CN110628679B - Raoultella ornithinolytica G10, enzyme production method, product and application - Google Patents
Raoultella ornithinolytica G10, enzyme production method, product and application Download PDFInfo
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- C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
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- C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
- C12Y305/01041—Chitin deacetylase (3.5.1.41)
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Abstract
The invention discloses marine-derived Raoultella ornithinolytica G10 with a preservation number of CCTCC NO: m2019485. The strain is gram-negative bacteria, short rod-shaped, encapsulated and immobile; when the beef extract peptone is used for culturing, the diameter of a bacterial colony is 1-3 mm, the optimal growth temperature is 30 ℃, and the optimal growth pH is 6. The invention also discloses a method for producing chitin deacetylase by using the strain and a product. The strain can produce various industrial and agricultural enzymes, including chitin deacetylase, xylanase, mannanase and cellulase. In the process of producing the chitin deacetylase, the Raoultella ornithinolytica G10 has the advantages of simple medium components, short culture time (24h) and low culture temperature (25 ℃). The enzyme activity is optimum at 30 deg.C and pH 5. The enzyme has good pH adaptability, has enzyme activity of more than 27% between the pH of 3-10, and has application in preparation of chitosan by an enzyme method.
Description
Technical Field
The invention relates to a microorganism, in particular to Raoultella ornithinolytica G10 which is separated from the Hongkong sea area of Lianhong in Jiangsu province of China and is preserved in the China center for type-III microorganism collection in 6 and 25 months in 2019, wherein the collection number is CCTCC NO: M2019485; the invention also relates to a method for producing the chitin deacetylase by using the strain, a product and application thereof.
Background
Chitin deacetylase (EC 3.5.1.41) hydrolyzes the N-acetyl group in Chitin polysaccharides. The chitosan can be called as chitosan if more than 55% of the N-acetyl in the chitin is removed. The chitosan has important application value in the fields of medicine, food, chemical industry, agriculture, environmental engineering, biomedical engineering and the like. The prior chitin deacetylation method adopted in the market is generally a concentrated alkali method, and has various defects, such as environmental pollution, long reaction time, unstable product quality and the like. The biological enzyme method, namely the chitin deacetylase, can solve the problems, and has the advantages that many chemical methods do not have, such as no degradation of the main chain part in chitin molecules, stable product quality, higher deacetylation degree, cost saving, short time consumption and the like. Although chitin deacetylase has important application value, no commercial enzyme preparation is available at home at present, so that the development potential of the chitin deacetylase is huge. The reported chitin deacetylase producing strain has the defects of long enzyme production fermentation time, high fermentation temperature, high enzyme production culture medium cost and the like. In conclusion, the wide screening of excellent strains with high enzyme yield and low production cost is an important way for developing the chitin deacetylase.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a novel Raoultella ornithinolytica G10 capable of producing chitin deacetylase.
Another objective of the invention is to provide a method for producing chitin deacetylase by Raoultella ornithinolytica G10.
The invention also aims to provide a chitin deacetylase product produced by Raoultella ornithinolytica G10.
Still another object of the present invention is to provide the use of the chitosan deacetylase produced by the aforementioned method;
it is still another object of the present invention to provide a hydrolysis application of Raoultella ornithine G10.
The purpose of the invention is realized by the following technical scheme. The invention relates to Raoultella ornithinolytica G10, which is characterized in that: the biological preservation number is as follows: CCTCC NO: M2019485.
The invention also discloses a method for producing chitin deacetylase by using the Raoultella ornithinolytica G10, which is characterized by comprising the following steps:
(1) seed culture: inoculating the Raoultella ornithinolytica G10 slant culture into a seed culture medium, rotating at 150-180 rpm, filling liquid at 20-30%, and culturing at 20-35 ℃ for 10-15 h to obtain a seed liquid; the seed culture medium consists of kelp juice, and the preparation method comprises the following steps: grinding dried herba Zosterae Marinae into 80 mesh powder, weighing herba Zosterae Marinae powder at a ratio of 20g/1000ml, adding into water, boiling for 20min, supplementing water loss, and filtering.
(2) Liquid shake flask fermentation: inoculating the seed solution into an enzyme production culture medium, culturing at 20-40 ℃ for 15-32 h at the liquid loading capacity of 30-50% at 150-200 rpm to obtain chitin deacetylase fermentation liquor; the enzyme production culture medium comprises the following components: 1-2% of shrimp shell powder, 0.1-0.5% of ammonium sulfate and kelp juice.
