CN108517329A - A kind of recombination Yarrowia lipolytica lipase and its expression bacterial strain and application - Google Patents
A kind of recombination Yarrowia lipolytica lipase and its expression bacterial strain and application Download PDFInfo
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- CN108517329A CN108517329A CN201810259811.1A CN201810259811A CN108517329A CN 108517329 A CN108517329 A CN 108517329A CN 201810259811 A CN201810259811 A CN 201810259811A CN 108517329 A CN108517329 A CN 108517329A
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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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/189—Enzymes
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
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- 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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Abstract
The invention discloses a kind of recombination Yarrowia lipolytica lipase and its expression bacterial strain with application, and the preparation method for expressing bacterial strain is：Lipase gene is connected on expression vector, recombinant plasmid is obtained；The lipase gene has such as SEQ ID NO:Nucleotide sequence shown in 1；Recombinant plasmid transformed is entered in bacillus subtilis, positive colony is selected, obtains recombination Yarrowia lipolytica fat expression of enzymes bacterial strain.Expression bacterial strain is carried out liquid fermentation for fermentation strain, Prepare restructuring Yarrowia lipolytica lipase, the vigor after culture in 16 hours is reached for 109.3U/mL.The safe and stable production for realizing feed lipase has significant application value in industries such as food, the energy, chemical industry and medicine.
Description
Technical field
The invention belongs to biotechnologys and feedstuff industry field, and in particular to a kind of recombination Yarrowia lipolytica lipase and
It expresses bacterial strain and application.
Background technology
Lipase (lipase) is a kind of typical carboxylic acid hydrolase, because energy catalyzing hydrolysis, esterification, transesterification etc. are a variety of anti-
It answers type and is widely used in the fields such as food, medicine, the energy, environment, but application and development of the lipase in terms of feed is opposite
Other feeding enzymes are still at an early stage.There are tolerance intestines by the feeding lipase of representative of Yarrowia lipolytica lipase Lip2
Feature needed for many feeding enzymes such as gastric environment, biological safety are good, pH and temperature performance are superior has very high development and application latent
Power.
By enzymatic biochemical reaction, the fat digestion for strengthening raising animal utilizes process, is ensuring to digest feeding lipase
Under the premise of road acidic environment stay in grade, improve the rate-limiting step that oil and fat assimilation absorbs in feed, solves energetic nourishing object oil
The digestibility and utilization efficiency of fat.Its specific effect is embodied in：(1) being added with for ectogenous fat enzyme is conducive to supplement cub because digesting machine
The deficiency of endogenous digestive enzyme activity and secretory volume caused by energy hypoplasia.The digestive system of adults is then because grease disappears
Change using more efficient and is lightened the burden；(2) by ecological improve animal digests and assimilates environment, can improve raising animal
Measurement techniques for quality detection of meat also can be reduced effectively because oil and fat assimilation Utilizing question induces the probability of happening of a variety of diseases.Such as：Add lipase
Animal trophism diarrhea caused by due to indigestion high in fat can be reduced, growth of animal is promoted；(3) addition of lipase can
To reduce the additive amount of grease, formulation space is saved, reduces feed cost；(4) lipase addition makes the energy profit of feedstuff
It is maximized with rate, reduces the excretion of nutriment in intensive culture, be of great significance to making green ecological aquaculture.
Bacillus subtilis is aerobic, is easy to cultivate, and growth cycle is shorter, is widely present in nature, acellular poison
Property, is easily isolated purifying, and transcription and translation is very stringent, is not likely to produce mistake, ideal type strain in microbe research.
Bacillus subtilis has the characteristics such as safe and free of pathogenicity, becomes GRAS (Generally by food Watch-dog committee of the U.S.
Recognized as Safe), and be probiotics.Secondly bacillus subtilis cell membrane is single layer, the albumen of secretion be easy recycling,
Purifying., it is easy to operate, and a variety of enzymes can be generated and be secreted into extracellularly；In addition, bacillus subtilis is easy to cultivate, it is right
Medium component requirement is not stringent, and gram use relative moderate medium component effectively reduces production cost, plasmid, bacteriophage
Cloning vector is all can be used as, without apparent codon preference, Transcription/Translation System is very tight, can be to avoid false folding
Protein and inclusion body, thus have preferable application prospect in fermentation arts.
