CN101486979A - Yersinia strain KM1, low temperature alkaline lipase prepared thereby and purification method thereof - Google Patents
Yersinia strain KM1, low temperature alkaline lipase prepared thereby and purification method thereof Download PDFInfo
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- CN101486979A CN101486979A CNA2008102336261A CN200810233626A CN101486979A CN 101486979 A CN101486979 A CN 101486979A CN A2008102336261 A CNA2008102336261 A CN A2008102336261A CN 200810233626 A CN200810233626 A CN 200810233626A CN 101486979 A CN101486979 A CN 101486979A
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- 238000000746 purification Methods 0.000 title claims description 17
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- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
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Abstract
The invention discloses a psychrotrophic bacteria strain Yersinia sp. KM1, a psychrotrophic alkaline lipase that is generated by the strain and resistant to organic solvents, and a separating and purifying method of the psychrotrophic alkaline lipase. The purifying method comprises the steps of ammonium sulfate precipitation of a fermented supernatant, centrifugal concentration with a 10KDa ultrafiltration pipe, Sephacry<TM> HRS-100 chromatography and Superdex G-75 chromatography, and finally obtains pure psychrotrophic alkaline lipase under electrophoresis. The purified psychrotrophic alkaline lipase has low reaction temperature, wider pH accommodation range and high resistance to organic solvents, and consequently has huge application potential in the fields of cleaning solvents, food processing, medicine production and biodiesel production, etc.
Description
Technical field:
The invention belongs to microbial technology field, particularly, relate to a strain psychrotropic bacteria bacterial strain, i.e. psychrotropic bacteria bacterial strain Yersinia (Yersinia sp.) KM1, and by this bacterial strain make feed purification and low-temperature alkali lipase and its separation purification method of organic solvent-resistant.
Background technology:
Lipase (Lipase, EC 3.1.1.3) is the enzyme that class catalysis natural fats and oils (triglyceride) on oil-water interface is degraded to glycerine and free fatty acids.Low-temperature lipase is generally produced by psychrophile secretion, and the optimal reactive temperature of enzyme generally all is lower than 40 ℃, lives and still have a high enzyme at 0 ℃.For high temperature and middle temperature lipase, low-temperature lipase has wideer pH subject range, and this has further widened its industrial applicability.
Low-temperature lipase has caused the extensive concern of various countries' scientists owing to have good catalytic activity and low temperature adaptability at low temperatures.At present used washing composition mostly needs could effectively remove greasy dirt under middle temperature even high temperature, and fabric there is certain destruction,, not only can effectively removes greasy dirt at low temperatures if adopt the washing composition that contains low-temperature lipase, save energy, and can not destroy fabric.When utilizing low-temperature lipase to carry out the low-temperature catalyzed degreasing of leather, need not heat and add other grease-removing agent, can not cause environmental pollution, can keep the natural appearance of leather to greatest extent yet, both save the energy, guarantee the quality of leather again.In Europe, Japan and the U.S., low-temperature lipase has begun to be used for detergent industry.
Low-temperature lipase is owing to have the low temperature high catalytic activity, organic solvent-resistant and to characteristics such as thermo-responsive, become a focus of enzymology in recent years, huge application potential arranged in that food, washing, pharmacy, lipid processing, low temperature environment reparation etc. are industrial.To the research of low-temperature lipase purifying, will provide certain reference for the basis and the action oriented research of China's low-temperature lipase.
Psychrotropic bacteria bacterial strain Yersinia (Yersinia sp.) KM1 is not arranged in the prior art, and by this bacterial strain make feed purification and the low-temperature alkali lipase of organic solvent-resistant and the report of its separation purification method.
Summary of the invention:
The present invention aims to provide the psychrotropic bacteria bacterial strain that a plant height produces alkaline lipase.
Another object of the present invention is to provide the low-temperature alkali lipase and the separation purification method thereof of the organic solvent-resistant that produces by above-mentioned bacterial strains.
