CN103881997A - Cubilose acid aldolase mutant as well as coding gene and application thereof - Google Patents
Cubilose acid aldolase mutant as well as coding gene and application thereof Download PDFInfo
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Abstract
The invention discloses a cubilose acid aldolase mutant as well as a coding gene and application thereof. The cubilose acid aldolase mutant is obtained from sequence 2 in a sequence list through point mutation, and the point mutation is at least one mutation at the 25th site and 275th site of the sequence. Through site directed mutation on the cubilose acid aldolase, cubilose acid aldolase mutant with high catalytic activity is finally obtained. Besides, the mutant uses N-acetylmannosamine, sodium pyruvate and trinosin (ATP) as the substrate and has the cubilose acid aldolase catalytic activity which is at least 50% higher than that of the parent. Thus, the cubilose acid aldolase mutant can be used for producing cubilose acid (sialic acid), the production cost is lowered, and the market competitiveness of the corresponding product is enhanced.
Description
Technical field
The present invention relates to molecular biology and biological technical field, relate in particular to a kind of Nidus collocaliae acid zymohexase mutant and encoding gene and application.
Background technology
Nidus collocaliae acid (English name: Sialic acid) claims again sialic acid or N-acetyl-neuraminate, its structure is as shown below, be that a class is extensively present in the natural sugar compounds in biosystem, in the much important physiology of life entity, biochemical reaction process, bringing into play indispensable effect.The structure of N-acetyl-neuraminate (Nidus collocaliae acid) as shown in Figure 1.
Nidus collocaliae acid is the bioactive major ingredient of tool in the traditional rare food bird's nest of China, is also one of female just Ruzhong perfect provided important component to the early stage brain development of baby and immune system.Owing to there being the Nidus collocaliae acid of high-content in bird's nest, so there is report that Nidus collocaliae acid is detected and evaluated and analysed for the Quality Control of bird's nest product.
A large amount of scientific research discoveries, Nidus collocaliae acid has many very important biological functions.Nidus collocaliae acid content in human brain cell be 2 times of other animal or more than, this is perhaps that the intelligence of human brain is far above the basic substance of other all animals.The Nidus collocaliae acid content of human neural cells film is the doubly a lot of of other cell of health, and obviously, Nidus collocaliae acid is the important substance that neurocyte information is transmitted.For confirming the vital role of Nidus collocaliae acid for children's intelligence development, Zelanian one group of scientist once carried out the follow-up study of 18 years by a definite date from be born to graduating from the high school to exceeding 1000 tested children, found that Nidus collocaliae acid can improve children's early intelligence developmental level.Result of study shows: breastfeeding children IQ test, standard testing, evaluation of teacher and at the mark of the general performance in middle school period all higher than the children of not breast fed.Due to not breast fed newborn baby, its Nidus collocaliae acid of taking in is fewer 20% than breast-feeding, and the breast-feeding time is longer, and children's integrated intelligence developmental level mark is higher.Therefore, scientists draws the following conclusions: infant is supplemented to Nidus collocaliae acid, can increase the concentration of Nidus collocaliae acid in brain and improve the learning capacity of brain, thereby improve the learning capacity of brain.
In addition, other major function of Nidus collocaliae acid has: with the growth at age, the Nidus collocaliae acid content on red blood cell surface progressively declines.The decline of red blood cell surface Nidus collocaliae acid content is easily degraded cell, thereby accelerates aging course, therefore that Nidus collocaliae acid can be used for is anti-ageing.The Nidus collocaliae acid of human epidermal cell has anti-microbial effect.Nidus collocaliae acid can protect skin to support bacterial-infection resisting, therefore Nidus collocaliae acid can be used for skin care.
Research is also found: the Nidus collocaliae acid content of alcoholic's red blood cell is starkly lower than normal people.Therefore, alcoholic's complexion is wax yellow, xerosis cutis and without color and luster.And be situated between a week after drinking, the Nidus collocaliae acid content of its red blood cell and normal people's indifference.So Nidus collocaliae acid is relevant with people's appearance.
And Nidus collocaliae acid and the medicine taking Nidus collocaliae acid as parent have been applied to clinical abroad, can be used for treating influenza, nervous system disease, inflammation, senile dementia, tumour etc.
