CN102127515B - Screening and application of L-proline high-producing Brevundimonas sp. (JNPP-1) - Google Patents
Screening and application of L-proline high-producing Brevundimonas sp. (JNPP-1) Download PDFInfo
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- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 title claims abstract description 38
- 229960002429 proline Drugs 0.000 title claims abstract description 38
- 229930182821 L-proline Natural products 0.000 title claims abstract description 37
- 241000061154 Brevundimonas sp. Species 0.000 title claims abstract description 10
- 238000012216 screening Methods 0.000 title abstract description 8
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- 238000000855 fermentation Methods 0.000 claims description 15
- 230000004151 fermentation Effects 0.000 claims description 15
- 230000001954 sterilising effect Effects 0.000 claims description 12
- 238000004659 sterilization and disinfection Methods 0.000 claims description 12
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- 239000008103 glucose Substances 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 8
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- 229910052799 carbon Inorganic materials 0.000 claims description 7
- 239000002054 inoculum Substances 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 244000005700 microbiome Species 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 6
- 239000001888 Peptone Substances 0.000 claims description 4
- 108010080698 Peptones Proteins 0.000 claims description 4
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- 229930006000 Sucrose Natural products 0.000 claims description 4
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- 235000019319 peptone Nutrition 0.000 claims description 4
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- 238000002156 mixing Methods 0.000 claims description 3
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- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of biology, and particularly relates to screening and application of an L-proline high-producing Brevundimonas sp. JNPP-1. An L-proline high-producing pseudomonad JNPP-1 is screened out for the first time; and by combining morphological characteristics and physiological biochemical characteristics, the strain JNPP-1 is identified as a Brevundimonas sp. The strain is preserved at China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC NO.3828. The strain can produce glucoses into L-proline, and the yield can be up to 32.67g/L. The L-proline produced by the strain is one of important amino acids for synthesizing human proteins, is an important material for amino acid transfusion, and has been widely used in multiple fields, such as food, medicine and other industries.
Description
Technical field
The invention belongs to biological technical field, be specifically related to screening and application that a plant height produces defective shortwave pseudomonas bacillus (Brevundimonas sp.) JNPP-1 of L-proline(Pro).
Background technology
The L-proline(Pro) has purposes widely at aspects such as medicine, agricultural, chemical industry and food; Pharmaceutically mainly as Branchamin; Be one of amino acid infusion solutions raw material, the protein that is used for after malnutrition, hypoproteinosis, serious intestines and stomach illness, scald and the surgical operation replenishes.It also is preparation captopril (Captopril)---the medicine material of treatment intractable hypertension and congestive heart failure.On compound probability, the L-proline(Pro) can participate in inducing asymmetric reaction, can be used as catalyst for reaction such as hydrogenation, polymerization, water Jie, has characteristics such as activity is strong, stereospecificity is good; Because the L-proline(Pro) has certain sweet taste, has begun to be applied to do in the foodstuffs industry additive in recent years abroad.At present, mainly adopt chemical method, natural protein hydrolysis method and Production by Microorganism Fermentation L-proline(Pro).Because the chemical synthesis operational path is longer, be difficult to go into operation; And from the natural protein hydrolyzed solution, extracting the L-proline(Pro), its cost is high, is inappropriate for modern chemical industry sparetime university to produce; Direct fermentation becomes the main path of producing the L-proline(Pro).The mikrobe that utilizes glucose fermentation to produce the L-proline(Pro) comprises corynebacterium glutamicum, brevibacterium flavum, chain Guan Shi bacillus, have a liking for bacterium such as acetate corynebacterium; Because the L-proline(Pro) is the mesostate in the metabolic process; Be difficult for accumulation; And the heteroacid that produces in the fermenting process is more, directly becomes the bottleneck in the L-proline(Pro) industrial production.Therefore, screening the mikrobe that a plant height produces the L-proline(Pro) has very important significance; And do not see the domestic literature report about the research of defective shortwave pseudomonas bacillus production L-proline(Pro).
Summary of the invention
(problem that will solve)
The objective of the invention is to the technological difficulties in the working method of existing L-proline(Pro) and the problem of existence; The defective shortwave pseudomonas bacillus of the novel high yield L-proline(Pro) of one strain is provided; But utilize the L-proline(Pro) of this strain fermentation production high density, this bacterial strain is the production bacterial strain that has the L-proline(Pro) of development research value.
