CN102876640B - Cyclodextrin glycosyltransferase mutant and application thereof - Google Patents
Cyclodextrin glycosyltransferase mutant and application thereof Download PDFInfo
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Abstract
The invention discloses a cyclodextrin glycosyltransferase mutant and application thereof. An amino acid sequence of the cyclodextrin glycosyltransferase mutant is shown as SEQ ID NO:1. Compared with enzyme before mutation, the mutant enzyme has the function of highly producing maltose. The mutant enzyme can be applied to the maltose production in which starch is taken as a raw material.
Description
Technical field
The present invention relates to a kind of cyclomaltodextrin glucanotransferase mutant, and this mutant enzyme is being that substrate is produced the application in the maltose with starch.
Background technology
Multifunctional amylase is the new class member of αDian Fenmei family (α-amylase family), comprise new Pullulanase, maltogenic amylase and cyclomaltodextrin glucanotransferase, they structurally have the zone of 4 total high conservatives of αDian Fenmei family, but on function, have the catalytic activity of glycoside hydrolase and glycosyltransferase again concurrently, though and different multifunctional amylases has certain similarity in structure and function, but the enzyme of different sources is in substrate specificity and hydrolysis, the aspects such as specificity that shift glycosidic link exist difference (Lee H S, Kim M S, Cho H S, et al.2002, Cyclomaltodextrinase, neopullulanase, and maltogenic amylase are nearly indistinguishable from each other.J.Biol.Chem., 277 (24): 21891-21897).
Cyclodextrin glucosyltransferase (CGTase:EC2.4.1.19) is the class of enzymes that is produced by the gemma bacterioid, mainly appear in the rod-shaped bacterium, in Bacillus macerans, separate at first, and at multiple microorganism discovery (Cristiane Moriwakia, Glauciane L.Costaa, Rubia Pazzettoa, et al.2007, Production and characterization of a new cyclodextrin glycosyltransferase from Bacillus firmus isolated from Brazilian soil, Process Biochemistry.42(10): 1384-l390).The reaction of cyclomaltodextrin glucanotransferase catalysis glycosyl generally includes following four kinds of modes of action (Qi Q S, Zimmermann W.2005, Cyclodextrin glucanotransferase from gene to application.Appl.Microbiol.Biotechnol., 66,475-485.): (1) cyclic action: α-(1 → 4) dextran molecule generates cyclodextrin by intramolecularly glycosyl shift reaction.(2) coupling: be the cyclic action reversed reaction, open the cyclodextrin ring, it is attached on the acceptors such as glucose and maltose.(3) disproportionation reaction: carry out the glycosyl transferance between the straight chain oligosaccharide molecular, generate the different various glycan molecules of the polymerization degree.(4) hydrolytic action: its hydrolysis rate is generally about cyclization speed ten thousand/15.
Maltogenic amylase (Maltogenic α-amylase, MAase) full name 1,4-α-D-dextran α-malt-base lytic enzyme [1,4-α-D-glucan α-maltohydrolase, EC3.2.1.133], be a class hydrolyzing alpha-1, the endo-amylase of 4-D-glucoside bond, act on starch and relevant saccharan, oligosaccharides, product is α-maltose.Maltose unit is hydrolyzed (Kim DY in the direction of the clock from the non-reduced end of starch chain, Cha CH, Oh WS, et al.2004, Expression of the promoter for the maltogenic amylase gene in Bacillus subtilis 168.J Microbiol, 42:319 ~ 327).The maltogenic amylase gene is mainly derived from subtilis (Bacillus subtilis), Bacillus licheniformis (Bacillus licheniformis), bacillus cereus (Bacillus cereus), bacstearothermophilus (Geobacillus stearothermophilus), Thermus (Thermus sp.), thermoactinomyces (Thermusvulgaris) etc.
Maltogenic amylase is mainly used in and bakes industry.In dough, add an amount of maltogenic amylase, the starch molecule that this enzyme decomposed in the dough in the early stage of the process of baking produces small molecules carbohydrates such as maltose, these small molecules carbohydrates can stop in the bread and form hinge between the protein network and starch granules, thereby delay the aging (Wang Xuedong of bread, Yang Hao, Yao Juan, etc. fungal alpha-amylase and maltogenic alpha-amylase enzyme are to the comparative studies .2006 of steamed bun powder quality-improving, food science and technology, 31 (10): 48-52.).On the other hand, maltogenic amylase directly catalysis starch transforms generation isomalto Oligosaccharide (IMO) (Park K H, Kim T J, Cheong T K, et al.2000, Structure, specificity and function of cyclomaltodextrinase, a multispecific enzyme of the α-amylase family.Biochim BiophyActa, 1478 (2): 165-185).Isomalto Oligosaccharide is effective bifidus factor, to keeping and strengthening HUMAN HEALTH, improve body immunity important effect is arranged.The excavation of maltogenic amylase and use certainly will greatly promote will more cheap starch to be converted into the technical development of expensive IMO, and (Park K H.2006, Function and tertiary-and quaternary-structure of cyclodextrin-hydrolyzing enzymes (CDase), a group of multisubstrate specific enzymes belonging to the α-amylase family.J Appl Glycosci, 53 (1): 35-44).
