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
- Publication number
- 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
- Authority
- CN
- China
- Prior art keywords
- yersinia
- temperature
- low
- lipase
- bacterial strain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108090001060 Lipase Proteins 0.000 title claims abstract description 61
- 102000004882 Lipase Human genes 0.000 title claims abstract description 60
- 239000004367 Lipase Substances 0.000 title claims abstract description 60
- 235000019421 lipase Nutrition 0.000 title claims abstract description 60
- 241000607734 Yersinia <bacteria> Species 0.000 title claims description 25
- 238000000746 purification Methods 0.000 title claims description 17
- 238000000034 method Methods 0.000 title abstract description 11
- 241000131891 Yersinia sp. Species 0.000 claims abstract description 24
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 22
- 239000006228 supernatant Substances 0.000 claims abstract description 21
- 239000003960 organic solvent Substances 0.000 claims abstract description 17
- 241000894006 Bacteria Species 0.000 claims abstract description 13
- 239000012505 Superdex™ Substances 0.000 claims abstract description 11
- 238000012870 ammonium sulfate precipitation Methods 0.000 claims abstract description 6
- 238000001962 electrophoresis Methods 0.000 claims abstract description 5
- 102000004190 Enzymes Human genes 0.000 claims description 74
- 108090000790 Enzymes Proteins 0.000 claims description 74
- 230000001580 bacterial effect Effects 0.000 claims description 43
- 230000000694 effects Effects 0.000 claims description 32
- 239000007788 liquid Substances 0.000 claims description 31
- 239000000872 buffer Substances 0.000 claims description 23
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 23
- 239000002953 phosphate buffered saline Substances 0.000 claims description 23
- 239000003513 alkali Substances 0.000 claims description 16
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 11
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 10
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 10
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 10
- 238000011067 equilibration Methods 0.000 claims description 10
- 108090000623 proteins and genes Proteins 0.000 claims description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 9
- 102000004169 proteins and genes Human genes 0.000 claims description 8
- 238000000855 fermentation Methods 0.000 claims description 7
- 230000004151 fermentation Effects 0.000 claims description 7
- 239000012141 concentrate Substances 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- 238000000926 separation method Methods 0.000 claims description 6
- 238000000502 dialysis Methods 0.000 claims description 5
- 238000011534 incubation Methods 0.000 claims description 5
- 238000013016 damping Methods 0.000 claims description 4
- 239000012530 fluid Substances 0.000 claims description 4
- 239000002994 raw material Substances 0.000 claims description 4
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 claims description 3
- 238000006243 chemical reaction Methods 0.000 abstract description 6
- 235000013305 food Nutrition 0.000 abstract description 2
- 238000012545 processing Methods 0.000 abstract description 2
- 238000004519 manufacturing process Methods 0.000 abstract 2
- 230000004308 accommodation Effects 0.000 abstract 1
- 239000003225 biodiesel Substances 0.000 abstract 1
- 238000004140 cleaning Methods 0.000 abstract 1
- 239000003814 drug Substances 0.000 abstract 1
- 239000002904 solvent Substances 0.000 abstract 1
- 238000000108 ultra-filtration Methods 0.000 abstract 1
- 239000000758 substrate Substances 0.000 description 7
- 241000607447 Yersinia enterocolitica Species 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 229940098232 yersinia enterocolitica Drugs 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 238000009413 insulation Methods 0.000 description 3
- 239000010985 leather Substances 0.000 description 3
- 230000000877 morphologic effect Effects 0.000 description 3
- 239000003921 oil Substances 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 238000005070 sampling Methods 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000001704 evaporation Methods 0.000 description 2
- 239000004744 fabric Substances 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- MEIRRNXMZYDVDW-MQQKCMAXSA-N (2E,4E)-2,4-hexadien-1-ol Chemical compound C\C=C\C=C\CO MEIRRNXMZYDVDW-MQQKCMAXSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 102100031375 Endothelial lipase Human genes 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 108010048581 Lysine decarboxylase Proteins 0.000 description 1
- OFOBLEOULBTSOW-UHFFFAOYSA-L Malonate Chemical compound [O-]C(=O)CC([O-])=O OFOBLEOULBTSOW-UHFFFAOYSA-L 0.000 description 1
- 102000052812 Ornithine decarboxylases Human genes 0.000 description 1
- 108700005126 Ornithine decarboxylases Proteins 0.000 description 1
- 241000237502 Ostreidae Species 0.000 description 1
- 108010044588 Phenylalanine decarboxylase Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 239000002551 biofuel Substances 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- HEBKCHPVOIAQTA-NGQZWQHPSA-N d-xylitol Chemical compound OC[C@H](O)C(O)[C@H](O)CO HEBKCHPVOIAQTA-NGQZWQHPSA-N 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000005238 degreasing Methods 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000003925 fat Substances 0.000 description 1
