CN103554214A - Extraction method of extracellular polymeric substance for keeping mycelial morphology of phanerochaete chrysosporium - Google Patents
Extraction method of extracellular polymeric substance for keeping mycelial morphology of phanerochaete chrysosporium Download PDFInfo
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- CN103554214A CN103554214A CN201310503190.4A CN201310503190A CN103554214A CN 103554214 A CN103554214 A CN 103554214A CN 201310503190 A CN201310503190 A CN 201310503190A CN 103554214 A CN103554214 A CN 103554214A
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Abstract
The invention discloses an extraction method of an extracellular polymeric substance for keeping a mycelial morphology of phanerochaete chrysosporium. The method comprises the following steps: (1) pretreatment, namely mixing a glutaraldehyde solution with the phanerochaete chrysosporium, carrying out heat preservation, filtering and separating the insulated mixture, rinsing by sterile water, so as to obtain insulated phanerochaete chrysosporium thalli; mixing the insulated phanerochaete chrysosporium thalli with the sterile water, carrying out heat preservation, so as to obtain a mixed liquor; (2) centrifugal treatment, namely carrying out high-speed centrifugal separation on the mixed liquor in the step (1) to obtain supernate; (3) dialyzing, namely putting the supernate obtained in the step (2) into a dialysis bag of which the molecular entrapment quantity is 3500-7000kDa to dialyze, so as to obtain the extracellular polymeric substance. The extraction method of the extracellular polymeric substance for keeping the mycelial morphology of the phanerochaete chrysosporium disclosed by the invention has the characteristics that cell rupture and bacteria hydrolysis are not caused; the original forms of the thalli can be kept; the extracellular polymeric substance also can be more completely extracted.
Description
Technical field
The present invention relates to extracellular polymeric and extract field, relate in particular to a kind of extracting method that keeps the extracellular polymeric of Phanerochaete chrysosporium thalli morphology.
Background technology
Phanerochaete chrysosporium, due to the processing power to xenobiontics of its uniqueness, can be processed the pollution of Persistent organic pollutants, also can be applied to the improvement of heavy metal contamination, and it becomes the study hotspot of environmental organism treatment technology gradually.Extracellular polymeric (EPS) is the matrix with stickiness and provide protection that a class is produced by the metabolism of microorganism own, is conventionally attached to microorganism cells periphery.Extracellular polymeric is mainly comprised of polysaccharide and protein substance, keeping microorganism cells form, interconnecting between reacting environment, organism being provided for extracellular enzyme, is especially utilizing the important roles such as microbiological treatment pollutent.Therefore the extracellular polymeric that, effectively, intactly extracts Phanerochaete chrysosporium is significant for advancing Phanerochaete chrysosporium to process the research of the outer mechanism of action of born of the same parents of pollutent.For more existing researchs of extraction of the extracellular polymeric of Phanerochaete chrysosporium, but all in all, all there is damage to a certain degree in Phanerochaete chrysosporium thalline in the leaching process of extracellular polymeric, as cell rupture, thalline hydrolysis and torsional deformation etc.
Summary of the invention
The technical problem to be solved in the present invention is to overcome the deficiencies in the prior art, provides a kind of step simple, feasible, effective, does not cause cell rupture, thalline hydrolysis, keeps the extracting method of the extracellular polymeric of Phanerochaete chrysosporium thalli morphology.
For solving the problems of the technologies described above, the technical scheme that the present invention proposes is a kind of extracting method that keeps the extracellular polymeric of Phanerochaete chrysosporium thalli morphology, comprises the following steps:
(1) pre-treatment: mix glutaraldehyde solution and Phanerochaete chrysosporium thalline, the concentration of described glutaraldehyde solution is preferably 0.01～0.012g/mL, in the Phanerochaete chrysosporium thalline of every 1g weight in wet base, the addition of glutaraldehyde solution is preferably 80～100mL, insulation, the holding temperature of glutaraldehyde solution and Phanerochaete chrysosporium thalline is 0～4 ℃, and soaking time is 4～6h; By the mixture filtering separation after above-mentioned insulation, aseptic water washing, obtains the Phanerochaete chrysosporium thalline after insulation; Phanerochaete chrysosporium thalline after insulation and sterilized water are mixed, insulation, the Phanerochaete chrysosporium thalline after insulation and the holding temperature of sterilized water are 30～35 ℃, soaking time is 1～2h, obtains mixed solution.
