CN103881997B - Sialic acid Aldolase mutant and encoding gene thereof and application - Google Patents
Sialic acid Aldolase mutant and encoding gene thereof and application Download PDFInfo
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Abstract
The open a kind of Sialic acid Aldolase mutant of the present invention and encoding gene thereof and application, this Sialic acid Aldolase mutant is by the sequence 2 in sequence table through point mutation gained, and described point mutation is at least one sudden change at the 25th and the 275th of this sequence.The present invention is by carrying out rite-directed mutagenesis to Sialic acid aldolase gene sequence, and final acquisition has the Sialic acid Aldolase mutant of high catalytic activity.And this mutant with N acetylmannosamine, Sodium Pyruvate and trinosin (ATP) be substrate have than parent exceed at least 50% Sialic acid aldolase catalysis activity.So that this Sialic acid Aldolase mutant can be used for producing Sialic acid (sialic acid), reduce production cost, improve the market competitiveness of corresponding product.
Description
Technical field
The present invention relates to molecular biology and biological technical field, particularly relate to a kind of Sialic acid Aldolase mutant and encoding gene thereof and application.
Background technology
Sialic acid (English name: Sialic acid) is also known as sialic acid or N-acetyl-neuraminate, its structure is as shown in Figure 1, it is the natural sugar compounds that is widely present in biosystem of a class, in the much important physiology of life entity, biochemical reaction process, plays indispensable effect.The structure of N-acetyl-neuraminate (Sialic acid) is as shown in Figure 1.
Sialic acid is to have bioactive Main Ingredients and Appearance in Chinese tradition rare food bird's nest, is also mother just Ruzhong to one of perfect provided important component of baby brain development and immune system in early days.Owing to bird's nest having the Sialic acid of high-load, so having been reported that and Sialic acid detection being used for bird's nest product Quality Control evaluation and analysis.
Substantial amounts of scientific research finds, Sialic acid has many highly important biological functions.Sialic acid content in human brain cell be 2 times of other animal or more than, this is perhaps the intelligence material base far above other all animals of human brain.The Sialic acid content of human neural cells film is a lot of times of other cell of health, it is clear that Sialic acid is the important substance of nerve cell information transmission.For confirming the important function that Sialic acid develops for children's intelligence, 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 more than 1000 tested children, found that Sialic acid can improve children's early intelligence development level.Result of study shows: breastfeeding children are above the children of NBF at IQ test, standard testing, evaluation of teacher and the mark in the general performance in middle school period.Due to NBF newborn baby, its Sialic acid taken in is than breast-feeding few 20%, and the breast-feeding time is the longest, and the integrated intelligence development level mark of children is the highest.Therefore, scientists is concluded that supplements Sialic acid to infant, can increase the concentration of Sialic acid in brain and improve the learning ability of brain, thus improving the learning ability of brain.
Additionally, other major function of Sialic acid has: with the growth at age, the Sialic acid content on red blood cell surface progressively declines.The decline of red blood cell surface Sialic acid content makes cell easily be degraded, thus accelerates aging course, therefore Sialic acid can be used for anti-aging.The Sialic acid of human epidermal cell has antibacterial action.Sialic acid can protect skin to support bacterial-infection resisting, therefore Sialic acid can be used for skin care.
Research is also found that the Sialic acid content of great drinker's red blood cell significantly lower than normal person.Therefore, great drinker's complexion is wax yellow, dry skin and without color and luster.And it is being situated between a week after drinking, the Sialic acid content of its red blood cell and normal person's indifference.So, Sialic acid is relevant with the appearance of people.
And Sialic acid and the medicine with Sialic acid as parent have been applied to clinic the most, can be used for treating influenza, neurogenic disease, inflammation, senile dementia, tumour etc..
