CN101440350B - A strain of Yarrowia lipolytica mutant strain capable of highly yielding lipase, cultivation method and use of enzyme thereof - Google Patents

A strain of Yarrowia lipolytica mutant strain capable of highly yielding lipase, cultivation method and use of enzyme thereof Download PDF

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CN101440350B
CN101440350B CN2008101721168A CN200810172116A CN101440350B CN 101440350 B CN101440350 B CN 101440350B CN 2008101721168 A CN2008101721168 A CN 2008101721168A CN 200810172116 A CN200810172116 A CN 200810172116A CN 101440350 B CN101440350 B CN 101440350B
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lipase
enzyme
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fat
culture
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李颖
王贵利
关国华
孟永宏
李月娟
徐志文
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Leading Bio-agricultural Co., Ltd.
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Qinhuangdao Leading Science And Technology Development Co ltd
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Abstract

The invention provides a Yarrowia lipolytica lipase high-yield strain LW1 (CGMCC No.2707) obtained through an ultraviolet mutagenesis method, a fermentation culture method, a lipase purification method, and a method for catalyzing the methyl esterification of fatty acid thereof. Yarrowia lipolytica CGMCC No.2707 is cultured for 97.5 h through fed-batch fermentation on a fermentation tank with thecapacity of 100L, the lipase production level is 15,500U/ml, and the activity of produced lipase powder is 30,000U/g. The obtained lipase powder or a zymotic fluid can be directly applied to a reaction of catalyzing the methyl esterification of the fatty acid. The conversion rate for 30h is more than 90 percent when the fatty acid and methanol are added according to the equal mol ratio and the using amount of the lipase in a system is 1,000-3,000U per gram of fatty acid.

Description

The separate inferior sieve yeast mutant of fat, cultural method and the enzyme thereof of one plant height yielding lipase are used
[technical field]
The present invention relates to biological technical field, what relate to high yielding lipase particularly separates the inferior sieve yeast strain of fat, fermentation culture method, and the fermentation lipase or the purposes of fermented liquid in the reaction of catalysis methyl esterification of fatty acid that obtain.
[background technology]
Separating the inferior sieve yeast strain of fat and claim to separate fat Ye Shi yeast (Yarrowia lipolytica) again, belong to the hemiascus yeast, is one of unconventional yeast, has been applied to produce lipase, single cell protein, citric acid, isopropylmolic acid etc.The lipase that it produces is widely used in industry, light industry, food, medicine and other fields.
Lipase can be free fatty acids, triglyceride, monoglyceride, glycerine with the glycerin fatty acid ester catalytic hydrolysis, in addition can also catalytic alcohol or ester and glyceryl ester between transesterification, esterification between lipid acid and the alcohol, be with a wide range of applications, become the third-largest industrial enzymes on the market.But to produce unit of enzyme on the low side for the inferior sieve yeast strain of fat of separating of existing yielding lipase, and the industrialization cost is higher.
Along with the aggravation of energy dilemma, environmental pollution, the biofuel energy as an alternative is to be raw material with the renewable resources, has the little advantage of environmental pollution simultaneously, becomes the focus of research and development, and has reached the practical application of certain scale.Lipase-catalyzed biofuel is synthetic, has that method is simple, a mild condition, low, the costly advantage of by product of energy consumption.At present, the lipase that is applied to production of biodiesel is developed few.The lipase cost height that NOVA company monopolizing manages is not suitable for catalyticreactor and further amplifies.Therefore, in the course of industrialization of China and even world's production of biodiesel, be badly in need of obtaining enzyme unit alive height, the industrialization lipase of low production cost is produced bacterial strain.
This patent uses the uv irradiating method that wild-type Yarrowia lipolytica starting strain is carried out mutagenesis, and conventional screening method is improved, and gets the mutant strain LW1 of high yielding lipase.The fermentation broth enzyme work that obtains through the described method fermentation of this patent is up to 15500u/ml.This bacterial strain and cultural method thereof have improved the generation of lipase, are fit to suitability for industrialized production, enzyme process catalyzed production biodiesel fields such as energy catalysis methyl esterification of fatty acid.The fermented liquid that the high enzyme that uses the described method of patent to obtain is lived can also be made the lipase preparation that different enzymes are lived, and can be used as product to market sale, therefore has higher economic value.
[summary of the invention]
[technical problem that will solve]
What first purpose of the present invention provided a kind of high yielding lipase separates inferior sieve yeast strain (Yarrowia lipolytica) LW1 (CGMCC No.2707) of fat, claims to separate fat Ye Shi yeast again.
Another object of the present invention provides the fermentation culture method of separating the inferior sieve yeast strain of fat LW1 of described high yielding lipase.
