP102A1-1L200R/L412R enzyme-containing supernatant prepared in preparation example 3. Detection of 25-hydroxyvitamin D in a System by HPLC 3 The content of (C) was 3.6g/L, and the conversion was 69.23%.
Comparative example 2
25-hydroxycholesterol was prepared as in example 1, except that glucose dehydrogenase was used, and the glucose dehydrogenase GDH-containing supernatant prepared in preparation example 7 was used instead of the glucose dehydrogenase GDH-containing supernatant prepared in preparation example 4 in an equal volume. The content of 25-hydroxycholesterol in the reaction system was 1.9g/L as determined by HPLC, and the conversion was 60.89%.
As can be seen from the results of the above examples and comparative examples, examples 1 to 9 use the technical scheme provided by the present invention to catalytically convert cholesterol or vitamin D by using the self-consistent P450 enzyme CYP102A1-1 enzyme-containing supernatant, the self-consistent P450 enzyme CYP102A1-1L200R enzyme-containing supernatant or the self-consistent P450 enzyme CYP102A1-1L200R/L412R enzyme-containing supernatant and glucose dehydrogenase GDH enzyme-containing supernatant 3 Production of 25-hydroxycholesterol or 25-hydroxyvitamin D 3 The conversion rate is more than 73%; comparative example 1 preparation of 25-hydroxyvitamin D from the enzyme-containing supernatant of the self-consistent P450 enzyme CYP102A1-1L200R/L412R prepared in preparation example 6 (using only IPTG inducer) instead of the enzyme-containing supernatant of the self-consistent P450 enzyme CYP102A1-1L200R/L412R prepared in preparation example 3 3 Comparative example 2 was conducted using preparation example 7 (cell OD in fermentation broth) 600 When the value reaches 25, adding 5-ALA and IPTG for induction culture), and replacing the glucose dehydrogenase GDH enzyme-containing supernatant prepared in preparation example 2 with the glucose dehydrogenase GDH enzyme-containing supernatant to prepare 25-hydroxycholesterol, wherein the conversion rate is obviously lowerDescending. The enzyme preparation mixture prepared by the method has higher enzyme activity, so that cholesterol or vitamin D is obtained 3 Has higher conversion rate. If the preparation process of glucose dehydrogenase is not properly selected, not only the conversion of the substrate is not promoted, but the progress of the conversion reaction may be inhibited.
In addition, in example 4, the amount of the cofactor glucose was changed when 25-hydroxycholesterol was prepared, and in example 5, the amount of the substrate cholesterol was changed when 25-hydroxycholesterol was prepared, and the conversion rate of cholesterol was reduced as compared with example 1; example 6 preparation of 25-hydroxyvitamin D 3 When the enzymatic conversion reaction conditions were changed, example 7 was conducted in the preparation of 25-hydroxyvitamin D 3 In this case, the ratio of the amounts of the enzyme-containing supernatant and glucose dehydrogenase GDH was changed from the self-consistent P450 enzyme CYP102A1-1L200R/L412R, and compared with example 2, vitamin D 3 The conversion of (c) is reduced. It is demonstrated that the cholesterol or vitamin D of the substrate can be further increased by selecting appropriate substrates, cofactors, enzymatic conversion reaction conditions and the ratio of the two enzymes in the enzyme preparation mixture 3 Is a conversion rate of (a).
In addition, the conversion rate of cholesterol in the case of example 8 using the self-consistent P450 enzyme CYP102A1-1 enzyme-containing supernatant and the conversion rate of cholesterol in the case of example 9 using the self-consistent P450 enzyme CYP102A1-1L200R enzyme-containing supernatant are smaller than the conversion rate of cholesterol in the case of example 1 using the self-consistent P450 enzyme CYP102A1-1L200R/L412R enzyme-containing supernatant. Example 8 compared to example 9, the conversion of cholesterol was higher in example 9 using the self-consistent P450 enzyme CYP102A1-1L200R enzyme-containing supernatant than in example 8 using the self-consistent P450 enzyme CYP102A1-1 enzyme-containing supernatant. Thus, the self-consistent P450 enzyme CYP102A1-1L200R and the self-consistent P450 enzyme CYP102A1-1L200R/L412R have the highest enzyme activity, and the self-consistent P450 enzyme CYP102A1-1L200R/L412R is inferior to the self-consistent P450 enzyme CYP102A1-1L 200R.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, a number of simple variants of the technical solution of the invention are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the invention, all falling within the scope of protection of the invention.