So, we have been mutated into maltogenic amylase Mcgt by enzyme molecular modification technology with cyclomaltodextrin glucanotransferase Pcgt, make that the cyclisation function of mutant enzyme Mcgt has bigger reduction, large increase--the output of maltose accounts for 66.39% of total reaction product in the hydrolytic activity acquisition.Like this, the character of high yield maltose makes that mutant enzyme Mcgt can also be for starch being substrate production high maltose syrup.
High maltose syrup is a kind of maltose content height, the medium invert sugar slurry that glucose content is low.Its feature is: limpid transparent, infusion temperature height is compared with glucose syrup, and it is soft also to have a sweet taste, and moisture retention is good, the thermostability height, and therefore premium propertiess such as resistive connection crystalline substance have a wide range of applications in foodstuffs industry.High maltose syrup is used for sweet food water conservation, protects fragrant; Can reduce its sugariness for marsh-mallow, whipped ice cream; Be used for filling class sweet food, render palatable, Bao Se and rot-resistant effect; Be used for food such as rice cake, glutinous rice group and can keep its pliability, prolong the shelf time; Can keep its freshness for jelly based foods such as fish jelly, improve quality.High maltose syrup not only is used for foodstuffs industry, and also has been widely used in zymin and medicine industry.
Be keyword lookup State Intellectual Property Office patent retrieval database with the maltogenic amylase, occur 10 patents of invention altogether.Having only 1 about maltogenic amylase, is by HNO2 natural maltogenic amylase to be carried out chemically modified, has obtained the lower maltogenic amylase of optimal pH; Other 9 patents of invention are to describe the application of maltogenic amylase in field of food.
Be keyword lookup State Intellectual Property Office patent retrieval database with the cyclomaltodextrin glucanotransferase, occur 16 patents of invention altogether.Wherein have only two to suddenly change about cyclomaltodextrin glucanotransferase, these two patents of invention all are that the CGT enzyme from Peanibacillus macerans JFB05-01 (CCTCC NO:M208063) is carried out the sudden change of a plurality of amino acid sites respectively, have obtained the mutant of the cyclomaltodextrin glucanotransferase of high yield alpha-cylodextrin and high yield beta-cyclodextrin ability respectively.
Summary of the invention
The objective of the invention is by the transformation to cyclomaltodextrin glucanotransferase, make it to obtain new cyclomaltodextrin glucanotransferase mutant, it is that substrate is produced maltose that this mutant enzyme can be used for starch.
New cyclomaltodextrin glucanotransferase mutant Mcgt(SEQ ID NO:1 of the present invention), it obtains by molecular modification beta-cyclodextrin glucosyl transferase gene Pcgt, gene Pcgt is 201110171181.0 from number of patent application, specifically be by the amino acid 200 of Pcgt molecule sudden changes and 201, be mutated into phenylalanine Phe and Threonine Thr by leucine Leu and tyrosine Tyr, added 5 aminoacid sequences simultaneously, transformed and obtain a new cyclomaltodextrin glucanotransferase mutant.This cyclomaltodextrin glucanotransferase mutant is compared with protoenzyme, can be raw material high yield maltose with starch.
The protein of SEQ ID NO:1 is cyclomaltodextrin glucanotransferase mutant Mcgt, is made up of 697 amino acid.
The invention still further relates to the host of containing cyclomaltodextrin glucanotransferase mutant of the present invention.
Cyclomaltodextrin glucanotransferase mutant of the present invention is being to have purposes widely in the raw material production maltose with starch.
The preparation method of cyclomaltodextrin glucanotransferase mutant of the present invention comprises the clone of beta-cyclodextrin glucosyl transferase gene, the acquisition of cyclomaltodextrin glucanotransferase mutant gene, the expression of cyclomaltodextrin glucanotransferase mutant gene and the steps such as mensuration of purifying and cyclomaltodextrin glucanotransferase mutant hydrolyzed starch product.
