CN103232981B - A group of cyclodextrine glucosyltransferases and encoding gene and application thereof - Google Patents
A group of cyclodextrine glucosyltransferases and encoding gene and application thereof Download PDFInfo
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- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
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- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a group of cyclodextrine glucosyltransferases and an encoding gene and application of the group of cyclodextrine glucosyltransferases.. The cyclodextrine glucosyltransferases provided by the invention are characterized in that a protein as shown in a sequence 1 in a sequence table is the protein obtained by mutating as shown in (a) and (b): (a) mutating from tyrosine to histidine since the 167th amino acid residue at the N tail end as shown in the sequence 1 in the sequence table; and (b) mutating from the 195th amino acid residue at N tail end as shown in the sequence 1 in the sequence table. By adoption of the protein, the influence of the 195th amino acid in a catalytic center on the catalyzing specificity of Alpha-CGTase is researched, which brings a significant value for the study on the catalytic mechanism of the CGTase.
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 link links, 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.
The generation of cyclodextrin, is mainly to take starch as raw material, through the glycosyl transformation of cyclodextrine Transglucosylase (cyclodextringlucano-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.
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, the three-dimensional arrangement of α-CD is cylindrical, and (one end is large, one end is little), there is the singularity of " outer hydrophilic; interior hydrophobic " and nontoxic premium properties, can 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, some very useful functions are provided.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.
In three kinds of cyclodextrin, beta-cyclodextrin is most widely used.Beta-cyclodextrin (β-Cyclodextyin) is the ring texture compound being become continuously by 7 glucose molecules, a main body configuration picture middle cylinder that has cavity, two ends not to seal.In empty structure, in cavity, owing to being subject to the shielding effect of c h bond, formed hydrophobic region.Larger opening end (upper end) consists of the secondary hydroxyl of C2 and C3, and smaller opening end (lower end) consists of the primary hydroxyl of C6, has wetting ability.There is good thermostability, be heated to approximately 200 ℃ and start to decompose.Because it is without reducing end, there is no reductibility; Easily form various stable hydrates, no hygroscopicity; Can crystallization well in alcohol and the aqueous solution.The cyclodextrin of cyclodextrin interlinkage can being take on polymkeric substance carries out chemical modification as monomer carries out polymerization Huo Jiang functional group interlinkage on cyclodextrin molecular.Utilize this special barrel structure; beta-cyclodextrin can be combined into host-guest inclusion thing with many inorganic, organic molecules; and can change by the chemistry of inclusion compound and physical properties; there is the characteristic of protection, stable, solubilising guest molecule and selectivity oriented molecule, thereby be all widely used at aspects such as food, environment, medicine, Polymer Synthesizing, toiletry, chemical detection.
γ-cyclodextrin has the cavity larger than beta-cyclodextrin, so the guest molecule that cavity can inclusion is wider.γ-cyclodextrin has better more water-soluble than beta-cyclodextrin, and during 25 ℃ of room temperatures, γ-cyclodextrin solubleness is 25.6g/100ml, and alpha-cylodextrin solubleness is 12.7g/100ml water, and beta-cyclodextrin solubleness only has 1.88g/100ml water.γ-cyclodextrin application prospect is more more wide than beta-cyclodextrin, but at present because industrial scale is less, so price is higher, has limited production and the application of γ-cyclodextrin.In 3 kinds of cyclodextrin, γ-cyclodextrin inner chamber is maximum, has the ability of embedding guest molecule widely, and its higher water-soluble, outstanding emulsification property and security, makes it in fields such as food, pharmacy and makeup, have very large application space.
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 is that protein shown in the sequence of sequence table 1 is carried out to (a) and (b) two protein that sudden change obtains as follows:
(a) sequence of sequence table 1 is sported to Histidine from N-terminal the 167th amino acids residue by tyrosine;
(b) sequence of sequence table 1 is suddenlyd change from N-terminal the 195th amino acids residue.
