CN106032531B - A kind of cyanalcohol lyases, preparation method and application - Google Patents
A kind of cyanalcohol lyases, preparation method and application Download PDFInfo
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- CN106032531B CN106032531B CN201510481026.7A CN201510481026A CN106032531B CN 106032531 B CN106032531 B CN 106032531B CN 201510481026 A CN201510481026 A CN 201510481026A CN 106032531 B CN106032531 B CN 106032531B
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Abstract
The present invention provides a kind of cyanalcohol lyases, preparation method and applications.Specifically, a kind of cyanalcohol lyases of mutation is obtained, the experimental results showed that, the catalytic activity of the cyanalcohol lyases of the mutation is 200% or more of the cyanalcohol lyases of wild type.
Description
Technical field
The invention belongs to field of biotechnology, specifically, the present invention relates to a kind of cyanalcohol lyases and its applications.
Background technique
Cyanalcohol lyases is a kind of highly useful industrial enzymes in Chemical Manufacture, and natural activity is catalysis cyanalcohol
It cracks and releases hydrogen cyanide.Cyanalcohol lyases can be catalyzed back reaction, i.e. the addition of HCN and aldehyde ketone obtain living with optics
α-cyanalcohol product of property.
Natural S- cyanalcohol lyases is present in a few plant tissue such as rubber, cassava and sorghum, and abundance is low, pure
It is big to change difficulty.Nineteen ninety-five, Wajant isolated cassava cyanalcohol lyases MeHNL from cassava using five step method of purification
(Plant Sci.,1995,108,1)；White et al. is extracted MeHNL using three-step approach from cassava leaves, using saltouing and thoroughly
The mode of analysis has obtained enzyme solution, but the stereoselectivity for being applied to chemical catalysis it is not high (Plant Physiol 1998,116,
1219).Cyanalcohol lyases (MeHNL) from cassava (Manihot esculenta) is a kind of S- cyanalcohol lyases, is had
MeHNL is used to be catalyzed the chemical synthesis of S- type chiral cyanohydrin by document report, and ee value > 99% has important application value, example
If S- 3-phenoxy-benzaldehyde cyanalcohol is the versatile intermediates of novel pyrethrin pesticide.1993, Wajant et al. reported volume
The cDNA sequence and protein sequence of code MeHNL.
It as host strain is that rapid, high volume obtains the effective ways of cyanalcohol lyases using microorganism.Effenberger
Et al. recombinant expression of the MeHNL in Escherichia coli is reported within 1996 in Escherichia coli, but albumen is mostly inclusion body, can
Dissolubility protein content is lower (Angew 1996,35,437).Chen Shuhua etc. 2001 is by MeHNL gene cloning to plasmid
PPic9K realizes the expression (bioengineering journal, 2001,17 (1), 78) in saccharomycete, but enzyme activity is still not high enough,
It is difficult to reach the requirement of practical application.
Summary of the invention
The purpose of the present invention is to provide a kind of cyanalcohol lyases, preparation method and applications.
The first aspect of the present invention, provides a kind of cyanalcohol lyases of mutation, and the cyanalcohol lyases of the mutation is out of office
The 60th glutamic acid (E) corresponding to SEQ ID NO.:1 of the cyanalcohol lyases of raw type, the 128th tryptophan (W), the 220th
Position isoleucine (I) mutates.
In another preferred example, the cyanalcohol lyases is cassava cyanalcohol lyases (MeHNL).
In another preferred example, the 60th glutamic acid (E) sports glycine (G)；And/or
128th tryptophan (W) sports alanine (A)；And/or
220th isoleucine (I) sports leucine (L).
In another preferred example, the amino acid sequence of the cyanalcohol lyases of the mutation is as shown in SEQ ID NO.:5.
In another preferred example, the catalytic activity of the cyanalcohol lyases of the mutation is corresponding wild type under equal conditions
Cyanalcohol cracks the 150%~350% of enzymatic activity, it is therefore preferable to 180%~220%.
The second aspect of the present invention provides a kind of polynucleotide molecule, and the polynucleotides are selected from the group:
(a) polynucleotides of the polypeptide as shown in SEQ ID NO.:5 are encoded；
(b) sequence polynucleotides as shown in SEQ ID NO.:2 or 7；
(c) homology >=95% (preferably >=98%) of nucleotide sequence and sequence shown in SEQ ID NO.:2 or 7, and
Encode the polynucleotides of polypeptide shown in SEQ ID NO.:1 or 5；
(d) polynucleotides complementary with any polynucleotides of (a)-(c).
