CN102559568A - Acidic heat-resisting isoamylase genetic engineering bacterium and application thereof - Google Patents
Acidic heat-resisting isoamylase genetic engineering bacterium and application thereof Download PDFInfo
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Abstract
The invention relates to an acidic heat-resisting isoamylase genetic engineering bacterium and an application thereof, belonging to the field of enzyme engineering. According to the invention, a Tfu_1891 encoding gene sequence of isoamylase is obtained by chemical synthesis; culture is carried out in an industrial fermentation culture medium by taking pT7-7 as an expression vector and E.coli BL21 (DE3) as an expression host; and by virtue of inductive recombination, the fermentation vigor of the isoamylase reaches 3185U/mL. The recombinase has hydrolysis activity of a starch alpha-1,6 glucoside bond; the optimum temperature is 40-50 DEG C, and the optimum pH value is 5.5; after heat preservation is carried out for 30 hours at the temperature of 50 DEG C, the enzyme activity of the recombinase still maintains more than 50%; the recombinase has starch debranching activity and can hydrolyze branched starch in starch so as to form amylose; and the recombinase is combined with alpha-cyclodextrin glucosyltransferase to be used, thereby obviously improving the conversion rate of cyclodextrin and being suitable for industrial production of cyclodextrin.
Description
Technical field
The present invention relates to heat-resisting isoamylase genetic engineering bacterium of a kind of acidity and application thereof, belong to the enzyme engineering field.
Background technology
Isoamylase (E.C.3.2.1.68) be one type can hydrolyzing alpha-1,6 glucoside bond starch-debranching enzyme, this endonuclease capable hydrolysis glycogen, pulullan, α-limit dextrin and β-limit dextrin, hydrolysate is linear Fructus Hordei Germinatus oligose.Although plant and mikrobe can both produce isoamylase, because mikrobe has advantages such as growth is rapid, simple to operate and with low cost, so business-like isoamylase is generally by microorganisms producing.It is pseudomonas (Pseudomonas.sp.) that isoamylase produces the maximum of bacterial strain research, secondly is other bacterium and fungi.The isoamylase of different sources has different substrate specificities, hydrolysate, ph optimum, optimum temperuture etc.Isoamylase is mainly used in hydrolyzed starch and produces foodstuff additive, like high glucose syrup, SANMALT-S, trehalose, Schardinger dextrins and Resistant starch etc.
Because the huge applications potentiality of isoamylase in the starch hydrolysis have all been carried out a large amount of research to isoamylase both at home and abroad.Harada etc. discover that the ph optimum of the isoamylase that Pseudomonas.sp. originates is between 5 to 6.Olemposka-Beer etc. have reported that the isoamylase ph optimum in Pseudomonas amyloderaosa source is between 3 to 5.And the ph optimum of the isoamylase in genus bacillus source differs greatly, the ph optimum that has be tart at PH 5.0, the isoamylase ph optimum in pH9.0. yeast source that has plenty of alkalescence is generally 6.0 to 7.0.Aspect thermostability, the isoamylase of different sources also has than big-difference.The isoamylase thermostability in yeast source is relatively poor; And the isoamylase in pseudomonas source generally has thermostability preferably; Yokobayashi etc. have reported that a kind of isoamylase that derives from pseudomonas has stability preferably at 45 ℃, but when temperature reaches 60 ℃ inactivation rapidly.The starch hydrolytic process is often carried out under acid and comparatively high temps (50 to 60 ℃) in the industry.Has better active isoamylase often still less and have simultaneously under acid and comparatively high temps.Unique business-like isoamylase is produced by the former Co., Ltd. of Japanese woods at present, and it produces bacterial classification is that this strain fermentation unit reaches 5100U/mL through the Pseudomonas amyloderaosa of mutagenesis improvement.Along with the development of genetic engineering technique, increasing isoamylase has obtained successful expression in intestinal bacteria or yeast, but fermentation level is lower at present, cost is higher, can't reach the suitability for industrialized production requirement.To derive from isoamylase encoding sox successful expression in yeast saccharomyces cerevisiae of Pseudomonas amyloderaosa like Chen etc., the enzyme work in 4 days of fermenting reaches 86U/mL.
