CN102250931B - Gene for coding beta-cyclodextrin glucosyltransferase and application thereof - Google Patents
Gene for coding beta-cyclodextrin glucosyltransferase and application thereof Download PDFInfo
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Abstract
The invention discloses a gene Pcgt for coding a beta-cyclodextrin glucosyltransferase, and the nucleotide sequence of the gene Pcgt is represented by a SEQIDNO.1. And the invention also discloses a beta-cyclodextrin glucosyltransferase SEQIDNO.2 coded by the gene and the application of the enzyme in the production of the beta-cyclodextrin by taking starch as raw material.
Description
Technical field
The present invention relates to the gene of a coding beta-cyclodextrin glucanotransferase, the protein available starches of this genes encoding is that raw material is directly produced beta-cyclodextrin.
Background technology
Cyclomaltodextrin glucanotransferase (Cyclodextrin Glycosyltransferase, CGTase) is the important enzyme in the foodstuffs industry.CGTase can 4 kinds of different enzyme reactions of catalysis, are respectively hydrolysis, cyclisation, disproportionation and linked reaction.Cyclomaltodextrin glucanotransferase (EC 2.4.1.19) is the important member of glycosyl hydrolase alpha-amylase family (glycosyl hydrolase family 13), and its unique function is that starch is converted into cyclodextrin.
Cyclodextrin (Cyclodextrin, be called for short CD) is the ring-type α that generated under the cyclization catalysis of cyclomaltodextrin glucanotransferase by starch or starch derivative-Isosorbide-5-Nitrae-dextran, is the malto-oligosaccharide of irreducibility.CD usually by 6-12 D-glucopyranosyl with α-1, the 4-glucoside bond is formed by connecting, wherein most study, most widely used be by 6,7 and 8 cyclodextrin that glucosyl residue forms, be called α-, β-and γ-CD (Ronan M.Kelly, Lubbert Dijkhuizen, Hans Leemhuis.2009.The evolution of cyclodextrin glucano-transferase product specificity.Appl.Microbiol.Biotechnol., 84 (1): 119-133.).
The spatial disposition of glucose unit in the cyclodextrin makes its outer rim hydrophilic and inner chamber is hydrophobic, thereby cyclodextrin can be as host's molecule and various hydrophobic guest molecule in conjunction with forming the envelope title complex, thereby changes the character of guest molecule.Therefore, cyclodextrin has widely purposes (Del Valle EMM.2004.Cyclodextrins and their uses:a review.Process Biochem., 39 (9): 1033-1046.) in association areas such as food, medicine, makeup, agriculturals.
At present, cyclodextrin mainly is for foodstuffs industry (Lajos Szente, Jozsef Szejtli.2004.Cyclodextrins as food ingredients.Trends Food Sci.Tech., 15 (3-4): 137-142.).Its function mainly comprises the following aspects: some composition in (1) stabilizing food product.(2) remove stink and bitter taste in the food.(3) can make some food form milk sap steady in a long-term.(4) foaming power of increase food.(5) liquid foodstuff is become pressed powder, be convenient to packing, transportation, store and use.As with cyclodextrin powdered wine processed, stable high, it was identical with original wine taste to add cold-water solution.(6) use with sanitas, can improve preservative effect.Cyclodextrin itself can not be anticorrosion, but use with sanitas, can make sanitas permanently effective.(7) as the foodstuff production auxiliary material.In addition, the at present popular a kind of application of cyclodextrin is Cholesterol removal from animals products such as egg and milk preparation, process through cyclodextrin, can remove in the goods 80% cholesterol, also can from fat, remove free fatty acid, thereby improve the fried performance of fat, as reducing oil smoke and bubble, being difficult for brown stain etc.; Fruits and vegetables juice also can be processed by cyclodextrin and remove the phenolic compound that can cause enzymatic browning.Because cyclodextrin is nontoxic or unusual low toxicity, to healthy without any harm, can be used for safely foodstuffs industry (C.Munro, P.M.Newberne, V.R.Young.et al.2004.Safety assessment of γ-cyclodextrin.Regul.Toxicol.Pharmacol.39 (Suppl.1): 3-13.).
