CN110396505A - Ketone group pantoic acid lactone reductase and its application - Google Patents
Ketone group pantoic acid lactone reductase and its application Download PDFInfo
- Publication number
- CN110396505A CN110396505A CN201910366828.1A CN201910366828A CN110396505A CN 110396505 A CN110396505 A CN 110396505A CN 201910366828 A CN201910366828 A CN 201910366828A CN 110396505 A CN110396505 A CN 110396505A
- Authority
- CN
- China
- Prior art keywords
- ketone group
- acid lactone
- pantoic acid
- reductase
- lactone
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Abstract
The present invention relates to technical field of microbial genetic engineering, more specifically to a kind of completely new from the ketone group pantoic acid lactone reductase of fission yeast and its application.The ketone group ketopantoate reductase has excellent enzymatic activity to ketone group pantoic acid lactone, can be catalyzed ketone group pantoic acid lactone asymmetric reduction and generate D-pantoyl lactone.The present invention also constructs the double enzyme reaction systems of a bacterium, the ketone group pantoic acid lactone reductase and glucose dehydrogenase coupling that double enzyme recombinant cell inductions generate, catalysis substrate ketone group pantoic acid lactone asymmetric reduction generates D-pantoyl lactone, by the dehydrogenation of auxiliary substrate glucose by oxidized coenzyme NADP+It is reduced into NADPH, to realize that coenzyme recycles.Since the catalytic efficiency of glucose dehydrogenase is much higher than ketone group pantoic acid lactone reductase, reduction reaction can be promoted to be carried out at high speed, greatly improve reaction efficiency, reduce cost.
Description
Technical field
The present invention relates to technical field of microbial genetic engineering, are originated from fission yeast more particularly, to one kind
The ketone group pantoic acid lactone reductase of (Schizosaccharomyces pombe) and its application.
Background technique
D-VB5 calcium is also known as vitamin B5, is the component part of coacetylase, have been widely used for medicine, food, feed and
The industries such as cosmetics.D-pantoyl lactone is the important source material for synthesizing D-VB5 calcium.In industrialized production, chemical method is first passed through
DL- pantoic acid lactone is synthesized, recycles the D-pantoyl lactone in lactone hydrolase selective hydrolysis mixed pantoic acid lactone raw
At Pantothenic acid, Pantothenic acid and L- pantoic acid lactone are then separated, it is general that the acidified cyclization of the Pantothenic acid after separation forms D-
Solution acid lactone, and the reuse after racemization of L- pantoic acid lactone.Therefore, hydrolyze the chiral separation of enzymatic although technique at
It is ripe, but still there are problems that longer, the soda acid consumption height of step etc..In consideration of it, exploitation is more direct, efficient, the general solution of D- of environmental protection
Acid lactone method of asymmetric synthesis substitutes existing chiral resolution technology will be with important application value.D-pantoyl lactone can
By oxidation-reduction method asymmetric syntheses, this method includes two kinds of approach, the first is using the DL- pantoic acid lactone of mixed the bottom of as
Object is generated in ketone group pantoic acid using the dehydrogenase catalyzed L- pantoic acid lactone dehydrogenation of the single-minded L- pantoic acid lactone of stereoselectivity
Ester, then ketone group pantoic acid lactone is asymmetric in the case where D- ketone group pantoic acid lactone restores enzymatic generates D-pantoyl lactone；The
Two kinds of approach are also first to generate ketone group pantoic acid lactone with the dehydrogenase catalyzed L- pantoic acid lactone dehydrogenation of L- pantoic acid lactone, then
Ketone group pantoic acid lactone spontaneous hydrolysis forms ketone group pantoic acid, and it is general that D- is then generated under the action of D- ketone group ketopantoate reductase
Solution acid, then closed loop forms D-pantoyl lactone to Pantothenic acid under the action of an acid.The process is more simple and direct with first access,
Compared with existing hydrolysis enzymatic access, technique is more simple, and the substrate of mixed directly obtains optical voidness through biocatalysis and produces
Object, does not need racemization step, does not need the separating step of lactone and acid yet；The coenzyme circulatory system is constructed in genetic engineering bacterium,
Additional coenzyme can not be needed；The purification procedures of enzyme are not needed using genetic engineering bacterium as intact cell catalysis agent.Therefore, it aoxidizes
The method of reductase asymmetric syntheses D-pantoyl lactone is a kind of very promising biological hydrolysis enzyme process replacer.
The step of ketone group pantoic acid lactone asymmetric reduction generation D-pantoyl lactone is its key step in oxidation-reduction method
One of rapid, D- ketone group pantoic acid lactone reductase is the key enzyme for being catalyzed the reaction.Existing research report, from saccharomyces cerevisiae gram
Grand to express 2 ketone groups pantoic acid lactone reductase SceCPR1 and SceAKR3C1, wherein SceAKR3C1 belongs to aldehyde ketone reductase
Family (Zhao M, Gao L, Zhang L, et al.Asymmetric reduction of ketopantolactone
using a strictly(R)-stereoselective carbonylreductase through efficient NADPH
regeneration and the substrate constant-feeding strategy.Biotechnology
Letters,2017,39:1741-1746).Microorganism aldehyde ketone reductase is the main composition of aldehyde ketone reductase family, usually
NADP (H) specificity dependent form contains highly conserved (α/β) 8 barrel-like structure.Microorganism aldehyde ketone reductase has the rich of source
The characteristics such as the diversity of richness and catalysis substrate have in terms of enzymatic clarification chiral alcohol as biocatalyst and successfully answer
With.The enzyme with D- ketone group pantoic acid lactone reductase vitality is excavated from microorganism aldehyde ketone reductase family, is one feasible
Strategy.
Currently, there is not yet the aldehyde ketone reductase for being originated from fission yeast has the report of D- ketone group pantoic acid lactone reductase vitality
Road also has no that the aldehyde ketone reductase of fission yeast is used for the report of D-pantoyl lactone asymmetric syntheses.
Summary of the invention
The purpose of the present invention is to provide a kind of completely new ketone group pantoic acid lactone reductases and its application.
First aspect present invention provides the ketone group pantoic acid lactone reductase of separation, the ketone group pantoic acid lactone reduction
Enzyme source is in fission yeast, and amino acid sequence is as shown in SEQ ID No:3.
Another aspect of the present invention, provides the polynucleotides of separation, and the polynucleotides are the coding ketone group pantoic acid
The polynucleotides of lactone reductase.
In a preferred embodiment, the nucleotide sequence of the polynucleotides is selected from the group: (i) SEQ ID No:1 or SEQ
Nucleotide sequence shown in ID No:2；(ii) nucleotide sequence complementary with the nucleotide sequence described in (i).
Another aspect of the present invention, provides a kind of carrier, and the carrier contains the polynucleotides.
Another aspect of the present invention, provides a kind of recombinant cell, and the recombinant cell contains in the carrier or genome
It is integrated with the polynucleotides.
Another aspect of the present invention provides the preparation method of the ketone group pantoic acid lactone reductase, comprising:
(a) Fiber differentiation is carried out to the recombinant cell；And
(b) the ketone group pantoic acid lactone reductase is separated from culture.
