CN103409394A - Method for separating and purifying Lecanicillium lecanii chitinase - Google Patents
Method for separating and purifying Lecanicillium lecanii chitinase Download PDFInfo
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- CN103409394A CN103409394A CN2013102958097A CN201310295809A CN103409394A CN 103409394 A CN103409394 A CN 103409394A CN 2013102958097 A CN2013102958097 A CN 2013102958097A CN 201310295809 A CN201310295809 A CN 201310295809A CN 103409394 A CN103409394 A CN 103409394A
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Abstract
The invention discloses a method for separating and purifying entomogenous fungus Lecanicillium lecanii strain chitinase. According to the method, Lecanicillium lecanii is fermented and cultured in an optimized enzyme-producing culture medium, and Lecanicillium lecanii trehalase in uniform SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) electrophoresis in fermentation liquor is obtained by the steps of ammonium sulfate precipitation, DEAE (Diethyl-Aminoethanol)-52 anion exchange column chromatography, SephadexG-100 gel filtration chromatography and the like. The process provided by the invention has the advantages of high specificity, simple process, easy control, difficult protein inactivation, good purifying effect, high recovery rate, low cost and the like, and has good popularization and application values.
Description
Technical field
The present invention relates to the separation purifying technique of chitinase.More specifically, the present invention relates to a kind of separation purification method of lecanium bacterium chitinase.
Background technology
The lecanium bacterium is subordinate to Hyphomycetes (Hyphomycetes), is the important insecticidal microorganism of a class, generally by body wall, penetrates mode and infects also lethal host, and they are the synthesis result of the Physiology and biochemistry effect between host and pathogenic bacteria to host's invasion.Infecting position is the epidermis of host insect.The chitin of insect cuticle is one natural cover for defense of fungi Invasive insect species, insect cuticle and peritrophic important composition composition, it is by the high-molecular biologic polymer that N-acetyl-β-D-glucosamine residue β-Isosorbide-5-Nitrae glycosidic link is formed by connecting, and accounts for epidermis dry weight 17%-50%.All need a certain amount of chitin each period of insect growth, growth.The chitin metabolism changes with the different growth and development stages of insect and keeps relative stable, it is vital that the normal growth of insect is grown, simultaneously, penetrating body wall is one of significant process infected at insect pathogenic fungus the host, therefore, the chitinase lytic enzyme has been brought into play vital role in its pathogenic host insects process.
This bacterium chitinase is carried out to the preliminary study of separation and purification and zymologic property thereof, can and guide its production application to lay the foundation for final clarification entomogenous fungi pathogenesis, can be the further investigation of fungi pest control in the future scientific basis is provided.
Summary of the invention
The object of the present invention is to provide the separation purification method of the circumscribed chitinase of a kind of lecanium bacterium, for the clonal expression of chitinase gene with disclose the entomogenous fungi pathogenesis and and guide its production application to lay the foundation;
To achieve these goals, the invention provides following technical scheme:
(1) (culture medium prescription is the culture medium culturing bacterial strain filtered out by optimization Test: 1% (w/v) Zulkovsky starch, 1% w peptone, 4 * 10
-4Mol/L Ba
2+, 0.01% w Vc, the initial pH=7.0 of substratum), utilize DNS reagent (3,5-dinitrosalicylic acid) colorimetry, every 24h, get bacteria culture fluid and measure, in the chitinase activity, reach when the highest and get fermented liquid;
(2) lecanium bacterium chitinase, on above-mentioned fermented liquid basis, is obtained by following purification process:
(2.1) preparation of crude enzyme liquid: by fermented liquid centrifugal 10min under the 9500rpm condition, the supernatant liquor vacuum filtration, collect filtrate.To adding solid ammonium sulfate to saturation ratio in filtrate, be 60%wt, 4 ℃ of hold over night.95000rpm, 4 ℃ of centrifugal 15min collecting precipitations, with same phosphate buffered saline buffer dissolution precipitation extremely fully.95000rpm, 4 ℃ of centrifugal 15min remove insoluble object, and supernatant liquor is loaded on dialysis tubing, after 0.02mol/L phosphate buffered saline buffer (pH6.5) dialysis, then concentrate and obtain crude enzyme liquid with polyoxyethylene glycol PEG20000.(namely concentrated with polyoxyethylene glycol PEG20000 again, until the supernatant liquor volume after dialysis no longer reduces, thereby obtain crude enzyme liquid.)