Sequence listing
SEQ ID NO. 1P 450 enzyme CYP102A1-1 amino acid sequence from bacillus subtilis (Bacillus subtilis)
MDKKVSAIPQPKTYGPLGNLPLIDKDKPTLSFIKLAEEYGPIFRMQTLSDTIIVVSGHELVAEVCDETRFDKSIEGALAKVRAFAGDGLFTSETHEPNWKKAHNILMPTFSQRAMKDYHAMMVDIAVQLVQKWARLNPNENVDVPEDMTRLTLDTIGLCGFNYRFNSFYRETPHPFITSMTRALDEAMHQLQRLDIEDKLMWRTKRQFQHDIQSMFSLVDNIIAERKSSENQEENDLLSRMLNVQDPETGEKLDDENIRFQIITFLIAGHETTSGLLSFAIYFLLKNPDKLKKAYEEVDRVLTDSTPTYQQVMKLKYIRMILNESLRLWPTAPAFSLYAKEDTVIGGKYPIKKGEDRISVLIPQLHRDKDAWGDDVEEFQPERFEELDKVPHHAYKPFGNGQRACIGMQFALHEATLVMGMLLQHFEFIDYEDYQLDVKQTLTLKPDDFKIRIVPRNQTISHTTVLAPTDEKLKNREIKQQVQKTPSIIGADNLSLLVLYGSDTGVAEGIARELADTASLEGVQTEVTALNDRIGSLPKEGAVLIVTSSYNGKPPSNAGQFVQWLEELKPDELKGVQYAVFGCGDHNWASTYQRIPRYIDEQMAQKGATRFSTRGEADASGDFEEQLEQWKQSMWSDAMKAFGLELNKNIEKERSTLSLQFVSRLGGSPLARTYEAVYASILENRELQSSSSERSTRHIEISLPEGATYKEGDHLGVLPINSEKNVNRILKRFGLNGKDQVILSASGRSVNHIPLDSPVRLYDLLSYSVEVQEAATRAQIREMVAFTACPPHKKELESLLEDGIYHEQILKKRISMLDLLEKYEACEIRFERFLELLPALKPRYYSISSSPLVAQDRLSITVGVVNAPAWSGEGTYEGVASNYLAQRHNKDEIICFIRTPQSNFQLPENPETPIIMVGPGTGIAPFRGFLQARRVQKQKGMKVGEAHLYFGCRHPEKDYLYRTELENDERDGLVSLHTAFSRLEGHPKTYVQHVIKEDRIHLISLLDNGAHLYICGDGSKMAPDVEDTLCQAYQEIHEVSEQEARNWLDRLQEEGRYGKDVWAGI
SEQ ID NO 2P450 enzyme CYP102A1-1L200R amino acid sequence
MDKKVSAIPQPKTYGPLGNLPLIDKDKPTLSFIKLAEEYGPIFRMQTLSDTIIVVSGHELVAEVCDETRFDKSIEGALAKVRAFAGDGLFTSETHEPNWKKAHNILMPTFSQRAMKDYHAMMVDIAVQLVQKWARLNPNENVDVPEDMTRLTLDTIGLCGFNYRFNSFYRETPHPFITSMTRALDEAMHQLQRLDIEDKRMWRTKRQFQHDIQSMFSLVDNIIAERKSSENQEENDLLSRMLNVQDPETGEKLDDENIRFQIITFLIAGHETTSGLLSFAIYFLLKNPDKLKKAYEEVDRVLTDSTPTYQQVMKLKYIRMILNESLRLWPTAPAFSLYAKEDTVIGGKYPIKKGEDRISVLIPQLHRDKDAWGDDVEEFQPERFEELDKVPHHAYKPFGNGQRACIGMQFALHEATLVMGMLLQHFEFIDYEDYQLDVKQTLTLKPDDFKIRIVPRNQTISHTTVLAPTDEKLKNREIKQQVQKTPSIIGADNLSLLVLYGSDTGVAEGIARELADTASLEGVQTEVTALNDRIGSLPKEGAVLIVTSSYNGKPPSNAGQFVQWLEELKPDELKGVQYAVFGCGDHNWASTYQRIPRYIDEQMAQKGATRFSTRGEADASGDFEEQLEQWKQSMWSDAMKAFGLELNKNIEKERSTLSLQFVSRLGGSPLARTYEAVYASILENRELQSSSSERSTRHIEISLPEGATYKEGDHLGVLPINSEKNVNRILKRFGLNGKDQVILSASGRSVNHIPLDSPVRLYDLLSYSVEVQEAATRAQIREMVAFTACPPHKKELESLLEDGIYHEQILKKRISMLDLLEKYEACEIRFERFLELLPALKPRYYSISSSPLVAQDRLSITVGVVNAPAWSGEGTYEGVASNYLAQRHNKDEIICFIRTPQSNFQLPENPETPIIMVGPGTGIAPFRGFLQARRVQKQKGMKVGEAHLYFGCRHPEKDYLYRTELENDERDGLVSLHTAFSRLEGHPKTYVQHVIKEDRIHLISLLDNGAHLYICGDGSKMAPDVEDTLCQAYQEIHEVSEQEARNWLDRLQEEGRYGKDVWAGI
