CN103695407B - A kind of raising is dwelt the method for hot bacterium TreP content - Google Patents
A kind of raising is dwelt the method for hot bacterium TreP content Download PDFInfo
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Abstract
The present invention relates to a kind of raising to dwell the method for hot bacterium TreP content, step is as follows: the liquid seeds of hot bacteria strain of dwelling is inoculated in liquid nutrient medium by (1), cultivates, obtained strain cell nutrient solution; (2) strain cell nutrient solution is placed in pressure reactor, ferments under stress, obtained fermented liquid; (3) fermented liquid is centrifugal, collect somatic cells, smudge cells, obtained cytoclasis liquid, adopt two phase aqueous extraction system to prepare TreP cytoclasis liquid.Method of the present invention can improve the TreP content of dwelling in hot mycetocyte, reduces the production cost of enzyme; Method of the present invention can realize serialization, the mass-producing of TreP production, and the production technique of enzyme is easy.
Description
Technical field
The present invention relates to a kind of raising to dwell the method for hot bacterium TreP content, belong to technical field of bioengineering.
Background technology
TreP (Trehalosesynthase, EC5.4.99.16) is glucuronosyltransferases in a kind of molecule, the α-Isosorbide-5-Nitrae glycosidic link of catalysis maltose specifically can be converted into α-1,1 glycosidic link and generate trehalose.Trehalose is by a kind of irreducibility disaccharide of two glucose molecules by hemiacetal hydroxyl condensation; have and protect the microbial film of organism and the specific function of biomacromolecule under adverse circumstance; the ability of the severe environment such as opposing nutritive deficiency, high temperature, high osmotic pressure, drying, toxic substance such as lower animal, plant and microorganism can be given, thus have a wide range of applications in fields such as biological products, food, medicine, crop breeding and fine chemistry industries.
Enzyme optrode is one of main method of producing trehalose, be that the transglycosylation that utilizes zymin to have acts on the substrate such as maltose, sucrose in vitro and produces trehalose, the method has higher specificity, fast gentle, the feature such as production cost is low.Production by Enzymes trehalose has multiple biosynthetic pathway, can by trehalose synthesis such as different enzymes difference catalytic starch, glucose, maltose, oligosaccharides.
TreP is utilized to be that the flow process of substrate trehalose synthesis only needs single step reaction with maltose, the productive rate of trehalose is high and not by the impact of substrate maltose concentration, technique is simple, be easy to regulation and control, be the most simple approach of Production by Enzymes trehalose, thus TreP has been widely used in the suitability for industrialized production of trehalose.M. smegmatics (
mycobacteriumsmegmatis), pimelobacter sp (
pimelobactersp.), Arthrobacter nicotinovorus (
arthrobacternicotinovorus), pseudomonas putida (
pseudomonasputida) and some hot bacterium that dwell (
thermus) etc. all found TreP in bacterial strain, but above-mentioned bacterial strains intracellular TreP content is low, the activity of enzyme is low, causes the production cost of enzyme higher, and therefore how improving the content of TreP, reducing production cost is the technical problem being badly in need of at present solving.
Research finds, when being in the external stimuluss such as hunger, high temperature, high osmotic pressure when microorganism cells, stress reaction to occur, and improves the activity of trehalose synthesis enzyme system in cell, thus increases trehalose output to resist stress from outside.At present certain research is had to the change of part microorganism cell intracellular trehalose and TreP under these conditions, as Duan Zuoying (biological processing, 2007.5) when carrying out the influence research of heat shock treatment on trehalose synthase of Pseudomonas putida S 1, adopting the method for thermal stimulus that the enzyme of TreP is lived and improve 76%; As Chinese patent literature CN12190715A(application number 03129701.3) disclose a kind of method taking glucose as matrix two-step fermentation and produce yeast extracellular-trehalose, first the methods such as nitrogen hunger, heat-shocked, osmotic stress are adopted, induction yeast cell produce more stress enzyme-TreP system, then utilize the TreP system in yeast cell to synthesize extracellular-trehalose in a large number; Ambient pressure is also a kind of incentive condition, the pressure of bio-reactor also has remarkably influenced to the content of specific product in extracellular microbial, as Chinese patent literature CN1480535A(application number 03130410.9) disclose the method utilizing high pressure to improve yeast trehalose output, the method comparatively under normal pressure collating condition intracellular trehalose content improve 20 ~ 70%.At present about utilizing pressure to improve to dwell hot bacterium TreP also there is no relevant report or patent containing quantifier elimination.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, a kind of method that hot bacterium TreP content is dwelt in raising is provided.
