CN103540535A - Intracellular lipase producing strain as well as application, screening method and using method thereof - Google Patents
Intracellular lipase producing strain as well as application, screening method and using method thereof Download PDFInfo
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Abstract
The invention discloses an intracellular lipase producing strain, an application, a screening method and a using method thereof. The preservation number of the strain is CCTCC (China Center For Type Culture Collection) No. M2012538. The intracellular lipase producing strain is separated from oil-rich soil, is used in an organic phase to react for high selectively synthesizing 1, 3-diglyceride, the reaction selectivity of the intracellular lipase producing strain is high, the content of a product-1, 3-diglyceride in diglyceride reaches above 80%, enzyme does not need to be purified, the intracellular lipase producing strain can be directly used for biological catalysis, and the reaction process is green and healthy.
Description
Technical field
The present invention relates to bio-science field, particularly a kind of intracellular lipase produces bacterium and application and screening method and using method.
Background technology
1,3-triglyceride is 1 of glycerine, the triglyceride that 3 hydroxyls and lipid acid are connected to form, it is the natural component of grease, its mouthfeel, color and luster, local flavor and common triglyceride level are as good as, the more important thing is the Absorption And Metabolism mode of 1,3-DAG in human body and the difference of triglyceride level and 1,2-triglyceride.Triglyceride level and 1,2-triglyceride are after digestive ferment digestion and generate mono-glycerides and free fatty acids, and the two absorption enters after blood, very major part synthetic glycerine three esters again.And 1,3-DAG generates glycerine and free fatty acids after digestive ferment effect, the two changes into energy in vivo.So edible 1,3-DAG can reduce interior fat, suppress body weight, increase, reduce the effects such as blood fat, because of but desirable healthy grease.As the substitute of natural fats and oils, 1,3-DAG can effectively prevent mainly because of the fat various diseases causing, as hypertension, coronary heart disease, cerebrovascular disease, diabetes, hyperlipidaemia, hyperuricemia, cholecystitis etc.And 1,3-DAG is also further applied to containing lipid food, composite structure triglyceride level, as the synthetic raw material of medicine intermediate etc.So the research to 1,3-DAG is paid close attention to widely.
Though 1,3-DAG is the natural component of grease, its content is very low, so open up new way, to prepare 1,3-DAG extremely important.The method of synthetic 1,3-DAG has chemical method and biological process.With chemical method, obtain 1,3-DAG, its shortcoming is to use poisonous and hazardous catalyzer, unfriendly to environment, with people's day by day craving for mutually and conflicting green food.With biological Lipids Enzymatic, obtain 1,3-DAG because of its green health, meet the day by day pursuit of people to high-quality food, by a trend that is development.And the domestic report that does not also have the synthetic 1,3-DAG of Whole-cell lipase nonaqueous phase.Produce intracellular lipase and can be used as Whole-cell lipase catalyzer, can save purification and the immobilization of enzyme, therefore can simplify technique cost-saving, be conducive to promote its commercial application.
Summary of the invention
Technical problem to be solved by this invention is: provide a kind of intracellular lipase to produce bacterium and application and screening method and using method, it is for the synthetic 1,3-DAG of lipase organic phase, and synthesis mode is simple, and the object selectivity of product of reaction is high.
Of the present inventionly be achieved in that intracellular lipase produces bacterium, described bacterial strain deposit number is CCTCC No. M2012538.
Intracellular lipase produces bacterium in the application of high selectivity 1,3-DAG.
Intracellular lipase produces the screening method of bacterium, first from rich oil soil sampling, intracellular lipase in sample is produced to bacterium after enrichment culture, adopting sweet oil emulsion is substrate, after enrichment culture, dilution spread is selected on substratum solid rhodamine B, and the microorganism of usining is decomposed the ability of sweet oil as primary dcreening operation index, and the bacterium colony of picking tool fluorescent ring is three ride purifying on flat board, after purifying, picking list bacterium colony moves and connects slant medium preservation, thereby realizes primary dcreening operation; Shake flask fermentation sieves again afterwards, and the synthetic thin layer result of the zymin making with microbial cells catalysis 1,3-DAG in organic phase is for sieving again for the first time index; The synthetic high performance liquid phase of the zymin making with microbial cells again catalysis 1,3-DAG in organic phase detects yield for sieving again for the second time index.