SEQ ID NO 3P450 enzyme CYP102A1-1L412R amino acid sequence
MDKKVSAIPQPKTYGPLGNLPLIDKDKPTLSFIKLAEEYGPIFRMQTLSDTIIVVSGHELVAEVCDETRFDKSIEGALAKVRAFAGDGLFTSETHEPNWKKAHNILMPTFSQRAMKDYHAMMVDIAVQLVQKWARLNPNENVDVPEDMTRLTLDTIGLCGFNYRFNSFYRETPHPFITSMTRALDEAMHQLQRLDIEDKLMWRTKRQFQHDIQSMFSLVDNIIAERKSSENQEENDLLSRMLNVQDPETGEKLDDENIRFQIITFLIAGHETTSGLLSFAIYFLLKNPDKLKKAYEEVDRVLTDSTPTYQQVMKLKYIRMILNESLRLWPTAPAFSLYAKEDTVIGGKYPIKKGEDRISVLIPQLHRDKDAWGDDVEEFQPERFEELDKVPHHAYKPFGNGQRACIGMQFARHEATLVMGMLLQHFEFIDYEDYQLDVKQTLTLKPDDFKIRIVPRNQTISHTTVLAPTDEKLKNREIKQQVQKTPSIIGADNLSLLVLYGSDTGVAEGIARELADTASLEGVQTEVTALNDRIGSLPKEGAVLIVTSSYNGKPPSNAGQFVQWLEELKPDELKGVQYAVFGCGDHNWASTYQRIPRYIDEQMAQKGATRFSTRGEADASGDFEEQLEQWKQSMWSDAMKAFGLELNKNIEKERSTLSLQFVSRLGGSPLARTYEAVYASILENRELQSSSSERSTRHIEISLPEGATYKEGDHLGVLPINSEKNVNRILKRFGLNGKDQVILSASGRSVNHIPLDSPVRLYDLLSYSVEVQEAATRAQIREMVAFTACPPHKKELESLLEDGIYHEQILKKRISMLDLLEKYEACEIRFERFLELLPALKPRYYSISSSPLVAQDRLSITVGVVNAPAWSGEGTYEGVASNYLAQRHNKDEIICFIRTPQSNFQLPENPETPIIMVGPGTGIAPFRGFLQARRVQKQKGMKVGEAHLYFGCRHPEKDYLYRTELENDERDGLVSLHTAFSRLEGHPKTYVQHVIKEDRIHLISLLDNGAHLYICGDGSKMAPDVEDTLCQAYQEIHEVSEQEARNWLDRLQEEGRYGKDVWAGI
SEQ ID NO 4P450 enzyme CYP102A1-1L200R/L412R amino acid sequence
MDKKVSAIPQPKTYGPLGNLPLIDKDKPTLSFIKLAEEYGPIFRMQTLSDTIIVVSGHELVAEVCDETRFDKSIEGALAKVRAFAGDGLFTSETHEPNWKKAHNILMPTFSQRAMKDYHAMMVDIAVQLVQKWARLNPNENVDVPEDMTRLTLDTIGLCGFNYRFNSFYRETPHPFITSMTRALDEAMHQLQRLDIEDKRMWRTKRQFQHDIQSMFSLVDNIIAERKSSENQEENDLLSRMLNVQDPETGEKLDDENIRFQIITFLIAGHETTSGLLSFAIYFLLKNPDKLKKAYEEVDRVLTDSTPTYQQVMKLKYIRMILNESLRLWPTAPAFSLYAKEDTVIGGKYPIKKGEDRISVLIPQLHRDKDAWGDDVEEFQPERFEELDKVPHHAYKPFGNGQRACIGMQFARHEATLVMGMLLQHFEFIDYEDYQLDVKQTLTLKPDDFKIRIVPRNQTISHTTVLAPTDEKLKNREIKQQVQKTPSIIGADNLSLLVLYGSDTGVAEGIARELADTASLEGVQTEVTALNDRIGSLPKEGAVLIVTSSYNGKPPSNAGQFVQWLEELKPDELKGVQYAVFGCGDHNWASTYQRIPRYIDEQMAQKGATRFSTRGEADASGDFEEQLEQWKQSMWSDAMKAFGLELNKNIEKERSTLSLQFVSRLGGSPLARTYEAVYASILENRELQSSSSERSTRHIEISLPEGATYKEGDHLGVLPINSEKNVNRILKRFGLNGKDQVILSASGRSVNHIPLDSPVRLYDLLSYSVEVQEAATRAQIREMVAFTACPPHKKELESLLEDGIYHEQILKKRISMLDLLEKYEACEIRFERFLELLPALKPRYYSISSSPLVAQDRLSITVGVVNAPAWSGEGTYEGVASNYLAQRHNKDEIICFIRTPQSNFQLPENPETPIIMVGPGTGIAPFRGFLQARRVQKQKGMKVGEAHLYFGCRHPEKDYLYRTELENDERDGLVSLHTAFSRLEGHPKTYVQHVIKEDRIHLISLLDNGAHLYICGDGSKMAPDVEDTLCQAYQEIHEVSEQEARNWLDRLQEEGRYGKDVWAGI