Schardinger dextrins (Cyclodextrins; Usually abbreviate CD as); Be one type by starch or polysaccharide under the cyclomaltodextrin glucanotransferase effect, generate by the D-glucopyranose units through α-1; The general name of the end to end ring compound of 4-glycosidic link, the common molecule that 6,7 and 8 glucose units are arranged, be called respectively α-, β-and γ-Huan Hujing.Because alpha-cylodextrin solubleness is bigger, so the chemical method preparation is relatively more difficult, and can cause bigger pollution to environment, biological process is considered to have the method for application potential at present.At present the technology of producing alpha-cyclodextrins by biological method generally is to adopt AMS that starch is carried out partial liquefaction, adds alpha-cyclodextrin glucosyl transferase then and carries out cyclization and prepare alpha-cylodextrin.Because raw material contains the pulullan of 75-85% in the starch, pulullan is hyperbranched polysaccharide, and every in its structure just have a tapping point that is made up of α-1,6 glycosidic link at a distance from 17-28 glucose unit.And alpha-cyclodextrin glucosyl transferase does not have α-1,6 glycosidic link hydrolytic activity, and it is difficult to cross α-1,6 glycosidic link, cause the cycle long, starch utilization ratio is not high, the alpha-cylodextrin transformation efficiency is often about 50% when adopting this technology at present.
Because the starch debranching endonuclease capable cuts off the tapping point in the starch, quickens the reaction of follow-up enzyme, shorten the reaction times, improve the transformation efficiency of starch, thereby increase output, reduce production costs.Reports such as Rendleman adopt debranching factor (comprising Pullulanase and isoamylase) that the waxy corn pulullan is taken off earlier and prop up, and add alpha-cyclodextrin glucosyl transferase again 15 ℃ of reactions 5 days, can make transformation efficiency reach 76%.Employing Pullulanases such as Ivan Pishtiyski take off Zhi Houzai to yam starch, W-Gum etc. and adopt enzymatic conversion preparation in 20 hours Schardinger dextrins, find that W-Gum concentration is that 2.5% o'clock transformation efficiency is the highest, are 65%.Therefore serious problems such as yet these technologies exist raw material too expensive or concentration of substrate is too low, and reaction time is long are difficult to realize suitability for industrialized production.
Summary of the invention
The technical problem that the present invention will solve provides the heat-resisting isoamylase genetic engineering bacterium of a kind of acidity, is to carry the intestinal bacteria E.coli BL21 (DE3) of the heat-resisting isoamylase gene of expression of recombinant plasmid.
Another technical problem that the present invention will solve provides a kind of method that makes up said genetic engineering bacterium; Arrive the pT7-7 carrier through clone's Tfu_1891 encoding sox (Genbank accession number NC_007333.1); With E.coli BL21 (DE3) is expressive host, has realized efficiently expressing of acid heat-resisting isoamylase.
Said recombinant plasmid is a pUC series, pET series, pT7-7, or among the pGEX any one.
Another technical problem that the present invention will solve provides a kind of production technique of production of alpha-cylodextrin, comprises the steps: that carrying out starch according to the concentration of 5-20% sizes mixing, and insulation is 5-15 minute under 50-90 ℃ of condition; 30 ℃-60 ℃ of attemperation, transfer pH5.0-6.0 after, add the isoamylase of 2-100U and the alpha-cyclodextrin glucosyl transferase of 10-100 unit according to every gram starch, add the organic solvent of 5% volume after, fully reacted 4-10 hour; Reclaim organic solvent, adopt crystallization method to obtain alpha-cylodextrin.
Said isoamylase is to add synchronously with alpha-cyclodextrin glucosyl transferase, two kinds of enzymes act on simultaneously starch reach take off, cyclisation carries out synchronously.
In addition, isoamylase can add earlier earlier and starch to be carried out part takes off and prop up, and adds alpha-cyclodextrin glucosyl transferase again and carries out cyclization.
Described organic solvent is an ethanol, Virahol, propyl carbinol, in the nonylcarbinol any.
