CN102559568B - 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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- CN102559568B CN102559568B CN201110459137.XA CN201110459137A CN102559568B CN 102559568 B CN102559568 B CN 102559568B CN 201110459137 A CN201110459137 A CN 201110459137A CN 102559568 B CN102559568 B CN 102559568B
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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 a kind of acidic heat-resisting isoamylase genetic engineering bacterium and application thereof, belong to enzyme engineering field.
Background technology
Isoamylase (E.C.3.2.1.68) is that a class can hydrolyzing alpha-1, the starch-debranching enzyme of 6 glucoside bonds, and this enzyme can be hydrolyzed glycogen, amylopectin, α-limit dextrin and β-limit dextrin, and hydrolysate is linear Fructus Hordei Germinatus oligose.Although plant and microorganism can both produce isoamylase, because microorganism has the growth advantage such as rapid, simple to operate and with low cost, so business-like isoamylase is generally by microorganisms producing.The maximum of isoamylase producing bacterial strain research are pseudomonas (Pseudomonas.sp.), are secondly other bacterium and fungi.The isoamylase of different sources has different substrate specificities, hydrolysate, optimal pH, optimum temperuture etc.Isoamylase is mainly used in hydrolyzed starch and produces foodstuff additive, as high glucose slurry, maltose, trehalose, cyclodextrin and Resistant starch etc.
Due to the huge applications potentiality of isoamylase in Starch Hydrolysis, all isoamylase is carried out to a large amount of research both at home and abroad.The optimal pH of the isoamylase in the research such as Harada discovery Pseudomonas.sp. source is between 5 to 6.Olemposka-Beer etc. have reported that the isoamylase optimal pH in Pseudomonas amyloderaosa source is between 3 to 5.And the optimal pH of the isoamylase in genus bacillus source differs greatly, some optimal pHs are acid at PH 5.0, have plenty of the isoamylase optimal pH in alkaline pH9.0. yeast source generally 6.0 to 7.0.Aspect thermostability, the isoamylase of different sources also has larger difference.The isoamylase thermostability in yeast source is poor, and the isoamylase in pseudomonas source generally has good thermostability, Yokobayashi etc. have reported that a kind of isoamylase that derives from pseudomonas has good stability at 45 DEG C, but in the time that temperature reaches 60 DEG C inactivation rapidly.Industrial Starch Hydrolysis process is often carried out under acid and comparatively high temps (50 to 60 DEG C).And have simultaneously under acid and comparatively high temps, there is better activity isoamylase often still less.At present unique business-like isoamylase is produced by Japanese Lin Yuan Co., Ltd., 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, cannot reach industrialization production requirements.As Chen etc. will derive from isoamylase encoding gene successful expression in yeast saccharomyces cerevisiae of Pseudomonas amyloderaosa, the enzyme work in 4 days of fermenting reaches 86U/mL.
Cyclodextrin (Cyclodextrins, conventionally referred to as CD), be a class generated under cyclomaltodextrin glucanotransferase effect by starch or polysaccharide by D-glucopyranose units by α-1, the general name of the end to end ring compound of 4-glycosidic link, common are the molecule of 6,7 and 8 glucose units, be called α-, β-and γ-cyclodextrin.Because alpha-cylodextrin solubleness is larger, so chemical method preparation is more difficult, and can cause larger pollution to environment, biological process is considered to have the method for application potential at present.The technique of producing alpha-cyclodextrins by biological method is generally to adopt α-amylase to carry out partial liquefaction to starch at present, then adds alpha-cyclodextrin glucosyl transferase to carry out cyclization and prepares alpha-cylodextrin.Due to the amylopectin that starch Raw contains 75-85%, amylopectin is hyperbranched polysaccharide, in its structure, just has one by α-1 every 17-28 glucose unit, the tapping point that 6 glycosidic links form.And alpha-cyclodextrin glucosyl transferase does not have α-1,6 hydrolysis of glycoside bond activity, it is difficult to cross α-1, and 6 glycosidic links cause that the cycle is long, starch utilization ratio is not high, and while adopting this technique at present, alpha-cylodextrin transformation efficiency is often in 50% left and right.
