CN102071164A - Gene engineering bacterium for producing glucosamine and application thereof - Google Patents
Gene engineering bacterium for producing glucosamine and application thereof Download PDFInfo
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- CN102071164A CN102071164A CN 201010578702 CN201010578702A CN102071164A CN 102071164 A CN102071164 A CN 102071164A CN 201010578702 CN201010578702 CN 201010578702 CN 201010578702 A CN201010578702 A CN 201010578702A CN 102071164 A CN102071164 A CN 102071164A
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Abstract
The invention discloses a gene engineering bacterium for producing glucosamine and application thereof. The gene engineering bacterium is the gene engineering bacterium E.coloK-12-glms which is obtained by introducing the gene (glms) of glucosamine synthetase into Escherichia coli E.coliK-12. The bacterial strain is used for producing the glucosamine by fermentation, and has the advantages of short fermentation time, high productivity, low production cost, environmental friendliness, no anaphylactic reaction and the like; and the produced glucosamine can be widely applied to the fields of medicine, food and the like.
Description
Technical field
The present invention relates to a kind of product glucosamine genetic engineering bacterium and application method thereof, belong to technical field of bioengineering.
Background technology
(Glucosamine 2-amino-2-deoxy-D-glucose) is compound after a hydroxyl of glucose is replaced by amino to glucosamine.Almost in existence and all organisms, comprising bacterium, yeast, filamentous fungus, plant and animal body, is the main moiety of glycoprotein and proteoglycan.Glucosamine can act on joint cartilage specifically, recover the normal metabolic function of chondrocyte, can stimulate the chondrocyte to produce protein-polysaccharide with normal polymer structure, the enzyme that suppresses the damage cartilage, delay the pathologic process of osteoarthritis and the progress of disease, improve joint motion, alleviating pain.Therefore, clinically osteoarthritis that are used for the treatment of more.In addition, glucosamine also has liver kidney detoxifcation, the performance anti-inflammatory, protects the effect of liver; Stimulate the hyperplasia of bifidus bacillus in the baby intestinal; As antibacterial-anti-inflammatory drug, be used for the treatment of rheumatic arthritis and gastric ulcer pharmaceutically.In food, be a kind of important micro-carbohydrate content that add in the infant formulas Ruzhong, the starting raw material of still synthetic VB6 and riboflavin intermediate also can be used in makeup and the fodder additives in addition.
The chitin hydrolysis method is the main method that China produces glucosamine at present, and chitin hydrolysis method transformation efficiency is low, produces a large amount of acid waste waters, thereby product cost height, seriously polluted.At present, less to the report of biological process production glucosamine both at home and abroad, fermentation yield is also lower, does not reach industrial production requirement as yet.
Summary of the invention
Technical problem to be solved by this invention provides a kind of product glucosamine genetic engineering bacterium, and this bacterial strain can be used for the fermentative production glucosamine.
Described genetic engineering bacterium be with the gene of glucosamine synthetic enzyme (
GlmS) import the genetic engineering bacterium that intestinal bacteria obtain.
The construction process of described genetic engineering bacterium is: by the clone
E. coliThe gene of glucosamine synthetic enzyme in BL21 (DE3) genome (GenBank No. CP001509.3)
GlmSSegment is used restriction enzyme
SacI and
HindIII is right
GlmSGene segment and pET-28a (+) carry out enzyme and cut, and will by ligation
GlmSGene segment inserts plasmid pET-28a's (+)
SacI and
HindBetween the III site, obtain recombinant plasmid pET-28a (+)-
GlmS, will carry again
GlmSPlasmid pET-28a (+)-
GlmSTransformed into escherichia coli
E. coliObtain genetic engineering bacterium among the K-12
E. coliK-12-
GlmS
Described
E. coliK-12 is available from ATCC(ATCC No:25947), described plasmid is pET-28a (+).
The technical problem that the present invention also solves has provided a kind of method of producing glucosamine genetic engineering bacterium fermentative production glucosamine.