In described (b), described sudden change can be any one in (1) to (16) as follows:
(1) sequence of sequence table 1 is sported to arginine from N-terminal the 195th amino acids residue by tyrosine;
(2) sequence of sequence table 1 is sported to Methionin from N-terminal the 195th amino acids residue by tyrosine;
(3) sequence of sequence table 1 is sported to leucine from N-terminal the 195th amino acids residue by tyrosine;
(4) sequence of sequence table 1 is sported to α-amino-isovaleric acid from N-terminal the 195th amino acids residue by tyrosine;
(5) sequence of sequence table 1 is sported to Isoleucine from N-terminal the 195th amino acids residue by tyrosine;
(6) sequence of sequence table 1 is sported to l-asparagine from N-terminal the 195th amino acids residue by tyrosine;
(7) sequence of sequence table 1 is sported to Serine from N-terminal the 195th amino acids residue by tyrosine;
(8) sequence of sequence table 1 is sported to halfcystine from N-terminal the 195th amino acids residue by tyrosine;
(9) sequence of sequence table 1 is sported to glycine from N-terminal the 195th amino acids residue by tyrosine;
(10) sequence of sequence table 1 is sported to L-glutamic acid from N-terminal the 195th amino acids residue by tyrosine;
(11) sequence of sequence table 1 is sported to tryptophane from N-terminal the 195th amino acids residue by tyrosine;
(12) sequence of sequence table 1 is sported to aspartic acid from N-terminal the 195th amino acids residue by tyrosine;
(13) sequence of sequence table 1 is sported to Histidine from N-terminal the 195th amino acids residue by tyrosine;
(14) sequence of sequence table 1 is sported to proline(Pro) from N-terminal the 195th amino acids residue by tyrosine;
(15) sequence of sequence table 1 is sported to methionine(Met) from N-terminal the 195th amino acids residue by tyrosine;
(16) sequence of sequence table 1 is sported to phenylalanine from N-terminal the 195th amino acids residue by tyrosine.
Said mutation if no special instructions, is single amino acids residue.
The gene of code for said proteins also belongs to protection scope of the present invention.
Described gene can be protein shown in the sequence of sequence table 2 is carried out to (c) and (d) two DNA moleculars that sudden change obtains as follows:
(c) sequence of sequence table 2 is sported to CAC from 5 ' end 499-501 position Nucleotide by TAC;
(d) sequence of sequence table 2 is suddenlyd change from 5 ' end 583-585 position Nucleotide.
Described sudden change in described (d) can be any one in (1) to (16) as follows:
(1) sequence of sequence table 2 is sported to CGC from 5 ' end 583-585 position Nucleotide by TAC;
(2) sequence of sequence table 2 is sported to AAA from 5 ' end 583-585 position Nucleotide by TAC;
(3) sequence of sequence table 2 is sported to CTA from 5 ' end 583-585 position Nucleotide by TAC;
(4) sequence of sequence table 2 is sported to GTA from 5 ' end 583-585 position Nucleotide by TAC;
(5) sequence of sequence table 2 is sported to ATA from 5 ' end 583-585 position Nucleotide by TAC;
(6) sequence of sequence table 2 is sported to AAC from 5 ' end 583-585 position Nucleotide by TAC;
(7) sequence of sequence table 2 is sported to AGC from 5 ' end 583-585 position Nucleotide by TAC;
(8) sequence of sequence table 2 is sported to TGC from 5 ' end 583-585 position Nucleotide by TAC.
(9) sequence of sequence table 2 is sported to GGG from 5 ' end 583-585 position Nucleotide by TAC;
(10) sequence of sequence table 2 is sported to GAA from 5 ' end 583-585 position Nucleotide by TAC;
(11) sequence of sequence table 2 is sported to TGG from 5 ' end 583-585 position Nucleotide by TAC;
(12) sequence of sequence table 2 is sported to GAC from 5 ' end 583-585 position Nucleotide by TAC;
(13) sequence of sequence table 2 is sported to CAC from 5 ' end 583-585 position Nucleotide by TAC;
(14) sequence of sequence table 2 is sported to CCG from 5 ' end 583-585 position Nucleotide by TAC;
(15) sequence of sequence table 2 is sported to ATG from 5 ' end 583-585 position Nucleotide by TAC;
(16) sequence of sequence table 2 is sported to TTC from 5 ' end 583-585 position Nucleotide 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 a kind of described method of protein of preparing, and is to cultivate described recombinant bacterium, obtains described protein.The process of described cultivation is specific as follows: described recombinant bacterium is seeded to 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.
