CN103589697A - One group of cyclodextrine glucosyltransferase as well as coding gene and application thereof - Google Patents
One group of cyclodextrine glucosyltransferase as well as coding gene and application thereof Download PDFInfo
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Abstract
The invention discloses one group of cyclodextrine glucosyltransferase as well as a coding gene and application thereof. The cyclodextrine glucosyltransferase is derived from bacillus macerans. The gene sequence of the cyclodextrine glucosyltransferase is mutated, so that four mutant proteins are obtained. The research and comparison of the mutant protein in degrading starch and preparing cyclodextrine are carried out. Results show that the cyclodextrine glucosyltransferase obtained through mutation of certain sites has high specificity in alpha-CD production, has higher production efficiency, and has great value in industrial production.
Description
Technical field
The present invention relates to one group of cyclodextrine Transglucosylase and encoding gene and application.
Background technology
The English of cyclodextrine is cyclodextrin, is called for short CD.Cyclodextrin is the product that cyclodextrin transglucosylase (CGTase) acts on starch, that six above glucose are with α-1, the member cyclic oligosaccharides that 4 glycosidic links link, wherein the most common, most study is alpha-cylodextrin (α-CD), beta-cyclodextrin (β-CD), γ-cyclodextrin (γ-CD), six, seven and eight glucose molecules, consisting of respectively, is the molecule of relatively large and relative flexibility.
Cyclohexaamylose is the high-valued product of a class of starch deep processing, the cyclic oligosaccharide being formed by connecting by α-Isosorbide-5-Nitrae glucoside bond by six glucosyl groups.Structurally, α-CD presents the three-dimensional arrangement of round shape (great ， one end, one end is little), has the special property of " outer hydrophilic, interior hydrophobic ".Rely on its special three-dimensional arrangement, in function, make it adopt proper method to carry out inclusion with multiple guest compound, thereby change by physico-chemical properties such as the solubleness of inclusion material, volatility and chemical reactivities, its safety non-toxic, makes it have some very useful functions in addition.
α-CD is a kind of potential foodstuff additive, particularly can be used as the substitute products of the cycloheptaamylose (β-CD) of having produced at present.β-CD has scale operation, but in use finds that it has the risk of internal organs deposition, and the problem of inclusion inefficiency.
Sum up, α-CD has following application prospect in foodstuffs industry: (1) makes by water-insoluble or the low compound of solubleness the encapsulation compound that water solubility is high; (2) compound that makes to seal has satisfactory stability (as protected look, protect fragrant, heat-resisting, acidproof, hydrolysis, anti-oxidant, volatilization prevention etc.); (3) shielding effect (covering unhappy smell and bitter taste in food); (4) remove unwanted composition (as caffeine, cholesterol etc.) in food; (5) there is emulsification, foaming effect, can make emulsifying agent and whipping agent; (6) liquid, oily, volatile material are solidified.
At present, the biggest obstacle of α-CD development and application is to lack to have the zymin that high alpha-cylodextrin transforms vigor, makes the cost and price of this product exceed three times of β-CD, has limited its large-scale application.
The generation of α-CD, is mainly to take starch as raw material, through the glycosyl transformation of cyclodextrine Transglucosylase (cyclodextrin glucano-transferase is called for short CGTases).The specificity of CGTases is lower, in most of the cases produces tri-kinds of products of α-CD, β-CD and γ-CD simultaneously.Three kinds of proportion of products differences that adopt different CGTases to produce, the main converted product of existing CGTases is β-CD, also produces a certain amount of α-and γ-CD simultaneously.Existing research direction is mainly two aspects: the first is sought catalytic effect better or the higher enzyme of specificity from natural resources; Thereby it two is existing zymoprotein to be carried out to directional transformation obtain the better or higher enzyme of specificity of catalytic effect.
Summary of the invention
The object of this invention is to provide one group of cyclodextrine Transglucosylase and encoding gene and application.
