CN107245484A - A kind of nitrilase and its application - Google Patents
A kind of nitrilase and its application Download PDFInfo
- Publication number
- CN107245484A CN107245484A CN201611207294.0A CN201611207294A CN107245484A CN 107245484 A CN107245484 A CN 107245484A CN 201611207294 A CN201611207294 A CN 201611207294A CN 107245484 A CN107245484 A CN 107245484A
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- Prior art keywords
- nitrilase
- mutant
- reaction
- ala
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- 108010033272 Nitrilase Proteins 0.000 title claims abstract description 56
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
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- 241000894006 Bacteria Species 0.000 claims description 22
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- 208000012839 conversion disease Diseases 0.000 abstract description 6
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- 108010045269 tryptophyltryptophan Proteins 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
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Abstract
The present invention relates to biological technical field, in particular to a kind of nitrilase and its application, the amino acid sequence such as SEQ ID NO of the nitrilase:Shown in 1, the nitrilase that the present invention is provided can be in the 12h reaction time, altogether by 600mM substrate complete hydrolysis, the diluting effect regulated and controled due to bottoms stream plus with pH, the accumulative product o-chloromandelic acid for generating 112g/L.Reaction conversion ratio is maintained at more than 90% in whole course of reaction.React to stop flowing after 10h and add, continue to react substrate after 2h and be totally converted.Reaction rate is very fast, and reaction conversion ratio is also very high.
Description
Technical field
The present invention relates to biological technical field, in particular to a kind of nitrilase and its application.
Background technology
(R)-(-)-o-chloromandelic acid is a kind of medicine intermediate and fine chemical product with extensive use, is mainly used in
The synthesis of anti-platelet aggregation medicinal clopidogrel.Clopidogrel is as whole world weight pound level medicine, and global marketing volume is near within 2010
10000000000 dollars, 2011 and 2012 of sales volume is also all more than 9,000,000,000 dollars.As clopidogrel on May 17th, 2012 is special
Profit expires, and the whole world has started one imitated upsurge.And it is used as (R)-(-)-o-chloromandelic acid consequently also water of its important intermediate
Rise height, and domestic and international demand constantly increases, therefore how research obtains high concentration, (R)-(-)-o-chloromandelic acid of high-purity is incited somebody to action
With important market significance.
(R)-(-)-o-chloromandelic acid is prepared it has been reported that but quantity is few by nitrilase enzyme process both at home and abroad.Zhang Chen
Win to screen from Stappiaaggregata by traditional genome mine locating technology and there is enantiomer choosing to adjacent chlorine benzaldehyde cyanohydrin
The nitrilase LaN of selecting property, the ee values of product (R)-(-)-o-chloromandelic acid are 96.5%, but vigor is not high.BASF AG's structure
The different mutants of nitrilase Y296 from A.faecalis ATCC 8750, wherein Y296A mutant pair are built
The relative Rate activity of racemic adjacent chlorine benzaldehyde cyanohydrin is nearly 10 times of Y296 wild types.Y296C mutant is each to what is detected
Individual substituted benzaldehyde cyanohydrin all has at a relatively high vigor, and does not change then for the benzaldehyde cyanohydrin vigor not replaced.
In view of this, it is special to propose the present invention.
The content of the invention
It is an object of the invention to provide a kind of new restructuring nitrilase, to solve the above problems.
In order to realize the above-mentioned purpose of the present invention, spy uses following technical scheme：
A kind of nitrilase, its amino acid sequence such as SEQ ID NO:Shown in 1.
Application of the nitrilase in R- o-chloromandelic acids are catalyzed and synthesized as described above.
Application of the nitrilase in the intermediate product for catalyzing and synthesizing R- o-chloromandelic acids as described above.
Nitrilase (bll6402, NP_ of the invention to come from Bradyrhizobiumjaponicum USDA110
773042) amino acid sequence is that template is transformed, the amino acid sequence such as SEQ ID of the new restructuring nitrilase of acquisition
Shown in NO.1.The present invention can promote application of the nitrilase in the synthesis of chiral medicinal intermediate, the greatly adjacent chlorine almonds of reduction R-
The production cost of acid, and then the price of clopidogrel is reduced, with good exemplary role, it will produce good social benefit
With economic benefit.And will significantly reduce energy consumption and three wastes discharge amount by the mild reaction conditions of biological catalysis.Project it is suitable
Profit is implemented not only realize the green production of R- o-chloromandelic acids, can also be urged to set up the biology based on nitrilase
The technology platform that change method prepares the chipal compounds such as chirality pharmaceutical intermediate compound provides technical support, it is possible to increase the biology of China,
Chemical industry, the chiral drug of medicine and other fields, the R ＆ D Level of chiral food additives and Chiral pesticide and its intermediate etc., favorably
The development of green production, industrial biotechnology in China, has broad application prospects, the sustainable development to national economy
It is significant.
The gene of the nitrilase described in coding claim 1 is also claimed in the present invention.
It is preferred that, the nucleotide sequence such as SEQ ID NO of the gene:Shown in 2.
It should be noted that the amino acid for the nitrilase that those skilled in the art can provide according to the present invention, according to close
Degeneracy principle of numeral etc. voluntarily adjusts its nucleotide sequence and function equivalence body, and these sequences being adjusted are also at this
In the range of invention is claimed.
In addition, be also claimed under strict conditions can be with SEQ ID NO by the present invention:Nucleotide sequence hybridization shown in 2
Nucleotide sequence；And the nucleotide sequence complementary with above-mentioned sequence.