The technical solution used in the present invention is:
Raising is dwelt the method for hot bacterium TreP content, and it is characterized in that, step is as follows:
(1) be inoculated in liquid seed culture medium from the hot bacteria strain of dwelling of picking 1-2 ring test tube slant, 65 ~ 70 DEG C, cultivate 12 ~ 16h under 100 ~ 150r/min, liquid seeds after obtained activation, then by liquid seeds by volume 3% ~ 5% inoculum size be inoculated in liquid nutrient medium, 60 ~ 70 DEG C, cultivate 20 ~ 24h under 150 ~ 200r/min, obtained strain cell nutrient solution;
(2) be placed in pressure reactor by strain cell nutrient solution, at 0.5 ~ 2.0MPa pressure bottom fermentation, 1 ~ 6h, fermentation condition is 60 ~ 70 DEG C, 150 ~ 200r/min, obtained fermented liquid;
(3) fermented liquid is centrifugal, collect somatic cells, smudge cells, obtained cytoclasis liquid, then adopt two phase aqueous extraction system to prepare TreP cytoclasis liquid.
Hot bacteria strain of dwelling in described step (1) be thermus aquaticus (
thermusaquaticus) ATCC33923 or Thermus bacterium (
thermussp.) ATCC43815, above bacterial strain is all preserved in American Type Culture preservation center (ATCC), can be obtained by the mode bought.
The component of the liquid seed culture medium in described step (1) is: glucose 10g/L, yeast extract paste 10g/L, peptone 10g/L, NaCl5g/L, pH7.5, distilled water 1000mL.
The component of the liquid nutrient medium in described step (1) is: glucose 20g/L, maltose 5 ~ 10g/L, yeast extract paste 5g/L, peptone 5g/L, KH
2pO
42g/L, MgSO
41g/L, pH7.5, distilled water 1000mL.
The step that employing two phase aqueous extraction system in described step (3) prepares TreP is as follows: in cytoclasis liquid, add ammonium sulfate and molecular weight is the polyoxyethylene glycol of 2000, in total system, make that the mass percent concentration of ammonium sulfate is 8wt% ~ 10wt%, the mass percent concentration of polyoxyethylene glycol is 10wt% ~ 12wt%, mix, leave standstill phase-splitting, phase solution and containing phase solution on the polyoxyethylene glycol of TreP under obtained ammonium sulfate; Ammonium sulfate is added in phase solution on polyoxyethylene glycol, make the mass percent concentration of ammonium sulfate in total system be 5wt% ~ 6wt%, the pH value of regulator solution is 7.0 ~ 8.0, mixes, leave standstill phase-splitting, obtained containing phase solution on phase solution under the ammonium sulfate of TreP and polyoxyethylene glycol; By phase solution under obtained ammonium sulfate, adopt ultrafiltration membrance filter to concentrate, collect trapped fluid, lyophilize, obtained TreP.
Beneficial effect
(1) what the present invention used dwells containing TreP in hot mycetocyte, and this enzyme can be converted into trehalose and catalysis productive rate is high for catalysis maltose specifically, is the zymin of most application prospect in trehalose Production by Enzymes.
(2) method of the present invention utilizes pressure as a kind of external stimulus factor, and the TreP content of dwelling under pressure in hot mycetocyte increases, enzymic activity raises, thus reduces the production cost of TreP.
(3) method of the present invention can realize serialization, the mass-producing of TreP production, and the production technique of enzyme is easy.
Embodiment
Be specifically described below in conjunction with embodiment technical scheme of the present invention or be described further, object is that methods of this invention will be better understood, but protection scope of the present invention is not limited to following embodiment.
The pressure reactor adopted in the present invention is the stainless steel compressive reaction device that a set of pressure with whipping appts can measure and control, and its volume is 3000mL, and sample usable syringes injects.Use high pure air is pressure medium, boosts with the speed of 0.05MPa/min, and pressurize is depressurized to normal pressure with the speed of 0.05MPa/min.In operation, the force value measured is the tank pressure value of reactor.