The invention also discloses a chitin deacetylase, which is characterized in that: the chitin deacetylase is prepared by fermenting the Raoultella ornithine G10 according to the technical scheme. Or the chitin deacetylase is prepared by the enzyme production method of the technical scheme. The chitin deacetylase can be used for preparing chitin deacetylated products such as chitosan. The chitin deacetylase produced by the method has the optimum pH value of 5, the optimum temperature of the enzyme activity of 30 ℃ and the activity within the pH range of 3-10.
The Raoultella ornithinolytica G10 is separated from sea mud in park of Haoyuang, Liyuanchong, Jiangsu province, China. Screening the culture medium to obtain the Raoultella ornithinolytica. The screening medium consists of: chitin powder 1%, agar 2%, and sea tangle juice. And performing shake flask culture on the strain, and performing chitin deacetylase activity identification on a fermentation broth of the strain.
The Raoultella ornithinolytica G10 is identified as Raoultella ornithinola through 16S rDNA sequence analysis and combined with colony morphology, cell microscopic morphology and physiological and biochemical reaction results. The physiological and biochemical tests show that the strain has the capability of producing various industrial and agricultural enzymes, including: chitin deacetylase, xylanase, mannanase and cellulase.
The invention also discloses a method for hydrolyzing mannan by using Raoultella ornithinolytica G10, which is characterized by comprising the following steps: inoculating Raoultella ornithinolytica G10 in a mannanase enzyme production culture medium, culturing at 32 ℃ for 34h at 180rpm with liquid loading of 40%, centrifuging and precipitating cells, and collecting supernatant to obtain mannanase extract. Mannase enzyme production medium: 0.5% of konjak fine powder, 0.5% of peptone, 0.3% of yeast extract and seawater.
The invention also discloses a method for hydrolyzing xylan by using Raoultella ornithinolytica G10, which is characterized by comprising the following steps: inoculating Raoultella ornithinolytica G10 in xylanase enzyme-producing culture medium, culturing at 30 deg.C and 180rpm for 34h with liquid loading amount of 40%, centrifuging to precipitate cells, and collecting supernatant to obtain xylanase extract. The xylanase enzyme production culture medium comprises the following components: peptone 1%, yeast powder 0.3%, magnesium sulfate 0.05%, K2HPO4 0.015％，KH2PO40.6 percent, xylan 1 percent and seawater.
The invention also discloses a method for hydrolyzing cellulose by using Raoultella ornithinolytica G10, which is characterized by comprising the following steps: inoculating Raoultella ornithinolytica G10 in cellulase enzyme production culture medium, culturing at 38 deg.C and 180rpm with liquid loading amount of 50% for 22 hr, centrifuging to precipitate cells, and collecting supernatant to obtain cellulase extractive solution. The cellulase-producing culture medium comprises the following components: 1% of sodium carboxymethylcellulose, 1% of peptone, 0.05% of yeast extract, 0.05% of sodium chloride, 0.1% of potassium dihydrogen phosphate, 0.05% of magnesium sulfate and tap water.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention provides a novel Raoultella ornithinolytica G10 for producing chitin deacetylase. The produced enzyme has wide application range.
2. The culture medium for producing the chitin deacetylase by using the Raoultella ornithinolytica G10 has simple components and low cost, and only contains three raw materials of shrimp shell, ammonium sulfate and kelp. The strain has short enzyme production culture time, high enzyme activity of the fermentation liquid, and the enzyme activity of the fermentation liquid is 187.34U/ml after only 24 hours of culture at 25 ℃. The chitin deacetylase product produced by the strain has the optimum pH value of 5, the optimum temperature of the enzyme activity of 30 ℃, the activity in the pH range of 3-10 and wide application range. The produced chitin deacetylase can be applied to the preparation of chitosan.
3. The strain Raoultella ornithinolytica G10 also produces various industrial and agricultural enzymes including xylanase, mannase and cellulase, and has wide application potential.
