CN108251406A - L- rhamnulose-1-phosphate aldolases and its application in rare sugared D-Psicose is catalyzed and synthesized - Google Patents
L- rhamnulose-1-phosphate aldolases and its application in rare sugared D-Psicose is catalyzed and synthesized Download PDFInfo
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Abstract
Application the invention discloses 1 phosphate aldolase of L rhamnuloses and its in rare sugar D psicoses are catalyzed and synthesized, gene order are to pass through the 29th, 188,266 or 152 saturation site-directed mutagenesis of its amino acid respectively by 1 phosphate aldolase of wild type L rhamnuloses that SEQ ID NO.1 are encoded.The present invention overcomes wild type RhaD to generate D psicoses:D sorbose=1：1 does not have the defects of stereoselectivity so that catalysis reaction only generates D psicoses or based on D psicoses, and the enzyme activity of the RhaD by mutation is higher.The RhaD of rite-directed mutagenesis prepared by the method for the present invention prepares D psicoses, combined coefficient is high, simple for process, cost-effective, D psicose catalytic efficiencies are up to 100%, and realizing industrialization RhaD catalysis D glyceraldehyde using the method for the present invention largely synthesizes D psicoses.
Description
Technical field
The invention belongs to biotechnologies, and in particular to L- rhamnulose-1-phosphate aldolases and its catalysis close
Application into rare sugared D-Psicose.
Background technology
D-Psicose (D-psicose) belongs to a kind of rare monosaccharide, as a kind of zero energy, the table sugar not being digested
Substitute has zero calory due to D-Psicose, it is difficult to the characteristics such as digest and assimilate by enteron aisle, be evaluated by U.S.'s food Navigation Network
It is now to be cited as novel low-calorie sweeteners for most potential sucrose succedaneum more.Meanwhile D-Psicose can also be made
For liver lipidase and the inhibitor of enteron aisle alpha-glucosidase, fat accumulation is reduced.Many results of study show that D-Psicose passes through
Inhibit the generation of ROS in testis tissue, the injury of testis that phthalic acid two (2- ethyls) ethyl ester is prevented to induce.In addition D- A Luo
Ketose is other than the Apoptosis induced 6- hydroxyl dopamines plays the role of neuroprotection, moreover it is possible to high concentration glucose be inhibited to lure
The expression of monocyte chemoattractant protein MCP-1 under leading.Based on the numerous physiological function of D-Psicose, in following food, doctor
The application in the fields such as medicine and cosmetics will be very extensive.
D-Psicose functional activity and human health are closely related, and the high yield system of the D-Psicose of Erecting and improving is compeled
In the eyebrows and eyelashes.The research of D-Psicose at present, bottleneck are mainly concentrated in expanding industrialization production.Early stage D-Psicose
Synthetic method is chemical conversion, and building-up process is not only cumbersome also to be had some limitations, and not meeting current green more can hold
The social theory of supervention exhibition.At present production D-Psicose mainly using enzyme fix conversion method, i.e., to the clonal expression of enzyme with
And the separation and purification of product.Whether chemical synthesis or biotransformation method prepare D-Psicose, and all there are a disasters
Topic, product separation are very difficult with refining.D-Psicose in crude product is often mixed with other sugar, not only yield
It reduces, and D-Psicose and sugared physicochemical property that it mixes are nearly identical, so it is difficult to refined with purifying.In order to right
Current D-Psicose synthetic system optimizes, and must just solve this problem.
Early-stage study shows the L- rhamnuloses -1- for belonging to phosphoric acid dihydroxyacetone (DHA) (DHAP) dependent form aldolase family
Phosphate aldolase (hereinafter referred to as RhaD) loses and the solid of the aldehyde receptor is selected when using D- glyceraldehyde as aldehyde receptor
Property, wild type generation two kinds of rare sugar of D-Psicose and D- sorboses, the two ratio is close to 1：1.
Invention content
The purpose of this part is to summarize some aspects of the embodiment of the present invention and briefly introduce some preferably to implement
Example.It may do in this section and the description of the application and the title of the invention a little simplified or omit to make our department
Point, the purpose of abstract of description and denomination of invention obscure, and this simplification or omit and cannot be used for limiting the scope of the invention.
In view of above-mentioned technological deficiency, it is proposed that the present invention.
Therefore, the one aspect as the present invention, the present invention overcome the deficiencies in the prior art, provide fixed point
The L- rhamnulose-1-phosphate aldolases of mutation.
