CN104774794A - Strain capable of producing D-mannose isomerase and method for producing D-mannose isomerase by using same - Google Patents
Strain capable of producing D-mannose isomerase and method for producing D-mannose isomerase by using same Download PDFInfo
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- CN104774794A CN104774794A CN201510195854.4A CN201510195854A CN104774794A CN 104774794 A CN104774794 A CN 104774794A CN 201510195854 A CN201510195854 A CN 201510195854A CN 104774794 A CN104774794 A CN 104774794A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/24—Preparation of compounds containing saccharide radicals produced by the action of an isomerase, e.g. fructose
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Abstract
The invention provides a strain capable of producing D-mannose isomerase and a method for producing D-mannose isomerase by using the same, belonging to the food biotechnical field. The invention provides a pseudomonas sp. SK27.016 screened from rotted juicy peach and collected at China Center for Type Culture Collection with a collection number of CCTCC NO.M2014411 and further provides a method for producing D-mannose isomerase through fermentation by using the strain. The method comprises the following steps: (a) fermenting the strain in a fermentation culture medium to obtain the thalli of pseudomonas sp. SK27.016; (b) converting D-fructose by using intracellular D-mannose isomerase produced by pseudomonas sp. SK27.016 to produce D-mannose; and (c) separating and purifying D-mannose. According to the method, D-mannose isomerase produced by the strain is used for efficiently producing D-mannose from D-fructose; therefore, the method is suitable for mass production and a new method is provided for enzyme-method industrial preparation of D-mannose.
Description
Technical field
The present invention relates to produce D-MANNOSE isomerase a kind of pseudomonas (
pseudomonas sp.) SK 27.016 screening and utilize this strain fermentation thalline transformation of D-levulose to produce the method for D-MANNOSE, belong to technical field of food biotechnology.
Background technology
D-MANNOSE is a kind of saccharic nutrient substance be uniquely used at present clinically, is distributed widely in body fluid and tissue, especially in nerve, skin, testis, retina, liver and intestines.It is directly utilized synthesis glycoprotein, participates in immunomodulatory.In addition, in recent years find that D-MANNOSE has Tumor suppression to grow and transfer, increase the effect of cancer survival rate, and it has incomparable advantage in treatment urinary tract infections.D-MANNOSE, can recommend to be used as that the supplement therapy of immunoenzyme technics is a kind of rare is called the not enough disease of phosphorylation mannose isomerase, this is called as carbohydrate defect glycoprotein syndrome 1b type (CDGS Ib type), show as intestinal protein to run off, hepatic diseases, hypoglycemia, and blood coagulation disorders etc.The market requirement of D-MANNOSE grows with each passing day, but its natural content is very low, therefore relies on merely the extraction of crude substance to can not meet the demand in market far away.
The method of current production D-MANNOSE mainly contains chemical synthesis and biological synthesis process.Biological synthesis process is divided into again plants enriched method and microbe fermentation method.Comparatively speaking, chemical synthesis is with a long history to be swift in response, but the expensive raw material price used, toxicity are large, and reaction conditions is violent, poor stability, and solvent corrosion used is strong, there is potential safety hazard, should not be used for foodstuffs industry.Plants enriched method cost is high, productive rate is low, have larger limitation.Because microorganism has that the speed of growth fast period is short, growth conditions is gentle, metabolic process is simple and the advantage such as widely distributed, so Production by Microorganism Fermentation D-MANNOSE is not by the restriction of space, environment, resource, have cost low, without remarkable advantages such as chemical residual, output are high, be the desirable route producing medicine and food grade D-MANNOSE.
Summary of the invention
The object of this invention is to provide a kind of new microorganism pseudomonas, after adopting fermentation, thalline transformation of D-levulose generates D-MANNOSE.
Another object of the present invention is to provide a kind of method utilizing above-mentioned pseudomonas strains to produce D-MANNOSE.
Another object of the present invention is to provide a kind of from the method containing separation and purification D-MANNOSE the conversion fluid of D-MANNOSE.
