CN104046601A - Culture medium for producing extracellular lipase by using yarrowia lipolytica W29 - Google Patents

Culture medium for producing extracellular lipase by using yarrowia lipolytica W29 Download PDF

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Publication number
CN104046601A
CN104046601A CN201410248592.9A CN201410248592A CN104046601A CN 104046601 A CN104046601 A CN 104046601A CN 201410248592 A CN201410248592 A CN 201410248592A CN 104046601 A CN104046601 A CN 104046601A
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lipase
yarrowia lipolytica
culture medium
oil
substratum
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CN201410248592.9A
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吴兰
葛刚
张小琴
余祥单
刘以珍
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Nanchang University
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Nanchang University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01003—Triacylglycerol lipase (3.1.1.3)

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention discloses a culture medium for producing extracellular lipase by using yarrowia lipolytica W29. The culture medium is characterized in that a culture medium solution contains 0.1% of sucrose, 0.1% of urea, 2.5% of peptone, 0.1% of magnesium sulfate, 1.5% of sodium chloride, 0.1% of tween-80 and 7g/L oil, wherein the pH value is natural. By using the culture medium, the capacity for producing lipase by using microbial cells is improved; a great number of lipases are generated while salad oil and olive oil wastewater can be treated, so that a basis is laid for recycling wastewater.