For addressing the above problem, concrete grammar is as follows:
With genetic engineering bacterium
E. coliK-12-
GlmSCultivate 12 h under 37 ℃ of conditions, shaking speed is 200 rpm, treats OD
600Reach at 0.8 o'clock, add lactose (make the lactose final concentration is 5 g/L in the fermented liquid) according to 10% concentration and induce.
Described substratum consists of:
Seed culture medium: peptone 12 g, yeast extract paste 24 g, glycerine 4ml adds kantlex according to 50 μ g/ml, the pH nature;
Fermention medium: peptone 12 g, yeast extract paste 24 g, glucose 10 g add kantlex according to 50 μ g/ml, the pH nature.
The present invention adopts genetic engineering bacterium fermentative production glucosamine, has the fermentation time weak point, the production intensity height, and production cost is low, and environmental pollution is little, advantages such as no anaphylaxis.The glucosamine that production obtains can be widely used in fields such as medicine, food.
Embodiment
Embodiment 1:
E. coliK-12-
GlmSConstruction process
The design primer:
Last primer: 5 '-C
GA GCT C(the underscore sequence is represented the restriction enzyme recognition site to AT GTG TGG AAT TGT TGG C-3 '
SacI)
Following primer: 5 '-CCC
AAG CTT(the underscore sequence is represented restriction enzyme site to TTA CTC AAC CGT AAC CGA-3 '
HindIII)
The glucosamine synthase gene
GlmSBe to utilize
E. coliBL21 (DE3) genome (GenBank No. CP001509.3) carries out as masterplate that PCR obtains.Use restriction enzyme
SacI and
HindIII is right
GlmSGene segment and pET-28a (+) carry out enzyme and cut, and will by ligation
GlmSGene segment inserts plasmid pET-28a's (+)
SacI and
HindBetween the III site, obtain recombinant plasmid pET-28a (+)-
GlmSWith recombinant plasmid pET-28a (+)-
GlmSTransform
E. coliK-12(ATCC No:25947), obtain recombination bacillus coli
E. coliK-12-
GlmS
Embodiment 2: the detection method of glucosamine;
The glucosamine typical curve is drawn: accurately take by weighing glucosamine 0.0100 g, add 20.0 ml distilled water, be mixed with the DAS of 0.5000 g/l, being diluted to concentration then respectively is 0.2500 g/l, 0.1250g/l, 0.0625g/l, the solution of 0.0313 g/l.Get respective concentration DAS 0.5 ml respectively in the glass stopper test tube, add methyl ethyl diketone reagent 1.0 ml, 90 ℃ of water bath processing 1h, be cooled to room temperature, slowly add 96%(v/v) ethanol 10.0mL(do not stir), add DMAB reagent 1.0 ml then, mix.Because of reaction system becomes acidity by alkalescence, noted that great amount of carbon dioxide gas produces.Mix back room temperature placement and stablized the 530nm colorimetric in 1 hour.With the sample solution concentration is X-coordinate, and the OD value is an ordinate zou drawing standard curve.
The detection of glucosamine: get fermented liquid 5.0 ml, add 6 mol/l HCl, 8.0 ml, again thalline suspends, this bacteria suspension is transferred in the tool plug test tube, 100 ℃ of water-bath 4.0 h, be cooled to room temperature, NaOH with 12 mol/L transfers pH to 7.0, mend distilled water to 20.0 ml, get 0.5 ml and add methyl ethyl diketone reagent 1.0 ml, 90 ℃ of water bath processing 1h, be cooled to room temperature, slowly adding 96% ethanol, 10 ml(does not stir), add then dimethylin phenyl aldehyde (DMAB) reagent 1.0 ml, mix.Mix the back room temperature and placed 1 hour, 530nm place colorimetric is calculated thalline glucosamine concentration according to typical curve.
Embodiment 3:
E. coliK-12-
GlmSFermentation process
Cultivate intestinal bacteria with the LB slant medium
E. coliK-12-
GlmS, cultivate behind 12 h and to get 1 articulating with transfering loop and go into to be equipped with in the triangular flask of 250mL of 20 mL seed culture mediums and carry out seed culture.Seed culture medium is: seed culture medium: peptone 12 g, and yeast extract paste 24 g, glycerine 4ml adds kantlex according to 50 μ g/ml, the pH nature.Incubation time is 12 h, 37 ℃ of temperature, shaking speed 200 rpm.