The enrichment medium that described enrichment culture adopts, calculates by weight, comprises (NH
4)
2sO
40.05～0.5 part, NaNO
30.01～0.2 part, 0.05 ~ 0.6 part of NaCl, MgSO
40.01～0.2 part, K
2hPO
40.001～0.03 part, 0.5～2 part, sweet oil, pH is 6～8.
Described solid rhodamine B is selected substratum, and concentration is calculated by mass percentage, comprises glucose 0.2～2%, Zulkovsky starch 0.3～3%, peptone 0.1～4%, Yeast diffusion juice 0.1～5%, K
2hPO
40.01～1%, MgSO
47H
2o 0.01～1%, NaNO
30.1～4% and agar powder 2%, pH is 6～8; In above-mentioned mixing solutions, add polyvinyl alcohol-sweet oil emulsion 9～14ml, sterilizing, is cooled to that after 60 ℃, to add the concentration of filtration sterilization be 1～3mg/ml rhodamine B, 1～4ml.
Intracellular lipase produces the using method of bacterium, will after thalline lyophilize, make Whole-cell lipase zymin, and Whole-cell lipase zymin is reacted to high selectivity 1,3-DAG for organic phase; The glycerolysis reaction that organic phase reaction is triglyceride level and glycerine, or the ester synthesis reaction of glycerine and lipid acid.Also can be used for that other ester of catalysis is synthetic, ester hydrolysis, transesterify etc.
Described glycerolysis reaction system is comprised of Whole-cell lipase zymin, triglyceride level, glycerine, organic solvent, the concentration of Whole-cell lipase zymin is 20～60 mg/mL, the mol ratio of triglyceride level and glycerine is 4:1～1:2, triglyceride concentration is 10～300 mmol/L, mixed solvent is normal hexane and the part of mixing of the trimethyl carbinol, and the mol ratio of normal hexane and the trimethyl carbinol is 49:1～20:1,30～50 ℃ of temperature of reaction, rotating speed 100～250 r/min, times 12～48 h.
In order to verify technique effect of the present invention, carried out following experiment:
the collection of sample:
In 2011, from Baiyun District, Guiyang City, Guizhou Province, oil pressing factory collected sample.
enrichment culture:
1g sample is dissolved in 10mL distilled water, fully shakes up, 30 ℃, a hour shaking culture of 180r/min, after solids precipitation, get supernatant liquid 5mL in the 250mL triangular flask of 50mL liquid enrichment medium is housed, 30 ℃, after 180r/min shaking culture 2 days, enrichment medium is fully shaken up, get 5mL and be transferred in new enrichment medium, carry out secondary enrichment, under similarity condition, shaking culture is 2 days, carries out enrichment culture for the third time.
enrichment medium(mass percent %):
(NH
4)
2sO
40.05～0.5, NaNO
30.01～0.2, NaCl 0.05～0.6, MgSO
40.01～0.2, K
2hPO
40.001～0.03, sweet oil 0.5～2.pH?6～8。
primary dcreening operation:
The enrichment bacterium liquid obtaining through above-mentioned cultivation is diluted to suitable multiple with sterilized water, getting 0.1mL bacterium liquid coats on solid rhodamine B selection substratum, cultivate 1 ~ 2 day for 30 ℃, picking has the bacterium colony three ride purifying of fluorescent ring under 254nm luminescent lamp, is seeded in slant tube and preserves afterwards.
Solid rhodamine B is selected substratum (mass percent %): glucose 0.2～2, Zulkovsky starch 0.3～3, peptone 0.1～4, Yeast diffusion juice 0.1～5, K
2hPO
40.01～1, MgSO
47H
2o 0.01～1, NaNO
30.1～4, agar powder 2, pH6～8; Polyvinyl alcohol (PVA)-sweet oil emulsion 9～14ml, sterilizing, is cooled to that after 60 ℃, to add the concentration of filtration sterilization be 1～3mg/ml rhodamine B, 1～4ml.