At present Nidus collocaliae acid be all substantially that production level is lower by fermenting or synthetic obtaining, not environmental protection, again or cost is higher.When Production by Microorganism Fermentation Nidus collocaliae acid, conventionally first produce poly-Nidus collocaliae acid, poly-Nidus collocaliae acid carries out after acid hydrolysis or enzymic hydrolysis again, and separation and purification obtains Nidus collocaliae acid.The main drawback of fermentative Production Nidus collocaliae acid is: fermentation production rate is low and purifying is more difficult.It is the sylvite condensation that adopts ManNAc and di-t-butyl oxo succinic acid that chemical method is produced Nidus collocaliae acid, then decarboxylation can generate N-acetyl-neuraminate under the catalysis of alkali; Again or in acidity alcohol solution, under the catalysis of indium, ManNAc is carried out to propenyl with A-brooethyl vinylformic acid, then carry out ozone decomposed and obtain N-acetyl-neuraminate.Chemical method production Nidus collocaliae acid chemical method production Nidus collocaliae acid yield is not high, not environmental protection again.Nidus collocaliae acid also can extract from natural product, and as Lekh etc. extracts Nidus collocaliae acid from the chalaza of birds, beasts and eggs and membrane of yolk, the people such as Feng's ten thousand samples, Gao Jianfeng extract Nidus collocaliae acid from pig blood.Because Nidus collocaliae acid content in natural matter is lower, also more complicated of separating-purifying process, so the rate of recovery is low, and causes larger environmental pollution.Production by Enzymes Nidus collocaliae acid have transformation efficiency high, extract simple, product purity advantages of higher.As far back as 1988, the people such as Simon
[5]with ManNAc, Sodium.alpha.-ketopropionate and ATP synthetic N-acetyl-neuraminate under the catalysis of Nidus collocaliae acid zymohexase.Isafumi etc.
[6]first N-Acetyl-D-glucosamine is converted into ManNAc with N-acyl glucose amine 2-epimerase, the latter makes Nidus collocaliae acid through Nidus collocaliae acid zymohexase again, has realized Nidus collocaliae acid through two enzymic synthesiss, and has simplified the purifying process of Nidus collocaliae acid.But because the vigor of Nidus collocaliae acid zymohexase is lower, technical scale transforms ManNAc and Sodium.alpha.-ketopropionate while producing Nidus collocaliae acid, and low conversion rate, causes production cost too high.
Therefore, prior art has yet to be improved and developed.
Summary of the invention
In view of above-mentioned the deficiencies in the prior art, the object of the present invention is to provide a kind of Nidus collocaliae acid zymohexase mutant and encoding gene and application, be intended to solve in current Production by Enzymes Nidus collocaliae acid Nidus collocaliae acid zymohexase catalysis activity low, low conversion rate, the problem that production cost is high.
Technical scheme of the present invention is as follows:
A kind of Nidus collocaliae acid zymohexase mutant, wherein, by the sequence 2 in sequence table, through point mutation gained, described point mutation is the 25th and at least one sudden change of the 275th in this sequence.
Described Nidus collocaliae acid zymohexase mutant, wherein, described Nidus collocaliae acid zymohexase mutant also comprises its variant, comprising conservative replacement form, the increase in other site except the 25th and the 275th in aminoacid sequence shown in described sequence 2 or lack one or several amino acid form, aminoterminal clipped form, carboxyl terminal clipped form, and the part or all of series connection of described sequence 2 repeats form.
Described Nidus collocaliae acid zymohexase mutant, wherein, described point mutation is specially: the alanine mutation of the 25th of described sequence 2 is L-glutamic acid.
Described Nidus collocaliae acid zymohexase mutant, wherein, described point mutation is specially: the glycine mutation of the 275th of described sequence 2 is L-Ala.
Described Nidus collocaliae acid zymohexase mutant, wherein, described Nidus collocaliae acid zymohexase mutant has the aminoacid sequence as shown in sequence in sequence table 3 or sequence 4.
The encode gene of Nidus collocaliae acid zymohexase mutant as above, wherein, described gene by the nucleotide sequence shown in sequence in sequence table 1 through rite-directed mutagenesis gained.
