CN101633944B - Application of trichosporon behrend for producing glutathione - Google Patents
Application of trichosporon behrend for producing glutathione Download PDFInfo
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- CN101633944B CN101633944B CN2009101842171A CN200910184217A CN101633944B CN 101633944 B CN101633944 B CN 101633944B CN 2009101842171 A CN2009101842171 A CN 2009101842171A CN 200910184217 A CN200910184217 A CN 200910184217A CN 101633944 B CN101633944 B CN 101633944B
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Abstract
The invention discloses application of trichosporon montevideense for producing glutathione while degrading phenol, aiming to provide a method for simultaneously degrading the phenol and producing the glutathione by using phenol-degradation bacteria. The phenol-degradation bacteria are trichosporon montevideense with the preservation number of CGMCC NO.3144. The method utilizes the trichosporon montevideense with the conservation number of CGMCC NO.3144 to carry out phenol biodegradation and simultaneously obtain yeast cells to extract the glutathione. The process comprises the following steps: carrying out conventional culture medium slant seed culture and at least secondary seed culture; carrying out phenol degradation by transferring a sewage treatment device or a fermentation tank; and collecting the yeast to extract the glutathione. The technology has the advantages of reducing pollutant emission, effectively utilizing resources, reducing production cost of the glutathione, and the like.
Description
Technical field
The invention belongs to biological chemical field, aim to provide the application of a kind of trichosporon Trichosporon montevideense CGMCCNo.3144 in degradation of phenol production gsh.
Background technology
Phenol is the raw material commonly used of organic synthesis, phenolic compound extensively is present in the industry waste water such as the synthetic and petrochemical complex of coking, pharmacy, papermaking, dyestuff, resol, phenol is the principal pollutant of phenolic wastewater, is about 3000 tons according to State Bureau of Environmental Protection statistics China's aldehydes matter quantity discharged in 2003.Phenol belongs to the protoplasma poisonous substance, biology had toxic action, to especially serious (the Paula M.et al. of human nervous system's harm, Isolation and characterization of phenol-degradingdenitrifying bacteria[J], Appl.Environ.Microbiol., 1998,64 (7): 2432-2438), use with regard to unable to drink when content of phenolic compounds reaches 0.005mg/L in the water, the irrigation water that contains phenol amount＞100mg/L will cause crop production reduction and withered (Li Shubin etc., the isolation identification of bacillus cereus strain Jp-A and degradation of phenol characteristic [J] thereof, Chinese Journal of Applied Ecology, 2006,15 (2): 920-924), so phenol is listed in " priority pollutants Black List in the water " that State Bureau of Environmental Protection was passed through in 1989.
The processing of phenol sewage has (Zhang Jins etc. such as physics, chemical oxidation and biodegradation method, the harm of phenolic wastewater and the application characteristic of treatment process [J], environmental engineering, 2001,83 (2): 36-37), for the wastewater containing phenol that is difficult to reclaim, biological degradation has gentleness, efficient height, applied range, processing power is big, equipment is simple and do not produce characteristics such as secondary pollution.It is multiple that bibliographical information has screened the microorganism of degradable phenol, wherein majority is acinetobacter calcoaceticus (Acinetobacter sp.) (Chen Ming, Zhang Wei, Acinetobacter calcoaceticus PHEA-2 Pyrogentisinic Acid's degradation characteristic research [J], China Environmental Science, 2001,21 (3): 226-229), pseudomonas (Pseudomonas sp.) (Arinjay Kumar et al., Biodegradaion kinetics of phenol and catecholusing Pseudomonas putida MTCC1194[J], Biochem.Eng.J., 2004,22:151-159), rhodococcus (Rhodococcus sp.) (Shen Xihui etc., the isolation identification of phenol degrading bacterium rhodococcus PNAN5 bacterial strain, degradation characteristic and open loop dioxygenase property research [J] thereof, ACTA Scientiae Circumstantiae, 2004,24:482-486), micrococci (Micrococcus sp.) (Pan Lihua etc., screening of phenol degrading bacterium and the preliminary study of degradation characteristic [J] thereof, the microbiology circular, 2003,30 (5): 78-81), skin shape trichosporon (Trichosporon cutaneum) (Andras Gaal ﹠amp; Halina Y.Neujahr, Metabolism of phenoland resorcinol in Trichosporon cutaneum[J], J.Bacteriol., 1979,137 (1): 12-31), candiyeast (Candida sp.) (Jiang Yan et al., The biodegradation of phenol at high initial concentration by theyeast Candida tropicalis[J], Biochem.Eng.J., 2005,24:243-247) and filamentous fungus (Santos V.L.﹠amp; Linardi V.R., Biodegradation of phenol by a filamentous fungi isolated from industrial effluents:identification and degradation potential[J], Process Biochem., 2004,39:1001-1006) etc., wherein the anti-phenol concentration of candiyeast can reach 2000mg/L, add the dominant microflora that in active sludge, can become Phenol-Containing Wastewater Treatment, the phenol degrading rate can reach more than 95%, do not add the low 60% (Zhu Yongguang etc. of its contrast phenol degrading rate of active sludge of bacterial classification, Sludge System is handled the biological reinforced effect [J] of phenolic waste water, use and the environmental organism journal, 2006,12 (4): 559-561), the anti-phenol concentration of skin shape trichosporon (Trichosporon cutaneum) also can reach 2000mg/L (Andras Gaal﹠amp; Halina Y.Neujahr, Metabolism of phenol and resorcinol in Trichosporon cutaneum[J], J.Bacteriol., 1979,137 (1): 12-31).