Another aspect of the present invention, the ketone group pantoic acid lactone reductase are applied to catalysis and generate D-pantoyl lactone
In reaction.
Another aspect of the present invention, provides a kind of double enzyme recombinant cells, and double enzyme recombinant cell inductions generate the ketone
Base pantoic acid lactone reductase and glucose dehydrogenase.
In a preferred embodiment, the glucose dehydrogenase derives from Exiguobacterium sp, amino acid sequence such as SEQ ID
Shown in No:5.
Further, the polynucleotide sequence for encoding the glucose dehydrogenase is selected from the group: (i) SEQ ID No:4 institute
The nucleotide sequence shown；(ii) nucleotide sequence complementary with the nucleotide sequence described in (i).
Another aspect of the present invention provides a kind of construction method of double enzyme recombinant cells, comprising:
The polynucleotides of the coding ketone group pantoic acid lactone reductase are inserted into first vector, obtain the first recombination load
The polynucleotides of the coding glucose dehydrogenase are inserted into Second support, obtain the second recombinant vector by body；And
First recombinant vector and the second recombinant vector are imported into host cell, obtain double enzyme recombinant cells.
In a preferred embodiment, the first vector is pET-28b, and the Second support is pACYCDuet-1, the place
Chief cell is E.coli BL21 (DE3).
Another aspect of the present invention, provides a kind of method for preparing D-pantoyl lactone, and the substrate supplemented by glucose utilizes
In the ketone group pantoic acid lactone reductase and glucose dehydrogenase catalysis ketone group pantoic acid that double enzyme recombinant cell inductions generate
Ester generates D-pantoyl lactone.
In a preferred embodiment, the ketone group pantoic acid lactone and glucose are added in reaction system with fed-batch mode carries out
Reaction.
In another preferred example, the reaction temperature of the catalysis reaction is 25~50 DEG C, and pH is 4.5~7.5.
In another preferred example, the initial molar ratio of the ketone group pantoic acid lactone and glucose is 1:1~1:2.5.
Compared with prior art, the present invention beneficial effect is mainly reflected in: the present invention provides a kind of high vigor ketone group is general
Acid lactone reductase, encoding gene, carrier and recombinant cell are solved, the ketone group pantoic acid lactone reductase is to ketone group pantoic acid lactone
With excellent enzymatic activity, ketone group pantoic acid lactone asymmetric reduction can be catalyzed and generate D-pantoyl lactone.In addition, of the invention
A kind of double enzyme recombinant cells are additionally provided, the double enzyme reaction systems of a bacterium are constructed, the ketone group that double enzyme recombinant cell inductions generate is general
Acid lactone reductase and glucose dehydrogenase coupling are solved, catalysis substrate ketone group pantoic acid lactone asymmetric reduction generates Pantothenic acid
Lactone, meanwhile, by the dehydrogenation of auxiliary substrate glucose by oxidized coenzyme NADP+It is reduced into NADPH, to realize that coenzyme follows
Ring.Since the catalytic efficiency of glucose dehydrogenase is much higher than ketone group pantoic acid lactone reductase, reduction reaction high speed can be promoted
It carries out, greatlys improve reaction efficiency.The present invention also passes through the low rate constant speed fed-batch mode of substrate and auxiliary substrate, solves bottom
The problem of object spontaneous hydrolysis, and optimize reaction system, it is determined that optimal reaction condition.Ketone group pantoic acid proposed by the present invention
Lactone asymmetric reduction reaction adds to react with the constant speed stream that glucose dehydrogenation reaction is mutually coupled can greatly improve reduzate
The yield of D-pantoyl lactone, and avoid the additional addition of coenzyme.In 10mL catalyst system, the glucose of 1M is added,
500mM ketone group pantoic acid lactone can obtain final product D-pantoyl lactone 470mM, obtain after the constant speed stream of 10h adds catalysis to react
For rate up to 94%, product e.e. value is greater than 99.9%.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
The embodiment of invention for those of ordinary skill in the art without creative efforts, can also basis
The attached drawing of offer obtains other attached drawings；
Fig. 1 is the reaction principle figure that enzymatic ketone group pantoic acid lactone of the present invention generates D-pantoyl lactone；
Fig. 2 is that the SDS-PAGE of fission yeast ketone group pantoic acid lactone reductase in the embodiment of the present invention 2 detects figure,
In: the correspondence of swimming lane 1 does not induce bacterium solution；The corresponding fission yeast ketone group pantoic acid lactone reductase after purification of swimming lane 2；
Fig. 3 is that the SDS-PAGE in the embodiment of the present invention 5 after double enzyme recombinant cell inducing expressions detects figure, in which: swimming lane 1
Double enzyme recombinant cells after corresponding Fiber differentiation；Double enzyme recombinant cells of the corresponding non-Fiber differentiation of swimming lane 2；
Fig. 4 is the gas phase color of D-pantoyl lactone, L- pantoic acid lactone, ketone group pantoic acid lactone in the embodiment of the present invention 6
Spectrogram；
Fig. 5 is influence of the different temperatures to catalysis reaction in the embodiment of the present invention 7, in which: corresponds to efficiency of pcr product；■ pairs
Answer substrate hydrolysis rate；DPL, D-pantoyl lactone；KPL, ketone group pantoic acid lactone；
Fig. 6 is influence of the difference pH to catalysis reaction in the embodiment of the present invention 7, in which: the corresponding efficiency of pcr product of and zero；■
With ● corresponding substrate hydrolysis rate；DPL, D-pantoyl lactone；KPL, ketone group pantoic acid lactone；
Fig. 7 is influence of the different catalysts additive amount to catalysis reaction in the embodiment of the present invention 7, in which: corresponds to product
Yield；■ corresponds to substrate hydrolysis rate；DPL, D-pantoyl lactone；KPL, ketone group pantoic acid lactone；
Fig. 8 is influence of the different coenzyme additive amounts to catalysis reaction in the embodiment of the present invention 7；DPL, D-pantoyl lactone；
KPL, ketone group pantoic acid lactone；
Fig. 9 is influence of the different auxiliary substrate additive amount to catalysis reaction in the embodiment of the present invention 7, in which: corresponds to product
Yield；■ corresponds to substrate hydrolysis rate；DPL, D-pantoyl lactone；KPL, ketone group pantoic acid lactone.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
Fig. 1~9 is please referred to, Fig. 1 is the reaction original that enzymatic ketone group pantoic acid lactone of the present invention generates D-pantoyl lactone
Reason figure；Fig. 2 is that the SDS-PAGE of fission yeast ketone group pantoic acid lactone reductase in the embodiment of the present invention 2 detects figure；Fig. 3 is this
SDS-PAGE in inventive embodiments 5 after double enzyme recombinant cell inducing expressions detects figure；Fig. 4 is that D- is general in the embodiment of the present invention 6
Solve the gas chromatogram of acid lactone, L- pantoic acid lactone, ketone group pantoic acid lactone；Fig. 5 is different temperatures in the embodiment of the present invention 7
Influence to catalysis reaction；Fig. 6 is influence of the difference pH to catalysis reaction in the embodiment of the present invention 7；Fig. 7 is the embodiment of the present invention
Influence of the different catalysts additive amount to catalysis reaction in 7；Fig. 8 is coenzyme additive amounts different in the embodiment of the present invention 7 to catalysis
The influence of reaction；Fig. 9 is influence of the different auxiliary substrate additive amount to catalysis reaction in the embodiment of the present invention 7.