Summary of the invention
Contriver of the present invention obtains the soil sample separation screening from domestication factory and obtains the novel defective shortwave of strain product L-proline(Pro) pseudomonas bacillus JNPP-1, and this strain bacterium has been carried out fermentation research.
Bacterial strain provided by the invention is to obtain the soil sample separation screening from domestication factory to obtain a strain tyrothricin, according to morphological specificity and physio-biochemical characteristics, it is accredited as Brevundimonas sp., is numbered JNPP-1.This bacterium is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on May 11st, 2010, and deposit number is CGMCC No.3828.
Morphological specificity and the physiological and biochemical property of defective shortwave pseudomonas bacillus JNPP-1 provided by the invention are:
This strain morphology is a rod-short, bacterium colony circle, smooth surface, moistening, opaque, the color pearl of lawn on the LB solid medium; Single, paired or short chain shape is arranged one-sided flagellum, no gemma.Oxydase reaction is positive, and the V.P. reaction is negative glucose capable of using, SANMALT-S; Can not utilize pectinose, wood sugar, sucrose, Citrate trianion to produce acid, can not hydrolyzed starch, casein, gelatin, can utilize urea, inorganic carbon source.Physiological and biochemical property is seen table 1:
Table 1
Defective shortwave pseudomonas bacillus JNPP-1 provided by the invention is the novel production bacterial strain with product L-proline(Pro) of good prospect, and this bacterial strain can ooze in the broth culture at height grows.Height oozes the broth culture prescription: glucose 20-50g/L; Peptone 5-20g/L; Yeast powder 4-10g/L; NaC1 5-20g/L; Ammonium chloride 50-100g/L; Agar powder 10-20g/L; Replenish zero(ppm) water to 1L, transfer the pH to 7.0-7.4 of substratum before the sterilization, sterilization 20min under 115 ℃ of HP steams;
Above-mentioned defective shortwave pseudomonas bacillus JNPP-1 can adopt fermentative Production L-proline(Pro).
Use mentioned microorganism and produce the method for L-proline(Pro), its step is following:
1) the employing preserving number is that the defective shortwave pseudomonas bacillus JNPP-1 of CGMCC No.3828 produces bacterial classification, carries out activation culture according to ordinary method to obtain seed liquor, and this seed liquor is applied on the meat soup solid medium;
2) the cell liquid culture of the defective shortwave pseudomonas bacillus JNPP-1 bacterial strain of preparation: JNPP-1 bacterial strain one ring on the meat soup solid medium of picking step (1); Be inoculated in the 250mL Erlenmeyer flask that the 20-60mL substratum is housed; Culture temperature is 30-40 ℃; Put on the shaking table with the rotating speed of 90-200r/min and cultivate 9-12h, promptly obtain the cell liquid culture of JNPP-1 bacterial strain to logarithmic growth mid-term;
3) fermentation culture: the amount of pressing the bottled 20-60mL of 250mL triangle; Drop into fermention medium; The cell liquid culture of the defective shortwave pseudomonas bacillus JNPP-1 bacterial strain of inoculation step (2), inoculum size is 5-10% (w/w), culture temperature is 30-40 ℃; Cultivating 72-84h under with the rotating speed of 90-200r/min on the shaking table, obtain fermented liquid; Or drop into described fermention medium by fermentor tank volumetrical 40-80%; Keep 30min with the 0.07-0.1MPa high pressure steam sterilization; Be cooled to 30-40 ℃, insert the cell liquid culture of the described defective shortwave of step (2) pseudomonas bacillus JNPP-1 bacterial strain, inoculum size 5-10% (w/w); Culture temperature is 30-40 ℃, and the control air flow is 1: 0.5-2vvm, and the mixing speed of control fermentor tank is 200-600r/min; Fermentation time is 72-84h, obtains fermented liquid;
4) product detects: with fermented liquid centrifugal 5-10min under 8000-12000r/min of step (3), collect the content that supernatant carries out L-proline(Pro) in the automatic analyzer for amino acids check and analysis fermented liquid
Wherein
The composition and the proportioning of the broth culture described in step (1) or (2) are: glucose 20-50g/L; Peptone 5-20g/L; Yeast powder 4-10g/L; NaCl 5-20g/L; Agar powder 10-20g/L; Replenish zero(ppm) water to 1L, transfer the pH to 7.0-7.4 of substratum before the sterilization, sterilization 20min under 115 ℃ of HP steams;
The composition and the proportioning of the said fermention medium of step (3) are: carbon source 100-150g/L, nitrogenous source 20-50g/L, (NH
4)
2SO
420-30g/L; K
2HPO
40.1-5.0g/L; MgSO
40.1-5g/L; ZnSO
40.01-0.10g/L; MnSO
40.01-0.10g/L; Sodium Glutamate 40-60g/L; Vitamin H 50-200 μ g/L; VitB1 50-200 μ g/L; Replenish zero(ppm) water to 1L, transfer the pH to 6.5-7.4 of substratum before the sterilization, sterilization 20min under 115 ℃ of HP steams.