Cyclodextrine Transglucosylase provided by the invention, from soft rotten genus bacillus (Bacillus macerans), is that protein shown in the sequence of sequence table 1 is carried out to following (a) and (b), (c) and (d) four protein that sudden change obtains:
(a) sequence of sequence table 1 is sported to three continuous Histidines from N-terminal the 167th amino acids residue by tyrosine
(b) sequence of sequence table 1 is sported to three continuous Histidines from N-terminal the 167th amino acids residue by tyrosine, the 195th amino acids residue is sported to phenylalanine by tyrosine simultaneously;
(c) sequence of sequence table 1 is sported to arginine from N-terminal the 89th amino acids residue by tyrosine, the 167th amino acids residue is sported to three continuous Histidines by tyrosine simultaneously, and the 195th amino acids residue is sported to phenylalanine by tyrosine.
(d) sequence of sequence table 1 is sported to arginine from N-terminal the 89th amino acids residue by tyrosine, the 167th amino acids residue is sported to three continuous Histidines by tyrosine simultaneously, by the 180th amino acids residue, by glycine mutation, be leucine simultaneously, and the 195th amino acids residue is sported to phenylalanine by tyrosine.
The gene of code for said proteins also belongs to protection scope of the present invention.
Described gene specifically can be protein shown in the sequence of sequence table 2 is carried out as follows to (e), (f), (g) and (h) four DNA moleculars that sudden change obtains:
(e) sequence of sequence table 2 is sported to CACCACCAC from 5 ' end 499-501 position Nucleotide by TAC
(f) sequence of sequence table 2 is sported to CACCACCAC from 5 ' end 499-501 position Nucleotide by TAC, 583-585 position Nucleotide is sported to CGA by TAT simultaneously;
(g) sequence of sequence table 2 is sported to CGA from 5 ' end 265-267 position Nucleotide by TAT, 499-501 position Nucleotide is sported to CACCACCAC by TAC simultaneously, and 583-585 position Nucleotide is sported to TTC by TAC.
(h) sequence of sequence table 2 is sported to CGA from 5 ' end 265-267 position Nucleotide by TAT, 499-501 position Nucleotide is sported to CACCACCAC by TAC simultaneously, 538-540 position Nucleotide is sported to CTC by GGG, and 583-585 position Nucleotide is sported to TTC by TAC.
The recombinant expression vector that contains described gene, expression cassette, transgenic cell line or recombinant bacterium all belong to protection scope of the present invention.
Described recombinant expression vector specifically can be the recombinant plasmid that the multiple clone site of described gene insertion vector pET-22b (+) is obtained.Described recombinant expression vector specifically can be the recombinant plasmid obtaining between the BamHI of described gene insertion vector pET-22b (+) and XhoI restriction enzyme site.
Described recombinant bacterium specifically can be described recombinant expression vector is imported to the recombinant bacterium that intestinal bacteria obtain.Described intestinal bacteria specifically can be e. coli bl21 (DE3).
The present invention also protects the application of described protein, is following (I) or (II) or (III):
(I) prepares cyclodextrine Transglucosylase;
(II) degraded starch (as Zulkovsky starch);
(III) produces cyclohexaamylose.
Protein provided by the invention, the specificity of producing α-CD is higher, and production efficiency is higher, has great value in industrial production.
Accompanying drawing explanation
Fig. 1 is the structural representation of recombinant plasmid first.
Fig. 2 is α-CD typical curve.
Fig. 3 is β-CD typical curve.
Fig. 4 is γ-CD typical curve
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment, results averaged for three times.Zulkovsky starch: modern east, Beijing fine chemicals company limited, lot number: 20070216.Carrier pET-22b (+): Novagen, catalog number is 69744-3.E. coli bl21 (DE3): Beijing Quanshijin Biotechnology Co., Ltd, catalog number is CD601-01.
TB substratum (g/L): Tryptones 12, yeast extract cream 24, glycerine 4mL, distilled water constant volume.
