CN108220171B - Schizochytrium limacinum and application thereof in producing amylase - Google Patents
Schizochytrium limacinum and application thereof in producing amylase Download PDFInfo
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
- C12N9/2411—Amylases
Abstract
The invention discloses Schizochytrium WZYU003 and application thereof in producing amylase. The method uses schizochytrium to rapidly produce amylase in a culture medium containing cheap raw materials such as glucose, peptone, yeast powder and sea salt for the first time, so that the production cost is greatly reduced; and the whole process flow is simple, the equipment requirement is low, the controllability is strong, and the method is suitable for industrial production. The amylase prepared by the preparation method has better temperature and pH stability, and can meet the requirement of industrial production of the amylase.
Description
(I) technical field
The invention relates to preparation of amylase, in particular to a method for producing amylase by utilizing schizochytrium limacinum.
(II) background of the invention
Schizochytrium sp is heterotrophic microorganism of marine fungi, is mostly distributed in marine habitat with variable environment, can fully utilize own multienzyme system to efficiently decompose various matrixes in the environment, and is suitable for rapid growth of environmental factors such as temperature, pH and the like. However, no enzyme preparation derived from Schizochytrium and having industrial value has been reported.
Amylase is a general term for enzymes which hydrolyze starch and glycogen substances, is the enzyme which is the most widely applied and can realize industrial production at the earliest, is widely applied to the fields of food, textile, pharmacy, medical treatment, washing, paper making, feed and the like, and also becomes one of the enzymes with the largest industrial consumption. At present, industrial amylase is mainly derived from microorganisms such as bacillus and archaea, but at present, the amylase still has defects, such as insufficient temperature and pH stability, higher fermentation cost and the like.
Disclosure of the invention
The invention aims to provide a new strain-schizochytrium sp WZYU003 and a method for efficiently producing high-stability amylase by using the schizochytrium sp WZYU 003. The invention uses the marine microorganism schizochytrium limacinum which passes food safety certification for the first time, adopts cheap raw materials to efficiently produce the amylase, effectively reduces the production cost of the amylase, has higher activity in a wide temperature and pH range, solves the problem of insufficient stability of industrial amylase, has simple whole process flow, low equipment requirement and strong controllability, and is suitable for industrial production.
The technical scheme adopted by the invention is as follows:
the invention provides a new strain-schizochytrium sp WZYU003, which is preserved in China center for type culture Collection with the preservation number: CCTCC NO: M2016618, preservation date is: 2016, 11/7/2016, and the preservation address is Wuhan, Wuhan university, Wuhan, Japan, and the zip code 430072.
The invention also provides a method for producing amylase by using schizochytrium WZYU003, which comprises the following steps: inoculating schizochytrium WZYU003 into a fermentation medium, performing shaking culture at 20-35 ℃ and 50-600rpm to obtain a fermentation broth, centrifuging the fermentation broth, removing cell precipitate to obtain a crude enzyme solution containing amylase, and separating and purifying the crude enzyme solution to obtain the amylase; the fermentation medium comprises the following components: 20-120g/L of glucose, 10-60g/L of yeast powder, 0-20g/L of peptone (0 refers to the condition without peptone, and the content is 5-20g/L when peptone is contained), 20g/L of sea salt, water as solvent and natural pH value.
Further, before inoculation of the schizochytrium WZYU003, plate activation and seed expansion culture are carried out: (1) plate culture: inoculating Schizochytrium WZYU003 to plate culture medium, and culturing at 30 deg.C for 2-3 days; the plate culture medium comprises the following components: 20g/L of glucose, 10g/L of yeast powder, 5g/L of peptone, 20g/L of sea salt, 20g/L of agar and water as a solvent, wherein the pH value is natural; (2) seed culture: inoculating the flat plate thallus into a 500mL triangular flask containing 100mL seed culture medium, and performing shake culture at 30 ℃ and 200rpm for 1-2 days to obtain a seed solution; the seed culture medium comprises the following components: 60g/L glucose, 10g/L yeast powder, 5g/L peptone, 20g/L sea salt and water as solvent, and the pH value is natural.