(2.2) DEAE A-52 anion-exchange chromatography: crude enzyme liquid is after polyoxyethylene glycol PEG20000 is concentrated, by initial damping fluid equilibrate overnight, be splined on the DEAE A-52 anion-exchange chromatography post after abundant balance, with the 0.02mol/L phosphate buffered saline buffer (pH=6.5) that contains 0-1mol/LNaCl, carry out continuous wash-out again, collect elutriant with Fraction Collector.Elution requirement is set to: 0.5mL/min, every 10 minutes one pipes.
(2.3) Sephadex G-100 gel permeation chromatography: with the Sephadex G-100 gel chromatography column (1.8 * 100 cm) of the abundant balance of 0.02mol/L phosphate buffered saline buffer (pH=6.5), after the enzyme liquid loading that back is collected, with identical damping fluid, carry out wash-out again, collect elutriant with Fraction Collector.Elution requirement is set to: flow velocity 0.25mL/min, and, every 20 minutes one pipes.
In step (2.1) (2.2) (2.3), phosphate buffering liquid concentration is 0.02mol/L, and the phosphate buffering liquid concentration that pH=6.5 contains 0-1mol/L NaCl is 0.02mol/L, pH=6.5.The purification result contrast situation of each purification process is in Table 1.
(2.4) molecular weight detection of purifying chitinase: adopt DNS reagent (3,5-dinitrosalicylic acid) colorimetry, survey respectively walks the enzyme size alive after purifying, as table 1.After comparative analysis Sephadex G-100 as can be known gel permeation chromatography, be that specific activity or the purity of chitinase has all reached higher value.Therefore, we adopt degree of purification and the molecular weight of discontinuous vertical slab electrophoresis systems measurement chitinase, and resolving gel concentration is 10%, and concentrated gum concentration is 5%, and the gel size is 100 mm * 70mm * 0.75mm.Standard protein used is: phosphorylase B 97 200; Bovine serum albumin 66 400; Actin muscle 44 300; Carbonic anhydrase 29 000; STI 20 100; Whey-protein 14 400.After electrophoresis, show protein band with the Xylene Brilliant Cyanine G Faxian, with the degree of purification of checking chitinase, determine its molecular size range.
The Verticillium lecanii that the present invention adopts is the lecanium bacterium
L. lecaniiFJ28(Qiu Jun will etc., the impact of metal ion on lecanium bacterium chitinase activity, laser biology journal, in February, 2009).
This technique specificity is high, and technique is simple, and protein is difficult for inactivation, and purification effect is good, and the rate of recovery is high, and cost is low, has good application value.
The accompanying drawing explanation
The DEAE A-52 anion-exchange chromatography elution profile of Fig. 1 chitinase liquid
The Sephadex G-100 gel permeation chromatography elution profile of Fig. 2 chitinase liquid
In the circumscribed chitinase purge process of Fig. 3 lecanium bacterium, respectively walk activeconstituents SDS-PAGE collection of illustrative plates
Swimming lane 2 is standard protein;
Swimming lane 4 is 60% (NH
4)
2SO
4The precipitation crude enzyme liquid.
Embodiment
In order to make content of the present invention more be convenient to understand, below in conjunction with embodiment, technical solutions according to the invention are described further, but the present invention is not limited only to this.