The discovery of embodiment 1, mutain and encoding gene thereof
From nature, screen the soft rotten genus bacillus of a strain (Bacillus macerans), can produce Maltose 4-glucosyltransferase, Maltose 4-glucosyltransferase gene in this bacterial strain as shown in the sequence 2 of sequence table, the protein shown in the sequence 1 of code sequence list.
By the α-CGTase-1 of protein called after shown in sequence 1 albumen, by its encoding gene called after α-CGTase-1 gene (as shown in sequence 2).By the α-CGTase-WT of protein called after shown in sequence 3 albumen (disclosed sequence in NCBI), by its encoding gene called after α-CGTase-WT gene (as shown in sequence 4).
The double-stranded DNA shown in the sequence 1 of sequence table of take is template, and 167 amino acids are suddenlyd change, and obtains a mutant; 167 and 195 amino acids are carried out to rite-directed mutagenesis simultaneously, obtain a mutant; 89,167 and 195 amino acids are carried out to rite-directed mutagenesis simultaneously, obtain a mutant; 89,167,180 and 195 amino acids are suddenlyd change simultaneously, obtain a mutant.By the albumen of each genetic expression in mutant library is carried out, enzyme is alive to be identified, has found that the product specificity of a series of enzymes is different from mutain and the encoding gene thereof of the albumen of α-CGTase-WT shown in sequence 1.
Embodiment 2, enzyme are lived and are identified
One, the following double chain DNA molecule of difference synthetic:
(1) double chain DNA molecule 1: with the difference of DNA molecular shown in the sequence 2 of sequence table be will be from 5 ' end 499-501 position Nucleotide by TAC sudden change for CACCACCAC; Accordingly the 167th amino acids residue in sequence 1 is sported to three continuous Histidines by tyrosine.The albumen called after albumen 1 of double chain DNA molecule 1 coding.
(2) double chain DNA molecule 2: be by TAC, to sport CACCACCAC from 5 ' end 499-501 position Nucleotide with the difference of DNA molecular shown in the sequence 2 of sequence table, and will by TAT, sport CGA from 5 ' end 583-585 position Nucleotide; Accordingly the 167th amino acids residue in sequence 1 is sported to three continuous Histidines by tyrosine, and the 195th amino acids residue is sported to phenylalanine by tyrosine.The albumen called after albumen 2 of double chain DNA molecule 2 codings.
(3) double chain DNA molecule 3: be by TAT, to sport CGA from 5 ' end 265-267 position Nucleotide with the difference of DNA molecular shown in the sequence 2 of sequence table, to by TAC, sport CACCACCAC from 5 ' end 499-501 position Nucleotide simultaneously, and will by TAC, sport TTC from 5 ' end 583-585 position Nucleotide; Accordingly the 89th amino acids residue in sequence 1 is sported to arginine by tyrosine, the 167th amino acids residue is sported to three continuous Histidines by tyrosine simultaneously, and the 195th amino acids residue is sported to phenylalanine by tyrosine.The albumen called after albumen 3 of double chain DNA molecule 3 codings.
(4) double chain DNA molecule 4: be by TAT, to sport CGA from 5 ' end 265-267 position Nucleotide with the difference of DNA molecular shown in the sequence 2 of sequence table, to by TAC, sport CACCACCAC from 5 ' end 499-501 position Nucleotide simultaneously, and will by GGG, sport CTC from 5 ' end 538-540 position Nucleotide, and will by TAC, sport TTC from 5 ' end 583-585 position Nucleotide; Accordingly the 89th amino acids residue in sequence 1 is sported to arginine by tyrosine, the 167th amino acids residue is sported to three continuous Histidines by tyrosine simultaneously, by the 180th amino acids residue, by glycine mutation, be leucine, and the 195th amino acids residue is sported to phenylalanine by tyrosine.The albumen called after albumen 3 of double chain DNA molecule 3 codings.
(5) double chain DNA molecule 5: i.e. double chain DNA molecule shown in the sequence 2 of sequence table.