In order to solve the above technical problems, the present invention provides following technical solutions：L- rhamnuloses-the 1- of rite-directed mutagenesis
Phosphate aldolase, wherein：The L- rhamnulose-1-phosphate aldolases of the rite-directed mutagenesis are by SEQ ID by gene order
The wild type L- rhamnulose-1-phosphate aldolases of NO.1 codings pass through the 29th, 188,266 or 152, its amino acid respectively
Saturation site-directed mutagenesis and the mutant generated.
A kind of preferred embodiment of L- rhamnulose-1-phosphate aldolases as rite-directed mutagenesis of the present invention, wherein：
Described the 29th, 188,266 or 152 saturation site-directed mutagenesis of its amino acid and the mutant generated, including L- rhamnuloses -1-
Mutant Y152A, mutant N29K, mutant P188C, mutant P188T, mutant P188F, the mutant of phosphate aldolase
N29I。
A kind of preferred embodiment of L- rhamnulose-1-phosphate aldolases as rite-directed mutagenesis of the present invention, wherein：
The wild type L- rhamnulose-1-phosphate aldolases by SEQ ID NO.1 codings derive from Escherichia coli MG1655 bacterium
Strain.
As another aspect of the present invention, the present invention overcomes the deficiencies in the prior art, provides DNA molecular.
In order to solve the above technical problems, the present invention provides following technical solutions：DNA molecular, wherein, the DNA molecular
Encode the L- rhamnulose-1-phosphate aldolases of the rite-directed mutagenesis described in claim 2.
As another aspect of the present invention, the present invention overcomes the deficiencies in the prior art, provides plasmid vector.
In order to solve the above technical problems, the present invention provides following technical solutions：Plasmid vector, wherein：The plasmid carries
Body is loaded with the DNA molecular described in claim 4.
As a kind of preferred embodiment of plasmid vector of the present invention, wherein：The plasmid vector is in pET-28a carriers
On be loaded with DNA molecular described in claim 4.
As another aspect of the present invention, the present invention overcomes the deficiencies in the prior art, provides rite-directed mutagenesis
Application of the L- rhamnulose-1-phosphate aldolases in rare sugared D-Psicose is catalyzed and synthesized.
As another aspect of the present invention, the present invention overcomes the deficiencies in the prior art, provides rite-directed mutagenesis
The preparation method of L- rhamnulose-1-phosphate aldolases.
In order to solve the above technical problems, the present invention provides following technical solutions：L- rhamnuloses-the 1- of rite-directed mutagenesis
The preparation method of phosphate aldolase, including,
Build mutant strain recombinant plasmid：Wild type L- rhamnulose-1-phosphate aldolase genetic fragments are connected to matter
On grain carrier, wild type L- rhamnulose-1-phosphate aldolase gene recombination plasmids are obtained, with the wild type L- sandlwood trees
Glycocoll -1- phosphate aldolases gene recombination plasmid is masterplate, to L- rhamnulose-1-phosphate aldolase gene coding amino acids
The 29th, 188,266 or 152 amino acids saturation site-directed mutagenesis are carried out respectively, respectively obtain mutant strain recombinant plasmid；
Mutant strain is expressed：By the mutant strain recombinant plasmid transformed Escherichia coli, picking individual colonies inoculation, 16 DEG C of IPTG
The L- rhamnulose-1-phosphate aldolases of induced expression rite-directed mutagenesis.
One kind of the preparation method of L- rhamnulose-1-phosphate aldolases as rite-directed mutagenesis of the present invention is preferably
Scheme further includes, protein purification：By the L- rhamnulose -1- phosphoric acid aldehyde of the rite-directed mutagenesis by mutant strain expression
Contracting enzyme is purified.
One kind of the preparation method of L- rhamnulose-1-phosphate aldolases as rite-directed mutagenesis of the present invention is preferably
Scheme, wherein：The plasmid vector be pET-28a carriers, the mutant strain recombinant plasmid include pET28a-rhaDN29I,
pET28a-rhaDN29K、pET28a-rhaDP188C、pET28a-rhaDP188F、pET28a-rhaDP188T、pET28a-
RhaDT266A, pET28a-rhaDY152A, it is described by mutant strain recombinant plasmid transformed Escherichia coli, to convert Escherichia coli
BL21 bacterial strains.