(1) substratum filtered out by optimization Test (1% (w/v) Zulkovsky starch, 1% w peptone, 4 * 10
-4Mol/L Ba
2+, 0.01% w Vc, the initial pH=7.0 of substratum) and cultivation lecanium bacterium
L. lecaniiFJ28, utilize DNS reagent (3,5-dinitrosalicylic acid) colorimetry, every 24h, gets bacteria culture fluid and measure, and in the chitinase activity, reach when the highest and get fermented liquid, the centrifugal 10min of 9500rpm, the supernatant liquor vacuum filtration, collect filtrate.To adding solid ammonium sulfate to saturation ratio in filtrate, be 60%wt, 4 ℃ of hold over night.95000rpm, 4 ℃ of centrifugal 15min collecting precipitations, with identical damping fluid dissolution precipitation extremely fully.95000rpm, 4 ℃ of centrifugal 15min remove insoluble object, supernatant liquor is loaded on dialysis tubing, after 0.02mol/L phosphate buffered saline buffer (pH6.5) dialysis, adds polyoxyethylene glycol PEG20000 to concentrate, until the dialysis after the supernatant liquor volume no longer reduce, thereby obtain crude enzyme liquid.
(2) above-mentioned crude enzyme liquid is fully dialysed through 0.02mol/L phosphate buffered saline buffer (pH6.5), and after with PEG20000, suitably concentrating, be loaded to the DEAE A-52 anion-exchange chromatography post that abundant balance is good, chromatography column connects Ultraviolet Detector outward and shows elution peak.The 0.02mol/L phosphate buffered saline buffer (pH6.5) of using with balance glue post carries out wash-out, with the 0.02mol/L phosphate buffered saline buffer (pH6.5) that contains 0-1mol/L NaCl, carry out the continuous wash-out of salt ion gradient again, above wash-out is all 0.5mL/min, every 10 minutes one pipes; Co-elute goes out 2 protein peaks (Fig. 1).DNS reagent (3,5-dinitrosalicylic acid) colorimetric method for determining is collected active discovery of chitinase of liquid, in above-mentioned collection liquid, only contains a circumscribed chitinase Peak Activity, mainly concentrates in the pipe that the 600-800min time collects.
(3) 600-800min collection obtained collects liquid, is loaded to the Sephadex G-100 gel column that abundant balance is good.The 0.02mol/L phosphate buffered saline buffer (pH6.5) of using with balance glue post carries out wash-out, flow velocity 0.25mL/min, and, every 20 minutes one pipes.By being connected in the Ultraviolet Detector on gel column outward, observing co-elute and go out a protein peak (Fig. 2).
(4) above-mentioned Peak Activity SDS-PAGE electrophoresis, show as single band (Fig. 3), shows that the chitinase after purifying is one-component, reached electrophoresis pure.As shown in Figure 3, pure enzyme and standard protein are measured through SDS-PAGE, and this enzyme molecular weight is about 32 kDa.