Technical scheme of the present invention: the bacterial strain of D-MANNOSE isomerase is produced in a strain, be separated to obtain from the honey peach of Yangshan, Wuxi, according to the mensuration of the mushroom character of certain rule, the analysis of biochemical property and 16sRNA sequence draw its Classification And Nomenclature be pseudomonas (
pseudomonas sp.) SK 27.016, be preserved in China typical culture collection center, deposit number is: CCTCC NO:M 2014411.
With the microorganism pseudomonas of described product D-MANNOSE isomerase (
pseudomonas sp.) SK 27.016 produces the method for D-MANNOSE, with pseudomonas (
pseudomonas sp.) SK 27.016 is starting strain, produce the extraction of D-MANNOSE, D-MANNOSE through seed culture, fermentation culture, thalline transformation of D-levulose, concrete steps are:
(1) seed culture
Seed culture medium: ammonia chloride 1 ~ 12 g/L, Sodium phosphate dibasic 1 ~ 6 g/L, potassium primary phosphate 0.1 ~ 1 g/L, magnesium chloride 0.05 ~ 0.1 g/L, sodium sulfate 0.1 ~ 1 g/L, D-Fructose 1 ~ 20 g/L, deionized water is prepared, and adjusts pH 7.0;
Seed culture condition: select on flat board and cultivate good single bacterium colony SK 27.016 in 25 ~ 37 DEG C, shaking speed 200 rpm, cultivate 14 ~ 20 h in seed culture medium.
(2) fermentation culture
Fermention medium: ammonia chloride 1 ~ 12 g/L, Sodium phosphate dibasic 1 ~ 6 g/L, potassium primary phosphate 0.1 ~ 1 g/L, magnesium chloride 0.05 ~ 0.1 g/L, sodium sulfate 0.1 ~ 1 g/L, D-Fructose 1 ~ 20 g/L, deionized water is prepared, and adjusts pH 7.0;
Fermentation condition: inoculum size 1% ~ 10%, temperature 25 ~ 37 DEG C, shaking speed 200 rpm, ferment in fermention medium 10 ~ 24 h.
(3) thalline transforms
First undertaken centrifugal by fermentation culture: get 1 L cultivation and fermentation liquid, centrifugal 5 ~ 25 min of 5000 ~ 8000 rpm, get and precipitate to obtain thalline;
Thalline transforms again, conversion condition: adopt D-Fructose content to be that the resuspended thalline of phosphate buffer soln of 10 ~ 600 g/L, pH 7.5 is to OD
600=2, temperature 40 DEG C transforms 1 ~ 72 h, obtains the mixed sugar liquid of D-Fructose and D-MANNOSE;
(4) extraction of D-MANNOSE
To transform that gained mixed sugar liquid is centrifugal, flocculation, Plate Filtration, decolouring, ion-exchange, vacuum concentration and spraying dry, obtain the D-MANNOSE of purifying.
During the extraction of D-MANNOSE, described centrifugal be centrifugal 5 ~ 25 min of 2000 ~ 4000 rpm.
Described flocculation be centrifugal clear liquid at 30 ~ 80 DEG C, add 100 ~ 300 mg/L chitosans stir flocculation.
Described Plate Filtration is by flocculate with chitosan liquid circulation press-in flame filter press filter cloth, till filtrate becomes clearly.
Described decolouring adopts activated carbon decolorizing.
Described ion-exchange, with 1 ~ 3 times of bed volume flow velocity upper prop, after ion exchange resin absorption is saturated, adopts 2 ~ 5 mol/L ammoniacal liquor wash-outs.
Described vacuum concentration adopts outer circulation type (vacuum ranges 86.66 ~ 93.33 KPa), and total solid reaches 20 % ~ 60 % to be stopped.
Described spraying dry: inlet temperature is 100 ~ 200 DEG C, air outlet temperature is 50 ~ 100 DEG C.