Another object of the present invention provides the preparation method of described lipase.
Another object of the present invention provides described lipase and the purposes of described fermented liquid in its catalysis methyl esterification of fatty acid method.
[technical scheme]
The present invention is achieved through the following technical solutions.
The present invention relates to the ultraviolet mutagenesis seed selection and obtain separating inferior sieve yeast strain (Yarrowialipolytica) LW1 of fat.
According to the present invention, with from China Agricultural University's dining room surrounding environment, separate obtain to separate the inferior sieve yeast of fat wild-type be test strain, method by ultraviolet mutagenesis, transparent circle screening method with improvement carries out primary dcreening operation, shakes the multiple sieve of bottle and has obtained the lipase high yield and separate inferior sieve yeast strain (Yarrowia lipolytica) LW1 of fat.
High yielding lipase of the present invention separate inferior sieve yeast strain (Yarrowialipolytica) LW1 of fat, this bacteria strain is on October 14th, 2008, be deposited at China Committee for Culture Collection of Microorganisms common micro-organisms center, address: Datun Road, Chaoyang District, Beijing City, numbering CGMCC No.2707, it has following Microbiological Characteristics:
1), malt extract medium: growth is after 48 hours down at 28 ℃, and cell is oblong candiyeast shape.
2), the slide culture on the PDA solid medium: good pseudomycelium forms.
3), no sporulation.
4), streak culture: greyish white, the top projection, the surface is configured with fold.
5), the suitableeest culture temperature: 28 ℃
6), the carbon source that can utilize: utilize glucose, glycerine, do not utilize starch, sucrose.
7), kNO 3Assimilation: do not have
The invention still further relates to described high yielding lipase and separate the mutagenic breeding method of the inferior sieve yeast strain of fat LW1, it is characterized in that the step of this method is as follows:
A, preparation wild-type starting strain bacteria suspension
To separate the inferior sieve yeast of fat be starting strain to separate the wild-type obtain from China Agricultural University's dining room surrounding environment, activate 1 time down at 28 ℃ through the PDA solid medium successively, the PDA liquid nutrient medium activates 2 times down at 28 ℃, inoculate liquid nutrient medium according to 5% inoculum size by weight then into PDA, cultivate 12h down for 28 ℃ in temperature, get 1ml bacterium liquid then and be added in the sterilized 0.86%NaCl by weight of the 10ml aqueous solution, obtain the wild type strain bacteria suspension.Described PDA substratum is identical with following PDA slant medium.
It is the culture apparatus that those skilled in the art know that described wild-type is separated the employed culture apparatus of the inferior sieve yeast culture of fat.It is constant incubator that for example described wild-type is separated the inferior sieve yeast solids of fat culture apparatus; It for example is constant temperature vibration shaking table that described wild-type is separated the inferior sieve yeast of fat liquid culture apparatus.
The employed equipment of described sterilization is the equipment that those skilled in the art know, for example Shenan Medical Appliances Factory, Shanghai's trade(brand)name LDZX-50KB vertical pressure steam sterilizer.
The seed selection of B, ultraviolet mutagenesis and superior strain
The method that adopts those skilled in the art to know, the method for ultraviolet mutagenesis for example will be in steps A) the resulting wild-type viable count of separating the inferior sieve yeast of fat starting strain bacteria suspension is adjusted to 1.85 * 10 6Individual/ml, described suspension is placed on the sheet glass, its suspension layer thickness is 0.2cm, is allowed to condition at apart from the following irradiation of 20 watts of ultraviolet lamps of ultraviolet lamp 25cm to carry out ultraviolet mutagenesis in 90 seconds; Ultraviolet mutagenesis is coated bacterium liquid on the PDA solid medium flat board after finishing immediately, cultivates 90 hours down for 28 ℃ in temperature.
The wavelength region of the ultraviolet lamp that uses is 250-265nm; Treating that the illuminance on the suspension laminar surface of ultraviolet mutagenesis is 1850uw/cm 2, described ultraviolet lamp for example is that Great Wall, Changping, Beijing air-purification unit engineering corporation is with trade(brand)name Bechtop product sold.
The PDA solid medium flat board of cultivating after the mutagenesis is selected single colony inoculation on primary dcreening operation isolation medium A, cultivates 48 hours down for 28 ℃ in temperature.