Claims (3)
1. method from lecanium bacterium separation and purification chitinase is characterized in that comprising the following steps:
(a) preparation of crude enzyme liquid: the culture medium culturing lecanium bacterium filtered out by optimization Test
L. lecaniiFJ28, utilize DNS reagents ratio color method, every 24h, gets bacteria culture fluid and measure, and to get fermented liquid centrifugal when the chitinase activity reaches when the highest, and the supernatant liquor vacuum filtration, collect filtrate; To adding solid ammonium sulfate to saturation ratio in filtrate, be 60%wt, 4 ℃ of hold over night; The recentrifuge collecting precipitation, with the phosphate buffered saline buffer dissolution precipitation extremely fully; Centrifugal lysate is removed insoluble object, and supernatant liquor is loaded on dialysis tubing, after the phosphate buffered saline buffer dialysis, then concentrates and obtains crude enzyme liquid with polyoxyethylene glycol PEG20000;
(b) DEAE A-52 anion-exchange chromatography: crude enzyme liquid is after polyoxyethylene glycol PEG20000 is concentrated, use the phosphate buffered saline buffer equilibrate overnight, be splined on the DEAE A-52 anion-exchange chromatography post after abundant balance, with phosphate buffered saline buffer wash-out foreign protein, in the process of collecting elutriant, by the wash-out situation of effective ultraviolet spectrophotometer check foreign protein, after baseline starts to walk to put down, use the phosphate buffered saline buffer that contains 0-1mol/L NaCl instead and carry out continuous wash-out, collect elutriant;
(c) Sephadex G-100 gel permeation chromatography: with the abundant balance Sephadex G-100 of phosphate buffered saline buffer gel chromatography column; By the sample loading after step (b), then carry out wash-out with the 0.02mol/L phosphate buffered saline buffer, collect elutriant;
Step (a) (b) (c) middle phosphate buffering liquid concentration is 0.02mol/L, pH=6.5; The phosphate buffering liquid concentration that contains 0-1mol/L NaCl in step (b) is 0.02mol/L, pH=6.5.
2. according to the separation purification method of the described lecanium bacterium of right 1 chitinase, it is characterized in that in step (a), three centrifugal rotational speeds are 95000rpm, but centrifugal first be at normal temperatures, the time is 10min, latter twice is 4 ℃ of centrifugal 15min of condition.
3. according to the separation purification method of the described lecanium bacterium of right 1 chitinase, it is characterized in that during step (b) (c) that elution requirement is set to respectively: 0.5mL/min, every 10 minutes one pipes; 0.25mL/min, every 20 minutes one pipes.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104894086A (en) * | 2015-05-18 | 2015-09-09 | 昆明理工大学 | Purification method of Baozhu pear chitinase |
| CN106011099A (en) * | 2016-06-23 | 2016-10-12 | 华东理工大学 | Separation and purification method of insect transglutaminase |
| CN110511915A (en) * | 2019-09-27 | 2019-11-29 | 泉州师范学院 | A kind of method and application isolating and purifying prawn internal organ chitin excision enzyme |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102994483A (en) * | 2012-12-17 | 2013-03-27 | 福建农林大学 | Culture medium and method for producing chitinase by Lecanicillium |
| CN102994484A (en) * | 2012-12-17 | 2013-03-27 | 福建农林大学 | Culture medium and method for fermentation production of chitinase by aschersonia placenta |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102994483A (en) * | 2012-12-17 | 2013-03-27 | 福建农林大学 | Culture medium and method for producing chitinase by Lecanicillium |
| CN102994484A (en) * | 2012-12-17 | 2013-03-27 | 福建农林大学 | Culture medium and method for fermentation production of chitinase by aschersonia placenta |
Non-Patent Citations (3)
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| 邱君志等: "有机溶剂对蜡蚧菌几丁质酶的影响", 《激光生物学报》, 15 April 2009 (2009-04-15) * |
| 邱君志等: "金属离子对蜡蚧菌几丁质酶活力的影响", 《激光生物学报》, 15 February 2009 (2009-02-15) * |
| 郭婷婷: "座壳孢菌海藻糖酶的纯化及酶学性质研究", 《全国优秀硕士学位论文全文数据库 基础科学辑》, 15 May 2011 (2011-05-15) * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104894086A (en) * | 2015-05-18 | 2015-09-09 | 昆明理工大学 | Purification method of Baozhu pear chitinase |
| CN104894086B (en) * | 2015-05-18 | 2019-02-05 | 昆明理工大学 | A kind of purification process of mound pears chitinase |
| CN106011099A (en) * | 2016-06-23 | 2016-10-12 | 华东理工大学 | Separation and purification method of insect transglutaminase |
| CN110511915A (en) * | 2019-09-27 | 2019-11-29 | 泉州师范学院 | A kind of method and application isolating and purifying prawn internal organ chitin excision enzyme |
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Application publication date: 20131127 |