At present Sialic acid is essentially all and is obtained by fermentation or synthesis, and the level of production is relatively low, not environmentally, again or relatively costly.During Production by Microorganism Fermentation Sialic acid, the most first produce poly-Sialic acid, after poly-Sialic acid carries out acid hydrolysis or enzyme hydrolysis again, isolated and purified obtain Sialic acid.Fermentation method produces the major defect of Sialic acid: fermentation production rate is low and purifies more difficult.It is the sylvite condensation using ManNAc and di-t-butyl oxo succinic acid that chemical method produces Sialic 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 A-bromomethyl acrylic acid is carried out acrylic, then carry out ozone decomposed and obtain N-acetyl-neuraminate.It is the highest, the most not environmentally that chemical method produces Sialic acid chemical method production Sialic acid yield.Sialic acid also can extract from natural products, and as Lekh etc. extracts Sialic acid from the chalaza and membrane of yolk of birds, beasts and eggs, Feng ten thousand sample, Gao Jianfeng et al. extract Sialic acid from pig blood.Owing to Sialic acid comparision contents in natural material is low, separating-purifying process is the most more complicated, so the rate of recovery is low, and causes bigger environmental pollution.Production by Enzymes Sialic acid has conversion ratio height, extracts simple, product purity advantages of higher.As far back as 1988, Simon et al.[5]Under the catalysis of Sialic acid aldolase, N-acetyl-neuraminate is synthesized with ManNAc, Sodium Pyruvate and ATP.Isafumi etc.[6]With N-acyl glucose amine 2-epimerase, N-Acetyl-D-glucosamine being first converted into ManNAc, the latter prepares Sialic acid through Sialic acid aldolase again, it is achieved that Sialic acid is through two enzymatic synthesis, and simplifies the purifying process of Sialic acid.But owing to the vigor of Sialic acid aldolase is relatively low, ManNAc and Sodium Pyruvate are converted when producing Sialic acid by commercial scale, and conversion ratio is low, 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, it is an object of the invention to provide a kind of Sialic acid Aldolase mutant and encoding gene thereof and application, aim to solve the problem that in current Production by Enzymes Sialic acid, Sialic acid aldolase catalysis activity is low, the problem that conversion ratio is low, production cost is high.
Technical scheme is as follows:
A kind of Sialic acid Aldolase mutant, wherein, by the sequence 2 in sequence table through point mutation gained, described point mutation is at least one sudden change at the 25th and the 275th of this sequence.
Described Sialic acid Aldolase mutant, wherein, described Sialic acid Aldolase mutant also includes its variant, including the conservative replacement form in other site in addition to the 25th and the 275th in amino acid sequence shown in described sequence 2, the part or all of form of tandem repeats that increases or lack one or several amino acid form, aminoterminal clipped form, c-terminus clipped form, and described sequence 2.
Described Sialic acid Aldolase mutant, wherein, described point mutation is particularly as follows: the alanine mutation of the 25th of described sequence 2 is glutamic acid.
Described Sialic acid Aldolase mutant, wherein, described point mutation is particularly as follows: the glycine mutation of the 275th of described sequence 2 is alanine.
Described Sialic acid Aldolase mutant, wherein, described Sialic acid Aldolase mutant has such as sequence 3 or the amino acid sequence shown in sequence 4 in sequence table.
A kind of gene encoding Sialic acid Aldolase mutant as above, wherein, described gene by the nucleotide sequence shown in sequence in sequence table 1 through rite-directed mutagenesis gained.
The application of a kind of Sialic acid Aldolase mutant as above, wherein, described Sialic acid Aldolase mutant is for preparing Sialic acid with ManNAc, Sodium Pyruvate and trinosin for substrate.
Beneficial effect: the present invention provides a kind of Sialic acid Aldolase mutant and encoding gene thereof and application, by Sialic acid aldolase gene sequence is carried out rite-directed mutagenesis, final acquisition has the Sialic acid Aldolase mutant of high catalytic activity.And this mutant with ManNAc, Sodium Pyruvate and trinosin (ATP) be substrate have than parent exceed at least 50% Sialic acid aldolase catalysis activity.So that this Sialic acid Aldolase mutant can be used for producing Sialic acid (sialic acid), reduce production cost, improve the market competitiveness of corresponding product.
