CN102634495A - Alpha-transglucosidase - Google Patents
Alpha-transglucosidase Download PDFInfo
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- CN102634495A CN102634495A CN2011100360200A CN201110036020A CN102634495A CN 102634495 A CN102634495 A CN 102634495A CN 2011100360200 A CN2011100360200 A CN 2011100360200A CN 201110036020 A CN201110036020 A CN 201110036020A CN 102634495 A CN102634495 A CN 102634495A
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- transglucosidase
- taotang
- alpha
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Abstract
The invention relates to the technical field of enzyme engineering or biological engineering. Alpha-transglucosidase is a key enzyme in oligoisomaltose production; no commercial production occurs currently in the country; and existing commercial products have a low glycoside transfer rate. Aspergillus niger strains are used as original strains; by adding an inducer of N-methyl-D-glucosamine into a fermentation medium, the aspergillus niger is induced to secrete a lot of alpha-transglucosidase, and thus alpha-transglucosidase fermentation broth with a high density is prepared. The enzyme activity of the fermentation broth is up to 12000 u/ml; the alpha-transglucosidase is applicable to oligoisomaltose production, and has a glycoside transfer rate of up to 65% which is much higher than that of commercial products.
Description
Technical field
The invention belongs to enzyme engineering or technical field of bioengineering, be specifically related to a kind of preparation method of α-Pu Taotang transglucosidase of high commentaries on classics glycosides ability.
Technical background
The α-Pu Taotang transglucosidase can hydrolysis SANMALT-S and the malto-oligosaccharide molecular structure in α-1; 4 glycosidic links; And a glucosyl residue that will dissociate out transfers on α-1,6 in another glucose molecule or the maltose molecule, is the zymin of most critical during oligomeric isomaltose is produced.
The α-Pu Taotang transglucosidase is extensively to be present in plant-animal and the intravital class of enzymes of mikrobe.Except that minority derives from part plant and Mammals, mainly come from the mikrobe at present.Some bacterial strain such as bacterium, mould and yeast can be secreted this enzyme, and what the product enzyme was higher is black mold (Aspergillus niger) and aspergillus oryzae (Aspergillus oryzae).According to investigation; The α-Pu Taotang transglucosidase that Japan Amono Pharmacevtical Co., Ltd. produces derives from black mold; The Hung of Cornell Univ USA selects for use smelly aspergillus (A.fotidus NRRL 337), platform sugar institute to select carbon black aspergillus (Acarbonarius CCRC 30414) for use, move slope selects for use black mold (ATCC 6257, and ATCC 9642) to wait to produce the α-Pu Taotang transglucosidase.Investigation shows that domestic research to the α-Pu Taotang transglucosidase at present mainly concentrates on screening and the optimization of reaction conditions and the research aspect of zymologic property of bacterial strain, does not also form commercial production.
From industriallization,, also there is the deficiency of two aspects though China's isomaltose output is very high: the first, the consumption of α-Pu Taotang transglucosidase is very big, but does not have industriallization because of domestic, so the complete dependence on import of this enzyme; The second, the α-Pu Taotang transglucosidase is in oligomeric isomaltose is produced, and transformation efficiency is lower, generally below 50%.Even external first hand zymin manufacturing enterprise-Japanese Amono Pharmacevtical Co., Ltd.; The α-Pu Taotang transglucosidase of producing (α-transglusidase LAmano is called for short TGLA) it is reported; Be used in the oligomeric isomaltose production; Transformation efficiency also only has 50%, must extract the method for processing purifying through the back, just can obtain highly purified oligomeric isomaltose.
The object of the present invention is to provide a kind of preparation method of α-Pu Taotang transglucosidase of high commentaries on classics glycosides ability.The α-Pu Taotang transglucosidase crude enzyme liquid that this method of using makes; Be used for committed step-commentaries on classics glycosides stage that oligomeric isomaltose is produced, can obviously improve speed of reaction, for whole process of production has shortened the reaction times; Reduced production cost for enterprise simultaneously; Oligomeric isomaltose content will be higher than existing report far away in the product, has also practiced thrift cost for follow-up purification process, has solved the low problem of yield in the oligomeric isomaltose production.
The α-Pu Taotang transglucosidase that utilizes the present invention to make has following characteristic:
1, pH stability: this enzyme can be stablized 30min during pH scope 4.5-7.5, ph optimum 6.5 55 ℃ of temperature;
2, thermostability: this enzyme is under pH value 6.0 conditions, and respectively with treatment of different temperature 30min gained result: this enzyme still keeps 90% of its initial activity in the time of 55 ℃, then is 70% in the time of 70 ℃.
Summary of the invention
In order to solve the low problem of yield in the oligomeric isomaltose production, the object of the present invention is to provide a kind of preparation method of α-Pu Taotang transglucosidase of high commentaries on classics glycosides ability.
To achieve these goals; Technical scheme of the present invention is: utilize Aspergillus niger strain (Chinese industrial microbial strains preservation administrative center preservation; Deposit number is 2450); Through slant culture, shake that bottle spreads cultivation, liquid submerged fermentation, and high speed centrifugation stage, make the high α-Pu Taotang transglucosidase preparation that changes the glycosides ability, the concrete operations step is following:
(1) slant culture
In the test tube that the Cha Shi solid medium is housed, insert Aspergillus niger strain, 30 ℃ leave standstill cultivation 3-5d, make spore suspension;
(2) shake a bottle enlarged culturing
Insert slant pore suspension-s in that shaking in the bottle of PDY substratum is being housed, under 28-32 ℃, 80-150r/min condition, cultivate, must shake phialosporae suspension-s;
(3) liquid submerged fermentation
Shaking the liquid fermentation medium that the amount of phialosporae suspension-s is inoculated in behind autoclaving by 5-10% cultivates;
Wherein: the preparation of the suspension-s of aspergillus niger spore described in the rejuvenation of spawn is the aspergillus niger spore on the substratum to be washed with sterilized water make; The prescription of described fermention medium is by sucrose 5-12%, peptone 2-8%, yeast extract 0.1-0.5%, K
2HPO
40.1-1%, N-methyl D-glycamine 0.01-0.05%, NaCl 0.1-1%, MgSO
40.01-0.05%, all the other moisture make through mixing; Said shake flask fermentation temperature is at 28-35 ℃, 100-160r/min.