Further, the fermentation culture method of the schizochytrium WZYU003 comprises the following steps: inoculating the seed solution into a fermentation tank containing fermentation medium, and culturing at 20-35 deg.C at rotation speed of 50-600rpm, ventilation rate of 20-100L/min and dissolved oxygen amount of 10-60% for 1-6 days to obtain fermentation liquid, preferably fermentation conditions: at 25-32 deg.C, rotation speed of 100-.
Further, the seed solution is inoculated to a fermentation medium in an inoculation amount of 1-20% of the volume concentration.
The amylase produced by the schizochytrium WZYU003 is characterized in that: the enzyme has the optimum action pH of 6.0, and still retains more than 80% of activity of the optimum activity within the range of pH4.0-8.0; the enzyme has better pH stability within the range of pH5.0-10.0, and the relative enzyme activity can be kept above 80%; the optimum temperature of the enzyme is 60 ℃, and more than 80% of activity is still preserved within the range of 40-100 ℃; treating the enzyme at 60 deg.C for 120min, and still retaining over 90% of enzyme activity; and the enzyme activity can be kept more than 80% by treating at 100 ℃ for 120 min.
Compared with the prior art, the invention has the following beneficial effects:
the schizochytrium has many advantages compared with other amylase producing strains: 1) the safety is high, and the food can be widely applied to the field of foods; 2) the traditional cheap raw materials such as glucose, yeast powder, peptone and the like can be utilized, and the sea salt component in the fermentation medium can replace inorganic salt necessary for producing amylase by fermenting other strains, so that the production cost is greatly reduced; 3) the mode for producing the amylase is extracellular secretion, so that the extraction and separation processes of the amylase are greatly simplified, and the method is suitable for industrial production; 4) the produced amylase has higher temperature and pH stability and is suitable for industrial application.
(IV) description of the drawings
FIG. 1 is a transparent circle of Schizochytrium WZYU003 on an amylase screening plate
FIG. 2 is the colony morphology of the Schizochytrium WZYU003 strain plate.
FIG. 3 is the microscopic colony morphology of Schizochytrium WZYU003 strain.
FIG. 4 is a graph of the effect of pH on the relative activity of the Schizochytrium WZYU003 amylase.
FIG. 5 is the pH stability of the Schizochytrium WZYU003 amylase.
FIG. 6 is a graph of the effect of temperature on the relative activity of the Schizochytrium WZYU003 amylase.
FIG. 7 is the temperature stability of Schizochytrium WZYU003 amylase.
(V) detailed description of the preferred embodiments
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
example 1 Schizochytrium WZYU003 screening and identification
(1) Strain WZYU003 screening
A leaf mold sample is collected at seaside of Leqing gulf of Wenzhou, Zhejiang, cleaned, placed in YP culture solution (1g/L yeast powder, 1g/L peptone, 50mg/L ampicillin, 50mg/L streptomycin, water preparation, pH6.0) with 0.1g of pollen pini suspended on the surface, cultured in the dark at 30 ℃ for 1 week, 50 microliter of culture is taken and spread on YP plates (1g/L yeast powder, 1g/L peptone, 50mg/L ampicillin, 50mg/L streptomycin, agar 20g/L, water preparation, pH6.0), cultured at 28 ℃ for 3 days until a single colony grows out, and streaking GYP plate purification culture is repeated for three times to obtain a pure strain. Pure colonies were spotted on an amylase screening plate, inverted cultured at 30 ℃ for 3 days, observed in a transparent circle, and labeled with strain WZYU 003.
GYP plate culture medium comprises (g/L) glucose 20, yeast powder 10, dried egg white 5, sea salt 20, agar 20, and solvent water, and has natural pH value, sterilized at 115 deg.C for 30min, and poured into plate.
The amylase screening plate comprises the following components in percentage by mass (g/L): soluble starch 15, yeast powder 10, protein 5, sea salt 20, agar 20, Trimeryl blue 0.05, water as solvent, pH7.0, sterilizing at 115 deg.C for 30min, and pouring into plate.
(2) Identification of Strain WZYU003
The morphological characteristics of the cells are as follows:
inoculating strain WZYU003 on GYP plate, and culturing at 28 deg.C for 2 days to obtain yellow-white round colony with wet and smooth surface and slight bulge (FIG. 2); under the microscope, the cells are round or oval, the diameter is 5-20 mu m, the cell surface is bright, the cells have scaly structures, the edges are clear, the cells divide and proliferate, and an even number of cells are gathered together (figure 3).