But defective shortwave pseudomonas bacillus JNPP-1 bacterial strain transforming glucose of the present invention directly generates the L-proline(Pro), and the L-concentration of proline is high in the fermented liquid, and transformation efficiency is high, is the production bacterial strain that a strain has the L-proline(Pro) of development research value.
Description of drawings
Fig. 1 is the result of shortwave pseudomonas bacillus Brevundimonas sp.JNPP-1 bacterial strain of the present invention fermentative prodn L-proline(Pro) in the glucose fermentation substratum.
[beneficial effect]
A kind of strain excellent with production L-proline(Pro) of industrial application potentiality is provided.
The biological material specimens preservation
Shortwave pseudomonas bacillus (Brevundimonas sp.) JNPP-1, preservation date on May 11st, 2010, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number are CGMCC No.3828.
Embodiment
The isolation identification of embodiment 1 shortwave pseudomonas bacillus JNPP-1 and the preservation of bacterial strain
(1) separation of shortwave pseudomonas bacillus JNPP-1, screening
The present invention obtains soil sample from domestication factory, takes by weighing the 1.0-2.0g soil sample in the 250mL triangular flask, suspends with the 10-50mL sterilized water; Place water-bath then, handle 10min, carry out gradient dilution after the cooling for 60 ℃; Getting the 0.2mL diluent coats on the meat soup solid plate; Behind the 30-40 ℃ of cultivation 24-36h, picking list bacterium colony carries out the oxydase experiment and detects.Select the checking of fermenting of oxydase reaction male bacterial strain.After picking oxydase reaction male bacterial strain list colony inoculation is cultivated 12h in the fresh meat soup liquid nutrient medium; The cell liquid culture is connected in the triangular flask of the 250mL that the 30mL fermention medium is housed with 5% (w/w) inoculum size, 30-40 ℃, cultivates with the speed oscillation of 90-200r/min; Collect fermented liquid behind the 36h; The centrifugal 5-10min of 8000-12000r/min collects supernatant, adopts automatic analyzer for amino acids to detect L-proline(Pro) output.
(2) evaluation of shortwave pseudomonas bacillus JNPP-1
Physiology and biochemistry is identified: with reference to the outstanding Bacteria Identification handbook of uncle the 8th edition
Strain morphology is a rod-short, bacterium colony circle, smooth surface, moistening, opaque, the color pearl of lawn on the LB solid medium; Single, paired or short chain shape is arranged one-sided flagellum, no gemma.Oxydase reaction is positive, and the V.P. reaction is negative glucose capable of using, SANMALT-S; Can not utilize pectinose, wood sugar, sucrose, Citrate trianion to produce acid, can not hydrolyzed starch, casein, gelatin, can utilize urea, inorganic carbon source.
Bacterial strain is preserved: picking one ring JNPP-1 inoculation is in the LB liquid nutrient medium, and 30-40 ℃, behind the 200r/min shaking culture 24-30h, the cell culture fluid of getting 0.5mL changes in 60% the glycerine preservation pipe that 0.5mL is housed ,-20 ℃ of freezings.This bacterium was stored in China Committee for Culture Collection of Microorganisms common micro-organisms center on May 11st, 2010, and its preserving number is CGMCC No.3828.
Embodiment 2 radiothermy pseudomonas bacillus fermentative prodn L-proline(Pro)
(1) the cell liquid culture of preparation defective shortwave pseudomonas bacillus JNPP-1 bacterial strain
Defective shortwave pseudomonas bacillus JNPP-1 bacterial strain one ring on the picking solid broth culture; Be seeded in the 250mL Erlenmeyer flask that 50mL LB substratum is housed; Under 30 ℃ of-40 ℃ of conditions; Place on the shaking table with the rotating speed of 200r/min and cultivate 9-12h, promptly make the cell liquid culture of defective shortwave pseudomonas bacillus JNPP-1 bacterial strain to logarithmic growth mid-term.