In medicine industry, CD is a kind of new drug carrier that development in recent years is got up, it can utilize its hollow round tube type special construction that all or part of inclusion of drug molecule is formed nonbonding class mixture, the similar microcapsule of its formulation, but cyclodextrin is the single molecules level inclusion to medicine, so claim again molecular capsule, this technology can produce microcapsule and the unexistent special role of nanocapsule (Mark E.Davis, Marcus E.Brewster.2004.Cyclodextrin-based pharmaceutics:past, present and future.Nature reviews, 3 (12): 1023-1035.).Cyclodextrin has had commonplace application at aspects such as cardiovascular medicament, antitubercular agent, hypertension drug, gastroenteropathy medicines.In medicine industry, cyclodextrin is mainly used in following aspect: (1) increases the solubleness of medicine.(2) stability of increase medicine.(3) pungency, the Side effect of reduction medicine are covered bitter taste.(4) powdered of volatile liquid and solid, oily liquids.(5) bioavailability of raising medicine.(6) prevent interaction between medicine and medicine, medicine and the additive.
In cosmetic field, cyclodextrin is mainly used in solubilising essence, prevents essence volatilization loss; Control increases the makeup transparent feel to skin tensio-active agent usage quantity excitatory, does not produce the oil content segregation phenomenon; The activeconstituents inclusion of makeup is got up, make it be difficult for oxidized and decomposition, play stable effect (Malton, Peter James, Holland, Lynette Anne Makins, Rizzi, George.2000.Cosmetic compositions comprising cyclic oligosaccharides and fragrance.Wo Pat, 2000/067716.).At present, some famous cosmetics companies all are used in the personal care product with cyclodextrin basically in the world, mainly be inclusion and the slowly-releasing characteristics of utilizing cyclodextrin, these active substances and fragrance inclusion are got up, the temperature and humidity that runs into human body skin plays later on trigger action, active substance and fragrance begin slowly balancedly to discharge from the cyclodextrin endoporus, and cyclodextrin produces body surface in the molecule inspiration endoporus of body odour, become the body odour absorption agent, thereby eliminated body odour, this is a dynamic process.
Except being applied in food, medicine and the cosmetic industry, cyclodextrin also has in fields such as biotechnology, agricultural, analytical chemistry, environmental protection and weavings very widely to be used.At biological technical field, because the cyclodextrin molecular cavity has certain selective recognition ability to guest molecule, so cyclodextrin can be used for molecular recognition, and has the function of analogue enztme; Agriculturally, cyclodextrin can be used as plant-growth regulator, can improve dissolution rate and the solubleness of agricultural chemicals simultaneously, increases biological effect, covers the agricultural chemicals peculiar smell; In analytical chemistry, because cyclodextrin can affect the absorption spectrum of coloured molecule, color reaction there are enhanced sensitivity, solubilising and increase steady effect, and can identify chiral molecules, can be used for all kinds of chromatograms, spectrum and electrochemical analysis; Aspect environmental protection, cyclodextrin plays solublization to low-pole organic pollutants such as the trieline in the media such as soil, chlorobenzene, naphthalene, anthracene and DDT, and can promote their biological degradation, CD also can extract organic pollutant and then utilize microorganism to degrade from contaminated soil.
Three kinds of CD of α, β and γ are white crystalline powder, without certain fusing point, begin when being heated to 200 ℃ of left and right sides to decompose, wherein the water solubility of β-CD is minimum, be easy to crystallization and purification, so its purposes noticeable (research of seed selection, fermentation condition optimization and the zymologic property of the big .2007. cyclomaltodextrin glucanotransferase of Guo Li superior strain MSUD5. Northwest University's Master's thesis, 3-65.).And because the hollow circuit cylinder radius of cycloheptaamylose is moderate, and has again the security the same with starch and dextrin, various aspects of performance is the best, has the most widely industrial use in CD.
The production of CGTase has two kinds of approach, the first adopts natural bacterial strain to come fermentative production, be mainly bacterium (Hans Leemhuis, Ronan M.Kelly, Lubbert Dijkhuizen.2010.Engineering of cyclodextrin glucanotransferases and the impact for biotechnological applications.Appl.Microbiol.Biotechnol.85:823-835.); It two is to make up genetic engineering bacterium fermentative production CGTase, compare with other expression system, intestinal bacteria (Escherichia coli) expression system have genetic background clear, easy and simple to handle, can the large scale fermentation cultivation etc. advantage, be the most frequently used expression system of present stage.In general, output and production intensity that natural strain fermentation produces CGTase are all lower, and this also is the one of the main reasons that causes the cyclodextrin production cost higher.
