CN108410850A - L- rhamnulose-1-phosphate aldolases and its application in catalyzing and synthesizing rare sugar D- sorboses - Google Patents
L- rhamnulose-1-phosphate aldolases and its application in catalyzing and synthesizing rare sugar D- sorboses Download PDFInfo
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Abstract
Application the invention discloses 1 phosphate aldolase of L rhamnuloses and its in catalyzing and synthesizing rare sugar D sorboses, gene order are to pass through its amino acid the 115th, 116,148,188 or 152 saturation site-directed mutagenesis respectively by 1 phosphate aldolase of wild type L rhamnuloses of SEQ ID NO.1 codings.The present invention carries out rite-directed mutagenesis by the amino acid sites to RhaD, overcoming wild type RhaD and catalyzing and synthesizing D sorboses does not have the defect of stereoselectivity, it is stereoselectivity when aldehyde receptor synthesizes D sorboses to realize RhaD catalysis D glyceraldehyde, so that catalytic reaction products, based on D sorboses, 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 sorboses, and combined coefficient is high, simple for process, cost-effective, D sorbose stereoselectivity catalytic efficiencies are up to 91.7%.
Description
Technical field
The invention belongs to biotechnologies, and in particular to L- rhamnulose-1-phosphate aldolases and its catalysis close
At the application in rare sugar D- sorboses.
Background technology
Rare sugar is that content is few in nature and important physiological function is played in diet, health care, medicine and other fields.
D- sorboses (D-sorbose) are exactly a kind of rare sugar, and are reducing hyperglycemia, hypertension, and hyperlipidemia etc. plays suitable
Important role.
Content is few in nature and is difficult to synthesize by chemistry route for D- sorboses, and it is main to produce D- sorboses at present
It is to fix conversion method, the i.e. separation and purification to the clonal expression of enzyme and product using enzyme.But the D- sorboses in crude product are past
Past to be mixed with other sugar, not only yield reduces, but also the sugared physicochemical property almost phase that D- sorboses mix with it
Together, so it is difficult to refined and purification.In order to be optimized to current D- sorbose synthetic systems, must just improve in product
The ratio of D- sorboses.Early-stage study shows the L- sandlwood trees for belonging to phosphoric acid dihydroxyacetone (DHA) (DHAP) dependent form aldolase family
Glycocoll -1- phosphate aldolases (hereinafter referred to as RhaD) lose the solid to the aldehyde receptor when using D- glyceraldehyde as aldehyde receptor
Selectively, two kinds of rare sugar of D-Psicose and D- sorboses are generated, the two ratio approaches and 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 a little simplified or be omitted to avoid our department is made in this section and the description of the application and the title of the invention
Point, the purpose of abstract of description and denomination of invention it is fuzzy, 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 present invention is therein overcomes the deficiencies in the prior art, provides the L- rhamnuloses-of rite-directed mutagenesis
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
Phosphate aldolase, wherein：The L- rhamnulose-1-phosphate aldolases of the rite-directed mutagenesis are by SEQ ID by gene order
NO.1 coding wild type L- rhamnulose-1-phosphate aldolases respectively pass through its amino acid the 115th, 116,148,188 or
152 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：
Its described amino acid the 115th, 116,148,188 or 152 saturation site-directed mutagenesis and the mutant generated, including L- sandlwood natural gum
The mutant Y152W of sugar -1- phosphate aldolases, mutant Y152Q, mutant Y152N, mutant P188Y, mutant T115A,
Mutant S116A, mutant I148A.
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 catalyzing and synthesizing rare sugar D- sorboses.
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 comprising,
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 aldolase gene recombination plasmids are masterplate, to L- rhamnulose-1-phosphate aldolase gene coding amino acids
The 115th, 116,148,188 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-rhaDT115A,
pET28a-rhaDS116A、pET28a-rhaDI148A、pET28a-rhaDP188Y、pET28a-rhaDY152W、pET28a-
RhaDY152Q, pET28a-rhaDY152N, 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:Sorbose=1 D-：It is sweet to realize RhaD catalysis D- for 1, the i.e. not no defect of stereoselectivity
Oily aldehyde is stereoselectivity when aldehyde receptor synthesizes D- sorboses so that catalytic reaction products are based on D- sorboses, 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- sorboses, and combined coefficient is high, technique is simple
Single, cost-effective, D- sorbose stereoselectivity catalytic efficiencies are up to 91.7% (i.e. product D- sorboses：D- A Luo ketone
Sugar=11：1).
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, drawings in the following description are only some embodiments of the 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 be rite-directed mutagenesis of the present invention L- rhamnulose-1-phosphate aldolase mutant T115A, S116A,
I148A, P188Y, Y152N, Y152Q, Y152W and wild type L- rhamnulose-1-phosphate aldolases catalyze and synthesize the mountains D-
The HPLC atlas analysis schematic diagrames of pears sugar.
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- sorboses.