The present invention provides a kind of ketone group pantoic acid lactone reductases, derive from fission yeast
(Schizosaccharomyces pombe) has excellent the enzyme activity to ketone group pantoic acid lactone, it is general can be catalyzed ketone group
It solves acid lactone and generates D-pantoyl lactone.
Present invention further teaches encode the polynucleotides of the ketone group pantoic acid lactone reductase, the load of the polynucleotides
Body and recombinant cell additionally provide a kind of ketone group pantoic acid lactone reductase in catalysis and generate the application in D-pantoyl lactone.
The present invention will be used for the regenerated enzyme of coenzyme NADP 11 and be integrated into recombinant cell, construct the double enzyme systems of a bacterium, thus
Coenzyme circulation is realized, the efficiency of catalysis reaction is greatly improved, reduces production cost.Present invention further teaches double enzyme weights
The construction method of group cell, and the side using double enzyme recombinant cell catalysis ketone group pantoic acid lactones generation D-pantoyl lactones
Method.
Ketone group pantoic acid lactone reductase, its coded polynucleotide, carrier and recombinant cell
Present invention discloses it is a kind of new, that there is the enzyme activity to ketone group pantoic acid lactone, can be catalyzed that generate D- general
Solve the ketone group pantoic acid lactone reductase of acid lactone.The ketone group pantoic acid lactone reductase derives from fission yeast
(Schizosaccharomyces pombe), amino acid sequence is as shown in SEQ ID No:3.
Enzyme of the invention can be natural, active peptides recombinantly or synthetically.The active peptides can be native purified
Product or chemically synthesized product, or using recombinant technique from protokaryon (for example, Escherichia coli) or eucaryon host (example
Such as, yeast, higher plant) in generate product.
Polynucleotides of the invention can be DNA form or rna form.The DNA can be single-stranded or double-strand.It is single-stranded
DNA can be coding strand or noncoding strand.Present invention discloses the polynucleotides of coding ketone group pantoic acid lactone reductase, preferably
Ground, the polynucleotide sequence is as shown in SEQ ID No:1 or SEQ ID No:2.
The polynucleotides of coding ketone group pantoic acid lactone reductase in the present invention usually can be by PCR amplification or artificial
Synthetic method obtains.
The invention also includes it is described coding ketone group pantoic acid lactone reductase polynucleotides variant, coding with
The polypeptide or polypeptide derivative of SEQ ID No:3 same amino acid sequence.The variant of the polynucleotides can be natural hair
The variant that raw variant or non-natural occur.The variant of the polynucleotides can be to be occurred by one or more bases
Replace, variant caused by missing and/or insertion.The polypeptide of the variant coding of the polynucleotides will not be lost to ketone group
The enzyme activity of pantoic acid lactone.
Enzyme and polynucleotides in the present invention preferably provide in a separate form, more preferably pass through certain purifying.
The invention also includes the carriers of the polynucleotides to pass through cloning vector reality such as cloning vector and expression vector
The duplication of existing correlated series, the expression of gene function is realized by expression vector.
The invention also includes will integrate in the recombinant cell or genome that generate after the vector introduction host cell
The recombinant cell of polynucleotides is stated, the recombinant cell is for expressing ketone group pantoic acid lactone reductase or derivative.The place
Chief cell can be prokaryote or eukaryotic cells, such as Escherichia coli, yeast etc..
It preferably, can to carry out codon first excellent by the polynucleotides of the coding ketone group pantoic acid lactone reductase
To change, the sequence after optimization is connect with carrier pET-28b, then is transferred to E. coli BL21 (DE3), by Fiber differentiation,
Efficiently express ketone group pantoic acid lactone reductase.
Ketone group pantoic acid lactone reductase generates the application in D-pantoyl lactone in catalysis
Ketone group pantoic acid lactone reductase in the present invention, which has for ketone group pantoic acid lactone to be catalyzed, to be generated in Pantothenic acid
The zymologic property of ester.Catalyst in reaction can be the ketone group pantoic acid lactone reductase by isolating and purifying, and be also possible to
By the recombinant cell of Fiber differentiation.When the recombinant cell is as catalyst, the wet thallus separated after culture can be, it can also
To be cell freeze-dried powder.Whether which kind of above-mentioned form, essence are all in the ketone group pantoic acid generated using recombinant cell
Ester reductase is catalyzed ketone group pantoic acid lactone.
Double enzyme recombinant cells, polynucleotides and double enzyme recombinant cell construction methods
It, being capable of inducing expression ketone group pantoic acid lactone reductase and auxiliary simultaneously present invention further teaches double enzyme recombinant cells
Help the regenerated enzyme of NADPH.Preferably, the auxiliary regenerated enzyme of NADPH is the glucose dehydrogenase from Exiguobacterium sp,
Amino acid sequence is as shown in SEQ ID No:5.
The invention also includes the polynucleotides for encoding the glucose dehydrogenase.Preferably, the polynucleotide sequence is such as
Shown in SEQ ID No:4；It further include the variant of the polynucleotides, coding and SEQ ID No:5 same amino acid sequence
Polypeptide or polypeptide derivative.The variant of the polynucleotides can be the change that the variant naturally occurred or non-natural occur
Allosome.The variant of the polynucleotides can be to be occurred to replace, caused by missing and/or insertion as one or more bases
Variant.
Present invention further teaches the construction methods of double enzyme recombinant cells, comprising:
The polynucleotides for encoding ketone group pantoic acid lactone reductase are inserted into first vector, obtain the first recombination load by S11
The polynucleotides for encoding glucose dehydrogenase are inserted into Second support, obtain the second recombinant vector by body.Preferably, described first
Carrier is pET-28b, and the Second support is pACYCDuet-1；
First recombinant vector and the second recombinant vector are imported host cell by S12, obtain double enzyme recombinant cells.Institute
Stating host cell can be prokaryote or eukaryotic cells, such as Escherichia coli, yeast etc..Preferably, the place
Chief cell is E.coli BL21 (DE3).
The method that double enzyme recombinant cell catalysis generate D-pantoyl lactone
The invention also includes a kind of method for preparing D-pantoyl lactone, the ketone group generated using the induction of double enzyme recombinant cells
Pantoic acid lactone reductase and glucose dehydrogenase catalysis ketone group pantoic acid lactone generate Pantothenic acid lactones.Reaction principle is as schemed
Shown in 1, substrate is ketone group pantoic acid lactone, and auxiliary substrate is glucose, and NADPH is hydrogen donor, and glucose dehydrogenase constantly will
NADP+It is converted into NADPH, while convert glucose is gluconic acid.This method will be aoxidized by the dehydrogenation of auxiliary substrate glucose
Type coenzyme NAD P+It is reduced into NADPH, to realize that coenzyme recycles, not only increases catalysis reaction efficiency, and can be greatly
Reduce production cost.
