CN105861341A - Debaryomyces hansenii bacterial strain and method for preparing 3-hydroxypropionic acid by fermenting debaryomyces hansenii bacterial strain - Google Patents
Debaryomyces hansenii bacterial strain and method for preparing 3-hydroxypropionic acid by fermenting debaryomyces hansenii bacterial strain Download PDFInfo
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Abstract
The invention discloses a Debaryomyces hansenii bacterial strain. The Debaryomyces hansenii bacterial strain is Debaryomyces hansenii. IS451, and the preservation number is CGMCC No.11893. A method for preparing 3-hydroxypropionic acid by fermenting the bacterial strain comprises the following steps that an agar slant culture medium is inoculated with the IS451 bacterial strain to activate the bacterial strain, then a seed medium is inoculated with the activated IS451 bacterial strain, the IS451 bacterial strain is cultured in a constant-temperature oscillator to a logarithmic phase, and IS451 bacterial strain seed liquid is prepared; a fermentation culture medium is inoculated with the cultured IS451 bacterial strain seed liquid according to the volume ratio of 10%, and the pH is regulated to 6.0; the IS451 bacterial strain seed liquid is cultured in the constant-temperature oscillator, fermenting is conducted for 5 days, and then fermentation liquid is prepared; it is detected that the content of 3-hydroxypropionic acid in the fermentation liquid reaches 48.96 g/L and is three times or above higher than that of a method for producing 3-hydroxypropionic acid through microbial fermentation in literature reports, and the wide application prospect is achieved.
Description
Technical field
The present invention relates to a strain Chinese inferior Dbaly yeast bacterial strain and the method preparing 3-hydracrylic acid of fermenting thereof, belong to biological
Chemical industry and field of fermentation engineering.
Background technology
Being affected by world petroleum resource, price, environmental protection and Global climate change etc., exploitation bio-based source has become perhaps
Energy resource safety, GHG emissions mitigation, the important measures of reply climate change improve in many countries.3-hydracrylic acid (3-hp)
Being a kind of important chemical intermediate of rising in recent years, 3-hp has hydroxyl and carboxyl Liang Zhong functional group, is that a lot of optics is lived
Property material precursor, be a kind of important platform chemicals, its yield directly affects the production of many high valuable chemicals.
3-hp can be as producing malonic acid, 1,3-PD (PDO), succinic acid, specialty polyesters and acrylic acid raw material, it is also possible to system
Standby multiple important fine chemical product.At present, one of chemical products being classified as most potentiality to be exploited by USDOE.Tradition system
Standby 3-hp is chemical synthesis, and the 3-hydroxyl nitrile generated with potassium cyanide effect such as adjacent halohydrin, by hydrolysis or Reformatsky
Reaction prepares.It is catalyzed 3 monohydroxy propionic aldehyde oxidations by platinum and produces 3-hp and the technique of salt thereof.Use solid acid catalyst ZSM5
Zeolite propene hydrate acid produces 3-hp.Employing chemical synthesis produces, and difficulty is relatively big, and separation and purification of products is more complicated, raw
Product cost is high.
By contrast, biological method can be effectively prevented from these unfavorable factors, has the spy such as low cost, mild condition
Point.Therefore, preparing 3-hp with biological method, particularly genetic engineering bacterium method has become the modern study hotspot gazed at most
One of.Cargill-Dow company develops and utilizes genetic engineering bacterium to express alanine 2,3, aminomutase, with carbon hydrate
The technique that thing produces 3-hp.Korea S scholar produces 3-hp to bioanalysis in recent years and is studied: table in recombination bacillus coli
Reaching malonyl-CoA reductase, acetyl-CoA decarboxylase, biotin enzyme and transhydrogenase gene, the genetic engineering bacterium of structure can
To utilize glucose to produce the 3-hp of 1.2 mmol/L for substrate；Construct the recombination bacillus coli with glycerol as substrate, express
Glycerol dehydratase and acetaldehyde dehydrogenase gene, 3-hp yield is 0.58 g/L；Additionally by knocking out glycerol dehydrogenase and mistake
Expressing acetaldehyde dehydrogenase gene, the 3-hp yield of recombinant bacterium is 2.07 g/L.Domestic Southern Yangtze University uses similar thinking to build
Recombination bacillus coli, with glycerol as substrate, 3-hp yield is 4.92 g/L, and other relevant bioanalysises produce the report of 3-hp
The most little.