At present, abroad to the research and comparison of CGTase deeply, and China mainly concentrates on the wild strain aspect that produces CGTase to the research of this enzyme, comprises screening wild mushroom, induction mutation of bacterium, fermentation condition optimization etc.And it is generally lower that the wild strain of the product β-CGTase that screens from occurring in nature produces enzyme level, and extraction and purification process is loaded down with trivial details, is difficult to satisfy the demand of suitability for industrialized production.
At present, domestic the beta-cyclodextrin glucosyl transferase gene to source basophilia Bacillus sp.N-227 and these two bacterial strains of Paenibacillus sp. carried out the research of heterogenous expression, to derive from basophilia Bacillus sp.N-227 β-CGTase gene such as people such as Tang Shanghua and be expressed in respectively (Tang Shanghua among E.coli and the B.subtlis (subtilis), Feng is short of money, Huang Ruixin, Deng .1990. Bacillus alcalophilus N-227 cycloheptaamylose glycosyltransferase gene cloning and Expression in Escherichia coli thereof. industrial microorganism, 20:1-5. China in the Tang, Wang Lei, the integrative gene expression of Zhou Xinyu .1995. Bacillus alcalophilus N-227 cyclodextrin glucanotransferase gene in Bacillus subtilus. industrial microorganism, 25:1-4.); The people such as Wang Yuan also express (Wang Yinyu with this gene in E.coli, Wang Yuan, Zhang Hongfa waits the Bacillus alcalophilus N-227 beta-cyclodextrin glycosyltransferase genetic analysis of .2007. source and the abduction delivering in intestinal bacteria. industrial microorganism .7 (3): 10-14.).The people such as the Xie Zhenrong beta-cyclodextrin glucosyl transferase gene that series bacillus belongs to of will originating has carried out clonal expression (Xie Zhenrong in intestinal bacteria, Zhao Sanjun, Tang Xianghua, clone and Expression in Escherichia coli Deng the beta-cyclodextrin glucosyl transferase gene of .2010. source Paenibacillus sp.. food science and technology, 35 (11): 16-19.).Chinese patent 200910029153.8 discloses " mutant and mutation method with cyclomaltodextrin glucanotransferase of high yield beta-cyclodextrin ability ", the Chen Jian seminar of Southern Yangtze University is by having carried out a series of sudden change to the alpha-cyclodextrin glucosyl transferase that derives from softening series bacillus (Peanibacillus macerans) FB05-01, obtained the alpha-cyclodextrin glucosyl transferase mutant enzyme K47T of high yield beta-cyclodextrin, beta-cyclodextrin accounts for 66.4% of total cyclodextrin amount in the cyclodextrin product of this mutant enzyme.
Summary of the invention
The present invention is gene clone to a beta-cyclodextrin glucanotransferase from the Ke Shi series bacillus GX-4 (Paenibacillus cookii GX-4) of Nanning screening, in host cell, express this genes produce beta-cyclodextrin glucanotransferase, and take starch as the raw material production beta-cyclodextrin.
The present invention relates to a kind of gene Pcgt of beta-cyclodextrin glucosyl group enzyme, its nucleotide sequence shown in SEQ ID NO.1, be from laboratory screening to Ke Shi series bacillus GX-4 separate and obtain.The conserved regions of cyclodextrin glucosyl transferase gene sequence, the foreign DNA with described base sequence of SEQ ID NO.1 in the sequence table are by 2135 based compositions, open reading frame (the Open Reading Frame that contains complete beta-cyclodextrin glucosyl transferase gene Pcgt, ORF), the initiation codon of Pcgt gene is ATG, and terminator codon is TAA.The gene for raw-starch-digesting amylase precursor homology of Pcgt and bacillus Bacillus sp.B1018 is the highest, both similaritys=2013/2043 (99%).
The protein of SEQ ID NO.2 is the beta-cyclodextrin glucanotransferase product P cgt of gene Pcgt coding, formed by 692 amino acid, with Pcgt catalysis territory homology the highest be the Precursor of Bacillus circulans (Bacillus circulans), cyclomaltodextrin glucanotransferase, both similaritys=672/693 (97%), homogeny=679/693 (98%).