Drawings
FIG. 1 is a morphological diagram (1000-fold; gram stain) of Raoultella ornithinolytica G10 cells;
FIG. 2 is a morphogram of a single colony of Raoultella ornithinolytica G10;
FIG. 3 is a diagram showing the phylogenetic relationship of Raoultella ornithinolytica G10 constructed based on 16S rDNA;
FIG. 4 is a graph showing the effect of different temperatures on the growth of Raoultella ornithinolytica G10;
FIG. 5 is a graph showing the effect of different pH on the growth of Raoultella ornithinolytica G10;
FIG. 6 is a graph showing the effect of different NaCl concentrations on the growth of Raoultella ornithinolytica G10;
FIG. 7 is a graph showing the effect of different reaction temperatures on enzyme activity;
FIG. 8 is a graph showing the thermostability of the enzyme at 30 ℃;
FIG. 9 is a graph showing the effect of different reaction pH on enzyme activity;
FIG. 10 is a graph showing the pH stability of the enzyme at pH 5.
The Raoultella ornithinolytica G10 (Raoultella ornithinolytica G10 for short) is preserved in the China center for type-III microorganism collection in 2019 and 6 and 25 months, the preservation number is CCTCC NO: M2019485, and the preservation addresses are as follows: contact telephone, Wuhan university collection center of Lojia mountain of Wuchang, Wuhan city, Hubei province: 027-68754052.
Detailed Description
The invention is further illustrated by the following examples to enable a person skilled in the art to better understand the invention without constituting a limitation to the rights of the invention.
Example 1, Raoultella ornithinolytica G10 (hereinafter, referred to as Raoultella ornithinolytica G10 or strain G10) has a biological preservation number of CCTCC NO: M2019485. The separation of the Raoultella ornithinolytica G10 comprises the following steps:
collecting sea mud samples in a low-tide area of a sea park in Hongyuan harbor, diluting the sea mud samples with sterile distilled water, coating the diluted sea mud samples on a screening culture medium plate, culturing the sea mud samples at 25 ℃ for 48-72 h, and selecting bacterial colonies with transparent rings. Selecting bacterial colony, inoculating to enzyme production culture medium, culturing at 25 deg.C for 24 hr, centrifuging the culture at 8000rpm for 3min to obtain supernatant, and detecting chitin deacetylase activity. Adding 3ml of 0.05mol/L phosphate buffer solution into a 10ml centrifuge tube, adding 1ml of fermentation supernatant, adding 1ml of 0.2g/L paranitroacetanilide solution, placing in a 30 ℃ water bath kettle for enzymatic reaction for 15min, and after the enzymatic reaction is finished, boiling in boiling water for inactivation for 10min to terminate the enzymatic reaction. 1ml of the same concentration of enzyme that had been inactivated by boiling water bath was used as a control. Measuring the absorbance value of the reaction solution under the condition of 400nm by using a spectrophotometer, calculating the enzyme activity by using a regression equation of a p-nitroaniline standard curve, and selecting a strain with the highest enzyme activity by comparing the enzyme activity. The enzyme activity was defined as the amount of enzyme required to produce 1. mu.g of p-nitroaniline per hour under the above reaction conditions as one enzyme unit.
Composition of the screening medium: 1% of chitin powder, 2% of agar powder and kelp juice. The preparation method of the kelp juice comprises the following steps: grinding dried herba Zosterae Marinae into 80 mesh powder, adding 20g into 1000ml tap water, boiling for 20min, supplementing water loss, and filtering.
The identification of Raoultella ornithinolytica G10 comprises the following steps:
(1) the result of microscopic observation of Raoultella ornithinolytica G10 is as follows: the strain is gram-negative rod-shaped bacteria; observed under an oil lens: the cells are short rod-shaped, have no spores, have capsules and cannot move; the size (width. times. length) of the cells is about 0.57 + -0.03 μm × 1.09 + -0.05 μm; either alone or in a chain-like arrangement with two cells, see figure 1.
(2) Colony morphology observation is carried out on the Raoultella ornithinolytica G10, and after the Raoultella ornithinolytica G10 is cultured at 30 ℃ for 24 hours, the colony characteristics of the strain G10 on a beef extract peptone culture medium are as follows: the diameter of a single colony is 1-3 mm, the colony is semitransparent, the surface is moist and glossy, the colony is circular, the colony is raised into a hemisphere, the edge is regular, and the diameter is shown in figure 2. The colony morphology is similar to the above when growing on a screening medium, but the colony diameter is increased to 5-10 mm.
Beef extract peptone medium: beef extract 0.3%, peptone 1%, NaCl 0.5%, distilled water, pH 7.0.