Sweet oil emulsion preparation method: 3% polyvinyl alcohol: sweet oil=3:1.With hollander mixing 6min, become emulsion.
Slant preservation substratum: fungi PDA substratum, bacterium bacterium perfect medium.
sieve again for the first time:
The synthetic thin layer result of the zymin making with microbial cells catalysis 1,3-DAG in organic phase is for sieving again for the first time index.
thalline fermentation:
Test tube bacterium two rings of picking primary dcreening operation, the enrichment medium 15mL/250mL that joins sterilizing cultivates 24h, and access fermention medium is cultivated 24h ~ 72h, and inoculum size is 1% ~ 2%.10000r/mn is centrifugal ten minutes afterwards, collects thalline and makes lyophilized powder.
Seed culture medium (%): extractum carnis 0.1 ~ 0.3; Peptone 0.1 ~ 1; Glucose 0.1 ~ 1; Sodium-chlor 0.1 ~ 1.pH?6~8。
Fermention medium (%): soybean cake powder 0.5 ~ 3; Corn steep liquor 0.5 ~ 3; Zulkovsky starch 0.1 ~ 2; K
2hPO
40.1 ~ 1; NaNO
30.1 ~ 1; KCl 0.01 ~ 0.1; MgSO
40.01 ~ 0.1; Sweet oil 0.5 ~ 2.pH?6~8。
glycerolysis reaction:
Glycerolysis reaction system is comprised of full cell lyophilized powder, triglyceride level, glycerine, organic solvent, full cell lyophilized powder concentration 20 mg/mL ~ 60 mg/mL, the mol ratio of triglyceride level and glycerine is 4:1 ~ 1:2, triglyceride concentration: 10 mmol/L ~ 300 mmol/L, the ratio of mixed solvent (normal hexane: the trimethyl carbinol) scope 49:1 ~ 20:1,30 ~ 50 ℃ of temperature of reaction, rotating speed 100 ~ 250 r/min, time 12-48 h
thin-layer chromatography:
Thin layer plate preparation: 1.2366g boric acid, 0.15g Xylo-Mucine, heating in water bath is dissolved in 50mL distilled water.Join afterwards in 17.5g silica gel, after furnishing pasty state, be evenly laid on the sheet glass of 10cm * 20cm.Dry in the shade latter 105 ℃ and dry half an hour, in 1 ~ 2 day, use.
Point sample: be taken into the reactant 5uL point sample of reaction system, each some interval 2cm.In chromatography cylinder, add in advance 47ml chloroform, 3ml acetone, the saturated 30min of 0.5ml dehydrated alcohol.After point sample finishes, thin layer plate is put into chromatography cylinder and launch, while waiting solvent front to thin layer plate forward position 2cm, taking-up is put into large beaker, iodo steam displaing color after drying.
Point is from top to bottom followed successively by: triglyceride level, 1,3-DAG, 1-2-triglyceride, lipid acid, Tegin 55G.Choose second darker bacterial enzyme preparation of point and prepare against for the second time sieve again.
sieve again for the second time:
thalline fermentation and glycerolysis reactionthe same.
high performance liquid phase detects:
Be taken into the yield that high performance liquid chromatography after the reacting liquid filtering after reaction system detects 1,3-DAG, the synthetic high performance liquid phase detected result of the zymin making with microbial cells catalysis 1,3-DAG in organic phase is for sieving again for the second time index.
Draw the reaction solution of 1ml, excessively after the organic membrane of 0.45um, prepare high performance liquid phase and detect.Testing conditions is: in the time of 0 minute, and acetonitrile: Virahol (%)=80:20 ~ 60:40; In the time of 27 minutes, acetonitrile: Virahol (%)=40:60 ~ 20:80; 30 ℃ of temperature; Flow velocity: 0.6 ~ 1mL/min.