(2) centrifugal treating: the mixed solution that step (1) is obtained carries out high speed centrifugation separation, obtains supernatant liquor, and the rotating speed of centrifugal treating is controlled at 10000～12000rpm, the time of centrifugal treating is 10～12min.
(3) dialysis: the supernatant liquor that step (2) is obtained is placed in the dialysis tubing that molecular retention amount is 3500～7000kDa and dialyses, and obtains extracellular polymeric, dialysis temperature is 0～4 ℃, dialysis time is 24～48h.It is 3500～7000kDa that the dialysis tubing specification of using in the present invention is preferably molecular weight cut-off, can reach the object in the chemical interference source in decontamination of biological chemical analysis process, make again dialysis procedure reach minimum to the impact of extracellular polymeric extracted amount, to extract more extracellular polymeric.
The invention described above extracting method in, the preparation method of Phanerochaete chrysosporium thalline can adopt following steps to make, by Phanerochaete chrysosporium (Phanerochaete chrysosporium) bacterial strain (as Phanerochaete chrysosporium (Phanerochaete chrysosporium) BKMF-1767 bacterial strain, in the center preservation of Chinese Typical Representative culture collection, deposit number is CCTCC AF96007, commercial) be inoculated in potato nutrient agar, under the temperature condition of 25～30 ℃, cultivate 5～7 days; Spore on the above-mentioned potato nutrient agar of scraping is dissolved in to be made spore suspension and is inoculated into liquid nutrient medium in sterilized water, under the speed conditions of the temperature of 30～35 ℃, 120～150rpm, shaking culture is 5～7 days; Thalline after above-mentioned shaking culture and nutrient solution are carried out to filtering separation, and with aseptic water washing, obtain Phanerochaete chrysosporium thalline.
The invention described above extracting method in, the suspension that the spore suspension spore that to be Phanerochaete chrysosporium produce after 5～7 days go down to posterity cultivated is made.
The invention described above extracting method in, the thalline after shaking culture is that spore suspension grows to the thalline of logarithmic phase middle and later periods through the shaking culture of 5～7 days.
In step (1), the washing time of sterilized water is 1～2 time, and the object of aseptic water washing is to remove and is adsorbed on glutaraldehyde solution unnecessary on Phanerochaete chrysosporium thalline, avoids its mensuration to all kinds of materials in extracellular polymeric to exert an influence.
In Phanerochaete chrysosporium yeast culture process, the washing time of sterilized water is 1～2 time, the object of aseptic water washing is to remove and is adsorbed on the liquid nutrient medium on Phanerochaete chrysosporium thalline, avoids glucide wherein to exert an influence to the extracellular polymeric extracting.
Compared with prior art, the invention has the advantages that:
The present invention has utilized the effect of glutaraldehyde solution protection thalli morphology; first with glutaraldehyde solution, Phanerochaete chrysosporium thalline to be carried out to pre-treatment specifically; extract again extracellular polymeric, solved the impaired problems of thalline such as the cell rupture easily causing, thalline hydrolysis and torsional deformation that exist in the method for existing extraction extracellular polymeric.The present invention not only has provide protection to Phanerochaete chrysosporium thalli morphology, can keep its original form, and can also be more complete, maximum extract its extracellular polymeric.
Accompanying drawing explanation
Fig. 1 is the microtexture scanning electron microscope (SEM) photograph of the Phanerochaete chrysosporium thalline being left intact of the present invention.
Fig. 2 be Phanerochaete chrysosporium thalline through sterilized water pre-treatment, extract the microtexture scanning electron microscope (SEM) photograph after extracellular polymeric.
Fig. 3 be Phanerochaete chrysosporium thalline through phosphate buffered saline buffer (PBS) pre-treatment, extract the microtexture scanning electron microscope (SEM) photograph after extracellular polymeric.
Fig. 4 be Phanerochaete chrysosporium thalline through glutaraldehyde solution pre-treatment, extract the microtexture scanning electron microscope (SEM) photograph after extracellular polymeric.
