CN104328062A - Pseudomonas mutant strain and application of pseudomonas mutant strain to production of (R)-3-hydroxybutyrate - Google Patents

Pseudomonas mutant strain and application of pseudomonas mutant strain to production of (R)-3-hydroxybutyrate Download PDF

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CN104328062A
CN104328062A CN201410260125.8A CN201410260125A CN104328062A CN 104328062 A CN104328062 A CN 104328062A CN 201410260125 A CN201410260125 A CN 201410260125A CN 104328062 A CN104328062 A CN 104328062A
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hydroxybutyrate
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李凡
陈珊
张春雨
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Northeast Normal University
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Abstract

The invention belongs to the technical field of fermentation engineering, and relates to a mutant strain (pseudomonas sp.DS1001a) and application of depolymerizing enzymes produced by the strain to conversion production of (R)-3-hydroxybutyrate. According to the mutant strain, pseudomonas DS1001 screened from soil in the Jilin province serves as an original strain; and the high-yield poly-3-hydroxybutyrate depolymerizing enzymes are bred through ultraviolet mutation. The poly-3-hydroxybutyrate depolymerizing enzymes produced by the strain can be efficiently degraded into poly-3-hydroxybutyrate in vitro; (R)-3-hydroxybutyrate monomers can be generated through conversion; the 8 h reaction yield can reach 2.848 g/L; and the production efficiency is obviously higher than the efficiency for producing 3-hydroxybutyrate through biological enzymes in vivo. In addition, a strain fermentation carbon source and an enzymatic conversion substrate provided by the pseudomonas mutant strain can be poly-3-hydroxybutyrate waste or industrial leftovers; large-amount low-cost production of the poly-3-hydroxybutyrate is easily achieved; and high-valued conversion of poly-3-hydroxybutyrate materials can be achieved.

Description

One pseudomonas mutagenic strain and the application in production (R)-3-hydroxybutyrate thereof
 
Technical field
The invention belongs to fermentation engineering field, be specifically related to the mutagenic strain that a plant height produces poly-3-hydroxybutyrate depolymerizing enzyme psedomonassp.DS1001a and utilize this mutagenic strain psedomonasthe poly-3-hydroxybutyrate of depolymerizing enzyme that sp.DS1001a produces degraded transforms produces (R)-3-hydroxybutyrate monomer.
 