(4) fermented liquid is got supernatant and is got α-Pu Taotang transglucosidase crude enzyme liquid through high speed centrifugation.
Description of drawings
Figure of description is the process flow sheet that oligomeric isomaltose is produced.
Embodiment
Embodiment 1
By above-mentioned working method, black mold is processed spore suspension behind slant culture 4d; Access is equipped with in the PDY culture media shaking vase; At 30 ℃, cultivate 48h under the 130r/min condition, the ratio in 7% is shaken at the 500ml of the liquid nutrient medium that 100ml is housed and is inserted spore suspension in the bottle.Wherein the liquid fermentation medium prescription is by sucrose 7%, peptone 3%, yeast extract 0.2%, K
2HPO
40.3%, NaCl 0.2%, MgSO
40.01%, all the other moisture make through mixing, and initial pH6.0 at 32 ℃, cultivates 42h under the 150r/min condition.The crude enzyme liquid that makes is 1000u/ml through the actual measurement vigor.
Embodiment 2
Be with embodiment 1 difference: the liquid fermentation medium prescription is by sucrose 7%, peptone 3%, yeast extract 0.2%, K
2HPO
40.3%, N-methyl D-glycamine 0.02%, NaCl 0.2%, MgSO
40.01%, all the other moisture make through mixing.Press aforementioned production method, the crude enzyme liquid that makes is 12000u/ml through the actual measurement vigor.
Embodiment 3
With the W-Gum is raw material, through steps such as liquefaction, saccharification, commentaries on classics glycosides, produces oligomeric isomaltose, and concrete technical process is shown in accompanying drawing:
Check analysis: the IMO finished product that takes a morsel and make; Other gets the commercial like product and compares; According to oligomeric isomaltose content assaying method among the GB/T20881-2007, detect with regard to the content of oligomeric isomaltose, the content that draws oligomeric isomaltose in the finished product is 65%; And the content of oligomeric isomaltose is merely 50% in the commercially available prod.
Claims (4)
1. the preparation method of the α-Pu Taotang transglucosidase of one kind high commentaries on classics glycosides ability through slant culture, shake a bottle enlarged culturing, liquid submerged fermentation, high speed centrifugation stage, makes α-Pu Taotang transglucosidase preparation efficiently.
2. according to the preparation method of the α-Pu Taotang transglucosidase described in the claim 1, it is characterized in that: fermentative medium formula is by sucrose 5-12%, peptone 2-8%, yeast extract 0.1-0.5%, K
2HPO
40.1-1%, inductor, NaCl 0.1-1%, MgSO
40.01-0.05%, all the other moisture make through mixing.
3. according to the preparation method of the α-Pu Taotang transglucosidase described in the claim 2, it is characterized in that: the inductor in the fermentative medium formula is N-methyl D-glycamine.
4. according to the preparation method of the α-Pu Taotang transglucosidase described in the claim 2, it is characterized in that: the preferred addition of inductor is 0.01-0.05% in the fermentative medium formula.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011100360200A CN102634495A (en) | 2011-02-11 | 2011-02-11 | Alpha-transglucosidase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011100360200A CN102634495A (en) | 2011-02-11 | 2011-02-11 | Alpha-transglucosidase |
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|---|---|
| CN102634495A true CN102634495A (en) | 2012-08-15 |
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ID=46619074
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|---|---|---|---|
| CN2011100360200A Pending CN102634495A (en) | 2011-02-11 | 2011-02-11 | Alpha-transglucosidase |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105505896A (en) * | 2016-02-16 | 2016-04-20 | 上海青瑞食品科技有限公司 | Preparation method of transglucosidase |
| CN105505787A (en) * | 2015-12-25 | 2016-04-20 | 青岛蔚蓝生物集团有限公司 | Aspergillus niger mutant strain for producing transglucosidase |
| CN107304431A (en) * | 2016-04-20 | 2017-10-31 | 顶尚(香港)有限公司 | Polynucleotide passage, the expression vector comprising it and aspergillus niger genetic engineering strain and its application |
| CN109055461A (en) * | 2018-08-28 | 2018-12-21 | 广州双桥股份有限公司 | A kind of production method of oligoisomaltose |
-
2011
- 2011-02-11 CN CN2011100360200A patent/CN102634495A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105505787A (en) * | 2015-12-25 | 2016-04-20 | 青岛蔚蓝生物集团有限公司 | Aspergillus niger mutant strain for producing transglucosidase |
| CN105505896A (en) * | 2016-02-16 | 2016-04-20 | 上海青瑞食品科技有限公司 | Preparation method of transglucosidase |
| CN107304431A (en) * | 2016-04-20 | 2017-10-31 | 顶尚(香港)有限公司 | Polynucleotide passage, the expression vector comprising it and aspergillus niger genetic engineering strain and its application |
| CN109055461A (en) * | 2018-08-28 | 2018-12-21 | 广州双桥股份有限公司 | A kind of production method of oligoisomaltose |
| CN109055461B (en) * | 2018-08-28 | 2021-12-17 | 广州双桥股份有限公司 | Production method of isomaltooligosaccharide |
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Application publication date: 20120815 |