The present invention also protects the application of described protein, is following (I), (II), (III) or (IV):
(I) prepares cyclodextrine Transglucosylase;
(II) degraded starch (as Zulkovsky starch);
(III) produces cyclooctaamylose;
(IV) produces cyclohexaamylose.
Protein provided by the invention, has studied and has been positioned at catalytic center 195 amino acids for the narrow spectrum impact of α-CGTase catalysis, and the catalytic mechanism research of CGTase is had to great value.
Accompanying drawing explanation
Fig. 1 is the structural representation of recombinant plasmid.
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: Tryptones 12g, yeast extract cream 24g, glycerine 4mL, and distilled water is settled to 1L.
α-CD standard substance: the MB3077 of Dalian Mei Lun biotech firm.
β-CD standard substance: the MB1895 of Dalian Mei Lun biotech firm.
γ-CD standard substance: the MB5536 of Dalian Mei Lun biotech firm.
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).
Sequence 1 is only the 590th amino acids residue (M/V) and the 677th amino acids residue (S/G) with the difference of sequence 3.Sequence 2 is only the 1768th Nucleotide (A/G) and the 2029th Nucleotide (A/G) with the difference of sequence 4.
The double-stranded DNA shown in the sequence 1 of sequence table of take is template, carries out 195 saturation mutations, obtains comprising 17 mutant of deletion mutantion.By the albumen that each mutant gene is expressed, carry out enzyme evaluation alive, found that the product specificity of a series of enzymes is different from the mutain of the albumen of α-CGTase-1 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: be will be from 5 ' end 583-585 position Nucleotide (TAC) disappearance with the difference of DNA molecular shown in the sequence 2 of sequence table, and will by TAC, sport CAC from 5 ' end 499-501 position Nucleotide; Accordingly the 195th tyrosine in sequence 1 is lacked, and the 167th amino acids residue is sported to Histidine by tyrosine.The albumen called after albumen 1 of double chain DNA molecule 1 coding.
(2) double chain DNA molecule 2: with the difference of DNA molecular shown in the sequence 2 of sequence table be will be from 5 ' end 583-585 position Nucleotide by TAC sudden change for TTC, and will by TAC, sport CAC from 5 ' end 499-501 position Nucleotide; Accordingly the 195th amino acids residue in sequence 1 is sported to phenylalanine by tyrosine, and the 167th amino acids residue is sported to Histidine by tyrosine.The albumen called after albumen 2 of double chain DNA molecule 2 codings.
(3) double chain DNA molecule 3: with the difference of DNA molecular shown in the sequence 2 of sequence table be will be from 5 ' end 583-585 position Nucleotide by TAC sudden change for CAC, and will by TAC, sport CAC from 5 ' end 499-501 position Nucleotide; Accordingly the 195th amino acids residue in sequence 1 is sported to Histidine by tyrosine, and the 167th amino acids residue is sported to Histidine by tyrosine.The albumen called after albumen 3 of double chain DNA molecule 3 codings.
(4) double chain DNA molecule 4: with the difference of DNA molecular shown in the sequence 2 of sequence table be will be from 5 ' end 583-585 position Nucleotide by TAC sudden change for CCG, and will by TAC, sport CAC from 5 ' end 499-501 position Nucleotide; Accordingly the 195th amino acids residue in sequence 1 is sported to proline(Pro) by tyrosine, and the 167th amino acids residue is sported to Histidine by tyrosine.The albumen called after albumen 4 of double chain DNA molecule 4 codings.