The 18SrDNA sequence of the strain WZYU003 is shown in SEQ ID No. 1. The 18SrDNA sequence similarity with strains such as Aurantiochytrium sp. KRS101 and ST-2012 can reach 99%, so the strain WZYU003 is identified as schizochytrium sp, named as schizochytrium sp WZYU003, deposited in the China center for type culture Collection with the deposit number: CCTCC NO: M2016618, preservation date is: 2016, 11/7/2016, and the preservation address is Wuhan, Wuhan university, Wuhan, Japan, and the zip code 430072.
Example 2 production of Amylase by liquid fermentation of Schizochytrium WZYU003
Transferring the Schizochytrium WZYU003 strain stored at-80 deg.C to slant culture medium, and static culturing at 30 deg.C for 2 days; inoculating the thalli into a 500mL triangular flask containing 100mL seed culture medium, and performing shake culture at 30 ℃ and 200rpm for 1 day to obtain seed liquid; transferring the seed solution into a fermentation tank containing 3L fermentation medium according to the inoculum size of 4% of volume concentration, culturing for 3 days at 30 deg.C, rotation speed of 300rpm, ventilation capacity of 80L/min, and dissolved oxygen amount of 60% to obtain fermentation broth; centrifuging the fermentation liquid at 5000rpm and 4 deg.C for 5min, removing cell precipitate to obtain crude enzyme solution with enzyme activity of 436.2U/mL.
Amylase activity was measured by the DNS (3, 5-dinitrosalicylic acid) method. Enzyme activity definition (U) is the amount of enzyme required to enzymatically hydrolyze starch to produce 1. mu.g of reducing sugars (in terms of glucose) within 1min at 60 ℃ and pH 6.0.
The slant culture medium comprises (g/L) glucose 20, yeast powder 10, protein 5, sea salt 20, agar 20, and solvent water, and has natural pH value and sterilized at 115 deg.C for 30 min; the seed culture medium (g/L) comprises glucose 60, yeast powder 10, protein 5, sea salt 20, and water as solvent, and has natural pH value and sterilized at 115 deg.C for 30 min; the fermentation medium (g/L) comprises glucose 60, yeast powder 10, peptone 0, sea salt 20, and solvent water, with natural pH value, and sterilizing at 115 deg.C for 30 min.
Example 3 production of Amylase by liquid fermentation of Schizochytrium WZYU003
Transferring the Schizochytrium WZYU003 strain stored at-80 deg.C to slant culture medium, and static culturing at 30 deg.C for 2 days; inoculating the thalli into a 500mL triangular flask containing 100mL seed culture medium, and performing shake culture at 30 ℃ and 200rpm for 1 day to obtain seed liquid; transferring the seed solution into a fermentation tank containing 3L fermentation medium according to the inoculum size of 1% of volume concentration, culturing for 6 days at 35 deg.C, 600rpm, 20L/min of ventilation capacity and 10% of dissolved oxygen to obtain fermentation broth; centrifuging the fermentation liquor at 5000rpm and 4 deg.C for 5min, removing cell precipitate to obtain crude enzyme solution with enzyme activity of 525.1U/mL.
Amylase activity was measured by the DNS (3, 5-dinitrosalicylic acid) method. Enzyme activity definition (U) is the amount of enzyme required to enzymatically hydrolyze starch to produce 1. mu.g of reducing sugars (in terms of glucose) within 1min at 60 ℃ and pH 6.0.
The composition of slant culture medium and seed culture medium is the same as that of example 2, the fermentation culture medium (g/L) comprises glucose 120, yeast powder 60, peptone 20, sea salt 20, and solvent water, the pH value is natural, and sterilization is carried out at 115 ℃ for 30 min.
Example 4 production of Amylase by liquid fermentation of Schizochytrium WZYU003
Transferring the Schizochytrium WZYU003 strain stored at-80 deg.C to slant culture medium, and static culturing at 30 deg.C for 2 days; inoculating the thalli into a 500mL triangular flask containing 100mL seed culture medium, and performing shake culture at 30 ℃ and 200rpm for 1 day to obtain seed liquid; transferring the seed solution into a fermentation tank containing 3L fermentation medium according to the inoculum size of 20% of volume concentration, culturing for 1 day at 20 deg.C, rotation speed of 50rpm, ventilation capacity of 100L/min, and dissolved oxygen amount of 60% to obtain fermentation broth; centrifuging the fermentation liquor at 5000rpm and 4 deg.C for 5min, removing cell precipitate to obtain crude enzyme solution with enzyme activity of 210.2U/mL.