Accompanying drawing explanation
Fig. 1 is Sialic acid schematic arrangement.
Fig. 2 is the polyacrylamide gel electrophoresis figure of Sialic acid aldolase parent and mutant G275A.
Detailed description of the invention
The present invention provides a kind of Sialic acid Aldolase mutant and encoding gene thereof and application, and for making the purpose of the present invention, technical scheme and effect clearer, clear and definite, the present invention is described in more detail below.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 present invention provides a kind of Sialic acid Aldolase mutant, wherein, the sequence 2(parental array by sequence table) through point mutation gained, described point mutation is at least one sudden change at the 25th and the 275th of this sequence.
Additionally, described Sialic acid Aldolase mutant also includes its variant, including the conservative replacement form in other site in addition to the 25th and the 275th in amino acid sequence shown in described sequence 2, the part or all of form of tandem repeats that increases or lack one or several amino acid form, aminoterminal clipped form, c-terminus clipped form, and described sequence 2.
Further, described point mutation is particularly as follows: the alanine mutation of described parental array the 25th is glutamic acid, and/or the glycine mutation of parental array the 275th is alanine.The alanine mutation that parental array is the 25th is the amino acid sequence shown in sequence 3 in glutamic acid formation sequence table.The glycine mutation that parental array is the 275th is the amino acid sequence that alanine forms as shown in sequence 4 in sequence table.
The present invention also provides for the DNA, the DNA of described gene of a kind of gene encoding Sialic acid Aldolase mutant as above by the nucleotide sequence shown in sequence in sequence table 1 through rite-directed mutagenesis gained.By the Sialic acid Aldolase mutant described in the transcribed expression of the DNA of this gene.Mainly obtain is the amino acid sequence shown in sequence 3 in sequence table, sequence 4.It is that substrate produces Sialic acid (sialic acid) that this Sialic acid Aldolase mutant can be used for ManNAc, Sodium Pyruvate and trinosin (ATP)
The substantially process of the preparation of described Sialic acid Aldolase mutant is: first build the vector plasmid containing parent's Sialic acid aldolase gene, then set rite-directed mutagenesis site and sudden change after amino acid classes, synthesize suitable primer again, with the described vector plasmid containing parent's Sialic acid aldolase gene as template, DNA fragmentation and PCR that pcr amplified DNA fragment, assembling are expanded expand total length mutator.Then this total length mutator it is cloned on suitable carrier and converts suitable host cell, filtering out the positive colony with Sialic acid aldolase activity through cultivation.Last extraction DNA from positive colony, carries out determined dna sequence analysis, to determine the sudden change of introducing, after determining that purpose fragment is inserted on carrier, can pass through LB Screening of Media, thus obtain the Sialic acid Aldolase mutant of tool high catalytic activity.In foregoing description, wherein, parent refers to from Escherichia coli
The Sialic acid aldolase of BL21 (DE3), (with reference to GenBank shown in sequence 1 in its nucleotide sequence such as sequence table
NC_012971), its amino acid sequence is such as (reference GenBank YP_003055660) shown in sequence 2
In above-mentioned preparation method, the carrier used can be prokaryotic expression carrier, such as pRSET and pES21 etc.;Can also be cloning vector, such as pUC18/19 and pBluscript-SK.
Further, described Sialic acid Aldolase mutant gene can be at prokaryotic or eukaryotic intracellular expression, the most also can be in prokaryotic or eukaryotic extracellular expression.
Further, the host cell of described carrier is prokaryotic or eukaryotic.Described prokaryotic can be Escherichia coli.Described eukaryotic can be saccharomyces cerevisiae or finish red saccharomyces pastorianus.