(3) The physiological and biochemical performance of the Raoultella ornithine G10 were measured, and the results are shown in Table 1:
TABLE 1 physiological and biochemical properties of Raoultella ornithinolytica G10
Note: +: positive; -: and (4) negativity.
(4) Bacterial 16S rDNA sequence universal primers 27F and 1492R are used for amplification by taking the genomic DNA of the strain G10 as a template. 16S rDNA amplification products are obtained through PCR reaction and sequenced to obtain a sequence with the length of 1506bp (see a sequence table). Species G10 was phylogenetically analyzed based on the 16S rDNA sequence using Mega 5 software, see fig. 3.
The strain G10 was identified as Raoultella ornithinolytica (Raoultella ornithinolytica) by a combination of colony morphology, cell morphology and 16S rDNA sequence analysis.
The growth characteristics of the Raoultella ornithinolytica G10 on a beef extract peptone medium are determined by the following steps:
(1) preparation of strain G10 cell suspension:
the strain G10 was cultured for 24h at 30 ℃ using beef extract peptone medium to obtain a cell suspension.
(2) Effect of temperature on growth of strain G10:
inoculating the strain G10 cell suspension into beef extract peptone medium at an inoculation amount of 0.5% (v/v), standing and culturing at different temperatures respectively, and measuring the OD value of the cell suspension at 600nm after 48 h. The result shows that the growth temperature range of the strain is 4-45 ℃, the optimal growth temperature is 30 ℃, and the figure is 4.
Beef extract peptone medium: beef extract 0.3%, peptone 1%, NaCl 0.5%, distilled water, pH 7.0.
(3) Effect of pH on growth of strain G10:
preparing a beef extract peptone culture medium with the pH value of 2-8 by using a phosphate buffer solution, and preparing a culture medium with the pH value of 9-12 by using a boric acid buffer solution. G10 cell suspension was inoculated at 0.5% inoculum size to media of different pH, incubated at 30 ℃ for 48h, and cell concentration was determined. The result shows that the growth pH range of the strain is 4-10, the optimum growth pH is 6, and the figure is 5.
(4) Effect of NaCl on growth of strain G10:
preparing beef extract peptone culture media with different NaCl concentrations to ensure that the concentration of the beef extract peptone culture media is 0-20% (w/v). G10 cell suspension was inoculated at 0.05% inoculum size to media of different NaCl concentrations, incubated at 30 ℃ for 48h, and cell concentration was determined. The result shows that the NaCl concentration for the strain to grow is 0-5%, and the NaCl concentration for the optimal growth is 0, which is shown in figure 6.
Example 2, the method of producing chitin deacetylase from Raoultella ornithinolytica G10 described in example 1 was performed by the following steps:
(1) preparing a seed solution: inoculating the strain G10 into a seed culture medium from a strain G10 slant, rotating at 180rpm, filling 20%, and culturing at 30 ℃ for 12h to obtain a seed solution; wherein the seed culture medium is kelp juice, and the preparation method comprises: grinding dried herba Zosterae Marinae into 80 mesh powder, adding 20g into 1000ml tap water, boiling for 20min, supplementing water loss, and filtering.
(2) Enzyme production culture: inoculating the seed solution into an enzyme production culture medium with the inoculation amount of 1% (v/v), culturing at 25 ℃ for 24h at 200rpm and the liquid loading amount of 30%, centrifuging or filtering cells, and collecting supernatant to obtain the chitin deacetylase fermentation liquor. The enzyme activity of the fermentation broth was measured to be 187.34U/ml according to the method in example 1.
Enzyme production culture medium: 1 percent of shrimp shell powder, 0.2 percent of ammonium sulfate and kelp juice.
The property determination of the chitin deacetylase produced by Raoultella ornithinolytica G10 comprises the following steps:
(1) effect of reaction temperature on enzyme activity:
the enzyme activity was measured at 5 temperatures of 25 deg.C, 30 deg.C, 35 deg.C, 40 deg.C and 45 deg.C. The results are shown in FIG. 7, the optimum action temperature of the enzyme is 30 ℃, and the enzyme activity is more than 60% in the temperature range of 25-35 ℃.
(2) Thermostability of the enzyme:
the chitin deacetylase fermentation broth is subjected to heat preservation at 30 ℃ for 48h, a group of samples are taken out every 4h, the samples are rapidly frozen and stored in a refrigerator at the temperature of-20 ℃, the residual enzyme activity is uniformly determined after the heat preservation is finished, the enzyme activity of the fermentation broth which is not subjected to temperature treatment is set as 100%, and the result is shown in figure 8. The chitin deacetylase generated by the strain G10 has high thermal stability, and the residual enzyme activity is 39% after heat preservation is carried out for 12h at the temperature of 30 ℃.