(5) double chain DNA molecule 5: with the difference of DNA molecular shown in the sequence 2 of sequence table be will be from 5 ' end 583-585 position Nucleotide by TAC sudden change for TGG, and will by TAC, sport CAC from 5 ' end 499-501 position Nucleotide; Accordingly the 195th amino acids residue in sequence 1 is sported to tryptophane by tyrosine, and the 167th amino acids residue is sported to Histidine by tyrosine.The albumen called after albumen 5 of double chain DNA molecule 5 codings.
(6) double chain DNA molecule 6: with the difference of DNA molecular shown in the sequence 2 of sequence table be will be from 5 ' end 583-585 position Nucleotide by TAC sudden change for GGG, and will by TAC, sport CAC from 5 ' end 499-501 position Nucleotide; Accordingly the 195th amino acids residue in sequence 1 is sported to glycine by tyrosine, and the 167th amino acids residue is sported to Histidine by tyrosine.The albumen called after albumen 6 of double chain DNA molecule 6 codings.
(7) double chain DNA molecule 7: with the difference of DNA molecular shown in the sequence 2 of sequence table be will be from 5 ' end 583-585 position Nucleotide by TAC sudden change for GTA, and will by TAC, sport CAC from 5 ' end 499-501 position Nucleotide; Accordingly the 195th amino acids residue in sequence 1 is sported to α-amino-isovaleric acid by tyrosine, and the 167th amino acids residue is sported to Histidine by tyrosine.The albumen called after albumen 7 of double chain DNA molecule 7 codings.
(8) double chain DNA molecule 8: with the difference of DNA molecular shown in the sequence 2 of sequence table be will be from 5 ' end 583-585 position Nucleotide by TAC sudden change for CTA, and will by TAC, sport CAC from 5 ' end 499-501 position Nucleotide; Accordingly the 195th amino acids residue in sequence 1 is sported to leucine by tyrosine, and the 167th amino acids residue is sported to Histidine by tyrosine.The albumen called after albumen 8 of double chain DNA molecule 8 codings.
(9) double chain DNA molecule 9: with the difference of DNA molecular shown in the sequence 2 of sequence table be will be from 5 ' end 583-585 position Nucleotide by TAC sudden change for ATA, and will by TAC, sport CAC from 5 ' end 499-501 position Nucleotide; Accordingly the 195th amino acids residue in sequence 1 is sported to Isoleucine by tyrosine, and the 167th amino acids residue is sported to Histidine by tyrosine.The albumen called after albumen 9 of double chain DNA molecule 9 codings.
(10) double chain DNA molecule 10: with the difference of DNA molecular shown in the sequence 2 of sequence table be will be from 5 ' end 583-585 position Nucleotide by TAC sudden change for AAA, and will by TAC, sport CAC from 5 ' end 499-501 position Nucleotide; Accordingly the 195th amino acids residue in sequence 1 is sported to Methionin by tyrosine, and the 167th amino acids residue is sported to Histidine by tyrosine.The albumen called after protein 10 of double chain DNA molecule 10 codings.
(11) double chain DNA molecule 11: with the difference of DNA molecular shown in the sequence 2 of sequence table be will be from 5 ' end 583-585 position Nucleotide by TAC sudden change for CGC, and will by TAC, sport CAC from 5 ' end 499-501 position Nucleotide; Accordingly the 195th amino acids residue in sequence 1 is sported to arginine by tyrosine, and the 167th amino acids residue is sported to Histidine by tyrosine.The albumen called after protein 11 of double chain DNA molecule 11 codings.
(12) double chain DNA molecule 12: with the difference of DNA molecular shown in the sequence 2 of sequence table be will be from 5 ' end 583-585 position Nucleotide by TAC sudden change for GAC, and will by TAC, sport CAC from 5 ' end 499-501 position Nucleotide; Accordingly the 195th amino acids residue in sequence 1 is sported to aspartic acid by tyrosine, and the 167th amino acids residue is sported to Histidine by tyrosine.The albumen called after protein 12 of double chain DNA molecule 12 codings.
(13) double chain DNA molecule 13: with the difference of DNA molecular shown in the sequence 2 of sequence table be will be from 5 ' end 583-585 position Nucleotide by TAC sudden change for GAA, and will by TAC, sport CAC from 5 ' end 499-501 position Nucleotide; Accordingly the 195th amino acids residue in sequence 1 is sported to L-glutamic acid by tyrosine, and the 167th amino acids residue is sported to Histidine by tyrosine.The albumen called after albumen 13 of double chain DNA molecule 13 codings.