This mutant can be purified by Histag purification schemes, find after enzyme activity determination the Sialic acid Aldolase mutant of the present invention have than parent exceed at least 50% Sialic acid aldolase catalysis activity.
It addition, the higher catalytic activity of the Sialic acid aldolase of present invention offer makes it can use with thick enzyme form with the most purified, it is also possible to be the enzyme through partially purified or Economical Purification.Certainly, it is also with curing technology and Sialic acid Aldolase mutant of the present invention is made the solidification enzyme of immobilized enzyme or solid phase cell form.
Amino acid trigram used in the application text or single-letter expression way, use the amino acid code (Eur. that IUPAC specifies
J. Biochem., 138:9-37, 1984)。
Sialic acid Aldolase mutant and the performance thereof prepared the present invention below by way of specific embodiment illustrate.The following example is merely to illustrate the present invention and should not be taken as limiting the scope of the invention.Unreceipted actual conditions person in embodiment, the condition of condition or manufacturer's suggestion is carried out routinely.
Embodiment 1
The amplification of parent's Sialic acid aldolase encoding gene and clone:
Primer EC-F and EC-R is designed according to gene pool (GenBank NC_012971) gene order.With primer, EC-F and EC-R is expanded Sialic acid aldolase encoding gene from Escherichia coli BL21 (DE3).
Amplification condition is: 20 mM Tris-HCl (pH 8.8), 10 mM
KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1%
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 oese picking a little Escherichia coli
BL21 (DE3) thalline, then adjust reaction volume to 50 ml with sterilized water.
Pcr amplification reaction program is: 95 DEG C 3 minutes, 40 circle circulation: 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 is digested with through as the carrier pRSET-A (being derived from Invitrogen, USA) that is digested of restriction enzyme NdeI and AscI connect, obtain plasmid pRSET-EC.Through DNA sequencing, determining the nucleotide sequence of this Sialic acid aldolase being cloned, be specifically shown in sequence 1 in sequence table, corresponding amino acid sequence is the sequence 2 in sequence table.
Table 1
Embodiment 2
The rite-directed mutagenesis in Sialic acid aldolase site 25
Obtaining mutant A25E in order to the Ala (A) in the 25th site in parent amino acid sequence sports Glu (E), with the plasmid pRSET-EC in embodiment 1 as template, design primer is to 25EF and 25ER (as shown in table 1).
With primer to EC-F and 25ER, expanding F-ER fragment, primer, to 25EF and EC-R, expands EF-R fragment.The particular 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 (NH4)2SO4, 2 mM MgSO4, 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 primer 2 5ER, or 400 nM primer 2 5EF and 400 nM primer EC-R, 1.5 U Pfu archaeal dna polymerases
(Promega, USA), 20 ng pRSET-EC, adjust reaction volume to 50 microlitres with sterilized water.
Pcr amplification reaction program is: 95 DEG C 3 minutes, 35 circle circulation: 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, respectively obtain F-ER fragment and EF-R fragment.Then full-length gene is expanded.
Amplification reaction condition is: 20 mM Tris-HCl (pH 8.8), 10 mM
KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 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 polymerase, 20 ng F-ER fragments and 20 ng
EF-R fragment, adjusts reaction volume to 50 microlitres with sterilized water.
Pcr amplification reaction program is: 95 DEG C 3 minutes, 40 circle circulation: 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 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 amino acid sequence of gained mutant is shown in the sequence 3 in sequence table.
Embodiment 3
Rite-directed mutagenesis to Sialic acid Aldolase mutant site 275
Obtaining mutant G275A in order to the Gly (G) in the 275th site in parent amino acid sequence sports Ala (A), with the plasmid pRSET-EC in embodiment 1 as template, design primer is to 275AF and 275AR (as shown in table 1).