Obtain a strain and in multiple sieve, there is the higher higher bacterial strain of ester synthesis of dynamic again.Wherein CCTCC No. M2012538 synthesis capability is higher.In organic synthesis, the synthetic ratio of 1,3-glycerine 20 is also higher.
the evaluation of bacterial strain:
The genus of identifying bacterial classification is other, understands its essential characteristic, to effectively applying document, instructing further research and application to play an important role.For this reason, according to the methods involving pair of document: CCTCC No. M2012538(GZUF36) carry out identification by morphological characters and 18sRNA sequence gene and identify.
morphological specificity:
Aspergillus niger strain is inoculated on PDA substratum to 30 ℃ of cultivations.On PDA substratum, colony growth is quick, and dark-brown is Powdered.First the velvet-like mycelia of hairiness grows, and then to surrounding, spreads, and is hair-like.The mycelia initial stage is yellow, becomes lividity after aging, and has a large amount of black Sporulations.Its colonial morphology is shown in Fig. 1 ((1) and (2)).
The top capsule that can be observed aspergillus niger mycelia conidial head under slide glass cultivation opticmicroscope is flask shape, stigma individual layer, is covered with top capsule and sees Fig. 2.
the decorum is grown tree and is built:
By the 18SrRNA sequence login NCBI(National center for Biotechnology information of GZUF36) database, by online BLAST(Basic Local Alignment Search Tool), search altogether 100 gene fragments higher with this sequence homology, use MEGA5.0 software to carry out the decorum and grow analysis, with Neighbour-joining method, build Molecular Phylogeny tree, the check Bootstrap value of bootstrapping that mark repeats for 1000 times to obtain on branch, with evaluating system, grow the degree of confidence of tree, thereby determine sibship and the classification position of bacterial strain.
The bacterial strain comprising in this bacterium place maximum branch is aspergillus niger (Fig. 3), and in this bacterium and Genbank, to register sequence be that the 3 strain aspergillus nigers of AM270052.1, FR774047.1 and AM270052.1 belong in a minimum branch.
Therefore, according to its morphological specificity, 18sRNA sequence with according to the Analysis and Identification of the constructed phylogenetic systematics tree (Fig. 3) of 18SRNA, it more approaches aspergillus niger, and according to the classification of fungi system of Ainsworth, the classification of proposal is called aspergillus niger GZUF36(Asperglillus niger
GZUF36), belong to Deuteromycotina, hyphomycetes, hyphomycetales, Moniliaceae, Aspergillus, aspergillus niger kind.The Chinese Typical Representative culture collection center (CCTCC) that has been preserved in Wuhan University, address is Wuhan, China university, postcode 430072, date saved is on December 18th, 2012, its deposit number is: CCTCC No.M2012538.
conclusion:
The present invention obtains 1,3-DAG because of its green health with biological Lipids Enzymatic, meets the day by day pursuit of people to high-quality food, by a trend that is development.The aspergillus niger GZUF36(CCTCC No.M2012538 that the present invention screens), can utilize its character of producing intracellular lipase to make Whole-cell lipase catalyzer for synthetic 1 of highly selective, 3-triglyceride, its content in triglyceride reaches more than 80%.And a full cell benefit making catalyzer is can be without enzyme purification, and be directly used in biocatalysis, so simple and practical.Therefore the bacterial strain GZUF36 that, the present invention screens has important application prospect at the synthetic 1,3-DAG of selectivity.Meanwhile, bacterial strain of the present invention also can be applicable to other glyceride synthetic, glyceryl ester hydrolysis and other ester is synthetic, in transesterification and ester hydrolysis reaction.
Owing to having adopted technique scheme, compared with prior art, the present invention's separation from rich oil soil obtains a kind of intracellular lipase and produces bacterium, uses it for and in organic phase, reacts high selectivity 1,3-DAG, the selectivity of its reaction is high, the content of the 1,3-DAG of product in triglyceride reaches more than 80%, without enzyme purification, can be directly used in biocatalysis, reaction process green health.Material source of the present invention is extensive, and method is simple, and result of use is very good.
Accompanying drawing explanation
The flat board that accompanying drawing 1 is bacterial strain of the present invention is observed photo;
The microscopic examination photo that accompanying drawing 2 is bacterial strain of the present invention;
The phylogenetic tree that accompanying drawing 3 is bacterial strain of the present invention;
Accompanying drawing 4 is embodiments of the invention 1 plate screening results;
Accompanying drawing 5 is the thin-layer chromatography of mixture after embodiments of the invention 1 reaction.