(3) Effect of reaction pH on enzyme activity:
buffer solutions with different pH values are used for preparing a reaction system, the concentration of a substrate and the concentration of enzyme are kept to be the same, and the enzyme activity is measured at 30 ℃. The results are shown in FIG. 9, where the enzyme activity is highest at pH 3. The enzyme activity is more than 60% between pH 2-4.
(4) pH stability of the enzyme:
adjusting the pH value of the chitin deacetylase fermentation liquor to 5 by using a buffer solution, placing the mixture in a refrigerator at the temperature of 4 ℃, preserving the heat, taking out one sample every 4 hours for freezing and storing, and finally uniformly measuring the enzyme activity. The results are shown in FIG. 10, and the enzyme has better pH stability.
Example 3 hydrolysis of mannan with Raoultella ornithinolytica G10, the procedure was as follows:
inoculating Raoultella ornithinolytica G10 in a mannanase enzyme production culture medium, culturing at 32 ℃ for 34h at 180rpm with liquid loading of 40%, centrifuging and precipitating cells, and collecting supernatant to obtain mannanase extract. 0.4g of konjak powder was added to 100ml of 0.05mol/L phosphate buffer (pH 7.0), and the mixture was dissolved by heating with a glass rod under stirring to prepare a substrate solution. Taking 0.9ml of substrate solution, putting the substrate solution into a 10ml centrifuge tube, adding 0.1ml of enzyme extracting solution, shaking up, putting the substrate solution into a water bath kettle with the temperature of 55 ℃, and carrying out enzymatic reaction for 10 min. After the reaction is finished, 2ml of DNS solution is added, and the mixture is placed into an electromagnetic oven to react in boiling water for 5 min. The absorbance value at 540nm was measured with a spectrophotometer. The enzyme extract inactivated by boiling water bath was used as a control group. And calculating according to a mannose standard curve to obtain the mannase extracting solution with the enzyme activity of 13.1U/ml. Definition of enzyme activity: the amount of enzyme required to hydrolyze 1. mu. mol of reducing sugars released by degradation in the beta-mannan solution per minute was one unit of activity at a pH of 7.0 and a temperature of 55 ℃.
Mannase enzyme production medium: 0.5% of konjak fine powder, 0.5% of peptone, 0.3% of yeast extract and seawater.
Example 4 hydrolysis of xylan with Raoultella ornithinolytica G10, the procedure was as follows:
inoculating Raoultella ornithinolytica G10 in xylanase enzyme-producing culture medium, culturing at 30 deg.C and 180rpm for 34h with liquid loading amount of 40%, centrifuging to precipitate cells, and collecting supernatant to obtain xylanase extract. Adding 0.2ml of xylanase extracting solution into 1.8ml of 1% xylan substrate solution (the 1% xylan substrate solution is prepared by pH 5 and 0.05mol/L citric acid-sodium citrate buffer solution), reacting at 30 ℃ for 30min, adding 1ml of DNS reagent, carrying out boiling water bath for 10min, cooling and fixing the volume to 20 ml. The absorbance values were measured at 540 nm. The enzyme extract inactivated by boiling water bath is used as a control group. The enzyme activity of the mannase extracting solution is calculated to be 73.2U/ml according to the xylose standard curve. The xylanase activity unit (U) is defined as: the amount of enzyme required to hydrolyze xylan to 1. mu. mol xylose per minute under the conditions mentioned above.
Xylanase enzyme production cultureBase: peptone 1%, yeast powder 0.3%, magnesium sulfate 0.05%, K2HPO4 0.015％，KH2PO40.6 percent, xylan 1 percent and seawater.
