CN117050959A - Biliverdin reductase mutant and application thereof in bilirubin synthesis - Google Patents
Biliverdin reductase mutant and application thereof in bilirubin synthesis Download PDFInfo
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- CN117050959A CN117050959A CN202311005734.4A CN202311005734A CN117050959A CN 117050959 A CN117050959 A CN 117050959A CN 202311005734 A CN202311005734 A CN 202311005734A CN 117050959 A CN117050959 A CN 117050959A
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- alanine
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Abstract
The invention relates to a biliverdin reductase mutant and application thereof in bilirubin synthesis. In particular discloses a biliverdin reductase and a mutant with improved activity, a coding gene and an amino acid sequence thereof, a recombinant expression vector and a recombinant expression transformant containing the gene sequence, a preparation method of a recombinant biliverdin reductase catalyst, and application of the recombinant biliverdin reductase catalyst in bilirubin preparation. Compared with other methods for producing bilirubin, the enzymatic reaction has the advantages of high substrate concentration, mild reaction condition, environmental friendliness, simplicity in operation, high yield and the like, and has good industrial application prospect.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a bacterium for producing biliverdin reductase, a mutant and application of the bacterium in bilirubin synthesis.
Background
Bilirubin is the main pigment in bile, is a monoclinic crystal of orange yellow color, and is a metabolite of heme in mammals. Bilirubin is one of the main components of bezoar, and bezoar currently on the market includes natural bezoar, in vitro cultured bezoar, artificial bezoar and the like. Natural bezoar is gall-stone of cattle, bezoar is a precious rare traditional Chinese medicinal material, and has medicinal history for over two thousand years in China. The bezoar medicines in Chinese pharmacopoeia reach more than 50, such as Angong bezoar pills, bezoar detoxicating tablet, bezoar supernatant capsule, etc. However, natural bezoar is rare, and in order to alleviate the problem of shortage of natural bezoar medicine sources, chinese scholars succeed in developing artificial bezoar in the 70 th century according to the ingredients of natural bezoar. However, the bilirubin content in artificial bezoar is only 0.7%, which is far lower than the content standard of natural bezoar of 25% and in vitro cultured bezoar of 35%, and the effect is naturally different, so that the improvement of bilirubin content and purity is beneficial to the improvement of the quality of artificial bezoar. Besides, bilirubin is also the main component of 100 Chinese patent medicines recorded in pharmacopoeia, and has the effects of tranquilizing, relieving convulsion, reducing blood pressure, relieving fever, treating diseases such as serum hepatitis, liver cirrhosis and the like, and has large market demand.
Because of the complex spatial structure of bilirubin, its chemical synthesis process has not yet been applied to maturity. The current bilirubin production methods mainly comprise two types: firstly, the extraction method is to extract natural bilirubin from bile of animals such as pig gall, which is the main production method of bilirubin in the market at present. However, the content of natural bilirubin in animal bile is very small, for example, bilirubin content in pig bile is about 0.05%, and 5 tons of pig bile is required for extracting 1 kg of bilirubin, resulting in high cost for producing bilirubin. Subsequently, a technique for draining bile by fistulization of live pigs was developed in China, which solves the problem of bile source, but the related problems of animal protection and animal ethics cause great social disputes. Another method is enzymatic synthesis. The biosynthesis method has the advantages of mild reaction conditions, environmental friendliness, high optical purity of the product, no need of toxic chemical reagents and the like, has good industrial application prospect, and is expected to gradually replace the traditional extraction method.
In 2012, chinese patent CN 103114110A reports that 0.1mM heme is catalyzed to bilirubin using immobilized heme oxygenase and biliverdin reductase, the conversion rate reaches 80%, but 1mM NADP is required to be added as cofactor during the reaction, thereby resulting in high cost. On the basis, chinese patent CN 114891707A reports a three-enzyme cascade system consisting of heme oxygenase, biliverdin reductase and glucose dehydrogenase, and the concentration of heme substrate can reach 1g/L.
In 2020, chinese patent CN 112143691A discloses an escherichia coli and its application in preparing biliverdin reductase, which can convert biliverdin into bilirubin in one step by engineering bacteria fermentation (Bioprocess and Biosystems Engineering,2022, 45:563-571). Since the activity of the obtained biliverdin reductase was only 1.84U/mg, only 0.44g/L of the substrate biliverdin (CN 113186235A) could be converted in the subsequent application.
In 2022, chinese patent CN 115044565A disclosed a mutant of biliverdin reductase derived from Rattus norvegicus, which can catalyze bioconversion of 0.5-1g/L biliverdin, but the reaction process requires the addition of 1-1.5g/L of expensive NADPH as a coenzyme, thus resulting in excessive cost and limiting its industrial application.
In conclusion, the route for synthesizing bilirubin by using biliverdin reductase as a catalyst is shortest, and the industrial prospect is wide. However, the currently reported biliverdin reductase has the problems of few varieties, low catalytic activity, low product concentration, low production efficiency and the like. Therefore, there is also a need to develop an enzyme catalyst with better catalytic performance to meet the technical requirements of industrial bilirubin production.
Disclosure of Invention
The invention aims to overcome the problems and the defects of the prior art, and provides a novel biliverdin reductase, which is used for catalyzing a substrate biliverdin reduction reaction to synthesize bilirubin by using the enzyme and a mutant thereof, and has the remarkable advantages of high concentration of a reaction substrate, mild reaction condition, simple process, high product yield and the like.
The aim of the invention can be achieved by the following technical scheme:
in a first aspect of the present invention, a biliverdin reductase is provided that catalyzes a biliverdin reduction reaction effective to produce bilirubin.
The biliverdin reductase of the present invention is derived from Nostoc sp or nochloropsis sp or hydrocystis sp. The Nostoc sp, the nococappa sp or the hydrocystis sp can catalyze the reduction reaction of bilirubin to produce bilirubin as a target product by culturing. The corresponding biliverdin reductase in the three bacteria was obtained by gene alignment and excavation methods and was named NoBVR, glBVR, hyBVR, respectively. The amino acid sequences of the biliverdin reductase NoBVR, glBVR, hyBVR are respectively shown as SEQ ID No.2, SEQ ID No.4 and SEQ ID No. 6. Methods for preparing the biliverdin reductase of the present invention include, but are not limited to: 1) Direct extraction and isolation from cells of genus Nostoc (Nostoc sp.) or genus nochloropsis (gloeocap sp.) or genus hydrocystis (Hydrococcus sp.); 2) Obtained by heterologous recombinant expression of the gene encoding the biliverdin reductase according to techniques conventional in the art.
In a second aspect of the invention, there are provided a plurality of biliverdin reductase mutants, each having increased activity compared to the parent;
further, the stability of the partial mutants was also improved.
The invention takes a biliverdin reductase from Nostoc (Nostoc sp.) as a female parent, and carries out molecular modification on the biliverdin reductase to obtain a mutant of the biliverdin reductase.
Further, the biliverdin reductase mutant is obtained by random mutation.
The biliverdin reductase mutant is a derivative protein of a novel amino acid sequence, wherein one or more amino acids of phenylalanine at position 26, glutamic acid at position 48, alanine at position 61, glycine at position 97, alanine at position 122, arginine at position 156, threonine at position 159, methionine at position 182, valine at position 198, serine at position 228, lysine at position 255, phenylalanine at position 260, asparagine at position 263, isoleucine at position 276, aspartic acid at position 298, tyrosine at position 303, alanine at position 318 or methionine at position 331 of the amino acid sequence shown in SEQ ID No.2 are replaced by other amino acids.