Ketopantoic acid lactone easily hydrolyzes, in order to reduce spontaneous hydrolysis of the substrate in catalytic process, substrate and auxiliary substrate
Addition use constant speed fed-batch model.In order to avoid auxiliary substrate glucose is oxidized to catalyst system acidity caused by gluconic acid
Variation, the Na that reaction system is preferably used with 1M2CO3The mode of titration control pH value is catalyzed.Constant speed stream adds the mode of control pH to make
Catalytic efficiency greatlys improve.
According to experimental verification, the reaction temperature for being catalyzed reaction is 25~50 DEG C, and pH is 4.5~7.5.Substrate ketone group pantoic acid
The initial molar ratio of lactone and auxiliary substrate glucose is 1:1~1:2.5.
In order to further illustrate the present invention, with reference to embodiments to ketone group pantoic acid lactone reductase provided by the invention
And its application is described in detail.In the following examples, the experimental methods for specific conditions are not specified, usually leads according to molecular biosciences
Domain routine experiment method carries out, such as J. Pehanorm Brooker is write, Molecular Cloning:A Laboratory guide, the third edition, Science Press,
Experimental method described in 2002, or according to experimental method proposed by manufacturer.
The building of 1 fission yeast ketone group pantoic acid lactone reductase gene engineering bacteria of embodiment and expression
Fission yeast ketone group pantoic acid lactone reductase gene KPR is with fission yeast (Schizosaccharomyces
Pombe, derive from Chinese industrial Microbiological Culture Collection administrative center, bacterium numbering: CICC 1056) genomic DNA be mould
Plate utilizes the primers F 1 (nucleotide sequence is as shown in SEQ ID NO:6) and R1 (nucleotide sequence such as SEQ ID NO:7 of design
It is shown) PCR amplification is carried out, amplification system is shown in Table 1.PCR reaction process are as follows: 95 DEG C of initial denaturation 2min；Later, become with 95 DEG C
Property 20sec, 55 DEG C of renaturation 20sec, 72 DEG C of holding 20sec be a circulation, repeat circulation 35 times in this way；Finally, 72 DEG C of holdings
5min.PCR product is detected and is recycled through 1% agarose gel electrophoresis.PCR product and carrier pEASY-Blunt E1 after purification
It is connected, connection product is transferred in Escherichia coli Trans1-T1 competent cell.It is detected using colony polymerase chain reaction (PCR) method and determines the positive
Clone extracts plasmid from positive clone molecule.Target gene is sequenced, nucleotide sequence is as shown in SEQ ID NO:1.
1 pcr amplification reaction system of table
Containing one that excessive rare codon is considered as low expression level and the incomplete product of generation in target gene
Reason.It finds that the objective gene sequence contains 15 rare codons altogether through Rare Codon Caltor analysis, passes through codon
Optimization software Codon adaptation tool (http://www.jcat.de/) carries out codon optimization, and entrusts through Shanghai
JaRa bioengineering (GENEray Biotechnology) Co., Ltd synthesizes the ketone group pantoic acid lactone after codon optimization also
Nitroreductase gene, nucleotide sequence is as shown in SEQ ID NO:2, the corresponding amino acid sequence such as SEQ ID NO:3 institute for expressing albumen
Show.By after optimization ketone group pantoic acid lactone reductase gene insertion pET-28b expression vector on, insertion point be Nde I with
BamH I, construction recombination plasmid pET-28b-KPR.Recombinant plasmid pET-28b-KPR is transferred to 100 μ L E.coli BL21
(DE3) in competent cell, recombinant bacterium E.coli BL21 (DE3)/pET-28b-KPR is constructed.By recombinant bacterium E.coli BL21
(DE3)/pET-28b-KPR is coated in the LB resistant panel containing 100 μ g/mL kanamycins, overnight incubation in 37 DEG C of incubators.
Picking positive clone molecule is inoculated in LB liquid medium of the 50mL containing 100 μ g/mL kanamycins, in 37 DEG C and 200rpm condition
Lower culture 12h, as seed liquor.Then expand culture by 1.5% inoculum concentration of volumetric concentration and be seeded to 150mL containing 100 μ g/mL cards
In the LB liquid medium of that mycin, cultivated under the same conditions to bacterial concentration (OD600) be 0.6~0.8 when, addition induction
Agent IPTG (isopropyl-beta D-thio galactopyranoside) to final concentration of 0.1mM continues to cultivate under 19 DEG C and 140rpm
14h overexpression destination protein, medium centrifugal collect wet thallus.Furthermore it is also possible to which wet thallus is cold under the conditions of -80 DEG C
Dry 48h is lyophilized, is prepared into cell freeze-dried powder.
2 fission yeast ketone group pantoic acid lactone reductase of embodiment isolates and purifies
The wet thallus collected in embodiment 1 is added into 15mL 50mM Tris-HCl buffer (pH8.0) by 1g wet thallus
Appropriate Tris-HCl (pH 8.0) buffer is added in ratio, at 500W after ultrasonication 20min (work 2s, interval 6s), breaks
Broken liquid is centrifuged 10min under 4 DEG C and 10000rpm, and repeated centrifugation obtains supernatant crude enzyme liquid afterwards three times.
According to Ni-NTA metal chelate affinity chromatography operation instruction, take supernatant crude enzyme liquid loading to pre-equilibrating Ni2+In column, then
Successively with imidazoles containing 5mM, 40mM imidazoles, 100mM imidazoles, 250mM imidazoles elution buffer (imidazoles of respective concentration and
The sodium chloride of 300mM is dissolved in the Tris-HCl buffer of 50mM, pH 8.0) elute foreign protein and destination protein.Purpose egg
After Bai Jing contains the elution buffer elution of 100mM imidazoles, it is concentrated by ultrafiltration with the Tris-HCl buffer of 50mM, pH 8.0
Desalination, resulting desalination enzyme solution, as fission yeast ketone group pantoic acid lactone reductase, enzyme activity are 250U/mg (according to embodiment
3 methods measurement)；The ultra-filtration conditions refer to that enzyme solution is added in the super filter tube of molecular cut off 10kDa, at 5000rpm
Centrifugation 20 minutes, then collects the enzyme solution of retention.
Crude enzyme liquid obtains the higher purpose fission yeast ketone group pantoic acid of purity through the purifying of Ni-NTA affinity protein purification
Lactone reductase is detected as single band through SDS- polyacrylamide gel electrophoresis (SDS-PAGE).Recombinating KPR is soluble egg
White, molecular size range is apparently 32kDa.Since the aim sequence after codon optimization does not design terminator codon, in conjunction with
Carrier sequence, destination protein size are about 36kDa, therefore are consistent with theory, as shown in Figure 2.