Function equivalence body sequence also includes and SEQ ID NO：Sequence shown in 2 has at least 90%, 95%, 96%, 97%,
98% or 99% sequence identity, and the gene order with nitrilase activity, can separate from any plant and obtain.
Wherein, the percentage of sequence identity can be obtained by known bioinformatics, including Myers and Miller is calculated
Method (Bioinformatics, 4 (1)：11-17,1988), Needleman-Wunsch overall comparisons method (J.Mol.Biol., 48
(3)：443-53,1970), Smith-Waterman Local Alignments method (J.Mol.Biol., 147：195-197,1981),
Pearson and Lipman similarity-searching (PNAS, 85 (8)：2444-2448,1988), Karlin and Altschul algorithm
(Altschul etc., J.Mol.Biol., 215 (3)：403-410,1990；PNAS, 90：5873-5877,1993).This is for this
It is known for art personnel.
A kind of expression vector, it includes the sequence of gene as described above.
It is preferred that, the expression vector is pQE30.
A kind of engineering bacteria, it is converted by expression vector as described above.
It is preferred that, the engineering bacteria is Escherichia coli.
It is furthermore preferred that the engineering bacteria is M15 coli strains.
Compared with prior art, the nitrilase that the present invention is provided can be in the 12h reaction time, altogether by 600mM substrate
Complete hydrolysis, the diluting effect regulated and controled due to bottoms stream plus with pH, the accumulative product o-chloromandelic acid for generating 112g/L.Entirely
Reaction conversion ratio is maintained at more than 90% in course of reaction.React to stop flowing after 10h and add, continue to react substrate after 2h and be totally converted
It is complete.Reaction rate is very fast, and reaction conversion ratio is also very high.
Brief description of the drawings
, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical scheme of the prior art
The accompanying drawing used required in embodiment or description of the prior art is briefly described, it should be apparent that, in describing below
Accompanying drawing is some embodiments of the present invention, for those of ordinary skill in the art, before creative work is not paid
Put, other accompanying drawings can also be obtained according to these accompanying drawings.
Fig. 1 is OD in fermentation process600Growth pattern；
The growth pattern that Fig. 2 is DCW in fermentation process；
Fig. 3 is the growth pattern of production of enzyme in fermentation process；
Fig. 4 is that continuous stream adds reaction to prepare R- o-chloromandelic acid proceeding results figures.
Embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the present invention.It is unreceipted specific in embodiment
Condition person, the condition advised according to normal condition or manufacturer is carried out.Agents useful for same or the unreceipted production firm person of instrument, be
The conventional products that can be obtained by commercially available purchase.
The gene excavating of 1.1 new nitrilases
1.1.1 sequence screening
To come from Bradyrhizobiumjaponicum USDA110 nitrilase (bll6402, NP_773042) ammonia
Base acid sequence is template, passes through BLASTP (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) program searches
The potential benzaldehyde cyanohydrin nitrilase of rope.BLASTP result is screened we set up three standards.Amino acid sequence first
Row uniformity is higher than 90% or being all removed less than 30%, will have perfect homology and dissimilarity with bll6402
Enzyme is filtered out.Enzyme with identical sequence identity only retains one to two.Second standard is the source of sequence.From same
The sequence of one kind only retains one.Because they often have very high sequence identity and identical enzymatic property.Come
All different sequences for coming from same bacterial strain all retain.3rd standard is the availability of source bacterial strain.
Three standards above are removed, it is next that there is the sequence of nitrilase activity to be all picked for all process experimental verifications.This
The substrate specificity of a little enzymes largely all have passed through experimental verification, and classification foundation is provided for phylogenetic analysis below.
ScanProsite programs on EXPASY proteomics servers are used for detecting whether the sequence filtered out has Glu-Lys-
Cys catalytic triads.
1.1.2 data analysis
The amino acid sequence screened first carries out clustering with ClustalW.Data after cluster are soft with MEGA 4.0
Adjacent method in part bag carries out phylogenetic analysis.Bootstrap values are set as 1000.
The orthogenesis of 1.2 nitrilases
1.2.1 muton library construction
Mutant primer contains restriction enzyme site (BamHI/HindIII) and protection base.Using plasmid BCJ2315/pQE30 as
Template, is expanded using the fallibility PCR kit of day bounties.PCR primer with mutating alkali yl is double through BamHI/HindIII
The linear carrier pQE30 through same digestion, conversion Escherichia coli M15 are connected in after digestion.
1.2.2 muton is screened
Picking monoclonal (8000 transformants), is inoculated in 24 well culture plates from flat board, 30 DEG C of culture 36h.Centrifugation
Collect thalline.Culture medium uses self-induction culture medium.Thalline after above-mentioned centrifugation is cleaned one time with 1mL physiological saline, centrifuged.
Thalline is suspended with 1mL 100mM sodium phosphate buffers (pH8.0), and 30 DEG C incubate the adjacent chlorine benzaldehyde cyanohydrin that 20 μ L 1M are added after 10min
Initial action.100 μ L 2M HCl terminating reactions are added after reaction 12h.200 μ L of supernatant liquid 13,000g are taken to be used after centrifuging 10min
Ammonia process detection enzyme activity is surveyed, active reaction solution detects ee values by HPLC.
1.3 results and analysis
React PCR conditions by optimizing fallibility, frequency of mutation control in 3-7 bases/kb, transformation efficiency be 1000 clones/
10 μ L connection liquid, effective cloning efficiency reaches more than 70%.