Fig. 5 is the column comparison diagram of protein, carbohydrate and DNA content in the extracellular polymeric solution that extracts respectively after sterilized water, phosphate buffered saline buffer (PBS), glutaraldehyde solution pre-treatment of Phanerochaete chrysosporium thalline.
Embodiment
By specific embodiment and Figure of description, the present invention will be described in more detail below.Embodiment is only to a kind of explanation of the present invention, and is not construed as limiting the invention.
Embodiment 1:
By Phanerochaete chrysosporium (Phanerochaete chrysosporium) bacterial strain (as BKMF-1767 bacterial strain, the center preservation of Chinese Typical Representative culture collection, deposit number is CCTCC AF96007, commercial) be inoculated in potato nutrient agar, under the temperature condition of 25 ℃, cultivate 7 days; Spore on the above-mentioned potato nutrient agar of scraping is dissolved in to be made spore suspension and is inoculated into liquid nutrient medium in sterilized water, under the speed conditions of the temperature of 30 ℃, 150rpm, shaking culture is 5 days; Thalline after above-mentioned shaking culture and nutrient solution are carried out to filtering separation, and with aseptic water washing, obtain Phanerochaete chrysosporium thalline.
Get the Phanerochaete chrysosporium thalline of 50mL, 0.01g/mL glutaraldehyde solution mixing 0.5g weight in wet base, insulation, wherein holding temperature is 4 ℃, soaking time is 5h; By the mixture filtering separation after insulation, use aseptic water washing 2 times, then join in sterilized water water bath heat preservation 1h at 30 ℃, the Phanerochaete chrysosporium thalline after being incubated and the mixed solution of sterilized water; Under the condition that is 10000rpm at rotating speed by above-mentioned mixed solution, carry out high speed centrifugation separation, centrifugation time is 10min, obtains supernatant liquor; Dialysis tubing dialysis 48h by supernatant liquor obtained above through 3500kDa, dialysis temperature is 4 ℃, obtains the extracellular polymeric of Phanerochaete chrysosporium.
Embodiment 2:
By Phanerochaete chrysosporium (Phanerochaete chrysosporium) bacterial strain (as BKMF-1767 bacterial strain, the center preservation of Chinese Typical Representative culture collection, deposit number is CCTCC AF96007, commercial) be inoculated in potato nutrient agar, under the temperature condition of 30 ℃, cultivate 5 days; Spore on the above-mentioned potato nutrient agar of scraping is dissolved in to be made spore suspension and is inoculated into liquid nutrient medium in sterilized water, under the speed conditions of the temperature of 35 ℃, 120rpm, shaking culture is 7 days; Thalline after above-mentioned shaking culture and nutrient solution are carried out to filtering separation, and with aseptic water washing, obtain Phanerochaete chrysosporium thalline.
Get the Phanerochaete chrysosporium thalline of 100mL, 0.012g/mL glutaraldehyde solution mixing 1.25g weight in wet base, insulation, wherein holding temperature is 0 ℃, soaking time is 6h; By the mixture filtering separation after insulation, use aseptic water washing 1 time, then join in sterilized water water bath heat preservation 2h at 35 ℃, the Phanerochaete chrysosporium thalline after being incubated and the mixed solution of sterilized water; Under the condition that is 12000rpm at rotating speed by above-mentioned mixed solution, carry out high speed centrifugation separation, centrifugation time is 12min, obtains supernatant liquor; Dialysis tubing dialysis 24h by supernatant liquor obtained above through 7000kDa, dialysis temperature is 0 ℃, obtains the extracellular polymeric of Phanerochaete chrysosporium.
Embodiment 3:
By Phanerochaete chrysosporium (Phanerochaete chrysosporium) bacterial strain (as BKMF-1767 bacterial strain, the center preservation of Chinese Typical Representative culture collection, deposit number is CCTCC AF96007, commercial) be inoculated in potato nutrient agar, under the temperature condition of 25 ℃, cultivate 6 days; Spore on the above-mentioned potato nutrient agar of scraping is dissolved in to be made spore suspension and is inoculated into liquid nutrient medium in sterilized water, under the speed conditions of the temperature of 32 ℃, 140rpm, shaking culture is 6 days; Thalline after above-mentioned shaking culture and nutrient solution are carried out to filtering separation, and with aseptic water washing, obtain Phanerochaete chrysosporium thalline.