The enzyme activity determination of 3 fission yeast ketone group pantoic acid lactone reductase of embodiment
Enzyme activity determination method: standard enzyme vitality test system (2.5mL) contains respectively: 0.15mM NADPH, 0.6 μ g/mL
Fission yeast ketone group pantoic acid lactone reductase, (ketone group pantoic acid lactone is with pH2.0's for 10mM ketone group pantoic acid lactone
0.2MKH2PO4The form that HCl solution is formulated as 1M substrate solution is added, and substrate solution additional amount is with ketone group pantoic acid lactone
Meter, the final concentration of 10mM of ketone group pantoic acid lactone in reaction system), with 0.2MNa2HPO4-NaH2PO4Buffer (pH 6.0)
For reaction medium.The concentration refers both to the final concentration in measurement system.Reaction carries out at 55 DEG C, and NADPH and substrate are last
It is added, enzyme activity (the mole coefficient ε of NADPH is determined by the light absorption value variation per minute at 340nm of detection reaction system340
=6.22mM-1cm-1).Unit of activity (U) is defined as enzyme amount needed for consuming 1 μm of ol NADPH per minute.
The acquisition of 4 Exiguobacterium sp glucose dehydrogenase gene of embodiment
Utilize the published dehydrogenase coding genes from Exiguobacterium sp (Exiguobacterium sibiricum)
(GenBank accession number is ACB59697.1), after codon optimization, artificial synthesized (Suzhou Jin Weizhi biotechnology is limited
Company provides gene chemical synthesis service) Exiguobacterium sp glucose dehydrogenase encoding gene, nucleotide sequence such as SEQ ID NO:4 institute
Show.
By III site EcoR I and Hind of Exiguobacterium sp glucose dehydrogenase encoding gene insertion carrier pACYCDuet-1
Between, obtain recombinant plasmid pACYCDuet-1-EsGDH.
The building and inducing expression of 5 pairs of enzyme recombinant cells of embodiment
By the encoding gene (nucleosides of the fission yeast ketone group pantoic acid lactone reductase after the optimization obtained in embodiment 1
Acid sequence is as shown in SEQ ID NO:2) it is synthesized on carrier pET-28b, by the Exiguobacterium sp after the optimization obtained in embodiment 4
Glucose dehydrogenase encoding gene (nucleotide sequence is as shown in SEQ ID NO:4) is synthesized in carrier pACYCDuet-1, and will
Above-mentioned recombinant plasmid imports competent cell E.coli BL21 (DE3) jointly to get the reduction of fission yeast ketone group pantoic acid lactone
Enzyme and Exiguobacterium sp glucose dehydrogenase coexpression Recombinant organism, be named as E.coli BL21 (DE3)/
pET-28b-KPR/pACYCDuet-1-GDH.Genetically engineered E.coli BL21 (DE3)/pET 28b- containing recombinant vector
The construction and expression of KPR/pACYCDuet1-GDH is in addition to antibiotic used is chloramphenicol and kanamycins, other processes and reality
The construction and expression process for applying ketone group pantoic acid lactone reductase recombinant bacterium in example 1 is consistent.The wet thallus of preparation is in -80 DEG C of conditions
Lower freeze-drying 48h, it is spare to be prepared into cell freeze-dried powder.
Induced cultures are detected through SDS- polyacrylamide gel electrophoresis (SDS-PAGE), in fission yeast ketone group pantoic acid
Ester reductase and Exiguobacterium sp glucose dehydrogenase molecular size range are respectively 36kD and 29kD, are consistent with theory, such as Fig. 3 institute
Show.
6 pairs of enzyme recombinant cell catalysis ketone group pantoic acid lactones of embodiment generate D-pantoyl lactone
Catalystic converter system: total volume 5mL, respectively include: 100mM ketone group pantoic acid lactone (use by ketone group pantoic acid lactone
The form that water is formulated as 1M substrate solution is added, and substrate solution additional amount is with the meter of ketone group pantoic acid lactone, in reaction system
The final concentration of 100mM of ketone group pantoic acid lactone), 200mM glucose, 3% (w/v, similarly hereinafter) embodiment 5 preparation cell freeze-drying
Powder, with 0.2M phosphate buffer (pH 6.0) for reaction medium.
Catalysis reaction carries out in three neck round bottom flask, in the slow current adding substrate ketone group pantoic acid of the speed of 10 μ L/min
1M Na is added dropwise in ester and auxiliary substrate glucose2CO3Maintain reaction system pH constant, controlled at 45 DEG C, magnetic agitation is reacted.
Stream plus reaction 1h, are during which sampled every 15min.Reaction solution is extracted with ethyl acetate, and organic phase obtained is through anhydrous sulphur
After sour sodium is dry, substrate ketone group pantoic acid lactone and product D-pantoyl lactone are quantitative determined using gas chromatography.
Gas-chromatography (GC-2014) testing conditions of substrate ketone group pantoic acid lactone and product D-pantoyl lactone are as follows:
0.25 μm of μ m of chiral column BGB-174,30m × 250；Detector: FID, 250 DEG C；Carrier gas: N2, 1.0mL/min；Air mass flow:
400mL/min；Hydrogen flowing quantity: 40mL/min；Split ratio: 1: 30；Sample volume: 1 μ L；Injector temperature: 220 DEG C.Column temperature condition:
175 DEG C of holding 7min.As shown in figure 4, under above-mentioned chromatographic condition, D-pantoyl lactone, L- pantoic acid lactone, ketone group pantoic acid
Lactone appearance time is respectively 5.32min, 5.53min and 5.78min.
7 pairs of enzyme recombinant cell catalytic reaction condition optimizations of embodiment
Double enzyme recombinant cells recycle driving force as coenzyme using the glucose dehydrogenase for being originated from Exiguobacterium sp, to be originated from fragmentation
The ketone group pantoic acid lactone reductase of yeast is general as catalyst building enzyme process asymmetric reduction ketone group pantoic acid lactone generation D-
Solve the catalyst system (Fig. 1) of acid lactone.Catalyst system as described in Example 6 maintains substrate additive amount constant, by changing it
The additive amount of his each component and the condition of reaction, optimize catalystic converter system, finally determine optimal catalyst system.
The factor investigated in optimization process includes: reaction temperature (25~55 DEG C), reaction pH (4.5~7.5), cell freeze-dried powder concentration
(0.5%~3.5%), coenzyme additive amount (0~0.8mM), substrate and glucose additive amount ratio (1:0~1:2.5).
Influence of the temperature to asymmetric syntheses D-pantoyl lactone within the scope of 25~55 DEG C is probed into, as a result such as Fig. 5 institute
Show.Within the scope of 25~45 DEG C, efficiency of pcr product grows steadily temperature as the temperature rises, and in view of the efficient catalytic of enzyme, bottom
Object percent hydrolysis also gradually reduces.With continuing growing for temperature, substrate is due to its own property, and ketone group pantoic acid lactone is also in addition
Protoenzyme heated denaturalization, thus catalytic efficiency dramatic decrease, therefore ketone group pantoic acid lactone percent hydrolysis linearly increases.To sum up, In
Under the conditions of Whole cell catalytic system, it is preferable that reaction temperature is 35~45 DEG C, it is highly preferred that optimum temperature is 45 DEG C.