An application for Nidus collocaliae acid zymohexase mutant as above, wherein, described Nidus collocaliae acid zymohexase mutant is for preparing Nidus collocaliae acid taking ManNAc, Sodium.alpha.-ketopropionate and Sodium ATP as substrate.
Beneficial effect: the invention provides a kind of Nidus collocaliae acid zymohexase mutant and encoding gene and application, by Nidus collocaliae acid aldolase gene sequence is carried out to rite-directed mutagenesis, the final Nidus collocaliae acid zymohexase mutant with high catalytic activity that obtains.And this mutant has the Nidus collocaliae acid zymohexase catalytic activity that exceeds at least 50% than parent taking ManNAc, Sodium.alpha.-ketopropionate and Sodium ATP (ATP) as substrate.Thereby make this Nidus collocaliae acid zymohexase mutant can be used for producing Nidus collocaliae acid (sialic acid), reduced production cost, improved the market competitiveness of corresponding product.
Brief description of the drawings
Fig. 1 is Nidus collocaliae acid schematic arrangement.
Fig. 2 is the polyacrylamide gel electrophoresis figure of Nidus collocaliae acid zymohexase parent and mutant G275A.
Embodiment
The invention provides a kind of Nidus collocaliae acid zymohexase mutant and encoding gene and application, for making object of the present invention, technical scheme and effect clearer, clear and definite, below the present invention is described in more detail.Should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not intended to limit the present invention.
The invention provides a kind of Nidus collocaliae acid zymohexase mutant, wherein, by the sequence 2(parental array in sequence table) through point mutation gained, described point mutation is the 25th and at least one sudden change of the 275th in this sequence.
In addition, described Nidus collocaliae acid zymohexase mutant also comprises its variant, comprising conservative replacement form, the increase in other site except the 25th and the 275th in aminoacid sequence shown in described sequence 2 or lack one or several amino acid form, aminoterminal clipped form, carboxyl terminal clipped form, and the part or all of series connection of described sequence 2 repeats form.
Further, described point mutation is specially: the alanine mutation that described parental array is the 25th is L-glutamic acid, and/or the glycine mutation of the 275th of parental array is L-Ala.The alanine mutation that parental array is the 25th is the aminoacid sequence shown in sequence 3 in L-glutamic acid formation sequence table.The glycine mutation that parental array is the 275th is that L-Ala forms the aminoacid sequence as shown in sequence in sequence table 4.
The present invention also provides a kind of DNA of gene of the Nidus collocaliae acid zymohexase mutant as above of encoding, the DNA of described gene by the nucleotide sequence shown in sequence in sequence table 1 through rite-directed mutagenesis gained.By the Nidus collocaliae acid zymohexase mutant described in the transcribed expression of DNA of this gene.What mainly obtain is the aminoacid sequence shown in sequence 3 in sequence table, sequence 4.This Nidus collocaliae acid zymohexase mutant can be used for producing taking ManNAc, Sodium.alpha.-ketopropionate and Sodium ATP (ATP) as substrate Nidus collocaliae acid (sialic acid)
The roughly process of the preparation of described Nidus collocaliae acid zymohexase mutant is: first build the vector plasmid that contains parent's Nidus collocaliae acid aldolase gene, then set rite-directed mutagenesis site and sudden change after amino acid kind, the primer that resynthesis is suitable, taking the described vector plasmid containing parent's Nidus collocaliae acid aldolase gene as template, DNA fragmentation and pcr amplification total length mutator gene that pcr amplified dna fragment, assembling are increased.Then this total length mutator gene be cloned on suitable carrier and transform suitable host cell, filtering out the positive colony with Nidus collocaliae acid aldolase activity through cultivating.Finally from positive colony, extract plasmid DNA, carry out determined dna sequence analysis, to determine the sudden change of introducing, after definite object fragment is inserted on carrier, can pass through LB Screening of Media, thereby obtain the Nidus collocaliae acid zymohexase mutant of tool high catalytic activity.In foregoing description, wherein, parent refers to the Nidus collocaliae acid zymohexase from Escherichia coli BL21 (DE3), its nucleotide sequence is (with reference to GenBank NC_012971) as shown in sequence in sequence table 1, and its aminoacid sequence is (with reference to GenBank YP_003055660) as shown in sequence 2
In above-mentioned preparation method, the carrier adopting can be prokaryotic expression carrier, as pRSET and pES21 etc.; Also can be cloning vector, as pUC18/19 and pBluscript-SK.