Because starch-debranching enzyme can cut off the tapping point in starch, accelerate the reaction of follow-up enzyme, Reaction time shorten, improves the transformation efficiency of starch, thereby increases output, reduce production costs.The reports such as Rendleman first adopt debranching factor (comprising Pullulanase and isoamylase) that waxy corn amylopectin is taken off and propped up, then add alpha-cyclodextrin glucosyl transferase 15 DEG C of reactions 5 days, can make transformation efficiency reach 76%.The employing Pullulanases such as Ivan Pishtiyski take off Zhi Houzai to yam starch, W-Gum etc. and adopt enzymatic conversion within 20 hours, to prepare cyclodextrin, and when discovery W-Gum concentration is 2.5%, transformation efficiency is the highest, is 65%.But there are the serious problems such as raw material is too expensive or concentration of substrate is too low, and reaction time is long in these techniques, is therefore difficult to realize suitability for industrialized production.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of acidic heat-resisting isoamylase genetic engineering bacterium, is the intestinal bacteria E.coli BL21 (DE3) that carries the heat-resisting isoamylase gene of expression of recombinant plasmid.
Another technical problem that the present invention will solve is to provide a kind of method that builds described genetic engineering bacterium, arrive pT7-7 carrier by clone's Tfu_1891 encoding gene (Genbank accession number NC_007333.1), taking E.coli BL21 (DE3) as expressive host, realize the high efficient expression of acid heat-resisting isoamylase.
Described recombinant plasmid is pUC series, pET series, pT7-7, or any one in pGEX.
Another technical problem that the present invention will solve is to provide 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 is incubated 5-15 minute under 50-90 DEG C of condition; Regulate 30 DEG C-60 DEG C of temperature, adjust after pH5.0-6.0, add the isoamylase of 2-100U and the alpha-cyclodextrin glucosyl transferase of 10-100 unit according to every gram of starch, add after the organic solvent of 5% volume, fully react 4-10 hour; Reclaim organic solvent, adopt crystallization method to obtain alpha-cylodextrin.
Described isoamylase is to synchronize and add with alpha-cyclodextrin glucosyl transferase, and two kinds of enzymes act on starch simultaneously and reach de-, cyclisation and synchronously carry out.
In addition, isoamylase can first add first starch is carried out to de-of part, then adds alpha-cyclodextrin glucosyl transferase and carry out cyclization.
Described organic solvent is ethanol, Virahol, propyl carbinol, in 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 DEG C, optimal pH 5.5 is incubated 30h at 50 DEG C, and enzyme is lived and still reached more than 50%.Carry out starch according to the concentration of 5-20% and size mixing, under 50-90 DEG C of condition, be incubated 5-15 minute; Regulate 30 DEG C-60 DEG C of temperature, adjust after pH5.0-6.0, add the heat-resisting isoamylase of acidity of 2-100U and the alpha-cyclodextrin glucosyl transferase of 10-100 unit according to every gram of starch, add after the organic solvent of 5% (v/v), fully reaction 4-10 hour, cyclodextrin total conversion rate reaches 76.95%, for current domestic and foreign literature is openly reported highest level.
Brief description of the drawings
The acid heat-resisting isoamylase separation and purification SDS-PAGE figure of Fig. 1.
1, sample after Sephadex G50 purifying; 2, sample after DEAE-Sepharose purifying; M, standard protein molecular weight.
The acid heat-resisting isoamylase optimum temperuture of Fig. 2.
The acid heat-resisting isoamylase optimal pH 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, according to the gene order (Genbank NC_007333.1) of the Tfu_1891 of the upper login of 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 and transform e. coli jm109, converted product coating is containing the LB flat board of 100mg/L penbritin.Through 37 DEG C of overnight incubation, choosing colony, access LB liquid nutrient medium, extracts plasmid after 8～10h, and called after Tfu_1891/pMD18-T simple, carries out sequencing by this plasmid.Result shows that Insert Fragment is the DNA fragmentation of a 2124bp, encodes one and contains 708 amino acid whose protein.
2, be pT7-7 for building the plasmid of coli expression carrier, with T7 promotor.PT7-7 plasmid and Tfu_1891/pMD18-T simple are carried out respectively to Nde I and HindIII double digestion, enzyme is cut after product rubber tapping recovery, connect with T4 ligase enzyme again, connect product Transformed E .coli JM109 competent cell, cultivate 8h through 37 DEG C, 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,, by plasmid Tfu_1891/pT7-7 Transformed E .coliBL21 (DE3) Host Strains, coating, containing on the LB flat board of penbritin (100mg/L), is cultivated 8h for 37 DEG C.Choose single bacterium colony to liquid LB, 37 DEG C of overnight incubation, preserve glycerine pipe.
Embodiment 2: fermentative production isoamylase
37 DEG C of liquid culture in glycerine pipe bacterial strain switching LB substratum are spent the night, rear access TB (glycerine 5g/L, peptone 12g/L, yeast extract paste 24g/L, K
2hPO
412.54g/L, KH
2pO
42.31g/L) fermentation broth, 30 DEG C are cultured to OD and reach 0.6 rear use final concentration 0.01 μ M isopropylthio-β-D-galactoside induction, constant temperature or be cooled to 25 DEG C cultivate 36 to 48 hours, centrifugal thalline, with pH5.50.1M phosphoric acid-citrate buffer solution suspension cell, ultrasonication, isoamylase vigor in centrifugal rear mensuration supernatant liquor, restructuring isoamylase fermentative activity reaches 3185U/mL.