SEQ ID NO. 5 nucleic acid sequence of the P450 enzyme CYP102A1-1 from Bacillus subtilis (Bacillus subtilis) optimized according to the codon preference of E.coli
ATGGATAAAAAAGTGAGCGCGATTCCGCAGCCGAAAACGTATGGCCCGCTGGGCAACCTGCCGCTGATTGATAAAGATAAACCGACCCTGAGCTTTATTAAACTGGCGGAAGAATATGGCCCGATTTTTCGCATGCAGACCCTGAGCGATACCATTATTGTGGTGAGCGGCCATGAACTGGTGGCGGAAGTGTGCGATGAAACCCGCTTTGATAAAAGCATTGAAGGCGCGCTGGCGAAAGTGCGCGCGTTTGCGGGCGATGGCCTGTTTACGAGCGAAACCCATGAACCGAACTGGAAAAAAGCCCATAACATTCTGATGCCGACCTTTAGTCAGCGCGCGATGAAAGATTATCATGCGATGATGGTGGATATTGCGGTGCAGCTGGTGCAGAAATGGGCGCGCCTGAACCCGAACGAAAACGTGGATGTGCCGGAAGATATGACCCGCCTGACCCTGGATACCATTGGCCTGTGCGGCTTTAACTATCGCTTTAACAGCTTTTATCGCGAAACCCCGCATCCGTTTATTACGAGCATGACCCGCGCGCTGGATGAAGCGATGCATCAGCTGCAGCGCCTGGATATTGAAGATAAACTGATGTGGCGCACCAAACGTCAGTTTCAGCATGATATTCAGAGCATGTTTAGCCTGGTGGATAACATTATTGCGGAACGCAAAAGCAGCGAAAACCAAGAAGAAAACGATCTGCTGAGCCGCATGCTGAACGTGCAAGATCCGGAAACCGGCGAAAAACTGGATGATGAAAACATTCGCTTTCAGATTATTACCTTTCTGATTGCGGGCCATGAAACCACGAGCGGCCTGCTGAGCTTTGCGATTTATTTTCTGCTGAAAAACCCGGATAAACTGAAAAAAGCGTATGAAGAAGTGGATCGCGTGCTGACCGATAGCACCCCGACCTATCAGCAAGTGATGAAACTGAAATATATTCGCATGATTCTGAACGAAAGCCTGCGCCTGTGGCCGACCGCGCCGGCGTTTAGCCTGTATGCGAAAGAAGATACCGTGATTGGCGGCAAATATCCGATTAAAAAAGGCGAAGATCGCATTAGCGTGCTGATTCCGCAGCTGCATCGCGATAAAGATGCGTGGGGCGATGATGTGGAAGAATTTCAGCCGGAACGCTTTGAAGAACTGGATAAAGTGCCGCATCATGCGTATAAACCGTTTGGCAACGGTCAGCGCGCGTGCATTGGCATGCAGTTTGCGCTGCATGAAGCGACCCTGGTGATGGGCATGCTGCTGCAGCATTTTGAATTTATTGATTATGAAGATTATCAGCTGGATGTGAAACAGACCCTGACCCTGAAACCGGATGACTTTAAAATTCGCATTGTGCCGCGCAATCAGACCATTAGCCATACCACCGTGCTGGCGCCGACCGATGAAAAACTGAAAAACCGCGAAATTAAACAGCAAGTGCAGAAAACCCCGAGCATTATTGGCGCGGATAACCTGAGCCTGCTGGTGCTGTATGGCAGCGATACCGGCGTGGCGGAAGGCATTGCGCGCGAACTGGCGGATACCGCGAGCCTGGAAGGCGTGCAGACCGAAGTGACCGCGCTGAACGATCGCATTGGCAGCCTGCCGAAAGAAGGCGCGGTGCTGATTGTGACGAGCAGCTATAACGGCAAACCGCCGAGCAACGCGGGTCAGTTTGTGCAGTGGCTGGAAGAACTGAAACCGGACGAACTGAAAGGCGTGCAGTATGCGGTGTTTGGCTGCGGCGATCATAACTGGGCGAGCACCTATCAGCGCATTCCGCGCTATATTGATGAACAGATGGCGCAGAAAGGCGCGACCCGCTTTAGCACCCGCGGCGAAGCGGATGCGAGCGGCGATTTTGAAGAACAGCTGGAACAGTGGAAACAGAGCATGTGGAGCGATGCGATGAAAGCGTTTGGCCTGGAACTGAACAAAAACATTGAAAAAGAACGCAGCACGCTGAGTCTGCAGTTTGTGAGCCGCCTGGGCGGCAGCCCATTAGCGCGCACCTATGAAGCGGTGTATGCGAGCATTCTGGAAAACCGCGAACTGCAGAGCAGTAGCAGCGAACGCAGCACCCGCCATATTGAAATTAGCCTGCCGGAAGGCGCGACCTATAAAGAAGGCGATCATCTGGGCGTGCTGCCGATTAACAGCGAAAAAAACGTGAACCGCATTCTGAAACGCTTTGGCCTGAACGGCAAAGATCAAGTGATTCTGAGCGCGAGCGGCCGCAGCGTGAACCATATTCCGCTGGATAGCCCGGTGCGCCTGTATGATTTATTAAGCTATAGCGTGGAAGTGCAAGAAGCGGCGACCCGCGCGCAGATTCGCGAAATGGTGGCGTTTACCGCGTGCCCGCCGCATAAAAAAGAACTGGAAAGCCTGCTGGAAGATGGCATTTATCATGAACAGATTCTGAAAAAACGCATTAGCATGCTGGATCTGCTGGAAAAATATGAAGCGTGCGAAATTCGCTTTGAACGCTTTCTGGAACTGCTGCCGGCGCTGAAACCGCGCTATTATAGCATTAGCAGTAGCCCGTTAGTGGCGCAAGATCGCCTGAGCATTACCGTGGGCGTGGTGAACGCGCCGGCGTGGAGCGGCGAAGGCACCTACGAAGGTGTGGCGAGCAACTATCTGGCGCAGCGCCATAACAAAGATGAAATTATTTGCTTTATTCGCACCCCGCAGAGCAACTTTCAGCTGCCGGAAAACCCGGAAACCCCGATTATTATGGTGGGCCCGGGCACCGGCATTGCGCCGTTTCGCGGCTTTCTGCAAGCGCGCCGCGTGCAGAAACAGAAAGGCATGAAAGTGGGCGAAGCCCATCTGTATTTTGGCTGCCGCCATCCGGAAAAAGATTATCTGTATCGCACGGAGCTGGAAAACGATGAACGCGATGGCCTGGTGAGCCTGCATACCGCGTTTAGCCGCCTGGAAGGCCATCCGAAAACCTATGTGCAGCATGTGATTAAAGAAGACCGTATTCATCTGATTAGCCTGCTGGATAACGGCGCGCATCTGTATATTTGCGGCGATGGCAGCAAAATGGCGCCGGATGTGGAAGATACCCTGTGCCAAGCGTATCAAGAAATTCATGAAGTGAGCGAACAAGAAGCGCGCAACTGGCTGGATCGCCTGCAAGAAGAAGGCCGCTATGGCAAAGATGTGTGGGCGGGCATTTAA
Claims (12)
1. An enzyme preparation mixture, characterized in that the enzyme preparation mixture comprises a self-consistent P450 enzyme and a glucose dehydrogenase; wherein, the liquid crystal display device comprises a liquid crystal display device,
the self-consistent P450 enzyme is the enzyme according to any one of (a) to (e):
(a) Has the sequence shown in SEQ ID NO:2 or SEQ ID NO:3, an enzyme having an amino acid sequence shown in FIG. 3;
(b) SEQ ID NO:2 or SEQ ID NO:3, and the enzyme shown in the amino acid sequence still has self-consistent P450 enzyme activity through substitution, deletion or addition of at least one amino acid residue;
(c) And SEQ ID NO:2 or SEQ ID NO:3, and has more than 80% homology, and has the enzyme shown in the amino acid sequence of self-consistent P450 enzyme activity;
(d) An enzyme represented by an amino acid sequence having a tag attached to the amino terminus and/or the carboxyl terminus of the amino acid sequence of (a), (b) or (c);