Genetic engineering bacterium inulinase-producing activity provided by the invention is high, reaches 3185U/mL, and it is applied to the preparation of alpha-cylodextrin: acid heat-resisting isoamylase has starch side chain hydrolytic activity; Optimum temperuture is 50 ℃; Ph optimum 5.5 is incubated 30h down at 50 ℃, and enzyme is lived and still reached more than 50%.Carry out starch according to the concentration of 5-20% and size mixing, insulation is 5-15 minute under 50-90 ℃ of condition; 30 ℃-60 ℃ of attemperation; After transferring pH5.0-6.0; Add the acid heat-resisting isoamylase of 2-100U and the alpha-cyclodextrin glucosyl transferase of 10-100 unit according to every gram starch, behind the organic solvent of adding 5% (v/v), fully reacted 4-10 hour; The Schardinger dextrins total conversion rate reaches 76.95%, is present domestic and foreign literature public reported highest level.
Description of drawings
The acid heat-resisting isoamylase separation and purification SDS-PAGE figure of Fig. 1.
1, sample behind the Sephadex G50 purifying; 2, sample behind the DEAE-Sepharose purifying; M, standard protein molecular weight.
The acid heat-resisting isoamylase optimum temperuture of Fig. 2.
The acid heat-resisting isoamylase ph optimum of Fig. 3.
The acid heat-resisting isoamylase thermostability of Fig. 4.
The acid heat-resisting isoamylase pH stability of Fig. 5.
Embodiment
Embodiment 1: the structure of genetic engineering bacterium
1, goes up the gene order (Genbank NC_007333.1) of the Tfu_1891 of login according to NCBI; Adopt chemical total synthesis method to synthesize heat-resisting isoamylase gene order; Be cloned into pMD18-T simple carrier (commercialization tool carrier); Connect product transformed into escherichia coli JM109, the converted product coating contains the LB flat board of 100mg/L penbritin.Through 37 ℃ of overnight cultures, choosing colony, insert the LB liquid nutrient medium, extract plasmid behind 8～10h, called after Tfu_1891/pMD18-T simple carries out sequencing with this plasmid.The result shows that inserting fragment is the dna fragmentation of a 2124bp, encodes one and contains 708 amino acid whose protein.
2, the plasmid that is used to make up coli expression carrier is pT7-7, has the T7 promotor.PT7-7 plasmid and Tfu_1891/pMD18-T simple are carried out Nde I and HindIII double digestion respectively; Enzyme connects with the T4 ligase enzyme after cutting product rubber tapping recovery again, connects product Transformed E .coli JM109 competent cell; Cultivate 8h through 37 ℃; Choose transformant shaking culture in the LB that contains 100mg/L penbritin liquid, extract plasmid, enzyme is cut checking and is obtained expression plasmid Tfu_1891/pT7-7.
3, with plasmid Tfu_1891/pT7-7 Transformed E .coliBL21 (DE3) host bacterium, coating contains on the LB flat board of penbritin (100mg/L), cultivates 8h for 37 ℃.Choose single bacterium colony to liquid LB, 37 ℃ of overnight cultures are preserved the glycerine pipe.
Embodiment 2: the fermentative prodn isoamylase
37 ℃ of liquid culture in the glycerine pipe bacterial strain switching LB substratum are spent the night, and TB (glycerine 5g/L, peptone 12g/L, yeast extract paste 24g/L, K are inserted in the back
2HPO
412.54g/L, KH
2PO
42.31g/L) fermentation broth; 30 ℃ be cultured to OD reach 0.6 the back induce with final concentration 0.01 μ M isopropylthio-, constant temperature or be cooled to 25 ℃ cultivations 36 to 48 hours, centrifugal thalline; With pH5.50.1M phosphoric acid-citrate buffer solution suspension cell; Ultrasonication, isoamylase vigor in the supernatant is measured in centrifugal back, and reorganization isoamylase fermentative activity reaches 3185U/mL.
Embodiment 3: the purifying and the zymologic property of reorganization isoamylase
In 4 ℃, 40000 * g is centrifugal, and 20min removes cell debris with the supernatant of ultrasonication.Adding 20% solid ammonium sulfate is saltoutd and is spent the night in supernatant, and 4 ℃, the centrifugal 20min of 40000 * g, taking precipitate are with an amount of pH 5.5, and 20mM phosphoric acid-citrate buffer solution dissolving is through processing all article behind the 0.4 μ m membrane filtration.After sample joins Sephadex G50 purifying again behind the DEAE-Sepharose anionite-exchange resin purifying, obtain electrophoretically pure isoamylase, the protein SDS-PAGE electrophorogram is seen Fig. 1 in the purge process.