The third aspect of the present invention, provides a kind of carrier, and the carrier contains nucleic acid described in second aspect of the present invention
Molecule.
In another preferred example, the carrier is pET28.
In another preferred example, the carrier further includes trp promoter Trp2；The preferably described trp promoter
The nucleotide sequence of Trp2 is as shown in SEQ ID NO.:6
The fourth aspect of the present invention, provides a kind of host cell, and the host cell contains third aspect present invention institute
The carrier or chromosomal integration stated have nucleic acid molecules described in second aspect of the present invention.
In another preferred example, the host cell is prokaryotic cell or eukaryocyte.
In another preferred example, the prokaryotic cell is Escherichia coli.
The fifth aspect of the present invention provides a kind of cyanalcohol lyases for preparing mutation described in first aspect present invention
Method, which is characterized in that comprising steps of
(i) under the suitable conditions, host cell described in fourth aspect present invention is cultivated, to give expression to described dash forward
The cyanalcohol lyases of change；With
(ii) the cyanalcohol lyases of the separation mutation.
The sixth aspect of the present invention, provides a kind of enzyme preparation, and the enzyme preparation includes to dash forward described in first aspect present invention
The cyanalcohol lyases of change.
In another preferred example, the enzyme preparation further includes one or more components selected from the group below: citric acid, apple
Acid and succinic acid.
The seventh aspect of the present invention, the cyanalcohol lyases for providing mutation described in first aspect present invention, such as present invention
The purposes of enzyme preparation described in 6th aspect, is used to prepare S- cyanalcohol product with optical activation.
In another preferred example, the purposes further includes being catalyzed the addition reaction of HCN and aldehyde ketone.
The eighth aspect of the present invention provides a kind of method for preparing S- cyanalcohol, comprising steps of
(1) the cyanalcohol lyases of mutation described in first aspect present invention is contacted with reaction substrate, carries out catalysis reaction,
To generate the S- cyanalcohol；
(2) it separates and purifies the S- cyanalcohol product.
In another preferred example, in the step (1), the reaction substrate includes 3-phenoxy-benzaldehyde and/or acetone
Cyanalcohol.
In another preferred example, in the step (1), the temperature for being catalyzed reaction is 0-20 DEG C.
It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention
It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, In
This no longer tires out one by one states.
Detailed description of the invention
Figure 1A shows the gel electrophoresis spectrum of pcr amplification product in embodiment 1.
Figure 1B shows the gel electrophoresis spectrum for the cyanalcohol lyases that the present invention is mutated.
Fig. 2 shows that catalytic activity measures curve.
Fig. 3 shows the HPLC testing result of this hair catalysis reaction.
Fig. 4 shows plasmid map constructed by the present invention.
Specific embodiment
The present inventor unexpectedly obtains a kind of cyanalcohol lyases of mutation, experimental result by extensive and in-depth research
The catalytic activity for showing the cyanalcohol lyases of the mutation is 200% or more of the cyanalcohol lyases of wild type.The present invention also mentions
The purposes of the cyanalcohol lyases of the mutation is supplied.
Specifically, the present invention has carried out codon optimization for Escherichia coli to the sequence of MeHNL, and according to directed evolution
Method introduce random mutation in the DNA sequence dna of MeHNL, screening mutant library obtains optimal sequence, finds to therein
60 sport G, and 128 amino acids sport A, and 220 sport L.And the sequence is cloned into escherichia coli plasmid, it opens
Mover is trp promoter Trp2, and conversion imports Escherichia coli, carries out high density fermentation.It obtains in this way
MeHNL, specific enzyme activity are up to 300u/mL, and higher than the cyanalcohol lyases of known heterogenous expression, and fermentation period is short, do not need to lure
Agent is led, it is at low cost.
Before describing the present invention, it should be understood that the present invention is not limited to the specific method and experiment conditions, because this
Class method and condition can change.It should also be understood that its purpose of the term as used herein is only that description specific embodiment, and
And it is not intended to be restrictive, the scope of the present invention will be limited only by the claims which follow.
Unless otherwise defined, otherwise whole technologies used herein and scientific term all have such as fields of the present invention
The normally understood identical meanings of those of ordinary skill.As used herein, in use, term in mentioning the numerical value specifically enumerated
" about " mean that the value can change not more than 1% from the value enumerated.For example, as used herein, statement " about 100 " includes 99 Hes
101 and between whole values (for example, 99.1,99.2,99.3,99.4 etc.).