With primer to EC-F and 275AR, expanding F-AR fragment, primer, to 275AF and EC-R, expands AF-R fragment.The particular 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 (NH4)2SO4, 2 mM MgSO4, 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 primer 2 75AR, or 400 nM primer 2 75AF and 400 nM primer EC-R, 1.5 U Pfu archaeal dna polymerases
(Promega, USA), 20 ng pRSET-EC, adjust reaction volume to 50 microlitres with sterilized water.
Above-mentioned pcr amplification reaction program is: 95 DEG C 3 minutes, 35 circle circulation: 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, respectively obtain F-AR fragment and AF-R fragment.Then full-length gene is expanded.
Amplification reaction condition is: 20 mM Tris-HCl (pH 8.8), 10 mM
KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 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 polymerase, 20 ng F-AR fragments and 20 ng
AF-R fragment, adjusts reaction volume to 50 microlitres with sterilized water.
Pcr amplification reaction program is: 95 DEG C 3 minutes, 35 circle circulation: 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 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 amino acid sequence of G275A is shown in the sequence 4 in sequence table.
Embodiment 4
The extraction of Sialic acid aldolase parent
The extraction and purification detailed process of Sialic acid aldolase is as follows:
By the plasmid pRSET-EC transformed competence colibacillus bacterial cell E. coli HB101 containing Sialic acid aldolase gene, at Luria
Broth (LB) flat board is (containing 100
Mg/L kanamycins) upper 37 DEG C cultivate 24 hours.Inoculate single being cloned in 5 milliliters of LB fluid nutrient mediums (containing 100 mg/L kanamycins) to 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 ultrasonic treatment bacterial cell is used.Centrifugal (10 DEG C, 17,800 g, 10 minutes) and collect supernatant, it is thick leach protein (or claiming crude extract).
As in figure 2 it is shown, A district shows the result of the polyacrylamide gel electrophoresis of the Sialic acid thick leach protein of aldolase parent of restructuring, show that Sialic acid aldolase (object tape size is about 33kD) has relatively high expression level in Escherichia coli HB101.
Embodiment 5
The extraction of Sialic acid Aldolase mutant
The extraction and purification detailed process of Sialic acid aldolase is as follows:
By the plasmid pRSET-A25E containing Sialic acid aldolase gene or pRSET-G275A transformed competence colibacillus bacterial cell E.
Coli HB101, cultivates 24 hours upper 37 DEG C of Luria broth (LB) flat board (containing 100 mg/L kanamycins).Inoculate single being cloned in 5 milliliters of LB fluid nutrient mediums (containing 100 mg/L kanamycins) to 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 ultrasonic treatment bacterial cell is used.Centrifugal (10 DEG C, 17,800 g, 10 minutes) and collect supernatant, it is thick leach protein (or claiming crude extract).
As in figure 2 it is shown, B district shows the result of the polyacrylamide gel electrophoresis of the thick leach protein of Sialic acid Aldolase mutant G275A of restructuring, show that Sialic acid aldolase (object tape size is about 33kD) has relatively high expression level in Escherichia coli HB101.
Embodiment 6
The mensuration of Sialic acid aldolase activity:
Preparation substrate solution: ManNAc, the adenosine disodium triphosphate (ATP) of 5mM, the Pyruvic acid sodium salt of 40mM, 100mM Tris hydrochloride buffer and the MgCl of final concentration of 10mM containing 50mM2, adjust pH to 7.5.Take substrate solution 400 microlitre, be subsequently adding the 100 microlitre thick leach proteins of Sialic acid aldolase, carry out reaction in 30 minutes in 32 DEG C.Product boils 5 minutes to terminate reaction in 100 DEG C.Centrifugal (10 DEG C, 17,800 g, 15 minutes) and collect supernatant.The content of N-acetyl-neuraminate (Sialic acid) in gained supernatant is measured by high pressure liquid chromatography (HPLC).Zymoprotein concentration is measured with sds polyacrylamide gel electrophoresis.It is the enzyme amount needed for N-acetyl-neuraminate (Sialic acid) that one unit specific enzyme activity is defined as conversion one micromole's ManNAc the most per minute.As a result, the specific vigor of Sialic acid aldolase (thick leach protein) is 1.03U/mg.