(14) double chain DNA molecule 14: with the difference of DNA molecular shown in the sequence 2 of sequence table be will be from 5 ' end 583-585 position Nucleotide by TAC sudden change for AGC, and will by TAC, sport CAC from 5 ' end 499-501 position Nucleotide; Accordingly the 195th amino acids residue in sequence 1 is sported to Serine by tyrosine, and the 195th amino acids residue is sported to Histidine by tyrosine.The albumen called after protein 14 of double chain DNA molecule 14 codings.
(15) double chain DNA molecule 15: with the difference of DNA molecular shown in the sequence 2 of sequence table be will be from 5 ' end 583-585 position Nucleotide by TAC sudden change for ATG, and will by TAC, sport CAC from 5 ' end 499-501 position Nucleotide; Accordingly the 195th amino acids residue in sequence 1 is sported to methionine(Met) by tyrosine, and the 167th amino acids residue is sported to Histidine by tyrosine.The albumen called after protein 15 of double chain DNA molecule 15 codings.
(16) double chain DNA molecule 16: be by TAC, to sport AAC from 5 ' end 583-585 position Nucleotide with the difference of DNA molecular shown in the sequence 2 of sequence table, and will by TAC, sport CAC from 5 ' end 499-501 position Nucleotide; Accordingly the 195th amino acids residue in sequence 1 is sported to l-asparagine by tyrosine, and the 167th amino acids residue is sported to Histidine by tyrosine.The albumen called after protein 16 of double chain DNA molecule 16 codings.
(17) double chain DNA molecule 17: be by TAC, to sport TGC from 5 ' end 583-585 position Nucleotide with the difference of DNA molecular shown in the sequence 2 of sequence table, and will by TAC, sport CAC from 5 ' end 499-501 position Nucleotide; Accordingly the 195th amino acids residue in sequence 1 is sported to halfcystine by tyrosine, and the 167th amino acids residue is sported to Histidine by tyrosine.The albumen called after protein 17 of double chain DNA molecule 17 codings.
(18) double chain DNA molecule 18: i.e. double chain DNA molecule shown in the sequence 2 of sequence table.
(19) double chain DNA molecule 19: 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 19 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 6 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.
According to sequencing result, recombinant plasmid 7 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 7.
According to sequencing result, recombinant plasmid 8 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 8.
According to sequencing result, recombinant plasmid 9 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 9.
According to sequencing result, recombinant plasmid 10 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 10.
According to sequencing result, recombinant plasmid 11 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 11.
According to sequencing result, recombinant plasmid 12 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 12.
According to sequencing result, recombinant plasmid 13 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 13.
According to sequencing result, recombinant plasmid 14 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 14.
According to sequencing result, recombinant plasmid 15 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 15.
According to sequencing result, recombinant plasmid 16 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 16.
According to sequencing result, recombinant plasmid 17 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 17.
According to sequencing result, recombinant plasmid 18 is carried out to structrual description as follows: between the BamHI of carrier pET-22b (+) and XhoI restriction enzyme site, inserted the double chain DNA molecule shown in the sequence 2 of sequence table.
According to sequencing result, recombinant plasmid 19 is carried out to structrual description as follows: between the BamHI of carrier pET-22b (+) and XhoI restriction enzyme site, inserted the double chain DNA molecule shown in the sequence 4 of sequence table.