Several genes is related to owing to building genetic engineering bacterium, and these genes stability in host cell, enzyme
Activity expression and the faces enormous challenge in actual production such as cellular metabolism flow control, still have many technical problems to have
To be solved.At present report the natural bacterium of glycerol production 3-hp can be utilized less, yield is relatively low.Known can fermenting and producing 3-
The wild strain of hp mainly has: Han-senuamiso, Fusarium merismoides, Candida rugosa,
Byssochlamys sp., Rhodococcus eryt-hropolis LG12 and Klebsiella terrigena etc..Wherein,
Li Bing and Pei Jiang gloomy from natural environment screening can metabolism produce 3-hp microbial strains Klebsiella terrigena
(Klebsiella terrigena) can produce 3-hp with glycerol for sole carbon source, and acid producing ability is 1% (10 g/L), for the first time
Realize wild-type strain and be directly converted into 3-hp from glycerol.
Fan Junying selection-breeding obtains a strain 3-HP and efficiently synthesizes bacterial strain, they with gather soil and fecal specimens for research material
Material, therefrom
Screening obtains a strain and propionic fermentation can be utilized to produce the yeast Y-11 of 3-HP, identifies and 18S rDNA sequence through Physiology and biochemistry
Row are analyzed, and primarily determine that the bacterial strain of screening is Candida sp. (candida mycoderma).Again with Y-11 as starting strain, through ultraviolet-
Nitrosoguanidine-60Co γ complex mutation, further screening has obtained the mutant character and has stablized and heritable superior strain 5-13B,
The yield of this bacterial strain 3-HP is 11.78 g/L, is 2.46 times of starting strain.Fermentable is produced 3-HP's by this result
Research brings up to new level.In the abundantest microbial resources storehouse, due to natural environment and the difference of other physico chemical factor
Different, still there is the microorganism that much can produce 3-hydracrylic acid undiscovered, be separated to now is also only a drop in the ocean.Therefore,
The microbial strains producing 3-hydracrylic acid also needs further to find and study.
Country flourishing in the world has been realized in bio-based and converts the industrialization of 3-hp, and produces as bio-based platform
Other important fine chemical product, has been widely used in medicine, biomaterial, functional material, bio-fuel and other has been fine
The industries such as chemicals, become indispensable important raw and processed materials during national economy produces.In the world, use microorganism convert and
Engineering bacteria synthesis 3-hp has become mainstream technology, and utilizing fermentable to produce 3-hp can effectively avoid chemical synthesis to be brought
Unfavorable factor, and have with short production cycle, abundant raw material, environmental protection, the advantages such as production cost is low, can be with production economy valency
It is worth higher chemical intermediate.Constantly carry out improvement and the innovation of prior art, solve the environmental conservation in producing, reduce life
Produce cost and expansion industrialized scale etc. and become the research emphasis in this technical field current.
Screen new 3-hydracrylic acid producing strains, biological fermentation process can be enriched and prepare the kind of 3-hydracrylic acid bacterial strain, for
Biochemical industry and the research of biofermentation and application provide foundation, for building the resource that 3-hp high efficiency engineering bacterium provides new, benefit
The mankind, solve the problems such as energy security and have very important significance.
Summary of the invention
The present invention, for building the microorganism that 3-hydracrylic acid (3-hp) high efficiency engineering bacterium provides new, specifically provides a strain Chinese
Inferior Dbaly yeast bacterial strain, the described Chinese inferior Dbaly yeast bacterial strain is the Chinese inferior Dbaly yeast bacterium (Debaryomyces
Hansenii.) IS451, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center；Address: north
North Star West Road, Jing Shi Chaoyang District 1 No. 3 Institute of Microorganism, Academia Sinica of institute；Preservation date: on December 18th, 2015；Preservation
Number it is: CGMCC No.11893.