In order to realize above-mentioned purpose of the present invention, the invention provides following technical scheme:
Psychrotropic bacteria Yersinia (Yersinia sp.) KM1, its preserving number is: CGMCC No.2637.
The low-temperature alkali lipase of the organic solvent-resistant that produces by psychrotropic bacteria Yersinia (Yersinia sp.) KM1 bacterial strain.
This low-temperature alkali lipase has following zymologic property:
(1) the suitableeest enzyme of the Yersinia of purifying (Yersinia sp.) KM1 low-temperature lipase that bacterial strain produces temperature alive is 37 ℃;
(2) optimal pH is 9.0, keeps stable at pH7.2-10.0;
(3) molecular weight of low-temperature lipase is 34.3Kda;
(4) low-temperature lipase can tolerate the part organic solvent of 50%-80%, as methyl alcohol, ethanol and DMSO.
Low-temperature alkali lipase is a raw material with Yersinia (Yersinia sp.) KM1 bacterial strain, is obtained by following purification process:
(1) preparation crude enzyme liquid: the fermentation culture conditions of Yersinia (Yersinia sp.) KM1 bacterial strain is 13 ℃ of temperature, pH7.2, and incubation time 52 hours, fermented liquid is centrifugal, get supernatant and be crude enzyme liquid;
(2) ammonium sulfate precipitation: crude enzyme liquid adds ammonium sulfate to the 30-40% saturation ratio, and the centrifuging and taking supernatant continues to add ammonium sulfate to the 70-80% saturation ratio, and the centrifugal supernatant of abandoning is deposited in dialysed overnight in the phosphate buffered saline buffer;
(3) Sephacry
TMThe HRS-100 chromatography: the enzyme liquid of dialysis is centrifugal, and supernatant concentrates, and is splined on the Sephacry that the phosphate buffered saline buffer pre-equilibration is crossed
TMThe HRS-100 chromatography column, wash-out is collected the enzymic activity part, and concentrated frozen is preserved;
(4) Superdex G-75 chromatography: spissated target protein after centrifugal treating, is splined on the Superdex G-75 chromatography column that the phosphate buffered saline buffer pre-equilibration is crossed, and wash-out is collected the enzymic activity part, and dialysed overnight is pure enzyme.
The purification process of low-temperature alkali lipase is a raw material with Yersinia (Yersinia sp.) KM1 bacterial strain, comprising:
(1) preparation crude enzyme liquid: the fermentation culture conditions of Yersinia (Yersinia sp.) KM1 bacterial strain is 13 ℃ of temperature, pH7.2, and incubation time 52 hours, fermented liquid is centrifugal, get supernatant and be crude enzyme liquid;
(2) ammonium sulfate precipitation: crude enzyme liquid adds ammonium sulfate to the 30-40% saturation ratio, and the centrifuging and taking supernatant continues to add ammonium sulfate to the 70-80% saturation ratio, and the centrifugal supernatant of abandoning is deposited in dialysed overnight in the phosphate buffered saline buffer;
(3) Sephacry
TMThe HRS-100 chromatography: the enzyme liquid of dialysis is centrifugal, and supernatant concentrates, and is splined on the Sephacry that the phosphate buffered saline buffer pre-equilibration is crossed
TMThe HRS-100 chromatography column, wash-out is collected the enzymic activity part, and concentrated frozen is preserved;
(4) Superdex G-75 chromatography: spissated target protein after centrifugal treating, is splined on the Superdex G-75 chromatography column that the phosphate buffered saline buffer pre-equilibration is crossed, and wash-out is collected the enzymic activity part, and dialysed overnight is pure enzyme.
Fermentation condition optimization in the above-mentioned purification process, step (1); The used damping fluid of separation and purification is the 25mM phosphate buffered saline buffer in the step (2), pH7.0-8.0; The further electrophoresis purifying of the pure enzyme of gained in the step (4), resolving gel concentration is 12.5%, concentrated gum concentration is 5%.