Beneficial effects of the present invention：The present invention carries out rite-directed mutagenesis by the amino acid sites to RhaD, overcomes wild
Type RhaD generates D-Psicose:D- sorbose=1：1, that is, there is no the defects of stereoselectivity, it is sweet to realize RhaD catalysis D-
Oily aldehyde synthesizes stereoselectivity during D-Psicose for aldehyde receptor so that catalysis reaction only generate D-Psicose or with D- Ah
Based on the ketose of Lip river, the enzyme activity of the RhaD by mutation is high.The RhaD of rite-directed mutagenesis prepared by the method for the present invention prepares D- A Luo ketone
Sugar, combined coefficient is high, simple for process, cost-effective, D-Psicose stereoselectivity catalytic efficiency is up to 100%, profit
Industrialization RhaD catalysis D- glyceraldehyde, which is realized, with the method for the present invention largely synthesizes D-Psicose.
Description of the drawings
In order to illustrate the technical solution of the embodiments of the present invention more clearly, required use in being described below to embodiment
Attached drawing be briefly described, it should be apparent that, the accompanying drawings in the following description is only some embodiments of the present invention, for this
For the those of ordinary skill of field, without having to pay creative labor, it can also be obtained according to these attached drawings other
Attached drawing.Wherein：
Fig. 1 is L- rhamnulose-1-phosphate aldolase mutant N29I, N29K of rite-directed mutagenesis of the present invention, N188C,
P188T, T266A, Y152A, P188F and wild type L- rhamnulose-1-phosphate aldolases catalyze and synthesize D-Psicose
HPLC atlas analysis schematic diagrames.
Fig. 2 be rite-directed mutagenesis of the present invention L- rhamnulose-1-phosphate aldolase mutant F211C, F170A,
F263A, N29A-Y152A catalyze and synthesize the HPLC atlas analysis schematic diagrames of D-Psicose.
Specific embodiment
In order to make the foregoing objectives, features and advantages of the present invention clearer and more comprehensible, with reference to specific embodiment pair
The specific embodiment of the present invention is described in detail.
Many details are elaborated in the following description to facilitate a thorough understanding of the present invention, still the present invention can be with
Implemented using other different from other manner described here, those skilled in the art can be without prejudice to intension of the present invention
In the case of do similar popularization, therefore the present invention is not limited by following public specific embodiment.
Secondly, " one embodiment " or " embodiment " referred to herein refers to may be included at least one realization side of the present invention
A particular feature, structure, or characteristic in formula." in one embodiment " that different places occur in the present specification not refers both to
Same embodiment, nor the individual or selective embodiment mutually exclusive with other embodiment.
In order to describe the convenience of the present invention, the conventional and unconventional abbreviation of various amino acid residues is used.These abbreviations are
Familiar to the person skilled in the art, but in order to clearly be listed below：
Asp=D=aspartic acids；Ala=A=alanine；Arg=R=arginine；
Asn=N=asparagines；Gly=G=glycine；Glu=E=glutamic acid；
Gln=Q=glutamine；His=H=histidines；Ile=I=isoleucines；
Leu=L=leucines；Lys=K=lysines；Met=M=methionines；
Phe=F=phenylalanines；Pro=P=proline；Ser=S=serines；
Thr=T=threonines；Trp=W=tryptophans；Tyr=Y=tyrosine；
Val=V=valines；Cys=C=cysteines.
Mutant name of the present invention, according to those skilled in the art's routine naming method, such as：Mutant
Y152A represents that the tyrosine (Y) of the 152nd, L- rhamnulose-1-phosphate aldolases (RhaD) its amino acid is fixed through supersaturation
Point mutation is alanine (A).
In the present invention, the L- rhamnulose-1-phosphate aldolases (L-Rhamnulose- in Escherichia coli MG1655 sources
1-phosphate Aldolase, RhaD) by 825 gene codes, gene order is optimized, RhaD encoding genes are inserted into
Expression vector pET-28a obtains recombinant expression plasmid pET-28a-rhaD.Using plasmid pET-28a-rhaD as template, respectively to L-
The active region site of rhamnulose-1-phosphate aldolase RhaD carries out saturation site-directed mutagenesis and carries out more than two
The combination site mutation in RhaD active regions site, this research using wild type RhaD recombinant expression plasmids pET-28a-rhaD as
Blank control.Escherichia coli MG1655 of the present invention is this laboratory (Biological Engineering College of Southern Yangtze University carbohydrate chemistry and biology
Key lab of technical education portion) preservation strain.