Get the Phanerochaete chrysosporium thalline of 150mL, 0.01g/mL glutaraldehyde solution mixing 1.5g weight in wet base, insulation, holding temperature is 2 ℃, soaking time is 4h; By the mixture filtering separation after insulation, use aseptic water washing 2 times, then join in sterilized water water bath heat preservation 1h at 35 ℃, the Phanerochaete chrysosporium thalline after being incubated and the mixed solution of sterilized water; Under the condition that is 11000rpm at rotating speed by above-mentioned mixed solution, carry out high speed centrifugation separation, centrifugation time is 11min, obtains supernatant liquor; Dialysis tubing dialysis 36h by supernatant liquor obtained above through 5000kDa, dialysis temperature is 2 ℃, obtains the extracellular polymeric of Phanerochaete chrysosporium.
Below by comparative analysis, illustrate that the present invention extracts the advantage of extracellular polymeric.
The Phanerochaete chrysosporium thalline being left intact of take is reference examples, then adopt respectively 0.01g/mL glutaraldehyde solution, sterilized water, the phosphate buffered saline buffer of 0.01M and pH=7.0, original Phanerochaete chrysosporium thalline is incubated to processing, other conditions are identical with embodiment 1, obtain embodiment 1, comparative example 1, comparative example 2.
The Phanerochaete chrysosporium thalline that reference examples is obtained adds respectively in sterilized water with the Phanerochaete chrysosporium thalline that embodiment 1, comparative example 1, comparative example 2 obtain after high speed centrifugation is separated, make respectively to organize Phanerochaete chrysosporium thalline and recover shape, then put into refrigerator at-20 ℃ of freezing 5h, then carry out respectively vacuum freezedrying for weighing and the scanning electron microscope analysis of dry cell weight.Under environmental scanning electronic microscope, observe the microtexture of above-mentioned four kinds of Phanerochaete chrysosporium thalline, as shown in Fig. 1～4.As shown in Figure 1, the Phanerochaete chrysosporium thalline that does not extract extracellular polymeric is full cylinder thread (reference examples).As shown in Figure 2, there is serious distortion, shrivelled, distortion (comparative example 1) in the Phanerochaete chrysosporium thalline after extracellular polymeric is extracted in sterilized water pre-treatment.As shown in Figure 3, than sterilized water pre-treatment, extract the thalline after extracellular polymeric, the impact that phosphate buffered saline buffer (PBS) produces Phanerochaete chrysosporium thalli morphology is relatively smaller, but thalline still exists torsional deformation (comparative example 2) to a certain degree.As shown in Figure 4; Phanerochaete chrysosporium thalline after extracellular polymeric is extracted in glutaraldehyde solution pre-treatment still keeps relatively full cylinder filamentary texture (embodiment 1); this explanation is in the leaching process of the extracellular polymeric of Phanerochaete chrysosporium; glutaraldehyde solution pre-treatment can play a protective role for Phanerochaete chrysosporium thalli morphology, can make thalline keep original form.
Finally, with anthrone-H
2sO
4colorimetry, with glucose preparation standard curve, the content of glucide in the Phanerochaete chrysosporium ecto polymer of mensuration embodiment 1, comparative example 1 and comparative example 2 preparations; Use Xylene Brilliant Cyanine G method, with bovine serum albumin preparation standard curve, the content of protein in Phanerochaete chrysosporium ecto polymer prepared by mensuration the present embodiment; The content of DNA in Phanerochaete chrysosporium ecto polymer prepared by optical density method mensuration the present embodiment.Measuring result as shown in Figure 5.