Although can be used in implementation or test of the invention and heretofore described similar or of equal value any method
And material, place enumerates preferred method and material herein.
Cyanalcohol lyases
Cyanalcohol lyases (Hydroxynitrile lyase) is mainly derived from several plantations such as rubber, cassava and sorghum
Object tissue.Specifically include that cassava cyanalcohol lyases (MeHNL), lacquer tree cyanalcohol lyases (HbHNL), almond cyanalcohol lyases
(PaHNL)。
In the preferred embodiment of the present invention, the cyanalcohol lyases is cassava cyanalcohol lyases.
In the preferred embodiment of the present invention, preferably cassava cyanalcohol lyases wild-type sequence is as follows:
In the preferred embodiments of the present invention, the sequence of the cyanalcohol lyases of the mutation is as follows:
Cyanalcohol lyase gene sequence optimisation
In the present invention, provide optimization, be particularly suitable for the cyanalcohol expressed in Bacillus coli cells cracking zymoprotein
Nucleic acid coding sequence.
The present inventor selects Escherichia coli preferred codons, cracks under the premise of not changing its amino acid sequence to cyanalcohol
The DNA sequence dna of enzyme is optimized.However, the inventors discovered that, the optimization and endless obtained only in accordance with codon frequency
It is suitble to entirely in expression in escherichia coli.Therefore present inventor has performed double optimizations, are unfavorable for the two of expression including eliminating
Level structure (such as hairpin structure), change A+T composition in gene, change G+C content etc..
By largely testing and screening, the present inventor obtains the cyanalcohol that one especially optimizes in numerous optimizations and splits
Solve enzyme coded sequence.The nucleotide sequence of the cyanalcohol lyases of the optimization as shown in SEQ ID NO:2,
In a preferred embodiment of the present invention, the polynucleotides of the cyanalcohol lyases of saltant type of the invention are encoded
Sequence is as follows:
Carrier and host cell
The present invention also provides a kind of carriers of cyanalcohol lyase gene comprising optimization of the invention, and contain the load
The host cell of body.
In a preference of the invention, the carrier of stating has in Escherichia coli (more preferably in e. coli bl21
(DE3) bacterial strain) in express ability.
The conventional method that those skilled in the art can be used obtains the cyanalcohol lyases base of optimization of the invention
Because of sequence, such as complete artificial synthesized or PCR method synthesis.A kind of preferred synthetic method is asymmetric PCR method.Asymmetric PCR method is
With the pair of primers of inequality, a large amount of single stranded DNA (SSDNA) is generated after PCR amplification.This is referred to as unrestricted draw to primer
Object and restricted primer, ratio are generally 50-100: 1.In the initial 10-15 circulation of PCR reaction, amplified production master
If double-stranded DNA, but after restricted primer (low concentration primer) runs out of, non-limiting primer (high density primer) guidance
PCR will generate a large amount of single stranded DNA.Primer for PCR can be appropriate according to the sequence information of invention disclosed herein
Ground selection, and available conventional method synthesis.The DNA/RNA piece of amplification such as can be separated and purified by gel electrophoresis with conventional method
Section.
Destination protein can be expressed or be produced to polynucleotide sequence of the invention by the recombinant dna technology of routine, including
Step:
(1) with the polynucleotides (or variant) for encoding albumen of the present invention, or with containing the polynucleotide recombinant expression
Carrier converts or suitable host cell of transduceing, preferably Bacillus coli cells；
(2) host cell is cultivated in suitable culture medium；
(3) it is separated from culture medium or cell, protein purification.
Method well-known to those having ordinary skill in the art can be used to construct DNA sequences encoding containing albumen of the present invention and suitable
Transcription/translation control signal expression vector, preferably commercially available carrier: pET28.These methods include recombinant DNA technology in vi,
DNA synthetic technology, In vivo recombination technology etc..The DNA sequence dna can be effectively connected in the appropriate promoter in expression vector,
To instruct mRNA to synthesize.Expression vector further includes the ribosome bind site and transcription terminator of translation initiation.In addition, expression
Carrier preferably comprises one or more selected markers, to provide the phenotypic character for selecting the host cell of conversion.