By the screenings of 14 wheel totally 8000 mutons, we have obtained 4 beneficial mutations strains, ee values 95% with
On.Found by being sequenced, four mutant are at least containing a muton.Wherein mutant 18 and 1332, its 49 Soviet Union's ammonia
Acid is all mutated.In mutant 18, the threonine of 49 has been mutated into serine.In mutant 1332,49
Threonine is mutated into isoleucine.In mutant 338 and 1332, the isoleucine of 113 has all been mutated into asparagine.Though
Right ee values are all improved, but four mutant all show the decline of vigor in the adjacent chlorine benzaldehyde cyanohydrin of hydrolysis.
Nitrilase BCJ2315 mutant and single-point mutants enzyme activity and enantio-selectivity that the random mutation of table 1 is produced
Compare
In order to verify of the beneficial mutation in four mutant, by simple point mutation, we have been obtained in four mutant
All mutons and enzyme is purified.The hydrolysis vigor and ee values of all mutons are as shown in table 6.4.In mutant 18
In, only one of which mutational site, relative enzyme activity is that 87%, ee values bring up to 95.9% after the mutation of the mutational site.Mutant 338
In, A91S is nonsense mutation, and I113N improves the ee values of product, but vigor has all declined.In mutant 101, M98K and
R345C is nonsense mutation.Y199C and T310P show the raising of ee values.Wherein T310P relative activity is 33%,
Y199C vigor has then brought up to 201%.In mutant 1332, Y85F is nonsense mutation.T49I and I113N are shown
The raising and the decline of vigor of ee values.
Nitrilase BCJ2315 catalytic triads are E48-K130-C164, the beneficial mutant obtained by us
In, the site nearest from catalytic triads is the T of the 49th.And another site influential on enantio-selectivity is
T310, site catalytic active center away from nitrilase on amino acid sequence.Therefore, close and remote nitrilase is urged
The amino acid mutation at change center can have influence on the enantio-selectivity of enzyme.Researcher is often inclined to by near to catalytic center
Site carry out point mutation and improve the purpose of enzyme some property to reach because to carrying out mutation ratio close to the site of catalytic center
Carrying out mutation to the site away from catalytic center has higher efficiency.We are more likely to reach raising by random mutation
The purpose of BCJ2315 selectivity.Because the site close to catalytic center is limited after all, and random mutation acts on whole base
Because of sequence, more beneficial mutations away from catalytic active center can be produced.
By rite-directed mutagenesis, have 4 sites and be made that beneficial tribute in terms of BCJ2315 enantio-selectivities are improved
Offer.Wherein I113N shows highest ee values, and it is minimum that T49I ee values are improved, and is 95.7%.Work as in all mutons
In, Y199C shows highest with respect to enzyme activity.But Y199C ee values only have 94.3%.Consider vigor and enantiomer choosing
Selecting property, in order to obtain a more preferable muton, 4 sites have been carried out saturation mutation, surveyed using whole-cell catalytic by us
Performance of the fixed all mutons in terms of enzyme activity and ee values.
The nitrilase BCJ2315 single-point saturated mutant enzyme activity of table 2 and enantio-selectivity compare
During beneficial mutant is screened, vigor and ee values be all it is contemplated that factor.Ee values are more than 95%
The mutant that vigor does not decline will be picked out preferentially.In all mutant of T49, only T49N shows vigor
Faint raising, but its ee value have dropped much compared to wild type BCJ2315, only 76.1%.Mutant higher than 95% has 8
Individual (T49A, T49E, T49F, T49G, T49I, T49M, T49S, T49Y), but the relative activity of this 8 mutant all have dropped,
Highest only has wild type BCJ2315 87% (T49S).In all mutant of T310, only T310P of the ee values higher than 95%,
T310P shows good enantio-selectivity, and ee values are 98.1%.But relative activity only has the 32% of wild type.
We do not have found beneficial mutation in the saturation mutation in T49 and T310 sites.Most I113 mutant all show excellent
Elegant enantio-selectivity, wherein the ee values of 7 mutant are both greater than 99.9%.Regrettably most of mutant is all shown
The reduction of relative enzyme activity, the relative activities of this 7 mutation only have the 1%-6% of wild type, show extremely low relative enzyme
It is living.Two I113 mutant show of a relatively high enzyme activity, I113M and I113L.It is respectively 138% He with respect to enzyme activity
188%.And I113M enantio-selectivity is very well, product ee values are 97.2%.Therefore I113M mutant be selected for into
One step research.From unlike I113 mutant, all mutant of Y199 overwhelming majority shows the raisings of relative enzyme activity,
But it is little that enantio-selectivity is improved.Ee values only 3 higher than 95%, Y199A, Y199E and Y199G, ee value are respectively
95.6%, 95.1% and 96.7%.Therefore these mutant also are selected out further studied.