Distinguish the above-mentioned wild type of picking, by RhaD active regions saturation site-directed mutagenesis or the monoclonal of combination rite-directed mutagenesis
LB fluid nutrient mediums (50 μ g/mL kanamycins) are inoculated into, are incubated overnight.It draws 2mL culture solutions and is transferred to TB fluid nutrient mediums
(50 μ g/mL kanamycins) is cultivated.When OD600 is 0.6~0.8, IPTG (final concentration 0.2mM), 16 DEG C of induction 22h are added in
Afterwards, stop culture, thalline were collected by centrifugation, abandons supernatant.Thalline ultrasonication crushes liquid high speed centrifugation, and obtained supernatant is used
25 protein purification instrument of Avant purifies destination protein.First with equilibration buffer to Ni2+Affinity column is balanced (10CV)
Loading afterwards, after loading, then with equilibration buffer to Ni2+Affinity column is balanced (5CV)；It is eluted with washing buffer
Destination protein is simultaneously collected, and eluting liquid is concentrated with super filter tube and desalination.
Embodiment 1：
Using BamHI, RhaD encoding genes are inserted into expression vector pET-28a and obtain recombinant expression matter by SacI double digestions
Grain pET-28a-rhaD.Using plasmid pET-28a-rhaD as template, respectively to L- rhamnulose-1-phosphate aldolases RhaD's
Active region site carries out saturation site-directed mutagenesis.The gene order of wild type L- rhamnulose-1-phosphate aldolases is shown in SEQ
ID NO.1。
Primer sequence is respectively：
Wild type rhaD amplimers：
Sense primer rhaD-F GCGCGGATCC(underlined sequences are BamHI digestions position to ATGCAAAACATTACTCAG
Point)；
Downstream primer rhaD-R GCGCGAGCTC(underlined sequences are SacI digestions position to TTACAGCGCCAGCGCACTG
Point).
And the combination site mutation in two or more RhaD active regions site is carried out, this research recombinates wild type RhaD
Expression plasmid pET-28a-rhaD is as blank control.It chooses respectively in the site of the saturation site-directed mutagenesis：RhaD the 29th, 188,
266th, 152,170,211 or 263 amino acids sites and site mutation is combined, i.e., above-mentioned single mutational site difference
Combination of two chooses 29＆188 combination of amino acids site mutations, 29＆266 combination of amino acids site mutations, 29＆152 amino acids
Combine site mutation.
Picking contains recombinant plasmid pET28a-rhaD respectivelyN29I、pET28a-rhaDN29K、pET28a-rhaDP188C、
pET28a-rhaDP188F、pET28a-rhaDP188T、pET28a-rhaDT266A、pET28a-rhaDY152A、pET28a-rhaDT170A、
pET28a-rhaDF211C、pET28a-rhaDF263AOr pET28a-rhaDN29A-Y152AMonoclonal be inoculated into 5mL LB Liquid Cultures
Base (50 μ g/mL kanamycins), 37 DEG C, 220rpm are incubated overnight.It draws 2mL culture solutions and is transferred to 200mLTB fluid nutrient mediums
(50 μ g/mL kanamycins), 37 DEG C, 220rpm.When OD600 is 0.6~0.8, addition IPTG (final concentration 0.2mM), 16 DEG C
After inducing 22h, stop culture, centrifugation (8000g, 3min, 4 DEG C) collects thalline, abandons supernatant.It is high to crush liquid for thalline ultrasonication
Speed centrifugation (12000g, 30min, 4 DEG C), obtained supernatant 25 protein purification instrument of Avant purify destination protein.It uses first
Equilibration buffer 50mM Tris-HCl (pH 7.5) are balanced Ni2+ affinity columns (10CV) loading afterwards, and loading finishes
Afterwards, then with equilibration buffer 50mM Tris-HCl (pH 7.5) Ni2+ affinity columns are balanced (5CV)；It is slow with washing
(50mM Tris-HCl (pH 7.5), 25mM NaCl, 60mM imidazoles wash off the smaller foreign protein of adsorption capacity on chromatographic column to fliud flushing；
Finally destination protein is eluted with elution buffer (50mM Tris-HCl (pH 7.5), 25mM NaCl, 500mM imidazoles) and receive
Collection, eluting liquid is concentrated with super filter tube and desalination.