Background technology
(R)-3-hydroxybutyrate monomer is the ketoboidies with chiral acti ve, and its molecular formula is C 4h 8o 3, molecular weight is 104.1g/mol, water-soluble and ethanol equal solvent.(R)-3-hydroxybutyrate has antibacterial, desinsection and antiviral activity; It can synthesize fine chemicals as handedness module, as microbiotic, VITAMIN, aromatic hydrocarbon and pheromone etc.; Because (R)-3-hydroxybutyrate is people's Half-life in vivo is short and human body has good tolerance to it, therefore directly oral pharmaceutical can be used as; Under the condition of scarce sugar, (R)-3-hydroxybutyrate can partly be protected and stable neurocyte; Also have some evidences to show, (R)-3-hydroxybutyrate can be used as the efficiency strengthening heart, prevents brain injury, shows huge potential pharmaceutical use.
At present, the main production process of chiral hydroxyl group lipid acid is chemical method, comprises chemical process and directly synthesizes (R)-3-hydroxybutyrate and chemical process degraded poly 3-hydroxy butyrate production (R)-3-hydroxybutyrate.Chemical process is directly synthesized, and (R)-3-hydroxybutyrate needs the reaction conditions of high temperature, high pressure and the chiral metal catalyst etc. of costliness; And the poly-3-hydroxybutyrate of chemical process degraded produces (R)-3-hydroxybutyrate, need the organic solvent of at substantial, longer reaction times and highly purified poly 3-hydroxy butyrate as initiator, and very easily produce racemization.The research of Biological preparation (R)-3-hydroxybutyrate in recent years receives much concern, the people such as Lee construct the genetic engineering bacterium gathering 3-hydroxybutyrate synthesis and degradation pathway containing allos, report this bacterial strain using glucose as carbon source, to utilize in body biological enzyme first synthesizing polyhydroxyalkanoateby make it be degraded to (R)-3-hydroxybutyrate monomer further, output can reach 0.18g/L h.Also in succession have similar report to occur afterwards, but the method is also in laboratory level at present, if enter industrialized production still there is the problems such as cost is high, productive rate is low.
From the sixties in last century, have benefited from the attention of the whole world to environmental problem, the research about Biodegradable polymer materials such as poly-3-hydroxybutyrates is subject to extensive concern, and is rapidly developed in the nearly more than ten years.The synthesis of current PHB and modified technique are ripe, and its product is applied in industry, agricultural and the field such as medical; And people have been separated to the multiple bacterial strain poly-3-hydroxybutyrate to Degradation from occurring in nature in degraded, and its degrading enzyme and degradation mechanism are conducted in-depth research.It is contemplated that if the poly-3-hydroxybutyrate degrading enzyme utilizing these bacterial strains to produce on this basis, poly-3-hydroxybutyrate waste or poly-3-hydroxybutyrate industrial waste are degraded, make it be converted into (R)-3-hydroxybutyrate monomer, then greatly can reduce the production cost of (R)-3-hydroxybutyrate.This laboratory Wu Xiao rock etc. has carried out the exploration of this respect work, utilizes pseudomonas mendocina DSWY0601 to confirm the feasibility of the method, and after fermented liquid and poly-3-hydroxybutyrate powder complete reaction 48h, 3-hydroxybutyrate output can reach 1.087g/L.
Mutagenic strain provided by the invention psedomonassp. DS1001a comparatively original strain and pseudomonas mendocina DSWY0601 activity higher, its degrading enzyme produced reacts merely through 8h, just can produce (R)-3-hydroxybutyrate 2.848g/L, be equivalent to 0.356g/L h, production efficiency produces the efficiency of (R)-3-hydroxybutyrate apparently higher than utilize biological enzyme or the external biological enzyme in body described above; And strain fermentation provided by the invention when producing enzyme with poly-3-hydroxybutyrate waste or poly-3-hydroxybutyrate industrial waste for carbon source, without the need to adding other carbon source or inductor, conversion of substrate also can be poly-3-hydroxybutyrate industrial waste, therefore compare with glucose is that carbon source transforms and produces the method for (R)-3-hydroxybutyrate and namely saved cost, can realize again utilizing the secondary recovery of the poly-3-hydroxybutyrate consumer's goods, environmental protection more, achieves the high-valued conversion of poly-3-hydroxybutyrate material.
 
Summary of the invention
An object of the present invention is to provide the mutagenic strain pseudomonas DS1001a that a strain has more poly-3-hydroxybutyrate degrading activity, another object is to provide the poly-3-hydroxybutyrate depolymerizing enzyme utilizing this bacterial strain to produce and degrades poly-3-hydroxybutyrate in vitro to obtain the application in (R)-3-hydroxybutyrate.
The object of the invention is to be achieved through the following technical solutions:
1. adopt ultraviolet mutagenesis technology breeding high-yield to gather the bacterial strain of 3-hydroxybutyrate depolymerizing enzyme
In aseptic plate, to pour 5mL into be in logarithmic phase psedomonassp. DS1001 bacterium liquid, is placed on magnetic stirring apparatus and stirs, under the 15W ultraviolet lamp (wavelength is 254nm) that vertical range is 30cm, irradiate 0 respectively, 30,60,90,120,150s.After the bacterium liquid gradient dilution of different irradiation time, coat in poly-3-hydroxybutyrate sole carbon source substratum, 37 DEG C of lucifuges cultivate 48h, calculate lethality rate and observe the size of periphery of bacterial colonies transparent circle.Select lethality rate about 75%, and transparent circle diameter and the larger bacterial strain of colony diameter ratio, be inoculated into in the poly-3-hydroxybutyrate liquid nutrient medium that is sole carbon source, gather 3-hydroxybutyrate depolymerizing enzyme vigor every 24h sampling and measuring, choose the bacterial strain that vigor is the highest.
2. the preparation method of poly-3-hydroxybutyrate depolymerizing enzyme
Being linked into 5% inoculum size by bacterial strain DS1001a produces in enzyme liquid nutrient medium, 27.5 DEG C, 130rpm constant-temperature shaking culture, and medium component is: poly-3-hydroxybutyrate 0.2%, NH 4cl 0.15%, Na 2hPO 412H 2o/KH 2pO 41.45/0.48%, MgSO 40.07%, CaCl 22H 2o 0.0005%, the initial pH8.3 of substratum, liquid amount 116.5/500mL triangular flask.
After fermentation reaches 30h fermented liquid is placed in centrifugal 10min under 14000rpm whizzer 4 DEG C of conditions, gained supernatant liquor is crude enzyme liquid.
3. the poly-3-hydroxybutyrate of poly-3-hydroxybutyrate depolymerizing enzyme crude enzyme liquid degraded produces the condition of (R)-3-hydroxybutyrate
In 50mL centrifuge tube, add 30mL crude enzyme liquid and 200mg gathers 3-hydroxybutyrate powder, after 50 DEG C of vibration insulation 8h, adopt high-performance liquid chromatogram determination (R)-3-hydroxybutyrate content, replaceable enzyme liquid continues conversion reaction simultaneously.
 