Psychrotropic bacteria provided by the invention separates in the freezer in a tame slaughterhouse.According to morphological feature, physiological and biochemical property and 16S rRNA gene order result, with a bacterial strain of its Yersinia that is accredited as (Yersinia sp.), called after Yersinia (Yersinia sp.) KM1.The pH scope that this bacterium produces enzyme is 4.0-9.5, and optimal pH is 7.2; The temperature range of producing enzyme is 4-37 ℃, and the suitableeest product enzyme temperature is 13 ℃; Producing the enzyme time range is 18-66 hour, and the best product enzyme time is 54 hours.
New bacterial strain Yersinia of the present invention (Yersinia sp.) KM1 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.2637.
Description of drawings:
Fig. 1 is the temperature of reaction and the relative enzyme relation curve alive of the low-temperature lipase of KM1 bacterial strain generation;
Fig. 2 is the thermostability curve of the low-temperature lipase of KM1 bacterial strain generation;
Fig. 3 is reaction pH and the relative enzyme relation alive and the pH beta stability line of the low-temperature lipase of KM1 bacterial strain generation;
Embodiment:
Embodiment 1:
The separation screening and the evaluation of yersinia entero-colitica (Yersinia enterocolitica) KM1 bacterial strain:
Yersinia entero-colitica (Yersinia enterocolitica) KM1 bacterial strain separates the freezer from slaughterhouse, eastern station, Kunming.The bacterial strain screening substratum is (g/L): Tryptones, 10; Yeast extract, 5; NaCl, 10; The sweet oil emulsion, 2%; Agar, 20; PH7.0-7.2.
Adopt the bacterial system authentication method that Yersinia (Yersinia sp.) KM1 bacterial strain is identified.The morphological feature of this bacterium is as follows: bacterium colony is circular, and neat in edge is moistening, oyster white, thickness, easily picking; Cell is a rod-short, Gram-negative bacteria.The pH scope of producing enzyme is 4.0-9.5, and optimal pH is 7.2; The temperature range of producing enzyme is 4-37 ℃, and the suitableeest product enzyme temperature is 13 ℃; Producing the enzyme time range is 18-66 hour, and the best product enzyme time is 54 hours.This bacterium physiological and biochemical property is as follows: can grow as sole carbon source with glucose, sorbyl alcohol, Pentitol, malonate and western Meng Shi citrate, can not grow as sole carbon source with lactose and galactitol, can not utilize urea, can not produce H
2S, lysine decarboxylase, ornithine decarboxylase and phenylalanine decarboxylase reaction are negative.
According to morphological feature, physiological and biochemical property and 16S rRNA gene order result, with a bacterial strain of its Yersinia that is accredited as (Yersinia sp.), called after Yersinia (Yersinia sp.) KM1.
This new bacterial strain Yersinia (Yersinia sp.) KM1 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.2637.
Embodiment 2:
The purifying of low-temperature lipase
(1) preparation crude enzyme liquid: the fermentation culture conditions of Yersinia (Yersinia sp.) KM1 bacterial strain is 13 ℃ of temperature, pH7.2, incubation time 52 hours.Fermented liquid is centrifugal, get supernatant and be crude enzyme liquid.
(2) ammonium sulfate precipitation: crude enzyme liquid adds ammonium sulfate to the 30-40% saturation ratio, and the centrifuging and taking supernatant continues to add ammonium sulfate to the 70-80% saturation ratio, and the centrifugal supernatant of abandoning is deposited in 25mM, dialysed overnight in the phosphate buffered saline buffer of pH7.0-8.0.