The present invention also provides recombinant vector, it includes the MeHNL DNA sequence dnas by optimization of the invention.Preferred
In embodiment, the promoter downstream of the recombinant vector includes multiple cloning sites or at least one restriction enzyme site.It needs to express
When target gene, target gene is connected into suitable multiple cloning sites or restriction enzyme site, thus the purpose base that is operably connected
Cause and promoter.
In another preferred embodiment, the recombinant vector includes: promoter, target gene on the direction 5' to 3'
And terminator.If desired, the recombinant vector can also include following elements: protein purification label；The acidification of 3' polymerized nucleoside
Signal；Untranslated nucleic acid sequence；Transhipment and targeting nucleic acid sequence；Selected marker (antibiotics resistance gene, fluorescin etc.)；Increase
Hadron；Or operator.
In a preferred embodiment of the invention, the recombinant vector includes trp promoter Trp2, preferably
Ground, sequence are as follows:
CCATGGGCCGACATCATAACGGTTCTGGCAAATATTCTGAAATGAGCTGTTGACAATTAATCATCGAA
CTAGTTAACTAGTACGCAAGTTCACGTAAAAAGGGTATGTCGACGGCCGACATCATAACGGTTCTGGCAAATATTC
TGAAATGAGCTGTTGACAATTAATCATCGAACTAGTTAACTAGTACGCAAGTTCACGTAAAAAGGGTATCGATCAT ATG, SEQ ID NO.:6.
The method for being used to prepare recombinant vector is well known to those of ordinary skill in the art.Expression vector can be bacterium
Plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus or other carriers.In short, as long as it can
It replicates and stablizes in host, any plasmid and carrier can be used.
Those of ordinary skill in the art can contain promoter of the present invention and/or target gene using the building of well known method
The carrier of sequence.These methods include recombinant DNA technology in vi, DNA synthetic technology, In vivo recombination technology etc..
Expression vector of the invention can be used for converting host cell appropriate, so that host transcription purpose RNA or expression
Target protein.Host cell can be prokaryotic cell, such as Escherichia coli, Corynebacterium glutamicum, brevibacterium flavum, streptomycete
Belong to, Agrobacterium: or low eukaryocyte, such as yeast cells；Or higher eucaryotic cells, such as plant cell.This field is general
Technical staff is aware that how to select carrier and host cell appropriate.This field skill can be used with recombinant DNA conversion host cell
Routine techniques known to art personnel carries out.When host is prokaryotes (such as Escherichia coli), CaCl can be used2Method processing,
It can be carried out with electroporation.When host is eucaryote, following DNA transfection method: calcium phosphate precipitation can be selected, it is conventional
Mechanical means (such as microinjection, electroporation, liposome packaging).Conversion plant can also be used Agrobacterium-mediated Transformation or particle gun to turn
The methods of change, such as leaf disk method, rataria conversion method, bud infusion method etc..It can be with for the plant cell, tissue or organ of conversion
Plant is regenerated with conventional method, to obtain the plant of transgenosis.
Term " being operatively connected " refers to that the target gene that will prepare transcriptional expression is connected with a kind of usual manner of this field
Its control sequence is connected to be expressed.
The culture and destination protein fermenting and producing of engineering bacteria
After obtaining engineering cell, can culturing engineering cell under the suitable conditions, express gene order of the invention
Encoded albumen.According to the difference of host cell, culture medium used in culture can be selected from various conventional mediums, be suitable for
It is cultivated under conditions of host cell growth.After host cell growth is to cell density appropriate, (such as with suitable method
Temperature transition or chemical induction) promoter that induces selection, cell is further cultured for a period of time.Engineering cell can be quick benefit
With methanol type (Mut+) or at a slow speed utilize methanol type (Muts)。
In the present invention, conventional fermentation condition can be used.Representative condition includes (but being not limited to):
(a) for temperature, the fermentation of cyanalcohol lyases and inducing temperature are maintained at 25-35 DEG C；
(b) for the pH value of induction period, induction period pH is controlled in 3-9；
(c) for dissolved oxygen (DO), DO control can use oxygen/air mixed gas in 20-90%, the maintenance of dissolved oxygen
It is passed through to solve；
(d) for feed supplement, feed supplement type preferably includes the carbon sources such as glycerol, methanol, glucose, can individually feed supplement or mixing be mended
Material；
(e) for induction period IPTG concentration, conventional induced concentration can be used in the present invention, and usual iptg concentration control exists
0.1-1.5mM；
(f) it for induction time, is not particularly limited, usually 2-20 hours, preferably 5-15 hours.