The biliverdin reductase mutant has one of the following sequences:
(1) Substitution of phenylalanine at position 26 of the amino acid sequence shown as SEQ ID No.2 in the sequence table with glutamic acid and substitution of alanine at position 61 with threonine;
(2) The 48 th glutamic acid of the amino acid sequence shown as SEQ ID No.2 in the sequence table is replaced by glycine;
(3) Substitution of threonine for glutamic acid at position 48 and threonine for alanine at position 61 of the amino acid sequence shown in SEQ ID No.2 of the sequence Listing;
(4) Substitution of threonine for glutamic acid at position 48, threonine for alanine for threonine for 61, and histidine for asparagine for 263 of the amino acid sequence shown in SEQ ID No.2 of the sequence Listing;
(5) Substitution of phenylalanine at position 26 of the amino acid sequence shown as SEQ ID No.2 in the sequence table with tyrosine and isoleucine at position 276 with valine;
(6) Substitution of phenylalanine at position 26 for tyrosine, isoleucine at position 276 for valine, aspartic acid at position 298 for serine in the amino acid sequence shown in SEQ ID No.2 of the sequence Listing;
(7) The 97 th glycine of the amino acid sequence shown as SEQ ID No.2 in the sequence table is replaced by threonine, the 122 th alanine is replaced by threonine, the 156 th arginine is replaced by lysine, the 159 th threonine is replaced by asparagine, and the 182 th methionine is replaced by leucine;
(8) Replacing alanine at 122 th site of an amino acid sequence shown as SEQ ID No.2 in a sequence table with threonine;
(9) The 122 th alanine of the amino acid sequence shown as SEQ ID No.2 in the sequence table is replaced by threonine, and the 228 th serine is replaced by alanine;
(10) Substitution of alanine at position 122 for threonine, serine at position 228 for alanine, asparagine at position 263 for lysine in the amino acid sequence shown in SEQ ID No.2 of the sequence Listing;
(11) The 26 th phenylalanine of the amino acid sequence shown as SEQ ID No.2 in the sequence table is replaced by tyrosine, the 48 th glutamic acid is replaced by threonine, the 61 th alanine is replaced by threonine, the 97 th glycine is replaced by threonine, the 122 th alanine is replaced by threonine, the 156 th arginine is replaced by lysine, the 182 th methionine is replaced by leucine, the 198 th valine is replaced by serine, the 228 th serine is replaced by glutamic acid, the 255 th lysine is replaced by isoleucine, the 260 th phenylalanine is replaced by valine, the 263 th asparagine is replaced by serine, the 276 th isoleucine is replaced by valine, the 303 th tyrosine is replaced by serine, and the 331 st methionine is replaced by alanine;
(12) Substitution of alanine at position 61 for threonine, glycine at position 97 for threonine, arginine at position 156 for lysine, and isoleucine at position 276 for valine of the amino acid sequence shown in SEQ ID No.2 of the sequence Listing;
(13) The 61 st alanine of the amino acid sequence shown as SEQ ID No.2 in the sequence table is replaced by threonine, the 97 th glycine is replaced by threonine, the 122 th alanine is replaced by threonine, the 156 th arginine is replaced by lysine, the 182 th methionine is replaced by leucine, the 198 th valine is replaced by serine, the 228 th serine is replaced by glutamic acid, the 255 th lysine is replaced by isoleucine, the 260 th phenylalanine is replaced by valine, the 276 th isoleucine is replaced by valine, and the 303 th tyrosine is replaced by serine;
(14) The 122 th alanine of the amino acid sequence shown as SEQ ID No.2 in the sequence table is replaced by threonine, the 159 th threonine is replaced by asparagine, the 198 th valine is replaced by serine, and the 318 th alanine is replaced by proline;
(15) The 182 th methionine of the amino acid sequence shown as SEQ ID No.2 in the sequence table is replaced by leucine, the 198 th valine is replaced by serine, and the 228 th serine is replaced by histidine;
(16) Substitution of methionine at 182 th position for leucine, valine at 198 th position for serine, asparagine at 263 th position for isoleucine of the amino acid sequence shown in SEQ ID No.2 of the sequence Listing;
(17) Substitution of serine at 228 th site of the amino acid sequence shown as SEQ ID No.2 in the sequence table with glutamic acid and substitution of lysine at 255 th site with isoleucine;
(18) Replacing lysine at 255 th site of an amino acid sequence shown as SEQ ID No.2 in a sequence table with isoleucine;
(19) The 26 th phenylalanine of the amino acid sequence shown as SEQ ID No.2 in the sequence table is replaced by tyrosine, the 48 th glutamic acid is replaced by threonine, the 61 th alanine is replaced by threonine, the 97 th glycine is replaced by threonine, the 122 th alanine is replaced by threonine, the 198 th valine is replaced by serine, the 228 th serine is replaced by glutamic acid, the 255 th lysine is replaced by isoleucine, the 260 th phenylalanine is replaced by valine, the 263 th asparagine is replaced by serine, the 276 th isoleucine is replaced by valine, the 298 th aspartic acid is replaced by serine, and the 318 th alanine is replaced by alanine;
(20) The 198 th valine of the amino acid sequence shown as SEQ ID No.2 in the sequence table is replaced by isoleucine, and the 303 rd tyrosine is replaced by phenylalanine;
(21) The 122 th alanine of the amino acid sequence shown as SEQ ID No.2 in the sequence table is replaced by threonine, the 198 th valine is replaced by serine, the 228 th serine is replaced by glutamic acid, the 255 th lysine is replaced by isoleucine, the 263 th asparagine is replaced by serine, the 276 th isoleucine is replaced by valine, the 298 th aspartic acid is replaced by serine, and the 318 th alanine is replaced by alanine;
(22) Substitution of phenylalanine at 260 th position with valine and substitution of asparagine at 263 th position with serine of the amino acid sequence shown as SEQ ID No.2 in the sequence table;
(23) Substitution of phenylalanine at position 260 with valine, asparagine at position 263 with serine, aspartic acid at position 298 with serine in the amino acid sequence shown in SEQ ID No.2 of the sequence table;
(24) The 263 rd asparagine of the amino acid sequence shown as SEQ ID No.2 in the sequence table is replaced by serine, the 276 rd isoleucine is replaced by valine, the 303 rd tyrosine is replaced by serine, and the 331 st methionine is replaced by alanine;
(25) Isoleucine 276 of the amino acid sequence shown as SEQ ID No.2 in the sequence table is replaced by valine, and tyrosine 303 is replaced by serine.
In a third aspect of the present invention, there is provided nucleic acids encoding the above-described biliverdin reductase and mutants thereof.
Sources of nucleic acids encoding the biliverdin reductase and mutants thereof include: the encoding genes of the biliverdin reductase and the mutant thereof are obtained through a gene cloning technology, or the encoding genes of the biliverdin reductase and the mutant thereof are obtained through an artificial full sequence synthesis method.
In a fourth aspect of the invention, there is provided a recombinant expression vector comprising a nucleic acid sequence of a biliverdin reductase or a mutant thereof according to the invention.
The recombinant expression vector can be constructed by ligating the nucleotide sequence of the biliverdin reductase gene of the present invention to various vectors by a conventional method in the art. The vector may be any of various plasmid vectors conventional in the art, preferably plasmid pET28a. Preferably, the recombinant expression vector of the present invention can be prepared by the following method: the gene sequence DNA fragment of the biliverdin reductase or the mutant thereof obtained by PCR amplification is digested with restriction enzymes EcoR I and Xho I, simultaneously the empty plasmid pET28a is digested with restriction enzymes EcoR I and Xho I, the digested biliverdin reductase DNA fragment and plasmid are recovered, and the recombinant expression vector containing the gene of the biliverdin reductase or the mutant thereof is constructed by using T4 DNA ligase.
In a fifth aspect of the present invention, there is provided a recombinant expression transformant comprising the biliverdin reductase gene of the present invention or a recombinant expression vector thereof.