In the present invention, the enzyme activity determination step of TreP is as follows:
Get cell culture fluid centrifugal 20min under 5000r/min, collect thalline, with distilled water wash thalline three times, obtained wet thallus.Get 1.0g wet thallus to join in the phosphoric acid buffer of 0.03mol/L, pH7.2 of 19.6mL, then add the toluene of 0.4mL, at 35 DEG C, process 2h, obtained cell treatment solution.Get cell treatment solution at 4 DEG C, centrifugal 15min under 12000r/min, collect supernatant liquor, supernatant liquor, through the filtering with microporous membrane of 0.45 μm, collects filtered liquid, obtained crude enzyme liquid.Getting 10.0mL crude enzyme liquid joins in the 10wt% maltose solution of 10.0mL, 50 DEG C, under 150r/min, reaction 10h, reaction solution is incubated 10min again in boiling water bath.Extract reaction solution centrifugal 20min under 5000r/min, collect supernatant liquor.Get supernatant liquor, adopt high performance liquid chromatograph (purchased from Shanghai Tian Pu Analytical Instrument Co., Ltd) to measure the content of trehalose.
The enzyme activity unit of TreP is defined as: under above-mentioned reaction conditions, and the enzyme amount transforming generation lmg trehalose per hour is 1U.
Raw material sources: toluene, ammonium sulfate all purchased from Tianjin Zhi Yuan chemical reagent company limited, polyoxyethylene glycol purchased from American Dow Chemical.
Embodiment 1
Raising is dwelt the method for hot bacterium TreP content, and step is as follows:
(1) be inoculated in liquid seed culture medium from picking test tube slant 2 ring hot bacteria strain of dwelling, 70 DEG C, cultivate 16h under 150r/min, liquid seeds after obtained activation, then by liquid seeds by volume 5% inoculum size be inoculated in liquid nutrient medium, 70 DEG C, cultivate 24h under 200r/min, obtained strain cell nutrient solution;
(2) be placed in pressure reactor by the strain cell nutrient solution that step (1) is obtained, at 1.5MPa pressure bottom fermentation 4h, fermentation condition is 70 DEG C, 200r/min, obtained fermented liquid;
(3) by fermented liquid 1000mL centrifugal 20min under 5000r/min obtained for step (2), collect somatic cells, somatic cells is resuspended in deionized water, obtained cell mass concentration is the suspension of 30wt%, suspension is cooled to about 10 DEG C, the APV-2000-1 type high-pressure cell crusher adopting German APV company to produce carries out three cytoclasis, and crusher working pressure is 60MPa, the obtained cytoclasis solution 216mL containing TreP; Ammonium sulfate is added and molecular weight is the polyoxyethylene glycol of 2000 in cytoclasis liquid, in total system, make that the mass percent concentration of ammonium sulfate is 10wt%, the mass percent concentration of polyoxyethylene glycol is 11wt%, mix, leave standstill phase-splitting, phase solution and containing phase solution on the polyoxyethylene glycol of TreP under obtained ammonium sulfate; Ammonium sulfate is added in phase solution on polyoxyethylene glycol, make the mass percent concentration of ammonium sulfate in total system be 5wt%, the pH value of regulator solution is 7.0, mixes, leave standstill phase-splitting, obtained containing phase solution on phase solution under the ammonium sulfate of TreP and polyoxyethylene glycol; By phase solution under obtained ammonium sulfate, employing molecular weight cut-off is that the ultrafiltration membrance filter of 20000Dal concentrates, and collects trapped fluid, lyophilize, obtained TreP 932mg.
After testing, the TreP vigor of dwelling in hot mycetocyte is 82.5U/g wet cell.
Hot bacteria strain of dwelling in described step (1) be thermus aquaticus (
thermusaquaticus) ATCC33923, this bacterial classification purchased from American Type Culture Collection (ATCC).
The component of the liquid seed culture medium in described step (1) is: glucose 10g/L, yeast extract paste 10g/L, peptone 10g/L, NaCl5g/L, pH7.5, distilled water 1000mL.
The component of the liquid nutrient medium in described step (1) is: glucose 20g/L, maltose 10g/L, yeast extract paste 5g/L, peptone 5g/L, KH
2pO
42g/L, MgSO
41g/L, pH7.5, distilled water 1000mL.