(6) double chain DNA molecule 6: i.e. double chain DNA molecule shown in the sequence 4 of sequence table.
Two, the structure of recombinant plasmid (structural representation is shown in Fig. 1)
1, design pair of primers, is comprised of S2 and A3.
S2：5’-CGC
GGATCCG
-3’；
A3：5'-CGG
CTCGAG 
-3’。
In S2, underscore mark BamHI restriction endonuclease recognition sequence, the region that square frame mark is corresponding with target sequence.In A3, underscore mark XhoI restriction endonuclease recognition sequence, the region that square frame mark is corresponding with target sequence.
2, take respectively each synthetic double chain DNA molecule of step 2 is template, with the primer pair of S2 and A3 composition, carries out pcr amplification, obtains pcr amplification product.
3, with the pcr amplification product of restriction enzyme BamHI and XhoI double digestion step (1), obtain enzyme and cut product.
4, with restriction enzyme BamHI and XhoI double digestion carrier pET-22b (+), reclaim carrier framework (about 5.4kb).
5, the enzyme of step (3) is cut to product and be connected with the carrier framework of step (4), obtain recombinant plasmid.
According to the numbering of double chain DNA molecule, called after recombinant plasmid 1 is to recombinant plasmid 6 successively.
According to sequencing result, recombinant plasmid 1 is carried out to structrual description as follows: between the BamHI of carrier pET-22b (+) and XhoI restriction enzyme site, inserted double chain DNA molecule 1.
According to sequencing result, recombinant plasmid 2 is carried out to structrual description as follows: between the BamHI of carrier pET-22b (+) and XhoI restriction enzyme site, inserted double chain DNA molecule 2.
According to sequencing result, recombinant plasmid 3 is carried out to structrual description as follows: between the BamHI of carrier pET-22b (+) and XhoI restriction enzyme site, inserted double chain DNA molecule 3.
According to sequencing result, recombinant plasmid 4 is carried out to structrual description as follows: between the BamHI of carrier pET-22b (+) and XhoI restriction enzyme site, inserted double chain DNA molecule 4.
According to sequencing result, recombinant plasmid 5 is carried out to structrual description as follows: between the BamHI of carrier pET-22b (+) and XhoI restriction enzyme site, inserted double chain DNA molecule 5.
According to sequencing result, recombinant plasmid 5 is carried out to structrual description as follows: between the BamHI of carrier pET-22b (+) and XhoI restriction enzyme site, inserted double chain DNA molecule 6.
Three, the structure of recombinant bacterium
Each recombinant plasmid that step 2 is built imports respectively e. coli bl21 (DE3), obtains each recombinant bacterium.According to the numbering of recombinant plasmid, called after recombinant bacterium 1 is to recombinant bacterium 6 successively.
Four, the fermentation of recombinant bacterium
Each recombinant bacterium that step 3 is built is seeded to respectively in TB substratum, and 37 ℃, 220rpm shaking culture are to OD600
nm=0.6 (0.6-0.8 all can); Add IPTG, making its concentration in culture system is 0.01mM, then 16 ℃, 220rpm shaking culture 96h; By 4 ℃ of culture systems, centrifugal 10 minutes of 8000rpm, collect supernatant liquor.
According to the numbering of recombinant bacterium, called after supernatant liquor 1 is to supernatant liquor 6 successively.
Five, enzyme is lived and is identified and product analysis
1, enzyme activity determination
(1) the Zulkovsky starch aqueous solution of preparation 0.25g/100mL.
(2) experimental group: add the 0.4mL Zulkovsky starch aqueous solution in test tube, be incubated 15min in 40 ℃ of water-baths, then add 0.1mL solution to be measured; Control group group: first added the 1.5mL0.1mol/L HCl aqueous solution before adding solution to be measured, other same experimental group; Experimental group and control group mix to be placed in 40 ℃ of waters bath with thermostatic control and are incubated 10min, and then experimental group is added the 1.5mL0.1mol/L HCl aqueous solution; Add 3mL0.1mol/L I
2liquid (solvent is water) and add 5mL distilled water, mixes, and rapidly in 700nm place photometry absorption value, and records experimental data.