Description

Yarrowia lipolytica W 29produce the substratum of extracellular lipase
Technical field
The present invention relates to a kind of microbiological culture media, relate in particular to a kind of Yarrowia lipolytica W 29substratum, belongs to the production technical field of production by biological lipase.
Background technology
Lipase (EC3.1.1.3) is the important lytic enzyme that a class acts on ester bond, to be hydrolyzed the interfacial enzymes that long-chain fat acid glyceride generates free longer chain fatty acid, glycerine, monoglyceride and triglyceride or this reaction reversed reaction, be can be in oil-water interface or fat-soluble system the biological catalyst that reacts of the number of chemical such as catalytic hydrolysis, acidolysis, alcoholysis, aminolysis, esterification, transesterification, transesterify.It has the features such as chemo-selective, stereoselectivity, site selectivity and Substratspezifitaet, become one of important industrial enzymes, be widely used in the traditional industry fields such as feed, food, washing, oil wastewater processing, and bioenergy is as modern industry fields such as biofuel, chiral drug synthesize, biosensors.
Lipase is mainly present in animal, plant and microorganism, and in microorganism yielding lipase most species, found to have at least 65 microbes producing cellulases to belong to, comprising 28 genus of bacterium, 10 genus of yeast, 4 genus of actinomycetes, other fungies totally 23 genus.Due to microbial lipase not only can catalysis ester compound the reactions such as the synthetic and transesterify of alcoholysis, hydrolysis, esterification, ester, and in reaction process, do not need coenzyme, reaction conditions gentle, consume energy low, substrate starting material are extensive, byproduct is few, and easily obtain and carry out scale operation, have than the animals and plants lipase advantage such as action pH and thermal adaptability widely simultaneously, become the important sources of industrial enzymes.Lipase is as important industrial enzymes, is widely used, demand is large, and therefore by optimization of fermentation condition, improving strain enzyme-producing ability is a very urgent job.
Solution ester formula yeast ( yarrowia lipolitica) be a kind of unconventional property, the strict gentle ascus yeast of aerobic, growth conditions, it can be grown as carbon source with extensive cheap materials such as carbohydrate, lipid and hydro carbons.Our early-stage Study shows: through the Yarrowia lipolytica W of our laboratory improvement 29salad oil, food and drink waste water are had to higher degradation capability, in temperature, be that 25 ~ 30 ℃, pH 6.0 ~ 7.0, incubation time are under the condition of 50 h, shaking speed 200 r/min, the degradation rate of the salad oil of oil-contg≤2000 mg/L and food and drink waste water has been reached to 93.3% and 85.1%.Yarrowia lipolytica W 29the ability that has very strong degraded grease, and degraded grease is mainly undertaken by producing lipase.Although Yarrowia lipolytica energy yielding lipase has bibliographical information, and has known y.lipoliticahave 8 kinds of different genes encoding lipase, the report of still producing lipase in processing oil wastewater process is relatively less.Research shows that the lipase gene that different inductors, carbon source and different culture condition express is different, the people such as Gerardo find with Semen Maydis oil do carbon source and inductor, reduction air flow can make y.lipoliticaimprove the output of lipase, so the composition of substratum is just right y.lipoliticaexpress that lipase and output and have vital impact.
Summary of the invention
The object of the present invention is to provide a kind of Yarrowia lipolytica W 29produce the substratum of extracellular lipase, to improve Yarrowia lipolytica W 29the output of yielding lipase.
The present invention is achieved like this, a kind of Yarrowia lipolytica W 29produce the substratum of extracellular lipase, it is characterized in that described substratum comprises 0.1% sucrose, 0.1% urea, 2.5% peptone, 0.1% magnesium sulfate, 1.5% sodium-chlor, 0.1% tween-80,7g/L oil, pH nature, described in be the grams that per-cent refers to contained solute in 100mL solution.Be that every 100ml culture medium solution is water containing 0.1g sucrose, 0.1g urea, 2.5g peptone, 0.1g magnesium sulfate, 1.5g sodium-chlor, 0.1g tween-80,0.7g oil, surplus.
The present invention be directed to Yarrowia lipolytica W 29the in the situation that in existing substratum (salad oil water culture medium, salad oil+YPD substratum, quote substratum), yield of enzyme being all very low (being low to moderate 0.52 U/mL), by the optimization of culture medium prescription, additional 0.1% sucrose of the oil of take as carbon source, take urea+peptone as nitrogenous source, add that a small amount of inorganic salt, tween-80 tensio-active agent are activator, make Yarrowia lipolytica ( y.lipolitica)w 29utilize grease for carbon source and to degraded in, significantly improved fermentation level, fermentation 60h reaches enzymatic activity high (sweet oil of take reaches 8.70 U/mL as substrate, and the salad oil of take reaches 4.04 U/mL as substrate).The lipase activity producing is still than the Yarrowia lipolytica W of people's reports such as Lopes 29decompose sweet oil waste water production lipase activity and exceed 40 times.The present invention fills a prescription by Optimal Medium, and the ability that makes microorganism cells produce lipase improves, and a large amount of lipase that produces when processing salad oil, sweet oil waste water, for the recycling of waste water is laid a good foundation.
Embodiment
For the ease of understanding, below in conjunction with embodiment, the present invention is further described.The percentage sign relating in embodiment (%) unless otherwise noted, refers to the grams of contained solute in 100mL solution.In following embodiment with y.lipoliticaw 29(being deposited in University Of Nanchang's molecular biology and genetically engineered key lab) is bacterial classification, and after the activation of YPD substratum, being inoculated into fermention medium carries out fermentation culture, and after cultivating, centrifugal acquisition supernatant liquor is comprised extracellular lipase.This research be take temperature as 40 ℃, under the condition of pH=7.0, produces the required enzyme amount of the free fat enzyme acid of 1 U/moL be defined as the U of Yi Gemeihuo unit, international unit (U/mL) with lipase hydrolysis triolein per minute.Described oil is sweet oil (Chemical Reagent Co., Ltd., Sinopharm Group) or salad oil (golden imperial fish board 1:1:1 mixed oil, commercially available).
Embodiment 1:
Use in accordance with the following steps and separate ester formula yeast W 29produce extracellular lipase:
(1) seed culture: will separate ester formula yeast ( y.lipolitica) W 29access contains in 1% yeast extract paste, 2% peptone, 2% glucose, the natural YPD liquid nutrient medium of pH carries out shaking table cultivation, uses 250mL triangular flask, liquid amount 50mL, and 28 ℃ of culture temperature, shaking speed 150 r/min, cultivate 18 h.
(2) inoculum size access fermentation culture: according to 2.5%(V:V) contains 0.1% sucrose, 0.1% urea, 2.5% peptone, 0.1% MgSO 47H 2o, 1.5% NaCl, 0.1% tween-80,7g/L sweet oil, carry out fermentation culture in the natural substratum of pH.Use 500mL triangular flask, liquid amount 150mL, 30 ℃ of culture temperature, shaking speed 150 r/min, aerobic is cultivated 60h.After cultivation finishes, fermented liquid is at 4 ℃, and the supernatant liquor obtaining after the centrifugal 10min of 5000rpm has comprised required lipase, and the lipase activity of mensuration is 8.70 U/mL.Simultaneously to quote substratum (2% peptone, 0.5% sucrose, 0.7% sweet oil, 0.1% (NH 4) 2sO 4, 0.1%MgSO 47H 2o, 0.2%K 2hPO 4) be contrast, the lipase activity of production has improved 2.8 times.
Embodiment 2:
Use in accordance with the following steps and separate ester formula yeast W 29produce extracellular lipase:
(1) seed culture: will separate ester formula yeast ( y.lipolitica) W 29in access (1% yeast extract paste, 2% peptone, 2% glucose, pH nature) YPD liquid nutrient medium, carry out shaking table cultivation, use 250mL triangular flask, liquid amount 50mL, 28 ℃ of culture temperature, shaking speed 150 r/min, cultivate 18 h.
Fermentation culture: according to 2.5%(V:V) inoculum size access contains 0.1% sucrose, 0.1% urea, 2.5% peptone, 0.1% MgSO 4.7H 2o, 1.5% NaCL, 0.1% tween-80, carry out fermentation culture in the substratum of pH nature, 7g/L salad oil.Use 500mL triangular flask, liquid amount 150mL, 30 ℃ of culture temperature, shaking speed 150 r/min, aerobic is cultivated 60h.After cultivation finishes, fermented liquid is at 4 ℃, and the supernatant liquor obtaining after the centrifugal 10min of 5000rpm has comprised required lipase, and the lipase activity of mensuration is 4.04 U/mL.Take 7g/L salad oil+water culture medium as contrast, and the lipase activity of production has improved 8 times simultaneously.