Gene Pcgt can be take starch as the raw material production cyclodextrin at the recombinant products Pcgt of expression in escherichia coli.
The invention still further relates to the expression vector that contains gene of the present invention, and be used for transforming the host of gene of the present invention.
The invention provides a beta-cyclodextrin glucosyl transferase gene, the beta-cyclodextrin glucanotransferase of this coded by said gene has widely purposes in take starch as the raw material production beta-cyclodextrin.
Description of drawings
Fig. 1 be screening contain beta-cyclodextrin glucosyl transferase gene Pcgt recombinant bacterial strain Congo red+the selection lithograph of starch.
Fig. 2 is beta-cyclodextrin glucanotransferase Pcgt produces cyclodextrin take starch as substrate HPLC figure.
Recognize that from Fig. 1 the recombinant bacterial strain that contains beta-cyclodextrin glucosyl transferase gene Pcgt can form transparent circle at the selection flat board of Congo red+starch.
A among Fig. 2, B and C are respectively the standard specimens of glucose, alpha-cylodextrin and beta-cyclodextrin, and D is the product of Pcgt converted starch.
From the D of Fig. 2, recognize, beta-cyclodextrin glucanotransferase Pcgt can change into starch alpha-cylodextrin, beta-cyclodextrin and γ-cyclodextrin, wherein the output of beta-cyclodextrin accounts for 63.42% of total cyclodextrin in the cyclodextrin product, be shown in Table 1, table 1 is the proportion of products analytical table of Pcgt converted starch.
Embodiment
Following implementation method is for better explanation the present invention, and the purpose that should not be construed as limiting the invention.
Used material comprises in an embodiment of the present invention: intestinal bacteria (Escherichia coli) strain XL1-Blue, carrier pMD19-T (available from Dalian TaKaRa company); Expression vector pSE380 is available from Stratagene company, available from reagent such as the restriction enzyme of TaKaRa, MBI, modifying enzymes.
The below will be described in detail the present invention by embodiment:
1, the cyclodextrin glucosyl transferase gene sequential analysis that belongs to from series bacillus
Find two gene orders that belong to the coding cyclomaltodextrin glucanotransferase from series bacillus having announced in the Genbank database.Use the construction package of the protein of these two genes encodings of SMART software on-line analysis, use Vector NTI10.0 software that the cyclodextrin glucosyl transferase gene sequence is carried out the Alignment compare of analysis, thereby obtain the homologous sequence of cyclodextrin glucosyl transferase gene sequence gene.
2, the acquisition of beta-cyclodextrin glucosyl transferase gene conserved regions dna fragmentation
According to homologous sequence, the design pair of primers.
P-cgt1：5‘-CAACAAGCAGAANTTCAGCAC-3’
P-cgt4：5‘-TTAAGGCTGCCAGTTCAC-3’
Take the total DNA of Ke Shi series bacillus GX-4 that screens from the Nanning as template, polymerase chain reaction (PCR) amplification obtains the conserved regions dna fragmentation, and size is about 2kb.The PCR product directly is connected with the pMD19-T carrier, connects 12hr in 16 ℃.Connect product all for CaCl
2Method transforms intestinal bacteria XL
1In-Blue the competent cell, be applied on the LA flat board of the penbritin that contains X-gal, IPTG and 100 μ g/mL, 37 ℃ of overnight incubation, after growing single bacterium colony, the white colony of 3 of pickings is inoculated in the LB liquid nutrient medium that contains 100 μ g/mL penbritins respectively, 37 ℃ of shaking culture 14hr, prepare in a small amount plasmid DNA, use respectively again restriction enzyme EcoRI complete degestion, carry out 0.7% agarose gel electrophoresis analysis, the equal enzyme of the plasmid DNA of these three white colonies is cut into the DNA band that size is about 4.8kb as a result.So it is logical that one of them plasmid DNA is carried out the sequence survey, called after P3-6.Then deliver Shanghai and give birth to worker bio-engineering corporation mensuration dna nucleotide sequence.