Enzyme (U/mL)=(a-b)/a * 100 * extension rate alive; A is the light absorption value of control group, the light absorption value that b is experimental group.
2, product analysis
Each supernatant liquor respectively step 4 being obtained (solution to be measured) is tested as follows: the Zulkovsky starch aqueous solution of 10g/100mL is boiled to 10min; After cooling, get 2mL and add in new EP pipe, then by 400U/g substrate, add solution to be measured, with distilled water, be settled to 4mL, then in 40 ℃ of water-baths standing 24 hours; Then boil 10min, the centrifugal 10min of 12000rpm, gets supernatant; Supernatant is carried out to HPLC analysis with getting 20uL after 0.45um ultrafiltration membrance filter.
The parameter that HPLC analyzes: Waters600HPLC chromatographic instrument, Waters manual injector, chromatographic column Lichrosorb NH2 (4.6mm * 150mm), Waters2414 differential detector, moving phase is comprised of 73 parts by volume acetonitriles and 27 parts by volume water, flow velocity 1mL/min, 40 ℃ of column temperatures.
Use respectively α-CD standard substance, β-CD standard substance and γ-CD standard substance production standard curve.The appearance time of α-CD standard substance is 8.5min, and typical curve is shown in Fig. 2, and typical curve equation is y=1081101x-3448, R2=0.9999, and x represents the concentration (g/100ml) of α-CD, y represents peak area.The appearance time of β-CD standard substance is 9.8min, and typical curve is shown in Fig. 3, and typical curve equation is y=1080914x-4309, R2=0.9998, and x represents the concentration (g/100ml) of β-CD, y represents peak area.The appearance time of γ-CD standard substance is 10.7min, and typical curve is shown in Fig. 4, and typical curve equation is y=1040306x-7683, R2=0.9982, and x represents the concentration (g/100ml) of γ-CD, y represents peak area.The concentration ÷ of concentration=α-CD of α-CD (concentration of concentration+γ-CD of concentration+β-CD of α-CD) * 100%.The concentration ÷ of concentration=β-CD of β-CD (concentration of concentration+γ-CD of content+β-CD of α-CD) * 100%, the concentration ÷ of concentration=γ-CD of γ-CD (concentration of concentration+γ-CD of content+β-CD of α-CD) * 100%.
Adopt supernatant liquor 1 to carry out step 5 and obtain in supernatant, the percentage composition that the percentage composition that the percentage composition of α-CD is 79.5 ± 3.7%, β-CD is 14.2 ± 3.2%, γ-CD is 6.1% ± 0.9%.The percentage composition ratio of α-CD and β-CD is 5.8 ± 0.5.（Y167HHH）
Adopt supernatant liquor 2 to carry out step 5 and obtain in supernatant, the percentage composition that the percentage composition that the percentage composition of α-CD is 79.9 ± 0.7%, β-CD is 13.6 ± 0.9%, γ-CD is 6.5% ± 0.4%.The percentage composition ratio of α-CD and β-CD is 5.9 ± 0.4.（Y167HHH/Y195F）
Adopt supernatant liquor 3 to carry out step 5 and obtain in supernatant, the percentage composition that the percentage composition that the percentage composition of α-CD is 83.2 ± 1.9%, β-CD is 12.8 ± 1.4%, γ-CD is 4.0% ± 0.6%.The percentage composition ratio of α-CD and β-CD is 6.6 ± 0.8.(Y89R/Y167HHH/Y195F)
Adopt supernatant liquor 4 to carry out step 5 and obtain in supernatant, the percentage composition that the percentage composition that the percentage composition of α-CD is 72.1 ± 1.3%, β-CD is 23.9 ± 1.2%, γ-CD is 4.0% ± 0.2%.The percentage composition ratio of α-CD and β-CD is 3.0 ± 0.1%.(Y89R/Y167HHH/G180L/Y195F)
Adopt supernatant liquor 5 to carry out step 5 and obtain in supernatant, the percentage composition that the percentage composition that the percentage composition of α-CD is 65.4 ± 1.5%, β-CD is 21.2 ± 0.2%, γ-CD is 13.1 ± 0.8%.The percentage composition ratio of α-CD and β-CD is 3.1 ± 0.7%.（Y167）?。