The primer sequence carried out involved by rite-directed mutagenesis RhaD recombinant expression plasmids is as follows：
Mutant P188F sense primers：5'-tttgccgtggatggtgttcGgcacggacgaaatc-3'(underscore sequences
It is classified as mutational site),
Mutant P188F downstream primers：3'-aaacggcacctaccacaagCcgtgcctgctttag-5'(underscore sequences
It is classified as mutational site).
Mutant P188T sense primers：5'-ccgtggatggtgaccGgcacggacg-3'(underlined sequences are mutation
Site),
Mutant P188T downstream primers：3'-ggcacctaccactggCcgtgcctgc-5'(underlined sequences are mutation
Site).
Mutant P188C sense primers：5'-gccgtggatggtgtgcGgcacggacgaa-3'(underlined sequences are prominent
Become site),
Mutant P188C downstream primers：3'-cggcacctaccacacgCcgtgcctgctt-5'(underlined sequences are prominent
Become site).
Mutant N29K sense primers：5'-ggctgggatgagcgcaagGgcggcaac-3'(underlined sequences are mutation
Site),
Mutant N29K downstream primers：3'-ccgaccctactcgcgttcCcgccgttg-5'(underlined sequences are mutation
Site).
Mutant Y152A sense primers：5'-aacctgatcgccctcaccgctUnder gtacttgaaaacgacacc-3'(
Underlined sequence is mutational site),
Mutant Y152A downstream primers：3'-ttggactagcgggagtggcgaUnder catgaacttttgctgtgg-5'(
Underlined sequence is mutational site).
Mutant N29I sense primers：5'-gctgggatgagcgcatcGgcggca-3'(underlined sequences are mutation position
Point),
Mutant N29I downstream primers：3'-cgaccctactcgcgtagCcgccgt-5'(underlined sequences are mutation position
Point).
Embodiment 2：
RhaD aldolases wild type and mutant Enzyme activity assay：
In order to measure enzyme activity, 50 μ L total volume reaction systems are as follows：DHAP (0.2M, 5 μ L, 0.02M)；D- glyceraldehyde
(0.5M, 2 μ L, 0.02M), aldolase RhaD wild types or mutant (10mg/mL, 2.5 μ L, 0.5mg/mL) add 50mM
Tris-HCl (pH8.0) to 50 μ L of volume.30 DEG C stand reaction overnight, and after being 5 with 3M HCl adjusting pH, it is acid to add in 0.25 μ L
Phosphatase AP, 30 DEG C stand reaction overnight.Reaction terminates to adjust pH as 7 using 3M NaOH for terminating reaction, then 15000g
It centrifuges 10min and collects supernatant.HPLC is detected, as shown in Figures 1 and 2.
Chromatographic condition of the present invention is：Hitachi's Composition distribution (HITACHI RI), chromatographic column Bio-Rad Aminex HPX-
87H, mobile phase are the dilute sulfuric acid aqueous solution of 5mmol/L, and flow velocity 0.5mL/min, sample size is 20 μ L.Using the same terms into
The drafting of the standard curve of row D-Psicose and D- sorboses.
From fig. 1, it can be seen that under identical HPLC detection architectures, measure two kinds of sugared standard items (i.e. complete standard curve with
The concentration of sugar is as abscissa, using peak area as ordinate).HPLC profiling results analysis shows, wild type RhaD-WT catalysis
D- glyceraldehyde, the rare sugared D-Psicose (appearance time 12.5 or so) of product and the D- sorboses that reaction terminating ultimately generates
(appearance time 11.23 or so), the rare monosaccharide ratio of two kinds of output keep 1：1 or so.Meanwhile research finds HPLC profiling results
RhaD mutant Y152A, N29K, P188C, P188T, P188F show D-Psicose yield and are significantly higher than D- sorboses
As a result, the i.e. above-mentioned RhaD by above-mentioned five site mutations show it is excellent for catalysis generation D-Psicose
Stereoselectivity, the yield of D- sorboses is seldom, and the enzyme activity of the RhaD by mutation is high；And RhaD mutant N29I and T266A
Also the stereoselectivity of RhaD catalysis generation D-Psicoses is shown, but still some D- sorbose generates.Wherein, it is mutated
, only there is a product peak in body P188F reaction solution HPLC atlas analysis, and standard of comparison understands that this product is D-
Psicose, i.e. mutant P188F show the stereoselectivity of RhaD catalysis generation D-Psicose completely, without generating D-
Sorbose, and the enzyme activity of the RhaD by mutation is higher.