The recombinant expression transformant can be prepared by transforming the above recombinant expression vector into a corresponding host cell by a conventional technique in the art. The host cell is a conventional host cell in the art, so long as the recombinant expression vector can stably and automatically replicate, and the encoded biliverdin reductase gene can be effectively expressed. The host cell is preferably E.coli, more preferably E.coli BL21 (DE 3).
In a sixth aspect of the invention, there is provided a recombinant biliverdin reductase catalyst in a form including, but not limited to:
(1) Culturing the recombinant expression transformant of the present invention, and isolating a transformant cell containing the biliverdin reductase;
(2) Culturing the recombinant expression transformant of the present invention, and separating a crude enzyme solution containing the biliverdin reductase;
(3) And drying the crude enzyme solution of the biliverdin reductase to obtain crude enzyme powder.
Wherein the culture medium used for the culture of the recombinant expression transformant may be selected from conventional culture media in the art, provided that the recombinant expression transformant can be grown and the recombinant biliverdin reductase of the present invention can be produced. For the host cell, the preferred medium is LB medium: 10g/L peptone, 5g/L yeast extract, 10g/L NaCl and pH 6.5-7.0. The preferred cultivation method is: recombinant E.coli constructed as described above was inoculated into LB medium containing kanamycin, and cultured overnight at 37℃with shaking at 180 rpm. Inoculating 1-2% (v/v) of the strain into a 2L Erlenmeyer flask containing 400mL of LB medium (containing kanamycin), shaking at 37deg.C with 180rpm, and culturing to OD 600 When the concentration reaches 0.6-0.8, adding isopropyl-beta-D-thiohemi-with the final concentration of 0.1-0.5mmol/LAfter inducing for 16-24 hours at 16-25 ℃, the culture solution is centrifuged, the precipitate is collected, and then washed twice with physiological saline to obtain recombinant expression transformant cells. Suspending the obtained recombinant cells in buffer solution with the volume of 5-10 times (v/w), performing ultrasonic crushing, centrifuging and collecting supernatant, thus obtaining the crude enzyme solution of the recombinant biliverdin reductase. And (3) placing the collected crude enzyme solution at the temperature of minus 80 ℃ for freezing, and then drying at low temperature by using a vacuum freeze dryer to obtain the freeze-dried enzyme powder. The obtained freeze-dried enzyme powder is stored in a refrigerator at the temperature of 4 ℃ and can be conveniently used.
The method for measuring the activity of the biliverdin reductase comprises the following steps: to a 5mL reaction system (100 mmol/L potassium phosphate buffer, pH 7.0) containing 0.15g/L substrate (biliverdin), 10mM glucose, 0.2mM NADP+, and 1.5g/L glucose dehydrogenase, an appropriate amount of biliverdin reductase was added, and the reaction was carried out at 30℃for 15 minutes, and then a sample was taken and subjected to liquid chromatography analysis for the conversion. 1 enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the production of 1. Mu. Mol of product per minute under the above conditions.
The seventh aspect of the invention also provides an application method of the biliverdin reductase catalyst in bilirubin preparation.
The application is that the biliverdin reductase is added into a solution containing biliverdin to catalyze the biliverdin reduction reaction to generate bilirubin.
In the aforementioned applications, the concentration of biliverdin may be 0.1 to 1g/L, the concentration of glucose may be 1 to 10g/L, the amount of recombinant biliverdin reductase may be selected to be 0.5 to 5g/L, the amount of glucose dehydrogenase may be 0.5 to 1.5g/L, NADP + The dosage can be 0.05-0.2 mM, and the buffer solution is phosphate buffer solution with pH of 6.0-9.0. The glucose dehydrogenase is derived from Bacillus megaterium Bacillus megaterium (Journal of Biological Chemistry,1989, 264:6381-6385). The reaction temperature is 20 to 35 ℃, preferably 30 ℃. The reaction time is based on the time when the reaction conversion rate stops increasing, and the reaction time is preferably less than 12 hours. The reaction conversion was analyzed by liquid chromatography under the following conditions: c18 column (250 mm. Times.4.6 mm), mobile phase acetonitrile/0.1% aqueous trifluoroacetic acid solution=95:5 (v/v), column temperature 30 ℃, flow rate 1.0mL/min, detection wavelength 450nm.
On the basis of the common general knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the present invention.
The reagents and materials used in the present invention are commercially available.
Compared with the prior art, the improvement and progress effect of the invention is as follows: provides a novel biliverdin reductase and mutant thereof, which can efficiently catalyze the reduction of biliverdin and prepare bilirubin. The biliverdin reductase can realize the conversion rate of more than 95% when the concentration of the substrate biliverdin is up to 1g/L, so that the concentration of the bilirubin is also greatly improved. Compared with other bilirubin synthesis methods, the enzymatic reaction has the advantages of high enzymatic activity, mild reaction conditions, large substrate loading amount, high yield and the like, and has good industrial application prospect.
Detailed Description
The individual reaction or detection conditions described in the context of the present invention may be combined or modified in accordance with common general knowledge in the art and may be verified experimentally. The technical solutions and technical effects of the present invention will be clearly and completely described in the following with reference to specific embodiments, but the scope of the present invention is not limited to these embodiments, and all changes or equivalent substitutions without departing from the concept of the present invention are included in the scope of the present invention.
The sources of materials in the following examples are:
Plasmid vector pET28a was purchased from Novagen.
E.coli DH 5. Alpha. And E.coli BL21 (DE 3) competent cells, 2X Taq PCR MasterMix, agarose gel DNA recovery kit were purchased from Beijing Tiangen Biochemical technology Co.
The restriction enzymes EcoR I, xho I, and T4 DNA ligase are commercial products of the company New England Biolabs (NEB).
EXAMPLE 1 Gene cloning of biliverdin reductase
The invention predicts the biliverdin reductase gene which may have obvious activity on biliverdin through a bioinformatics analysis method, sorts out the biliverdin reductase gene for clone expression, and verifies the function of the biliverdin reductase gene. By adopting the method, three novel encoding genes of the biliverdin reductase (NoBVR, glBVR, hyBVR respectively) are cloned from Nostoc sp, gloeocappsa sp and hydrocystis sp respectively, wherein the nucleotide sequence of the encoding gene of the biliverdin reductase NoBVR is shown as SEQ ID No.1, and the amino acid sequence of the biliverdin reductase NoBVR is shown as SEQ ID No. 2; the nucleotide sequence of the coding gene of the biliverdin reductase GlBVR is shown as SEQ ID No.3, and the amino acid sequence of the biliverdin reductase GlBVR is shown as SEQ ID No. 4; the nucleotide sequence of the coding gene of the biliverdin reductase HyBVR is shown as SEQ ID No.5, and the amino acid sequence of the biliverdin reductase HyBVR is shown as SEQ ID No. 6.
Illustratively, the complete nucleic acid molecule encoding the biliverdin reductase is obtained using genomic DNA of Nostoc (Nostoc sp.) as a template, using methods conventional in the art, such as polymerase chain reaction PCR. The synthetic primers are preferably shown as SEQ ID No.7 and SEQ ID No.8:
the upstream primer SEQ ID No.7: CCG (CCG)GAATTC ATGTACAACAGCGAAG
The downstream primer SEQ ID No.8: CCG (CCG)CTCGAG GATGTCTTTCATAAAAATG
Wherein the sequence shown by the upper primer underline is EcoR I cleavage site and the sequence shown by the lower primer underline is Xho I cleavage site.