The method that a kind of Chinese as described above inferior Dbaly yeast strain fermentation prepares 3-hydracrylic acid, as follows
Preparation:
Step 1) by inferior for Chinese Dbaly yeast bacterium (Debaryomyces hansenii.) IS451 inoculation in slant culture
Base activates, 30 DEG C of constant temperature culture 24h, take 2 ring inclined-plane IS451 bacterial strains, i.e. connect inclined-plane bacterial strain with 2 points of inoculating loop；
In described slant medium, each component and consumption thereof are: glucose 20.00 g/L, yeast extract 10.00 g/L, albumen
Peptone 20.00 g/L, agar powder 20.00 g/L.
Accessing seed culture medium again, culture medium liquid amount is 50mL/250mL, cultivates, rotating speed 180 r/ in constant temperature oscillator
Min, cultivates 48h at 30 DEG C, cultivates to logarithmic (log) phase, prepare IS451 bacterial strain seed liquor；
In described seed culture medium, each component and consumption thereof are: glucose, 20.00 g/L；Yeast extract, 10.00 g/L；
(NH4)2SO4, 10.00 g/L；KH2PO4 , 5.00 g/L； K2HPO4, 2.00 g/L；MgSO4·7 H2O, 1.00 g/L；
FeSO4·7 H2O, 0.03 g/L；TES 5 mL；
Step 2) it is 10% to be inoculated in cultured for step 1) IS451 bacterial strain seed liquor in fermentation medium according to volume ratio, adjust
Joint pH to 6.0；Culture medium liquid amount is 100mL/250mL, cultivates, rotating speed 180 r/min, 30 DEG C in constant temperature oscillator, fermentation
5 days time, prepare fermentation liquid；
In described fermentation medium, each component and consumption thereof are: glucose 25.00-35.00 g/L；Yeast extract 10.00 g/
L；KH2PO4, 5.00 g/L；K2HPO4, 2.00 g/L；(NH4)2SO4, 10.00-25.00 g/L；MgSO4·7H2O, 1.00
g/L；FeSO4·7 H2O, 0.03 g/L；Propanoic acid, 10.00-25.00 g/L；Glycerol, 5.00-15.00 g/L；TES, 5 mL；
Step 3) detecting step 2) prepare fermentation liquid in 3-hydroxypropionic acid content；
In above-mentioned steps, in described TES, each component and consumption thereof are: HCl, 3.65 g/L；H3BO4, 0.30 g/L；CuCl2·
6H2O, 0.20 g/L；ZnSO4·7H2O, 0.10 g/L；MnSO4 , 0.03 g/L；NaMoO4·2KH2PO4, 0.03 g/L；
NiCl2·6H2O, 0.02 g/L；CuSO4·5H2O, 0.01 g/L.
Described step 2) in fermentation medium each component and consumption thereof be optimized for further: glucose 30.00 g/L；
Yeast extract 10.00 g/L；KH2PO4, 5.00 g/L；K2HPO4, 2.00 g/L；(NH4)2SO4, 15.00 g/L；MgSO4·7H2O,
1.00 g/L；FeSO4·7 H2O, 0.03 g/L；Propanoic acid, 15.00 g/L；Glycerol, 10.00 g/L；TES, 5 mL；
In described TES, each component and consumption thereof are: HCl, 3.65 g/L；H3BO4, 0.30 g/L；CuCl2·6H2O, 0.20
g/L；ZnSO4·7H2O, 0.10 g/L；MnSO4 , 0.03 g/L；NaMoO4·2KH2PO4, 0.03 g/L；NiCl2·6H2O,
0.02 g/L；CuSO4·5H2O, 0.01 g/L.Technical scheme is summarized as follows:
The method have the benefit that
The present invention filters out a plant height from orchard soil and human feces and produces the Chinese inferior Dbaly yeast bacterium of 3-hydracrylic acid
(Debaryomyces hansenii.) IS451, it is relatively strong that this bacterial strain adapts to ability, and breeding is fast, with short production cycle；There is fermentation
Cultivation abundant raw material is various, inexpensive while also environmental protection, simultaneously can the advantage such as large-scale production continuously；In optimum culture scheme
Under, this strain fermentation produces the amount of 3-hydracrylic acid and is up to 48.96 g/L, produces 3-hydracrylic acid than document report fermentable
Content exceeds more than 3 times, and this bacterial strain is with a wide range of applications.