PH has been probed into the shadow of asymmetric syntheses D-pantoyl lactone with 0.2M phosphate buffer (pH 4.5~7.5)
It rings, as a result as shown in Figure 6.Under the conditions of Whole cell catalytic system, it is preferable that reaction pH is 5.5~6.5, it is highly preferred that most suitable
Reacting pH is 6.0.
Selecting the 0.2M phosphate buffer that pH is 6.0 is reaction medium, and cell freeze-drying is investigated at 45 DEG C, under the conditions of 400rpm
Powder additive amount is respectively 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5% pair of catalytic reaction products yield and bottom
The influence of object percent hydrolysis, as a result as shown in Figure 7.With the increase of biocatalyst usage amount, its product is obtained in lower range
Rate linearly rises.And the usage amount for working as cell freeze-dried powder increases to 3.0%, is further continued for increasing its dosage, efficiency of pcr product
It is not highly improved.Therefore, in the Whole cell catalytic system, cell freeze-dried powder additive amount is 3.0%.
Influence of the coenzyme additive amount to asymmetric syntheses D-pantoyl lactone is investigated, as a result as shown in Figure 8.Addition is different
The NADP of concentration+, substrate hydrolysis rate gradually reduces with the increase of coenzyme concentration, and the rising that efficiency of pcr product is presented slightly becomes
Gesture, but its advantage is not fairly obvious.When coenzyme final concentration increases to 0.8mM by 0.4mM, efficiency of pcr product only from
95.65% increases to 95.89%.Therefore, under the conditions of Whole cell catalytic system, it is contemplated that cost factor, it is not necessary to additional addition
Coenzyme NAD P+。
Substrate and influence of the auxiliary substrate adding proportion to asymmetric syntheses D-pantoyl lactone are probed into, as a result such as Fig. 9 institute
Show.Under the conditions of existing for no auxiliary substrate glucose, substrate hydrolysis rate has nearly reached 100%, and efficiency of pcr product is
0.05%.When auxiliary substrate glucose adding proportion is higher than substrate ketone group pantoic acid lactone, the increase of glucose additive amount can pole
The earth promotes the progress of catalysis reaction, in ketone group pantoic acid lactone: glucose reaches peak when being 1:2 (molar concentration rate, similarly hereinafter)
Value, and in ketone group pantoic acid lactone: glucose is fallen after rise when being 1:2.5, and substrate hydrolysis rate tends towards stability after being gradually reduced.
It is therefore preferred that the ratio of the substrate of whole-cell catalytic reaction system and auxiliary substrate be 1:1~1:2, it is highly preferred that substrate with
The ratio of auxiliary substrate is 1:2.
Embodiment 8 is catalyzed in optimal conditions generates D-pantoyl lactone
Catalyst system (5mL) after optimization are as follows: 100mM ketone group pantoic acid lactone (ketone group pantoic acid lactone pH 2.0,
0.2M KH2PO4The form that-HCl is formulated as 1M substrate solution is added, and substrate solution additional amount is with the amount of ketone group pantoic acid lactone
Meter, the final concentration of 100mM of ketone group pantoic acid lactone in reaction system), 3% cell freeze-dried powder (preparation of embodiment 5), 200mM
Glucose, substrate and auxiliary substrate are added after being sufficiently mixed with 10 μ L/min speed streams, anti-with 0.2M phosphate buffer (pH 6.0)
Answer medium；Asymmetric reduction reaction carries out in three neck round bottom flask, and controlled at 45 DEG C, 1M Na is added dropwise2CO3Maintain reaction
System pH constant 6.0.After reacting 1h, find that the yield of D-pantoyl lactone reaches 96.88% by gas chromatographic detection, product
E.e. value is greater than 99.9%.
9 concentration of substrate of embodiment increases to 500mM catalysis and generates D-pantoyl lactone
Constant speed stream when concentration of substrate 500mM adds catalyst system to be (10mL): (ketone group is general for 500mM ketone group pantoic acid lactone
Solve acid lactone pH 2.0,0.2M KH2PO4The form that-HCl is formulated as 1M substrate solution is added, and substrate solution additional amount is with ketone
The meter of base pantoic acid lactone, the final concentration of 500mM of ketone group pantoic acid lactone in reaction system), 3% cell freeze-dried powder it is (real
It is prepared by example 5), 1M glucose (ketone group pantoic acid lactone and glucose are flowed together by the ratio of molar concentration rate 1:2 to be added), with 0.2M
Phosphate buffer (pH 6.0) reaction medium；Substrate and auxiliary substrate be sufficiently mixed after with 10 μ L/min flow accelerations, with 1M's
Na2CO3It is added dropwise and keeps catalytic pH 6.0, asymmetric reduction reaction carries out in three neck round bottom flask, controlled at 45 DEG C.
After reacting 10h, up to 470mM (corresponding yield is 94%), the e.e. value of product is greater than the yield of D-pantoyl lactone
99.9%.
The foregoing description of the disclosed embodiments enables those skilled in the art to implement or use the present invention.
Various modifications to these embodiments will be readily apparent to those skilled in the art, as defined herein
General Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore, of the invention
It is not intended to be limited to the embodiments shown herein, and is to fit to and the principles and novel features disclosed herein phase one
The widest scope of cause.
Sequence table
<110>Hangzhou Xin Fu Science and Technology Ltd.