The isoamylase of will recombinating carries out the test of different substrate hydrolysis, finds its hydrolyzable Glutinous Semen Maydis pulullan, can not hydrolysis glycogen and pulullan polysaccharide, and the result shows that reorganization isoamylase encoding sox realizes efficiently expressing.The optimum temperuture of acid heat-resisting isoamylase is 50 ℃ (Fig. 2), ph optimum 5.5 (Fig. 3), and under 50 ℃ of conditions, the transformation period can reach 30h (Fig. 4).
Embodiment 4: the production of alpha-cylodextrin
Present embodiment adopt acid heat-resisting isoamylase to starch earlier part take off and add alpha-cyclodextrin glucosyl transferase again and carry out cyclisation.
Carry out starch according to the concentration of 5-20% and size mixing, insulation is 5-15 minute under 50-90 ℃ of condition; 30 ℃-60 ℃ of attemperation, behind the accent pH5.0-6.0, the acid heat-resisting isoamylase according to every gram starch adding 2-100U reacted 2-6 hour.Add the alpha-cyclodextrin glucosyl transferase of 10-100 unit then by every gram starch, behind the nonylcarbinol of adding 5% (v/v), fully reacted 4-10 hour again, the Schardinger dextrins total conversion rate reaches 74.5%, and wherein the alpha-cylodextrin transformation efficiency 59.6%.
Embodiment 5: the production of alpha-cylodextrin
Present embodiment adopts acid heat-resisting isoamylase and alpha-cyclodextrin glucosyl transferase while converted starch to prepare alpha-cylodextrin.
Carry out starch according to the concentration of 5-20% and size mixing, insulation is 5-15 minute under 50-90 ℃ of condition; 30 ℃-60 ℃ of attemperation; After transferring pH5.0-6.0; Add the acid heat-resisting isoamylase of 2-100U and the alpha-cyclodextrin glucosyl transferase of 10-100 unit according to every gram starch, add the nonylcarbinol of 5% (v/v) again after, fully reacted 4-10 hour; The Schardinger dextrins total conversion rate reaches 76.9%, and wherein the alpha-cylodextrin transformation efficiency 61.7%.
Claims (7)
1. the heat-resisting isoamylase genetic engineering bacterium of acidity is characterized in that, this genetic engineering bacterium is to carry the intestinal bacteria E.coli BL21 (DE3) of the heat-resisting isoamylase gene of expression of recombinant plasmid.
2. genetic engineering bacterium according to claim 1 is characterized in that said recombinant plasmid is a pUC series, pET series, pT7-7, or among the pGEX any one.
3. make up the method for claim 1 or 2 said genetic engineering bacteriums; It is characterized in that cloning the Tfu_1891 encoding sox; Genbank accession number NC_007333.1 is expressive host to the pT7-7 carrier with E.coli BL21 (DE3), has realized efficiently expressing of acid heat-resisting isoamylase.
4. the said genetic engineering bacterium of claim 1 is applied to the production of alpha-cylodextrin, it is characterized in that carrying out starch according to the concentration of 5-20% sizes mixing, and insulation is 5-15 minute under 50-90 ℃ of condition; 30 ℃-60 ℃ of attemperation, transfer pH5.0-6.0 after, add the isoamylase of 2-100U and the alpha-cyclodextrin glucosyl transferase of 10-100 unit according to every gram starch, add the organic solvent of 5% volume after, fully reacted 4-10 hour; Reclaim organic solvent, adopt crystallization method to obtain alpha-cylodextrin.
5. working method according to claim 4 is characterized in that said isoamylase is to add synchronously with alpha-cyclodextrin glucosyl transferase, two kinds of enzymes act on simultaneously starch reach take off, cyclisation carries out synchronously.
6. working method according to claim 4, it is characterized in that described isoamylase can add earlier earlier carries out part to starch and takes off and prop up, and adds alpha-cyclodextrin glucosyl transferase again and carries out cyclization.