Further, described Nidus collocaliae acid zymohexase mutant gene can be at prokaryotic cell prokaryocyte or eukaryotic cell intracellular expression, certainly also can be in prokaryotic cell prokaryocyte or eukaryotic cell extracellular expression.
Further, the host cell of described carrier is prokaryotic cell prokaryocyte or eukaryotic cell.Described prokaryotic cell prokaryocyte can be intestinal bacteria.Described eukaryotic cell can be yeast saccharomyces cerevisiae or finish red saccharomyces pastorianus.
This mutant can carry out purifying by Histag purification schemes, finds that Nidus collocaliae acid zymohexase mutant of the present invention has the Nidus collocaliae acid zymohexase catalytic activity that exceeds at least 50% than parent after enzyme activity determination.
In addition, the higher catalytic activity of Nidus collocaliae acid zymohexase provided by the invention can not purifiedly be used it with thick enzyme form, can be also the enzyme through partially purified or Economical Purification.Certainly, also can utilize curing technology Nidus collocaliae acid zymohexase mutant of the present invention to be made to the curing enzyme of solid enzyme or solid phase cells form.
Amino acid trigram or single-letter phraseology used in the application's text, the amino acid code (Eur. J. Biochem., 138:9-37,1984) that adopts IUPAC to specify.
Nidus collocaliae acid zymohexase mutant and the performance thereof by specific embodiment prepared by the present invention below describe.The following example only should not be considered as limiting scope of the present invention for the present invention is described.Unreceipted actual conditions person in embodiment, the condition of conditioned disjunction manufacturers suggestion is carried out routinely.
Embodiment 1
Amplification and the clone of parent's Nidus collocaliae acid zymohexase encoding gene:
According to gene pool (GenBank NC_012971) gene order design primer EC-F and EC-R.With primer pair EC-F and the EC-R Nidus collocaliae acid zymohexase encoding gene that increases from Escherichia coli BL21 (DE3).
Amplification condition is: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH
4)
2sO
4, 2 mM MgSO
40.1% Triton X-100,50 mM dATP, 50 mM dTTP, 50 mM dCTP, 50 mM dGTP, 400 nM primer EC-F, 400 nM primer EC-R, 1.0 U Pfu archaeal dna polymerase (Promega, USA), with a little Escherichia coli BL21 (DE3) thalline of transfering loop picking, then adjust reaction volume to 50 ml with sterilized water.
Pcr amplification reaction program is: 95 DEG C 3 minutes, 40 circle circulations: 95 DEG C 50 seconds, 50 DEG C 30 seconds and 72 DEG C 1 minute, last 72 DEG C 10 minutes.The product of amplification after restriction enzyme NdeI and AscI enzyme are cut with carrier pRSET-A (being derived from Invitrogen, the USA) connection of cutting through same restriction enzyme NdeI and AscI enzyme, obtain plasmid pRSET-EC.Through DNA sequencing, determine the nucleotide sequence of this Nidus collocaliae acid zymohexase being cloned, be specifically shown in sequence 1 in sequence table, corresponding aminoacid sequence is the sequence 2 in sequence table.
Table 1
Embodiment 2
The rite-directed mutagenesis in Nidus collocaliae acid zymohexase site 25
Obtain mutant A25E for the Ala (A) in the 25th site in parent's aminoacid sequence being sported to Glu (E), taking the plasmid pRSET-EC in embodiment 1 as template, design primer pair 25EF and 25ER (as shown in table 1).
With primer pair EC-F and 25ER, amplification F-ER fragment, primer pair 25EF and EC-R, amplification EF-R fragment.The concrete sequence of primer EC-F and EC-R, as shown in table 1.
Above-mentioned amplification reaction condition is: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH
4)
2sO
4, 2 mM MgSO
40.1% Triton X-100,50 mM dATP, 50 mM dTTP, 50 mM dCTP, 50 mM dGTP, 400 nM primer EC-F and 400 nM primer 2 5ER, or 400 nM primer 2 5EF and 400 nM primer EC-R, 1.5 U Pfu archaeal dna polymerase (Promega, USA), 20 ng pRSET-EC, with sterilized water tune reaction volume to 50 microlitre.
