CN102071181A - Method for preparing alkali protease from serratieae and special culture medium thereof - Google Patents
Method for preparing alkali protease from serratieae and special culture medium thereof Download PDFInfo
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- CN102071181A CN102071181A CN2010101725452A CN201010172545A CN102071181A CN 102071181 A CN102071181 A CN 102071181A CN 2010101725452 A CN2010101725452 A CN 2010101725452A CN 201010172545 A CN201010172545 A CN 201010172545A CN 102071181 A CN102071181 A CN 102071181A
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- serratia
- kmr
- culture medium
- serratieae
- alkali protease
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- 238000000034 method Methods 0.000 title claims description 20
- 108091005804 Peptidases Proteins 0.000 title abstract description 24
- 239000004365 Protease Substances 0.000 title abstract description 11
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 title abstract description 11
- 239000001963 growth medium Substances 0.000 title abstract description 9
- 239000003513 alkali Substances 0.000 title abstract 5
- 239000012138 yeast extract Substances 0.000 claims abstract description 22
- 241000607714 Serratia sp. Species 0.000 claims abstract description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 7
- 239000007788 liquid Substances 0.000 claims description 27
- 230000001580 bacterial effect Effects 0.000 claims description 24
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 22
- 235000015097 nutrients Nutrition 0.000 claims description 22
- 229940041514 candida albicans extract Drugs 0.000 claims description 20
- 241000607720 Serratia Species 0.000 claims description 14
- 108010046845 tryptones Proteins 0.000 claims description 14
- 239000011780 sodium chloride Substances 0.000 claims description 11
- 241000607715 Serratia marcescens Species 0.000 claims description 9
- 244000005700 microbiome Species 0.000 claims description 8
- 238000004321 preservation Methods 0.000 claims description 4
- 230000000694 effects Effects 0.000 abstract description 3
- 238000009776 industrial production Methods 0.000 abstract description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 239000004615 ingredient Substances 0.000 abstract 3
- 239000012137 tryptone Substances 0.000 abstract 2
- 238000009630 liquid culture Methods 0.000 abstract 1
- 230000001737 promoting effect Effects 0.000 abstract 1
- 229930000044 secondary metabolite Natural products 0.000 abstract 1
- 239000002609 medium Substances 0.000 description 22
- 102000035195 Peptidases Human genes 0.000 description 13
- 102000004190 Enzymes Human genes 0.000 description 11
- 108090000790 Enzymes Proteins 0.000 description 11
- 239000001888 Peptone Substances 0.000 description 6
- 108010080698 Peptones Proteins 0.000 description 6
- 235000019319 peptone Nutrition 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 239000000306 component Substances 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 3
- 238000000855 fermentation Methods 0.000 description 3
- 230000004151 fermentation Effects 0.000 description 3
- 238000005457 optimization Methods 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 239000002351 wastewater Substances 0.000 description 3
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000012803 optimization experiment Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 241000194108 Bacillus licheniformis Species 0.000 description 1
- 241000194103 Bacillus pumilus Species 0.000 description 1
- 241000726221 Gemma Species 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000000118 anti-neoplastic effect Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 239000012092 media component Substances 0.000 description 1
- 239000012533 medium component Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000002975 protease activity determination Methods 0.000 description 1
- 239000001054 red pigment Substances 0.000 description 1
- 238000012882 sequential analysis Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
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Abstract
The invention relates to a production method for preparing alkali protease from serratieae, which belongs to the technical field of biology. Serratia sp. KMR-3 strains are cultured for 24h at 25 DEG C and the pH value being 7 to 10 in an improved NaCl-free luria-bertani (LB) liquid culture medium with 10g/l tryptone, 2.5g/l yeast extracts and water as ingredients. The culture medium for preparing the alkali protease from the serratieae is the culture medium using the tryptone, the yeast extracts and the water as the ingredients. The culture medium has the characteristic of relatively little ingredient consumption, also has an effect of promoting the serratieae to fast produce the alkali protease, is applicable to the fields of germ secondary metabolites in modern biological engineering, laboratory culture and the like and can provide the study basis for the industrial production of the alkali protease.