Accompanying drawing explanation
Fig. 1 is the cellular morphology figure of the Chinese inferior Dbaly yeast bacterium (Debaryomyces hansenii.) IS451 bacterial strain；
Fig. 2 is the Chinese inferior Dbaly yeast bacterium (Debaryomyces hansenii.) IS451 bacterial strain colonial morphology；
Fig. 3 is the PCR amplification of the Chinese inferior Dbaly yeast bacterium (Debaryomyces hansenii.) IS451 bacterial strain；
Fig. 4 is that the Phylogenetic of the Chinese inferior Dbaly yeast bacterium (Debaryomyces hansenii.) IS451 bacterial strain divides
Analysis；
Fig. 5 is 3-hydracrylic acid mark product HPLC figure；
Fig. 6 is that under the present embodiment 2 fermentation condition, the Chinese inferior Dbaly yeast bacterium (Debaryomyces hansenii.) IS451 produces
The HPLC figure of 3-hydroxypropionic acid content；
Fig. 7 is that under the present embodiment 3 fermentation condition, the Chinese inferior Dbaly yeast bacterium (Debaryomyces hansenii.) IS451 produces
3-hydroxypropionic acid content HPLC schemes.
Detailed description of the invention
Below in conjunction with embodiment, method and the effect of the present invention are described further.
Embodiment 1: the separation screening of the Chinese inferior Dbaly yeast bacterium (Debaryomyces hansenii.) IS451 bacterial strain
With purification
The sample collecting of the Chinese inferior Dbaly yeast bacterium (Debaryomyces hansenii.) IS451 bacterial strain: be mainly derived from fruit
Garden gather return soil and acquisition human feces sample in isolated.
The method of its screening and separating is: will gather a small amount of sample of human feces of soil and the acquisition returned from orchard
It is soaked in respectively in physiological saline solution, stands 12 h；After 12 h, bacteria suspension being carried out gradient dilution, its gradient dilution concentration depends on
Secondary is 10-4, 10-5, 10-6, 10-7, 10-8, 10-9；Bacteria suspension after dilution, to be transferred；
Preparation propionic acid content (V/V) is 0.4%, 0.8%, 1.2%, 1.6%, and the screening culture medium of 2.0%, and regulate pH value with pH meter
Being 6.0, after sterilizing, be down flat plate, the amount of each plate screening culture medium controls, at 10 mL, to be screening flat board after cooling；
The bacterial suspension inoculation after 0.2 mL dilution is drawn respectively on the screening flat board cooled down, and with sterilized with liquid-transfering gun
Spreading rod is coated with so that sample distribution is uniform at screening planar surface gently, and each bacteria suspension dilution gradient is coated on different propanoic acid
On the screening flat board of content；
The screening flat board accessing strain diluent is placed in 30 DEG C of constant incubators inversion and cultivates 48h, observe microorganism raw
Long situation；For obtaining purpose bacterial strain further, the method needing to take line to separate.Constant temperature is inverted the flat board cultivated take out,
In an aseptic environment, with inoculating loop picking single bacterium colony therein in the flat lining out of above-mentioned screening, then proceed to cultivate, repeat this
Operate 3 to 5 times, cultivate about 2 d every time, until single bacterium colony occurs.
Described screening culture medium (g/L): glucose 20.00, yeast extract 10.00, (NH4)2SO410.00, KH2PO4
5.00, K2HPO4 2.00, MgSO4·7 H2O 1.00, FeSO4·7 H2O 0.03, TES 5 mL.
Described slant medium (g/L): glucose 20.00, yeast extract 10.00, peptone 20.00, agar powder
20.00。
TES(trace element solution g/L): HCl 3.65, H3BO4 0.30, CuCl2·6H2O 0.20, ZnSO4·7H2O
0.10, MnSO4 0.03, NaMoO4·2 KH2PO40.03, NiCl2·6H2O 0.02, CuSO4·5H2O 0.01。
Under this bacterial strain microscope, visible bacterial strain thalline is spherical, sees Fig. 1；Base plate is cultivated bacterium colony and presents greyish white
Color, spherical for projection, surface is soft and moistening, easily provoke, neat in edge, see Fig. 2.