As one of a kind of petrochemical complex derived products and chemical industry main raw material, phenol has toxicity when being present among the environment with waste formation, and a large amount of bacterial metabolisms propagation can produce the placement issue of mud behind the Wastewater Treated by Activated Sludge Process phenolic waste water.Therefore, need a kind of method of wastewater treatment of the utility value of the phenol in the phenolic wastewater that can utilize better of exploitation to utilize with reasonable at present with the saving that reaches resource.
Gsh, be γ-L-glutamy-L-cysteinyl-glycine (γ-L-glutamyl-L-cysteinyl-glycine, be called for short GSH), occurring in nature mainly is present in yeast, animal livers, muscle and the blood, is a kind of important physical active substance (A.Meister ﹠amp; M.E.Anderson, Glutathione, Annu.Rev.Biochem., 1983,52:711-760), have purposes (volume such as Hu Wenduo widely aspect clinical medicine, foodstuff additive and the nutrition in sport, national essential drugs and special medicine clinical guidelines [M], Tianjin: Tianjin Scientific English Translation publishing company, 1996,537).The production method of gsh mainly contains the synthetic and fermentation method of chemical synthesis, enzyme process etc.At present, from the yeast cell that is rich in gsh, extract gsh and become main flow.
The extraction separation of gsh and preparation method have many documents (Wang Miao etc., the gsh [J] in the base exchange method separation and purification yeast cell extracting solution, Food science, 2007,28 (4): 211-217; Liu Juan etc., the progress of fermentative Production gsh [J], microbiology circular, 2006,29 (6): 72-75; Su Xiaojin etc. use ultra-filtration technique and remove proteinic research [J] in the yeast extract that is rich in gsh, foodstuffs industry science and technology, 2006,27 (2): 136-139).Because product concentration is low in the born of the same parents, the production of gsh need be cultivated a large amount of thalline, and the production method of gsh exists raw material availability low at present, problems such as cost height.In the fermentative Production gsh, it is common producing gsh with the yeast mutation bacterial strain of mutagenic treatment acquisition homoglutathion content.The research of the optimization aspect of gsh fermentation condition and feed supplement mode there is a report in that the output that improves gsh more, Li Yin etc. have studied the fermentation condition of gsh, the sugared strategy of bottle benefit is shaken in utilization makes gsh output reach 119mg/L (Li Yin etc., the high-density culture engineering bacteria is produced gsh [J], Chinese Journal of Pharmaceuticals, 1999,30 (1): 1-4).Sakato etc. utilize simultaneously and mend sugar and ethanol control method, make gsh total amount and content reach 2360mg/L and 3.7% (K.Sakato respectively; H.Tanaka, Advanced Control of Glutathione Fermentation Process[J], Biotechnol Bioeng, 1992,40 (8): 904-912.).
If extract as the auxilliary product of producing, raw material availability is low to cause having optional part aspect the expensive problem alleviating.There is report to utilize yeast cell to produce the research of S-adenosine-L-methionine(Met) and gsh (Hui Liu et al. simultaneously, Co-production of S-adenosyl-L-methionine and glutathione with the spent brew ' s yeast cells[J] .Process Biochemistry, 2004,39 (12), 1993-1997), and as the phenol of organic toxic pollutants, the auxilliary gsh that produces when if can utilize phenol degrading bacterium degradation of phenol, then can reach the purpose of killing two birds with one stone: both eliminated the phenol pollution, but the product of production high added value again gsh.