Pcr amplification reaction program is: 95 DEG C 3 minutes, 35 circle circulations: 95 DEG C 50 seconds, 52 DEG C 30 seconds and 72 DEG C 3 minutes, last 72 DEG C 5 minutes.
Separate and use commercial reagents box to reclaim through 1% agarose gel electrophoresis, obtain respectively F-ER fragment and EF-R fragment.Then full-length gene increases.
Amplification reaction condition is: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH
4)
2sO
4, 2 mM MgSO
4, 0.1% Triton X-100,50 mM dATP, 50 mM dTTP, 50 mM dCTP, 50 mM dGTP, 400 nM primer EC-F and 400 nM EC-R, 1.5 U Pfu archaeal dna polymerases, 20 ng F-ER fragments and 20 ng EF-R fragments, with sterilized water tune reaction volume to 50 microlitre.
Pcr amplification reaction program is: 95 DEG C 3 minutes, 40 circle circulations: 95 DEG C 50 seconds, 52 DEG C 30 seconds and 72 DEG C 3 minutes, last 72 DEG C 5 minutes.
Separate and use commercial reagents box to reclaim through 1% agarose gel electrophoresis, obtain total length mutator gene A25E.A25E is connected with carrier pRSET-A, obtains plasmid pRSET-A25E.Plasmid pRSET-A25E is proceeded to competence bacterial cell E. coli BL21.Determine that through DNA sequencing the point mutation of introducing is errorless.The aminoacid sequence of gained mutant is shown in the sequence 3 in sequence table.
Embodiment 3
To the rite-directed mutagenesis in Nidus collocaliae acid zymohexase mutant site 275
Obtain mutant G275A for the Gly (G) in the 275th site in parent's aminoacid sequence being sported to Ala (A), taking the plasmid pRSET-EC in embodiment 1 as template, design primer pair 275AF and 275AR (as shown in table 1).
With primer pair EC-F and 275AR, amplification F-AR fragment, primer pair 275AF and EC-R, amplification AF-R fragment.The concrete sequence of primer EC-F and EC-R is as shown in table 1.
Above-mentioned amplification reaction condition is: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH
4)
2sO
4, 2 mM MgSO
40.1% Triton X-100,50 mM dATP, 50 mM dTTP, 50 mM dCTP, 50 mM dGTP, 400 nM primer EC-F and 400 nM primer 2 75AR, or 400 nM primer 2 75AF and 400 nM primer EC-R, 1.5 U Pfu archaeal dna polymerase (Promega, USA), 20 ng pRSET-EC, with sterilized water tune reaction volume to 50 microlitre.
Above-mentioned pcr amplification reaction program is: 95 DEG C 3 minutes, 35 circle circulations: 95 DEG C 50 seconds, 52 DEG C 30 seconds and 72 DEG C 3 minutes, last 72 DEG C 5 minutes.
Separate and use commercial reagents box to reclaim through 1% agarose gel electrophoresis, obtain respectively F-AR fragment and AF-R fragment.Then full-length gene increases.
Amplification reaction condition is: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH
4)
2sO
4, 2 mM MgSO
4, 0.1% Triton X-100,50 mM dATP, 50 mM dTTP, 50 mM dCTP, 50 mM dGTP, 400 nM primer EC-F and 400 nM EC-R, 1.5 U Pfu archaeal dna polymerases, 20 ng F-AR fragments and 20 ng AF-R fragments, with sterilized water tune reaction volume to 50 microlitre.
Pcr amplification reaction program is: 95 DEG C 3 minutes, 35 circle circulations: 95 DEG C 50 seconds, 52 DEG C 30 seconds and 72 DEG C 3 minutes, last 72 DEG C 5 minutes.
Separate and use commercial reagents box to reclaim through 1% agarose gel electrophoresis, obtain total length mutator gene G275A.G275A is connected with carrier pRSET-A (reference example 1), obtains plasmid pRSET-G275A.Plasmid pRSET-G275A is proceeded to competence bacterial cell E. coli BL21.Determine that through DNA sequencing the point mutation of introducing is errorless.The aminoacid sequence of G275A is shown in the sequence 4 in sequence table.