(3) Sephacry
TMThe HRS-100 chromatography: the enzyme liquid of dialysis is centrifugal, and supernatant concentrates, and is splined on 25mM, the Sephacry that the phosphate buffered saline buffer pre-equilibration of pH7.0-8.0 is crossed
TMHRS-100 chromatography column, flow velocity are 0.4ml/min, and wash-out is collected the enzymic activity part, and the evaporating pipe of using 10KDa is in 4 ℃ of centrifugal collection concentrated solutions, freezing preservation.Detect protein concentration and enzymic activity, the purifying multiple of enzyme is 23.2 times, and the rate of recovery is 30.2%.
(4) Superdex G-75 chromatography: with spissated target protein after centrifugal treating, be splined on 25mM, the Superdex G-75 chromatography column that the phosphate buffered saline buffer pre-equilibration of pH7.0-8.0 is crossed, flow velocity is 0.4ml/min, use 25mM, the phosphate buffered saline buffer wash-out of pH7.0-8.0 detects protein concentration and enzymic activity, collect the enzymic activity part, the evaporating pipe of using 10KDa is in 4 ℃ of centrifugal collection concentrated solutions.Spissated sample is carried out the SDS-PAGE electrophoretic analysis, and resolving gel concentration is 12.5%, and concentrated gum concentration is 5%, obtains single band, and it is pure that the low-temperature lipase that shows purifying has reached electrophoresis, and molecular weight is 34.3KDa.The purifying multiple that concentrates the back enzyme is 26 times, and the rate of recovery is 10.3%.
The active mensuration of low-temperature alkali lipase: adopt People's Republic of China's industry standard (Ministry of Light Industry of the People's Republic of China (PRC), 1993).The hydrolysis unit of activity of low-temperature alkali lipase is defined as: with the low-temperature alkali lipase hydrolysate oil, per minute produces the enzyme amount of 1mol low-temperature alkali lipid acid, is defined as a lipase activity unit of force.
The mensuration of protein concn: adopt the LOWRY method to measure (LOWRY.0H, 1951).
SDS-PAGE vertical gel electrophoresis: carry out (Laemmli U K, 1970) by the Laemmli method.
Embodiment 3:
The characteristic of low-temperature lipase:
1. the suitableeest enzyme temperature alive
With enzyme liquid be added to phosphate buffered saline buffer (the 25mM phosphate buffered saline buffer, pH7.2) in, be substrate with pNPB, in 0-70 ℃ of scope, measure the enzyme of KM1 bacterial strain institute yielding lipase under the differing temps and live.The result as shown in Figure 1, the suitableeest enzyme temperature alive that shows this low-temperature lipase is 37 ℃, 0 ℃ still has nearly 20% activity, when temperature surpasses 40 ℃, enzymic activity sharply descends, the residual enzyme activity is less than 10% when reaching 70 ℃, and this result shows that this lipase has the characteristic feature of cold-adapted enzyme, and promptly the temperature of reaction of enzyme is low.
2. thermostability
Enzyme liquid is added to phosphate buffered saline buffer (25mM phosphate buffered saline buffer, pH7.2) in, and in the water bath with thermostatic control of differing temps, be incubated, at different time (0-90min, the sampling interval is 15min) sampling, measure enzyme according to the activity determination method of standard then and live.Temperature is expressed as the per-cent of the shared original enzymic activity of remaining enzymic activity behind certain hour to the influence of enzyme stability, the thermally-stabilised curve of KM1 low-temperature lipase that bacterial strain produces as shown in Figure 2, show this low-temperature lipase stable enzymic activity of (13 ℃ and 25 ℃) maintenance at low temperatures, and be incubated 60min down at 37 ℃, activity of residual enzyme is 50% of a maximum enzyme activity, this illustrates the thermally-stabilised very poor of this enzyme, the feature of symbol cold-adapted enzyme.
3. optimal pH
With the different damping fluid dilution enzyme liquid of pH5.0 to pHl1.0, add the pNPB substrate of corresponding pH respectively, measure enzyme activities down at 37 ℃, the result shows that the enzymic activity of low-temperature lipase when pH9.0 that the KM1 bacterial strain produced is the highest as shown in Figure 3.