Summary of the invention
An object of the present invention is to provide the application of trichosporon Trichosporon montevideense CGMCC No.3144 in producing gsh.
Trichosporon Trichosporon montevideense CGMCC No.3144, growth has a large amount of mycelia, bacterium colony center protrusion, edge that dendroid radiation mycelia is arranged on phenol selectivity solid medium, and is non-wrinkled, oyster white, growth in about 30 ℃.Cell ellipsoid shape, bar-shaped has gemmation, and bar-shaped arthrospore is arranged.
Technical solution of the present invention is: the application of trichosporon Trichosporon montevideense CGMCC No.3144 in producing gsh.
Above-mentioned said application specifically is a kind of method of utilizing trichosporon Trichosporon montevideense CGMCC No.3144 to produce gsh, be: after trichosporon Trichosporon montevideense CGMCC No.3144 is carried out conventional medium slant seed culture and secondary seed cultivation, switching goes in the proliferated culture medium to cultivate propagation, collect the yeast thalline then and carry out the gsh extraction, make the gsh product through separation and purification.
Said proliferated culture medium is to contain the substratum that phenol concentration is not higher than 2500mg/L that contains that phenol concentration is not higher than the waste water of 2500mg/L or artificial preparation in the aforesaid method.
Wherein said artificial preparation contains the substratum that phenol concentration is not higher than 2500mg/L, can be: Sodium phosphate dibasic 6g/L, potassium primary phosphate 3g/L, sodium-chlor 0.5g/L, ammonium chloride 10g/L, sal epsom 0.24g/L, Calcium Chloride Powder Anhydrous 0.011g/L, phenol concentration be higher than 2500mg/L.
Said proliferated culture medium also contains the substratum that glucose is carbon source in the aforesaid method.As: Sodium phosphate dibasic 6g/L, potassium primary phosphate 3g/L, sodium-chlor 0.5g/L, ammonium chloride 10g/L, sal epsom 0.24g/L, Calcium Chloride Powder Anhydrous 0.011g/L, glucose concn is any.
Cultivating propagation in the aforesaid method and be in temperature is room temperature or 28-30 ℃, carries out under aerobic ventilation/stirring.
Extraction separation purifying gsh adopts ordinary method just can from yeast cell, as a concrete example, be: after the yeast washing of collection, utilize volume fraction to carry out gsh organic solvent infiltration extraction 2-3hrs for the 40-50% spirituous solution, perhaps use 80-90 ℃ of hot water to stir extracting 15min, extraction liquid is transferred pH 1.0-3.0, use the PES film room temperature ultrafiltration (0.2-0.3MPa) of molecular weight cut-off 10000 to remove behind the albumen through macroporous strong-acid cation S-9 resin-column after exchange separates, use the 5%NaOH eluant solution, the elutriant vacuum concentration, freeze-drying gets product.
Cultivate production model among the present invention for batch formula, stream adds or continuous mode carries out.
The present invention preferably uses phenolic wastewater as proliferated culture medium, as seed liquor being inserted phenol-containing wastewater treatment unit or fermentor tank, produces gsh in degradation of phenol, has significant resources conservation advantage.
Trichosporon montevideense CGMCC No.3144 bacterial strain, anti-phenol concentration reaches 2500mg/L, by with the phenol in the phenolic wastewater as carbon source, cell is bred, born of the same parents' glutathion inside output can be up to 1.1-1.2%, and the gsh volumetric production can increase with cell proliferation.From cell, extract gsh and isolation technique by using, can carry out the production of gsh.The present invention produces gsh by utilizing the phenol in the phenolic wastewater as carbon source, not only can turn waste into wealth the phenol in the phenolic wastewater, and greatly reduce the production cost of gsh.
By following accompanying drawing and detailed description, the feature and advantage of these aspects of the present invention will be more clear.
Description of drawings
Fig. 1 is batch the formula phenol degrading and the gsh production kinetics of Trichosporon montevideense bacterial strain.
Fig. 2 is that Trichosporon montevideense bacterial strain utilizes glucose to criticize formula growth and gsh production kinetics.
Fig. 3 is that Trichosporon montevideense bacterial strain adds degraded phenol production gsh output under the pattern at stream.