Existing technology can carry out the production of lipase using bio-carrier, such as Yan Jinyong is given birth to using Pichia anomala expression
Yielding lipase, such as Xu Li produce lipase using conversion Yarrowia lipolytica, and then fermentation time is long (being more than 72 hours),
Production cycle is partially long.
Invention content
Disadvantages described above in view of the prior art and Improvement requirement, the present invention provide a kind of recombination Yarrowia lipolytica fat
Enzyme and its expression bacterial strain reduce the cost of production to realize the production cycle for shortening feed lipase.
To achieve the above object, according to one aspect of the present invention, a kind of recombination Yarrowia lipolytica lipase is provided
Bacterial strain is expressed, is made by following methods：
(1) lipase gene is connected on expression vector, obtains recombinant plasmid；The lipase gene has such as SEQ
ID NO:Nucleotide sequence shown in 1；
(2) recombinant plasmid transformed is entered in bacillus subtilis, selects positive colony, obtain recombination Yarrowia lipolytica fat
Fat expression of enzymes bacterial strain.
Further, the carrier is pHT43.The bacillus subtilis is WB800N.
Second aspect, the present invention provides a kind of recombination Yarrowia lipolytica lipase, to express bacterial strain as fermentation strain
It carries out liquid fermentation, prepares lipase.
Further, the fermentation process is：Expression bacterial strain is placed in the culture shaking flask of liquid amount 20-40%, 200
With shaking table culture under the rotating speed of~220rpm, 37 DEG C of cultivation temperature, 6~16h of incubation time, each component content is in culture medium：
Starch 0.15wt%, glucose 0.5wt%, urea 0.1wt%, dipotassium hydrogen phosphate 0.3wt%, potassium dihydrogen phosphate 0.15wt%,
Magnesium sulfate 0.05wt%, yeast extract 0.02wt%, iron chloride 0.01wt%, calcium carbonate 0.01wt%, dregs of beans 1wt%, 30.8mg/
The manganese sulfate solution 0.1vol% of L concentration.
The liquid amount for cultivating shaking flask is 30%, and the yield of lipase is higher, and especially when incubation time is 16h, rotating speed is
200rpm, yield reach 109.3U/mL.
Lipase activity detection method is：4mL olives oil emulsion and 5mL Tris-HCl buffer solutions (pH 8.0) is taken to add
Into the conical flask of 50mL, 40 DEG C of water-baths preheat 5min, are added appropriate diluted enzyme supernatant 1mL, reaction system 10mL, and 40
DEG C, the termination reaction of 15mL terminate liquids is added after 10min is reacted in 200rpm shaking baths.The aliphatic acid that enzymatic hydrolysis reaction generates is logical
0.05M NaOH titration is crossed, the phenolphthalein solution that two drops 0.5% are added indicates titration end-point.It is free that hydrolysis per minute generates 1 μm of ol
Aliphatic acid is defined as a unit of activity U.Enzyme activity calculation formula is：
U=(V2-V1)*10-3*M*106*N/T
Wherein：V1：Control group reaches titration end-point consumption NaOH volumes (mL)；V2：Experimental group reaches titration end-point consumption
NaOH volumes (mL)；M：The concentration (M) of titration NaOH solution used；N：Enzyme supernatant extension rate；T：Reaction time (min).
The third aspect, the application the present invention provides a kind of recombination Yarrowia lipolytica lipase as feed addictive.
In general, through the invention it is contemplated above technical scheme is compared with the prior art, can obtain following has
The effect of benefit：
By genetic engineering recombinant technique, establish Yarrowia lipolytica lipase bacillus subtilis WB800N table
It reaches, finally realizes the efficient production of feed lipase, the yield of verified recombinant lipase is 109.3U/mL.It will be above-mentioned
It recombinates Yarrowia lipolytica lipase and is applied to Feed Manufacturing as feed addictive, biological safety is good, and fermentation period is short, energy
Consume low, the clear superiorities such as mild condition, the foreground of great agricultural application.
Description of the drawings
Fig. 1 is recombinant plasmid pHT43-Lip schematic diagrames.