4. pH stability
Enzyme liquid is added in the damping fluid of different pH, and,, measures enzyme according to the activity determination method of standard then and live at different time samplings 4 ℃ of insulations 10 hours.PH is expressed as the per-cent of the shared original enzymic activity of remaining enzymic activity behind certain hour to the influence of enzyme stability, and the pH stability of KM1 low-temperature lipase that bacterial strain produces shows that the low-temperature lipase that the KM1 bacterial strain is produced keeps stable at pH7.2-10.0 as shown in Figure 3.
5. organic solvent is to the influence of enzymic activity
Enzyme liquid is added in the organic solvent of different concns (0-80%), and 4 ℃ of insulations 10 hours, organic solvent was respectively methyl alcohol, ethanol, acetonitrile and dimethyl sulfoxide (DMSO) (DMSO).With pNPB is substrate, organic solvent is expressed as the per-cent of the shared original enzymic activity of remaining enzymic activity behind certain hour to the influence of enzymic activity, organic solvent is as shown in table 1 to the influence of KM1 low-temperature lipase that bacterial strain produces, the organic solvent of lower concentration (10%) has activation to this low-temperature lipase, and when the concentration of methyl alcohol reaches 50%, KM1 low-temperature lipase that bacterial strain produces still keeps 62.4% activity, so this enzyme is the good material of preparation biofuel.KM1 low-temperature lipase that bacterial strain produces makes it have certain application potential in organic chemical industry's compound probability to the tolerance of organic solvent.
6. enzyme kinetics
Enzyme liquid is added in the phosphate buffered saline buffer, and (4-60 ℃) insulation 10min measures corresponding enzyme and lives under different concentration of substrate (pNPB is 0.4-4.0mM, is spaced apart 0.4mM) and thermograde.Under differing temps, utilize the inverse of concentration of substrate and the inverse of speed of reaction to do curve, calculate corresponding Km and Kcat, and calculate activation energy (Ea).As shown in table 2, show Km minimum in the time of 37 ℃ of the low-temperature lipase that the KM1 bacterial strain is produced to illustrate that the binding ability of enzyme-to-substrate in the time of 37 ℃ is the strongest, and at 4-37 ℃, along with temperature raises, Km reduces gradually, this is the characteristic feature that cold-adapted enzyme has.The Ea value of the low-temperature lipase that the KM1 bacterial strain produced is 31.0KJmol
-1, being higher than the value of a lot of cold-adapted enzymes of present report, this shows that further the lipase of purifying is typical cold-adapted enzyme.
Table 1 organic solvent is to the influence of KM1 low-temperature lipase that bacterial strain produces
Table 2 is lived and activation energy at different concentration of substrate and the enzyme under the thermograde
Claims (8)
1, psychrotropic bacteria bacterial strain Yersinia (Yersinia sp.) KM1, its preserving number is: CGMCCNo.2637.
2, low-temperature alkali lipase is produced by psychrotropic bacteria bacterial strain Yersinia (Yersinia sp.) KM1.
3, low-temperature alkali lipase as claimed in claim 2 has following zymologic property:
(1) the suitableeest enzyme temperature alive is 37 ℃;
(2) optimal pH is 9.0, keeps stable at pH7.2-10.0;
(3) molecular weight of low-temperature lipase is 34.3Kda;
(4) low-temperature lipase can tolerate the part organic solvent of 50%-80%, as methyl alcohol, ethanol and DMSO.