The primary dcreening operation isolation medium A that improvement transparent circle screening method of the present invention is taked be in the PDA solid medium, add tributyrin to final concentration 2% (by weight), sodium succinate to final concentration 25mg/ml and nystatin to final concentration 20U/ml.Wherein the nystatin of Tian Jiaing is a kind of anti-mycotic agent, can combine with the ergosterol in the fungal cell membrane, cause that cell leakage changes, promote the absorption of thalline to nutritive substance, help the secretion of extracellular enzyme, therefore utilize mildew making usually to screen the hypertonicity variant extracellular enzyme output is further improved; In addition, when screening, add lipase substrate structure analogue---sodium succinate, can remove the feedback inhibition of meta-bolites, regulate variant in the hope of the metabolism that obtains releasing meta-bolites feedback inhibition by screening and improve production of enzyme (referring to document: Song Xin, Qu Yinbo " seed selection of alkali resistance lipase mutant strain and the research of zymologic property thereof ", industrial microorganism 1999 (29) 12; Zhang Wenxian, Jiang Yongmei etc., " resistant mutant strain sieve method seed selection lipase high yield bacterium ", industrial microorganism 2001 (31) 6).
The tributyrin that can decompose in the substratum owing to lipase produces transparent circle, so can select 17 bacterial strains that the hydrolysis transparent circle is big from above-mentioned primary dcreening operation separation and Culture flat board, in the substratum of 5% inoculum size access by weight B, under 28 ℃ of temperature and 200rpm condition, on shaking table, shake the multiple sieve of bottle, the sampling and measuring enzyme was lived in per 4 hours, until enzyme decline alive, obtain producing the highest high yielding lipase of enzyme level like this and separate the inferior sieve yeast strain of fat LW1.
The composition of described substratum B and proportion optimization are: bean powder 6%, soya-bean oil 4%, glucose 1%, (NH by weight 4) 2SO 40.2%, KH 2PO 40.1%, MgSO 47H 2O 0.05 %, pH 6.5.
In the present invention, the measuring method that described lipase enzyme is lived is the sweet oil volumetry, and the step of this method is as follows:
1) preparation of 0.05M NaOH: earlier with no CO 2Water preparation 5M NaOH liquid storage; After accurately diluting 50 times again, take by weighing 100 ℃ of Potassium Hydrogen Phthalate 0.38g that dry to constant weight, being dissolved in 80ml does not have CO 2In the water, adopt the method for acid base titration to calibrate its accurate concentration, extrapolate stock concentrations again; The no CO of on-the-spot use 2Water is mixed with 0.05MNaOH solution with described liquid storage;
2) preparation of the 2%PVA-1750 aqueous solution by weight: accurate weighing PVA-1750, be added in the distilled water, and make it dissolving with the boiling water bath heating, use three layers of filtered through gauze then, and constant volume, preserve down for 4 ℃ in temperature; PVA is a polyvinyl alcohol.
3) preparation of PVA-sweet oil emulsion substrate: with sweet oil and in above-mentioned steps 2) 2%PVA-1750 by weight that obtains was with 1: 3 (v/v) mixed, use laboratory high speed shear dispersion emulsifying machine (special electromechanical equipment Science and Technology Ltd. of Shanghai Bell, the B25 type, 220V, 50Hz) 10000rpm, emulsification 2-3 minute, obtain PVA-sweet oil emulsion substrate;
4) get 5ml in above-mentioned steps 3) the emulsion substrate and the 4ml 0.1M pH8.0 sodium phosphate buffer that obtain, they are added in the 150ml triangular flask, again triangular flask is placed the water bath with thermostatic control shaking table, incubation 5min under 37 ℃ of temperature and 130rpm;
5) get the suitably enzyme liquid for preparing in front of dilution of 1ml, join in the mixture of above-mentioned substrate and damping fluid, under 37 ℃ of temperature and 130rpm condition, react 10min, add 15ml dehydrated alcohol termination reaction then;
6) drip 4 phenolphthalein and make indicator, use the lipid acid of the 0.05M NaOH solution titration enzymolysis generation of step 1), stop to reaction solution is pink.
Blank operation method as previously described is identical, just will join in described substrate and the damping fluid behind fermented liquid and the dehydrated alcohol hybrid reaction 10min.
According to the present invention, described enzyme work is defined as under the condition determination of this sweet oil volumetry, and the enzyme amount that 1min discharges 1 μ mol lipid acid is an enzyme unit alive.
The invention still further relates to described cultivation of separating the inferior sieve yeast strain of fat LW1.It is as follows that described high yielding lipase is separated the cultural method of the inferior sieve yeast strain of fat LW1:
A, slant culture
The described inferior sieve yeast strain of the fat bacterial strain LW1 that separates is got a transfering loop lawn and is inoculated in PDA solid inclined-plane, 28 ℃-30 ℃ of initial pH6.0-pH7.0 and temperature following constant temperature culture 12-72 hour.