Claims (8)
1. protein shown in the sequence of sequence table 1 is carried out to following (a) and (b), (c) and (d) four protein that sudden change obtains:
(a) sequence of sequence table 1 is sported to three continuous Histidines from N-terminal the 167th amino acids residue by tyrosine;
(b) sequence of sequence table 1 is sported to three continuous Histidines from N-terminal the 167th amino acids residue by tyrosine, the 195th amino acids residue is sported to phenylalanine by tyrosine simultaneously;
(c) sequence of sequence table 1 is sported to arginine from N-terminal the 89th amino acids residue by tyrosine, the 167th amino acids residue is sported to three continuous Histidines by tyrosine simultaneously, and the 195th amino acids residue is sported to phenylalanine by tyrosine.
(d) sequence of sequence table 1 is sported to arginine from N-terminal the 89th amino acids residue by tyrosine, the 167th amino acids residue is sported to three continuous Histidines by tyrosine simultaneously, by the 180th amino acids residue, by glycine mutation, be leucine simultaneously, and the 195th amino acids residue is sported to phenylalanine by tyrosine.
2. the gene of protein described in the claim 1 of encoding.
3. gene as claimed in claim 2, is characterized in that: described gene carries out as follows (e), (f), (g) and (h) four DNA moleculars that sudden change obtains by protein shown in the sequence of sequence table 2:
(e) sequence of sequence table 2 is sported to CACCACCAC from 5 ' end 499-501 position Nucleotide by TAC
(f) sequence of sequence table 2 is sported to CACCACCAC from 5 ' end 499-501 position Nucleotide by TAC, 583-585 position Nucleotide is sported to CGA by TAT simultaneously;
(g) sequence of sequence table 2 is sported to CGA from 5 ' end 265-267 position Nucleotide by TAT, 499-501 position Nucleotide is sported to CACCACCAC by TAC simultaneously, and 583-585 position Nucleotide is sported to TTC by TAC.
(h) sequence of sequence table 2 is sported to CGA from 5 ' end 265-267 position Nucleotide by TAT, 499-501 position Nucleotide is sported to CACCACCAC by TAC simultaneously, 538-540 position Nucleotide is sported to CTC by GGG, and 583-585 position Nucleotide is sported to TTC by TAC.
4. the recombinant expression vector, expression cassette, transgenic cell line or the recombinant bacterium that contain gene described in claim 2 or 3.
5. recombinant expression vector as claimed in claim 4, is characterized in that: the recombinant plasmid of described recombinant expression vector for the multiple clone site of described gene insertion vector pET-22b (+) is obtained.
6. recombinant bacterium as claimed in claim 4, is characterized in that: described recombinant bacterium is that recombinant expression vector described in claim 7 is imported to the recombinant bacterium that intestinal bacteria obtain.
7. preparing a method of protein described in claim 1, is to cultivate recombinant bacterium described in claim 4 or 6, obtains protein described in claim 1.
8. the application of protein described in claim 1, is following (I) or (II) or (III):
(I) prepares cyclodextrine Transglucosylase;
(II) degraded starch;
(III) produces cyclohexaamylose.
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| CN103232981A (en) * | 2013-05-06 | 2013-08-07 | 中国科学院微生物研究所 | A group of cyclodextrine glucosyltransferases and encoding gene and application thereof |
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| CN103232981A (en) * | 2013-05-06 | 2013-08-07 | 中国科学院微生物研究所 | A group of cyclodextrine glucosyltransferases and encoding gene and application thereof |
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