Embodiment
Embodiments of the invention 1: intracellular lipase produces the screening of bacterium, in the soil sample of oil pressing factory, Baiyun District, Guiyang City, Guizhou Province: get from tens grams of left and right of soil, 5-10cm place, earth's surface, with polyethylene bag, seal, fetch after sample, set about mask work, by getting 1g sample after the sample blending collecting, be dissolved in 10mL distilled water, fully shake up, 30 ℃, a hour shaking culture of 180r/min, after solids precipitation, get supernatant liquid 5mL in 50mL liquid enrichment medium (composition (mass percent concentration %): (NH is housed
4)
2sO
40.05～0.5; NaNO
30.01～0.2; NaCl 0.05～0.6; MgSO
40.01～0.2; K
2hPO
40.001～0.03; Sweet oil 0.5～2, pH=6～8) in 250mL triangular flask, at 30 ℃, after 180r/min shaking culture 2 days, enrichment medium is fully shaken up, get 5mL and be transferred in new enrichment medium, carry out secondary enrichment, under similarity condition, shaking culture is 2 days, carries out enrichment culture for the third time.
The enrichment bacterium liquid obtaining through above-mentioned cultivation is diluted to suitable multiple with sterilized water, get 0.1mL bacterium liquid and coat solid rhodamine B selection substratum (composition (mass percent %): glucose 0.2～2, Zulkovsky starch 0.3～3, peptone 0.1～4, Yeast diffusion juice 0.1～5, K
2hPO
40.01～1, MgSO
47H
2o 0.01～1, NaNO
30.1～4, agar powder 2, pH6～8, polyvinyl alcohol (PVA)-sweet oil emulsion 9～14ml, sterilizing, is cooled to and after 60 ℃, adds rhodamine B 1 ~ 4ml that the concentration of filtration sterilization is 1mg/ml ~ 3mg/ml) upper, cultivate 1 ~ 2 day for 30 ℃, picking has the bacterium colony three ride purifying of fluorescent ring under 254nm luminescent lamp, is seeded in slant tube and preserves afterwards.The results are shown in Figure 4; Wherein, (A) colour developing of rhodamine B ultraviolet is enclosed; (B) yielding lipase bacterium separation and purification; (C) yielding lipase bacterium preservation.
Point sample: point sample amount is 5uL, each some interval 2cm.In chromatography cylinder, add in advance 47ml chloroform, 3ml acetone, the saturated 30min of 0.5ml dehydrated alcohol.After point sample finishes, thin layer plate is put into chromatography cylinder and launch, while waiting solvent front to thin layer plate forward position 2cm, taking-up is put into large beaker, iodo steam displaing color after drying.The results are shown in Figure 5.
Point is from top to bottom followed successively by: triglyceride level, and 1,3-DAG, 1-2-triglyceride, lipid acid, Tegin 55G, chooses second darker bacterial enzyme preparation of point and prepares against for the second time sieve again.
Embodiments of the invention 2: reaction system is comprised of full cell lyophilized powder, triglyceride level, glycerine, organic solvent, full cell lyophilized powder concentration 10～60 mg/mL, the mol ratio of triglyceride level and glycerine is 4:1～1:2, triglyceride concentration: 10～300 mmol/L, the mol ratio of mixed solvent (normal hexane: the trimethyl carbinol) be 49:1～20:1,30～50 ℃ of temperature of reaction, rotating speed 100～250 r/min, times 12～48 h.After reaction, triglyceride level glycerine solution becomes the transformation efficiency 10%～20% of 1,3-DAG, and in triglyceride, the shared proportional range of 1,3-DAG is 70%～90%.
Claims (7)
1. intracellular lipase produces a bacterium, it is characterized in that: described bacterial strain deposit number is CCTCC No. M2012538.
2. an intracellular lipase as claimed in claim 1 produces bacterium in the application of high selectivity 1,3-DAG.