Case is embodied
In order to keep the purpose of the present invention, technical solution more preferably clear, with reference to the accompanying drawings and embodiments, to the present invention
It is further elaborated.It should be appreciated that specific embodiment described herein is not used to only to explain the present invention
Limit the present invention.
Embodiment 1：
(1) (there is such as SEQ IDNO with the lipase Lip in Yarrowia lipolytica fat enzyme family:Shown in 1
Nucleotide sequence) it is that aimed aliphatic enzyme gene passes through the enzyme-linked construction recombination plasmid pHT43- of digestion using pHT43 as plasmid vector
Lip, restriction enzyme site position BamHI and SmaI；
(2) by recombinant plasmid transformed bacillus subtilis WB800N；
(3) on the solid LB media containing ammonia benzyl mycin, screening positive clone obtains the recombination solution fat Ye Shi of high yield
Yeast fat expression of enzymes bacterial strain.
Embodiment 2
The acquisition bacillus subtilis engineered strain of embodiment 1 is fermented in 1. number fluid nutrient medium, shaking flask liquid amount
The lipase of 20vol%, shaking speed 220rpm, 37 DEG C, incubation time 6h of cultivation temperature, acquisition have SEQ ID NO.1
Shown in nucleotide sequence, coding albumen have SEQ ID NO.2 shown in amino acid sequence.Lipase activity reaches
37.5U/mL,
1. the matrix of number fluid nutrient medium is deionized water, each component content is：Starch 0.15wt%, glucose
0.5wt%, urea 0.1wt%, dipotassium hydrogen phosphate 0.3wt%, potassium dihydrogen phosphate 0.15wt%, magnesium sulfate 0.05wt%, yeast
The manganese sulfate solution of cream 0.02wt%, iron chloride 0.01wt%, calcium carbonate 0.01wt%, dregs of beans 1wt%, 30.8mg/L concentration
0.1vol%.
Embodiment 3
The acquisition bacillus subtilis engineered strain of embodiment 1 is fermented in 1. number fluid nutrient medium, shaking flask liquid amount
The lipase of 30vol%, shaking speed 220rpm, 37 DEG C, incubation time 6h of cultivation temperature, acquisition have SEQ ID NO.1
Shown in nucleotide sequence, coding albumen have SEQ ID NO.2 shown in amino acid sequence.Lipase activity reaches
76.2U/mL。
Embodiment 4
The acquisition bacillus subtilis engineered strain of embodiment 1 is fermented in 1. number fluid nutrient medium, shaking flask liquid amount
The lipase of 40vol%, shaking speed 220rpm, 37 DEG C, incubation time 6h of cultivation temperature, acquisition have SEQ ID NO.1
Shown in nucleotide sequence, coding albumen have SEQ ID NO.2 shown in amino acid sequence.Lipase activity reaches
49.3U/mL。
Embodiment 5
The acquisition bacillus subtilis engineered strain of embodiment 1 is fermented in 1. number fluid nutrient medium, shaking flask liquid amount
The lipase of 20vol%, shaking speed 220rpm, 37 DEG C, incubation time 12h of cultivation temperature, acquisition have SEQ ID NO.1
Shown in nucleotide sequence, coding albumen have SEQ ID NO.2 shown in amino acid sequence.Lipase activity reaches
62.1U/mL。
Embodiment 6
The acquisition bacillus subtilis engineered strain of embodiment 1 is fermented in 1. number fluid nutrient medium, shaking flask liquid amount
The lipase of 30vol%, shaking speed 220rpm, 37 DEG C, incubation time 12h of cultivation temperature, acquisition have SEQ ID NO.1
Shown in nucleotide sequence, coding albumen have SEQ ID NO.2 shown in amino acid sequence.Lipase activity reaches
97.2U/mL。
Embodiment 7
The acquisition bacillus subtilis engineered strain of embodiment 1 is fermented in 1. number fluid nutrient medium, shaking flask liquid amount
The lipase of 40vol%, shaking speed 200rpm, 37 DEG C, incubation time 12h of cultivation temperature, acquisition have SEQ ID NO.1
Shown in nucleotide sequence, coding albumen have SEQ ID NO.2 shown in amino acid sequence.Lipase activity reaches
50.2U/mL。
Embodiment 8