Embodiment 7
The mensuration of Sialic acid Aldolase mutant A25E activity:
Preparation substrate solution: ManNAc, the adenosine disodium triphosphate (ATP) of 5mM, the Pyruvic acid sodium salt of 40mM, 100mM Tris hydrochloride buffer and the MgCl of final concentration of 10mM containing 50mM2, adjust pH to 7.5.Take substrate solution 400 microlitre, be subsequently adding the 100 microlitre thick leach proteins of Sialic acid Aldolase mutant G275A, carry out reaction in 30 minutes in 32 DEG C.Product boils 5 minutes to terminate reaction in 100 DEG C.Centrifugal (10 DEG C, 17,800 g, 15 minutes) and collect supernatant.The content of N-acetyl-neuraminate (Sialic acid) in gained supernatant is measured by high pressure liquid chromatography (HPLC).Zymoprotein concentration is measured with sds polyacrylamide gel electrophoresis.It is the enzyme amount needed for N-acetyl-neuraminate (Sialic acid) that one unit specific enzyme activity is defined as conversion one micromole's ManNAc the most per minute.As a result, the specific vigor of Sialic acid Aldolase mutant G275A (thick leach protein) is 1.85U/mg, higher by 79.6% than parent.
Embodiment 8
The mensuration of Sialic acid Aldolase mutant G275A activity
Preparation substrate solution: ManNAc, the adenosine disodium triphosphate (ATP) of 5mM, the Pyruvic acid sodium salt of 40mM, 100mM Tris hydrochloride buffer and the MgCl of final concentration of 10mM containing 50mM2, adjust pH to 7.5.Take substrate solution 400 microlitre, be subsequently adding the 100 microlitre thick leach proteins of Sialic acid Aldolase mutant G275A, carry out reaction in 30 minutes in 32 DEG C.Product boils 5 minutes to terminate reaction in 100 DEG C.Centrifugal (10 DEG C, 17,800 g, 15 minutes) and collect supernatant.The content of N-acetyl-neuraminate (Sialic acid) in gained supernatant is measured by high pressure liquid chromatography (HPLC).Zymoprotein concentration is measured with sds polyacrylamide gel electrophoresis.It is the enzyme amount needed for N-acetyl-neuraminate (Sialic acid) that one unit specific enzyme activity is defined as conversion one micromole's ManNAc the most per minute.As a result, the specific vigor of Sialic acid Aldolase mutant G275A (thick leach protein) is 4.10U/mg, higher by 298% than parent.
Embodiment 9
Sialic acid aldolase immobilization
Take the Sialic acid thick leach protein of aldolase parent, with washing enzyme buffer liquid (0.02M
Tris-HCl/0.001M EDTA, pH7.0 solution) it is diluted to protein content 5-10mg/ml.By enzyme dilution and PB solution (2.0mol/L potassium dihydrogen phosphate, pH7.5) equal-volume mixing, adding 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.With sock filtration after having reacted, clean 5-6 time with washing enzyme buffer liquid, obtain immobilization Sialic acid aldolase.