3. an intracellular lipase as claimed in claim 1 produces the screening method of bacterium, it is characterized in that: first from rich oil soil sampling, intracellular lipase in sample is produced to bacterium after enrichment culture, adopting sweet oil emulsion is substrate, after enrichment culture, dilution spread is selected on substratum solid rhodamine B, the microorganism of usining is decomposed the ability of sweet oil as primary dcreening operation index, the bacterium colony of picking tool fluorescent ring is three ride purifying on flat board, after purifying, picking list bacterium colony moves and connects slant medium preservation, thereby realizes primary dcreening operation; Shake flask fermentation sieves again afterwards, and the synthetic thin layer result of the zymin making with microbial cells catalysis 1,3-DAG in organic phase is for sieving again for the first time index; The synthetic high performance liquid phase of the zymin making with microbial cells again catalysis 1,3-DAG in organic phase detects yield for sieving again for the second time index.
4. intracellular lipase according to claim 5 produces the screening method of bacterium, it is characterized in that: the enrichment medium that described enrichment culture adopts, the mass percent concentration of its each component and component is: (NH
4)
2sO
40.05～0.5%, NaNO
30.01～0.2%, NaCl 0.05 ~ 0.6%, MgSO
40.01～0.2%, K
2hPO
40.001～0.03%, sweet oil 0.5～2%, pH is 6～8.
5. intracellular lipase according to claim 5 produces the screening method of bacterium, it is characterized in that: described solid rhodamine B is selected substratum, concentration is calculated by mass percentage, comprise glucose 0.2～2%, Zulkovsky starch 0.3～3%, peptone 0.1～4%, Yeast diffusion juice 0.1～5%, K
2hPO
40.01～1%, MgSO
47H
2o 0.01～1%, NaNO
30.1～4% and agar powder 2%, pH is 6～8; In above-mentioned mixing solutions, add polyvinyl alcohol-sweet oil emulsion 9～14ml, sterilizing, is cooled to that after 60 ℃, to add the concentration of filtration sterilization be 1～3mg/ml rhodamine B, 1～4ml.
6. an intracellular lipase as claimed in claim 1 produces the using method of bacterium, it is characterized in that: will after thalline lyophilize, make Whole-cell lipase zymin, Whole-cell lipase zymin is reacted to high selectivity 1,3-DAG for organic phase; The glycerolysis reaction that organic phase reaction is triglyceride level and glycerine, or the ester synthesis reaction of glycerine and lipid acid.
7. intracellular lipase according to claim 6 produces the using method of bacterium, it is characterized in that: glycerolysis reaction system is by Whole-cell lipase zymin, triglyceride level, glycerine, organic solvent forms, the concentration of Whole-cell lipase zymin is 20～60 mg/mL, the mol ratio of triglyceride level and glycerine is 4:1～1:2, triglyceride concentration is 10～300 mmol/L, mixed solvent is normal hexane and the part of mixing of the trimethyl carbinol, the mol ratio of normal hexane and the trimethyl carbinol is 49:1～20:1, 30～50 ℃ of temperature of reaction, rotating speed 100～250 r/min, times 12～48 h.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104749314A (en) * | 2015-04-13 | 2015-07-01 | 山东省花生研究所 | Thin layer chromatography detection method for triglyceride in yeast |
| CN110272827A (en) * | 2019-05-13 | 2019-09-24 | 自然资源部第三海洋研究所 | A kind of filamentous fungi Aspergillus niger strain producing bio-oil and its application |
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| CN103060286A (en) * | 2012-12-10 | 2013-04-24 | 华南理工大学 | Lipase made of aspergillus niger strains, and producing method and utilization thereof |
| CN103074388A (en) * | 2012-12-21 | 2013-05-01 | 江南大学 | Method for catalytic synthesis of monoglyceride and diacylglycerol by lipase under ultrahigh pressure |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104749314A (en) * | 2015-04-13 | 2015-07-01 | 山东省花生研究所 | Thin layer chromatography detection method for triglyceride in yeast |
| CN110272827A (en) * | 2019-05-13 | 2019-09-24 | 自然资源部第三海洋研究所 | A kind of filamentous fungi Aspergillus niger strain producing bio-oil and its application |
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| CN103540535B (en) | 2016-03-16 |
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