4, the low-temperature alkali lipase of claim 2 is a raw material with Yersinia (Yersinia sp.) KM1 bacterial strain, is obtained by following purification process:
(1) preparation crude enzyme liquid: the fermentation culture conditions of Yersinia (Yersinia sp.) KM1 bacterial strain is 13 ℃ of temperature, pH7.2, and incubation time 52 hours, fermented liquid is centrifugal, get supernatant and be crude enzyme liquid;
(2) ammonium sulfate precipitation: crude enzyme liquid adds ammonium sulfate to the 30-40% saturation ratio, and the centrifuging and taking supernatant continues to add ammonium sulfate to the 70-80% saturation ratio, and the centrifugal supernatant of abandoning is deposited in dialysed overnight in the phosphate buffered saline buffer;
(3) Sephacry
TMThe HRS-100 chromatography: the enzyme liquid of dialysis is centrifugal, and supernatant concentrates, and is splined on the Sephacry that the phosphate buffered saline buffer pre-equilibration is crossed
TMThe HRS-100 chromatography column, wash-out is collected the enzymic activity part, and concentrated frozen is preserved;
(4) Superdex G-75 chromatography: spissated target protein after centrifugal treating, is splined on the Superdex G-75 chromatography column that the phosphate buffered saline buffer pre-equilibration is crossed, and wash-out is collected the enzymic activity part, and dialysed overnight is pure enzyme.
5, the purification process of claim 2 low-temperature alkali lipase is a raw material with Yersinia (Yersinia sp.) KM1 bacterial strain, and its step comprises:
(1) preparation crude enzyme liquid: the fermentation culture conditions of Yersinia (Yersinia sp.) KM1 bacterial strain is 13 ℃ of temperature, pH7.2, and incubation time 52 hours, fermented liquid is centrifugal, get supernatant and be crude enzyme liquid;
(2) ammonium sulfate precipitation: crude enzyme liquid adds ammonium sulfate to the 30-40% saturation ratio, and the centrifuging and taking supernatant continues to add ammonium sulfate to the 70-80% saturation ratio, and the centrifugal supernatant of abandoning is deposited in dialysed overnight in the phosphate buffered saline buffer;
(3) Sephacry
TMThe HRS-100 chromatography: the enzyme liquid of dialysis is centrifugal, and supernatant concentrates, and is splined on the Sephacry that the phosphate buffered saline buffer pre-equilibration is crossed
TMThe HRS-100 chromatography column, wash-out is collected the enzymic activity part, and concentrated frozen is preserved;
(4) Superdex G-75 chromatography: spissated target protein after centrifugal treating, is splined on the Superdex G-75 chromatography column that the phosphate buffered saline buffer pre-equilibration is crossed, and wash-out is collected the enzymic activity part, and dialysed overnight is pure enzyme.
6, purification process according to claim 4 is characterized in that Optimizing Conditions of Fermentation in step (1).
7, purification process according to claim 4 is characterized in that at the used damping fluid of separation and purification in step (2) be the 25mM phosphate buffered saline buffer, pH7.0-8.0.
8, purification process according to claim 4 is characterized in that carrying out the SDS-PAGE electrophoretic analysis at the pure enzyme sample that further step (3) is obtained, and resolving gel concentration is 12.5%, and concentrated gum concentration is 5%.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103194433A (en) * | 2013-04-19 | 2013-07-10 | 昆明理工大学 | Low-temperature lipase Lip1 as well as gene and application thereof |
| CN113975465A (en) * | 2021-10-19 | 2022-01-28 | 吾奇生物医疗科技(江苏)有限公司 | Low-temperature degreasing method based on animal tissue and application thereof |
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| CN1301326C (en) * | 2004-12-03 | 2007-02-21 | 中国水产科学研究院黄海水产研究所 | New type hypothermal alkaline lipase and marine yeast suitable to cold for producing the lipase |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103194433A (en) * | 2013-04-19 | 2013-07-10 | 昆明理工大学 | Low-temperature lipase Lip1 as well as gene and application thereof |
| CN103194433B (en) * | 2013-04-19 | 2014-10-01 | 昆明理工大学 | Low-temperature lipase Lip1 as well as gene and application thereof |
| CN113975465A (en) * | 2021-10-19 | 2022-01-28 | 吾奇生物医疗科技(江苏)有限公司 | Low-temperature degreasing method based on animal tissue and application thereof |
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