Beneficial effect of the present invention: the present invention relates to a strain screen from the honey peach of Yangshan and come pseudomonas (
pseudomonas sp.) SK 27.016, be preserved in China typical culture collection center, deposit number is CCTCC NO:M 2014411.Through the fermentation of 10 ~ 24 h in the fermention medium taking D-Fructose as carbon source, transform through thalline again, be that the D-MANNOSE concentration that substrate can transform generation reaches 2.5 ~ 150 g/L with the D-Fructose of 10 ~ 600 g/L, the preparation of industrialization for D-MANNOSE provides new method.The D-MANNOSE that the present invention produces is safe and reliable, is a kind of functional product having very much market potential, is widely used in the industries such as food, makeup, medicine.The present invention can produce D-MANNOSE efficiently, is suitable for carrying out scale operation.
Biological material specimens preservation: the bacterial strain of D-MANNOSE isomerase is produced in a strain, its Classification And Nomenclature be pseudomonas (
pseudomonas sp.) SK 27.016, be preserved in China typical culture collection center, be called for short CCTCC, address: Wuhan, China Wuhan University, deposit number is CCTCC NO:M 2014411, preservation date on September 12nd, 2014.
Embodiment
Be below pseudomonas (
pseudomonas sp.) SK 27.016 carries out the embodiment of fermentative production D-MANNOSE, but technical scope of the present invention is not limited to listed several examples, under the prerequisite not changing its main points, can make various change and implement.In addition, technical scope of the present invention prolongs and the scope of equalization.
Embodiment 1 produces the separation of mannose isomerase bacterium
The peach adopting nature to rot or apple, get a little pulp and join (in g/L) in isolation medium: ammonia chloride 1 ~ 12 g/L, Sodium phosphate dibasic 1 ~ 6 g/L, potassium primary phosphate 0.1 ~ 1 g/L, magnesium chloride 0.05 ~ 0.1 g/L, sodium sulfate 0.1 ~ 1 g/L, D-Fructose 1 ~ 20 g/L, deionized water is prepared; Adjust pH 7.0; In 30 DEG C, 200 rpm cultivation 14 ~ 24 h, after dilution spread, obtain pure growth, cultivate 30 h for 30 DEG C, obtain the bacterial strain that 15 strain enzyme actives are high, a selected strain enzyme is lived higher bacterial strain, this D-MANNOSE isomery enzyme-producing bacteria is sent to Sangon Biotech (Shanghai) Co., Ltd. and measures the morphological specificity of this bacterium, physio-biochemical characteristics and the sequential analysis of 16s rna gene, through qualification result be pseudomonas (
pseudomonas sp.) SK 27.016.
The preparation of the thick enzyme powder of embodiment 2
The pseudomonas SK27.016 obtained, in the fermentation medium (in g/L): ammonia chloride 2 g/L, Sodium phosphate dibasic 6 g/L, potassium primary phosphate 0.5 g/L, magnesium chloride 0.05 g/L, sodium sulfate 0.1 g/L, D-Fructose 10 g/L, deionized water is prepared; Adjust pH 7.0; At 30 DEG C, 24 h are cultivated under the condition of 200 rpm, frozen centrifugation, obtained fermentation thalli is crude enzyme liquid, crude enzyme liquid is carried out concentrated after, concentrated enzyme liquid first being added ammonium sulfate to saturation ratio is that 40% precipitation removes most foreign protein, then increases the saturation ratio of ammonium sulfate to 80%, the protein frozen of collecting precipitation is dry, obtains thick enzyme powder.
Embodiment 3 enzyme reaction
Use the thick enzyme powder of embodiment 2, join the 50 mM phosphate buffered saline buffer (Na containing D-Fructose 600 g/L with 0.5 U/mL
2hPO
4-NaH
2pO
4) in, at 40 DEG C, carry out the enzyme reaction of 5 h, transform generation 150 g/L D-MANNOSE.
The mensuration of embodiment 4 seminose output
By the enzyme reaction solution carried out in embodiment 3 through adsorbing diluted film process, by Agilent liquid chromatograph, carry out quantitatively D-MANNOSE, D-Fructose content respectively, analysis condition is as follows: INSTRUMENT MODEL: Ag1260, chromatographic column: Sugar-Pak I, 6.5 × 300 mm; Moving phase: redistilled water; Flow velocity: 0.4 mL/min; Detector: differential refraction detector; Column temperature: 85 DEG C.