The PDA solid slant culture basigamy system method of using when the PDA solid slant culture is as follows: the 200g potato is after cleaning the peeling chopping, add water 1000ml and boil half hour, use filtered through gauze, its filtrate adds 20g glucose again, use deionized water liquid make-up volume to 1000ml behind the mixing, sterilized 15 minutes down for 115 ℃, obtain the PDA liquid nutrient medium in temperature, in this PDA liquid nutrient medium, add 15g agar, treat to obtain the PDA solid medium after agar dissolves, solidifies.
The described slant culture time selects to be in the growth curve bacterial classification of increased logarithmic phase according to the growth situation.
The instrument that described slant culture uses is the instrument that those skilled in the art know, as test tube and eggplant bottle.
B, shake-flask culture
With above-mentioned steps A) bacterial strain that obtains of slant culture gets a transfering loop lawn and inserts in the ferment-seeded substratum, under 28 ℃-30 ℃ of initial pH6.0-pH7.0, temperature, liquid amount 1: 5-1: 4, cultivate under shaking speed 180-300 rev/min the condition, the sampling and measuring enzyme was lived in every 4-24 hour, and living until enzyme descends.
The ferment-seeded substratum that uses when shake-flask culture is composed as follows: bean powder 4%-9%, soya-bean oil 2%-6%, glucose 1%-3%, (NH by weight 4) 2SO 40.1%-0.4%, KH 2PO 40.1%-0.4%, MgSO 47H 2O 0.05%-2%, pH6.0-pH 6.5.
C, ferment tank are cultivated
In the bacterial classification that 1-2 eggplant bottle slant culture obtains, add the 30-100ml sterilized water respectively, scrape lawn, be inoculated in the 100L fermentation tank culture medium, 28 ℃-30 ℃ of temperature, initial pH6.1-pH6.5, tank pressure 0.5-1.0 normal atmosphere and blowing air 1: 1-1: the fermentation culture conditions of 2 (v/m) was cultivated 90-140 hour down; Live by changing following condition raising fermenting enzyme in the fermenting process: rotating speed is brought up to 300 rev/mins gradually by 180 rev/mins; Regulate air flow and increase to 1: 2 gradually (v/m) by 1: 1 (v/m); Mend into the soya-bean oil of 1%-5% by weight according to the fermentation situation.Ferment by 97.5 hours, its enzyme work reaches the highest, produces enzyme level and reaches 15500U/ml, and its detected result is referring to accompanying drawing 1.
The fermention medium that uses when ferment tank is cultivated is composed as follows: bean powder 4%-15%, soya-bean oil 4%-12%, (NH by weight 4) 2SO 40.1%-0.4%, KH 2PO 40.1%-0.4%, MgSO 47H 2O 0.05%-2%, CaCO 31%-5%, defoamer (glycerin polyether) 1%-5%, pH6.0-pH 6.5.
Described enzyme activity determination method is previously described sweet oil volumetry.
Described fermentor tank is normally used in the art fermentor tank, for example the fermentor tank of the auspicious biotechnology of Zhenjiang lattice company limited sale.
A preferred embodiment of the invention, described fermentation culture method are according to producing the enzyme situation, progressively rotating speed being promoted to 240rpm by 180rpm and improving the product enzyme level.
A preferred embodiment of the invention, described fermentation culture method are according to producing the enzyme situation, add carbon source by miscarriage in batches and add in batches promptly by weight that the mode of 1%-5% soya-bean oil improves enzyme level.
The invention still further relates to high yielding lipase separates the inferior sieve yeast strain of fat LW1 fermented liquid and prepares lipase enzyme powder.Described high yielding lipase is separated the method that the inferior sieve yeast strain of fat LW1 fermented liquid prepares lipase enzyme powder, it is characterized in that the step of this method is as follows:
The high yielding lipase that aforesaid method 2 obtains is separated the inferior sieve yeast strain LW1 fermented liquid of fat centrifugal 10min under 3000 commentaries on classics/min, get its supernatant liquor.In supernatant liquor, add its volume 3-4 doubly 95%,-18 ℃ of ethanol, after adding whole ethanol, at freezer compartment of refrigerator in temperature-18 ℃--20 ℃ of coolings 0.5-1 hour down, descended centrifugal 2-10 minute at 2000 rev/mins then, incline and supernatant liquor, the throw out that obtains is with 95% cold washing with alcohol, its ethanol consumption is the 1/4-1/2 of described supernatant liquor volume, centrifugal again, repeated washing operation 2-3 time, the supernatant liquor that up to inclining becomes light yellow, use the washing with acetone of temperature-18 ℃ then, the 1/2-1 that its acetone consumption is the ethanol volume doubly, and is centrifugal again, repeated washing operation 2-3 time, colourless substantially until the acetone supernatant liquor, vacuum removal acetone obtains lipase enzyme powder, records the work of lipase enzyme powder lytic enzyme at 20000u/g-30000u/g.