Insert with 5% inoculum size in the triangular flask of 500 ml that 100 ml fermention mediums are housed and carry out fermentation culture.Fermention medium: peptone 12 g, yeast extract paste 24 g, glucose 10 g add kantlex according to 50 μ g/ml simultaneously, the pH nature.Incubation time is 12 h, 37 ℃ of temperature, and shaking speed 200 rpm treat OD
600Reach at 0.8 o'clock, add lactose according to 10% concentration and induce.Glucosamine concentration can reach 20g/L in the fermentation ends secondary fermentation liquid.
Though the present invention with preferred embodiment openly as above; but it is not in order to qualification the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, so protection scope of the present invention should be with being as the criterion that claims were defined.
Sequence table
＜110〉Southern Yangtze University
＜120〉a kind of product glucosamine genetic engineering bacterium and application thereof
<160> 2
<170> PatentIn?version?3.5
<210> 1
<211> 25
<212> DNA
＜213〉artificial synthesized sequence
<220>
＜223〉be designed for amplification according to gene order.
<400> 1
cgagctcatg?tgtggaattg?ttggc 25
<210> 2
<211> 27
<212> DNA
＜213〉artificial synthesized sequence
<220>
＜223〉be designed for amplification according to gene order.
<400> 2
cccaagcttt?tactcaaccg?taaccga 27
Claims (6)
1. one kind is produced the glucosamine genetic engineering bacterium, is that the gene with the glucosamine synthetic enzyme imports the genetic engineering bacterium that intestinal bacteria obtain.
2. according to the described genetic engineering bacterium of claim 1, it is characterized in that the gene source of described glucosamine synthetic enzyme is in GenBank No. CP001509.3 genome
GlmSSegment.
3. according to the described genetic engineering bacterium of claim 1, it is characterized in that the construction process of described genetic engineering bacterium is:
1) uses restriction enzyme
SacI and
HindIII is right
GlmSGene segment and pET-28a (+) carry out enzyme and cut, and will by ligation
GlmSGene segment inserts plasmid pET-28a's (+)
SacI and
HindBetween the III site, obtain recombinant plasmid pET-28a (+)-
GlmS
2) with plasmid pET-28a (+)-
GlmSTransformed into escherichia coli
E. coliObtain genetic engineering bacterium among the K-12
E. coliK-12-
GlmS
4. the fermentation process of the described genetic engineering bacterium of claim 1 is characterized in that, genetic engineering bacterium is cultivated 12 h under 37 ℃ of conditions, and shaking speed is 200 rpm, treats OD
600Reach at 0.8 o'clock, add concentration and be 10% lactose and induce, make lactose concn maintain 5 g/L.
5. method according to claim 4 is characterized in that, every liter of seed culture medium consists of: peptone 12 g, and yeast extract paste 24 g, glycerine 4ml adds kantlex according to 50 μ g/ml, the pH nature.