Advantage of the present invention and beneficial effect are: the mutagenic strain provided psedomonassp. to gather 3-hydroxybutyrate depolymerizing enzyme activity high for DS1001a, and through the optimization of ultraviolet mutagenesis and fermentation condition, specific activity original strain improves 7.9 times.The ability that vitro enzyme depolymerization 3-hydroxybutyrate produces (R)-3-hydroxybutyrate is stronger, and its degrading enzyme produced reacts through 8h, can produce (R)-3-hydroxybutyrate 2.848g/L, be equivalent to 0.356g/L h; And strain fermentation provided by the invention when producing enzyme with poly-3-hydroxybutyrate waste or poly-3-hydroxybutyrate industrial waste for carbon source, without the need to adding other carbon source or inductor, conversion of substrate also can be poly-3-hydroxybutyrate industrial waste, and a large amount of cheapnesss that therefore can realize (R)-3-hydroxybutyrate are produced.
Preservation illustrates:
Poly-3-hydroxybutyrate degradation bacteria strains pseudomonassp. DS1001,
One plant height produce poly-3-hydroxybutyrate depolymerizing enzyme mutagenic strain pseudomonas ( pseudomonassp.) DS1001a, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address on December 25th, 2013: Chaoyang District Beijing great Tun road, preserving number is CGMCC No. 8638.
 
Accompanying drawing explanation
The expression activitiy figure of Fig. 1 mutagenic strain and original strain.
The product enzyme curve of Fig. 2 mutagenic strain.
Fig. 3 strain enzyme-producing training systern.A: temperature; B: inoculum size; C: liquid amount; D:pH value.
Fig. 4 strain enzyme-producing substratum compositional optimization.A: carbon source; B: nitrogenous source; C: phosphorus source; D:Mg 2+.
The optimal reactive temperature of Fig. 5 crude enzyme liquid measures curve.
The optimal reaction pH value of Fig. 6 crude enzyme liquid measures curve.
The high-efficient liquid phase chromatogram of Fig. 7 (R)-3-hydroxybutyrate.
(R)-3-hydroxybutyrate content and pH value change curve in time in Fig. 8 reaction solution.
 
Specific embodiment
By the present invention of the following example more detailed description, but obviously, scope of the present invention is not limited to these embodiments.
the poly-3-hydroxybutyrate degradation bacteria strains that embodiment 1 filters out from soil psedomonassp. DS1001
Adopt sole carbon source method, from brew-house of Jilin Province soil, filter out the bacterial strain poly-3-hydroxybutyrate to Degradation---pseudomonas pseudomonassp. DS1001, is characterized in that bacterium colony is rounded, yellow-white, and smooth surface is without wrinkle, and edge is comparatively neat, and surface is more moistening, easily provokes; Thalline is rod-short, Gram-negative, amphitrichous, without gemma and pod membrane; Bacterial strain can grow for sole carbon source with poly(lactic acid), the poly-macromolecular compound such as 3-hydroxybutyrate or polycaprolactone, and has efficient Degradation to these compounds; On November 25th, 2010 in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, address: Chaoyang District Beijing great Tun road, preserving number is CGMCC No.4366.
 