Embodiment 2
Raising is dwelt the method for hot bacterium TreP content, and step is as follows:
(1) be inoculated in liquid seed culture medium from picking test tube slant 1 ring hot bacteria strain of dwelling, 65 DEG C, cultivate 12h under 100r/min, liquid seeds after obtained activation, then by liquid seeds by volume 3% inoculum size be inoculated in liquid nutrient medium, 60 DEG C, cultivate 20h under 150r/min, obtained strain cell nutrient solution;
(2) be placed in pressure reactor by the strain cell nutrient solution that step (1) is obtained, at 2.0MPa pressure bottom fermentation 1h, fermentation condition is 60 DEG C, 150r/min, obtained fermented liquid;
(3) by fermented liquid 1000mL centrifugal 20min under 5000r/min obtained for step (2), collect somatic cells, somatic cells is resuspended in deionized water, obtained cell mass concentration is the suspension of 35wt%, suspension is cooled to about 10 DEG C, the APV-2000-1 type high-pressure cell crusher adopting German APV company to produce carries out three cytoclasis, and crusher working pressure is 60MPa, the obtained cytoclasis solution 198mL containing TreP; Ammonium sulfate is added and molecular weight is the polyoxyethylene glycol of 2000 in cytoclasis liquid, in total system, make that the mass percent concentration of ammonium sulfate is 8wt%, the mass percent concentration of polyoxyethylene glycol is 12wt%, mix, leave standstill phase-splitting, phase solution and containing phase solution on the polyoxyethylene glycol of TreP under obtained ammonium sulfate; Ammonium sulfate is added in phase solution on polyoxyethylene glycol, make the mass percent concentration of ammonium sulfate in total system be 5wt%, the pH value of regulator solution is 8.0, mixes, leave standstill phase-splitting, obtained containing phase solution on phase solution under the ammonium sulfate of TreP and polyoxyethylene glycol.By phase solution under obtained ammonium sulfate, employing molecular weight cut-off is that the ultrafiltration membrance filter of 20000Dal concentrates, and collects trapped fluid, lyophilize, obtained TreP 663mg.
After testing, the TreP vigor of dwelling in hot mycetocyte is 54.1U/g wet cell.
The component of the liquid seed culture medium in described step (1) is: glucose 10g/L, yeast extract paste 10g/L, peptone 10g/L, NaCl5g/L, pH7.5, distilled water 1000mL.
The component of the liquid nutrient medium in described step (2) is: glucose 20g/L, maltose 5g/L, yeast extract paste 5g/L, peptone 5g/L, KH
2pO
42g/L, MgSO
41g/L, pH7.5, distilled water 1000mL.
Embodiment 3
Raising as described in Example 1 is dwelt the method for hot bacterium TreP content, and difference is:
In step (2), strain cell nutrient solution is placed in pressure reactor, at 0.5MPa pressure bottom fermentation 6h, obtained fermented liquid.
With this understanding, obtained TreP 862mg.
After testing, the TreP vigor of dwelling in hot mycetocyte is 78.1U/g wet cell.
Embodiment 4
Raising as described in Example 2 is dwelt the method for hot bacterium TreP content, and difference is:
In step (2), strain cell nutrient solution is placed in pressure reactor, at 1.0MPa pressure bottom fermentation 2h, obtained fermented liquid.
With this understanding, obtained TreP 824mg.
After testing, the TreP vigor of dwelling in hot mycetocyte is 73.2U/g wet cell.
Embodiment 5
Raising as described in Example 1 is dwelt the method for hot bacterium TreP content, and difference is:
In step (1), hot bacteria strain of dwelling be Thermus bacterium (
thermussp.) ATCC43815, this bacterial classification purchased from American Type Culture Collection (ATCC).
With this understanding, obtained TreP 918mg.
After testing, the TreP vigor of dwelling in hot mycetocyte is 84.2U/g wet cell.
Embodiment 6
Raising as described in Example 2 is dwelt the method for hot bacterium TreP content, and difference is:
In step (1), hot bacteria strain of dwelling be Thermus bacterium (
thermussp.) ATCC43815, this bacterial classification purchased from American Type Culture Collection (ATCC).
With this understanding, obtained TreP 657mg.
After testing, the TreP vigor of dwelling in hot mycetocyte is 52.2U/g wet cell.
Comparative example 1
Raising as described in Example 1 is dwelt the method for hot bacterium TreP content, and difference is:
In step (2), be placed in pressure reactor by strain cell nutrient solution, ferment 4h at ambient pressure, and fermentation condition is 70 DEG C, 200r/min, obtained fermented liquid.
With this understanding, obtained TreP 506mg.
After testing, the TreP vigor of dwelling in hot mycetocyte is 43.3U/g wet cell.
Comparative example 2
Raising as described in Example 6 is dwelt the method for hot bacterium TreP content, and difference is:
In step (2), get strain cell nutrient solution and be placed in pressure reactor, ferment 1h at ambient pressure, and fermentation condition is 60 DEG C, 150r/min, obtained fermented liquid.
With this understanding, obtained TreP 502mg.
