CN103409399A

CN103409399A - Method for separating and purifying verticillium lecanii proteases

Info

Publication number: CN103409399A
Application number: CN2013103189063A
Authority: CN
Inventors: 邱君志; 董冬; 涂洁; 李小霞; 孟丽雪; 何肖云; 曹丽萍; 张以盼; 郭庆丰; 姚灵丹
Original assignee: Fujian Agriculture and Forestry University
Current assignee: Fujian Agriculture and Forestry University
Priority date: 2013-07-27
Filing date: 2013-07-27
Publication date: 2013-11-27

Abstract

The invention discloses a method for separating and purifying verticillium lecanii proteases. The method comprises the following steps: fermenting verticillium lecanii spores in an optimized enzyme production culture medium, then performing ultrafiltration concentration, ammonium sulfate salting out, DEAESepharosefastflow anion exchange column chromatography, SephadexG-150 molecular sieve chromatography and other processes to obtain the SDS-PAGE verticillium lecanii proteases with uniform cataphoresis in fermentation liquor. The method is high in specificity, simple in process, good in purification effect, high in recovery rate and low in cost, and has excellent popularization and application value; by adopting the method, proteins are susceptible to inactivation.

Description

The method of a kind of lecanium mycoproteinase separation and purification

Technical field

The present invention relates to the separation purifying technique of proteolytic enzyme.More specifically, the present invention relates to a kind of separation purification method of lecanium mycoproteinase.

Background technology

The lecanium bacterium is subordinate to Hyphomycetes (Hyphomycetes), is distributed widely in the torrid zone, subtropics and temperate zone, can parasitic a red-spotted lizard class, Phytophthira, mite class and aleyrodid, and also can parasitic more lepidopterous insects and nematode, thrips etc.Since 20 century 70s, the states such as some countries of Europe and the U.S., Japan are to utilizing lecanium bacterium control greenhouse vegetable insect to give very big attention.The lecanium bacterium all can infect protection ground each worm state of aleyrodid, can be popular in the aleyrodid population.When being not suitable for propagation conditions, can in greenhouse, survive for some time.And the popular of lecanium bacterium is subjected to the restriction of aleyrodid population density little, be not subjected on artificial medium to turn the impact of culture number of times etc., virulence is stable, is difficult for variation.Therefore this bacterium is considered to prevent and treat the important pathogen microorganism of aleyrodid, and obtains research and development energetically.

Proteolytic enzyme is the enzyme that a class can the catalytic proteins hydrolysis reaction, extensively is present in various organisms.The process of the various complexity such as proteolysis, Growth of Cells and migration, activation of zymogen, hormone release under the normal physiological of their participation cells and abnormal pathological conditions.Protein is the important component part of insect body wall, is one of significant process infected at insect pathogenic fungus the host and penetrate body wall.But the insect cuticle of lecanium mycetocyte exoproteinase decomposing protein character, this helps the invasion of mycelia, and provides nutrition for mycelia.Proteolytic enzyme is carried out to the preliminary study of separation and purification and its proteolytic enzyme zymologic property, and guide its production application to lay the foundation for disclosing the 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 method for a kind of lecanium mycoproteinase separation and purification, for the clonal expression of proteinase 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) the culture medium culturing bacterial strain filtered out by optimization Test, reach when the highest and get fermented liquid at protease activity;

(2) lecanium mycoproteinase, on above-mentioned fermented liquid basis, is obtained by following purification process:

(2.1) preparation of crude enzyme liquid: at the centrifugal 10min of 8500rpm, the supernatant liquor vacuum filtration, collect filtrate by fermented liquid.To adding solid ammonium sulfate to saturation ratio in filtrate, be 60%wt, 4 ℃ of hold over night.11000rpm, 4 ℃ of centrifugal 15min collecting precipitations, with appropriate dissolution precipitation.12000rpm, 4 ℃ of centrifugal 15min remove insoluble object, and supernatant liquor 0.02mol/L Tris-HCl damping fluid (pH8.0) is through dialysis, then are crude enzyme liquid after concentrated with polyoxyethylene glycol.

(2.2) DEAE Sepharose fast flow anion exchange chromatography: with the abundant balance DEAE Sepharose fast flow anion exchange chromatography post of 0.02mol/L Tris-HCl damping fluid (pH8.0), loading is through the concentrated crude enzyme liquid of PEG20000, first use 0.02mol/L Tris-HCl damping fluid (pH8.0) to rinse chromatography column, after use 0-1mol/L NaCl instead 0.02mol/L Tris-HCl damping fluid (pH8.0) carry out continuous wash-out.Elution requirement is set to: flow velocity 30mL/h; The 5mL/ pipe, collect and have the enzymic activity part.

(2.3) Sephadex G-150 sieve chromatography: by the Sephadex G-150 molecular sieve chromatography (1.8 * 100) of the abundant balance of 0.02mol/L Tris-HCl damping fluid (pH8.0), 2.1 enzymic activitys that have of collecting partly are splined on to it, then carry out wash-out with identical damping fluid.Elution requirement is set to: flow velocity 15mL/h; The 5mL/ pipe.Collect zymophore.

(2.4) molecular weight detection of purifying protein enzyme: adopt degree of purification and the molecular weight of discontinuous vertical slab electrophoresis systems measurement proteolytic enzyme, resolving gel concentration is 10%, and concentrated gum concentration is 5%, and the gel size is 100 mm * 70 * 0.75mm.Standard protein used is: phosphorylase B 94 000; Bovine serum albumin 67 000; Actin muscle 43 000; Carbonic anhydrase 30 000; STI 20100; Whey-protein 14 400.After electrophoresis, show protein band with the Xylene Brilliant Cyanine G Faxian.