In view of the ee values and enzyme activity of mutant, totally four beneficial mutants are selected out.They are
I113M, Y199A, Y199E and Y199G.Four mutant all show the raising of vigor and ee values, and product ee values are up to
97.6%.In the saturated mutant in 4 sites, it is observed that the Y199 overwhelming majority all shows carrying for enzyme activity
The raising of high and ee values, and in the saturation mutation in the other three site, only I113M is selected out, shows ee values
With the dual raising of relative enzyme activity.The joint mutation in site would generally cause the synergistic effect of beneficial outcomes.Therefore we choose
I113M mutant, by 199 progress saturation mutations to I113M mutant, investigates the connection in two sites as template
Close whether mutation can further improve the relative enzyme activity and enantio-selectivity of enzyme.As a result as shown in table 6.6.Pass through 2 points of connection
Mutation is closed, we have obtained many beneficial mutation.All mutant ee values are all higher than 97%, and highest ee values are
98.7%, produced by mutant I113M/Y199G.Highest enzyme activity is also by mutant I113M/Y199G in mutant
Produce, be 3.76 times of wild type, therefore I113M/Y199G is selected as further research.
Many researchs existing at present are reported transforms nitrilase on gene level, and nitrile is changed by protein engineering
The substrate specificity of hydrolase, enantio-selectivity, pH tolerances, substrate tolerance and improve vigor of enzyme etc..Kiziak is sent out
Now removing Pseudomonas fluorescens EBC191 nitrilases 47-67 amino acid of C-terminal causes enzyme activity to reduce, acyl
The change increased with enantio-selectivity of amine product.Kiziak identifies a conservative amino acid sites H296, the site
Formation with Pseudomonas fluorescens EBC191 nitrilases enantio-selectivities and acid amides is relevant.DeSantis
Full genome saturation mutation is carried out to the nitrilase obtained by grand genome method, obtained mutant can be resistant to height
The 3-HGN (3M) of concentration and enantio-selectivity has brought up to 98.1%.From the aliphatic nitrile of Acidovorax facilis
Hydrolase, a single mutation (T210A) makes its activity to 3- hydroxyvaleronitriles improve 7.3 times.The joint of two points is dashed forward
The activity that becoming (F168V+L201N) makes it to hydroxyacetonitrile improves 15.3 times, and mutant enzyme activity, and volume productivity, product is received
Rate all meets industrial demand.From the nitrilase of prunosus red coccus, Y142 residues are proved to play substrate specificity
Effect.From Neurosporacrassa OR74A nitrilase, W168A mutant causes it in hydrolysis benzaldehyde cyanohydrin and 2-
More acid amides are produced during phenylpropanenitrile.And the enantio-selectivities of 2- phenylpropanenitriles is inverted completely (S is reversed to by R types
Type).So far, nitrilase is transformed by genetic engineering to enable the adjacent chlorine of high enantiomeric optional water racemic flat
There is not been reported for peach nitrile generation (R)-(-)-o-chloromandelic acid.Schreiner is derived from by directed evolution technologies
Alcaligenesfaecalis nitrilase NITAf mutant pHNIT45, the mutant is in pH4.5 with higher
Stability.PHNIT45 can hydrolyze adjacent chlorine benzaldehyde cyanohydrin generation (R)-(-)-neighbour's chlorine almond of R types under pH4.5 reaction condition
Acid, product ee values are more than 99%.But in pH7.0, NITAf and pHNIT45 selectivity are all very poor, respectively 76% He
19%.In our study, by the orthogenesis to nitrilase BCJ2315, obtained beneficial mutant in vigor and
All it is greatly improved in terms of ee, can the adjacent chlorine benzaldehyde cyanohydrin generation of hydrolysis of racemic under neutral and meta-alkalescence reaction condition
(R)-(-)-o-chloromandelic acid, theoretical yield is that 100%, ee values are 98.7%.
The I113M199 single-point saturated mutant enzyme activity of nitrilase BCJ2315 mutant of table 3 and enantio-selectivity ratio
Compared with
On the basis of I113M/Y199G mutant, for active site amino acid proceed iteration saturation dash forward
Become, during beneficial mutant is screened, screen one plant of beneficial mutation strain, find that G59A mutant exists by sequencing
On the basis of I113M/Y199G mutant, enzyme activity is further improved, therefore in follow-up R- o-chloromandelic acids preparation technology,
Biocatalyst is used as using G59A/I113M/Y199G (MGA) mutant.
2 nitrilases catalyze and synthesize R- o-chloromandelic acid technical studies
2.1.1 Shaking culture
Unless otherwise specified, in following incubation, addition Kan 0.05mM, Amp 0.1mM are both needed in LB culture mediums.
(1) go bail for the E.coli engineering bacteria strains being hidden at -80 DEG C, be inoculated in LB culture mediums, 37 DEG C of overnight incubations.
(2) stay overnight bacterium 0.5% (v/v) to be inoculated in 400mL LB culture mediums, 37 DEG C of cultures to OD600=0.6.
(3) thalline is collected after IPTG to final concentration 0.1mM, 30 DEG C of culture 16h are added in culture.
2.1.2 catalyst preparation
(1) culture that Shaking culture is obtained centrifuges 5min in 8000rpm at 4 DEG C.Collect thalline.
(2) thalline is resuspended in 100mM pH 8.0 Na2HPO4/NaH2PO4 buffer solutions, 4 DEG C of 8000rpm centrifugations
5min, collects thalline.
(3) repeat step (2) is once.
(4) thalline after buffer solution washing is saved backup in -20 DEG C.
2.1.3E.coli/MGA engineering bacterium fermentation
R- o-chloromandelic acids are prepared, it is necessary to which large batch of engineering bacteria is used as catalyst for technical grade, it is therefore necessary to built
Found the E.coli/MGA engineering bacterium fermentation techniques inexpensively stablized.High density fermentation refers in certain condition and cultivating system
Under, most cell concentrations are obtained, purpose product thus more or is more efficiently obtained.Realize that high density fermentation can not only subtract
Few volume of culture, reinforcing downstream separation is extracted, and can also shorten the production cycle, equipment investment is reduced, so as to reduce production cost.