Amylase activity was measured by the DNS (3, 5-dinitrosalicylic acid) method. Enzyme activity definition (U) is the amount of enzyme required to enzymatically hydrolyze starch to produce 1. mu.g of reducing sugars (in terms of glucose) within 1min at 60 ℃ and pH 6.0.
The composition of slant culture medium and seed culture medium is the same as that of example 2, and the fermentation culture medium comprises (g/L) glucose 20, yeast powder 10, peptone 0, sea salt 20, and solvent water, and has natural pH value and sterilized at 115 deg.C for 30 min.
Example 5 production of Amylase by liquid fermentation of Schizochytrium WZYU003
Transferring the Schizochytrium WZYU003 strain stored at-80 deg.C to slant culture medium, and static culturing at 30 deg.C for 2 days; inoculating the thalli into a 500mL triangular flask containing 100mL seed culture medium, and performing shake culture at 30 ℃ and 200rpm for 1 day to obtain seed liquid; transferring the seed solution into a fermentation tank containing 3L fermentation medium according to the inoculum size of 10% of volume concentration, culturing for 5 days at 28 deg.C, 500rpm, 70L/min of ventilation capacity and 50% of dissolved oxygen to obtain fermentation broth; centrifuging the fermentation liquid at 5000rpm and 4 deg.C for 5min, removing cell precipitate to obtain crude enzyme solution with enzyme activity of 352.1U/mL.
Amylase activity was measured by the DNS (3, 5-dinitrosalicylic acid) method. Enzyme activity definition (U) is the amount of enzyme required to enzymatically hydrolyze starch to produce 1. mu.g of reducing sugars (in terms of glucose) within 1min at 60 ℃ and pH 6.0.
The composition of slant culture medium and seed culture medium is the same as that of example 2, the fermentation medium is (g/L) glucose 100, yeast powder 20, peptone 5, sea salt 20, and solvent water, the pH value is natural, and sterilization is carried out at 115 ℃ for 30 min.
Example 6 Schizochytrium WZYU003 amylase optimum temperature, pH, and stability thereof
The amylase activity of the crude enzyme solution prepared by fermentation of the schizochytrium WZYU003 strain prepared in example 3 was measured by the DNS standard method using buffers of different pH values (0.1M glycine-hydrochloric acid buffer at pH 2.0; 0.1M citric acid-sodium citrate buffer at pH 3.0-6.0; 0.1M barbiturate-hydrochloric acid buffer at pH 3.0-6.0; 0.1M citric acid-sodium citrate buffer at pH 7.0-9.0) and 0.0 at concentrations of 5g/L as substrates, respectively, and the amylase activity of the crude enzyme solution prepared by fermentation of the schizochytrium WZYU003 strain prepared in example 3 was measured using the DNS standard method, with the optimum pH of the enzyme being 6.0 and this being taken as 100%, and the relative activities of the amylase corresponding to substrates of other pH values under the same conditions were compared to obtain the amylase activity (see fig. 4). The results showed that the enzyme had a high enzymatic activity (greater than 80% of the optimal activity) in the pH range of 4.0-8.0.
The pH values of the crude enzyme solutions prepared by fermentation of the Schizochytrium WZYU003 strain prepared in example 3 were adjusted to 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 respectively using buffers of different pH values (0.1M glycine-hydrochloric acid buffer at pH 2.0; 0.1M citric acid-sodium citrate buffer at pH 3.0-6.0; 0.1M barbiturate-hydrochloric acid buffer at pH 7.0-9.0; glycine-sodium hydroxide buffer at pH 10.0) and stored at 4 ℃ for 60min, and the amylase activity was measured by the DNS standard method, and the relative enzyme activities under different pH conditions were calculated using the original enzyme solution activity of natural pH as 100% to evaluate the pH stability of the enzyme (see FIG. 5). The result shows that the enzyme has better pH stability in the pH range of 5.0-10.0, and the relative enzyme activity can be kept above 80%.