Description of drawings
Fig. 1 is the SDS-PAGE figure of cyclomaltodextrin glucanotransferase mutant Mcgt partial purification thing.
Fig. 2 is the HPLC figure of the starch hydrolysate of cyclomaltodextrin glucanotransferase Pcgt before the sudden change.
Fig. 3 is the HPLC figure of the starch hydrolysate of cyclomaltodextrin glucanotransferase mutant Mcgt.
Embodiment
By the following examples technical scheme of the present invention is described further.
Material is prepared: intestinal bacteria (Escherichia coli) XL10-Gold, carrier pSE380 available from Invitrogen company, Ni-NTA histidine protein purification media available from Novagen company, polysaccharase available from TaKaRa company, modifying enzyme DpnI available from MBI company.
1) clone of cyclodextrin glucosyl transferase gene Pcgt
Use upstream primer 5 '-TCGCCATGGTGCACCACCACCACCACCACATTACGCCAGCTTGCATGCTGCAG-3 ' (comprising a Nco I restriction enzyme site and a 6xHis label at 5 ' end) and downstream primer 5 '-CACAAGCTTTTAAGGCTGCCAGTTCACGTTAATG-3 ' (comprising a Hind III restriction enzyme site), by polymerase chain reaction (PCR) amplification cyclodextrin glucosyl transferase gene Pcgt, after cutting cyclodextrin glucosyl transferase gene Pcgt with restriction enzyme Nco I and Hind III enzyme, be inserted into through Nco I and be connected with the expression vector pSE380 that Hind III enzyme is cut.The recombinant plasmid called after pSE-Pcgt that obtains.
2) acquisition of cyclomaltodextrin glucanotransferase mutant gene Mcgt
The Mcgt gene is to be mutated into phenylalanine TTC and Threonine ACT artificially and to have added 5 amino acid whose nucleotide sequences (CAGCTGGCTTCTCGC) by will encode in forward primer leucine CTG and tyrosine TAC according to Pcgt gene order information to transform.With 1) in the pSE-Pcgt that makes up be template, use forward primer: 5 '-TACAAAAACCTGTACGATCCAGCTGGCTTCTCGCTCGCCGACCTGAACCAT-3 ' and reverse primer 5 '-ATGGTTCAGGTCGGCGAGCGAGAAGCCAGCTGGATCGTACAGGTTTTTGTA-3 ' to carry out PCR, the PCR response procedures: 95 ℃ of the first steps 2 minutes; Second step was carried out 30 circulations, circulation be 98 ℃ 10 seconds, 68 ℃ 6.5 minutes; The 3rd the step 72 ℃ 10 minutes.The PCR product uses 1 μ L Dpn I to cut one hour at 37 ℃ of enzymes, uses CaCl then
2Chemical method transformed into escherichia coli XL10-Gold competent cell.Converted product clone delivers magnificent mcroorganism company and carries out the dna sequencing analysis and determine correct transformant.
3) partial purification of the expression of cyclomaltodextrin glucanotransferase mutant gene Mcgt and expression product
The recombination bacillus coli XL10-Gold inoculation that will contain plasmid pSE-Mcgt contains to 20mL in the LB substratum of 100 μ g/mL penbritins, and 37 ℃ of shaking culture are treated OD
600Be 0.6 o'clock, adding the IPTG(final concentration is 0.5mmol/L), L-sorbic alcohol (final concentration is 100mmol/L) and trimethyl-glycine (final concentration is 2.5mmol/L), induced 20 hours for 20 ℃.11000 leave heart 3min, collect thalline, with 4mL lysis buffer (50mmol/L NaH
2PO
4, 300mmol/L NaCl, 10mmol/L imidazoles, pH8.0) resuspended thalline, the broken born of the same parents of ultrasonic wave 9 minutes.12000 leave heart 10min, get the protein purification that supernatant carries out the back.Nickel affinity chromatography colloid by every 4ml supernatant liquor adding 1mL50% shook 60 minutes with 200 commentaries on classics at 4 ℃, and mixture is filled into pillar, collected effluent.Add 1ml dcq buffer liquid (50mmol/LNaH
2PO
4, 300mmol/LNaCl, the 20mmol/L imidazoles pH8.0) in pillar, slowly stirs, and collects effluent.Repeat rinse step 4 times.Add elution buffer (50mmol/L NaH
2PO
4, 300mmol/L NaCl, 250mmol/L imidazoles, pH8.0) elute protein.Collect the protein soln of wash-out, with polyacrylamide gel electrophoresis (SDS-PAGE) checking of sex change, find to have the protein band of purpose size.