3, the analysis of beta-cyclodextrin glucosyl transferase gene Pcgt conserved regions dna sequence dna
The external source clip size of P3-6 is 2135bp.Use Vector NTI10.0 software that the dna sequence dna of P3-6 is carried out open reading frame (Open Reading Frame, ORF) analysis, find to have an ORF from 51-2129bp, formed sequence such as SEQ ID NO.1 by 2079 Nucleotide.Wherein, the initiator codon of Pcgt gene is ATG, and terminator codon is TAA.
4, the amino acid sequence analysis of the product P cgt of beta-cyclodextrin glucosyl transferase gene Pcgt coding
One of beta-cyclodextrin glucosyl transferase gene Pcgt coding contains 692 amino acid whose protein, is 75282.16 dalton with the theoretical molecular size of this protein of DNAStar software prediction.
Use the aminoacid sequence of SMART on-line analysis Pcgt, find that there is not signal peptide sequence in this protein Pcgt.
5, the clone of beta-cyclodextrin glucosyl transferase gene Pcgt and expression
Use upstream primer 5 '-TCGCCATGGTGCACCACCACCACCACCACATTACGCCAGCTTGCATGCTGCAG-3 ' and downstream primer: 5 '-CACAAGCTTTTAAGGCTGCCAGTTCACGTTAATG-3 ', by pcr amplification beta-cyclodextrin glucosyl transferase gene Pcgt, after cutting beta-cyclodextrin glucosyl transferase gene Pcgt with restriction enzyme NcoI and HindIII enzyme, be connected with the expression vector pSE380 that is connected with the HindIII enzyme through NcoI.To connect again product and be transformed among the intestinal bacteria XL1-Blue, be applied on the LA flat board that contains 100 μ g/mL penbritins.On the LA flat board of picking transforms the single bacterium colony to 0.01% obtain Congo red+1% starch+0.5mmol/L IPTG+100 μ g/mL penbritin, place 37 ℃ of thermostat containers to cultivate 6hr flat board, then carry out the stifling broken born of the same parents of chloroform, place flat board 37 ℃ of thermostat containers to make the Pcgt enzyme-to-substrate reaction 2～3hr of expression.Observe and select flat board.
Then further extraction can form the plasmid DNA of clone's of transparent circle, and with its called after pSE-Pcgt, after cutting pSE-Pcgt with restriction enzyme NcoI and HindIII enzyme, carry out 0.7% agarose gel electrophoresis analysis, pSE-Pcgt also has a size to be about the dna fragmentation of 2kb except the dna fragmentation that a 4.1kb is arranged as a result.
The recombination bacillus coli XL1-Blue inoculation that will contain plasmid pSE-Pcgt contains to 20mL in the LB substratum of 100 μ g/mL penbritins, and 37 ℃ of shaking culture are treated OD
600Be 0.4 o'clock, adding final concentration is that 0.5mmol/L IPTG, final concentration are that 100mmol/L sorbyl alcohol and final concentration are the 2.5mmol/L trimethyl-glycine, induces 20hr for 20 ℃.The centrifugal 3min of 11000rpm collects thalline, and with the resuspended thalline of pH 7.0 phosphoric acid buffers of 4mL 100mmol/L, ultrasonic wave is broken born of the same parents 9min.The centrifugal 10min of 12000rpm, supernatant is the crude enzyme liquid that contains beta-cyclodextrin glucanotransferase Pcgt.
6, the mensuration of the amylatic vigor of beta-cyclodextrin glucanotransferase Pcgt
Get the crude enzyme liquid of 20 μ l beta-cyclodextrin glucanotransferase Pcgt, pH 7.0 phosphoric acid buffers that add 500 μ l 100mmol/L, mix with 480 μ l, 1% starch solution, 37 ℃ the effect 1 hour after, add 800 μ l DNS solution, place in the boiling water and reacted 5 minutes, the room temperature cooling is with spectrophotometric instrumentation absorbancy OD
530The glucose absorbancy canonical plotting of absorbance measurement value and different content is made comparisons and is calculated enzyme work.Described DNS reagent preparation: take by weighing the about 40mL ddH of 1 gram NaOH
2The O dissolving takes by weighing 1 gram dinitrosalicylic acid, 0.2 gram phenol, 0.05 gram sodium sulphite anhydrous 99.3,20 gram Rochelle salts again, and it is dissolved in about 30mL ddH
2Among the O, two kinds of solution mix, and constant volume is to 100mL.