Description
Technical field
The present invention relates to biological technical field, particularly, relate to the method that a kind of Serratia produces Sumizyme MP, be exclusively used in the substratum of this method, this substratum with peptone and yeast extract paste as C, N source and the energy.
Background technology
Sumizyme MP all is widely used in industries such as enzyme-containing detergent, process hides, silks, the main at present industrial bacterium producing multi enzyme preparation of using is Bacillus licheniformis and bacillus pumilus, but gemma class bacterial strain can produce a large amount of spores when producing enzyme, make troubles for the extraction of enzyme, aftertreatment is also more loaded down with trivial details.Serratia marcescens is the non-spore bacteria of a kind of Gram-negative, can produce Sumizyme MP, and this protease molecule is bigger, and purifying is the good candidate bacterial strain of industrial production Sumizyme MP easily; In addition, the proteolytic enzyme of Serratia generation also has antineoplastic property of medicine; But because the protease activities of its generation is lower, output is not high yet, therefore is not used widely in industrial production.
Screen the bacterial strain that a strain can be produced Sumizyme MP from smeltery, Kunming waste water on every side, this bacterial strain can produce red pigments, and has the various heavy resistance.Physiology and biochemistry experiment and 16S rDNA sequential analysis show that this bacterial strain belongs to the serratia marcescens in the serratia, and called after KMR-3 is a starting strain with this bacterial strain, and its fermentation condition that produces Sumizyme MP is optimized.
Summary of the invention
The present invention aims to provide a kind of safety, cost is low and can promote Serratia (Serratia sp.) the KMR-3 bacterial strain production method of the Serratia product Sumizyme MP of output Sumizyme MP rapidly and efficiently, and the liquid nutrient medium of this method special use.
To achieve these goals, the invention provides following technical scheme:
Serratia produces the method for Sumizyme MP, serratia marcescens (Serratia sp.) KMR-3 bacterial strain is being 5-20g/L with the Tryptones, yeast extract paste is that 2.5-10g/L and water are in the improvement LB liquid nutrient medium that does not contain NaCl of component, and 25 ℃, pH7-10 cultivates 6-48h down.
Described serratia marcescens (Serratia sp.) KMR-3 on August 26th, 2008 in that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center " preservation, preserving number is: CGMCCNo.2636.
Aforesaid method, preferred Tryptones is 10g/L, yeast extract paste is 2.5g/L; PH is 8, cultivates 24h.
Be exclusively used in described Serratia and produce the substratum of the method for Sumizyme MP, with Tryptones 10g/L, yeast extract paste 2.5g/L and water are component, do not contain the improvement LB liquid nutrient medium of NaCl.
The used technical scheme of the present invention is to use a kind of liquid nutrient medium, and it is formulated as substratum with Tryptones, yeast extract paste and water.
Serratia marcescens (Serratia sp.) the KMR-3 bacterial strain of haematochrome is produced in the strain that utilization of the present invention is separated to the waste water around the smeltery, Kunming, and its preserving number is: CGMCC No.2636.By optimizing the fermentation condition and the nutrient media components of substratum, improve its proteolytic enzyme output.
The serratia marcescens that the present invention relates to (Serratia sp.) KMR-3 bacterial strain on August 26th, 2008 in that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center " (BeiJing, China) preservation, preserving number is: CGMCC No.2636.
Description of drawings:
Fig. 1 is that different incubation time KMR-3 bacterial strains produce the proteolytic enzyme amount;
Fig. 2 is that different incubation time KMR-3 bacterial strains produce the proteolytic enzyme amount.