The desinsection lecanium bacterium that the present invention adopts is Verticillium lecanii 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 Sepharose fast flow anion exchange chromatography result of Fig. 1 liquid of protease

The Sephadex G-150 sieve chromatography result of Fig. 2 liquid of protease

In Fig. 3 lecanium mycoproteinase purge process, respectively walk activeconstituents SDS-PAGE collection of illustrative plates; Wherein swimming lane M is standard protein, and swimming lane 1 is 60% (NH ₄) ₂SO ₄The precipitation crude enzyme liquid, swimming lane 2 is the pure enzyme after Sephadex G-150 sieve chromatography.

Specific implementation method:

?(1) the culture medium culturing bacterial strain filtered out by optimization Test (culture medium prescription: wheat bran 1w%, peptone 1 w %, Mg ²⁺4 * 10 ^-4Mol/L, V _B60.01%, the initial pH=6.0 of substratum), when reaching the highest (enzyme work records by ultraviolet spectrophotometer), gets protease activity fermented liquid, the centrifugal 10min of 8500rpm, 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.11000rpm, 4 ℃ of centrifugal 15min collecting precipitations, with appropriate 0.02mol/L Tris-HCl damping fluid (pH8.0) damping fluid dissolution precipitation.12000rpm, 4 ℃ of centrifugal 15min remove insoluble object, and supernatant liquor 0.02mol/L Tris-HCl damping fluid (pH8.0) is through dialysis, then are crude enzyme liquid after concentrated with polyoxyethylene glycol.

(2) crude enzyme liquid is fully dialysed through 0.02mol/L Tris-HCl damping fluid (pH8.0) and suitably concentrated with PEG20000 after be loaded to the DEAE Sepharose fast flow anion exchange chromatography post that abundant balance is good.The damping fluid 0.02mol/L Tris-HCl damping fluid (pH8.0) of using with balance glue post carries out wash-out, then carries out the continuous wash-out of salt ion gradient, flow velocity 30mL/h with the 0.02mol/L Tris-HCl damping fluid (pH8.0) that contains 0.1mol/L NaCl; The 5mL/ pipe, collect and have the enzymic activity part, elutes 2 protein peaks (Fig. 1) (protein peak is recorded by Ultraviolet Detector, lower same).

(3) protease activity that measure to collect liquid is found, in above-mentioned collection liquid, only contains a protease activity peak, mainly concentrates in the pipe that the 300-420min time collects.Collect the collection liquid of 300-420min, PEG20000 is loaded to the Sephadex G-150 sieve chromatography that abundant balance is good after suitably concentrating.The damping fluid 0.02mol/L Tris-HCl damping fluid (pH8.0) of using with balance glue post carries out wash-out, flow velocity 15mL/h; The 5mL/ pipe, elute a protein peak (Fig. 2).

(4) above-mentioned Peak Activity shows as single band (Fig. 3) after the SDS-PAGE electrophoresis, shows that the proteolytic enzyme after purifying is one-component, has 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 45kDa.The purification result of proteolytic enzyme is in Table 1.

Figure 2013103189063100002DEST_PATH_IMAGE002

Claims

1. method from lecanium bacterium separation and purification proteolytic enzyme is characterized in that comprising the following steps:

(a) preparation of crude enzyme liquid: use the culture medium culturing Verticillium lecanii L. lecaniiThe FJ28 bacterial strain, reach when the highest at protease activity in cultivation and fermentation liquid that to get fermented liquid centrifugal, 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, use the damping fluid dissolution precipitation; Centrifugal lysate is removed insoluble object, and supernatant liquor is loaded on dialysis tubing, after the damping fluid dialysis, then concentrates and obtains crude enzyme liquid with polyoxyethylene glycol;

DEAE Sepharose fast flow anion exchange chromatography: crude enzyme liquid is splined on the DEAE Sepharose fast flow anion exchange chromatography after the abundant balance of damping fluid equilibrate overnight, with the 0.02mol/L Tris-HCl pH of buffer that contains 0-1mol/L NaCl=8.0 damping fluids, carry out continuous wash-out again, collect and have the enzymic activity part;

Sephadex G-150 sieve chromatography: with the abundant balance Sephadex G-150 of damping fluid molecular sieve chromatography, the collection that step (b) is collected has enzymic activity partly goes up in wherein, then carries out wash-out with damping fluid; Collection has the part of enzymic activity;

In step (a) and (b), (c), damping fluid is that damping fluid is 0.02mol/L Tris-HCl pH of buffer=8.0.

2. at the separation purification method according to the described lecanium mycoproteinase of right 1, it is characterized in that in step (a), three centrifugal rotational speeds are 11000 rpm, but first centrifugal be at normal temperatures the time be 10min, latter twice is 4 ℃ of centrifugal 15min of condition.

3. according to the separation purification method of the described lecanium mycoproteinase of right 1, it is characterized in that in step (b), elution requirement is set to: flow velocity 30mL/h; The 5mL/ pipe.

4. according to the separation purification method of the described lecanium mycoproteinase of right 1, it is characterized in that in step (c), elution requirement is set to: flow velocity 15mL/h; The 5mL/ pipe.