embodiment 2 with psedomonassp. DS1001 is that starting strain is gone out by Uv-induced screening psedomonassp. DS1001a
(1) growth selection is good psedomonassp. DS1001 slant strains access liquid nutrient medium, when growth is to logarithmic growth after date, gets 5mL bacterium liquid and joins in sterilized petri dishes;
(2) plate be placed on magnetic stirring apparatus stir, under the 15W ultraviolet lamp (wavelength is 254nm) that vertical range is 30cm, irradiate 0 respectively, 30,60,90,120,150s.After the bacterium liquid gradient dilution of different irradiation time, coat in poly-3-hydroxybutyrate sole carbon source substratum, 37 DEG C of lucifuges cultivate 48h;
(3) calculate lethality rate, select lethality rate about 75% 90s as mutation time, in the flat board of this mutation time, select transparent circle diameter and the larger bacterial strain of colony diameter ratio;
(4) by the inoculation selected in the liquid nutrient medium being sole carbon source with poly-3-hydroxybutyrate, gather 3-hydroxybutyrate depolymerizing enzyme vigor every 24h sampling and measuring, compare with original strain, choose the bacterial strain that vigor is the highest, called after psedomonassp. DS1001a.This bacterial strain bacterium colony is rounded, and color is white, and smooth surface is without wrinkle, and edge is comparatively neat, and surface is more moistening, easily provokes; Thalline is rod-short, Gram-negative, amphitrichous, without gemma and pod membrane; Mutagenic strain changes not quite on colonial morphology, but strengthens the degradation capability of poly-3-hydroxybutyrate, and the highest poly-3-hydroxybutyrate depolymerizing enzyme enzyme activity of its fermented liquid improves 51%(accompanying drawing 1 than original bacteria vigor).This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address on December 25th, 2013: Chaoyang District Beijing great Tun road, and preserving number is CGMCC No. 8638.
 