The hydrolyzed starch activity of beta-cyclodextrin glucanotransferase Pcgt is 212U.
7, beta-cyclodextrin glucanotransferase Pcgt produces the mensuration of cyclodextrin product
The beta-cyclodextrin glucanotransferase is produced the reaction of cyclodextrin take starch as substrate: get the crude enzyme liquid of 20 μ l beta-cyclodextrin glucanotransferase Pcgt, with 1% (w/v) starch solution (with the dissolving of 50mmol/L pH 7.0 phosphoric acid buffers), 37 ℃ of effects 3 hours in the reaction system of 500 μ l.After reaction times arrives, in 10 minutes termination reactions of 100 ℃ of heating.The product of Pcgt detects with high performance liquid chromatography (HPLC).The result that HPLC detects shows: have glucose, alpha-cylodextrin, beta-cyclodextrin and γ-cyclodextrin to exist in the product of Pcgt, wherein beta-cyclodextrin output accounts for 63.42% of total cyclodextrin in the cyclodextrin product.
HPLC condition: instrument: Agilent1100 chromatographic instrument; Chromatographic column: nh 2 column; Moving phase: acetonitrile: water (70: 30); Flow velocity: 1.0mL/min; Detector: RID (refraction detector).
The proportion of products analytical table of table 1 Pcgt converted starch
| Retention time (min) | Peak area | Peak area ratio (%) | Peak height | Ratio of peak (%) |
| 12.354 | 162098 | 26.90 | 5483 | 36.96 |
| 16.257 | 382101 | 63.42 | 8402 | 56.64 |
| 22.242 | 58341 | 9.68 | 950 | 6.40 |
| Amount to | 602540 | 100.00 | 14835 | 100 |
Claims (5)
1. the gene Pcgt of a coding beta-cyclodextrin glucanotransferase is characterized in that its nucleotide sequence is shown in SEQ ID NO.1.
2. according to claim 1 the protein of coded by said gene, its aminoacid sequence is shown in SEQ ID NO.2.
3. an expression vector is characterized in that, it contains gene claimed in claim 1.
4. a host cell is characterized in that, it is prokaryotic cell prokaryocyte or the eukaryotic cell that the described expression vector of claim 3 transforms.
5. the application of protein claimed in claim 2 in take starch as the raw material production beta-cyclodextrin.
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| CN102796712A (en) * | 2012-09-11 | 2012-11-28 | 云南师范大学 | Method for producing extracellular production and recombination beta-cyclodextrin transferase |
| CN102876640B (en) * | 2012-10-12 | 2013-09-11 | 广西科学院 | Cyclodextrin glycosyltransferase mutant and application thereof |
| CN103602641B (en) * | 2013-11-20 | 2015-05-20 | 广西大学 | Beta-cyclodextrin glucosyl tranferase enzyme mutant for producing gamma-cyclodextrin and use thereof |
| CN105936912A (en) * | 2016-06-28 | 2016-09-14 | 国家海洋局第三海洋研究所 | Cyclodextrin glycosyl transferase and preparation method thereof |
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| WO2002044350A2 (en) * | 2000-11-28 | 2002-06-06 | Henkel Kommanditgesellschaft Auf Aktien | Cyclodextrin glucanotransferase (cgtase), obtained from bacillus agaradherens (dsm 9948) and detergents and cleaning agents containing said novel cyclodextrin glucanotransferase |
| CN101503680A (en) * | 2009-01-06 | 2009-08-12 | 江南大学 | Mutant of cyclodextrin glucosyl transferase having highly beta-cyclodextrin yielding property and mutation method |
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| WO2002044350A2 (en) * | 2000-11-28 | 2002-06-06 | Henkel Kommanditgesellschaft Auf Aktien | Cyclodextrin glucanotransferase (cgtase), obtained from bacillus agaradherens (dsm 9948) and detergents and cleaning agents containing said novel cyclodextrin glucanotransferase |
| CN101503680A (en) * | 2009-01-06 | 2009-08-12 | 江南大学 | Mutant of cyclodextrin glucosyl transferase having highly beta-cyclodextrin yielding property and mutation method |
Non-Patent Citations (1)
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| Itkor,P. et al..Accession No.M33302.《Gene Bank》.1993,全文. * |
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