That there are Bacillus coli cells is intracellular for purpose of the present invention albumen cyanalcohol lyases, and it is thin to collect host by centrifuge
Then born of the same parents are crushed host cell by high pressure, machine force, lysed cells quilt or other method of cell disruption, discharge recombinant protein,
Preferably high-pressure process.Host cell lysis liquid can by saltouing, the methods of ultrafiltration carry out carrying out chromatographing again after preliminary purification it is pure
Change, can also directly carry out chromatographic purifying.
Chromatographic technique includes cation-exchange chromatography, anion-exchange chromatography, gel permeation chromatography, hydrophobic chromatography, affine
The technologies such as chromatography.Commonly chromatography method includes:
1. anion-exchange chromatography:
Anion-exchange chromatography medium includes but is not limited to: Q-Sepharose, DEAE-Sepharose.If fermentation
The salinity of sample is higher, the combination of influence and Ion Exchange Medium, then needs to reduce salinity before carrying out ion-exchange chromatography.
Sample can be balanced the replacement of buffer with means such as dilution, ultrafiltration, dialysis, gel permeation chromatographies, until with corresponding
Ion exchange column equilibrium liquid system is similar, then loading, carries out salinity or the gradient elution of pH.
2. hydrophobic chromatography:
Hydrophobic chromatoghaphy medium includes but is not limited to: Phenyl-Sepharose, Butyl-Sepharose, Octyle-
Sepharose.Sample passes through addition NaCl, (NH4)2SO4Etc. modes improve salinity, then loading, passing through reduces salinity side
Method elution.The foreign protein that hydrophobicity has larger difference is removed by hydrophobic chromatography.
3. gel permeation chromatography
Hydrophobic chromatoghaphy medium includes but is not limited to: Sephacryl, Superdex, Sephadex class.Pass through gel filtration
Chromatography replacement buffer system, or it is further consummate.
4. affinity chromatography
Affinity chromatography medium includes but is not limited to: HiTrapTM Heparin HP Columns。
Prepare combination of enzyme preparations object
The present invention also provides a kind of combination of enzyme preparations object, cracked in the combination of enzyme preparations object comprising cyanalcohol of the invention
Enzyme.
Combination of enzyme preparations object of the invention can also include: citric acid, and/or acetic acid.
The preparation method of S- cyanalcohol
The present invention also provides a kind of preparation method of S- cyanalcohol, the method includes the steps:
(1) the cyanalcohol lyases of mutation of the invention is contacted with reaction substrate, carries out catalysis reaction, thus described in generating
S- cyanalcohol；
(2) it separates and purifies the S- cyanalcohol product.
Be preferably carried out in mode in the present invention, in the step (1), the reaction substrate be 3-phenoxy-benzaldehyde,
With acetone cyanohydrin (or, hydrocyanic acid).
It is preferably carried out in mode in the present invention, in the step (1), the temperature for being catalyzed reaction is 0-20 DEG C.
Main advantages of the present invention are:
(1) the cyanalcohol lyases of mutation of the invention, catalytic reaction activity are much higher than the cyanalcohol lyases of wild type, are wild
Twice or so of raw type；
(2) polynucleotide sequence for encoding the cyanalcohol lyases of mutation of the invention, table high in expression in escherichia coli amount
Up to stabilization, the cost for preparing cyanalcohol lyases can be significantly reduced.
(3) using the cyanalcohol lyases of preparation mutation of the invention, the period is short, at low cost, is suitable for industrialized production.
Combined with specific embodiments below, the further old present invention in detail.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.The experimental method of detailed conditions is not specified in the following example, usually according to conventional strip
Part such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory
Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and
Number is calculated by weight.Experimental material used in following embodiment and reagent can obtain unless otherwise instructed from commercially available channel
.
The building of 1 mutant of embodiment
It is inclined according to the codon of Escherichia coli according to the protein sequence 1 (SEQ ID NO.:1) of the MeHNL of Wajant report
Good property has synthesized MeHNL DNA sequence dna 2 (SEQ ID NO.:2), is cloned into plasmid pET28 (purchased from Invitrogene company)
The site NdeI-HindIII.Using the plasmid as template, design primer is shown in sequence 3 and sequence 4.Using Random Mutagenesis Kit,
By changing MnSO4Concentration carries out multiple reactions, so that 1 to 3 point mutation is introduced MeHNL gene, makes MeHNL enzyme amino
1-3 amino acid is replaced in acid sequence.For amplification coding MeHNL enzyme, respectively with primer SEQ ID No.3 and SEQ ID
No.4 is as forward and reverse primer.Two primers, which all contain, passes through fixed point recombinant clone with Gateway Technology is used
The compatible site of the PCR amplification MeHNL genetic fragment of acquisition.The gel electrophoresis spectrum of pcr amplification product is as shown in Figure 1, obtain
Amplified production is consistent with expection.