After testing, the TreP vigor of dwelling in hot mycetocyte is 41.7U/g wet cell.
In above-described embodiment and comparative example, when hot bacteria strain of dwelling under stress ferments, its intracellular TreP content increases, and enzyme activity also raises.In embodiment 1 and comparative example 1, hot bacteria strain of dwelling to ferment 4h than normal pressure at 1.5MPa pressure bottom fermentation 4h, and its intracellular TreP content increases by 84.19%, enzyme activity raises 90.53%; In embodiment 6 and comparative example 2, hot bacteria strain of dwelling to ferment 1h than normal pressure at 2.0MPa pressure bottom fermentation 1h, and its intracellular TreP content increases by 30.88%, enzyme activity raises 25.18%.Show thus, pressure can significantly improve the content of the TreP of dwelling in hot mycetocyte.
Claims (4)
1. raising is dwelt a method for hot bacterium TreP content, and it is characterized in that, step is as follows:
(1) be inoculated in liquid seed culture medium from picking test tube slant 1-2 ring hot bacteria strain of dwelling, 65 ~ 70 DEG C, cultivate 12 ~ 16h under 100 ~ 150r/min, liquid seeds after obtained activation, then by liquid seeds by volume 3% ~ 5% inoculum size be inoculated in liquid nutrient medium, 60 ~ 70 DEG C, cultivate 20 ~ 24h under 150 ~ 200r/min, obtained strain cell nutrient solution;
(2) be placed in pressure reactor by strain cell nutrient solution, at 0.5 ~ 2.0MPa pressure bottom fermentation, 1 ~ 6h, fermentation condition is 60 ~ 70 DEG C, 150 ~ 200r/min, obtained fermented liquid;
(3) fermented liquid is centrifugal, collect somatic cells, smudge cells, obtained cytoclasis liquid, then adopt two phase aqueous extraction system to prepare TreP cytoclasis liquid;
The step that described employing two phase aqueous extraction system prepares TreP is as follows: in cytoclasis liquid, add ammonium sulfate and molecular weight is the polyoxyethylene glycol of 2000, in total system, make that the mass percent concentration of ammonium sulfate is 8wt% ~ 10wt%, the mass percent concentration of polyoxyethylene glycol is 10wt% ~ 12wt%, mix, leave standstill phase-splitting, phase solution and containing phase solution on the polyoxyethylene glycol of TreP under obtained ammonium sulfate; Ammonium sulfate is added in phase solution on polyoxyethylene glycol, make the mass percent concentration of ammonium sulfate in total system be 5wt% ~ 6wt%, the pH value of regulator solution is 7.0 ~ 8.0, mixes, leave standstill phase-splitting, obtained containing phase solution on phase solution under the ammonium sulfate of TreP and polyoxyethylene glycol; By phase solution under obtained ammonium sulfate, adopt ultrafiltration membrance filter to concentrate, collect trapped fluid, lyophilize, obtained TreP.
2. the method for claim 1, it is characterized in that, the hot bacteria strain of dwelling in described step (1) is thermus aquaticus (Thermusaquaticus) ATCC33923 or Thermus bacterium (Thermussp.) ATCC43815.
3. the method for claim 1, is characterized in that, the component of the liquid seed culture medium in described step (1) is: glucose 10g/L, yeast extract paste 10g/L, peptone 10g/L, NaCl5g/L, pH7.5, distilled water 1000mL.
4. the method for claim 1, is characterized in that, the component of the liquid nutrient medium in described step (1) is: glucose 20g/L, maltose 5 ~ 10g/L, yeast extract paste 5g/L, peptone 5g/L, KH
2pO
42g/L, MgSO
41g/L, pH7.5, distilled water 1000mL.
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| CN1216152C (en) * | 2003-07-18 | 2005-08-24 | 天津科技大学 | Method for raising yield of fucose produced from microzyme by using high pressure |
| CN103194432A (en) * | 2013-04-15 | 2013-07-10 | 山东天力药业有限公司 | Novel maltooligosyl trehalose synthase, gene of synthase, recombinant expression vector containing gene, and recombinant bacterium, and preparation of synthase |
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| CN1216152C (en) * | 2003-07-18 | 2005-08-24 | 天津科技大学 | Method for raising yield of fucose produced from microzyme by using high pressure |
| CN103194432A (en) * | 2013-04-15 | 2013-07-10 | 山东天力药业有限公司 | Novel maltooligosyl trehalose synthase, gene of synthase, recombinant expression vector containing gene, and recombinant bacterium, and preparation of synthase |
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