Described whizzer is normally used in the art whizzer, for example the flying pigeon board whizzer of Anting Scientific Instrument Factory, Shanghai's manufacturing.
The present invention relates to separate the purposes of the inferior sieve yeast strain LW1 fermented liquid of fat in the catalysis fatty acid response.
The invention still further relates to the described purposes of the inferior sieve yeast strain LW1 fermented liquid of fat in the catalysis fatty acid response of separating, it is characterized in that with lipid acid and methyl alcohol by etc. mol ratio add, wherein methyl alcohol adds once every 5h-10h, dividing 3-6 time adds, the lipase consumption is every gram lipid acid 1000-2000U in the esterification system, temperature 28-45 ℃ with 180-200 rev/min airbath shaking table in reacted 15-40 hour, the product that obtains detects through gas-chromatography and is confirmed to be fatty acid methyl ester, esterification yield 70-95%.
Detecting the vapor-phase chromatography of lipase-catalyzed reaction product carries out under the following conditions:
Tianjin, island GC2010 gas chromatograph, chromatographic column, DB-1HT 30 * 0.25 * 0.1; Hydrogen flame detector; Splitting ratio 30: 1, post flow 1.50ml/min, nitrogen purging flow 10.0ml/min, 380 ℃ of injector temperatures, retention time 25min, 365 ℃ of post oven temperature, degree, adopt temperature programming: 20 ℃/min, 4min; 15 ℃/min, 3min; 20 ℃/min, 7min, retention time 30min.Detector temperature is 385 ℃.
The present invention can also use other gas chromatograph to detect, but its testing conditions can be done suitably to adjust according to the situation of this instrument.
The invention still further relates to and separate the inferior sieve yeast strain of fat LW1 fermented liquid by described high yielding lipase and prepare the purposes of lipase enzyme powder in the catalysis fatty acid response.The purposes of the lipase enzyme powder that obtains with described method in the reaction of catalysis methyl esterification of fatty acid, it is characterized in that with lipid acid and methyl alcohol by etc. mol ratio add, wherein methyl alcohol adds once every 2h-10h, dividing 3-6 times adds, be every gram lipid acid 1000-3000U with the enzyme amount in the esterification system, 28-45 ℃, reacted 6-30 hour in 180-200 rev/min the airbath shaking table, the product that obtains detects through gas-chromatography and is confirmed to be fatty acid methyl ester, esterification yield 70%-95%.
Described methyl alcohol stream adds the time specifically to be regulated according to fermented liquid that uses in the reaction or the enzyme powder enzyme situation of living.
Described airbath shaking table is an equipment well known in the art, as the THZ-C constant temperature oscillator of Taicang, Jiangsu laboratory equipment factory production.
Described lipid acid is selected from the saturated or unsaturated fatty acids of C12-C18.
Preferably, described lipid acid is selected from the C16-C18 unsaturated fatty acids.
More preferably, described lipid acid is selected from oleic acid or plam oil.
The fatty acid methyl ester that adopts the inventive method to obtain can be widely used for industry, agricultural is used as biofuel.
[beneficial effect]
The invention provides the lipase high yield and separate inferior sieve yeast strain LW1 of fat and cultural method thereof, and the lipase that will adopt this method to obtain is applied to catalyzed chemical reaction.Adopt method of the present invention, 100L fermentor cultivation 97.5 hours is a substrate with the sweet oil, and the product enzyme level is 15500U/ml.The lipase fermented liquid that uses this method acquisition is with 1500U/g lipid acid consumption, 30 hours transformation efficiencys of catalysis oleic acid methanol esterification reaction are more than 75%, use lipase enzyme powder that this bacterial strain makes with 2500U/g lipid acid consumption, 30 hours transformation efficiencys of catalysis oleic acid methanol esterification reaction are more than 90%.The present invention lays a good foundation for the industrial applications of this enzyme.
[description of drawings]
Fig. 1 separates the inferior sieve yeast of fat LW1 fermentor cultivation enzyme and lives and the pH surveillance map.
Fig. 2 separates the inferior sieve yeast mutant of fat lipase fermented liquid catalysis oleic acid methanol esterification reaction vapor detection figure
Black lines is the fatty acid methyl ester color atlas, and the pink colour lines are the lipid acid color atlas of contrast
Fig. 3. separate the lipase enzyme powder catalysis methyl esterification of fatty acid of the inferior sieve yeast mutant of fat
A. Dui Zhao lipid acid color atlas, the fatty acid methyl ester color atlas after the B. esterification
[embodiment]
Embodiment 1
Will be from agricultural university's dining room surrounding environment isolating wild-type separate the inferior sieve yeast of fat successively through the PDA solid medium 28 ℃ of activation 1 time down, the PDA liquid nutrient medium activates 2 times down at 28 ℃, then according to the PDA of 5% inoculum size access by weight liquid nutrient medium, cultivate 12h down at 28 ℃, get then in the aqueous solution of 0.86%NaCl by weight that 1ml bacterium liquid is added to sterilized 10ml, obtain the bacteria suspension of starting strain.