Its physiological and biochemical property is: carbon assimilation glucose, sucrose, maltose, lactose, soluble starch；Nitrogen source is assimilated
Ammonium sulfate, potassium nitrate, ammonium nitrate；Carbamide can not be assimilated；The pH scope being suitable for this Yeast Growth is 4.0-6.0, the most suitable growth
Temperature is 30 DEG C.
Through detecting to obtain this bacterial strain 18S rDNA sequence, as shown in SEQ ID No.1 in sequence table.
By 18S rDNA sequence analysis, PCR amplification figure is shown in that Fig. 3, morphologic observation and Physiology and biochemistry are identified, determines
IS451 bacterium is the Chinese inferior Dbaly yeast bacterium (Debaryomyces hansenii.), and Fig. 4 is bacterial strain Debaryomyces
Hansenii. the phylogenetic tree analysis on Position of IS451.
Embodiment 2: under optimum fermentation condition, Debaryomyces hansenii. IS451 bacterial strain high yield 3-hydroxyl third
Acid situation
The method that the described Chinese inferior Dbaly yeast strain fermentation prepares 3-hydracrylic acid, is prepared as follows:
Step 1) by inferior for Chinese Dbaly yeast bacterium (Debaryomyces hansenii.) IS451 inoculation in slant culture
Base activates, 30 DEG C of constant temperature culture 24h, take 2 ring inclined-plane IS451 bacterial strains, i.e. connect inclined-plane bacterial strain with 2 points of inoculating loop；
In described slant medium, each component and consumption thereof are: glucose 20.00 g/L, yeast extract 10.00 g/L, peptone
20.00 g/L, agar powder 20.00 g/L.
Accessing seed culture medium again, culture medium liquid amount is 50mL/250mL, cultivates, rotating speed 180 r/ in constant temperature oscillator
Min, cultivates 48h at 30 DEG C, cultivates to logarithmic (log) phase, prepare IS451 bacterial strain seed liquor；
In described seed culture medium, each component and consumption thereof are: glucose, 20.00 g/L；Yeast extract, 10.00 g/L；
(NH4)2SO4, 10.00 g/L；KH2PO4 , 5.00 g/L； K2HPO4, 2.00 g/L；MgSO4·7 H2O, 1.00 g/L；
FeSO4·7 H2O, 0.03 g/L；TES 5 mL；
In described TES, each component and consumption thereof are: HCl, 3.65 g/L；H3BO4, 0.30 g/L；CuCl2·6H2O, 0.20
g/L；ZnSO4·7H2O, 0.10 g/L；MnSO4 , 0.03 g/L；NaMoO4·2KH2PO4, 0.03 g/L；NiCl2·6H2O,
0.02 g/L；CuSO4·5H2O, 0.01 g/L.
Step 2) it is 10% to be inoculated in fermentation medium by cultured for step 1) IS451 bacterial strain seed liquor according to volume ratio
In, regulate pH to 6.0；Culture medium liquid amount is 100mL/250mL, cultivates, rotating speed 180 r/min, 30 DEG C in constant temperature oscillator,
Fermentation time 5 days, prepares fermentation liquid；
In described fermentation medium, each component and consumption thereof are: glucose 30.00 g/L；Yeast extract 10.00 g/L；
KH2PO4, 5.00 g/L；K2HPO4, 2.00 g/L；(NH4)2SO4, 15.00 g/L；MgSO4·7H2O, 1.00 g/L；FeSO4·
7 H2O, 0.03 g/L；Propanoic acid, 15.00 g/L；Glycerol, 10.00 g/L；TES, 5 mL；
In described TES, each component and consumption thereof are: HCl, 3.65 g/L；H3BO4, 0.30 g/L；CuCl2·6H2O, 0.20
g/L；ZnSO4·7H2O, 0.10 g/L；MnSO4 , 0.03 g/L；NaMoO4·2KH2PO4, 0.03 g/L；NiCl2·6H2O,
0.02 g/L；CuSO4·5H2O, 0.01 g/L.