Zhejiang Polytechnical University
<120>ketone group pantoic acid lactone reductase and its application
<130> MP1908801
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 852
<212> DNA
<213>fission yeast (Schizosaccharomyces pombe)
<400> 1
atgcttatcg ctgctatggg ccctaagatt cccgtccctg cttatggggt tggtacagct 60
ttgtttaaga aagaaaaggg agaaatcaat cgaaccattg tagattcggt aaagaatgca 120
cttgctgctg gtttcattca tatcgactgc gcggaagtat atggcaacga agaggaagtt 180
ggggtggctt tgaaggaagc taatgttcca agaagcaaac tctttattac ttccaaggtt 240
atgcataatg tcgataacat tcctgaagcc ctcaacgaat ctttacgtaa gcttggtact 300
gactatttgg acctgtacct tttgcatagt cctattcctt tctatgaaaa gaaaataccc 360
atttctgaag gttggaaagc aatggaaacg gctttgggaa ccggtcttgt ccactcagtt 420
ggtgtttcta actttcgtat tcccgactta gaggagctgt tgaaaacttc aactattact 480
cctcgtgtca accaaatcga atttcaccct caagtataca aggccgcgaa gcctcttgta 540
gaattttgcc agagtaaagg aattatcgtt gagggttatg gccctctttc tcccttggtt 600
cgtgatgcgc aaggtcctgt tgctgaattc actaaatcat tggagagcaa gtaccatgtt 660
tctgatactc aaattcttct caagtgggcc tattctaaag gcgttatacc tatcactact 720
acctcaaaaa ttgagcgtat gaaggagtgc ttgaattttg attctttcac tctagacaag 780
gctgacattg acgaacttgg taccttaggt gttcaacatc acaagcgtac atttatgaag 840
cacatggatg ag 852
<210> 2
<211> 852
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
atgctgattg cagcaatggg tcctaaaatc ccggtgccag cgtatggtgt tggcacggcc 60
ctgtttaaaa aagaaaaagg cgaaattaat cgcaccatcg tggattcagt taaaaatgcc 120
ttagccgcgg gctttatcca tattgattgc gccgaagtgt atggcaacga ggaagaagtt 180
ggcgtggccc tgaaagaagc caatgtgcct cgtagtaaac tgtttattac gagcaaagtg 240
atgcataatg ttgataatat tccggaagcg ctgaatgagt ctctgcgcaa actgggtacg 300
gattatttgg atctgtatct gctgcattct cctattccgt tttatgagaa aaagatcccg 360
attagcgaag gttggaaagc tatggaaacc gctctgggta caggcttagt tcatagtgtt 420
ggtgtgagta attttcgcat cccagacctg gaagaactgc tgaaaacgtc taccatcaca 480
cctcgcgtta atcagatcga atttcatcca caggtgtaca aggccgccaa accgttagtg 540
gaattttgtc agtctaaagg tatcattgtg gaaggctatg gtcctctgtc tccactggtt 600
cgcgatgctc agggtccggt tgcggagttt accaaatcat tagaatctaa atatcatgtg 660
agcgatacac agattttact gaaatgggcc tatagcaaag gcgttattcc gatcacgacg 720
acatctaaaa tcgaacgcat gaaagaatgc ctgaattttg atagctttac gctggataaa 780
gcagacattg atgaactggg caccttaggc gtgcagcatc ataaacgtac ctttatgaaa 840
cacatggatg aa 852
<210> 3
<211> 284
<212> PRT
<213>fission yeast (Schizosaccharomyces pombe)
<400> 3
Met Leu Ile Ala Ala Met Gly Pro Lys Ile Pro Val Pro Ala Tyr Gly
1 5 10 15
Val Gly Thr Ala Leu Phe Lys Lys Glu Lys Gly Glu Ile Asn Arg Thr
20 25 30
Ile Val Asp Ser Val Lys Asn Ala Leu Ala Ala Gly Phe Ile His Ile
35 40 45
Asp Cys Ala Glu Val Tyr Gly Asn Glu Glu Glu Val Gly Val Ala Leu
50 55 60
Lys Glu Ala Asn Val Pro Arg Ser Lys Leu Phe Ile Thr Ser Lys Val
65 70 75 80
Met His Asn Val Asp Asn Ile Pro Glu Ala Leu Asn Glu Ser Leu Arg
85 90 95
Lys Leu Gly Thr Asp Tyr Leu Asp Leu Tyr Leu Leu His Ser Pro Ile
100 105 110
Pro Phe Tyr Glu Lys Lys Ile Pro Ile Ser Glu Gly Trp Lys Ala Met
115 120 125
Glu Thr Ala Leu Gly Thr Gly Leu Val His Ser Val Gly Val Ser Asn
130 135 140
Phe Arg Ile Pro Asp Leu Glu Glu Leu Leu Lys Thr Ser Thr Ile Thr
145 150 155 160
Pro Arg Val Asn Gln Ile Glu Phe His Pro Gln Val Tyr Lys Ala Ala
165 170 175
Lys Pro Leu Val Glu Phe Cys Gln Ser Lys Gly Ile Ile Val Glu Gly
180 185 190
Tyr Gly Pro Leu Ser Pro Leu Val Arg Asp Ala Gln Gly Pro Val Ala
195 200 205
Glu Phe Thr Lys Ser Leu Glu Ser Lys Tyr His Val Ser Asp Thr Gln
210 215 220
Ile Leu Leu Lys Trp Ala Tyr Ser Lys Gly Val Ile Pro Ile Thr Thr
225 230 235 240
Thr Ser Lys Ile Glu Arg Met Lys Glu Cys Leu Asn Phe Asp Ser Phe
245 250 255
Thr Leu Asp Lys Ala Asp Ile Asp Glu Leu Gly Thr Leu Gly Val Gln
260 265 270
His His Lys Arg Thr Phe Met Lys His Met Asp Glu
275 280
<210> 4
<211> 789
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
atgggttata attctctgaa aggcaaagtc gcgattgtta ctggtggtag catgggcatt 60
ggcgaagcga tcatccgtcg ctatgcagaa gaaggcatgc gcgttgttat caactatcgt 120
agccatccgg aggaagccaa aaagatcgcc gaagatatta aacaggcagg tggtgaagcc 180
ctgaccgtcc agggtgacgt ttctaaagag gaagacatga tcaacctggt gaaacagact 240
gttgatcact tcggtcagct ggacgtcttt gtgaacaacg ctggcgttga gatgccttct 300
ccgtcccacg aaatgtccct ggaagactgg cagaaagtga tcgatgttaa tctgacgggt 360
gcgttcctgg gcgctcgtga agctctgaaa tacttcgttg aacataacgt gaaaggcaac 420
attatcaata tgtctagcgt ccacgaaatc atcccgtggc ctactttcgt acattacgct 480
gcttctaagg gtggcgttaa actgatgacc cagactctgg ctatggaata tgcaccgaaa 540
ggtatccgca ttaacgctat cggtccaggc gcgatcaaca ctccaattaa tgcagaaaaa 600
ttcgaggatc cgaaacagcg tgcagacgtg gaaagcatga tcccgatggg caacatcggc 660
aagccagagg agatttccgc tgtcgcggca tggctggctt ctgacgaagc gtcttacgtt 720
accggcatca ccctgttcgc agatggtggc atgaccctgt acccgagctt tcaggctggc 780
cgtggttga 789
<210> 5
<211> 262
<212> PRT
<213>Exiguobacterium sp (Exiguobacterium sibiricum)
<400> 5
Met Gly Tyr Asn Ser Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly
1 5 10 15
Ser Met Gly Ile Gly Glu Ala Ile Ile Arg Arg Tyr Ala Glu Glu Gly
20 25 30
Met Arg Val Val Ile Asn Tyr Arg Ser His Pro Glu Glu Ala Lys Lys
35 40 45
Ile Ala Glu Asp Ile Lys Gln Ala Gly Gly Glu Ala Leu Thr Val Gln
50 55 60
Gly Asp Val Ser Lys Glu Glu Asp Met Ile Asn Leu Val Lys Gln Thr
65 70 75 80
Val Asp His Phe Gly Gln Leu Asp Val Phe Val Asn Asn Ala Gly Val
85 90 95
Glu Met Pro Ser Pro Ser His Glu Met Ser Leu Glu Asp Trp Gln Lys
100 105 110
Val Ile Asp Val Asn Leu Thr Gly Ala Phe Leu Gly Ala Arg Glu Ala
115 120 125
Leu Lys Tyr Phe Val Glu His Asn Val Lys Gly Asn Ile Ile Asn Met
130 135 140
Ser Ser Val His Glu Ile Ile Pro Trp Pro Thr Phe Val His Tyr Ala
145 150 155 160
Ala Ser Lys Gly Gly Val Lys Leu Met Thr Gln Thr Leu Ala Met Glu
165 170 175
Tyr Ala Pro Lys Gly Ile Arg Ile Asn Ala Ile Gly Pro Gly Ala Ile
180 185 190
Asn Thr Pro Ile Asn Ala Glu Lys Phe Glu Asp Pro Lys Gln Arg Ala
195 200 205
Asp Val Glu Ser Met Ile Pro Met Gly Asn Ile Gly Lys Pro Glu Glu
210 215 220
Ile Ser Ala Val Ala Ala Trp Leu Ala Ser Asp Glu Ala Ser Tyr Val
225 230 235 240
Thr Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Leu Tyr Pro Ser
245 250 255
Phe Gln Ala Gly Arg Gly
260
<210> 6
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
atgctgattg cagcaatg 18
<210> 7
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
ttcatccatg tgtttcataa ag 22
Claims (16)
1. isolated ketone group pantoic acid lactone reductase, which is characterized in that the ketone group pantoic acid lactone reductase, which derives from, to be split
Yeast is grown, amino acid sequence is as shown in SEQ ID No:3.