The viable count of above-mentioned resulting starting strain bacteria suspension is adjusted to 1.85 * 10 6Individual/ml, described suspension is placed on the sheet glass, its suspension layer thickness is 0.2cm, is allowed to condition at apart from the following irradiation of 20 watts of ultraviolet lamps of ultraviolet lamp 25cm to carry out ultraviolet mutagenesis in 90 seconds; Ultraviolet mutagenesis is coated bacterium liquid on the PDA solid medium flat board after finishing immediately, cultivates 90 hours down for 28 ℃ in temperature.
Select single colony inoculation on the primary dcreening operation isolation medium from the PDA solid medium flat board that mutagenesis is cultivated, promptly in the PDA solid medium, add tributyrin to final concentration 2% (by weight), sodium succinate to final concentration 25mg/ml and nystatin to final concentration 20U/ml.28 ℃, cultivated 48 hours.
Select 17 bacterial strains that the hydrolysis transparent circle is big from above-mentioned primary dcreening operation separation and Culture flat board, insert with single bacterium colony and shake bottle and sieve in the substratum (bean powder 6%, soya-bean oil 4%, glucose 1%, (NH by weight again 4) 2SO4 0.2%, KH 2PO 40.1%, MgSO 47H 2O 0.05%, and pH 6.5.) 28 ℃, 200rpm shakes the multiple sieve of bottle.
Got sample one time every 4 hours when sieving again, detect the lipase enzyme and live.17 bacterial strains that primary dcreening operation obtains are cultured to 125 hours respectively, get the highest mutant strain of a strain yielding lipase, called after LW1, its final enzyme 2850u/ml alive.Described enzyme work is to adopt the sweet oil titration measuring described in the present specification.
Embodiment 2
To separate the inferior sieve yeast mutant of fat LW1 cultivates after 24 hours in the eggplant bottle, to wherein adding the 50ml sterile purified water, wash lawn, direct inoculation is cultivated under the fermentation condition of 28 ℃ of temperature, initial pH 6.5,0.7 normal atmosphere of tank pressure and blowing air 1: 1 (v/m) in the 100L fermentor tank.Ferment by 63 hours, the fermentor tank rotating speed is brought up to 240 rev/mins by 180 rev/mins, this moment, tank pressure was increased to 1.0 normal atmosphere, air flow is brought up to 1: 2 (v/m) by 1: 1 (v/m), cultivated 100 hours, and adopted the enzyme work of the sweet oil titration measuring of describing in the present specification to reach 8250u/ml.
Embodiment 3
To separate the inferior sieve yeast mutant of fat LW1 cultivates after 24 hours in the eggplant bottle, to wherein adding the 50ml sterile purified water, wash lawn, direct inoculation is in the 100L fermentor tank, 28 ℃, initial pH 6.5, the fermentation culture of tank pressure 0.6 normal atmosphere and blowing air 1: 1 (v/m), enzyme rate of growth alive is slow when fermenting by 55 hours, mend 2% soya-bean oil by weight, rotating speed is brought up to 240 rev/mins by 180 rev/mins, when cultivating 75 hours, mend 2% soya-bean oil by weight again, this moment, tank pressure was elevated to 1.0 normal atmosphere, cultivated 97.5 hours, and adopted the enzyme work of the sweet oil titration measuring of describing in the present specification to reach 15500u/ml.
Embodiment 4
Separate the enzyme liquid of the inferior sieve yeast mutant of fat LW1 fermented liquid dilution with what embodiment 3 obtained for enzyme work 4000u/ml.Catalysis oleic acid methanol esterification reaction (reaction system: enzyme dosage 4000U, 2.82g oleic acid, 405 μ l methyl alcohol, wherein the every interval 10h of methyl alcohol adds 1/3rd i.e. 135 μ l of above-mentioned volume, 30 ℃, 190rpm) 30 hours.Reaction product adopts following condition to carry out gas chromatographic detection:
Tianjin, island GC2010 gas chromatograph, chromatographic column, DB-1HT 30 * 0.25 * 0.1; Hydrogen flame detector; Splitting ratio 30: 1, post flow 1.50ml/min, nitrogen purging flow 10.0ml/min, 380 ℃ of injector temperatures, retention time 25min, 365 ℃ of post oven temperature, degree, adopt temperature programming: 20 ℃/min, 4min; 15 ℃/min, 3min; 20 ℃/min, 7min, retention time 30min.Detector temperature is 385 ℃.Detection obtains Witconol 2301 content 75.17%.