Gene amplification was performed using polymerase chain reaction PCR using genomic DNA of Nostoc sp as a template. PCR System (50. Mu.L): 2X Taq PCR MasterMix. Mu.l, 1. Mu.l of genomic DNA (100 ng/. Mu.l), 2.5. Mu.l of each of the upstream and downstream primers (10. Mu.M), and sterilized distilled water were added to make up to 50. Mu.l. PCR reaction procedure: (1) pre-denaturation at 95℃for 5min; (2) denaturation at 94℃for 30s; (3) annealing at 55 ℃ for 30s; (4) extending at 72 ℃ for 2min; steps (2) - (4) are carried out for 30 cycles altogether; finally, the product is preserved at 72 ℃ for 10min and 4 ℃. And (3) performing agarose gel electrophoresis analysis and verification on the PCR product, then cutting and purifying, and recovering the target fragment by using a DNA recovery kit. The biliverdin reductase genes in the genus Gloeocappa (Gloeocappsa sp.) and the genus Cyanophyceae (Hydrococussp.) can be cloned separately by the same method.
EXAMPLE 2 preparation of recombinant expression vector and recombinant expression transformant for biliverdin reductase
The target gene of the biliverdin reductase obtained by PCR amplification in example 1 and the empty plasmid pET28a were digested simultaneously with restriction enzymes EcoRI and XhoI at 37℃for 12 hours, respectively. And (3) carrying out agarose gel electrophoresis analysis and verification on the double enzyme-digested product, then carrying out gel-digested purification and recovery, connecting the obtained linearized pET28a plasmid with the purified target gene fragment by using T4 DNA ligase at 16 ℃ overnight, converting the connected product into E.coli BL21 (DE 3) competent cells, uniformly coating the competent cells on an LB agar plate containing 50 mug/mL kanamycin, placing the competent cells in a 37 ℃ incubator for standing culture for about 12 hours, carrying out colony PCR verification on the grown colonies, and picking the colonies with positive colony PCR for sequencing verification. After verification of correctness by sequencing, the corresponding plasmid is extracted, and the recombinant expression vector pET28a-NoBVR of the biliverdin reductase is obtained. Recombinant expression vectors of biliverdin reductase in the genus Gloeocappa (Gloeocappsa sp.) and in the genus hydrocephappaZostera (hydrocephappaZostera sp.) can be obtained by the same method, respectively: pET28a-GlBVR, pET28a-HyBVR.
And further converting the obtained recombinant expression vector into E.coli BL21 (DE 3) competent cells, and selecting positive clones to obtain a recombinant expression transformant E.coli BL21 (DE 3)/pET 28a-NoBVR of the biliverdin reductase. Recombinant expression transformants of biliverdin reductase in the genus Gloeocapram (Gloeocappsa sp.) and the genus Cyanophyceae (Hydrococcus sp.) can be obtained by the same method, respectively: e.coli BL21 (DE 3)/pET 28a-GlBVR, E.coli BL21 (DE 3)/pET 28a-HyBVR.
EXAMPLE 3 construction of a mutant library of biliverdin reductase and high throughput screening
The invention uses NoBVR (Noston sp.) source biliverdin reductase as female parent, and adopts error-prone PCR technology to construct a random mutant library of biliverdin reductase: error-prone PCR was performed with rTaq DNA polymerase using pET28a-NoBVR as a template and gene sequences shown as SEQ ID No.7 and SEQ ID No.8 as primers to construct a random mutant library. PCR System (50. Mu.L): rTaq DNA polymerase 0.5. Mu.L, 10 XPCR buffer (Mg 2+ Plus) 5.0. Mu.L, dNTP mix (2.0 mM each) 4.0. Mu.L, mnCl at a final concentration of 100. Mu. Mol/L 2 pET28a-NoBVR plasmid 0.5ng, 2. Mu.L each of the upstream and downstream primers (10. Mu.M), and sterile distilled water was added to make up to 50. Mu.L. PCR reaction procedure: (1) pre-denaturation at 95℃for 5min; (2) denaturation at 94℃for 30s; (3) annealing at 55 ℃ for 30s; (4) extending at 72 ℃ for 2min; steps (2) - (4) are carried out for 30 cycles altogether; finally, the product is preserved at 72 ℃ for 10min and 4 ℃. The PCR product is purified and recovered by agarose gel electrophoresis analysis and verification, and the recovered target gene and empty plasmid pET28a are respectively subjected to double enzyme digestion for 12 hours at 37 ℃ by using restriction enzymes EcoR I and Xho I. And (3) performing agarose gel electrophoresis analysis and verification on the double-enzyme-digested product, then performing gel-digested purification and recovery, and connecting the obtained linearized pET28a plasmid with the purified target gene fragment at 16 ℃ by using T4 DNA ligase for overnight. The ligation product was transformed into E.coli BL21 (DE 3) competent cells, and uniformly spread on LB agar plates containing 50. Mu.g/ml kanamycin, and placed in a 37℃incubator for stationary culture for about 12 hours.
Transformants on the transformation plate were picked up into deep-well plates with 300. Mu.L of LB medium (containing 50. Mu.g/mL of kanapigenin), shake-cultured overnight at 37℃and then transferred to a secondary deep-well plate with 600. Mu.L of LB medium (containing 50. Mu.g/mL of kanapigenin), shake-cultured for 3 hours at 37℃and then added with IPTG at a final concentration of 0.2mmol/L, and induced for 24 hours at 16 ℃. Then, centrifugation was performed at 3500 Xg for 10min at 4℃and the upper medium was removed, 200. Mu.L of lysozyme solution (750 mg of lysozyme and 10mg of DNase were dissolved in 1L of deionized water) was added to each well, and the mixture was stirred and mixed well, and then, the mixture was subjected to shaking and treatment on a shaker at 37℃for 2 hours. Subsequently centrifuged at 3500 Xg at 4℃for 10min, 10. Mu.L of the appropriately diluted cell disruption supernatant was transferred to a 96-well ELISA plate containing 190. Mu.L of a reaction solution (reaction solution formulation: 100mM, pH 7.0 potassium phosphate buffer, 0.2g/L biliverdin, 0.1mM NADPH), the absorbance at 340nm was measured for a change in 1min at 30℃and the higher the activity of the mutant, the faster its absorbance at 340nm was changed, and thus mutants with improved performance were obtained by screening and sequencing the corresponding genes.
Mutants with significantly improved activity are obtained by screening, and the thermal stability of these mutants is further characterized, preferably a series of mutants with improved thermal stability, the sequences of these mutants and the activity and stability of these mutants are listed in table 1. In table 1, the sequence numbers correspond to the series of sequences following table 1, respectively. In the active column, a plus sign "+" indicates that mutant protein activity is increased 1-10 fold compared to parent NoBVR; two plus signs "++" indicate that the activity of the mutant protein is increased by 11-50 times; three plus signs "+." indicates mutant the activity of the protein is improved by 51-100 times. In the thermostability column, one plus "+" corresponds to a residual activity of the mutant protein remaining 5.0-30.0% after 30min incubation at 45 ℃; the two plus signs "++" correspond to a residual activity retention of the mutant protein of 30.1-50.0% after 30min incubation at 45 ℃; the three plus signs "++ + +" correspond to a residual activity of the mutant protein remaining 50.1-90.0% after 30min incubation at 50 ℃.