Step 3) detecting step 2) prepare fermentation liquid in 3-hydroxypropionic acid content；With high performance liquid chromatography (chromatographic column:
Ecosile C18 post (250 mm × 4.6 mm, 5 μm)；Flowing phase: 3% methanol, uses H3PO4Adjust pH to 2.0；Testing conditions:
UV-detector 210 nm, column temperature 35 DEG C, flow velocity 0.8 mL/ min, sample size 20 μ L) 3-hydroxyl in detecting step 2 fermentation liquid
The content of base propanoic acid.Now, after Debaryomyces hansenii. IS451 strain fermentation, the content of 3-hydracrylic acid reaches
48.96 g/L.3-hydracrylic acid mark product HPLC figure is shown in 3-hydroxypropionic acid content prepared by Fig. 5, the present embodiment, and its HPLC schemes, and sees
Fig. 6.
Embodiment 3: under general fermentation condition, Debaryomyces hansenii. IS451 bacterial strain produces 3-hydracrylic acid
Situation
The method that the described Chinese inferior Dbaly yeast strain fermentation prepares 3-hydracrylic acid, is prepared as follows:
Step 1) by inferior for Chinese Dbaly yeast bacterium (Debaryomyces hansenii.) IS451 inoculation in slant culture
Base activates, 30 DEG C of constant temperature culture 24h, take 2 ring inclined-plane IS451 bacterial strains, i.e. connect inclined-plane bacterial strain with 2 points of inoculating loop；
In described slant medium, each component and consumption thereof are: glucose 20.00 g/L, yeast extract 10.00 g/L, peptone
20.00 g/L, agar powder 20.00 g/L.
Accessing seed culture medium again, culture medium liquid amount is 50mL/250mL, cultivates, rotating speed 180 r/ in constant temperature oscillator
Min, cultivates 48h at 30 DEG C, cultivates to logarithmic (log) phase, prepare IS451 bacterial strain seed liquor；
In described seed culture medium, each component and consumption thereof are: glucose, 20.00 g/L；Yeast extract, 10.00 g/L；
(NH4)2SO4, 10.00 g/L；KH2PO4 , 5.00 g/L； K2HPO4, 2.00 g/L；MgSO4·7 H2O, 1.00 g/L；
FeSO4·7 H2O, 0.03 g/L；TES 5 mL.
Step 2) it is 10% to be inoculated in fermentation medium by cultured for step 1) IS451 bacterial strain seed liquor according to volume ratio
In, regulate pH to 6.0；Culture medium liquid amount is 100mL/250mL, cultivates, rotating speed 180 r/min, 30 DEG C in constant temperature oscillator,
Fermentation time 5 days, prepares fermentation liquid；
In described fermentation medium, each component and consumption thereof are: glucose 35.00 g/L；Yeast extract 10.00 g/L；KH2PO4
, 5.00 g/L；K2HPO4, 2.00 g/L；(NH4)2SO4, 10.00 g/L；MgSO4·7H2O, 1.00 g/L；FeSO4·7
H2O, 0.03 g/L；Propanoic acid, 10.00 g/L；Glycerol, 5.00 g/L；TES, 5 mL；
In described TES, each component and consumption thereof are: HCl, 3.65 g/L；H3BO4, 0.30 g/L；CuCl2·6H2O, 0.20
g/L；ZnSO4·7H2O, 0.10 g/L；MnSO4 , 0.03 g/L；NaMoO4·2KH2PO4, 0.03 g/L；NiCl2·6H2O,
0.02 g/L；CuSO4·5H2O, 0.01 g/L.
Step 3) detecting step 2) prepare fermentation liquid in 3-hydroxypropionic acid content；With high performance liquid chromatography (chromatographic column:
Ecosile C18 post (250 mm × 4.6 mm, 5 μm)；Flowing phase: 3% methanol, uses H3PO4Adjust pH to 2.0；Testing conditions:
UV-detector 210 nm, column temperature 35 DEG C, flow velocity 0.8 mL/ min, sample size 20 μ L) 3-hydroxyl in detecting step 2 fermentation liquid
The content of base propanoic acid.Now, after Debaryomyces hansenii. IS451 strain fermentation, the content of 3-hydracrylic acid reaches
36.39 g/L；3-hydracrylic acid mark product HPLC figure is shown in Fig. 5；3-hydroxypropionic acid content prepared by the present embodiment, its HPLC schemes, sees
Fig. 7.