The optimization of embodiment 5 enzyme reaction condition
Use from pseudomonas (
pseudomonas sp.) optimization of zymocyte liquid enzyme reaction condition of SK 27.016.According to the enzyme reaction condition in embodiment 3, have studied the suitableeest enzyme reaction condition during zymocyte liquid used from pseudomonas SK 27.016, find use 100 g/L D-Fructose, at 40 DEG C, under pH7.5 condition, react the D-MANNOSE that 2 h can obtain maximum yield 25%.
D-MANNOSE is produced in embodiment 6 cell transformation
Centrifugal: centrifugal 8000 rpm × 5 min of fermentation thalli.Conversion condition: adopt D-Fructose content to be that the phosphate buffer soln suspension thalline of 10 g/L, pH 7.5 is to OD
600=2, under temperature is 40 DEG C of conditions, transforms 2 h, 2.5 g/L D-MANNOSEs can be obtained.
Embodiment 7 refines D-MANNOSE
Conversion fluid, after frozen centrifugation, collects the supernatant liquor containing D-MANNOSE, adopts highly acidic cation and weak base anion-exchange resin series connection desalination bleaching, then passes through DTF-Ca
2+type ion exchange resin carries out separation and purification.By high-performance liquid chromatogram determination, being separated the D-MANNOSE purity obtained can reach 98%.
Claims (9)
1. the bacterial strain of D-MANNOSE isomerase is produced in a strain, its Classification And Nomenclature be pseudomonas (
pseudomonas sp.) SK 27.016, be preserved in China typical culture collection center, deposit number is CCTCC NO:M 2014411.
2. utilize the bacterial strain SK 27.016 of the product D-MANNOSE isomerase described in claim 1 to produce a method for D-MANNOSE, it is characterized in that with pseudomonas (
pseudomonas sp.) SK 27.016 is starting strain, produce the extraction of D-MANNOSE, D-MANNOSE through seed culture, fermentation culture, thalline transformation of D-levulose, concrete steps are:
(1) seed culture
Seed culture medium: ammonia chloride 1 ~ 12 g/L, Sodium phosphate dibasic 1 ~ 6 g/L, potassium primary phosphate 0.1 ~ 1 g/L, magnesium chloride 0.05 ~ 0.1 g/L, sodium sulfate 0.1 ~ 1 g/L, D-Fructose 1 ~ 20 g/L, deionized water is prepared, and adjusts pH 7.0;
Seed culture condition: select on flat board and cultivate good single bacterium colony SK 27.016 in 25 ~ 37 DEG C, shaking speed 200 rpm, cultivate 14 ~ 20 h in seed culture medium;
(2) fermentation culture
Fermention medium: ammonia chloride 1 ~ 12 g/L, Sodium phosphate dibasic 1 ~ 6 g/L, potassium primary phosphate 0.1 ~ 1 g/L, magnesium chloride 0.05 ~ 0.1 g/L, sodium sulfate 0.1 ~ 1 g/L, D-Fructose 1 ~ 20 g/L, deionized water is prepared, and adjusts pH 7.0;
Fermentation condition: inoculum size 1% ~ 10%, temperature 25 ~ 37 DEG C, shaking speed 200 rpm, ferment in fermention medium 10 ~ 24 h;
(3) thalline transforms:
First undertaken centrifugal by fermentation culture: get 1 L fermentation culture, centrifugal 5 ~ 25 min of 5000 ~ 8000 rpm, get and precipitate to obtain thalline;
Thalline transforms again, conversion condition: adopt D-Fructose content to be that the resuspended thalline of phosphate buffer soln of 10 ~ 600 g/L, pH 7.5 is to OD
600=2, temperature 40 DEG C transforms 1 ~ 72h, obtains the mixed sugar liquid of D-Fructose and D-MANNOSE;
(4) extraction of D-MANNOSE
To transform that gained mixed sugar liquid is centrifugal, flocculation, Plate Filtration, decolouring, ion-exchange, vacuum concentration and spraying dry, obtain the D-MANNOSE of purifying.