(e) An enzyme represented by an amino acid sequence wherein a signal sequence is linked to the amino terminus of the amino acid sequence of (a), (b) or (c).
2. The mixture according to claim 1, characterized in that the self-consistent P450 enzyme is a polypeptide identical to SEQ ID NO:2 or SEQ ID NO:3, preferably having more than 90%, more than 99%, homology, and having self-consistent P450 enzymatic activity;
preferably, the self-consistent P450 enzyme is a polypeptide having the sequence as set forth in SEQ ID NO: 2. SEQ ID NO:3 or SEQ ID NO:4, more preferably an enzyme having the amino acid sequence shown in SEQ ID NO:4, an enzyme having an amino acid sequence shown in FIG. 4;
preferably, the self-consistent P450 enzyme is derived from bacillus subtilis (Bacillus subtilis);
Preferably, the volume ratio of the self-consistent P450 enzyme to the glucose dehydrogenase is (3-5): 1.
3. a method for preparing an enzyme preparation, comprising the steps of:
(1) Fermenting and culturing the genetically engineered bacterium containing the coding gene of the self-consistent P450 enzyme or the seed of the genetically engineered bacterium containing the coding gene of the glucose dehydrogenase to obtain the OD of the fermentation liquid 600 When the value is 10-15, mixing the fermentation liquor with an inducer for induction culture to obtain a bacteria-containing fermentation liquor; wherein the inducer is 5-aminolevulinic acid hydrochloride and isopropyl thiogalactoside;
(2) Performing solid-liquid separation on the bacteria-containing fermentation liquid to obtain thalli, and breaking walls of the thalli to obtain wall-broken liquid;
(3) Separating the wall-broken liquid to obtain self-consistent P450 enzyme or glucose dehydrogenase;
(4) Mixing the self-consistent P450 enzyme and glucose dehydrogenase obtained by each;
preferably, the coding gene of the self-consistent P450 enzyme has a nucleotide sequence encoding a polypeptide having the sequence of SEQ ID NO: 2. SEQ ID NO:3 or SEQ ID NO:4, more preferably having a nucleotide sequence encoding an enzyme having the amino acid sequence shown in SEQ ID NO:4, and a nucleotide sequence of an enzyme having an amino acid sequence shown in seq id no.