Utilize general zymotechnique production Escherichia coli or the expression product with the genetic engineering bacterium of its structure, large intestine bar
The biomass of bacterium, expressing quantity, concentration of the metabolite in endobacillary zymotic fluid is all than relatively low, it is difficult to obtain preferable
Production efficiency.Recombination bacillus coli, which carries out high density fermentation, can obtain higher biomass, but have to fermentation condition very high
It is required that.Influence the factor of high density fermentation very many, growth inhibition in the nutriment, fermentation process as required for cell growth
The accumulation of thing, oxyty, induction mode, the pH value of zymotic fluid, feed profile and zymotic fluid rheological properties etc..
2.1.3.1
Culture medium
Culture medium can be divided into synthetic media, semisynthetic medium, complex medium by constituent.Its component bag
Include：C, H, O, N, S, P, Fe, Mg, K etc..Culture medium used in Escherichia coli high density fermentation is generally semisynthetic medium,
It is that addition can promote the different material of cell growth and metabolite formation on the basis of synthetic media, such as appropriate inorganic
Salt, amino acid, vitamin etc..Conventional carbon source has carbohydrate, grease, organic acid and low-carbon alcohols.Glucose is most easy profit in carbon source
Sugar, is often used as the main component of culture medium.Conventional nitrogen source can be divided into organic nitrogen source and inorganic nitrogen-sourced.Culture medium each group
The concentration and ratio divided are appropriate, especially the ratio of carbon source and nitrogen source.If carbon source is too high with nitrogen concentration, Su Anran ratios
Properly, fermentation starting can cause the amount reproduction of thalline, but fermentation later stage thalli growth is slow, and metabolic waste is excessive, increase hair
Ferment viscosity, influences dissolved oxygen concentration, easily causes the metabolic disorder of thalline.Therefore design a kind of culture medium of balance and be conducive to production
The expression of thing.
In the design process of nutrient media components, we use peptone and carbon nitrogen source based on yeast extract, because two
Person can not only provide the carbon nitrogen source needed for engineering bacteria growth course, and provide the trace contents such as trace element and growth factor
Matter.On this basis, the growth that a small amount of glycerine is conducive to fermentation thalline at initial stage as quick-acting carbon sources is added.The addition of potassium phosphate
The favourable pH for maintaining zymotic fluid is balanced.Mg2+Extra be added with the vigor for being beneficial to improve enzyme.
The fermentation basal medium component of table 4
The fermentation feed medium component of table 5
2.1.3.2 pH value
Stable pH value is the necessary condition for making thalline keep optimum growh state.Because extraneous pH value change can pass through
The change of weak acid or weak base and change the pH value in somatic cells, so as to influence the metabolic response of cell.Therefore in fermentation process
The change of middle pH value can influence the biomass of cell and the expression of gene outcome.On the other hand, the growth of microorganism can cause training
The change of nutrient solution pH value.Microorganism is cultivated in minimal medium, usual pH value can have greatly changed.Ammonia disappears in ammonium salt
Consumption, or the metabolite that is produced using carbon source of microorganism are the accumulation of organic acid, can all cause the decline of pH value, and organic acid
Consumption can then cause the rising of pH value.Cultivated in complex medium, when consuming carbon source, amino acid in culture medium by with
When alienation is metabolized, because of the release of ammonia, pH value can be caused to raise.The change of pH value is tracked in fermentation process, can be micro- to understand
The change of biological metabolism provides highly important information.In zymotechnique control, it is necessary to timely use the alkali or ammonia of low concentration
Water regulation pH value is allowed in the range of suitable pH, it is to avoid the unfavorable shadow that pH value acute variation cell growth and metabolism are caused
Ring.
In the fermentation process of E.coli/MGA engineering bacterias, we control pH 7.2 or so, are conducive under the conditions of the pH
The growth and the accumulation of MGA nitrilases of engineering bacteria.Because Escherichia coli produce the accumulation of acid, the drop of zymotic fluid pH value can be caused
Low, the most important means of maintenance of pH value are the additions of alkaline matter.In this fermentation, ammoniacal liquor regulates and controls with NaOH solution in pH
Within the limit of consideration of means.NaOH solution is used to regulate and control pH advantages and is the simplicity that sterilizes, non-volatile, and can be configured to high concentration
Solution, its shortcoming is, because NaOH is strong alkaline substance, local zymotic fluid moment can be caused to produce highly basic during instillation
Property environment, is damaged to thalline.The advantage that ammoniacal liquor is used to regulate and control pH is that ammoniacal liquor alkalescence relatively is weaker, and regulation is relatively warm
With, but ammoniacal liquor sterilizing is relatively cumbersome, needs filtration sterilization, and due to ammoniacal liquor highly volatile, endanger larger to operating personnel.
2.1.3.3 dissolved oxygen
Dissolved oxygen concentration is one of key factor of influence thalli growth in process of high-density fermentation of aerobic microorganism, and
It often turns into the governing factor of fermentation.This is mainly due to oxygen in water caused by solubility very little.Given birth in the logarithm of cell
If the dissolved oxygen in long-term oxygen feeding stop, zymotic fluid can exhaust within a few minutes.Biomass is very in phase after fermentation, reactor
Greatly, and fermentation broth viscosity increases, now limited by reactor oxygen delivery capacity, the dissolved oxygen concentration in zymotic fluid can be less than facing
Boundary's oxygen concentration.So, the dissolving and transmission of oxygen have become the conditioning step of the cell effect.