2. isolated polynucleotides, which is characterized in that the polynucleotides are in coding ketone group pantoic acid described in claim 1
The polynucleotides of ester reductase.
3. polynucleotides as claimed in claim 2, which is characterized in that the nucleotide sequence of the polynucleotides is selected from the group:
(i) nucleotide sequence shown in SEQ ID No:1 or SEQ ID No:2；(ii) core complementary with the nucleotide sequence described in (i)
Nucleotide sequence.
4. a kind of recombinant vector, which is characterized in that the recombinant vector contains polynucleotides as claimed in claim 2.
5. a kind of recombinant cell, which is characterized in that the recombinant cell contains recombinant vector or genome as claimed in claim 4
In be integrated with polynucleotides as claimed in claim 2.
6. a kind of preparation method of ketone group pantoic acid lactone reductase described in claim 1 characterized by comprising
(a) Fiber differentiation is carried out to the recombinant cell described in claim 5；And
(b) the ketone group pantoic acid lactone reductase is separated from culture.
7. ketone group pantoic acid lactone reductase described in claim 1 generates the application in D-pantoyl lactone in catalysis.
8. a kind of double enzyme recombinant cells, which is characterized in that it is general that double enzyme recombinant cell inductions generate ketone group described in claim 1
Solve acid lactone reductase and glucose dehydrogenase.
9. double enzyme recombinant cells as claimed in claim 8, which is characterized in that the glucose dehydrogenase derives from small bar
Bacterium, amino acid sequence is as shown in SEQ ID No:5.
10. double enzyme recombinant cells as claimed in claim 9, which is characterized in that encode the multicore glycosides of the glucose dehydrogenase
Acid sequence is selected from the group: nucleotide sequence shown in (i) SEQ ID No:4；(ii) complementary with the nucleotide sequence described in (i)
Nucleotide sequence.
11. the construction method of pair enzyme recombinant cell characterized by comprising
(a) polynucleotides as claimed in claim 2 are inserted into first vector, the first recombinant vector are obtained, by claim 10 institute
The polynucleotides insertion Second support stated, obtains the second recombinant vector；And
(b) first recombinant vector and the second recombinant vector are imported into host cell, obtains double enzyme recombinant cells.
12. construction method described in claim 11, which is characterized in that the first vector is pET-28b, the Second support
For pACYCDuet-1, the host cell is E.coli BL21 (DE3).
13. a kind of method for preparing D-pantoyl lactone, which is characterized in that the substrate supplemented by glucose is recombinated thin using double enzymes
Born of the same parents induce the ketone group pantoic acid lactone reductase generated and glucose dehydrogenase catalysis ketone group pantoic acid lactone to generate Pantothenic acid
Lactone.
14. method as claimed in claim 13, which is characterized in that the ketone group pantoic acid lactone and glucose are with fed-batch mode
It is added in reaction system and is reacted.
15. method as claimed in claim 13, which is characterized in that the reaction temperature of the catalysis reaction is 25~50 DEG C, pH
It is 4.5~7.5.
16. method as claimed in claim 13, which is characterized in that the initial molar of the ketone group pantoic acid lactone and glucose
Concentration ratio is 1:1~1:2.5.
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|---|---|---|---|---|
| CN111139270A (en) * | 2019-12-23 | 2020-05-12 | 浙江大学 | Enzyme combination for producing L-glufosinate-ammonium and L-glufosinate-ammonium production method |
| CN112195198A (en) * | 2020-11-30 | 2021-01-08 | 天津工微生物科技有限公司 | Method for preparing D-pantoic acid |
| CN113913399A (en) * | 2021-11-19 | 2022-01-11 | 万华化学集团股份有限公司 | Ketopantolactone reductase derived from Candida maltosa Xu316 |
| CN114085820A (en) * | 2021-11-19 | 2022-02-25 | 万华化学集团股份有限公司 | Ketopantolactone reductase derived from Candida viswanathii |
| CN114457129A (en) * | 2020-09-29 | 2022-05-10 | 安徽华恒生物科技股份有限公司 | Recombinant engineering bacterium and application thereof in efficient conversion of L-pantolactone |
| CN114934061A (en) * | 2022-05-20 | 2022-08-23 | 中国科学院微生物研究所 | Engineering bacteria and application thereof in producing D-pantolactone by full-cell catalysis of keto-pantolactone |
| CN115851642A (en) * | 2022-09-14 | 2023-03-28 | 杭州鑫富科技有限公司 | Ketone group pantoic acid lactone reductase and application thereof |
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2019
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111139270A (en) * | 2019-12-23 | 2020-05-12 | 浙江大学 | Enzyme combination for producing L-glufosinate-ammonium and L-glufosinate-ammonium production method |
| CN114457129A (en) * | 2020-09-29 | 2022-05-10 | 安徽华恒生物科技股份有限公司 | Recombinant engineering bacterium and application thereof in efficient conversion of L-pantolactone |
| CN112195198A (en) * | 2020-11-30 | 2021-01-08 | 天津工微生物科技有限公司 | Method for preparing D-pantoic acid |
| CN113913399A (en) * | 2021-11-19 | 2022-01-11 | 万华化学集团股份有限公司 | Ketopantolactone reductase derived from Candida maltosa Xu316 |
| CN114085820A (en) * | 2021-11-19 | 2022-02-25 | 万华化学集团股份有限公司 | Ketopantolactone reductase derived from Candida viswanathii |
| CN113913399B (en) * | 2021-11-19 | 2023-10-20 | 万华化学集团股份有限公司 | Ketopantolactone reductase from Candida maltosa Xu316 |
| CN114085820B (en) * | 2021-11-19 | 2023-10-20 | 万华化学集团股份有限公司 | Ketopantolactone reductase from Candida viswanathii |
| CN114934061A (en) * | 2022-05-20 | 2022-08-23 | 中国科学院微生物研究所 | Engineering bacteria and application thereof in producing D-pantolactone by full-cell catalysis of keto-pantolactone |
| CN115851642A (en) * | 2022-09-14 | 2023-03-28 | 杭州鑫富科技有限公司 | Ketone group pantoic acid lactone reductase and application thereof |
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