The acquisition bacillus subtilis engineered strain of embodiment 1 is fermented in 1. number fluid nutrient medium, shaking flask liquid amount
The lipase of 20vol%, shaking speed 200rpm, 37 DEG C, incubation time 16h of cultivation temperature, acquisition have SEQ ID NO.1
Shown in nucleotide sequence, coding albumen have SEQ ID NO.2 shown in amino acid sequence.Lipase activity reaches
74.1U/mL。
Embodiment 9
The acquisition bacillus subtilis engineered strain of embodiment 1 is fermented in 1. number fluid nutrient medium, shaking flask liquid amount
The lipase of 30vol%, shaking speed 200rpm, 37 DEG C, incubation time 16h of cultivation temperature, acquisition have SEQ ID NO.1
Shown in nucleotide sequence, coding albumen have SEQ ID NO.2 shown in amino acid sequence.Lipase activity reaches
109.3U/mL。
Embodiment 10
The acquisition bacillus subtilis engineered strain of embodiment 1 is fermented in 1. number fluid nutrient medium, shaking flask liquid amount
The lipase of 40vol%, shaking speed 200rpm, 37 DEG C, incubation time 16h of cultivation temperature, acquisition have SEQ ID NO.1
Shown in nucleotide sequence, coding albumen have SEQ ID NO.2 shown in amino acid sequence.Lipase activity reaches
64.2U/mL。
As it will be easily appreciated by one skilled in the art that the foregoing is merely illustrative of the preferred embodiments of the present invention, and do not have to one
Limitation the present invention, all within the spirits and principles of the present invention made by people and modifications, equivalent substitutions and improvements etc., should all include
Within protection scope of the present invention.
Sequence table
<110>The Hangzhou bio tech ltd Bai Putai
<120>A kind of recombination Yarrowia lipolytica lipase and its expression bacterial strain and application
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Met Lys Leu Ser Thr Ile Leu Phe Thr Ala Cys Ala Thr Leu Ala Ala
1 5 10 15
Ala Leu Pro Ser Pro Ile Thr Pro Ser Glu Ala Ala Val Leu Gln Lys
20 25 30
Arg Val Tyr Thr Ser Thr Glu Thr Ser His Ile Asp Gln Glu Ser Tyr
35 40 45
Asn Phe Phe Glu Lys Tyr Ala Arg Leu Ala Asn Ile Gly Tyr Cys Val
50 55 60
Gly Pro Gly Thr Lys Ile Phe Lys Pro Phe Asn Cys Gly Leu Gln Cys
65 70 75 80
Ala His Phe Pro Asn Val Glu Leu Ile Glu Glu Phe His Asp Pro Arg
85 90 95
Leu Ile Phe Asp Val Ser Gly Tyr Leu Ala Val Asp His Ala Ser Lys
100 105 110
Gln Ile Tyr Leu Val Ile Arg Gly Thr His Ser Leu Glu Asp Val Ile
115 120 125
Thr Asp Ile Arg Ile Met Gln Ala Pro Leu Thr Asn Phe Asp Leu Ala
130 135 140
Ala Asn Ile Ser Ser Thr Ala Thr Cys Asp Asp Cys Leu Val His Asn
145 150 155 160
Gly Phe Ile Gln Ser Tyr Asn Asn Thr Tyr Asn Gln Ile Gly Pro Lys
165 170 175
Leu Asp Ser Val Ile Glu Gln Tyr Pro Asp Tyr Gln Ile Ala Val Thr
180 185 190
Gly His Ser Leu Gly Gly Ala Ala Ala Leu Leu Phe Gly Ile Asn Leu
195 200 205
Lys Val Asn Asp His Asp Pro Leu Val Val Thr Leu Gly Gln Pro Ile
210 215 220
Val Gly Asn Ala Gly Phe Ala Asn Trp Val Asp Lys Leu Phe Phe Gly
225 230 235 240
Gln Glu Asn Pro Asp Val Ser Lys Val Ser Lys Asp Arg Lys Leu Tyr
245 250 255
Arg Ile Thr His Arg Gly Asp Ile Val Pro Gln Val Pro Phe Trp Asp
260 265 270
Gly Tyr Gln His Cys Ser Gly Glu Val Phe Ile Asp Trp Pro Leu Ile
275 280 285
His Pro Pro Leu Ser Asn Val Val Met Cys Gln Gly Gln Ser Asn Lys
290 295 300
Gln Cys Ser Ala Gly Asn Thr Leu Leu Gln Gln Val Asn Val Ile Gly
305 310 315 320
Asn His Leu Gln Tyr Phe Val Thr Glu Gly Val Cys Gly Ile
325 330
Claims (8)
1. a kind of recombination Yarrowia lipolytica fat expression of enzymes bacterial strain, which is characterized in that be made by following methods：
(1) lipase gene is connected on expression vector, obtains recombinant plasmid；The lipase gene has such as SEQ ID
NO:Nucleotide sequence shown in 1；
(2) recombinant plasmid transformed is entered in bacillus subtilis, selects positive colony, obtain recombination Yarrowia lipolytica lipase
Express bacterial strain.