0.5mL of a soluble starch solution prepared using 0.1M citric acid-sodium citrate buffer (pH6.0) and having a concentration of 5g/L and 0.5mL of a crude enzyme solution prepared by fermentation of the Schizochytrium WZYU003 strain prepared in example 3 were mixed, and the mixture was incubated at 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃, 80 ℃, 85 ℃, 90 ℃ and 100 ℃ for 30min, DNS was added thereto, and DNS was boiled to develop color, and the temperature gradients were determined by using the boiling-inactivated enzyme solution as a negative control and the enzyme activity of the 60 ℃ system as 100%, thereby calculating the relative activities of the systems (see FIG. 6). The results show that the optimum temperature of the enzyme is 60 ℃, and the enzyme still can keep more than 60 percent of the optimum activity within the range of 40-100 ℃.
The crude enzyme solution prepared by fermenting the Schizochytrium WZYU003 strain prepared in example 3 was subjected to water bath at 55 deg.C, 60 deg.C, 70 deg.C, 80 deg.C and 100 deg.C for 0, 10, 30, 40, 50, 60, 90 and 120min, respectively, and amylase activity was measured by DNS method. The thermal stability of the enzyme was evaluated by calculating the relative activity under other conditions, taking the activity of the crude enzyme solution without temperature treatment as 100%. The result shows that the enzyme can still retain more than 90 percent of activity after being preserved for 120min at the optimal temperature of 60 ℃; and when the temperature is kept at 100 ℃ for 120min, the enzyme activity can still be kept more than 80% (see figure 7).
Sequence listing
<110> Zhejiang industrial university
<120> Schizochytrium limacinum and application thereof in producing amylase
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>1771
<212>DNA
<213> Schizochytrium sp (Aurantiochytrium sp.)
<400>1
tcctgccagt agtcatatgc tcgtctcaaa gattaagcca tgcatgtgta agtataagcg 60
attgtactgt gagactgcga acggctcatt atatcagtaa taatttcttc ggtagtttct 120
tttatatgga tacctgcagt aattctggaa ataatacatg ctgtaagagc cctgtatggg 180
gctgcactta ttagattgaa gccgatttta ttggtgaatc atgataattg agcagattga 240
ctatttttgg tcgatgaatc gtttgagttt ctgccccatc agttgtcgac ggtagtgtat 300
tggactacgg tgactataac gggtgacgga gagttagggc tcgactccgg agagggagcc 360
tgagagacgg ctaccatatc caaggatagc agcaggcgcg taaattaccc actgtggact 420
ccacgaggta gtgacgagaa atatcgatgc gaagcgtgta tgcgttttgc tatcggaatg 480
agagcaatgt aaaaccctca tcgaggatca actggagggc aagtctggtg ccagcagccg 540
cggtaattcc agctccagaa gcatatgcta aagttgttgc agttaaaaag ctcgtagttg 600
aatttctggc atgggcgacc ggtgctttcc ctgaatgggg attgattgtc tgcgttgcct 660
tggccatctt tctcatgcta tttttggtat gagatctttc actgtaatca aagcagagtg 720
ttccaagcag gtcgtatgac cggtatgttt attatgggat gataagatag gacttgggtg 780
ctattttgtt ggtttgcacg cctgagtaat ggttaatagg aacagttggg ggtattcgta 840
tttaggagct agaggcgaaa ttcttggatt tccgaaagac gaactagagc gaaggcattt 900
accaagcatg ttttcattaa tcaagtacga aagtctgggg atcgaagatg attagatacc 960
atcgtagtct agaccgtaaa cgatgccgac ttgcgattgt tgggtgcttt attaatgggc 1020
ctcagcagca gcacatgaga aatcaaagtc tttgggttcc ggggggagta tggtcgcaag 1080
gctgaaactt aaaggaattg acggaagggc accaccagga gtggagcctg cggcttaatt 1140
tgactcaaca cgggaaaact taccaggtcc agacataggt aggattgaca gattgagagc 1200
tctttcatga ttctatgggt ggtggtgcat ggccgttctt agttggtgga gtgatttgtc 1260
tggttaattc cgttaacgaa cgagacctcg gcccactaaa tagtgcgtgg tatggcaaca 1320
tagtgcgttt ttacttctta gagggacatg tccggtttac gggcaggaag ttcgaggcaa 1380
taacaggtct gtgatgccct tagatgttct gggccgcacg cgcgctacac tgatgggttc 1440
atcgggtttt aatttcattt ttggaattga gtgcttggtc ggaaggcctg gctaatcctt 1500
ggaacgctca tcgtgctggg gctagatttt tgcaattatt aatctccaac gaggaattcc 1560
tagtaaacgc aagtcaccag cttgcattga atacgtccct gccctttgta cacaccgccc 1620
gtcgcaccta ccgattgaac ggtccgatga aaccatggga tgtttctgtt tggattgatt 1680
tttggacaga ggcagaactc gggtgaatct tattgtttag aggaaggtga agtcgtaaca 1740
aggtttccgt aggtgaacct gcagaggatc a 1771
Claims (8)
1. Schizochytrium sp WZYU003, deposited at the China center for type culture Collection with the deposit number: CCTCC NO: M2016618, preservation date is: 2016, 11/7/2016, and the preservation address is Wuhan, Wuhan university, Wuhan, Japan, and the zip code 430072.