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 19 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 (in practical application, 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 19 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 (each supernatant liquor that step 4 obtains); Control 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/LI
2liquid (solvent is water) and 5mL distilled water, mix, 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
Respectively solution to be measured (each supernatant liquor that step 4 obtains) is tested as follows: the Zulkovsky starch aqueous solution of 2g/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 LichrosorbNH
2(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, R
2=0.9999, x represents the concentration (g/100ml) of α-CD, and 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, R
2=0.9998, x represents the concentration (g/100ml) of β-CD, and 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, R
2=0.9982, x represents the concentration (g/100ml) of γ-CD, and y represents peak area.The concentration ÷ of percentage composition=α-CD of α-CD (concentration of concentration+γ-CD of concentration+β-CD of α-CD) * 100%.The concentration ÷ of percentage composition=β-CD of β-CD (concentration of concentration+γ-CD of content+β-CD of α-CD) * 100%, the concentration ÷ of percentage composition=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, HPLC does not detect cyclodextrin and generates.(disappearance)
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 83.4 ± 1.4%, β-CD is 10.3 ± 0.9%, γ-CD is 6.4 ± 0.6%.(phenylalanine)
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 80.6 ± 1.6%, β-CD is 10.4 ± 0.7%, γ-CD is 9.0 ± 2.0%.(Histidine)
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 82.2 ± 1.3%, β-CD is 12.1 ± 0.4%, γ-CD is 5.7 ± 1.4%.(proline(Pro))
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 61.9 ± 1.1%, β-CD is 21.3 ± 1.3%, γ-CD is 16.8 ± 0.6%.(tryptophane)
Adopt supernatant liquor 6 to carry out step 5 and obtain in supernatant, the percentage composition that the percentage composition that the percentage composition of α-CD is 58.8 ± 3.3%, β-CD is 17.7 ± 0.9%, γ-CD is 23.5 ± 2.9%.(glycine)
Adopt supernatant liquor 7 to carry out step 5 and obtain in supernatant, the percentage composition that the percentage composition that the percentage composition of α-CD is 42.8 ± 5.6%, β-CD is 21.3 ± 1.4%, γ-CD is 35.9 ± 4.4%.(α-amino-isovaleric acid)
Adopt supernatant liquor 8 to carry out step 5 and obtain in supernatant, the percentage composition that the percentage composition that the percentage composition of α-CD is 25.1 ± 1.1%, β-CD is 35.8 ± 0.7%, γ-CD is 39.1 ± 1.4%.(bright acid amides)
Adopt supernatant liquor 9 to carry out step 5 and obtain in supernatant, the percentage composition that the percentage composition that the percentage composition of α-CD is 28.4 ± 1.2%, β-CD is 36.7 ± 1.0%, γ-CD is 34.9 ± 1.6%.(Isoleucine)
Adopt supernatant liquor 10 to carry out step 5 and obtain in supernatant, the percentage composition that the percentage composition that the percentage composition of α-CD is 38.3 ± 4.1%, β-CD is 17.7 ± 1.8%, γ-CD is 44.0 ± 2.9%.(Methionin)
Adopt supernatant liquor 11 to carry out step 5 and obtain in supernatant, the percentage composition that the percentage composition that the percentage composition of α-CD is 35.1 ± 1.4%, β-CD is 14.7 ± 0.5%, γ-CD is 50.3 ± 1.9%.(arginine)
Adopt supernatant liquor 12 to carry out step 5 and obtain in supernatant, the percentage composition that the percentage composition that the percentage composition of α-CD is 67.7 ± 0.1%, β-CD is 17.6 ± 0.4%, γ-CD is 14.7 ± 0.6%.(aspartic acid)
Adopt supernatant liquor 13 to carry out step 5 and obtain in supernatant, the percentage composition that the percentage composition that the percentage composition of α-CD is 59.9 ± 1.9%, β-CD is 19.0 ± 1.2%, γ-CD is 21.1 ± 1.9%.(L-glutamic acid)
Adopt supernatant liquor 14 to carry out step 5 and obtain in supernatant, the percentage composition that the percentage composition that the percentage composition of α-CD is 63.1 ± 0.7%, β-CD is 33.3 ± 1.5%, γ-CD is 26.6 ± 0.8%.(Serine)
Adopt supernatant liquor 15 to carry out step 5 and obtain in supernatant, the percentage composition that the percentage composition that the percentage composition of α-CD is 40.1 ± 0.8%, β-CD is 10.5 ± 0.2%, γ-CD is 6.9 ± 1.8%.(methionine(Met))
Adopt supernatant liquor 16 to carry out step 5 and obtain in supernatant, the percentage composition that the percentage composition that the percentage composition of α-CD is 46.9 ± 2.2%, β-CD is 25.5 ± 1.8%, γ-CD is 27.6 ± 4.0%.(l-asparagine)
Adopt supernatant liquor 17 to carry out step 5 and obtain in supernatant, the percentage composition that the percentage composition that the percentage composition of α-CD is 59.3 ± 8.0%, β-CD is 15.5 ± 2.8%, γ-CD is 25.2 ± 9.3%.(halfcystine)
Adopt supernatant liquor 18 to carry out step 5 and obtain in supernatant, the percentage composition that the percentage composition that the percentage composition of α-CD is 82.6 ± 1.5%, β-CD is 10.5 ± 0.2%, γ-CD is 6.9 ± 1.8%.