<110>Xuzhou Engineering Institute
The method that 3-hydracrylic acid is prepared in<120>the one strain Chinese inferior Dbaly yeast bacterial strains and fermentation thereof
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 597
<212> DNA
<213>artificial sequence
<400> 1
AAAGAAACCA ACAGGGATTG CCTTAGTAAC GGCGAGTGAA GCGGCAAAAG CTCAAATTTG 60
AAATCTGGCG CCTTCGGTGT CCGAGTTGTA ATTTGAAGAA GGTAACTTTG GAGTTGGCTC 120
TTGTCTATGT TCCTTGGAAC AGGACGTCAC AGAGGGTGAG AATCCCGTGC GATGAGATGC 180
CCAATTCTAT GTAAAGTGCT TTCGAAGAGT CGAGTTGTTT GGGAATGCAG CTCTAAGTGG 240
GTGGTAAATT CCATCTAAAG CTAAATATTG GCGAGAGACC GATAGCGAAC AAGTACAGTG 300
ATGGAAAGAT GAAAAGAACT TTGAAAAGAG AGTGAAAAAG TACGTGAAAT TGTTGAAAGG 360
GAAGGGCTTG AGATCAGACT TGGTATTTTG CGATCCTTTC CTTCTTGGTT GGGTTCTCCG 420
CAGCTTACTG GGCCAGCATC GGTTTGGATG GTAGGATAAT GATTAAGGAA TGTGGCTCTA 480
CTTCGGTGGA GTGTTATAGC CTTGGTTGAT ACTGCCTGTC TAGACCGAGG ACTGCGTCTT 540
TGACTAGGAT GCTGGCATAA TGATCTTAAG CCACCCGTC 579
Claims (3)
1. a strain Chinese inferior Dbaly yeast bacterial strain, it is characterised in that: the described Chinese inferior Dbaly yeast bacterial strain is Han Xunde Bali
Yeast (Debaryomyces hansenii.) IS451, preserving number is: CGMCC No.11893.
2. the method that the Chinese as claimed in claim 1 inferior Dbaly yeast strain fermentation prepares 3-hydracrylic acid, its feature
It is: be prepared as follows:
Step 1) by inferior for Chinese Dbaly yeast bacterium (Debaryomyces hansenii.) IS451 inoculation in slant culture
Base activates, 30 DEG C of constant temperature culture 24h, take 2 ring inclined-plane IS451 bacterial strains, i.e. connect inclined-plane bacterial strain with 2 points of inoculating loop；
In described slant medium, each component and consumption thereof are: glucose 20.00 g/L, yeast extract 10.00 g/L, albumen
Peptone 20.00 g/L, agar powder 20.00 g/L；
Accessing seed culture medium again, culture medium liquid amount is 50mL/250mL, cultivates, rotating speed 180 r/min in constant temperature oscillator,
At 30 DEG C, cultivate 48h, cultivate to logarithmic (log) phase, prepare IS451 bacterial strain seed liquor；
In described seed culture medium, each component and consumption thereof are: glucose, 20.00 g/L；Yeast extract, 10.00 g/L；
(NH4)2SO4, 10.00 g/L；KH2PO4 , 5.00 g/L； K2HPO4, 2.00 g/L；MgSO4·7 H2O, 1.00 g/L；
FeSO4·7 H2O, 0.03 g/L；TES 5 mL；
Step 2) it is 10% to be inoculated in cultured for step 1) IS451 bacterial strain seed liquor in fermentation medium according to volume ratio, adjust
Joint pH to 6.0；Culture medium liquid amount is 100mL/250mL, cultivates, rotating speed 180 r/min, 30 DEG C in constant temperature oscillator, fermentation
5 days time, prepare fermentation liquid；
In described fermentation medium, each component and consumption thereof are: glucose 25.00-35.00 g/L；Yeast extract 10.00 g/
L；KH2PO4, 5.00 g/L；K2HPO4, 2.00 g/L；(NH4)2SO4, 10.00-25.00 g/L；MgSO4·7H2O, 1.00
g/L；FeSO4·7 H2O, 0.03 g/L；Propanoic acid, 10.00-25.00 g/L；Glycerol, 5.00-15.00 g/L；TES, 5 mL；
Step 3) detecting step 2) prepare fermentation liquid in 3-hydroxypropionic acid content；
In above-mentioned steps, in described TES, each component and consumption thereof are: HCl, 3.65 g/L；H3BO4, 0.30 g/L；CuCl2·
6H2O, 0.20 g/L；ZnSO4·7H2O, 0.10 g/L；MnSO4 , 0.03 g/L；NaMoO4·2KH2PO4, 0.03 g/L；
NiCl2·6H2O, 0.02 g/L；CuSO4·5H2O, 0.01 g/L.