6. method according to claim 4 is characterized in that, every liter of fermention medium consists of: peptone 12 g, and yeast extract paste 24 g, glucose 10 g add kantlex according to 50 μ g/ml, the pH nature.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102268399A (en) * | 2011-06-27 | 2011-12-07 | 江南大学 | High-yield glucosamine engineering bacterium with nagE being knocked-out by homologous recombination and construction method thereof |
| CN102286420A (en) * | 2011-06-27 | 2011-12-21 | 江南大学 | High-glucosamine-yield engineering bacterium with manX knocked out by homologous recombination and construction method thereof |
| CN103589696A (en) * | 2013-09-30 | 2014-02-19 | 安徽丰原发酵技术工程研究有限公司 | Escherichia coli glucosamine synthetase mutant and application thereof |
| CN105463041A (en) * | 2015-12-17 | 2016-04-06 | 安徽丰原发酵技术工程研究有限公司 | Preparation method of glucosamine |
| CN107739728A (en) * | 2017-10-19 | 2018-02-27 | 江南大学 | A kind of recombination bacillus coli of efficiently production Glucosamine and its application |
| CN108103126A (en) * | 2016-11-25 | 2018-06-01 | 北大方正集团有限公司 | A kind of composition for improving Glucosamine fermentation unit yield and its application |
| CN108410783A (en) * | 2018-05-29 | 2018-08-17 | 成都本则生科技有限公司 | A kind of method of high-density cultivation of Escherichia coli fermenting and producing Glucosamine |
| CN116731934A (en) * | 2023-08-08 | 2023-09-12 | 欧铭庄生物科技(天津)有限公司滨海新区分公司 | Escherichia coli and application thereof in production of glucosamine |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1081717A (en) * | 1993-06-14 | 1994-02-09 | 何林 | Biochemical identification carton for bacteria |
-
2010
- 2010-12-08 CN CN201010578702XA patent/CN102071164B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1081717A (en) * | 1993-06-14 | 1994-02-09 | 何林 | Biochemical identification carton for bacteria |
Non-Patent Citations (2)
| Title |
|---|
| 《中国优秀硕士学位论文全文数据库医药卫生科技辑》 20080915 费良润 结核分枝杆菌葡糖胺-6-磷酸合成酶的克隆表达及其性质研究 1-42 1-6 第2008年卷, 第09期 2 * |
| 《生命的化学》 20081231 费良润等 新抗生素和糖尿病治疗药物开发的靶标-葡糖胺-6-磷酸合酶 472-476 1-6 第28卷, 第4期 2 * |
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102286420A (en) * | 2011-06-27 | 2011-12-21 | 江南大学 | High-glucosamine-yield engineering bacterium with manX knocked out by homologous recombination and construction method thereof |
| CN102268399B (en) * | 2011-06-27 | 2013-05-22 | 江南大学 | High-yield glucosamine engineering bacterium with nagE being knocked-out by homologous recombination and construction method thereof |
| CN102268399A (en) * | 2011-06-27 | 2011-12-07 | 江南大学 | High-yield glucosamine engineering bacterium with nagE being knocked-out by homologous recombination and construction method thereof |
| CN103589696A (en) * | 2013-09-30 | 2014-02-19 | 安徽丰原发酵技术工程研究有限公司 | Escherichia coli glucosamine synthetase mutant and application thereof |
| CN103589696B (en) * | 2013-09-30 | 2015-10-14 | 安徽丰原发酵技术工程研究有限公司 | Escherichia coli glucosamine synthetase mutant and application thereof |
| CN105463041A (en) * | 2015-12-17 | 2016-04-06 | 安徽丰原发酵技术工程研究有限公司 | Preparation method of glucosamine |
| CN108103126B (en) * | 2016-11-25 | 2021-08-27 | 北大方正集团有限公司 | Composition for improving fermentation unit yield of glucosamine and application thereof |
| CN108103126A (en) * | 2016-11-25 | 2018-06-01 | 北大方正集团有限公司 | A kind of composition for improving Glucosamine fermentation unit yield and its application |
| CN107739728A (en) * | 2017-10-19 | 2018-02-27 | 江南大学 | A kind of recombination bacillus coli of efficiently production Glucosamine and its application |
| CN108410783B (en) * | 2018-05-29 | 2020-11-03 | 成都本则生科技有限公司 | Method for producing glucosamine by culturing escherichia coli and fermenting |
| CN108410783A (en) * | 2018-05-29 | 2018-08-17 | 成都本则生科技有限公司 | A kind of method of high-density cultivation of Escherichia coli fermenting and producing Glucosamine |
| CN116731934A (en) * | 2023-08-08 | 2023-09-12 | 欧铭庄生物科技(天津)有限公司滨海新区分公司 | Escherichia coli and application thereof in production of glucosamine |
| CN116731934B (en) * | 2023-08-08 | 2023-10-13 | 欧铭庄生物科技(天津)有限公司滨海新区分公司 | Escherichia coli and application thereof in production of glucosamine |
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Address after: No. 188, Beitang street, Liangxi District, Wuxi City, Jiangsu Province Patentee after: Jiangnan University Address before: 1800 No. 214122 Jiangsu city of Wuxi Province Li Lake Avenue Patentee before: Jiangnan University |