Embodiment 5
Separate the inferior sieve yeast mutant of fat LW1 in the fermentor tank top fermentation with what embodiment 1 mutagenesis obtained, the fermentation broth enzyme that obtains 12000u/ml alive, centrifugal 10min under 3000 commentaries on classics/min gets its supernatant liquor; The 95%-18 ℃ of ethanol that adds 3 times of its volumes in this supernatant liquor, after adding whole ethanol, at freezer compartment of refrigerator in temperature-20 ℃ cooling 0.5 hour down, descended centrifugal 4 minutes at 2000 rev/mins then, inclining supernatant liquor, and the throw out that obtains is 1/4 of a described supernatant liquor volume with 95% cold washing with alcohol, its ethanol consumption, centrifugal again, repeated washing operation 2 times, the supernatant liquor that up to inclining becomes light yellow, uses the washing with acetone of temperature-18 ℃ then, its acetone consumption is 1/2 of an ethanol volume, centrifugal again, repeated washing operation 2 times, colourless substantially until the acetone supernatant liquor, vacuum removal acetone obtains lipase enzyme powder.Adopt the sweet oil volumetry of describing in the present specification to record the enzyme powder lytic enzyme 24000u/g of being alive.
Get above-mentioned enzyme powder 0.2g catalysis oleic acid methylalcohol reaction system (reaction system: enzyme dosage 5000U, 2.82g oleic acid, 405 μ l methyl alcohol, wherein the every interval 10h of methyl alcohol adds 1/3rd i.e. 135 μ l of above-mentioned volume, and 30 ℃, 190rpm), behind the 30h, with reaction product through gas chromatographic detection.
Detection method is as described in the embodiment 4, and detecting and obtaining Witconol 2301 content is 89.18%.
Embodiment 6
With embodiment 1 mutagenesis obtain separate the inferior sieve yeast mutant of fat LW1 with embodiment 3 described methods in the fermentor tank top fermentation, the fermentation broth enzyme that the obtains 15500u/ml that lives.Obtain lipase enzyme powder to implement the enzyme powder, preparation method thereof described in sharp 5 again.Record the enzyme powder lytic enzyme 30000u/g of being alive.
Get above-mentioned enzyme powder catalysis plam oil methylalcohol reaction system, plam oil and methyl alcohol mol ratio such as are pressed and are added, wherein methyl alcohol adds once every 6h, dividing 4 times adds, enzyme dosage is every gram lipid acid 3000U, 30 ℃, reacts 24 hours in 180 rev/mins the airbath shaking table, the gas chromatographic detection that the product that obtains is described through embodiment 4, fatty acid methyl ester is 90.16%.

Claims (10)

  1. A high yielding lipase separate inferior sieve yeast mutant (Yarrowialipolytica) LW1 of fat, its deposit number is CGMCC No.2707.
  2. 2. high yielding lipase according to claim 1 is separated the cultural method of the inferior sieve yeast strain of fat LW1, it is characterized in that the step of this method is as follows:
    A.PDA solid slant culture basigamy system is as follows: the 200g potato is after cleaning the peeling chopping, add water 1000ml and boil half hour, use filtered through gauze, its filtrate adds 20g glucose again, use deionized water liquid make-up volume to 1000ml behind the mixing, sterilized 15 minutes down for 115 ℃, obtain the PDA liquid nutrient medium in temperature, in this PDA liquid nutrient medium, add 15g agar, treat to obtain the PDA solid medium after agar dissolves, solidifies;
    B. the ferment-seeded substratum is formulated as follows: incite somebody to action bean powder 4%-9%, soya-bean oil 2%-6%, glucose 1%-3%, (NH by weight 4) 2SO 40.1%-0.4%, KH 2PO 40.1%-0.4%, MgSO 47H 2O 0.05%-2% is mixed with the ferment-seeded substratum of pH6.0-pH 6.5;
    C. fermention medium is formulated as follows: incite somebody to action bean powder 4%-15%, soya-bean oil 4%-12%, (NH by weight 4) 2SO 40.1%-0.4%, KH 2PO 40.1%-0.4%, MgSO 47H 2O0.05%-2%, CaCO 31%-5%, defoamer glycerin polyether 1%-5% is mixed with the fermention medium of pH6.0-pH 6.5;
    D. culture condition and method
    (i) slant culture
    The described inferior sieve yeast strain of the fat bacterial strain LW1 that separates is got a transfering loop lawn and is inoculated in PDA solid inclined-plane, 28 ℃-30 ℃ of initial pH6.0-pH7.0 and temperature following constant temperature culture 12-72 hour;
    (ii) shake-flask culture
    With above-mentioned steps i) bacterial strain that obtains of slant culture gets a transfering loop lawn and inserts in the ferment-seeded substratum, under 28 ℃-30 ℃ of initial pH6.0-pH7.0, temperature, liquid amount 1: 5-1: 4, cultivate under shaking speed 180-300 rev/min of condition, the sampling and measuring enzyme was lived in every 4-24 hour, and living until enzyme descends.