2. A method for producing amylase by using the Schizochytrium WZYU003 in claim 1, which is characterized in that: inoculating schizochytrium WZYU003 into a fermentation medium, performing shaking culture at 20-35 ℃ and 50-600rpm to obtain a fermentation broth, centrifuging the fermentation broth, removing cell precipitate to obtain a crude enzyme solution containing amylase, and separating and purifying the crude enzyme solution to obtain the amylase; the fermentation medium comprises the following components: 20-120g/L of glucose, 10-60g/L of yeast powder, 0-20g/L of peptone, 20g/L of sea salt and water as a solvent, and the pH value is natural.
3. The method for producing amylase according to claim 2, wherein the Schizochytrium WZYU003 is inoculated with plate activation and seed expansion culture: (1) activating a flat plate: inoculating Schizochytrium WZYU003 into a plate culture medium, and culturing at 30 deg.C for 2-3 days to obtain plate thallus; the plate culture medium comprises the following components: 20g/L of glucose, 10g/L of yeast powder, 5g/L of peptone, 20g/L of sea salt, 20g/L of agar and water as a solvent, wherein the pH value is natural; (2) seed culture: inoculating the flat plate thallus into a 500mL triangular flask containing 100mL seed culture medium, and performing shake culture at 30 ℃ and 200rpm for 1-2 days to obtain a seed solution; the seed culture medium comprises the following components: 60g/L glucose, 10g/L yeast powder, 5g/L peptone, 20g/L sea salt and water as solvent, and the pH value is natural.
4. The method of claim 3 for producing amylase using Schizochytrium WZYU003, wherein the fermentation broth is prepared by: inoculating the seed solution into a fermentation tank containing fermentation culture medium, and culturing at 20-35 deg.C at rotation speed of 50-600rpm, ventilation rate of 20-100L/min, and dissolved oxygen amount of 10-60% for 1-6 days to obtain fermentation liquid.
5. The method for producing amylase according to claim 3, wherein the seed solution is inoculated into the fermentation medium at an inoculation amount of 1% -20% by volume concentration.
6. The method of claim 4 for producing amylase using Schizochytrium WZYU003, wherein the fermentation conditions are: the temperature is 25-32 ℃, the rotating speed is 100-.
7. The method for producing amylase of claim 2 using schizochytrium WZYU003 wherein the fermentation broth is centrifuged under conditions of: centrifuge at 5000rpm at 4 ℃ for 5 min.
8. The method of claim 2 for producing amylase using schizochytrium WZYU003, wherein the fermentation medium consists of: 100g/L glucose, 20g/L yeast powder, 5g/L peptone, 20g/L sea salt and water as solvent, and the pH value is natural.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2015292421A1 (en) * | 2014-07-25 | 2017-02-16 | Novogy, Inc. | Promoters derived from Yarrowia lipolytica and Arxula adeninivorans, and methods of use thereof |
| CN107245451A (en) * | 2016-12-31 | 2017-10-13 | 浙江工业大学 | Schizochytrium limacinum WZYU011 and its method for producing renin |
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