Ferment the incipient stage, control dissolved oxygen is more than 20%.The control of dissolved oxygen it is main by step up mixing speed with
And the mode of change throughput is controlled.Initial stage, mixing speed was set as 300rpm, and the final speed of speed of agitator is 700rpm.
2.1.3.4 temperature and inductive condition
Cultivation temperature is to influence the key factor of thalli growth and regulating cell metabolism.At the initial stage of fermentation, temperature is set as
37 DEG C, be conducive to fast-growth and the breeding of cell.After exponential phase is entered, induced to adjust engineering by IPTG
The gene expression dose of bacterium.Now to obtain larger amount of target protein i.e. MGA nitrilases as main target.Due to higher
At a temperature of in engineering bacteria MGA expression easily form more inactive inclusion bodies, therefore after induction starts, it is appropriate to need
The metaboilic level of engineering bacteria is reduced, temperature is now reduced to 30 DEG C and fermented.Be conducive to MGA at a lower temperature can
Dissolubility is expressed, and reduces the formation of inclusion body.
2.1.3.5 fermentation results
Summary condition, the fermentation to engineering bacteria E.coli/MGA processes, during the fermentation different time points carry out
OD600 in sampling analysis, detection fermentation process, DCW and production of enzyme growth pattern.Concrete outcome is shown in Fig. 1, Fig. 2, Fig. 3 knots
Shown in fruit.
It can see by above-mentioned three figure, by the design to fermentative medium formula, and pH during the fermentation, temperature
And the regulation and control of dissolved oxygen.Taken into account simultaneously by main target of production of enzyme under conditions of cell concentration, the fermentation concentration of engineering bacteria reaches
OD600 is arrived in 75 or so, DCW in 35g/L or so, production of enzyme reaches 35000kU/L.Production of enzyme is in nitrilase fermentation arts
Be in a leading position level.
2.2 catalytic reaction
Nitrilase catalyzing hydrolysis neighbour's chlorine benzaldehyde cyanohydrin prepares the reaction of R- o-chloromandelic acids using the anti-of continuous flow feeding
Pattern is answered, by maintaining concentration of substrate in relatively low level to slow down the speed that enzyme is inactivated, by continuous flow feeding, to reach
The high concentration accumulation of R- o-chloromandelic acids.
2.2.1 prepared by conversion fluid
Nitrilase catalyzing hydrolysis neighbour's chlorine benzaldehyde cyanohydrin prepares the conversion reaction of R- o-chloromandelic acids in 100mM pH's 8.0
Na2HPO4/NaH2PO4Carried out in buffer solution.
Wherein, ethanol 10% (v/v) is added in buffer solution and is used as cosolvent.
The resting cell of 20mg/ml final concentrations.
Conversion fluid after preparation, which is incubated to 30 DEG C, is used for subsequent reactions.
2.2.2 the preparation of current adding substrate
Adjacent chlorine benzaldehyde cyanohydrin for flowing plus reacting is dissolved in the stream liquid feeding that final concentration of 6M is configured in ethanol, and stream liquid feeding is in 30
It is incubated under the conditions of DEG C.
2.2.3 continuous feeding stream adds reaction
The adjacent chlorine benzaldehyde cyanohydrin of high concentration to nitrilase MGA inhibitory action clearly, it is easier to cause the inactivation of enzyme.
In order to obtain product (R)-(-)-o-chloromandelic acid of high concentration, we adjust reaction by using continuous current adding substrate pattern
In concentration of substrate, make reaction remain under a higher speed carry out, to reach that high concentration (R)-(-)-neighbour's chlorine is flat
The cumulative production of peach acid.During current adding substrate, by substrate dissolving in organic solvent, reaction system can be improved simultaneously
Organic cosolvent, with simultaneously reach improve product enantio-selectivity purpose.Organic solvent is added into reaction system also
The speed of reaction can be improved, the reaction time is reduced.We are used as cosolvent by selected ethanol.Substrate neighbour's chlorine benzaldehyde cyanohydrin is dissolved in ethanol
The mother liquor (6M) of high concentration is formed, the content of ethanol in reaction system is improved constantly while current adding substrate, makes the work of reaction
Power and ee values make up the inactivation of part enzyme in course of reaction all in the process of a growth.The addition of initial action ethanol is
20%, at this concentration, the ee values of generation product are 95.9%.By TLC monitoring reaction courses, substrate feed rate is adjusted.
Carried out in the 300mL three neck round bottom flask for reacting on 500mL, water-bath is incubated 30 DEG C, double blade stirring oar machineries
Speed of agitator 350rpm.14# flexible pipe current adding substrates, wherein flow acceleration 60mM/h, flexible pipe need 30 DEG C of insulations with stream liquid feeding.
PH electrodes monitoring reaction pH change, 6M ammoniacal liquor maintains pH between 7.95-8.10, wherein pH regulation and monitoring
Electrode links.
2.2.4 detection method
The monitoring of reaction process uses TLC on-line monitorings, the method that HPLC is quantitatively detected.Detection method detail is shown in point
Analysis method one is saved.
2.2.5 reaction result
Substrate is added to stop flowing after 10h with 60mM/h flow acceleration continuous stream and added, and continues to terminate reaction after extension 2h.