Embodiment 3: purifying and the zymologic property of restructuring isoamylase
By the supernatant liquor of ultrasonication, in 4 DEG C, 40000 × g is centrifugal, and 20min removes cell debris.Spend the night to adding 20% solid ammonium sulfate to saltout in supernatant liquor, 4 DEG C, the centrifugal 20min of 40000 × g, appropriate pH 5.5 for taking precipitate, 20mM phosphoric acid-citrate buffer solution dissolves, by making loading sample after 0.4 μ m membrane filtration.After DEAE-Sepharose anionite-exchange resin purifying, sample obtains electrophoretically pure isoamylase after joining Sephadex G50 purifying again, and in purge process, protein SDS-PAGE electrophorogram is shown in Fig. 1.
Restructuring isoamylase is carried out to different substrate hydrolysis tests, find its hydrolyzable Glutinous Semen Maydis amylopectin, can not be hydrolyzed glycogen and pulullan polysaccharide, the result isoamylase encoding gene that shows to recombinate is realized high efficient expression.The optimum temperuture of acid heat-resisting isoamylase is 50 DEG C (Fig. 2), optimal pH 5.5 (Fig. 3), and under 50 DEG C of conditions, the transformation period can reach 30h (Fig. 4).
Embodiment 4: the production of alpha-cylodextrin
The present embodiment adopts acid heat-resisting isoamylase to add alpha-cyclodextrin glucosyl transferase to carry out cyclisation to de-of the first part of starch again.
Carry out starch according to the concentration of 5-20% and size mixing, under 50-90 DEG C of condition, be incubated 5-15 minute; Regulate 30 DEG C-60 DEG C of temperature, adjust after pH5.0-6.0, add the heat-resisting isoamylase of acidity of 2-100U according to every gram of starch, reaction 2-6 hour.Then the alpha-cyclodextrin glucosyl transferase that adds 10-100 unit by every gram of starch, adds after the nonylcarbinol of 5% (v/v), more fully reacts 4-10 hour, and cyclodextrin total conversion rate reaches 74.5%, wherein alpha-cylodextrin transformation efficiency 59.6%.
Embodiment 5: the production of alpha-cylodextrin
The 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, under 50-90 DEG C of condition, be incubated 5-15 minute; Regulate 30 DEG C-60 DEG C of temperature, adjust after pH5.0-6.0, add the heat-resisting isoamylase of acidity of 2-100U and the alpha-cyclodextrin glucosyl transferase of 10-100 unit according to every gram of starch, add again after the nonylcarbinol of 5% (v/v), fully reaction 4-10 hour, cyclodextrin total conversion rate reaches 76.9%, wherein alpha-cylodextrin transformation efficiency 61.7%.
Claims (4)
1. the construction process of an acidic heat-resisting isoamylase genetic engineering bacterium, it is characterized in that cloning Tfu_1891 encoding gene, Genbank accession number NC_007333.1 is to pT7-7 carrier, taking E.coliBL21 (DE3) as expressive host, realize the high efficient expression of acid heat-resisting isoamylase.
2. the genetic engineering bacterium that method builds is according to claim 1 applied to the method for producing alpha-cylodextrin, it is characterized in that carrying out starch according to the concentration of 5-20% sizes mixing, and is incubated 5-15 minute under 50-90 DEG C of condition; Regulate 30 DEG C-60 DEG C of temperature, adjust after pH5.0-6.0, add the isoamylase of 2-100U and the alpha-cyclodextrin glucosyl transferase of 10-100 unit according to every gram of starch, add after the nonylcarbinol of 5% volume, fully react 4-10 hour; Reclaim nonylcarbinol, adopt crystallization method to obtain alpha-cylodextrin.
3. method according to claim 2, is characterized in that described isoamylase is to synchronize and add with alpha-cyclodextrin glucosyl transferase, and two kinds of enzymes act on starch simultaneously and reach de-, cyclisation and synchronously carry out.
4. method according to claim 2, is characterized in that described isoamylase first adds, and first starch is carried out to de-of part, then adds alpha-cyclodextrin glucosyl transferase and carry out cyclization.
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| CN102586312B (en) * | 2012-02-27 | 2014-07-09 | 江南大学 | Method for expressing intracellular protein matrix and application thereof |
| 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 |
| 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 |
| 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 |
| CN116656759B (en) * | 2023-05-25 | 2023-11-17 | 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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| 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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