Fallibility PCR amplification uses following temperature program(me)s: 94 DEG C of 2min, and 25 of 94 DEG C of 30s and 68 DEG C of 1min recycle, then
68℃10min.Fallibility PCR fragment is cloned into pDONR carrier (purchased from Invitrogene company) first, preparation is extensive
PENTR (being purchased from Invitrogene company) plasmid library, originates more than 20,000 bacterium colonies.Then using pDEST14 as carrier,
PENTR introduction plasmid library is configured to expression library.Then expression library is transferred to Competent E.coil BL21Star
(DE3) (Invitrogene company is purchased from), for expressing the MeHNL gene of mutation.
Convert e. coli jm109 (DE3), the transformant of obtained Kana resistance, be seeded to LB culture medium 37 DEG C into
Row culture.When between OD600=0.6-1.0, IPTG to final concentration 0.1mM is added, is cooled to 25-30 DEG C, it is small to continue culture 16
When, thalline were collected by centrifugation by 4000rpm.
Enzyme activity determination: method (the Analytical Biochemistry 166 (1987), 208- reported with reference to Selmar
211), 3-phenoxy-benzaldehyde 10mM, methanol 20uL, 20mM citrate buffer solution (pH5.0), acetone cyanohydrin 50mM, crude enzyme liquid
10uL.Above-mentioned reaction solution is incubated in 25 degree, and above-mentioned reaction solution is incubated in 25 degree, respectively in the absorbance of 1-5min measurement 250nm
Variation.Catalysis generates enzyme amount required for 1umole 3-phenoxy-benzaldehyde and is defined as 1 enzyme-activity unit in per minute.Variation speed
The most fast as highest mutant of enzyme activity of rate.
Testing result as shown in Fig. 2, ■ be mutant, ▲ be wild type, ◆ be check experiment (not enzyme).Mutant enzyme
Work/wild type enzyme activity=(0.2299-0.086)/(0.1577-0.086) * 100%=200.6%.
As can be seen from the figure the activity of mutant will be significantly higher than the activity of wild type, and catalytic activity is wild type
200%.The saltant type that sequencing obtains the high activity is sequence 5 (SEQ ID NO.:5).
The primer sequence is as follows in the present embodiment:
Forward primer: 5 '-GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT CGA AGG AGA TAG
AAC CAT GGT GAC CGC CCA TTT C-3'SEQ ID NO.:3；
Reverse primer: 5 '-GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC TTA AGC ATA AGC
ACG GCC-3’SEQ ID NO.:4。
The building of 2 strain of embodiment and high density fermentation
Synthesis contains the trp promoter of sequence 6 (SEQ ID NO.:6), and is connected to the NcoI and NdeI of pET28a
Then the coded polynucleotide of sequence 1 and 5 in embodiment 1 is connected to the site NdeI-XhoI above-mentioned respectively, obtained by site
To escherichia coli plasmid pTrp2-MeHNL1 and pTrp2-MeHNL5 (plasmid map such as Fig. 4 containing series connection trp promoter
It is shown).Plasmid is converted into e. coli jm109 (being purchased from Invitrogene company), is obtained in Kana resistant panel corresponding
Strain is inoculated into LB culture medium, and 37 DEG C are incubated overnight, and saves strain with 20% glycerol.
Strain is inoculated in the 1L shaking flask equipped with 200mL LB culture medium, in 37 DEG C, 180-220rpm cultivates 10-16h.
Above-mentioned cultured seed is inoculated on 3L (glucose 4g/L, phosphorus in tank fermentation medium (M9) in the ratio of 10% (v/v)
Sour disodium hydrogen 12.8g/L, potassium dihydrogen phosphate 3g/L, ammonium chloride 1g/L, sodium sulphate 0.5g/L, calcium chloride 0.0152g/L, six water chlorine
Change magnesium 0.41g/L.), it is cultivated under conditions of 25-35 DEG C, 300-800rpm, air mass flow 2-6L/min.After cultivating 6-10h,
Add the supplemented medium containing 60% glycerol with the rate stream of 5-20mL/h, continues to fermentation ends.Flow feeding culture radix is small
Up to OD600When reaching 80-100, tank is put, thalline were collected by centrifugation by 5000rpm.Enzyme activity, respectively 73U/mg are measured after cellular lysate
Crude protein and 143U/g crude protein.Detected through gel electrophoresis is consistent with expection.