the preparation of embodiment 3 crude enzyme liquid and the optimization of condition of enzyme production
Crude enzyme liquid preparation adopts liquid shake-flask fermentation method, and after fermentation 30h, fermented liquid is placed in centrifugal 10min under 14000rpm whizzer 4 DEG C of conditions, gained supernatant liquor is crude enzyme liquid.
The culture condition of liquid shake-flask fermentation and substratum composition are all optimized with single factor test and response surface laboratory method, and concrete optimizing process is as follows:
(1) mensuration of enzyme time curve is produced
Bacterial strain is at minimum medium (poly-3-hydroxybutyrate: 0.15%; Na 2hPO 412H 2o:1.194%; KH 2pO 4: 0.554%; NH 4cl:0.1%; MgSO 47H 2o:0.05%; CaCl 22H 2o:0.0005%, 121 DEG C of high pressure steam sterilization 20 min) middle fermentation, every 6 h get fermented liquid survey enzyme and live, and draw fermentation time-enzyme curve (Fig. 2) alive.In result display Initial stage of culture fermented liquid, poly-3-hydroxybutyrate depolymerizing enzyme is active rises rapidly, and when cultivating 30h, enzyme is lived the highest, and along with the prolongation of fermentation time, in fermented liquid, enzyme is lived and declined, and determines that producing the enzyme time is 30h.
(2) the single factor test optimization of strain culturing condition
Single factor test condition optimizing is carried out to the condition of enzyme production of bacterial strain, optimizes the initial pH of culture temperature, inoculum size, liquid amount and substratum.Experiment determines that the suitableeest product enzyme temperature of this bacterial strain is 30 DEG C, and the suitableeest product enzyme inoculum size is 5%, and the suitableeest product enzyme liquid amount is 100 mL/500 mL, and the suitableeest product enzyme pH value is 8.0(Fig. 3).The impact of inoculum size on strain enzyme-producing is less, and on enzymic activity impact comparatively greatly, bacterial strain all keeps higher activity at neutral and alkaline culture condition bottom fermentation liquid, but enzyme is lived lower in acid condition for temperature and liquid amount.
(3) the single factor test optimization of substratum
Be optimized the culture medium condition of strain enzyme-producing, experiment determines that the suitableeest culture medium condition of bacterial strain is: 0.15% PHB, 0.2% NH 4cl, 1.2/0.45% Na 2hPO 412H 2o/KH 2pO 4, 0.07% MgSO 4(Fig. 4), 0.0005% CaCl is also added in substratum in addition 22H 2o, but Ca 2+the impact of presence or absence on strain enzyme-producing little.
(4) response surface optimization of condition of enzyme production
According to the optimum result of single factor test condition, the central combination design of response surface is utilized further to optimize.The response surface analysis test of design Three factors five level, with culture temperature, pH value, liquid amount for independent variable(s), is set to X respectively 1, X 2, X 3, five level such as tables 1 of each independent variable(s) are encoded.
The scope of independent variable(s) in table 1 center combination test design
The scheme of center combination design and the results are shown in Table 2.
Table 2 center combination test design and result
Adopt Design-Expert software to carry out regression fit to the response value in upper table and each factor, obtain equation:
Y = 83.5 - 0.17X 1 + 1.15X 2 + 0.99X 3 - 1.15X 1X 2 - 1.72X 1X 3 - 1.27X 2X 3 - 2.02X 1 2 - 0.71X 2 2 - 0.8X 3 2
Wherein, Y is response value, i.e. depolymerizing enzyme vigor, X 1, X 2, X 3represent culture temperature, pH value and liquid amount respectively.The result of variance analysis shows, extremely significantly (P=0.0007), the matching of regression equation is successful for this regression equation model.Coefficient R 2=89.92%, illustrate that the change of response value has 89.92% to derive from selected variable, and then illustrate that this equation can express the relation between each factor and response value really.The test of significance of the coefficient of regression equation is shown in Table 3.
The coefficient test of significance of table 3 regression equation
From the test of significance of regression equation coefficient, once item X 2(p < 0.01) is extremely remarkable, X 3(p < 0.05) is remarkable, mutual item X 1x 2(p < 0.05) is remarkable, X 1x 3(p < 0.01) is extremely remarkable, X 2x 3(p < 0.05) is remarkable, quadratic term X 1 2(p < 0.01) is extremely remarkable, X 3 2(p < 0.05) is remarkable.Show that single factor test pH value, liquid amount live impact significantly to the enzyme of depolymerizing enzyme, the interaction of temperature, pH value, liquid amount three lives impact significantly to the enzyme of depolymerizing enzyme.
By to the nonlinear model Solving Equations single order local derviation returned, and make it equal 0, the maximum point of curved surface can be obtained, i.e. optimal culture condition combination: temperature 27.5 DEG C, pH value 8.31, liquid amount is 116.5mL/500 mL, and the work of theoretical prediction maximum enzyme is 84.2989 U/mL.Under predicted condition, carry out proof test, enzyme is lived and is reached 96.63 U/mL.Being 3.79 times before optimizing, is 1.15 times after single factor test is optimized.
(5) response surface optimization of substratum composition
Carbon source in culture medium condition, nitrogenous source and phosphorus source are carried out to the optimization of response surface.The response surface analysis test of design Three factors five level, with poly-in substratum 3-hydroxybutyrate content, NH 4cl content, Na 2hPO 412H 2o/KH 2pO 4content is independent variable(s), is set to X respectively 1, X 2, X 3, five level such as tables 4 of each independent variable(s) are encoded.
 
The scope of independent variable(s) in table 4 center combination test design
The scheme of center combination design and the results are shown in Table 5.
Table 5 center combination test design and result
Adopt Design-Expert software to carry out regression fit to the response value in upper table and each factor, obtain equation:
Y = 93.69 - 6.23X 1+ 7.99X 2+ 15.92X 3+ 7.88X 1X 2+ 8.43X 1X 3- 8.07X 2X 3- 12.6X 3 2- 7.3X 1X 2X 3
Wherein, Y is response value, i.e. depolymerizing enzyme vigor, X 1, X 2, X 3represent poly-3-hydroxybutyrate content, NH in substratum respectively 4cl content, Na 2hPO 412H 2o/KH 2pO 4content.The result of variance analysis shows, extremely significantly (P=0.0015), the matching of regression equation is successful for this regression equation.Coefficient R 2=84.75%, illustrate that the change of response value has 84.75% to derive from selected variable, and then illustrate that this equation can express the relation between each factor and response value really.The test of significance of the coefficient of regression equation is shown in Table 6.
 