(2) composition of fermention medium and proportioning are: carbon source 50-150g/L; Organic nitrogen source 6-30g/L; (NH
4)
2SO
420-30g/L; K
2HPO
40.1-5.0g/L; MgSO
40.1-5g/L; ZnSO
40.01-0.10g/L; MnSO
40.01-0.10g/L; Sodium Glutamate 40-60g/L; Vitamin H 50-200 μ g/L; VitB1 50-200 μ g/L; PH 6.5-7.4, sterilization 20min under 115 ℃ of HP steams.Described carbon source is selected from glucose, sucrose, SANMALT-S, one or both of molasses; Described organic nitrogen source is selected from steeping water, peptone, one or both in the yeast powder.
(3) shake flask fermentation: the cell liquid culture of the above-mentioned defective shortwave pseudomonas bacillus JNPP-1 bacterial strain inoculum size with 5-10% (w/w) is seeded in the 250mL Erlenmeyer flask of the 20-60mL fermention medium that sterilization is housed; Under 30-40 ℃ of condition; Place on the shaking table with the rotating speed of 90-200r/min and cultivate; Fermentation 72-84h, the output of L-proline(Pro) reaches 32.67g/L.
(4) ferment tank: the cell liquid culture of above-mentioned defective shortwave pseudomonas bacillus JNPP-1 bacterial strain is dropped into described fermention medium by fermentor tank volumetrical 40-80%; With 0.07-0.1Mpa sterilization 30min; Be cooled to 30-40 ℃, inoculum size is 5-10% (w/w), and culture temperature is 30-35 ℃; Air flow is 1: 0.5-2 (vvm), the mixing speed of fermentor tank are 200-600r/min; Fermentation time 72-84h.
Claims (2)
1. a plant height produces the bacterial strain of L-proline(Pro), this bacterial strain be the shortwave Zymomonas mobilis (
Brevundimonas sp.) JNPP-1, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on May 11st, 2010, be numbered CGMCC No.3828.
The described shortwave Zymomonas mobilis of claim 1 (
Brevundimonas sp.) fermentation process of JNPP-1, it is characterized by: substratum consists of carbon source 50-150 g/L, organic nitrogen source 6-30g/L, (NH
4)
2SO
420-30 g/L, K
2HPO
40.1-5.0 g/L, MgSO
40.1-5 g/L, ZnSO
40.01-0.10 g/L, MnSO
40.01-0.10g/L, Sodium Glutamate 40-60g/L, vitamin H 50-200 μ g/L, VitB1 50-200 μ g/L, pH 6.5-7.4, sterilization 20min under 115 ℃ of HP steams.
3. the described fermentation process of claim 2 is characterized by: described carbon source is selected from one or both of glucose, sucrose, SANMALT-S, molasses, and described organic nitrogen source is selected from one or both in steeping water, peptone, the yeast powder.
4. utilize the described shortwave Zymomonas mobilis of claim 1 (
Brevundimonas sp.) method of fermentative prodn L-proline(Pro) of JNPP-1, it is characterized by: the fermention medium of the bottled 20-60 mL of 250 mL triangles, inoculum size are 5-10% (w/w), culture temperature 30-40 ℃, and shaking speed 90-200 r/min, fermentation time 72-84h; Or drop into described fermention medium by fermentor tank volumetrical 40-80%, with 0.07-0.1 Mpa sterilization 30min, be cooled to 30-40 ℃; Inoculum size is 5-10% (w/w); Culture temperature is 30-35 ℃, and air flow is 1:0.5-2 vvm, and the mixing speed of fermentor tank is 200-600 r/min; Fermentation time 72-84 h.
5. the application of the described method of claim 4 in producing the L-proline(Pro).
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Non-Patent Citations (2)
| Title |
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| 刘党生 等.一株L-脯氨酸产生菌的选育.《沈阳医科大学学报》.1996,第13卷(第2期),112-116. * |
| 张伟国 等.L-脯氨酸发酵条件的研究.《江苏食品与发酵》.2000,(第4期),1-4. * |
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