It is however clear from fig. 2 that the RhaD of RhaD mutant T170A and F211C is without enzyme activity, i.e. T170A or
The mutation of F211C can cause RhaD enzyme activity to lose；And RhaD mutant F263A, F263A can generate a small amount of D- sorboses,
Its enzyme activity is very low, and albumen is unstable.Illustrate the mutation in T170A, F211C or F263A site for RhaD be catalyzed generation D- Ah
The stereoselectivity of Lip river ketose can not be realized.
The combinatorial mutagenesis that inventor also studied two sites is catalyzed RhaD the three-dimensional selection for generating D-Psicose
The influence of property, figure it is seen that the combination site mutation of N29A-Y152A still has relatively while D-Psicose is generated
The D- sorboses answered generate, therefore the combination site mutation of N29A-Y152A is catalyzed RhaD the solid of generation D-Psicose
Poor selectivity is in single site mutation.
Embodiment 3：
The stereoselectivity analysis of RhaD aldolases：
The study find that phosphoric acid dihydroxyacetone (DHA) (DHAP) the dependent form aldolase family in Escherichia coli MG1655 sources
The reaction substrate of L- rhamnulose-1-phosphate aldolases (RhaD) catalysis is two：First is phosphoric acid dihydroxyacetone (DHA)
(DHAP), second is aldehydes.When using D- glyceraldehyde as aldehyde receptor, the stereoselectivity to the aldehyde receptor is lost, is generated
Two kinds of rare sugar of D-Psicose and D- sorboses, the two ratio approach and 1：1.And when the rite-directed mutagenesis strain of MG1655 RhaD
When N29I, N29K, P188C, P188T, T266A, Y152A are using DHAP and D- glyceraldehyde as substrate, D-Psicose is mainly generated,
And the RhaD rite-directed mutagenesis strains P188F energy complete synthesis D-Psicoses of Escherichia coli MG1655 bacterial strains, specific experiment result are shown in
Table 1.
To RhaD, possible more amino acid site includes the 29th, 188,266,152,170,211,263 for this research
Fixed point saturation mutation is made in amino acids site, and screening obtains mutant N29I, N29K, P188C, P188T, P188F, Y152A
The analysis of HPLC enzyme activity shows as tendency synthesis D-Psicose.The table 1 summarized from research is analyzed, and mutant P188F only occurs
One product, i.e. D-Psicose, without generating D- sorboses.As known from Table 1, to L- rhamnulose-1-phosphate aldolases
(RhaD) gene coding amino acid makees the 29th, 188,266,152 amino acids progress saturation site-directed mutagenesis, mutant strain recombinant plasmid
pET28a-rhaDN29I、pET28a-rhaDN29K、pET28a-rhaDP188C、pET28a-rhaDP188F、pET28a-rhaDP188T、
pET28a-rhaDT266A、pET28a-rhaDY152AThe albumen catalytic reaction substrate aldehyde of translation is encoded, synthesizes D-Psicose：D-
The ratio of sorbose compares wild type (1：1) it is respectively 6：1、14：1、55：1、1：0、60：1、3.5：1、8：1.For same prominent
Become site, be different aminoacids by the site mutation, catalytic efficiency has very big difference, such as the catalytic efficiency of mutant P188F
For 100% (D-Psicose:D- sorbose=1:0), the D-Psicose of mutant P188C catalysis generations:D- sorboses
=55:1；And inventor another research shows that, mutant Y152W is then to catalyze and synthesize based on D- sorboses, mutant
The D-Psicose that Y152W is catalyzed and synthesized:D- sorbose=1：11.
1 RhaD differences mutational site of table catalyzes and synthesizes D-Psicose and D- sorbs candy output ratio
| Type | D-Psicose:D- sorboses |
| WT | 1：1 |
| RhaDY152A | 8：1 |
| RhaDN29I | 6：1 |
| RhaDN29K | 14：1 |
| RhaDP188C | 55：1 |
| RhaDP188T | 60：1 |
| RhaDP188F | 1:0 |
| RhaDT266A | 3.5:1 |
The present invention carries out rite-directed mutagenesis by the amino acid sites to RhaD, overcomes wild type RhaD and generates D- A Luo ketone
Sugar:D- sorbose=1：1, that is, there is no the defects of stereoselectivity, realize RhaD catalysis D- glyceraldehyde and synthesize D- for aldehyde receptor
Stereoselectivity during psicose so that catalysis reaction only generates D-Psicose or based on D-Psicose, by prominent
The enzyme activity of the RhaD of change is high.The RhaD of rite-directed mutagenesis prepared by the method for the present invention prepares D-Psicose, and combined coefficient is high, technique
Simply, cost-effective, D-Psicose stereoselectivity catalytic efficiency is up to 100% (i.e. product D-Psicose:D- mountains
Pears sugar=1：0), industrialization RhaD catalysis D- glyceraldehyde is realized using the method for the present invention largely synthesize D-Psicose.