4. The method according to claim 3, wherein the fermentation culture is performed in a fermentation medium comprising, based on 100mL of the total volume of the fermentation medium: 2-3g of glucose, 0.5-1g of peptone, 0.5-1g of yeast extract, 0.2-0.5g of monopotassium phosphate, 0.5-1g of ammonium chloride, 0.05-0.1g of magnesium sulfate heptahydrate, 0.005-0.01g of manganese chloride tetrahydrate and 0.005-0.01g of anhydrous ferric chloride;
preferably, the aeration ratio of each of the fermentation culture and the induction culture is independently 0.8 to 1VVM, and the DO value of each is independently 30% or more;
preferably, the temperature of the fermentation culture is 35-37 ℃;
preferably, the temperature of the induction culture is 22-25 ℃;
preferably, the pH of each of the fermentation culture and the induction culture is independently from 6.5 to 7;
preferably, the total time of the fermentation culture and the induction culture is 22-26 hours;
preferably, the amount of 5-aminolevulinic acid hydrochloride is 100-200mg and the amount of isopropyl thiogalactoside is 0.1-0.15mmol, based on 1L total volume of the fermentation broth.
5. The production method according to claim 3 or 4, characterized in that the production method further comprises: in the step (1), when the fermentation culture is carried out for 4-6 hours, continuously feeding a feed medium into the fermentation liquid;
Preferably, the feed medium comprises, based on a total volume of 100mL of the feed medium: glucose 40-60g and magnesium sulfate heptahydrate 0.5-1g;
preferably, the feeding speed of the feeding medium is 5-10mL/L/h.
6. The method according to any one of claims 3 to 5, wherein the genetically engineered bacterium containing a gene encoding a self-consistent P450 enzyme and the host cell of the genetically engineered bacterium containing a gene encoding a glucose dehydrogenase are each independently selected from escherichia coli or actinomycetes.
7. The method according to any one of claims 3 to 6, wherein in the step (1), the seed preparation method comprises: culturing the genetically engineered bacterium containing the coding gene of the self-consistent P450 enzyme or the genetically engineered bacterium containing the coding gene of the glucose dehydrogenase in a seed culture medium at the temperature of 30-35 ℃ for 3-5h to obtain the seeds.
8. The method according to any one of claims 3 to 7, wherein in the step (2), the method of breaking wall comprises: suspending the thalli by using phosphate buffer solution with pH value of 7.2-7.5 to obtain suspension, and breaking the wall of the suspension to obtain the wall breaking solution.
9. The method according to any one of claims 3 to 8, wherein in step (3), the method of separation comprises: mixing the wall-broken liquid with a polyethyleneimine solution with the concentration of 10-15wt% for flocculation precipitation, and then performing secondary centrifugation and collecting supernatant to obtain the self-consistent P450 enzyme or the glucose dehydrogenase.
10. An enzyme preparation mixture obtainable by the process of any one of claims 3 to 9.
11. 25-hydroxycholesterol or 25-hydroxyvitamin D 3 Is characterized in that the preparation method comprises the following steps: mixing enzyme preparation mixture, cosolvent, cofactor, substrate and solvent to obtain mixed solution, and then performing conversion reaction to obtain the 25-hydroxycholesterol or the 25-hydroxyvitamin D 3 ；
Wherein the substrate is cholesterol or vitamin D 3 The enzyme preparation mixture according to any one of claims 1, 2 and 10;
preferably, the temperature of the enzymatic conversion reaction is 25-35 ℃ and the time is 2-5h.
12. The method of claim 11, wherein the cofactor is glucose;
Preferably, the cosolvent is cyclodextrin;
preferably, the solvent is selected from at least one of absolute ethanol, ethyl acetate and diethyl ether;
preferably, the substrate is used in an amount of 0.1 to 0.5g, the cosolvent is used in an amount of 10 to 15g, and the cofactor is used in an amount of 2 to 5g, based on 100mL of the total volume of the mixed solution.
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