3. the method for production D-MANNOSE according to claim 2, it is characterized in that described centrifugal be centrifugal 5 ~ 25 min of 5000 ~ 8000 rpm.
4. the method for production D-MANNOSE according to claim 2, it is characterized in that described flocculation be centrifugal clear liquid at 30 ~ 80 DEG C, add 100 ~ 300 mg/L chitosans stir flocculation.
5. the method for production D-MANNOSE according to claim 2, is characterized in that described Plate Filtration is for being pressed into flame filter press filter cloth by the circulation of flocculate with chitosan liquid, till filtrate becomes clearly.
6. the method for production D-MANNOSE according to claim 2, is characterized in that described decolouring adopts activated carbon decolorizing.
7. the method for production D-MANNOSE according to claim 2, is characterized in that described ion-exchange is with 1 ~ 3 times of bed volume flow velocity upper prop, after ion exchange resin absorption is saturated, adopts 2 ~ 5 mol/L ammoniacal liquor wash-outs.
8. the method for production D-MANNOSE according to claim 2, it is characterized in that described vacuum concentration adopts outer circulation type, vacuum ranges 86.66 ~ 93.33 KPa, total solid reaches 20 % ~ 60 % to be stopped.
9. the method for production D-MANNOSE according to claim 2, is characterized in that described spraying dry: inlet temperature is 100 ~ 200 DEG C, and air outlet temperature is 50 ~ 100 DEG C.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105116088A (en) * | 2015-08-19 | 2015-12-02 | 汤臣倍健股份有限公司 | Detection method for mannose content in dendrobium officinale |
| CN109161514A (en) * | 2018-09-28 | 2019-01-08 | 江南大学 | A kind of recombination D-MANNOSE isomerase and its application in D-MANNOSE production |
| CN109321613A (en) * | 2018-09-30 | 2019-02-12 | 江南大学 | A method of producing D-MANNOSE |
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| CN1366059A (en) * | 2001-12-27 | 2002-08-28 | 杭州华大基因研发中心 | Refractory phosphomannose isomerase gene and its polypeptide coded by it and preparing process |
| CN101519640A (en) * | 2008-11-10 | 2009-09-02 | 江南大学 | Method for increasing yield of producing D-mannose isomerase through pseudomonas |
| CN103484392A (en) * | 2012-06-13 | 2014-01-01 | 中国海洋大学 | Application of pseudomonas fluorescens PGM37 strain to produce glucomannan |
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2015
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| US5124262A (en) * | 1990-02-28 | 1992-06-23 | Amano Pharmaceutical Co., Ltd. | Mannose isomerase and process for mannose production using it |
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| CN101519640A (en) * | 2008-11-10 | 2009-09-02 | 江南大学 | Method for increasing yield of producing D-mannose isomerase through pseudomonas |
| CN103484392A (en) * | 2012-06-13 | 2014-01-01 | 中国海洋大学 | Application of pseudomonas fluorescens PGM37 strain to produce glucomannan |
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| Title |
|---|
| DEAN SHINABARGER等: "Purification and Characterization of Phosphomannose Isomerase Guanosine Diphospho-D-mannose Pyrophosphorylase", 《THE JOURNAL OF BIOLOGICACLH EMISTRY》 * |
| P. ALLENZA等: "Conversion of Mannose to Fructose by Immobilized Mannose Isomerase from Pseudomonas cepacia", 《APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY》 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105116088A (en) * | 2015-08-19 | 2015-12-02 | 汤臣倍健股份有限公司 | Detection method for mannose content in dendrobium officinale |
| CN109161514A (en) * | 2018-09-28 | 2019-01-08 | 江南大学 | A kind of recombination D-MANNOSE isomerase and its application in D-MANNOSE production |
| CN109161514B (en) * | 2018-09-28 | 2020-08-04 | 江南大学 | Recombinant D-mannose isomerase and application thereof in D-mannose production |
| CN109321613A (en) * | 2018-09-30 | 2019-02-12 | 江南大学 | A method of producing D-MANNOSE |
| CN109321613B (en) * | 2018-09-30 | 2020-11-06 | 江南大学 | Method for producing D-mannose |
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