TABLE 1 biliverdin reductase NoBVR mutant sequences and corresponding list of Activity improvement
The amino acid sequences of the biliverdin reductase mutants corresponding to the sequence numbers are as follows:
(1) Substitution of phenylalanine at position 26 of the amino acid sequence shown as SEQ ID No.2 in the sequence table with glutamic acid and substitution of alanine at position 61 with threonine;
(2) The 48 th glutamic acid of the amino acid sequence shown as SEQ ID No.2 in the sequence table is replaced by glycine;
(3) Substitution of threonine for glutamic acid at position 48 and threonine for alanine at position 61 of the amino acid sequence shown in SEQ ID No.2 of the sequence Listing;
(4) Substitution of threonine for glutamic acid at position 48, threonine for alanine for threonine for 61, and histidine for asparagine for 263 of the amino acid sequence shown in SEQ ID No.2 of the sequence Listing;
(5) Substitution of phenylalanine at position 26 of the amino acid sequence shown as SEQ ID No.2 in the sequence table with tyrosine and isoleucine at position 276 with valine;
(6) Substitution of phenylalanine at position 26 for tyrosine, isoleucine at position 276 for valine, aspartic acid at position 298 for serine in the amino acid sequence shown in SEQ ID No.2 of the sequence Listing;
(7) The 97 th glycine of the amino acid sequence shown as SEQ ID No.2 in the sequence table is replaced by threonine, the 122 th alanine is replaced by threonine, the 156 th arginine is replaced by lysine, the 159 th threonine is replaced by asparagine, and the 182 th methionine is replaced by leucine;
(8) Replacing alanine at 122 th site of an amino acid sequence shown as SEQ ID No.2 in a sequence table with threonine;
(9) The 122 th alanine of the amino acid sequence shown as SEQ ID No.2 in the sequence table is replaced by threonine, and the 228 th serine is replaced by alanine;
(10) Substitution of alanine at position 122 for threonine, serine at position 228 for alanine, asparagine at position 263 for lysine in the amino acid sequence shown in SEQ ID No.2 of the sequence Listing;
(11) The 26 th phenylalanine of the amino acid sequence shown as SEQ ID No.2 in the sequence table is replaced by tyrosine, the 48 th glutamic acid is replaced by threonine, the 61 th alanine is replaced by threonine, the 97 th glycine is replaced by threonine, the 122 th alanine is replaced by threonine, the 156 th arginine is replaced by lysine, the 182 th methionine is replaced by leucine, the 198 th valine is replaced by serine, the 228 th serine is replaced by glutamic acid, the 255 th lysine is replaced by isoleucine, the 260 th phenylalanine is replaced by valine, the 263 th asparagine is replaced by serine, the 276 th isoleucine is replaced by valine, the 303 th tyrosine is replaced by serine, and the 331 st methionine is replaced by alanine;
(12) Substitution of alanine at position 61 for threonine, glycine at position 97 for threonine, arginine at position 156 for lysine, and isoleucine at position 276 for valine of the amino acid sequence shown in SEQ ID No.2 of the sequence Listing;
(13) The 61 st alanine of the amino acid sequence shown as SEQ ID No.2 in the sequence table is replaced by threonine, the 97 th glycine is replaced by threonine, the 122 th alanine is replaced by threonine, the 156 th arginine is replaced by lysine, the 182 th methionine is replaced by leucine, the 198 th valine is replaced by serine, the 228 th serine is replaced by glutamic acid, the 255 th lysine is replaced by isoleucine, the 260 th phenylalanine is replaced by valine, the 276 th isoleucine is replaced by valine, and the 303 th tyrosine is replaced by serine;
(14) The 122 th alanine of the amino acid sequence shown as SEQ ID No.2 in the sequence table is replaced by threonine, the 159 th threonine is replaced by asparagine, the 198 th valine is replaced by serine, and the 318 th alanine is replaced by proline;
(15) The 182 th methionine of the amino acid sequence shown as SEQ ID No.2 in the sequence table is replaced by leucine, the 198 th valine is replaced by serine, and the 228 th serine is replaced by histidine;
(16) Substitution of methionine at 182 th position for leucine, valine at 198 th position for serine, asparagine at 263 th position for isoleucine of the amino acid sequence shown in SEQ ID No.2 of the sequence Listing;
(17) Substitution of serine at 228 th site of the amino acid sequence shown as SEQ ID No.2 in the sequence table with glutamic acid and substitution of lysine at 255 th site with isoleucine;
(18) Replacing lysine at 255 th site of an amino acid sequence shown as SEQ ID No.2 in a sequence table with isoleucine;
(19) The 26 th phenylalanine of the amino acid sequence shown as SEQ ID No.2 in the sequence table is replaced by tyrosine, the 48 th glutamic acid is replaced by threonine, the 61 th alanine is replaced by threonine, the 97 th glycine is replaced by threonine, the 122 th alanine is replaced by threonine, the 198 th valine is replaced by serine, the 228 th serine is replaced by glutamic acid, the 255 th lysine is replaced by isoleucine, the 260 th phenylalanine is replaced by valine, the 263 th asparagine is replaced by serine, the 276 th isoleucine is replaced by valine, the 298 th aspartic acid is replaced by serine, and the 318 th alanine is replaced by alanine;
(20) The 198 th valine of the amino acid sequence shown as SEQ ID No.2 in the sequence table is replaced by isoleucine, and the 303 rd tyrosine is replaced by phenylalanine;
(21) The 122 th alanine of the amino acid sequence shown as SEQ ID No.2 in the sequence table is replaced by threonine, the 198 th valine is replaced by serine, the 228 th serine is replaced by glutamic acid, the 255 th lysine is replaced by isoleucine, the 263 th asparagine is replaced by serine, the 276 th isoleucine is replaced by valine, the 298 th aspartic acid is replaced by serine, and the 318 th alanine is replaced by alanine;
(22) Substitution of phenylalanine at 260 th position with valine and substitution of asparagine at 263 th position with serine of the amino acid sequence shown as SEQ ID No.2 in the sequence table;
(23) Substitution of phenylalanine at position 260 with valine, asparagine at position 263 with serine, aspartic acid at position 298 with serine in the amino acid sequence shown in SEQ ID No.2 of the sequence table;
(24) The 263 rd asparagine of the amino acid sequence shown as SEQ ID No.2 in the sequence table is replaced by serine, the 276 rd isoleucine is replaced by valine, the 303 rd tyrosine is replaced by serine, and the 331 st methionine is replaced by alanine;
(25) Isoleucine 276 of the amino acid sequence shown as SEQ ID No.2 in the sequence table is replaced by valine, and tyrosine 303 is replaced by serine.
Example 4 Induction expression of biliverdin reductase and fermentation Activity determination
The recombinant expression transformants obtained in examples 2 and 3 were inoculated into LB medium containing 50. Mu.g/ml kanamycin, shake-cultured at 37℃for 12 hours, then inoculated into a 2L Erlenmeyer flask containing 400ml of LB medium (containing 50. Mu.g/ml kanamycin) at an inoculum size of 1% (v/v), placed in a shaking table at 37℃and shake-cultured at 180rpm, and the OD of the culture solution was determined 600 When reaching 0.6, IPTG with the final concentration of 0.2mmol/L is added as an inducer to induce for 24 hours at 16 ℃. Centrifuging the culture solution at 8000 Xg for 10min, collecting cells, and washing twice with physiological saline to obtainTo resting cells. Cells obtained in 100ml of the culture were suspended in 10ml of potassium phosphate buffer (100 mM, pH 7.0) and subjected to the following ultrasonication in an ice-water bath: 400W power, 4s on, 6s off, 99 cycles, 12000 Xg centrifugation at 4℃for 40 min, and collection of crude enzyme supernatant, activity determination was performed as described in detail above. According to the determination, the mutant NoBVR of the biliverdin reductase of the invention M11 The activity of the crude enzyme solution is 75U/mL, and the activity of the freeze-dried enzyme powder is 5.8U/mg.
EXAMPLES 5-7 recombinant biliverdin reductase catalyzed Synthesis of bilirubin
The reaction was carried out in a 20mL jacketed reactor, 10mL of potassium phosphate buffer (100 mM, pH 7.0) containing 0.1g/L biliverdin, 1g/L glucose, 0.5g/L glucose dehydrogenase, 0.05mM NADP + 10U/mL of crude enzyme solution of recombinant biliverdin reductase NoBVR, glBVR or HyBVR. The reaction was magnetically stirred at 30 ℃. The pH of the reaction solution was maintained at 7.0 by controlling the dropwise addition of NaOH solution (1M) with an automatic potentiometric titrator. After 6 hours of reaction, substrate conversion was determined as described in detail in the previous description of the invention and the results are shown in Table 2.