- 235000021588 free fatty acids Nutrition 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- FBPFZTCFMRRESA-GUCUJZIJSA-N galactitol Chemical compound OC[C@H](O)[C@@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-GUCUJZIJSA-N 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 239000000413 hydrolysate Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 235000019626 lipase activity Nutrition 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 235000020636 oyster Nutrition 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 1
- 108010046845 tryptones Proteins 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Images
Landscapes
- Enzymes And Modification Thereof (AREA)
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%.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN2008102336261A CN101486979B (en) | 2008-11-24 | 2008-11-24 | Yersinia strain KM1, low temperature alkaline lipase prepared thereby and purification method thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN2008102336261A CN101486979B (en) | 2008-11-24 | 2008-11-24 | Yersinia strain KM1, low temperature alkaline lipase prepared thereby and purification method thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
CN101486979A true CN101486979A (en) | 2009-07-22 |
CN101486979B CN101486979B (en) | 2011-05-18 |
Family
ID=40890041
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN2008102336261A Expired - Fee Related CN101486979B (en) | 2008-11-24 | 2008-11-24 | Yersinia strain KM1, low temperature alkaline lipase prepared thereby and purification method thereof |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN101486979B (en) |
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 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
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 |
-
2008
- 2008-11-24 CN CN2008102336261A patent/CN101486979B/en not_active Expired - Fee Related
Cited By (3)
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 |
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 |
Also Published As
Publication number | Publication date |
---|---|
CN101486979B (en) | 2011-05-18 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Khusro et al. | Statistical optimization of thermo-alkali stable xylanase production from Bacillus tequilensis strain ARMATI | |
Apun et al. | Screening and isolation of a cellulolytic and amylolytic Bacillus from sago pith waste | |
CN101555461B (en) | Bacterial strain LT3 producing alkalescence cellulase and breeding method and initial optimization of cellulase production conditions thereof | |
Chen et al. | Improving microalgal oil collecting efficiency by pretreating the microalgal cell wall with destructive bacteria | |
Navya et al. | Production, statistical optimization and application of endoglucanase from Rhizopus stolonifer utilizing coffee husk | |
Begum et al. | Purification and characterization of intracellular cellulase from Aspergillus oryzae ITCC-4857.01 | |
Al-Shorgani et al. | Isolation of a Clostridium acetobutylicum strain and characterization of its fermentation performance on agricultural wastes | |
Sharma et al. | Fermentation of enzymatically saccharified sunflower stalks for ethanol production and its scale up | |
CN103060286B (en) | Lipase made of aspergillus niger strains, and producing method and utilization thereof | |
CN102533563B (en) | Celluase producing bacterium and application thereof | |
CN103710326B (en) | A kind of beta-glucosidase and application thereof | |
Abada et al. | Optimization of cellulase production from Bacillus albus (MN755587) and its involvement in bioethanol production | |
CN101486979B (en) | Yersinia strain KM1, low temperature alkaline lipase prepared thereby and purification method thereof | |
Sahil et al. | Enhanced biogas production from rice straw through pretreatment with cellulase producing microbial consortium | |
Chen et al. | Use of elephant grass (Pennisetum purpureum) acid hydrolysate for microbial oil production by Trichosporon cutaneum | |
CN101654697B (en) | Method for preparing rapeseed peptides by mixed fermentation | |
CN101928686B (en) | Bacillus subtilis secreting surfactant | |
Akintola et al. | Production and physicochemical properties of thermostable, crude cellulase from Enterobacter cloacae IP8 isolated from plant leaf litters of Lagerstroemia indica Linn. | |
CN101643708B (en) | Constitutive acidic incision cellulase high-yield strain | |
Seddouk et al. | Isolation and characterization of a mesophilic cellulolytic endophyte Preussia africana from Juniperus oxycedrus | |
CN102807958B (en) | Bacterial strain capable of secreting cellulase as well as cellulase extraction method and application thereof | |
CN103509742A (en) | Baclicus lincheniformis strain for generating elastase and screening method thereof | |
CN101654693B (en) | Method for preparing rapeseed peptides by microorganism fermentation | |
CN101412981B (en) | Serratieae WJ39 bacterial strain, low temperature proteinase obtained therefrom and purification method thereof | |
CN102994408B (en) | Carrageenan degrading bacterium and fermentation method and application thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C14 | Grant of patent or utility model | ||
GR01 | Patent grant | ||
C17 | Cessation of patent right | ||
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20110518 Termination date: 20131124 |