DNA column by Fig. 5 relatively can be found out, in three kinds of pretreatment processs, in the extracellular polymeric extract of sterilized water and phosphate buffered saline buffer (PBS) pre-treatment thalline, DNA all detected, this explanation Phanerochaete chrysosporium in the process of these two kinds of pre-treatment extraction extracellular polymerics has produced cell rupture to a certain degree, thalline hydrolysis, wherein so that sterilized water is pretreated, have the greatest impact, the DNA of 15.3mg/g detected, the DNA that contains 15.3mg in the extracellular polymeric (EPS) that the thalline that expression dry weight is 1g extracts.Secondly be phosphate buffered saline buffer (PBS) pre-treatment, the DNA of 12.1mg/g detected.The pretreated extracting method of glutaraldehyde solution does not detect DNA, and this has confirmed that the extracting method of the pretreated extracellular polymeric of glutaraldehyde solution has solved the problems such as the cell rupture that occurs in extracting method in the past, thalline hydrolysis equally further.In the extracellular polymeric that three kinds of pre-treatment extracting method extract, the detected result of protein and sugar class can be found out, in embodiment 1, comparative example 1 and comparative example 2, the protein content being more or less the same detected respectively, be respectively 30.8mg/g, 34.3mg/g, 36.5mg/g; What difference was large is the contents of saccharide extracting, glutaraldehyde solution pre-treatment extraction method is extracted the glucide (416.6mg/g) of maximum, next is sterilized water pre-treatment extraction method (223.2mg/g), is finally phosphate buffered saline buffer (PBS) pre-treatment extraction method (179.7mg/g).As can be seen here, the present invention's glutaraldehyde solution pre-treatment extraction method used not only has provide protection for Phanerochaete chrysosporium thalli morphology, and can also more completely extract extracellular polymeric.
The above is only the preferred embodiment of the present invention, and protection scope of the present invention is also not only confined to above-described embodiment, and all technical schemes belonging under thinking of the present invention all belong to protection scope of the present invention.Be noted that for those skilled in the art, improvements and modifications under the premise without departing from the principles of the invention, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (8)
1. an extracting method that keeps the extracellular polymeric of Phanerochaete chrysosporium thalli morphology, comprises the following steps:
(1) pre-treatment: mix glutaraldehyde solution and Phanerochaete chrysosporium thalline, insulation; By the mixture filtering separation after above-mentioned insulation, aseptic water washing, obtains the Phanerochaete chrysosporium thalline after insulation; Phanerochaete chrysosporium thalline after insulation and sterilized water are mixed, and insulation, obtains mixed solution;
(2) centrifugal treating: the mixed solution that step (1) is obtained carries out high speed centrifugation separation, obtains supernatant liquor;
(3) dialysis: the supernatant liquor that step (2) is obtained is placed in the dialysis tubing that molecular retention amount is 3500～7000kDa and dialyses, and obtains extracellular polymeric.
2. according to the extracting method described in claims 1, it is characterized in that, the concentration of described glutaraldehyde solution is 0.01～0.012g/mL.
3. according to the extracting method described in claims 1 or 2, it is characterized in that, in the Phanerochaete chrysosporium thalline of every 1g weight in wet base, the addition of glutaraldehyde solution is 80～100mL.
4. according to the extracting method described in claims 1 or 2, it is characterized in that, the holding temperature of glutaraldehyde solution and Phanerochaete chrysosporium thalline is 0～4 ℃.
5. according to the extracting method described in claims 1 or 2, it is characterized in that, the soaking time of glutaraldehyde solution and Phanerochaete chrysosporium thalline is 4～6h.
6. according to the extracting method described in claims 1 or 2, it is characterized in that, the Phanerochaete chrysosporium thalline in described step (1) after insulation and the holding temperature of sterilized water are 30～35 ℃, and soaking time is 1～2h.
7. according to the extracting method described in claims 1 or 2, it is characterized in that, the rotating speed of the high speed centrifugation separation of step (2) is controlled at 10000～12000rpm, and centrifugation time is 10～12min.
8. according to the extracting method described in claims 1 or 2, it is characterized in that, the dialysis temperature of step (3) is 0～4 ℃, and dialysis time is 24～48h.
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Cited By (2)
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| CN104845456A (en) * | 2015-04-28 | 2015-08-19 | 北京建筑大学 | Environment-friendly metal anti-corrosive paint and preparation method as well as application thereof |
| CN110257454A (en) * | 2019-06-19 | 2019-09-20 | 桂林理工大学 | Exocellular polysaccharide flocculant and its preparation method and application |
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| CN104845456A (en) * | 2015-04-28 | 2015-08-19 | 北京建筑大学 | Environment-friendly metal anti-corrosive paint and preparation method as well as application thereof |
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| CN110257454A (en) * | 2019-06-19 | 2019-09-20 | 桂林理工大学 | Exocellular polysaccharide flocculant and its preparation method and application |
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