  3. 3. cultural method according to claim 2 is characterized in that described method steps D condition is (ii) replaced with:
    (iii) fermentor cultivation
    In the bacterial classification that 1-2 eggplant bottle slant culture obtains, add the 30-100ml sterilized water respectively, scrape lawn, be inoculated in the 100L fermentation tank culture medium, 28 ℃-30 ℃ of temperature, initial pH 6.1-pH6.5, tank pressure 0.5-1.0 normal atmosphere and blowing air 1: 1-1: the fermentation culture conditions of 2 (v/m) was cultivated 90-140 hour down, obtained described bacterial strain LW1 fermented liquid.
  4. 4. method according to claim 3, it is characterized in that described fermentor tank rotating speed is brought up to 300 rev/mins gradually by 180 rev/mins, or regulate air flow and increase to 1: 2 gradually (v/m), or mend by weight the soya-bean oil of 1%-5% during the fermentation and improve fermenting enzyme and live by 1: 1 (v/m).
  5. 5. according to the described method of each claim among the claim 2-4, it is characterized in that described to separate the work of the inferior sieve yeast strain of fat LW1 fermentation broth enzyme be more than the 15500U/ml.
  6. 6. use the method that the inferior sieve yeast strain of fat LW1 fermented liquid prepares lipase enzyme powder of separating that the described method of claim 2 obtains, it is characterized in that this method steps is as follows:
    To separate the inferior sieve yeast strain LW1 fermented liquid of fat centrifugal 10min under 3000 commentaries on classics/min according to the high yielding lipase that the described method of claim 2 obtains, get its supernatant liquor, in supernatant liquor, add its volume 3-4 doubly 95%,-18 ℃ of ethanol, after adding whole ethanol, at freezer compartment of refrigerator in temperature-18 ℃--20 ℃ of coolings 0.5-1 hour down, descended centrifugal 2-10 minute at 2000 rev/mins then, inclining supernatant liquor, and the throw out that obtains is the 1/4-1/2 of described supernatant liquor volume with 95% cold washing with alcohol, its ethanol consumption, centrifugal again, repeated washing operation 2-3 time, the supernatant liquor that up to inclining becomes light yellow, uses the washing with acetone of temperature-18 ℃ then, the 1/2-1 that its acetone consumption is the ethanol volume doubly, centrifugal again, repeated washing operation 2-3 time, colourless substantially until the acetone supernatant liquor, vacuum removal acetone obtains lipase enzyme powder.
  7. 7. the lipase enzyme powder that obtains according to the described method of claim 6 is characterized in that the lytic enzyme work of this lipase enzyme powder is 20000u/g-30000u/g.
  8. 8. that uses that the described method of claim 2 obtains separates the purposes of the inferior sieve yeast strain of fat LW1 fermented liquid in the reaction of catalysis methyl esterification of fatty acid, it is characterized in that with lipid acid and methyl alcohol by etc. mol ratio add, wherein methyl alcohol adds once every 5h-10h, dividing 3-6 time adds, the lipase consumption is every gram lipid acid 1000-2000U in the esterification system, 28-45 ℃, reacted 15-40 hour in 180-200 rev/min the airbath shaking table, the product that obtains detects through gas-chromatography and is confirmed to be fatty acid methyl ester, its esterification yield 70-95%.
  9. 9. the purposes of lipase enzyme powder in the catalysis fatty acid response that obtains according to the described method of claim 6, it is characterized in that with lipid acid and methyl alcohol by etc. mol ratio add, wherein methyl alcohol adds once every 2h-10h, dividing 3-6 time adds, the lipase consumption is every gram lipid acid 1000-3000U in the esterification system, 28-45 ℃, reacted 6-30 hour in 180-200 rev/min the airbath shaking table, the product that obtains detects through gas-chromatography and is confirmed to be fatty acid methyl ester, its esterification yield 70%-95%.
  10. 10. according to Claim 8 or 9 described purposes, it is characterized in that described lipid acid is selected from the saturated or unsaturated fatty acids of C12-C18.
CN2008101721168A 2008-11-11 2008-11-11 A strain of Yarrowia lipolytica mutant strain capable of highly yielding lipase, cultivation method and use of enzyme thereof Active CN101440350B (en)

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