Embodiment 4
Nidus collocaliae acid zymohexase parent's extraction
The extraction and purification detailed process of Nidus collocaliae acid zymohexase is as follows:
By the plasmid pRSET-EC transformed competence colibacillus bacterial cell E. coli HB101 containing Nidus collocaliae acid aldolase gene, cultivate 24 hours upper 37 DEG C of Luria broth (LB) dull and stereotyped (containing 100 mg/L kantlex).Inoculate single being cloned in 5 milliliters of LB liquid nutrient mediums (containing 100 mg/L kantlex) and cultivate 20-24 hour in 30 DEG C.Centrifugal collection thalline, and be suspended in 1 milliliter of 100mM Tris hydrochloride buffer (pH 7.5).Then use ultrasonic treatment bacterial cell.Centrifugal (10 DEG C, 17,800 g, 10 minutes) also collect supernatant liquor, are thick leach protein (or claiming crude extract).
As shown in Figure 2, A district has shown the result of the polyacrylamide gel electrophoresis of the thick leach protein of Nidus collocaliae acid zymohexase parent of restructuring, shows that Nidus collocaliae acid zymohexase (object tape size is about 33kD) has compared with high expression level in Escherichia coli HB101.
Embodiment 5
The extraction of Nidus collocaliae acid zymohexase mutant
The extraction and purification detailed process of Nidus collocaliae acid zymohexase is as follows:
By plasmid pRSET-A25E or pRSET-G275A transformed competence colibacillus bacterial cell E. coli HB101 containing Nidus collocaliae acid aldolase gene, cultivate 24 hours upper 37 DEG C of Luria broth (LB) dull and stereotyped (containing 100 mg/L kantlex).Inoculate single being cloned in 5 milliliters of LB liquid nutrient mediums (containing 100 mg/L kantlex) and cultivate 20-24 hour in 30 DEG C.Centrifugal collection thalline, and be suspended in 1 milliliter of 100mM Tris hydrochloride buffer (pH 7.5).Then use ultrasonic treatment bacterial cell.Centrifugal (10 DEG C, 17,800 g, 10 minutes) also collect supernatant liquor, are thick leach protein (or claiming crude extract).
As shown in Figure 2, B district has shown the result of the polyacrylamide gel electrophoresis of the thick leach protein of Nidus collocaliae acid zymohexase mutant G275A of restructuring, shows that Nidus collocaliae acid zymohexase (object tape size is about 33kD) has compared with high expression level in Escherichia coli HB101.
Embodiment 6
The mensuration of Nidus collocaliae acid aldolase activity:
Preparation substrate solution: the MgCl that is 10mM containing Pyruvic acid sodium salt, 100mM Tris hydrochloride buffer and the final concentration of the adenosine disodium triphosphate (ATP) of the ManNAc of 50mM, 5mM, 40mM
2, adjust pH to 7.5.Get substrate solution 400 microlitres, then add the thick leach protein of 100 microlitre Nidus collocaliae acid zymohexase, carry out reaction in 30 minutes in 32 DEG C.Reaction product is boiled 5 minutes with termination reaction in 100 DEG C.Centrifugal (10 DEG C, 17,800 g, 15 minutes) also collect supernatant liquor.Measure the content of N-acetyl-neuraminate (Nidus collocaliae acid) in gained supernatant liquor by high pressure liquid chromatography (HPLC).By sds polyacrylamide gel electrophoresis mensuration zymoprotein concentration.One unit specific enzyme activity is defined as under these conditions per minute, and to transform micromole's ManNAc be the required enzyme amount of N-acetyl-neuraminate (Nidus collocaliae acid).As a result, Nidus collocaliae acid zymohexase (thick leach protein) specificity vigor is 1.03U/mg.