Table 2 Experimental results of recombinant biliverdin reductase catalyzed bilirubin synthesis
EXAMPLES 8-33 biliverdin reductase and its mutant conversion of biliverdin to bilirubin
The reaction was carried out in a 20mL jacketed reactor, 10mL of potassium phosphate buffer (100 mM, pH 7.0) containing 0.5g/L biliverdin, 5g/L glucose, 1g/L glucose dehydrogenase, 0.1mM NADP + 1g/L resting cell catalyst of the recombinant expression transformant as described in example 3. The reaction was magnetically stirred at 30 ℃. The pH of the reaction solution was maintained at 7.0 by controlling the dropwise addition of NaOH solution (1M) with an automatic potentiometric titrator. After 12 hours of reaction, substrate conversion was determined as described in detail in the previous description of the invention and the results are shown in Table 3.
TABLE 3 biliverdin reductase and mutant thereof catalyzing reduction of biliverdin
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EXAMPLE 34 recombinant NoBVR M11 Catalytic bilirubin synthesis by biliverdin reduction
The reaction was carried out in a 20mL jacketed reactor, 10mL of potassium phosphate buffer (100 mM, pH 7.0) containing 1g/L biliverdin, 10g/L glucose, 1.5g/L glucose dehydrogenase, 0.2mM NADP + 20U/mL recombinant biliverdin reductase NoBVR M11 Is a crude enzyme solution of (a). The reaction was magnetically stirred at 30 ℃. The pH of the reaction solution was maintained at 7.0 by controlling the dropwise addition of NaOH solution (1M) with an automatic potentiometric titrator. Intermittent sampling is carried out to detect the conversion rate of the reaction, and after the reaction is carried out for 12 hours, the bilirubin concentration in the water phase is detected to reach 0.92g/L.
EXAMPLE 35 recombinant NoBVR M11 Catalytic bilirubin synthesis by biliverdin reduction
The reaction was carried out in a 20mL jacketed reactor, 10mL of potassium phosphate buffer (100 mM, pH 7.0) containing 1g/L biliverdin, 10g/L glucose, 1.5g/L glucose dehydrogenase, 0.2mM NADP + 5g/L recombinant biliverdin reductase NoBVR M11 Is a freeze-dried enzyme powder. The reaction was magnetically stirred at 25 ℃. The pH of the reaction solution was maintained at 7.0 by controlling the dropwise addition of NaOH solution (1M) with an automatic potentiometric titrator. Intermittent sampling is carried out to detect the conversion rate of the reaction, and after the reaction is carried out for 8 hours, the bilirubin concentration in the water phase is detected to reach 0.95g/L.
EXAMPLE 36 1-L Scale recombinant NoBVR M11 Catalytic synthesis of bilirubin
The reaction was carried out in a 2L jacketed reactor, 1L potassium phosphate buffer (100 mM, pH 7.0) containing 1g/L biliverdin, 10g/L glucose, 1.5g/L glucose dehydrogenase, 0.2mM NADP + 5g/L recombinant biliverdin reductase NoBVR M11 Is a freeze-dried enzyme powder. The reaction was mechanically stirred at 25℃with a stirring speed of 250rpm. Drop-adding NaOH solution by automatic potentiometric titrator(1M), the pH of the reaction solution was maintained at 7.0. Intermittent sampling is carried out to detect the conversion rate of the reaction, and after the reaction is carried out for 8 hours, the bilirubin concentration in the water phase is detected to reach 0.97g/L.
EXAMPLE 37 1-L Scale recombinant NoBVR M11 Catalytic synthesis of bilirubin
The reaction was carried out in a 2L jacketed reactor, 1L potassium phosphate buffer (100 mM, pH 7.0) containing 1g/L biliverdin, 10g/L glucose, 1.5g/L glucose dehydrogenase, 0.1mM NADP + 5g/L recombinant biliverdin reductase NoBVR M11 Is a freeze-dried enzyme powder. The reaction was mechanically stirred at 25℃with a stirring speed of 250rpm. The pH of the reaction solution was maintained at 7.0 by controlling the dropwise addition of NaOH solution (1M) with an automatic potentiometric titrator. Intermittent sampling is carried out to detect the conversion rate of the reaction, and after the reaction is carried out for 12 hours, the bilirubin concentration in the water phase is measured to reach 0.96g/L.
The sequences applied in the above examples are specifically as follows:
SEQ ID No.1: the nucleotide sequence of the gene encoding the biliverdin reductase NoBVR is derived from the microorganism Nostoc sp.
ATGTACAACAGCGAAGCAACCCTGGAAACCGCAAAAGTTCGCGTTGGTCTGGTTGGTACCGGTTATGCGGCGAAATTTCGTACCGAAGCACTGCTGCACGACGAACGTTCTCAACTGGTTGCGATTGCAGGTCATACCCTGGAAAATACCCAGGCGTTTGCGAAAGACTATCAGGCAGAAGCGATTAGCTCTTGGCAGCAACTGGTTGAACGCGAAGATATCGACCTGGTCATCATTAGCACCATCAATCGCGATCACGGCGCTATTGCACGTGCAGCACTGACCCACGGTAAACACGTAATTGTCGAATATCCGCTGGCACTGGACGTTGAAGAAGCGAAAGAAATCATTGCGCTGGCGAAAGCACAGAACAAACTGCTGCACGTCGAGCATATCGAAATTCTGGGCGGTCTGCATCAGGCACTGAAACAGCATATCGAGAAAGTCGGCGACATCTTCTACGTCCGCTATAGCACCATCAGTCCGCGTTATCCGGCTCCGCGTAAATGGACCTACAACCACGAACTGTTTGGCTTTCCGCTGATGGGCGCACTGAGTCGTCTGCATCGTCTGACCGACCTGTTTGGCGAAGTTTTCACCGTCAACTGCCATCAGCGTTTTTGGCAGATCGAACCGGAATACTACCAGACCTGCTTCTGTCTGACCCAGCTGTGTTTTACCTCAGGTCTGCTGGCACAGGTTGTTTACGGTAAAGGCGAAACCCTGTGGCAACAGGAACGCAAATTCGAAGTTCACGGCGAAAAAGGCGGTCTGATTTTCGACGGCAATCAAGGCTTCTTTATGCAGGCAGAAGAAACCACCCCGATTGAAGTTGGTACCCGTCGCGGTCTGTTTGCTAAAGATACCACCATGGTCCTGGATCGCATTTTTGACGGTAGCCCGCTGTACGTTACCCCGGAAGCAAGTCTGTACACCCTGAAAGTTGCGGAAGCAGCGAAACGTGCAGCAGAAACCGGTCTGACCATTTTTATGAAAGACATC
SEQ ID No.2: the amino acid sequence of the biliverdin reductase NoBVR is derived from the microorganism Nostoc sp.
MYNSEATLETAKVRVGLVGTGYAAKFRTEALLHDERSQLVAIAGHTLENTQAFAKDYQAEAISSWQQLVEREDIDLVIISTINRDHGAIARAALTHGKHVIVEYPLALDVEEAKEIIALAKAQNKLLHVEHIEILGGLHQALKQHIEKVGDIFYVRYSTISPRYPAPRKWTYNHELFGFPLMGALSRLHRLTDLFGEVFTVNCHQRFWQIEPEYYQTCFCLTQLCFTSGLLAQVVYGKGETLWQQERKFEVHGEKGGLIFDGNQGFFMQAEETTPIEVGTRRGLFAKDTTMVLDRIFDGSPLYVTPEASLYTLKVAEAAKRAAETGLTIFMKDI
SEQ ID No.3: the nucleotide sequence of the coding gene of the biliverdin reductase GlBVR is derived from the microorganism Gloeocappa sp.