Embodiment 10
N-acetyl-neuraminate (Sialic acid) is prepared with immobilization Sialic acid aldolase
Preparation substrate solution: ManNAc, the adenosine disodium triphosphate (ATP) of 5mM, the Pyruvic acid sodium salt of 100mM, 100mM Tris hydrochloride buffer and the MgCl of final concentration of 10mM containing 100mM2, adjust pH to 8.0.Take substrate solution 10 milliliters, be subsequently adding 0.5 gram of immobilization Sialic acid aldolase, after carrying out reacting 2 hours in 32 DEG C, reaction temperature is reduced to 30 DEG C and adds a small amount of Pyruvic acid sodium salt.After reacting 2 hours, then reaction temperature is reduced to 28 DEG C and adds a small amount of Pyruvic acid sodium salt.After reacting 2 hours, then reaction temperature is reduced to 26 DEG C and adds a small amount of Pyruvic acid sodium salt.After reacting 2 hours, then reaction temperature is reduced to 20 DEG C and adds a small amount of Pyruvic acid sodium salt.Reaction is overnight.Centrifugal (10 DEG C, 17,800 g, 15 minutes) and collect supernatant.The content of N-acetyl-neuraminate (Sialic acid) in gained supernatant is measured by high pressure liquid chromatography (HPLC).As a result, ManNAc is converted into the conversion ratio of N-acetyl-neuraminate (Sialic acid) and reaches 80%.
The present invention provides a kind of Sialic acid Aldolase mutant and encoding gene thereof and application, and by Sialic acid aldolase gene sequence is carried out rite-directed mutagenesis, final acquisition has the Sialic acid Aldolase mutant of high catalytic activity.And this mutant with ManNAc, Sodium Pyruvate and trinosin (ATP) be substrate have than parent exceed at least 50% Sialic acid aldolase catalysis activity.So that this Sialic acid Aldolase mutant can be used for producing Sialic acid (sialic acid), reduce production cost, improve the market competitiveness of corresponding product.
It should be appreciated that the application of the present invention is not limited to above-mentioned citing, for those of ordinary skills, can be improved according to the above description or convert, all these modifications and variations all should belong to the protection domain of claims of the present invention.
Claims (4)
1. a Sialic acid Aldolase mutant, it is characterised in that by the sequence 2 in sequence table through point mutation gained, described point mutation is a sudden change at the 25th or the 275th of this sequence；
Described point mutation is particularly as follows: the alanine mutation of the 25th of described sequence 2 is glutamic acid；
Or the glycine mutation of the 275th of described sequence 2 is alanine.
Sialic acid Aldolase mutant the most according to claim 1, it is characterised in that described Sialic acid Aldolase mutant is specially such as sequence 3 or the amino acid sequence shown in sequence 4 in sequence table.
3. the gene of a coding Sialic acid Aldolase mutant as described in any one of claim 1-2, it is characterised in that described gene by the nucleotide sequence shown in sequence in sequence table 1 through rite-directed mutagenesis gained.
4. the application of the Sialic acid Aldolase mutant as described in any one of claim 1-2, it is characterised in that described Sialic acid Aldolase mutant is for preparing Sialic acid with ManNAc, Sodium Pyruvate and trinosin for substrate.
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| CN109402098B (en) * | 2018-11-06 | 2021-09-17 | 王喆明 | Threonine aldolase, mutant and application of mutant in preparation of substituted phenylserine derivative |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS62278982A (en) * | 1986-05-27 | 1987-12-03 | Kyowa Hakko Kogyo Co Ltd | Production of n-acetylneuraminic acid lyase |
| EP1484406A1 (en) * | 2002-02-28 | 2004-12-08 | Kyowa Hakko Kogyo Co., Ltd. | Process for producing n-acetylneuraminic acid |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS62278982A (en) * | 1986-05-27 | 1987-12-03 | Kyowa Hakko Kogyo Co Ltd | Production of n-acetylneuraminic acid lyase |
| EP1484406A1 (en) * | 2002-02-28 | 2004-12-08 | Kyowa Hakko Kogyo Co., Ltd. | Process for producing n-acetylneuraminic acid |
| US7329514B2 (en) * | 2002-02-28 | 2008-02-12 | Kyowa Hakko Kogyo Co., Ltd. | Process for producing n-acetylneuraminic acid |
Non-Patent Citations (1)
| Title |
|---|
| Synthesis of CMP-NeuAc from N-Acetylglucosamine: Generation of CTP from CMP Using Adenylate Kinase;Ethan S. Simon, 等;《Journal of the American Chemical Society》;19881231;7159-7164 * |
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