It should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention and it is unrestricted, although with reference to preferable
The present invention is described in detail in embodiment, it will be understood by those of ordinary skill in the art that, it can be to the technology of the present invention
Scheme is modified or replaced equivalently, and without departing from the spirit and scope of technical solution of the present invention, should all be covered in this hair
In bright right.
Claims (10)
1. the L- rhamnulose-1-phosphate aldolases of rite-directed mutagenesis, it is characterised in that：The L- sandlwoods natural gum of the rite-directed mutagenesis
Sugar -1- phosphate aldolases are the wild type L- rhamnulose-1-phosphate aldolases encoded by SEQ ID NO.1 by gene order
The mutant generated respectively by the 29th, 188,266 or 152 saturation site-directed mutagenesis of its amino acid.
2. the L- rhamnulose-1-phosphate aldolases of rite-directed mutagenesis as described in claim 1, it is characterised in that：It is described its
The the 29th, 188,266 or 152 saturation site-directed mutagenesis of amino acid and the mutant generated, including L- rhamnulose -1- phosphoric acid aldehyde
Mutant Y152A, mutant N29K, mutant P188C, mutant P188T, mutant P188F, the mutant N29I of contracting enzyme.
3. the L- rhamnulose-1-phosphate aldolases of rite-directed mutagenesis as claimed in claim 1 or 2, it is characterised in that：It is described
Escherichia coli MG1655 bacterial strains are derived from by the wild type L- rhamnulose-1-phosphate aldolases of SEQ ID NO.1 codings.
4.DNA molecules, it is characterised in that：The L- sandlwoods natural gum of rite-directed mutagenesis described in the DNA molecular coding claim 2
Sugar -1- phosphate aldolases.
5. plasmid vector, it is characterised in that：The plasmid vector is loaded with the DNA molecular described in claim 4.
6. plasmid vector as claimed in claim 5, it is characterised in that：The plasmid vector is loads on pET-28a carriers
DNA molecular described in claim 4.
7. in claims 1 to 3 the L- rhamnulose-1-phosphate aldolases of any rite-directed mutagenesis catalyze and synthesize it is dilute
There is the application in sugared D-Psicose.
8. the preparation method of the L- rhamnulose-1-phosphate aldolases of any rite-directed mutagenesis of claims 1 to 3,
It is characterized in that：Including,
Build mutant strain recombinant plasmid：Wild type L- rhamnulose-1-phosphate aldolase genetic fragments are connected to plasmid to carry
On body, wild type L- rhamnulose-1-phosphate aldolase gene recombination plasmids are obtained, with wild type L- sandlwoods natural gum
Sugar -1- phosphate aldolases gene recombination plasmid is masterplate, to L- rhamnulose-1-phosphate aldolases gene coding amino acid point
Not carry out the 29th, 188,266 or 152 amino acids saturation site-directed mutagenesis, respectively obtain mutant strain recombinant plasmid；
Mutant strain is expressed：By the mutant strain recombinant plasmid transformed Escherichia coli, picking individual colonies inoculation, IPTG16 DEG C of induction table
Up to the L- rhamnulose-1-phosphate aldolases of rite-directed mutagenesis.
9. preparation method as claimed in claim 8, it is characterised in that：It further includes,
Protein purification：The L- rhamnulose-1-phosphate aldolases of rite-directed mutagenesis by mutant strain expression are carried out pure
Change.
10. preparation method as claimed in claim 8 or 9, it is characterised in that：The plasmid vector is pET-28a carriers, described
Mutant strain recombinant plasmid includes pET28a-rhaDN29I、pET28a-rhaDN29K、pET28a-rhaDP188C、pET28a-
rhaDP188F、pET28a-rhaDP188T、pET28a-rhaDT266A、pET28a-rhaDY152A, it is described to turn mutant strain recombinant plasmid
Change Escherichia coli, to convert e. coli strain bl21.
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