Specific implementation mode
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 implementation mode 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 different from other manner described here using other, 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 that 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
The 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
Y152N indicates the tyrosine (Y) of the 152nd, L- rhamnulose-1-phosphate aldolases (RhaD) its amino acid through satiety
It is asparagine (N) with rite-directed mutagenesis.
In the present invention, the L- rhamnulose-1-phosphate aldolases (L-Rhamnulose- in the sources Escherichia coli MG1655
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 the active regions RhaD 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 the active regions RhaD saturation site-directed mutagenesis or the monoclonal of combination rite-directed mutagenesis
It is inoculated into LB liquid medium (50 μ g/mL kanamycins), is 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 is added
Afterwards, stop culture, thalline were collected by centrifugation, abandons supernatant.Thalline ultrasonication is crushed 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 carries out concentration and desalination with super filter tube.
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 the two or more active regions RhaD 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 115th,
116,148,188,152,170,211 or 263 amino acids site, and be combined site mutation, i.e., above-mentioned single mutation
Combination of two is distinguished in site, chooses 152＆188 combination of amino acids site mutations, 152＆263 combination of amino acids site mutations, 29＆
152 amino acids combine site mutation.
Picking contains recombinant plasmid pET28a-rhaD respectivelyT115A、pET28a-rhaDS116A、pET28a-rhaDI148A、
pET28a-rhaDP188Y、pET28a-rhaDY152W、pET28a-rhaDY152Q、pET28a-rhaDY152N、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 be crushed 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 (10CV) loading afterwards to Ni2+ affinity columns, 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；
It finally uses elution buffer (50mM Tris-HCl (pH7.5), 25mM NaCl, 500mM imidazoles) elution destination protein and collects,
Eluting liquid carries out concentration and desalination with super filter tube.
The primer sequence carried out involved by rite-directed mutagenesis RhaD recombinant expression plasmids is as follows：
Mutant T115A sense primers：5'-cgaagccgtccccgctTccgaacttcc-3'(underlined sequences are prominent
Become site),
Mutant T115A downstream primers：3'-gcttcggcaggggcgaAggcttgaagg-5'(underlined sequences are prominent
Become site).
Mutant S116A sense primers：5'-gaagccgtccccactgccGaacttccg-3'(underlined sequences are prominent
Become site),
Mutant S116A downstream primers：3'-cttcggcaggggtgacggCttgaaggc-5'(underlined sequences are prominent
Become site).
Mutant I148A sense primers：5'-cacgccaccaacctggccGccctcacctatgt-3'(underlined sequences
For mutational site),
Mutant I148A downstream primers：3'-gtgcggtggttggaccggCgggagtggataca-5'(underlined sequences
For mutational site).
Mutant P188Y sense primers：5'-attttgccgtggatggtgtatUnder ggcacggacgaaatcggc-3'(
Underlined sequence is mutational site),
Mutant P188Y downstream primers：3'-taaaacggcacctaccacataUnder ccgtgcctgctttagccg-5'(
Underlined sequence is mutational site).
Mutant Y152W sense primers：5'-acctgatcgccctcacctggLower stroke of gtacttgaaaacgacacc-3'(
Line sequence is classified as mutational site),
Mutant Y152W downstream primers：3'-tggactagcgggagtggaccLower stroke of catgaacttttgctgtgg-5'(
Line sequence is classified as mutational site).
Mutant Y152Q sense primers：5'-aacctgatcgccctcacccagUnder gtacttgaaaacgacacc-3'(
Underlined sequence is mutational site),
Mutant Y152Q downstream primers：3'-ttggactagcgggagtgggtcUnder catgaacttttgctgtgg-5'(
Underlined sequence is mutational site).
Mutant Y152N sense primers：5'-cctgatcgccctcaccaatGtacttgaaaacgac-3'(underscore sequences
It is classified as mutational site),
Mutant Y152N downstream primers：3'-ggactagcgggagtggttaCatgaacttttgctg-5'(underscore sequences
It is classified as mutational site).
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 adjustings pH, it is acid that 0.25 μ L are added
Phosphatase AP, 30 DEG C stand reaction overnight.Reaction terminates using 3M NaOH adjusting pH as 7 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 Y152W, Y152Q, Y152N show D- sorbose yield be significantly higher than D-Psicose as a result, on i.e.
It states and shows the excellent stereoselectivity for catalyzing and synthesizing D- sorboses, D- by the RhaD of above three site mutation
The yield of psicose is seldom, and the enzyme activity of the RhaD by mutation is high；And RhaD mutant P188Y, T115A, S116A, I148A
Also the stereoselectivity that preferable RhaD catalysis generates D- sorboses is shown.Wherein, the mountains D- that mutant Y152W is catalyzed and synthesized
The yield highest of pears sugar.
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；Although and RhaD mutant F263A, F263A can generate a small amount of D- sorbs
Sugar, but enzyme activity is very low, and albumen is unstable.Illustrate that the mutation in the site T170A, F211C or F263A catalyzes and synthesizes RhaD
The stereoselectivity of D- sorboses can not be realized.