Embodiment:
Concrete steps of the present invention are as follows: LB (Luria-Bertani) substratum is a most frequently used substratum in the microbiology experiment, is used to cultivate bacterium such as intestinal bacteria and is divided into liquid state or adds the solid medium that agar is made.Produce the initial substratum of proteolytic enzyme as serratia marcescens (Serratia sp.) KMR-3 strain fermentation with the LB liquid nutrient medium, leavening temperature is 25 ℃, the pH value is 8.0, by optimizing LB liquid nutrient medium (Tryptones 10g/L, yeast extract paste 5g/L, NaCl 10g/L) different components improves the output of this bacterial strain Sumizyme MP.The optimization experiment content is: the LB liquid nutrient medium, do not contain the improvement LB liquid nutrient medium of Tryptones, and do not contain the improvement LB liquid nutrient medium of yeast extract paste, do not contain the improvement LB liquid nutrient medium of NaCl; Estimate its output by the protease content of measuring in the supernatant of the centrifugal back of fermented liquid, the result shows: the improvement LB liquid nutrient medium that does not contain NaCl can improve proteolytic enzyme output (Fig. 1) to a certain extent, again Tryptones content and yeast extract paste content are optimized then, the result shows: contain Tryptones 10g/L, the substratum of yeast extract paste 2.5g/L can promote KMR-3 to produce Sumizyme MP in a large number, fast.(Fig. 2) (the protease activity property surveyed method is a forint phenol method.)
Below in conjunction with accompanying drawing, further specify essentiality content of the present invention with embodiment, but do not limit the present invention with this.
Embodiment 1: the detection of serratia (Serratia sp.) KMR-3 bacterial strain Sumizyme MP:
A strain that is separated to the waste water around the smeltery, Kunming has high resistance to contents of many kinds of heavy metal ion serratia (Serratia sp.) KMR-3 bacterial strain.This serratia (Serratia sp.) KMR-3 bacterial strain on August 26th, 2008 in that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center " (BeiJing, China) preservation, preserving number is: CGMCC No.2636.
The detection of KMR-3 bacterial strain Sumizyme MP: the KMR-3 bacterium liquid that is coated with 50 μ l fresh culture is in the LB liquid nutrient medium of different pH (1～14) 5mL, and 25 ℃, 150rpm cultivates 24h, observes the generation scope of thalli growth pH and proteolytic enzyme.Measure the protease-producing situation of KMR-3 bacterial strain under the condition of different pH by forint phenol method.The result shows: the KMR-3 bacterial strain can be grown in the LB of pH410 scope liquid nutrient medium, can both produce proteolytic enzyme in the pH7-10 scope, and is that 8.0 o'clock yield of enzyme are higher in the pH value, so the proteolytic enzyme that the KMR-3 bacterial strain produces is Sumizyme MP.
Embodiment 2: the optimization of medium component:
The initial optimization of the LB liquid nutrient medium of improvement: as initial substratum, leavening temperature is 15 ℃ with LB liquid nutrient medium (Tryptones 10g/L, yeast extract paste 5g/L, NaCl 10g/L), and the pH value is 8.0.The initial optimization experiment content is: the LB liquid nutrient medium, do not contain the improvement LB liquid nutrient medium of Tryptones, and do not contain the improvement LB liquid nutrient medium of yeast extract paste, the improvement LB liquid nutrient medium that does not contain NaCl is respectively at 0,6,12,24,36,48,60,72,84 and 96h fermented liquid is taken a sample, by the centrifuging and taking supernatant, pass through to measure OD according to the protease activity determination method then
660Value entry evaluation proteolytic enzyme output converts protein concentration at last to, and the result as shown in Figure 1; The result shows: the LB substratum that lacks NaCl can promote bacterial strain to produce enzyme in advance, and yield of enzyme is higher than the initial substratum of LB, shows that NaCl is the supressor that the KMR-3 bacterial strain produces proteolytic enzyme.And the substratum product enzyme that lacks yeast extract paste is minimum, lacks next of peptone, and the two is all low compared with the LB substratum yield of enzyme of beginning.Therefore, yeast extract paste and peptone are that the KMR-3 bacterial strain produces the needed nutritive substance of proteolytic enzyme, are indispensable in to LB Optimum of culture medium process.