ATGACCAACTTTCTGGTCGAAGGCCCGATTAAAGTTGGCATTGTTGGCACCGGTTACGCAGCAAGTAAACGCGCAGAAGCACTGCAAGCAGATCAACGCGCAGAACTGAAAAGCGTTGCAGGCTACACCGTTATCAGCAGCGAGAAATTCGCGCAGACCTATAGCATCGAACGCATTAACAGCTGGCAGACCCTGGTTAAACAACCGGATCTGGACCTGATCATCATCTGCAACATCAACCAAGATCACGGCGCAATTGCACAAGCAGCACTGTCTGCAGGCAAACACGTTGTTCTGGAATATCCGCTGGCACTGAAACCGCTGCAAGCAGAAGCACTGATTAACCTGGCGCGTCAGAATCAGAAACTGCTGCACGTCGAACACATCGAACTGATTGGCGGCGTTCATCAGGCAATTCGTCGTTATCTGCCGGAAATTGGCGAAGTCTTCTATGGTCGCTATAGCACCATTAGCCCGCAACAAAACGCACCGCGTCGTTGGACCTATCATCACGAAATGTTCGGCTTTCCGCTGATTGCAGCACTGAGCCGTATTCATCGCTTTACCGACCTGTTTGGCGCCGTTGATACCGTTAACTGTCAAACCCGCTTTTGGGACGCACCGGAAACCGGTTATTATCTGGCCTGTCTGTGCGAAGCACAACTGCGTTTTAGCAACGGCCTGATTGCACAGATCACCTACGGCAAAGGCGAGAAATTCTGGAGTTCAGATCGTACCCTGGAACTGTACGGCGATCGTGGTACCCTGTCTTTTGACGGTGAAACCGGTATCCTGATCAAAGGCGAAGAGAAAATCCCGCTGGAAGTTGTTAGCCGTCAGGGTCTGTTTGCTCAAGACACCCAAATGGTCCTGGATCATCTGGTTCACGGTAAACCGCTGTACCTGAAACCGGAAAGCAGCTACTACGCACTGACCGTTGCAGAAGCAGCACGTCAATCTGCAGCAACCGGTAGCATTATTAGCATTCCGCCGCAACTG
SEQ ID No.4: the amino acid sequence of the biliverdin reductase GlBVR is derived from the microorganism Gloeocaapssp.
MTNFLVEGPIKVGIVGTGYAASKRAEALQADQRAELKSVAGYTVISSEKFAQTYSIERINSWQTLVKQPDLDLIIICNINQDHGAIAQAALSAGKHVVLEYPLALKPLQAEALINLARQNQKLLHVEHIELIGGVHQAIRRYLPEIGEVFYGRYSTISPQQNAPRRWTYHHEMFGFPLIAALSRIHRFTDLFGAVDTVNCQTRFWDAPETGYYLACLCEAQLRFSNGLIAQITYGKGEKFWSSDRTLELYGDRGTLSFDGETGILIKGEEKIPLEVVSRQGLFAQDTQMVLDHLVHGKPLYLKPESSYYALTVAEAARQSAATGSIISIPPQL
SEQ ID No.5: the nucleotide sequence of the gene encoding the biliverdin reductase HyBVR is derived from the microorganism Hydrococcus sp.
ATGAAAAACTCTGCCGCCTTCACCATGAGCCATCCGATTAAAGTCGGCATCGTTGGTACCGGTTATGCAGCAAGTAAACGCGCAGAAGCATTTCAGGCAGACGATCGCGCACAGGTTATTGTTGTTGCAGGTAACACCCCGGAAAAAACCGAAGCGTTTTGCCAGGCATATAGCGTTGCACCGATTGATAGCTGGCAGCAACTGGTTTCTCATCCGGACGTTGACGTAGTTGTCATTAGCAACATTAATCGCGATCGCGCGTCTATTGCGCGTGCAGCACTGCTGGCAGGTAAACACGTTGTCACCGAATATCCGCTGGCACTGAGTTATCGCGAAGCAGAAGAAATTCTGGCACTGAGTCAGCAGCAGAACAAACTGCTGCACATCGAACACATTGAACTGCTGGGCGGTCTGCATCAAGCAATGCGTCAAAGTCTGCCGGAAATTGGCGACGTGTTTTATGCGCGTTACGCGACCATTACCCCGCAACGTCCGGTTCCGCGTCGTTGGACCTTCCACTACCAGATGTTTGGCTTTCCGCTGACCGCAGCACTGAGTCGTATTCATCGTCTGACCGACCTGTTTGGTACCGTTGCAAGCGTTACCTGTCAGAGTCGTTTTTGGGACGCACCGGAATCTGGCTATTATACCGCCTGTCTGTGCAACGCACAACTGCGTTTTACCAACGGTCTGATTGCGGACGTTATCTACGGTAAAGGCGACGCGTTTTGGGATAGCGATCGTACCTTTGAACTGCGCGGCGATCGCGGTACCCTGATTTTTGAAGGCGAAACCGGCAATCTGATTCGCGGCGAAGAAAAAACCGCGATTGAAGTCGTCAGCCGTAAAGGTCTGTTCGTCAAAGACACCCAGATGGTTCTGAATTACCTGACCCGCGGTACCCCGCTGTATATTAATCCGGAAGCCTCTCTGTATGCGCTGAAAGTTGCAGAAGCAGCAGAACGTAGCGCGATTACCGGTAAAACCATCGAACTGAGT
SEQ ID No.6: the amino acid sequence of the biliverdin reductase HyBVR is derived from the microorganism Hydrococcus sp.
MKNSAAFTMSHPIKVGIVGTGYAASKRAEAFQADDRAQVIVVAGNTPEKTEAFCQAYSVAPIDSWQQLVSHPDVDVVVISNINRDRASIARAALLAGKHVVTEYPLALSYREAEEILALSQQQNKLLHIEHIELLGGLHQAMRQSLPEIGDVFYARYATITPQRPVPRRWTFHYQMFGFPLTAALSRIHRLTDLFGTVASVTCQSRFWDAPESGYYTACLCNAQLRFTNGLIADVIYGKGDAFWDSDRTFELRGDRGTLIFEGETGNLIRGEEKTAIEVVSRKGLFVKDTQMVLNYLTRGTPLYINPEASLYALKVAEAAERSAITGKTIELS
SEQ ID No.7: upstream primer
CCG GAATTC ATGTACAACAGCGAAG
SEQ ID No.8: downstream primer
CCG CTCGAG GATGTCTTTCATAAAAATG
The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the teachings of this invention, should consider improvements and modifications made without departing from the scope of this invention as still within the scope of this invention.
Claims (10)
1. A biliverdin reductase is characterized in that the amino acid sequence is shown as SEQ ID No.2, SEQ ID No.4 or SEQ ID No. 6.
2. A mutant biliverdin reductase, characterized in that the mutant biliverdin reductase is the following protein:
one or more amino acids of 26 th phenylalanine, 48 th glutamic acid, 61 st alanine, 97 th glycine, 122 th alanine, 156 th arginine, 159 th threonine, 182 th methionine, 198 th valine, 228 th serine, 255 th lysine, 260 th phenylalanine, 263 th asparagine, 276 th isoleucine, 298 th aspartic acid, 303 th tyrosine, 318 th alanine or 331 st methionine of the amino acid sequence shown in SEQ ID No.2 are replaced by other amino acids, and the novel amino acid sequence corresponds to a protein with a bilirubin function generated by catalyzing the reduction reaction of biliverdin.