Embodiment 7
The mensuration of Nidus collocaliae acid zymohexase mutant A25E activity:
Preparation substrate solution: the MgCl that is 10mM containing Pyruvic acid sodium salt, 100mM Tris hydrochloride buffer and the final concentration of the adenosine disodium triphosphate (ATP) of the ManNAc of 50mM, 5mM, 40mM
2, adjust pH to 7.5.Get substrate solution 400 microlitres, then add the thick leach protein of 100 microlitre Nidus collocaliae acid zymohexase mutant G275A, carry out reaction in 30 minutes in 32 DEG C.Reaction product is boiled 5 minutes with termination reaction in 100 DEG C.Centrifugal (10 DEG C, 17,800 g, 15 minutes) also collect supernatant liquor.Measure the content of N-acetyl-neuraminate (Nidus collocaliae acid) in gained supernatant liquor by high pressure liquid chromatography (HPLC).By sds polyacrylamide gel electrophoresis mensuration zymoprotein concentration.One unit specific enzyme activity is defined as under these conditions per minute, and to transform micromole's ManNAc be the required enzyme amount of N-acetyl-neuraminate (Nidus collocaliae acid).As a result, Nidus collocaliae acid zymohexase mutant G275A (thick leach protein) specificity vigor is 1.85U/mg, higher by 79.6% than parent.
Embodiment 8
The mensuration of Nidus collocaliae acid zymohexase mutant G275A activity
Preparation substrate solution: the MgCl that is 10mM containing Pyruvic acid sodium salt, 100mM Tris hydrochloride buffer and the final concentration of the adenosine disodium triphosphate (ATP) of the ManNAc of 50mM, 5mM, 40mM
2, adjust pH to 7.5.Get substrate solution 400 microlitres, then add the thick leach protein of 100 microlitre Nidus collocaliae acid zymohexase mutant G275A, carry out reaction in 30 minutes in 32 DEG C.Reaction product is boiled 5 minutes with termination reaction in 100 DEG C.Centrifugal (10 DEG C, 17,800 g, 15 minutes) also collect supernatant liquor.Measure the content of N-acetyl-neuraminate (Nidus collocaliae acid) in gained supernatant liquor by high pressure liquid chromatography (HPLC).By sds polyacrylamide gel electrophoresis mensuration zymoprotein concentration.One unit specific enzyme activity is defined as under these conditions per minute, and to transform micromole's ManNAc be the required enzyme amount of N-acetyl-neuraminate (Nidus collocaliae acid).As a result, Nidus collocaliae acid zymohexase mutant G275A (thick leach protein) specificity vigor is 4.10U/mg, higher by 298% than parent.
Embodiment 9
The immobilization of Nidus collocaliae acid zymohexase
Get the thick leach protein of Nidus collocaliae acid zymohexase parent, be diluted to protein content 5-10mg/ml with washing enzyme buffer liquid (0.02M Tris-HCl/0.001M EDTA, pH7.0 solution).By enzyme diluent and PB solution (2.0mol/L potassium primary phosphate, pH7.5) equal-volume mixes, add epoxy type fixed enzyme vector LX-3000 (10 milligrams of enzyme/gram carriers), in shaking table (rotating speed 100rpm), 25 DEG C are reacted 20 hours.React rear with filter bag filtration, cleaned 5-6 time being fixed Nidus collocaliae acid zymohexase with washing enzyme buffer liquid.
Embodiment 10
Prepare N-acetyl-neuraminate (Nidus collocaliae acid) with immobilization Nidus collocaliae acid zymohexase
Preparation substrate solution: the MgCl that is 10mM containing Pyruvic acid sodium salt, 100mM Tris hydrochloride buffer and the final concentration of the adenosine disodium triphosphate (ATP) of the ManNAc of 100mM, 5mM, 100mM
2, adjust pH to 8.0.Get 10 milliliters of substrate solutions, then add 0.5 gram of immobilization Nidus collocaliae acid zymohexase, react after 2 hours in 32 DEG C, temperature of reaction is reduced to 30 DEG C and add a small amount of Pyruvic acid sodium salt.React after 2 hours, then temperature of reaction is reduced to 28 DEG C and add a small amount of Pyruvic acid sodium salt.React after 2 hours, then temperature of reaction is reduced to 26 DEG C and add a small amount of Pyruvic acid sodium salt.React after 2 hours, then temperature of reaction is reduced to 20 DEG C and add a small amount of Pyruvic acid sodium salt.Reaction is spent the night.Centrifugal (10 DEG C, 17,800 g, 15 minutes) also collect supernatant liquor.Measure the content of N-acetyl-neuraminate (Nidus collocaliae acid) in gained supernatant liquor by high pressure liquid chromatography (HPLC).As a result, the transformation efficiency that ManNAc is converted into N-acetyl-neuraminate (Nidus collocaliae acid) reaches 80%.