Peptone and yeast extract paste different content Optimum of culture medium: ferment with the substratum that only contains different concns peptone and yeast extract paste, be designated as LB, LB-1, LB-2, LB-3 and LB-4 respectively, the proportioning of substratum sees Table 1.Culture temperature is 25 ℃ (showing that through preliminary experiment KMR-3 can produce enzyme in advance at 25 ℃, carries out medium optimization so select 25 ℃ for use), and the pH value is 8.0, respectively at 0,6,12,24,36 and 48h fermented liquid is taken a sample OD then
660Measure the protease content in the supernatant of the centrifugal back of fermented liquid down, and change into protein concentration, the result as shown in Figure 2.During LB-1 culture medium culturing Serratia KMR-3 to 24h, protein concentration is the highest, reaches 817.6mg/ml as can be seen, and yield of enzyme is 1.3 times of basic medium.Final definite substratum of optimizing is the LB-1 substratum, and composition is Tryptones 10g/L, and yeast extract paste is 2.5g/L; Compare with initial LB substratum, assorted component of substratum and content all reduce, and have reduced cost, have improved KMR-3 proteolytic enzyme output simultaneously again.The various set of dispense ratios of table 1 substratum
| Substratum | Peptone (g/L) | Yeast extract paste (g/L) | Sodium-chlor (g/L) |
| LB | 10 | 5 | 10 |
| LB-1 | 10 | 2.5 | 0 |
| LB-2 | 10 | 10 | 0 |
| LB-3 | 5 | 5 | 0 |
| LB-4 | 20 | 5 | 0 |
Claims (6)
1. Serratia produces the method for Sumizyme MP, serratia marcescens (Serratia sp.) KMR-3 bacterial strain is being 5-20g/L with the Tryptones, yeast extract paste is that 2.5-10g/L and water are in the improvement LB liquid nutrient medium that does not contain NaCl of component, and 25 ℃, pH7-10 cultivates 6-48h down.
2. the method for claim 1, described serratia marcescens (Serratia sp.) KMR-3 on August 26th, 2008 in that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center " preservation, preserving number is: CGMCC No.2636.
3. the method for claim 1 is characterized in that Tryptones is 10g/L, and yeast extract paste is 2.5g/L.
4. the method for claim 1 is characterized in that pH is 8.
5. the method for claim 1 is characterized in that cultivating 24h.
6. be exclusively used in claim 1-5 Serratia and produce the substratum of the method for Sumizyme MP, with Tryptones 10g/L, yeast extract paste 2.5g/L and water are component, do not contain the improvement LB liquid nutrient medium of NaCl.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105670981A (en) * | 2016-04-22 | 2016-06-15 | 淮海工学院 | Serratia marcescens SPG-1 and method for producing chitosanase by adopting same |
| CN116355783A (en) * | 2022-07-15 | 2023-06-30 | 海南大学 | Serratia marcescens and application thereof |
-
2010
- 2010-05-14 CN CN2010101725452A patent/CN102071181A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105670981A (en) * | 2016-04-22 | 2016-06-15 | 淮海工学院 | Serratia marcescens SPG-1 and method for producing chitosanase by adopting same |
| CN105670981B (en) * | 2016-04-22 | 2020-01-07 | 淮海工学院 | Serratia marcescens SPG-1 and method for producing chitosanase by using same |
| CN116355783A (en) * | 2022-07-15 | 2023-06-30 | 海南大学 | Serratia marcescens and application thereof |
| CN116355783B (en) * | 2022-07-15 | 2024-03-29 | 海南大学 | Serratia marcescens and application thereof |
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Application publication date: 20110525 |