The combinatorial mutagenesis that inventor also studied two sites is catalyzed RhaD the stereoselectivity for generating D- sorboses
Influence, figure it is seen that the combination site mutation of N29A-Y152A still have while generating D- sorboses it is corresponding
D-Psicose generates, therefore the combination site mutation of N29A-Y152A is catalyzed the three-dimensional of generation D- sorboses for RhaD and selects
Property is worse than single site mutation.
Embodiment 3：
The stereoselectivity of RhaD aldolases is analyzed：
The study find that phosphoric acid dihydroxyacetone (DHA) (DHAP) the dependent form aldolase family in the sources Escherichia coli MG1655
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 MG1655RhaD
When Y152W, Y152Q, Y152N, P188Y, T115A, S116A, I148A are using DHAP and D- glyceraldehyde as substrate, D- is mainly generated
Sorbose, specific experiment the results are shown in Table 1.
This research includes the 115th, 116,148,188,152,170,211 to the possible more amino acid sites RhaD
Or 263 amino acids site make fixed point saturation mutation, screening obtain mutant Y152W, Y152Q, Y152N, P188Y, T115A,
The HPLC enzyme activity analysis of S116A, I148A show as tendency synthesis D- sorboses.As known from Table 1, to L- rhamnuloses -1-
Phosphate aldolase (RhaD) gene coding amino acid work the 115th, 116,148,188,152 amino acids carry out saturation fixed point and dash forward
Become, mutant strain recombinant plasmid pET28a-rhaDT115A、pET28a-rhaDS116A、pET28a-rhaDI148A、pET28a-
rhaDP188Y、pET28a-rhaDY152W、pET28a-rhaDY152Q、pET28a-rhaDY152NEncode the albumen catalytic reaction bottom of translation
Object aldehyde synthesizes D- sorboses：The ratio of D-Psicose compares wild type (1：1) it is respectively 4.3:1、5.5:1、4.5:1、
4.75:1、10.8:1、8:1、10:1.It is different aminoacids by the site mutation for identical mutation site, catalytic efficiency has very
It distinguishes greatly, such as the D- sorboses that mutant Y152W is catalyzed and synthesized：D-Psicose=11:1, and the mutant in same site
The D- sorboses that Y152Q is catalyzed and synthesized：D-Psicose=8:1；And another of inventor is studies have shown that mutant Y152A
Then to synthesize based on D-Psicose, D- sorboses that mutant Y152A is catalyzed and synthesized：D-Psicose=1:8.
Table 1RhaD differences mutational site catalyzes and synthesizes D- sorboses and D-Psicose yield ratio
| Type | D- sorboses：D-Psicose |
| WT | 1：1 |
| RhaDY152W | 11：1 |
| RhaDY152Q | 8：1 |
| RhaDY152N | 10：1 |
| RhaDP188Y | 4.75：1 |
| RhaDT115A | 4.3：1 |
| RhaDS116A | 5.5：1 |
| RhaDI148A | 4.5：1 |
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:Sorbose=1 D-：1, the i.e. not no defect of stereoselectivity, it is that aldehyde receptor synthesizes D- to realize RhaD catalysis D- glyceraldehyde
Stereoselectivity when sorbose so that for catalytic reaction products based on D- sorboses, the enzyme activity of the RhaD by mutation is high.This
The RhaD of rite-directed mutagenesis prepared by inventive method prepares D- sorboses, and combined coefficient is high, simple for process, cost-effective, D- sorbs
Sugared stereoselectivity catalytic efficiency is up to 91.7% (i.e. product D- sorboses：D-Psicose=11：1).
It should be noted that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although with reference to preferable
Embodiment describes the invention in detail, 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 of the technical scheme of the invention and range, 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- sandlwood natural gum of the rite-directed mutagenesis
Sugar -1- phosphate aldolases are by the wild type L- rhamnulose-1-phosphate aldolases of SEQ ID NO.1 codings by gene order
The mutant generated by its amino acid the 115th, 116,148,188 or 152 saturation site-directed mutagenesis respectively.
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
Amino acid the 115th, 116,148,188 or 152 saturation site-directed mutagenesis and the mutant generated, including L- rhamnuloses -1-
The mutant Y152W of phosphate aldolase, mutant Y152Q, mutant Y152N, mutant P188Y, mutant T115A, mutation
Body S116A, mutant I148A.
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- sandlwood 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 to be loaded 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- sorboses.
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 aldolase gene recombination plasmids are masterplate, to L- rhamnulose-1-phosphate aldolase gene coding amino acids point
Not carry out the 115th, 116,148,188 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, 16 DEG C of inductions of IPTG
Express the L- rhamnulose-1-phosphate aldolases of rite-directed mutagenesis.
9. preparation method as claimed in claim 8, it is characterised in that：Further include,
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-rhaDT115A、pET28a-rhaDS116A、pET28a-rhaDI148A、pET28a-
rhaDP188Y、pET28a-rhaDY152W、pET28a-rhaDY152Q、pET28a-rhaDY152N, described to turn mutant strain recombinant plasmid
Change Escherichia coli, to convert e. coli strain bl21.
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