The invention provides a kind of Nidus collocaliae acid zymohexase mutant and encoding gene and application, by Nidus collocaliae acid aldolase gene sequence is carried out to rite-directed mutagenesis, the final Nidus collocaliae acid zymohexase mutant with high catalytic activity that obtains.And this mutant has the Nidus collocaliae acid zymohexase catalytic activity that exceeds at least 50% than parent taking ManNAc, Sodium.alpha.-ketopropionate and Sodium ATP (ATP) as substrate.Thereby make this Nidus collocaliae acid zymohexase mutant can be used for producing Nidus collocaliae acid (sialic acid), reduced production cost, improved the market competitiveness of corresponding product.
Should be understood that, application of the present invention is not limited to above-mentioned giving an example, and for those of ordinary skills, can be improved according to the above description or convert, and all these improvement and conversion all should belong to the protection domain of claims of the present invention.
Claims (7)
1. a Nidus collocaliae acid zymohexase mutant, is characterized in that, by the sequence 2 in sequence table, through point mutation gained, described point mutation is the 25th and at least one sudden change of the 275th in this sequence.
2. Nidus collocaliae acid zymohexase mutant according to claim 1, it is characterized in that, described Nidus collocaliae acid zymohexase mutant also comprises its variant, comprising conservative replacement form, the increase in other site except the 25th and the 275th in aminoacid sequence shown in described sequence 2 or lack one or several amino acid form, aminoterminal clipped form, carboxyl terminal clipped form, and the part or all of series connection of described sequence 2 repeats form.
3. Nidus collocaliae acid zymohexase mutant according to claim 1, is characterized in that, described point mutation is specially: the alanine mutation of the 25th of described sequence 2 is L-glutamic acid.
4. Nidus collocaliae acid zymohexase mutant according to claim 1, is characterized in that, described point mutation is specially: the glycine mutation of the 275th of described sequence 2 is L-Ala.
5. Nidus collocaliae acid zymohexase mutant according to claim 1, is characterized in that, described Nidus collocaliae acid zymohexase mutant has the aminoacid sequence as shown in sequence in sequence table 3 or sequence 4.
6. a gene for the Nidus collocaliae acid zymohexase mutant of coding as described in claim 1-5 any one, is characterized in that, described gene by the nucleotide sequence shown in sequence in sequence table 1 through rite-directed mutagenesis gained.
7. an application for the Nidus collocaliae acid zymohexase mutant as described in claim 1-5 any one, is characterized in that, described Nidus collocaliae acid zymohexase mutant is for preparing Nidus collocaliae acid taking ManNAc, Sodium.alpha.-ketopropionate and Sodium ATP as substrate.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105063000A (en) * | 2015-08-28 | 2015-11-18 | 安徽丰原发酵技术工程研究有限公司 | Escherichia coli 2-deoxidation-D-ribose-5-phosphoric acid aldolase mutants and preparation method thereof |
| CN109402098A (en) * | 2018-11-06 | 2019-03-01 | 王喆明 | Threonine aldolase, mutant and its preparing the application in substituted benzene serine derivative |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105063000A (en) * | 2015-08-28 | 2015-11-18 | 安徽丰原发酵技术工程研究有限公司 | Escherichia coli 2-deoxidation-D-ribose-5-phosphoric acid aldolase mutants and preparation method thereof |
| CN105063000B (en) * | 2015-08-28 | 2018-07-13 | 安徽丰原发酵技术工程研究有限公司 | Escherichia coli 2-deoxy-D-ribose -5- phosphate aldolase mutant and preparation method thereof |
| CN109402098A (en) * | 2018-11-06 | 2019-03-01 | 王喆明 | Threonine aldolase, mutant and its preparing the application in substituted benzene serine derivative |
| CN109402098B (en) * | 2018-11-06 | 2021-09-17 | 王喆明 | Threonine aldolase, mutant and application of mutant in preparation of substituted phenylserine derivative |
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