Example 5 hydrolysis of cellulose with Raoultella ornithinolytica G10 the procedure was as follows:
inoculating Laurushibara ornithine G10 in a cellulase enzyme production culture medium, culturing at 38 ℃ and 180rpm for 22h with a liquid loading capacity of 50%, centrifuging and precipitating cells, and collecting supernatant to obtain a cellulase extracting solution. 0.5ml of cellulase extracting solution is taken, 0.5ml of 0.5% sodium carboxymethylcellulose solution (0.1M, prepared by phosphate buffer with pH 7) is added, after heat preservation is carried out for 20min at 50 ℃, 1ml of DNS reagent is added, the mixture is boiled in boiling water for 5min, after the mixture is cooled to room temperature, the mixture is diluted to 5ml by distilled water, and the absorbance value at 540nm is measured by a spectrophotometer. The enzyme extract inactivated by boiling water bath is used as a control group. And calculating to obtain the cellulase activity of the cellulase extracting solution of 43U/ml according to a glucose standard curve. Definition of enzyme activity: the amount of enzyme that hydrolyzes the substrate to produce 1. mu.g of glucose per minute under the above conditions was 1 enzyme activity unit.
Cellulase production medium: 1% of sodium carboxymethylcellulose, 1% of peptone, 0.05% of yeast extract, 0.05% of sodium chloride, 0.1% of potassium dihydrogen phosphate, 0.05% of magnesium sulfate and tap water.
Sequence listing
<110> university of oceanic Jiangsu
<120> Raoultella ornithinolytica G10, enzyme production method, product and application
<160> 1
<170> SIPOSequenceListing 1.0
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<211> 1506
<212> DNA
<213> Raoultella ornithinolytica (Raoultella ornithinolytica)
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agagtttgat cctggctcag attgaacgct ggcggcaggc ctaacacatg caagtcgagc 60
ggtagcacag agagcttgct ctcgggtgac gagcggcgga cgggtgagta atgtctggga 120
aactgcctga tggaggggga taactactgg aaacggtagc taataccgca taacgtcgca 180
agaccaaagt gggggacctt cgggccccat gccatcagat gtgcccagat gggattagct 240
agtaggtgag gtaatggctc acctaggcga cgatccctag ctggtctgag aggatgaccg 300
gccacactgg aactgagaca cggtccagac tcctacggga ggcagcagtg gggaatattg 360
cacaatgggc gcaagcctga tgcagccatg ccgcgtgtat gaagaaggcc ttcgggttgt 420
aaagtacttt cagcgaggag gaaggcgata aggttaataa ccttatcgat tgacgttact 480
cgcagaagaa gcaccggcta actccgtgcc agcagccgcg gtaatacgga gggtgcaagc 540
gttaatcgga attactgggc gcaaagcgca cgcaggcggt ctgtcaagtc ggatgtgaaa 600
tccccgggct caacctggga actgcatccg aaactggcag gctagagtct tgtagagggg 660
ggtagaattc caggtgtagc ggtgaaatgc gtagagatct ggaggaatac cggtggcgaa 720
ggcggccccc tggacaaaga ctgacgctca ggtgcgaaag cgtggggagc aaacaggatt 780
agataccctg gtagtccacg ctgtaaacga tgtcgacttg gaggttgttc ccttgaggag 840
tggcttccgg agctaacgcg ttaagtcgac cgcctgggga gtacggccgc aaggttaaaa 900
ctcaaatgaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgatgcaa 960
cgcgaagaac cttacctact cttgacatcc agagaactta gcagagatgc tttggtgcct 1020
tcgggaactc tgagacaggt gctgcatggc tgtcgtcagc tcgtgttgtg aaatgttggg 1080
ttaagtcccg caacgagcgc aacccttatc ctttgttgcc agcggttcgg ccgggaactc 1140
aaaggagact gccagtgata aactggagga aggtggggat gacgtcaagt catcatggcc 1200
cttacgagta gggctacaca cgtgctacaa tggcatatac aaagagaagc gacctcgcga 1260
gagcaagcgg acctcataaa gtatgtcgta gtccggatcg gagtctgcaa ctcgactccg 1320
tgaagtcgga atcgctagta atcgtggatc agaatgccac ggtgaatacg ttcccgggcc 1380
ttgtacacac cgcccgtcac accatgggag tgggttgcaa aagaagtagg tagcttaacc 1440
ttcgggaggg cgcttaccac tttgtgattc atgactgggg tgaagtcgta acaaggtagc 1500
cgtagg 1506
Claims (7)