The immobilization (cross-linked enzyme aggregate, CLEA) of embodiment 3MeHNL
The Escherichia coli 50g wet thallus containing sequence 1 and sequence 5 is taken respectively, is resuspended in 200mL citrate buffer solution
(50mM, pH 5.5), carrying out ultrasonic bacteria breaking, 14000rpm centrifugation 30min collect supernatant, ammonium sulfate solids are added to final concentration
60%, 15min is stirred on ice bath, and precipitating is collected by centrifugation in 14000rpm.Under ice bath stirring, albumen ammonium sulfate precipitation is added 10%
Precipitating is collected by centrifugation in the aqueous solution of glutaraldehyde, respectively obtains 4.1g (sequence 1) and 4.5g (sequence 5) immobilization MeHNL.
The use (pure organic phase) of embodiment 4-5 immobilised enzymes
In 100mL MTBE (methyl tert-butyl ether, methyl tertiary butyl ether(MTBE)) (embodiment 4) or isopropyl ether
In (embodiment 5), addition 10mL 3-phenoxy-benzaldehyde, 2.5g MeHNL immobilised enzymes (sequence 5), 5mL acetone cyanohydrin, 20 DEG C
It is stirred to react, mixing speed 400-500rpm.It reacts sampling in 4 hours and detects reaction conversion ratio with HPLC, fixation is recovered by filtration
Change enzyme, puts into next batch.The case where reuse, is shown in Table 1.
Note: Δ is saltant type；WT is wild type.Liquid chromatographic detection, conversion ratio calculation be, conversion ratio=product/
(product+substrate) * 100%.
The use (water+organic phase) of embodiment 6-7 immobilised enzymes
In 50mL citric acid solution (50mM, pH5), 10mL 3-phenoxy-benzaldehyde is added, 5g MeHNL (sequence 1) is solid
Surely change enzyme, 5mL hydrocyanic acid is separately added into 50mL MTBE (embodiment 6) or 50mL isopropyl ether (embodiment 7), and 20 DEG C of stirrings are anti-
It answers, mixing speed 400-500rpm.Sampling detects conversion ratio with HPLC after reaction 4 hours, after reaction was completed, is recovered by filtration solid
Surely change enzyme, put into next batch.The case where reuse, is shown in Table 2.
Table 2
According to embodiment 4-7's as a result, currently preferred reaction system be MTBE organic phase；Reaction condition are as follows: CLEA
Immobilised enzymes is catalyst, and MTBE is solvent, and substrate is 3-phenoxy-benzaldehyde, acetone cyanohydrin (or, hydrocyanic acid), 20 DEG C of reactions
3-5 hours, mixing speed 400-500rpm.In reaction system 3-phenoxy-benzaldehyde concentration be 0.1-1.2 mol/L, third
Ketone cyanalcohol concentration is 0.2-2 mol/L), and the concentration of acetone cyanohydrin is 1.5-3 times of 3-phenoxy-benzaldehyde concentration.
9 analysis detection of embodiment
Use the monitoring reaction of high performance liquid chromatography (HPLC) method: with water and acetonitrile (45:55) for mobile phase, chromatographic column is
ODS-18 reversed-phase column, Shimadzu LC-15C high performance liquid chromatography detect UV absorption under 210nm；Reaction system water and acetonitrile
(45:55) is diluted, sample detection after being centrifuged and being filtered with nylon membrane.In the present invention preferably reaction system, HPLC inspection
Survey reaction process (Fig. 3): after reaction 1 hour, detection, 17.3min is 3-phenoxy-benzaldehyde, and 17.5min is S- configuration cyanalcohol
Chiral purity is analyzed using 1260 liquid chromatogram of Agilent, testing conditions are as follows: Chiralpak AD-H column,
N-hexane: ethyl alcohol (0.1%DEA)=90:10,0.8mL/min, Detection wavelength 220nm.By comparing S- produced by the present invention
The product and target substance standard items (being purchased from Jiangxi Science Court Bioceuticals Inc.) of configuration are consistent.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Claims (12)
1. a kind of cyanalcohol lyases of mutation, which is characterized in that the amino acid sequence such as SEQ of the cyanalcohol lyases of the mutation
Shown in ID NO.:5.