7. according to the arbitrary described working method of claim 4-6, it is characterized in that described organic solvent is an ethanol, Virahol, propyl carbinol, in the nonylcarbinol any.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586312A (en) * | 2012-02-27 | 2012-07-18 | 江南大学 | Method for expressing intracellular protein matrix and application thereof |
| CN103074399A (en) * | 2013-01-17 | 2013-05-01 | 江南大学 | Production process of gamma-cyclodextrin through double-enzyme compounding |
| CN104673860A (en) * | 2015-03-20 | 2015-06-03 | 江苏农牧科技职业学院 | Method and device for preparing amylose on basis of isoamylase |
| CN104762248A (en) * | 2015-03-25 | 2015-07-08 | 江南大学 | Genetically engineered bacteria for highly yielding isoamylase and fermentation process thereof |
| EP3228705A4 (en) * | 2014-12-05 | 2018-06-27 | Godo Shusei Co., Ltd. | Heat-resistant isoamylase |
| CN112625984A (en) * | 2019-09-24 | 2021-04-09 | 南京盛德生物科技研究院有限公司 | Gene engineering bacterium of isoamylase and application thereof |
| CN116656759A (en) * | 2023-05-25 | 2023-08-29 | 江南大学 | Method for preparing beta-cyclodextrin |
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| CN1161716A (en) * | 1994-07-28 | 1997-10-08 | 孟山都公司 | Isoamylase gene from flaviobacterium sp. compositions contg. it, and method using it |
| CN1705750A (en) * | 2002-08-23 | 2005-12-07 | 纳幕尔杜邦公司 | Utilization of starch products for biological production by fermentation |
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Patent Citations (2)
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| CN1161716A (en) * | 1994-07-28 | 1997-10-08 | 孟山都公司 | Isoamylase gene from flaviobacterium sp. compositions contg. it, and method using it |
| CN1705750A (en) * | 2002-08-23 | 2005-12-07 | 纳幕尔杜邦公司 | Utilization of starch products for biological production by fermentation |
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586312B (en) * | 2012-02-27 | 2014-07-09 | 江南大学 | Method for expressing intracellular protein matrix and application thereof |
| CN102586312A (en) * | 2012-02-27 | 2012-07-18 | 江南大学 | Method for expressing intracellular protein matrix and application thereof |
| CN103074399A (en) * | 2013-01-17 | 2013-05-01 | 江南大学 | Production process of gamma-cyclodextrin through double-enzyme compounding |
| CN103074399B (en) * | 2013-01-17 | 2014-10-01 | 江南大学 | Production process of gamma-cyclodextrin through double-enzyme compounding |
| EP3228705A4 (en) * | 2014-12-05 | 2018-06-27 | Godo Shusei Co., Ltd. | Heat-resistant isoamylase |
| US10017751B2 (en) | 2014-12-05 | 2018-07-10 | Godo Shusei Co., Ltd. | Heat-resistant isoamylase |
| CN104673860A (en) * | 2015-03-20 | 2015-06-03 | 江苏农牧科技职业学院 | Method and device for preparing amylose on basis of isoamylase |
| CN104762248B (en) * | 2015-03-25 | 2018-02-23 | 江南大学 | A kind of genetic engineering bacterium and its zymotechnique of high yield isoamylase |
| CN104762248A (en) * | 2015-03-25 | 2015-07-08 | 江南大学 | Genetically engineered bacteria for highly yielding isoamylase and fermentation process thereof |
| CN112625984A (en) * | 2019-09-24 | 2021-04-09 | 南京盛德生物科技研究院有限公司 | Gene engineering bacterium of isoamylase and application thereof |
| CN112625984B (en) * | 2019-09-24 | 2023-11-24 | 南京盛德生物科技研究院有限公司 | Genetically engineered bacterium of isoamylase and application thereof |
| CN116656759A (en) * | 2023-05-25 | 2023-08-29 | 江南大学 | Method for preparing beta-cyclodextrin |
| CN116656759B (en) * | 2023-05-25 | 2023-11-17 | 江南大学 | Method for preparing beta-cyclodextrin |
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