The coefficient test of significance of table 6 regression equation
From the test of significance of regression equation coefficient, once item X 2(p < 0.05) is remarkable, X 3(p < 0.01) is extremely remarkable, quadratic term X 3 2(p < 0.01) is extremely remarkable.Show that nitrogenous source content in single factor test substratum, phosphorus source content are remarkable to poly-3-hydroxybutyrate depolymerizing enzyme activity influence, wherein phosphorus source is extremely remarkable on the impact that enzyme is alive.
By to the nonlinear model Solving Equations single order local derviation returned, and make it equal 0, can obtain the maximum point of curved surface, namely optimal medium conditional combination is poly-3-hydroxybutyrate 0.2%, NH 4cl 0.15 %, Na 2hPO 412H 2o/KH 2pO 41.45/0.48%.The work of theoretical prediction maximum enzyme is 104.922 U/mL.After optimizing, enzyme is lived and is reached 150.8 U/mL, is 5.91 times before optimizing, and is 1.80 times (table 7) after single factor test is optimized.
 
The comparison that front and back enzyme is lived optimized by table 7
The optimum condition that experimental result display strain fermentation produces poly-3-hydroxybutyrate depolymerizing enzyme is: inoculum size 5%, culture temperature 27.5 DEG C, the initial pH8.3 of substratum, liquid amount 116.5/500mL triangular flask, shaking speed 130rpm, incubation time 30h; Medium component is poly-3-hydroxybutyrate 0.2%, NH 4cl 0.15%, Na 2hPO 412H 2o/KH 2pO 41.45/0.48%, MgSO 40.07%, CaCl 22H 2o 0.0005%, after optimizing, enzymic activity improves 4.91 times before comparatively optimizing.
the mensuration of embodiment 4 crude enzyme liquid optimum reaction conditions
(1) mensuration of thick enzyme optimal reactive temperature
Poly-3-hydroxybutyrate emulsification substrate is placed in respectively water-bath preheating 10 min of 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C, 75 DEG C, 80 DEG C, 85 DEG C, then in substrate, a certain amount of crude enzyme liquid is added respectively, the ratio of enzyme-to-substrate is 1:3, be incubated 10 min at different temperatures, each thermograde do two parallel, crude enzyme liquid is replaced in contrast with distilled water, measure the enzyme activity of enzyme at differential responses temperature before and after insulation, draw thick enzyme optimal reactive temperature curve (Fig. 5).Experimental result show, enzyme activity is higher at 50-65 DEG C, wherein 50 DEG C time enzyme live the highest, therefore determine that the optimal reactive temperature of thick enzyme is 50 DEG C.
(2) the optimal reaction pH value of thick enzyme
Prepare the poly-3-hydroxybutyrate emulsification substrate (pH value 3-6 is citric acid-sodium citrate damping fluid, and pH value 6-8 is phosphate buffered saline buffer, and pH value 8-10 is Glycine-NaOH damping fluid) of different pH value (3-10), correct with pH meter.By the poly-3-hydroxybutyrate emulsification substrate of above-mentioned different pH value in 50 DEG C of water-bath preheating 10 min, then in substrate, a certain amount of crude enzyme liquid is added, the ratio of enzyme-to-substrate is 1:3,50 DEG C of insulation 10 min, crude enzyme liquid is replaced in contrast with distilled water, measure the enzyme activity of enzyme under differential responses pH value before and after insulation, draw thick enzyme reaction pH value-enzyme activity curve (Fig. 6).Experimental result shows, and when pH is acid, enzyme is lived lower, and enzyme is lived higher in neutral and alkaline conditions, and when pH 8.0, enzyme activity is maximum.
 