4) the amylatic product of cyclomaltodextrin glucanotransferase mutant Mcgt is measured
The amylatic reaction of cyclomaltodextrin glucanotransferase mutant Mcgt: get the partial purification thing of 3 μ l cyclomaltodextrin glucanotransferase mutant Mcgt, with 1% (w/v) starch solution (with the dissolving of 50mmol/L pH7.0 phosphoric acid buffer), 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 Mcgt detects with high performance liquid chromatography (HPLC).The result that HPLC detects shows: have glucose, maltose, alpha-cylodextrin and beta-cyclodextrin to exist in the product of Mcgt, wherein the output of maltose accounts for 66.39% of total hydrolysate in the hydrolysate.
HPLC condition: instrument: Agilent1100 chromatographic instrument; Chromatographic column: nh 2 column; Moving phase: acetonitrile: water (70:30); Flow velocity: 1.0mL/min; Detector: refraction detector RID.
Recognize that from Fig. 1 the molecular weight of cyclomaltodextrin glucanotransferase mutant Mcgt partial purification thing is 75.7kDa.
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.Recognize that from the D of Fig. 2 beta-cyclodextrin glucanotransferase Pcgt can change into starch glucose, alpha-cylodextrin, beta-cyclodextrin and γ-Huan Hujing.
A among Fig. 3, B, C and D are respectively the standard specimens of glucose, maltose, alpha-cylodextrin and beta-cyclodextrin, and E is the product of Mcgt converted starch.Recognize that from the E of Fig. 3 cyclomaltodextrin glucanotransferase mutant Mcgt can change into starch glucose, maltose, alpha-cylodextrin and beta-cyclodextrin.Wherein the output of maltose accounts for 66.39% of total hydrolysate in the hydrolysate, is shown in Table 1.
Table 1
| Retention time (min) | Peak area | Peak area ratio (%) | Peak height | Ratio of peak (%) |
| 5.990 | 563643 | 0.57 | 21408 | 0.75 |
| 7.770 | 65579569 | 66.39 | 1998615 | 70.36 |
| 13.484 | 23280061 | 23.57 | 615033 | 21.65 |
| 17.259 | 9355657 | 9.47 | 205454 | 7.23 |
| Amount to | 98778930 | 100.00 | 2840510 | 100 |
Claims (3)
1. cyclomaltodextrin glucanotransferase mutant, it is characterized in that: its aminoacid sequence is shown in SEQ ID NO:1.
2. a host cell is characterized in that, it is prokaryotic cell prokaryocyte or the eukaryotic cell of the described cyclomaltodextrin glucanotransferase mutant of claim 1.
3. the described mutein of claim 1 with starch is being application in the raw material production maltose.
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| KR20080072118A (en) * | 2007-02-01 | 2008-08-06 | 경북대학교 산학협력단 | Novel cyclodextrin glycosyltransferase specific for intermolecular transglycosylation of functional antioxidant bioflavonoid from bacillus sp. bl-31 and its thermostable muatant enzyme |
| 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 |
| CN102250931A (en) * | 2011-06-23 | 2011-11-23 | 广西大学 | Gene for coding beta-cyclodextrin glucosyltransferase and application thereof |
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| KR20080072118A (en) * | 2007-02-01 | 2008-08-06 | 경북대학교 산학협력단 | Novel cyclodextrin glycosyltransferase specific for intermolecular transglycosylation of functional antioxidant bioflavonoid from bacillus sp. bl-31 and its thermostable muatant enzyme |
| 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 |
| CN102250931A (en) * | 2011-06-23 | 2011-11-23 | 广西大学 | Gene for coding beta-cyclodextrin glucosyltransferase and application thereof |
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| Itkor,P. et al..Accession No.M33302.《Gene Bank》.1993, |
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| 一株产环糊精葡萄糖基转移酶的地衣芽孢杆菌的选育、产酶条件及酶学特性;陈龙然等;《微生物学报》;20050204(第01期);97-101 * |
| 郭利伟等.β-环糊精葡萄糖基转移酶高产菌株MS-UD2的选育及产酶条件的研究.《食品科学》.2008,(第12期), |
| 陈龙然等.一株产环糊精葡萄糖基转移酶的地衣芽孢杆菌的选育、产酶条件及酶学特性.《微生物学报》.2005,(第01期), |
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