2. a kind of polynucleotide molecule, which is characterized in that the polynucleotides are selected from the group:
(a) polynucleotides of the polypeptide as shown in SEQ ID NO.:5 are encoded；
(b) sequence polynucleotides as shown in SEQ ID NO.:7；
(c) homology >=95% of nucleotide sequence and sequence shown in SEQ ID NO.:7, and encode more shown in SEQ ID NO.:5
The polynucleotides of peptide.
3. a kind of carrier, which is characterized in that the carrier contains polynucleotide molecule as claimed in claim 2.
4. carrier as claimed in claim 3, which is characterized in that the carrier further includes trp promoter Trp2.
5. a kind of host cell, which is characterized in that the host cell contains carrier or chromosomal integration as claimed in claim 3
Polynucleotide molecule described in having the right to require 2.
6. a kind of method for the cyanalcohol lyases for preparing mutation described in claim 1, which is characterized in that comprising steps of
(i) under the suitable conditions, host cell described in claim 5 is cultivated, to give expression to the cyanalcohol of the mutation
Lyases；With
(ii) the cyanalcohol lyases of the separation mutation.
7. a kind of enzyme preparation, which is characterized in that the enzyme preparation includes the cyanalcohol lyases of mutation described in claim 1.
8. enzyme preparation as claimed in claim 7, which is characterized in that the enzyme preparation further includes selected from the group below one or more
Component: citric acid, malic acid and succinic acid.
9. a kind of purposes of the cyanalcohol lyases of mutation described in claim 1, which is characterized in that for m-phenoxy benzene first
Aldehyde is that substrate prepares S- cyanalcohol product with optical activation.
10. a kind of purposes of enzyme preparation as claimed in claim 7, which is characterized in that for using 3-phenoxy-benzaldehyde as substrate
Prepare S- cyanalcohol product with optical activation.
11. a kind of method for preparing S- cyanalcohol, which is characterized in that comprising steps of
(1) the cyanalcohol lyases of mutation described in claim 1 is contacted with reaction substrate, carries out catalysis reaction, to generate
The S- cyanalcohol；
(2) it separates and purifies the S- cyanalcohol product；Also, in the step (1), the reaction substrate is m-phenoxy benzene first
Aldehyde.
12. the method for preparing S- cyanalcohol as claimed in claim 11, which is characterized in that in the step (1), be catalyzed reaction
Temperature is 0-20 DEG C.
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| CN108277215A (en) * | 2017-01-06 | 2018-07-13 | 上海弈柯莱生物医药科技有限公司 | A kind of high activity S- cyanalcohols lyases and its application |
| CN113528498B (en) * | 2021-08-12 | 2022-05-03 | 江西科苑生物股份有限公司 | Preparation method of S-cyanohydrin lyase and product thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1203908A (en) * | 1998-05-04 | 1999-01-06 | 中国科学院上海有机化学研究所 | Catalytic synthesis of optical active cyanhydrin compound by using cyanhydrin catenase |
| CN101611141A (en) * | 2006-12-14 | 2009-12-23 | 帝斯曼知识产权资产管理有限公司 | R-HNL random variants and be used to prepare the purposes of optically pure sterically hindered cyanohydrins |
-
2015
- 2015-08-07 CN CN201510481026.7A patent/CN106032531B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1203908A (en) * | 1998-05-04 | 1999-01-06 | 中国科学院上海有机化学研究所 | Catalytic synthesis of optical active cyanhydrin compound by using cyanhydrin catenase |
| CN101611141A (en) * | 2006-12-14 | 2009-12-23 | 帝斯曼知识产权资产管理有限公司 | R-HNL random variants and be used to prepare the purposes of optically pure sterically hindered cyanohydrins |
Non-Patent Citations (2)
| Title |
|---|
| Identification of potential active-site residues in the hydroxynitrile lyase from Manihot esculenta by site-directed mutagenesis;Harald Wajant and Klaus Pfizenmaier;《J Biol Chem》;19961018;25830-25834 * |
| Substrate specificity of mutants of the hydroxynitrile lyase from Manihot esculenta;Bühler H et al.;《Chembiochem》;20030303;211-216 * |
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