Fig. 4 is phenol degrading and the gsh output of Trichosporon montevideense bacterial strain under the different concns phenol concentration.
Among the present invention, unless specialize, term " phenol degrading bacterium " is meant phenol to be the microorganism strains that the sole carbon source and the energy are grown, and promptly bacterial strain can be that carbonic acid gas and water are removed with phenol degrading in propagation or metabolic process.
Among the present invention, unless specialize, term " phenolic wastewater " or " phenolic waste water " are meant that what produce in the commercial run is to mainly contain the sewage that organic pollutants is formed with phenol.
Among the present invention, unless specialize, term " carbon source " is meant the available carbonaceous material of microorganism, and microorganism utilizes the carbonaceous material of synthetic self cell of this carbonaceous material.Term " energy " is that oxidable its of microorganism cells produces electronics, produces the material that ATP utilizes for cell in electron transfer process.
Embodiment
(1) bacterial strain
A strain yeast belong phenol degrading bacterium that obtains is screened in this laboratory from oil field sludge, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on June 26th, 2009, preserving number CGMCC No.3144, its anti-phenol concentration reaches 2500mg/L, reach more than 99% in the degradation rate 24hrs to the phenol of 1200mg/L, residual concentration behind this strains for degrading phenol can be lower than 0.5mg/L, qualified discharge.
(2) substratum
Prepare following substratum with deionized water.
Slant medium: Tryptones 20g/L, yeast extract paste 10g/L, sodium-chlor 20g/L, agar powder 2.0%
Seed culture medium: Tryptones 5g/L, yeast extract paste 5g/L, sodium-chlor 10g/L, glucose 10g/L.
Manually contain the phenol substratum: Sodium phosphate dibasic 6g/L, potassium primary phosphate 3g/L, sodium-chlor 0.5g/L, ammonium chloride 10g/L, sal epsom 0.24g/L, Calcium Chloride Powder Anhydrous 0.011g/L, phenol concentration is adjusted according to experiment.
Dextrose culture-medium: the above-mentioned phenol that manually contains in the phenol substratum is replaced by glucose.
(3) reduced glutathion test (Hui Liu et al., Co-production of S-adenosyl-L-methionine andglutathione with the spent brew ' s yeast cells[J] .Process Biochemistry, 2004,39 (12), 1993-1997)
Use ALLOXAN (tetraoxypyrimidine) reagent method, reduced glutathion (GSH) acts on ALLOXAN (tetraoxypyrimidine) under experiment condition, and the material that reaction generates has the highest absorption peak in ultraviolet region, 305nm place, uses ultraviolet spectrophotometer to measure.
(4) phenol concentration test: the direct light-intensity method of use 4-aminoantipyrene (State Bureau of Environmental Protection " water and effluent monitoring analytical procedure " editorial committee, " water and effluent monitoring analytical procedure " [M]. Beijing: the China Environmental Science Press, 1997,408-410).
(5) biomass test: adopt dry weight and OD
600Carry out, nutrient solution 5ml carries out centrifugal, and the lower floor thalline uses to be placed in the baking oven 80 ℃ behind the deionized water wash three times and dry to constant weight and survey dry cell weight; Nutrient solution 5ml carries out centrifugal, and supernatant liquor is tested the light absorption value of 600nm place nutrient solution as reference.
Yeast culture: bacterial classification inoculation seed culture medium, place 30 ℃, carry out shaking culture in the shaking table of 180r/min, get the certain volume seed culture fluid after cultivating 24hrs, centrifugal (4000rpm, down together), thalline is transferred in the artificial phenol substratum of the about 1200mg/L of phenol concentration after adopting the physiological saline washing centrifugal, and inoculum size 30% mixes, place 30 ℃, in the shaking table of 180r/min or inoculation 15L ferment at constant temperature jar, 28 ℃-30 ℃ of temperature, mixing speed 200-500rpm, air flow 0.5-1.5vvm cultivates 24hrs.Get nutrient solution 5ml and carry out centrifugally, contain the phenol substratum and get supernatant liquid and detect phenol concentration; Behind lower floor's thalline use deionized water wash three times, add equal-volume 45% ethanol and extract 3hrs, the concentration of analysis-reduction type gsh down for 30 ℃.The degraded of phenol and gsh output are as shown in Figure 1.Along with phenol degrading, cell is bred gradually, and gsh output increases gradually.