Reaction process result is shown in Fig. 4.
In the 12h reaction time, common 600mM substrate is by complete hydrolysis, and the dilution regulated and controled due to bottoms stream plus with pH is made
With the accumulative product o-chloromandelic acid for generating 112g/L.Reaction conversion ratio is maintained at more than 90% in whole course of reaction.Instead
Answer to stop flowing after 10h and add, continue to react substrate after 2h and be totally converted.
At present, on mass producing the document report of o-chloromandelic acid and few using nitrilase.Chen Shengli is opened to use
Will in 8h by water/toluene two-phase system containing the restructuring nitrilase cell from Labrenziaaggregata
300mM adjacent chlorine benzaldehyde cyanohydrin complete hydrolysis, yield is 94.5%, and product ee values are 96.5%.Arthrobacterium containing nitrilase
(Arthrobacter sp.) F-73 is by water/ethyl acetate two-phase system, by the way that adjacent chlorine benzaldehyde cyanohydrin (final concentration is added batch-wise
100mM), in 9h cotransformation 400mM adjacent chlorine benzaldehyde cyanohydrin, yield is 92%, product ee values be 98.5%.Product is recovered to lay equal stress on
Ee values can bring up to 99.3% after crystallization.
2.3 products are isolated and purified
The purifying of R- o-chloromandelic acids is carried out using crystallisation.Reaction solution after centrifugal treating, is carried out by dense HCl
PH 1 is acidified to, 1/3 volume is concentrated into by rotary evaporation, the crystallisation by cooling under the conditions of 10 DEG C collects crystal, concentration knot is repeated
After brilliant process 2 times, the crystal being collected into is dissolved in ethanol and recrystallized.Packed after recrystallizing crystal drying.
2.4 Product checking
R- o-chloromandelic acid testing results after purification are as follows：
1H NMR(400MHz,D2O)δ/ppm:5.530(s,1H),7.262–7.300(m,2H),7.322–7.356(m,
1H),7.382–7.415(m,1H).
[α]D25=-153 (c=0.5, ethanol)
ee>98%
After testing, product meets company standard.
Finally it should be noted that：Various embodiments above is merely illustrative of the technical solution of the present invention, rather than its limitations；To the greatest extent
The present invention is described in detail with reference to foregoing embodiments for pipe, but it will be understood by those within the art that：Its
The technical scheme described in foregoing embodiments can still be modified, or to which part or all technical characteristic
Carry out equivalent substitution；And these modifications or replacement, the essence of appropriate technical solution is departed from various embodiments of the present invention skill
The scope of art scheme.
SEQUENCE LISTING
<110>Zaozhuang Jienuo Enzyme Co., Ltd.
<120>A kind of nitrilase and its application
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 334
<212> PRT
<213>Artificial sequence
<400> 1
Met Gln Asp Thr Lys Phe Lys Val Ala Val Val Gln Ala Ala Pro Val
1 5 10 15
Phe Met Asp Ala Pro Ala Ser Val Ala Lys Ala Ile Gly Phe Ile Ala
20 25 30
Glu Ala Gly Ala Ala Gly Ala Lys Leu Leu Ala Phe Pro Glu Val Trp
35 40 45
Ile Pro Gly Tyr Pro Trp Trp Leu Trp Leu Ala Thr Pro Ala Trp Gly
50 55 60
Met Gln Phe Val Pro Arg Tyr His Ala Asn Ser Leu Arg Ala Asp Gly
65 70 75 80
Pro Asp Ile Leu Ala Leu Cys Ala Ala Ala Ala Glu Ala Lys Ile Asn
85 90 95
Val Val Met Gly Phe Ser Glu Ile Asp Gly Gly Thr Leu Tyr Leu Ser
100 105 110
Met Val Phe Ile Ser Asp Ala Gly Glu Ile Ile Phe Lys Arg Arg Lys
115 120 125
Leu Lys Pro Thr His Val Glu Arg Thr Leu Tyr Gly Glu Gly Asp Gly
130 135 140
Ser Asp Phe Arg Val Val Glu Ser Ser Val Gly Arg Leu Gly Ala Leu
145 150 155 160
Cys Cys Ala Glu His Ile Gln Pro Leu Ser Lys Tyr Ala Met Tyr Ser
165 170 175
Met Asn Glu Gln Val His Val Ala Ser Trp Pro Ser Phe Thr Leu Tyr
180 185 190
Arg Asp Lys Ala Tyr Ala Gly Gly His Glu Val Asn Leu Ala Ala Ser
195 200 205
Gln Ile Tyr Ala Leu Glu Gly Gly Cys Phe Val Leu His Ala Ser Ala
210 215 220
Ile Thr Gly Gln Asp Met Phe Asp Met Leu Cys Asp Thr Pro Glu Lys
225 230 235 240
Ala Asp Leu Leu Asn Ala Glu Gly Ala Lys Pro Gly Gly Gly Tyr Ser
245 250 255
Met Ile Phe Gly Pro Asp Gly Gln Pro Met Cys Glu His Leu Pro Gln
260 265 270
Asp Lys Glu Gly Ile Leu Tyr Ala Asp Val Asp Leu Ser Met Ile Ala
275 280 285
Ile Ala Lys Ala Ala Tyr Asp Pro Thr Gly His Tyr Ala Arg Gly Asp
290 295 300
Val Val Arg Leu Met Val Asn Arg Ser Pro Arg Arg Thr Ser Val Ser
305 310 315 320