2. expression bacterial strain as is described in the claims, which is characterized in that the carrier is pHT43.
3. expression bacterial strain as is described in the claims, which is characterized in that the bacillus subtilis is WB800N.
4. a kind of recombination Yarrowia lipolytica lipase, which is characterized in that the expression bacterial strain obtained using claim 1 is zymophyte
Strain carries out liquid fermentation, and prepares lipase.
5. lipase according to claim 4, which is characterized in that the fermentation process is：Expression bacterial strain is placed in dress liquid
In the culture shaking flask for measuring 20-40%, with shaking table culture, 37 DEG C of cultivation temperature, incubation time 6 under the rotating speed of 200~220rpm
~16h, each component content is in culture medium：Starch 0.15wt%, glucose 0.5wt%, urea 0.1wt%, dipotassium hydrogen phosphate
0.3wt%, potassium dihydrogen phosphate 0.15wt%, magnesium sulfate 0.05wt%, yeast extract 0.02wt%, iron chloride 0.01wt%, carbonic acid
The manganese sulfate solution 0.1vol% of calcium 0.01wt%, dregs of beans 1wt%, 30.8mg/L concentration.
6. lipase according to claim 5, which is characterized in that the liquid amount for cultivating shaking flask is 30%.
7. lipase according to claim 6, which is characterized in that incubation time 16h, rotating speed 200rpm.
8. application of the recombination Yarrowia lipolytica lipase as feed addictive described in a kind of claim 4.
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| CN201810259811.1A CN108517329A (en) | 2018-03-27 | 2018-03-27 | A kind of recombination Yarrowia lipolytica lipase and its expression bacterial strain and application |
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| CN201810259811.1A CN108517329A (en) | 2018-03-27 | 2018-03-27 | A kind of recombination Yarrowia lipolytica lipase and its expression bacterial strain and application |
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ID=63434276
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110760451A (en) * | 2019-10-21 | 2020-02-07 | 广东轻工职业技术学院 | Yarrowia lipolytica and application thereof in preparation of low-sugar low-fat desiccated coconut nutrition powder |
| CN112899177A (en) * | 2021-02-02 | 2021-06-04 | 中国海洋大学 | Recombinant yarrowia lipolytica expressing myrosinase TGG4 and application thereof |
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| CN102573516A (en) * | 2009-04-24 | 2012-07-11 | 丹尼斯科有限公司 | Feed supplement |
| CN103243117A (en) * | 2013-05-24 | 2013-08-14 | 江南大学 | Method for cloning and expressing Serratia marcescens lipase by utilizing recombinant Bacillus subtilis |
| CN107245470A (en) * | 2017-05-24 | 2017-10-13 | 华南理工大学 | A kind of lipase expression of recombinant e. coli bacterial strain, recombinant lipase and application |
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| YU,M. ET AL.: "ABG81956.1", 《GENBANK》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110760451A (en) * | 2019-10-21 | 2020-02-07 | 广东轻工职业技术学院 | Yarrowia lipolytica and application thereof in preparation of low-sugar low-fat desiccated coconut nutrition powder |
| WO2021077788A1 (en) * | 2019-10-21 | 2021-04-29 | 广东轻工职业技术学院 | Yarrowia lipolytica strain and use of same in preparation of low-sugar low-fat dessicated coconut nutritional powder |
| CN112899177A (en) * | 2021-02-02 | 2021-06-04 | 中国海洋大学 | Recombinant yarrowia lipolytica expressing myrosinase TGG4 and application thereof |
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