The method that the Chinese the most according to claim 2 inferior Dbaly yeast strain fermentation prepares 3-hydracrylic acid, its feature exists
In described step 2) in fermentation medium each component and consumption thereof be: glucose 30.00 g/L；Yeast extract 10.00 g/
L；KH2PO4, 5.00 g/L；K2HPO4, 2.00 g/L；(NH4)2SO4, 15.00 g/L；MgSO4·7H2O, 1.00 g/L；
FeSO4·7 H2O, 0.03 g/L；Propanoic acid, 15.00 g/L；Glycerol, 10.00 g/L；TES, 5 mL；
In described TES, each component and consumption thereof are: HCl, 3.65 g/L；H3BO4, 0.30 g/L；CuCl2·6H2O, 0.20 g/
L；ZnSO4·7H2O, 0.10 g/L；MnSO4 , 0.03 g/L；NaMoO4·2KH2PO4, 0.03 g/L；NiCl2·6H2O, 0.02
g/L；CuSO4·5H2O, 0.01 g/L.
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| CN106967708A (en) * | 2016-08-23 | 2017-07-21 | 徐州工程学院 | A kind of method of the secondary inferior Dbaly yeast of the immobilization Chinese of utilization composite |
| CN108949596A (en) * | 2018-08-22 | 2018-12-07 | 上海海洋大学 | A kind of preparation of composite ferment and the application in freezing flour-dough production |
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| CN109852605A (en) * | 2019-01-16 | 2019-06-07 | 徐州工程学院 | A kind of method of mutagenesis screening high yield 3- hydracrylic acid bacterial strain |
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| CN113817662A (en) * | 2021-10-08 | 2021-12-21 | 徐州工程学院 | Inducer for improving yield of 3-hydroxypropionic acid prepared by microbiological method |
| CN114806593A (en) * | 2022-05-05 | 2022-07-29 | 北京化工大学 | Preparation method of composite soil remediation agent containing 3-hydroxypropionic acid fermentation liquor |
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| CN106967708A (en) * | 2016-08-23 | 2017-07-21 | 徐州工程学院 | A kind of method of the secondary inferior Dbaly yeast of the immobilization Chinese of utilization composite |
| CN106967708B (en) * | 2016-08-23 | 2020-08-18 | 徐州工程学院 | Method for secondary immobilization of debaryomyces hansenii by using composite material |
| CN108949596A (en) * | 2018-08-22 | 2018-12-07 | 上海海洋大学 | A kind of preparation of composite ferment and the application in freezing flour-dough production |
| CN109749936A (en) * | 2019-01-16 | 2019-05-14 | 徐州工程学院 | A kind of screening technique of 3- hydracrylic acid producing bacterial strain |
| CN109852605A (en) * | 2019-01-16 | 2019-06-07 | 徐州工程学院 | A kind of method of mutagenesis screening high yield 3- hydracrylic acid bacterial strain |
| CN113817662A (en) * | 2021-10-08 | 2021-12-21 | 徐州工程学院 | Inducer for improving yield of 3-hydroxypropionic acid prepared by microbiological method |
| CN113817662B (en) * | 2021-10-08 | 2023-08-29 | 徐州工程学院 | Inducer for improving yield of 3-hydroxy propionic acid prepared by microorganism method |
| CN113774093A (en) * | 2021-10-11 | 2021-12-10 | 徐州工程学院 | Supplementary fermentation production method of 3-hydroxypropionic acid |
| CN114806593A (en) * | 2022-05-05 | 2022-07-29 | 北京化工大学 | Preparation method of composite soil remediation agent containing 3-hydroxypropionic acid fermentation liquor |
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