Claims (2)

1. a Yarrowia lipolytica W 29produce the substratum of extracellular lipase, it is characterized in that described culture medium solution comprises 0.1% sucrose, 0.1% urea, 2.5% peptone, 0.1% magnesium sulfate, 1.5% sodium-chlor, 0.1% tween-80,7g/L oil, pH nature, described in be the grams that per-cent refers to contained solute in 100mL solution.
2. a Yarrowia lipolytica W 29the method of producing extracellular lipase, is characterized in that Yarrowia lipolytica W29 according to 2.5%(V:V) inoculum size access claim 1 described in substratum in carry out fermentation culture; Use 500mL triangular flask, liquid amount 150mL, 30 ℃ of culture temperature, shaking speed 150 r/min, aerobic is cultivated 60h; After cultivation finishes, fermented liquid is at 4 ℃, and the supernatant liquor obtaining after the centrifugal 10min of 5000rpm has comprised required lipase.
CN201410248592.9A 2014-06-06 2014-06-06 Culture medium for producing extracellular lipase by using yarrowia lipolytica W29 Pending CN104046601A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108094714A (en) * 2017-12-01 2018-06-01 杭州柏普泰生物科技有限公司 A kind of feed addictive for promoting Acipenser baeri growth and preparation method and application

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CHIA-FENG LO，ET AL: "Optimization of lipase production by burkholderia sp. Using response surface methodology", 《INT.J.MOL SCI》 *
G.SEGHAL KIRAN，ET AL: "Optimization of extracellular psychrophilic alkaline lipase produced by marine Pseudomonas sp.(MSI057)", 《BIOPROCESS BIOSYST ENG》 *
GERARDO CORZO，ET AL: "Production and characteristic s of the lipase from Yarrowialipolytica 681", 《BIORESOURCE TECHNOLOGY》 *
张小琴 等: "解脂耶氏酵母W29产脂肪酶最佳条件", 《南昌大学学报》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108094714A (en) * 2017-12-01 2018-06-01 杭州柏普泰生物科技有限公司 A kind of feed addictive for promoting Acipenser baeri growth and preparation method and application

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