embodiment 5 is gathered 3-hydroxybutyrate depolymerizing enzyme and is transformed production (R)-3-hydroxybutyrate
(R)-3-hydroxybutyrate standard substance are diluted to different concns, bioassay standard curve.(R)-3-hydroxybutyrate detects and uses high performance liquid chromatography, chromatographic column is Shim-pack Vp-ODSC18 post (150 L × 4.6), moving phase is acetonitrile: water (v/v)=15:85, ultraviolet detection wavelength is 210 nm, sample size 20 μ L, flow velocity 1 mL/min, column temperature 10 DEG C, adopts peak area to characterize the concentration of (R)-3-hydroxybutyrate.Typical curve equation is y=355741x-16922, r=0.9997.(R)-3-hydroxybutyrate high-efficient liquid phase chromatogram is shown in accompanying drawing 7.
Substratum after optimization and culture condition bottom fermentation obtain crude enzyme liquid, measuring crude enzyme liquid pH and are about 8.0, meeting the pH scope that crude enzyme liquid is the suitableeest, therefore without the need to adjusting the pH of crude enzyme liquid.The thick enzyme of 30 mL is added in 50 mL centrifuge tubes, 200 mg gather 3-hydroxybutyrate powder, 50 DEG C of shaking culture, sample at set intervals, METHOD FOR CONTINUOUS DETERMINATION 8 h, utilize high-efficient liquid phase technique to detect the concentration of (R)-3-hydroxybutyrate in sample, draw (R)-3-hydroxybutyrate content time history plot (Fig. 8), and calculate productive rate.
Experimental result shows, in reaction system, (R)-3-hydroxybutyrate concentration increased gradually along with the time of insulation reaction, when after insulation reaction 8 h, the concentration of (R)-3-hydroxybutyrate can reach 2.848 mg/mL, the concentration of (R)-3-hydroxybutyrate no longer increases afterwards.Measure the pH value of reaction system, find that pH value reduces gradually, also illustrate that (the R)-3-hydroxybutyrate generated accumulates gradually, reaction need change enzyme liquid to obtain better hydrolysis result further.After sustained reaction reaches 48h, powder is degraded completely substantially, and (R)-3-hydroxybutyrate rate of recovery can reach more than 90%.

Claims (7)

1. a plant height produce poly-3-hydroxybutyrate depolymerizing enzyme mutagenic strain pseudomonas ( psedomonassp.) DS1001a, is characterized in that bacterium colony is rounded, white, and smooth surface is without wrinkle, and edge is comparatively neat, and surface is more moistening, easily provokes; Thalline is rod-short, Gram-negative, amphitrichous, without gemma and pod membrane; Stronger compared with the ability of the poly-3-hydroxybutyrate of original strain degraded, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address on December 25th, 2013: Chaoyang District Beijing great Tun road, and preserving number is CGMCC No. 8638.
2. pseudomonas according to claim 1 gathers the application in 3-hydroxybutyrate depolymerizing enzyme in fermentative production.
3. apply as claimed in claim 2, it is characterized in that, the medium component used that ferments is: poly-3-hydroxybutyrate 0.2%, NH 4cl 0.15%; Na 2hPO 412H 2o/KH 2pO 41.45/0.48%, MgSO 40.07%; CaCl 22H 2o 0.0005%.
4. the application as described in claim 2-3, is characterized in that, described fermentation condition is: inoculum size 5%, the initial pH 8.3 of culture temperature 27.5 DEG C, substratum, liquid amount 20%-30%, shaking speed 130rpm, incubation time 30h.
5. pseudomonas according to claim 1 is transforming the application of producing in (R)-3-hydroxybutyrate.
6. apply as claimed in claim 5, it is characterized in that, the crude enzyme liquid utilizing strain fermentation to obtain generates (R)-3-hydroxybutyrate monomer in optimum reaction conditions decline depolymerization 3-hydroxybutyrate substrate conversion.
7. the application as described in claim 5-6, is characterized in that, described optimum reaction conditions is temperature of reaction 50 DEG C, reaction pH is 8.0.
CN201410260125.8A 2014-06-13 2014-06-13 Pseudomonas mutant strain and application of pseudomonas mutant strain to production of (R)-3-hydroxybutyrate Pending CN104328062A (en)

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CN110904161A (en) * 2019-12-27 2020-03-24 浙江英玛特生物科技有限公司 Method for producing high-purity (R) - (-) -3-hydroxybutyric acid by adopting enzyme method

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