3. The mutant biliverdin reductase according to claim 2, wherein the mutant biliverdin reductase has one of the following sequences:
(1) Substitution of phenylalanine at position 26 of the amino acid sequence shown as SEQ ID No.2 in the sequence table with glutamic acid and substitution of alanine at position 61 with threonine;
(2) The 48 th glutamic acid of the amino acid sequence shown as SEQ ID No.2 in the sequence table is replaced by glycine;
(3) Substitution of threonine for glutamic acid at position 48 and threonine for alanine at position 61 of the amino acid sequence shown in SEQ ID No.2 of the sequence Listing;
(4) Substitution of threonine for glutamic acid at position 48, threonine for alanine for threonine for 61, and histidine for asparagine for 263 of the amino acid sequence shown in SEQ ID No.2 of the sequence Listing;
(5) Substitution of phenylalanine at position 26 of the amino acid sequence shown as SEQ ID No.2 in the sequence table with tyrosine and isoleucine at position 276 with valine;
(6) Substitution of phenylalanine at position 26 for tyrosine, isoleucine at position 276 for valine, aspartic acid at position 298 for serine in the amino acid sequence shown in SEQ ID No.2 of the sequence Listing;
(7) The 97 th glycine of the amino acid sequence shown as SEQ ID No.2 in the sequence table is replaced by threonine, the 122 th alanine is replaced by threonine, the 156 th arginine is replaced by lysine, the 159 th threonine is replaced by asparagine, and the 182 th methionine is replaced by leucine;
(8) Replacing alanine at 122 th site of an amino acid sequence shown as SEQ ID No.2 in a sequence table with threonine;
(9) The 122 th alanine of the amino acid sequence shown as SEQ ID No.2 in the sequence table is replaced by threonine, and the 228 th serine is replaced by alanine;
(10) Substitution of alanine at position 122 for threonine, serine at position 228 for alanine, asparagine at position 263 for lysine in the amino acid sequence shown in SEQ ID No.2 of the sequence Listing;
(11) The 26 th phenylalanine of the amino acid sequence shown as SEQ ID No.2 in the sequence table is replaced by tyrosine, the 48 th glutamic acid is replaced by threonine, the 61 th alanine is replaced by threonine, the 97 th glycine is replaced by threonine, the 122 th alanine is replaced by threonine, the 156 th arginine is replaced by lysine, the 182 th methionine is replaced by leucine, the 198 th valine is replaced by serine, the 228 th serine is replaced by glutamic acid, the 255 th lysine is replaced by isoleucine, the 260 th phenylalanine is replaced by valine, the 263 th asparagine is replaced by serine, the 276 th isoleucine is replaced by valine, the 303 th tyrosine is replaced by serine, and the 331 st methionine is replaced by alanine;
(12) Substitution of alanine at position 61 for threonine, glycine at position 97 for threonine, arginine at position 156 for lysine, and isoleucine at position 276 for valine of the amino acid sequence shown in SEQ ID No.2 of the sequence Listing;
(13) The 61 st alanine of the amino acid sequence shown as SEQ ID No.2 in the sequence table is replaced by threonine, the 97 th glycine is replaced by threonine, the 122 th alanine is replaced by threonine, the 156 th arginine is replaced by lysine, the 182 th methionine is replaced by leucine, the 198 th valine is replaced by serine, the 228 th serine is replaced by glutamic acid, the 255 th lysine is replaced by isoleucine, the 260 th phenylalanine is replaced by valine, the 276 th isoleucine is replaced by valine, and the 303 th tyrosine is replaced by serine;
(14) The 122 th alanine of the amino acid sequence shown as SEQ ID No.2 in the sequence table is replaced by threonine, the 159 th threonine is replaced by asparagine, the 198 th valine is replaced by serine, and the 318 th alanine is replaced by proline;
(15) The 182 th methionine of the amino acid sequence shown as SEQ ID No.2 in the sequence table is replaced by leucine, the 198 th valine is replaced by serine, and the 228 th serine is replaced by histidine;
(16) Substitution of methionine at 182 th position for leucine, valine at 198 th position for serine, asparagine at 263 th position for isoleucine of the amino acid sequence shown in SEQ ID No.2 of the sequence Listing;
(17) Substitution of serine at 228 th site of the amino acid sequence shown as SEQ ID No.2 in the sequence table with glutamic acid and substitution of lysine at 255 th site with isoleucine;
(18) Replacing lysine at 255 th site of an amino acid sequence shown as SEQ ID No.2 in a sequence table with isoleucine;
(19) The 26 th phenylalanine of the amino acid sequence shown as SEQ ID No.2 in the sequence table is replaced by tyrosine, the 48 th glutamic acid is replaced by threonine, the 61 th alanine is replaced by threonine, the 97 th glycine is replaced by threonine, the 122 th alanine is replaced by threonine, the 198 th valine is replaced by serine, the 228 th serine is replaced by glutamic acid, the 255 th lysine is replaced by isoleucine, the 260 th phenylalanine is replaced by valine, the 263 th asparagine is replaced by serine, the 276 th isoleucine is replaced by valine, the 298 th aspartic acid is replaced by serine, and the 318 th alanine is replaced by alanine;
(20) The 198 th valine of the amino acid sequence shown as SEQ ID No.2 in the sequence table is replaced by isoleucine, and the 303 rd tyrosine is replaced by phenylalanine;
(21) The 122 th alanine of the amino acid sequence shown as SEQ ID No.2 in the sequence table is replaced by threonine, the 198 th valine is replaced by serine, the 228 th serine is replaced by glutamic acid, the 255 th lysine is replaced by isoleucine, the 263 th asparagine is replaced by serine, the 276 th isoleucine is replaced by valine, the 298 th aspartic acid is replaced by serine, and the 318 th alanine is replaced by alanine;
(22) Substitution of phenylalanine at 260 th position with valine and substitution of asparagine at 263 th position with serine of the amino acid sequence shown as SEQ ID No.2 in the sequence table;
(23) Substitution of phenylalanine at position 260 with valine, asparagine at position 263 with serine, aspartic acid at position 298 with serine in the amino acid sequence shown in SEQ ID No.2 of the sequence table;
(24) The 263 rd asparagine of the amino acid sequence shown as SEQ ID No.2 in the sequence table is replaced by serine, the 276 rd isoleucine is replaced by valine, the 303 rd tyrosine is replaced by serine, and the 331 st methionine is replaced by alanine;
(25) Isoleucine 276 of the amino acid sequence shown as SEQ ID No.2 in the sequence table is replaced by valine, and tyrosine 303 is replaced by serine.
4. An isolated nucleic acid, characterized in that: the nucleic acid encoding the biliverdin reductase mutant according to any one of claims 2 or 3.
5. A recombinant expression vector comprising the nucleic acid of claim 4.
6. A recombinant expression transformant comprising the recombinant expression vector according to claim 5.
7. A biliverdin reductase catalyst, characterized in that the biliverdin reductase catalyst is in the form of:
(1) Culturing the recombinant expression transformant according to claim 6, and isolating a transformant cell containing the biliverdin reductase;
(2) Culturing the recombinant expression transformant according to claim 6, and isolating a crude enzyme solution containing the biliverdin reductase.
8. The bile chlorophyllin reductase catalyst of claim 7, wherein said crude enzyme solution of bile chlorophyllin reductase is dried to obtain crude enzyme powder.
9. The use of a biliverdin reductase catalyst as defined in claim 7, wherein the biliverdin reductase catalyst catalyzes the reduction of biliverdin to produce the corresponding bilirubin product.
10. The use according to claim 9, wherein the reduction reaction conditions are: the concentration of the biliverdin is 0.1-1 g/L, the dosage of glucose is 1-10 g/L, the dosage of the recombinant biliverdin reductase catalyst is 0.5-5 g/L, the dosage of glucose dehydrogenase is 0.5-1.5 g/L, and the dosage of NADP + The dosage is 0.05-0.2 mM, the buffer solution is phosphate buffer solution with pH of 6.0-9.0, the reaction temperature is 20-35 ℃, and the time of the reduction reaction is based on the time that the reaction conversion rate stops increasing.
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Application publication date: 20231114 |