Phe Ser Glu Asp Glu Asn Ala Ala Val Thr Phe Thr Glu Thr
325 330
<210> 2
<211> 1005
<212> DNA
<213>Artificial sequence
<400> 2
atgcaggaca cgaaattcaa agtcgcggtc gttcaggctg cgccggtatt catggatgcg 60
ccagcttccg tggccaaggc gattggtttc attgcggagg cgggtgcagc gggggcgaag 120
ctgctggcgt tcccggaggt ctggattccg ggctatcctt ggtggctttg gctcgggacg 180
ccggcttggg gaatgcagtt tgtaccccgc tatcacgcca attcgctgcg tgctgatggg 240
cccgacatcc tcgcactgtg tgcggccgcc gccgaagcga aaatcaatgt ggtgatgggc 300
ttctccgaaa tcgacggagg aacgctctac ctaagtcagg tctttattag cgatgcggga 360
gagatcatct tcaagcgccg aaagctcaag ccgacacacg tcgaacgtac gctctatggc 420
gaaggagatg ggtctgattt ccgcgtcgtc gaaagcagcg tcggacgtct cggagccttg 480
tgctgcgccg agcacattca gccgttgtcg aaatacgcca tgtactcgat gaacgagcaa 540
gttcacgtgg cgtcgtggcc atcttttacg ctgtatcgcg acaaggccta cgctttgggc 600
catgaggtga atctcgccgc cagccagatc tacgcgctag agggaggttg cttcgtcctg 660
catgcctcgg caattactgg tcaagatatg ttcgacatgc tttgcgatac tccggaaaag 720
gccgatttgc tgaacgcgga gggagcgaag ccgggtggag gctattcgat gatcttcggt 780
cccgatggtc agccgatgtg cgagcatctg ccgcaggaca aggaaggcat cctctatgct 840
gacgtggacc tgtcaatgat cgcaatcgcc aaagcggcct acgaccccac ggggcattac 900
gcccgcggcg atgtcgtccg tctcatggtc aatcgcagcc cgcgtcgcac gagcgtcagc 960
ttcagcgaag acgagaacgc ggcggtcact ttcaccgaga cttga 1005
Claims (10)
1. a kind of nitrilase, its amino acid sequence such as SEQ ID NO:Shown in 1.
2. application of the nitrilase in R- o-chloromandelic acids are catalyzed and synthesized described in claim 1.
3. application of the nitrilase in the intermediate product for catalyzing and synthesizing R- o-chloromandelic acids described in claim 1.
4. encode the gene of the nitrilase described in claim 1.
5. gene according to claim 4, its nucleotide sequence such as SEQ ID NO:Shown in 2.
6. a kind of expression vector, it includes the sequence of gene described in claim 4 or 5.
7. expression vector according to claim 6, it is characterised in that the expression vector is pQE30.
8. a kind of engineering bacteria, it is converted by the expression vector described in claim 6 or 7.
9. engineering bacteria according to claim 8, it is characterised in that the engineering bacteria is Escherichia coli.
10. engineering bacteria according to claim 9, it is characterised in that the engineering bacteria is M15 coli strains.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109312375A (en) * | 2018-04-25 | 2019-02-05 | 邦泰生物工程(深圳)有限公司 | A kind of preparation method of hesperetin, the preparation method of hesperetin intermediate and the biological enzyme for being used to prepare hesperetin |
| CN113025601A (en) * | 2019-12-25 | 2021-06-25 | 上海奥博生物医药技术有限公司 | Nitrilase promoter optimized expression and application |
| CN114410669A (en) * | 2022-03-28 | 2022-04-29 | 佛山市玉凰生态环境科技有限公司 | Production and immobilization method of recombinant nitrilase and application of recombinant nitrilase to degradation of acetonitrile |
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| WO2011011630A2 (en) * | 2009-07-23 | 2011-01-27 | Codexis, Inc. | Nitrilase biocatalysts |
| CN104774825A (en) * | 2015-03-23 | 2015-07-15 | 浙江工业大学 | Nitrilase mutant and application thereof |
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| WO2011011630A2 (en) * | 2009-07-23 | 2011-01-27 | Codexis, Inc. | Nitrilase biocatalysts |
| CN104774825A (en) * | 2015-03-23 | 2015-07-15 | 浙江工业大学 | Nitrilase mutant and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109312375A (en) * | 2018-04-25 | 2019-02-05 | 邦泰生物工程(深圳)有限公司 | A kind of preparation method of hesperetin, the preparation method of hesperetin intermediate and the biological enzyme for being used to prepare hesperetin |
| CN109312375B (en) * | 2018-04-25 | 2022-06-17 | 邦泰生物工程(深圳)有限公司 | Preparation method of hesperetin, preparation method of hesperetin intermediate and biological enzyme for preparing hesperetin |
| CN113025601A (en) * | 2019-12-25 | 2021-06-25 | 上海奥博生物医药技术有限公司 | Nitrilase promoter optimized expression and application |
| CN114410669A (en) * | 2022-03-28 | 2022-04-29 | 佛山市玉凰生态环境科技有限公司 | Production and immobilization method of recombinant nitrilase and application of recombinant nitrilase to degradation of acetonitrile |
| CN114410669B (en) * | 2022-03-28 | 2022-06-17 | 佛山市玉凰生态环境科技有限公司 | Production and immobilization method of recombinant nitrilase and application of recombinant nitrilase to degradation of acetonitrile |
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