1. Raoultella ornithinolytica G10, characterized by: the preservation number is CCTCC NO: m2019485.
2. A method of producing chitin deacetylase from Raoultella ornithinolytica G10 according to claim 1, comprising the steps of:
(1) preparing a seed solution: inoculating Raoultella ornithinolytica G10 into a seed culture medium, rotating at 150-180 rpm, filling liquid at 20-30%, and culturing at 20-35 ℃ for 10-15 h to obtain a seed liquid; the seed culture medium is kelp juice, and the preparation method comprises the following steps: grinding dried herba Zosterae Marinae into 80 mesh powder, adding water at a ratio of 20g/1000ml into herba Zosterae Marinae powder, boiling for 20min, supplementing water loss, and filtering;
(2) enzyme production culture: inoculating the seed solution into an enzyme production culture medium with the inoculation amount of 1% v/v, culturing at 20-40 ℃ for 15-32 h at 150-200 rpm and the liquid loading amount of 30-50% to obtain chitin deacetylase fermentation liquor; the enzyme production culture medium comprises the following components: 1-2% of shrimp shell powder and 0.1-0.5% of ammonium sulfate, and is prepared by adopting the kelp juice in the step (1).
3. A chitin deacetylase, comprising: the chitin deacetylase is prepared by fermenting Raoultella ornithinolytica G10 according to claim 1; or by the process of claim 2.
4. Use of the chitin deacetylase produced according to the method of claim 2 or the chitin deacetylase according to claim 3 for producing a chitin deacetylated product using said chitin deacetylase.
5. A method for hydrolyzing mannan using Raoultella ornithinolytica G10 according to claim 1, comprising the steps of: inoculating Raoultella ornithinolytica G10 in a mannase enzyme production culture medium, culturing at 32 ℃ for 34h at 180rpm with liquid loading capacity of 40%, centrifuging and precipitating cells, and collecting supernatant to obtain mannase extract; the mannase enzyme production culture medium comprises the following components: 0.5% of konjak fine powder, 0.5% of peptone, 0.3% of yeast extract and seawater.
6. A method for hydrolyzing xylan using Raoultella ornithinolytica G10 according to claim 1, comprising the steps of: inoculating Raoultella ornithinolytica G10 in xylanase enzyme production culture medium, culturing at 30 deg.C and 180rpm for 34h with liquid loading amount of 40%, centrifuging to precipitate cells, and collecting supernatant to obtain xylanase extract; the xylanase enzyme production culture medium comprises the following components: peptone 1%, yeast powder 0.3%, magnesium sulfate 0.05%, K2HPO40.015％，KH2PO40.6 percent, xylan 1 percent and seawater.
7. A method for hydrolyzing cellulose by using Raoultella ornithinolytica G10 according to claim 1, comprising the steps of: inoculating Raoultella ornithinolytica G10 in a cellulase enzyme production culture medium, culturing at 38 ℃, 180rpm for 22h with a liquid loading capacity of 50%, centrifuging and precipitating cells, and collecting supernatant to obtain a cellulase extracting solution; the cellulase-producing culture medium comprises the following components: 1% of sodium carboxymethylcellulose, 1% of peptone, 0.05% of yeast extract, 0.05% of sodium chloride, 0.1% of potassium dihydrogen phosphate, 0.05% of magnesium sulfate and tap water.
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| CN102391961A (en) * | 2011-11-02 | 2012-03-28 | 东华大学 | Raoultella ornithinolytica N-4 bacterial strains and preparation and application thereof |
| CN104498365A (en) * | 2014-11-17 | 2015-04-08 | 华南理工大学 | Bacterial strain capable of producing chitin deacetylase and application of bacterial strain in production of chitin deacetylase through fermentation |
| CN106978368A (en) * | 2017-03-31 | 2017-07-25 | 浙江工业大学 | Solve ornithine Raoul bacterium and its application |
| CN108546660A (en) * | 2018-04-13 | 2018-09-18 | 天津科技大学 | Chitin deacetylase superior strain and its application |
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| CN102391961A (en) * | 2011-11-02 | 2012-03-28 | 东华大学 | Raoultella ornithinolytica N-4 bacterial strains and preparation and application thereof |
| CN104498365A (en) * | 2014-11-17 | 2015-04-08 | 华南理工大学 | Bacterial strain capable of producing chitin deacetylase and application of bacterial strain in production of chitin deacetylase through fermentation |
| CN106978368A (en) * | 2017-03-31 | 2017-07-25 | 浙江工业大学 | Solve ornithine Raoul bacterium and its application |
| CN108546660A (en) * | 2018-04-13 | 2018-09-18 | 天津科技大学 | Chitin deacetylase superior strain and its application |
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