Adopt supernatant liquor 19 to carry out step 5 and obtain in supernatant, the percentage composition of α-CD is 83 ± 3.4.0%, and the percentage composition that the percentage composition of β-CD is 10 ± 0.6%, γ-CD is 7 ± 0.2%.(tyrosine)
Claims (11)
1. protein shown in the sequence of sequence table 1 is carried out to (a) and (b) two protein that sudden change obtains as follows:
(a) sequence of sequence table 1 is sported to Histidine from N-terminal the 167th amino acids residue by tyrosine;
(b) sequence of sequence table 1 is sported to Isoleucine from N-terminal the 195th amino acids residue 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 is for to carry out (c) and (d) two DNA moleculars that sudden change obtains as follows by gene shown in the sequence of sequence table 2:
(c) sequence of sequence table 2 is sported to CAC from 5 ' end 499-501 position Nucleotide by TAC;
(d) sequence of sequence table 2 is sported to ATA from 5 ' end 583-585 position Nucleotide by TAC.
4. the recombinant expression vector that contains 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. the expression cassette that contains gene described in claim 2 or 3.
7. the transgenic cell line that contains gene described in claim 2 or 3.
8. the recombinant bacterium that contains gene described in claim 2 or 3.
9. recombinant bacterium as claimed in claim 8, is characterized in that: described recombinant bacterium is that recombinant expression vector described in claim 5 is imported to the recombinant bacterium that intestinal bacteria obtain.
10. preparing a method of protein described in claim 1, is to cultivate recombinant bacterium described in claim 8 or 9, obtains protein described in claim 1.
The application of protein described in 11. claims 1 is following (I), (II), (III) or (IV):
(I) prepares cyclodextrine Transglucosylase;
(II) degraded starch;
(III) produces cyclooctaamylose;
(IV) produces cyclohexaamylose.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101294149A (en) * | 2008-05-14 | 2008-10-29 | 江南大学 | Alpha-cyclodextrin glucosyl transferase gene clone and expression |
| CN102827815A (en) * | 2012-08-23 | 2012-12-19 | 中国科学院微生物研究所 | A group of cyclodextrin glucosyltransferase, and coding gene and application thereof |
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| CN101294149A (en) * | 2008-05-14 | 2008-10-29 | 江南大学 | Alpha-cyclodextrin glucosyl transferase gene clone and expression |
| CN102827815A (en) * | 2012-08-23 | 2012-12-19 | 中国科学院微生物研究所 | A group of cyclodextrin glucosyltransferase, and coding gene and application thereof |
Non-Patent Citations (3)
| Title |
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| Structure-function relationship in cyclodextrin glycosyltransferase from bacillus circulans DF 9R;Costa H等;《Carbohydrate Research》;20080927;第344卷(第1期);74-79 * |
| 利用来源于Paenibacillus macerans的α-CGTase突变体Y89D制备α-环糊精;王宁等;《食品科学》;20110215;第32卷(第3期);165-170 * |
| 重组α-环糊精葡萄糖基转移酶表达条件的优化及其产物专一性的分析;宋炳红等;《重组α-环糊精葡糖基转移酶表达条件的优化及其产物专一性的分析》;20120131;第10卷(第1期);30-36 * |
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