Gsh separation and purification: after cell centrifugation is collected, washed, use 40% spirituous solution extraction 2hrs.Centrifugal then 4000rpm, supernatant liquor is transferred pH 1.0-3.0, after using the PES membrane ultrafiltration (0.2-0.3MPa) of molecular weight cut-off 10000, last macroporous strong-acid cation resin S-9 ion exchange column, adopt the 5%NaOH eluant solution, behind the elutriant vacuum concentration, get white powder after the lyophilize, make GSH purity 65-75%, yield is more than 60%.
Purification procedures is with embodiment 1, and just cell extraction is used 50% spirituous solution extraction 2hrs instead, GSH purity 68-77%, and yield is more than 62%.
Purification procedures is with embodiment 1, and just cell extraction is used 80-90 ℃ of hot water instead and stirred extracting 15min, GSH purity 70-80%, and yield is more than 65%.
Purification procedures is with embodiment 3, GSH purity 70-80%, and yield is more than 65%.
Purification procedures is with embodiment 3, GSH purity 50-60%, and yield is more than 50%.
Phenol is as carbonaceous material, the ability that provides the carbon source and the energy to make microorganism cells propagation for microorganism is provided, Pyrogentisinic Acid's degradation bacteria glutathion inside cell fully utilizes has the advantage that alleviates resource consumption and reduce the gsh production cost, make that simultaneously the pollution of phenol in wastewater is eliminated, have the advantage of killing two birds with one stone.Produce gsh when utilizing the phenol degrading bacterium to carry out phenol degrading, belong to the crossing domain of biological fermentation engineering and environmental engineering, have the energy-saving and emission-reduction advantage.In today of resource worsening shortages, the processing of phenolic wastewater should take in from the comprehensive utilization aspect, in the time of to the not high phenolic wastewater biodegrade of recovery value, a large amount of bacterial metabolisms can be bred after utilizing phenol, the placement issue of mud will be produced, as fully utilizing thalline, exploitation high added value biological product has the advantage of important energy-saving and emission-reduction.
Claims (7)
1. trichosporon
Trichosporon montevideenseThe application of CGMCC No. 3144 in producing gsh.
2. according to the described application of claim 1, it is characterized in that detailed process is: with trichosporon
Trichosporon montevideenseAfter CGMCC No. 3144 carried out conventional medium slant seed culture and secondary seed cultivation, switching went in the proliferated culture medium to cultivate propagation, collected the yeast thalline then and carried out the gsh extraction, made the gsh product through separation and purification.
3. according to the described application of claim 2, it is characterized in that: wherein said proliferated culture medium is the substratum that phenol concentration is not higher than 2500mg/L that contains of artificial preparation.
4. application according to claim 3, it is characterized in that, wherein said artificial preparation contains the substratum that phenol concentration is not higher than 2500mg/L, wherein: Sodium phosphate dibasic 6g/L, potassium primary phosphate 3g/L, sodium-chlor 0.5g/L, ammonium chloride 10g/L, sal epsom 0.24g/L, Calcium Chloride Powder Anhydrous 0.011g/L, phenol concentration is not higher than 2500mg/L.
5. application according to claim 2 is characterized in that: wherein said proliferated culture medium is to contain the substratum that glucose is carbon source.
6. application according to claim 5, it is characterized in that: wherein said glucose is in the substratum of carbon source: Sodium phosphate dibasic 6g/L, potassium primary phosphate 3g/L, sodium-chlor 0.5g/L, ammonium chloride 10g/L, sal epsom 0.24g/L, Calcium Chloride Powder Anhydrous 0.011g/L, glucose concn is any.
7. application according to claim 2 is characterized in that, culture propagation temperature is room temperature or 28 ℃-30 ℃, carries out under aerobic ventilation/stirring.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1468964A (en) * | 2002-07-17 | 2004-01-21 | 中国科学院微生物研究所 | Dermoid trichocyst yeast fermentation process of producing pyruvic acid |
| CN1844407A (en) * | 2005-04-06 | 2006-10-11 | 北京化工大学 | Method for simultaneous production of ergosterol and glutathione by yeast fermentation |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1468964A (en) * | 2002-07-17 | 2004-01-21 | 中国科学院微生物研究所 | Dermoid trichocyst yeast fermentation process of producing pyruvic acid |
| CN1844407A (en) * | 2005-04-06 | 2006-10-11 | 北京化工大学 | Method for simultaneous production of ergosterol and glutathione by yeast fermentation |
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