CN117247877A - Luminous bacillus rufimbriae and application thereof in preparing carrageenan oligosaccharides - Google Patents
Luminous bacillus rufimbriae and application thereof in preparing carrageenan oligosaccharides Download PDFInfo
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- CN117247877A CN117247877A CN202311542969.7A CN202311542969A CN117247877A CN 117247877 A CN117247877 A CN 117247877A CN 202311542969 A CN202311542969 A CN 202311542969A CN 117247877 A CN117247877 A CN 117247877A
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- kappa
- carrageenan
- luminous bacillus
- neokara
- oligosaccharides
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- 229920001525 carrageenan Polymers 0.000 title claims abstract description 66
- 229940113118 carrageenan Drugs 0.000 title claims abstract description 65
- 239000000679 carrageenan Substances 0.000 title claims abstract description 64
- 235000010418 carrageenan Nutrition 0.000 title claims abstract description 64
- 229920001542 oligosaccharide Polymers 0.000 title claims abstract description 60
- -1 carrageenan oligosaccharides Chemical class 0.000 title claims abstract description 45
- 241000193830 Bacillus <bacterium> Species 0.000 title claims abstract description 44
- 108090000790 Enzymes Proteins 0.000 claims abstract description 22
- 102000004190 Enzymes Human genes 0.000 claims abstract description 22
- 150000002016 disaccharides Chemical class 0.000 claims abstract description 20
- ZNOZWUKQPJXOIG-XSBHQQIPSA-L [(2r,3s,4r,5r,6s)-6-[[(1r,3s,4r,5r,8s)-3,4-dihydroxy-2,6-dioxabicyclo[3.2.1]octan-8-yl]oxy]-4-[[(1r,3r,4r,5r,8s)-8-[(2s,3r,4r,5r,6r)-3,4-dihydroxy-6-(hydroxymethyl)-5-sulfonatooxyoxan-2-yl]oxy-4-hydroxy-2,6-dioxabicyclo[3.2.1]octan-3-yl]oxy]-5-hydroxy-2-( Chemical compound O[C@@H]1[C@@H](O)[C@@H](OS([O-])(=O)=O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H]2OC[C@H]1O[C@H](O[C@H]1[C@H]([C@@H](CO)O[C@@H](O[C@@H]3[C@@H]4OC[C@H]3O[C@H](O)[C@@H]4O)[C@@H]1O)OS([O-])(=O)=O)[C@@H]2O ZNOZWUKQPJXOIG-XSBHQQIPSA-L 0.000 claims abstract description 13
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- 238000006731 degradation reaction Methods 0.000 claims description 32
- UHVMMEOXYDMDKI-JKYCWFKZSA-L zinc;1-(5-cyanopyridin-2-yl)-3-[(1s,2s)-2-(6-fluoro-2-hydroxy-3-propanoylphenyl)cyclopropyl]urea;diacetate Chemical compound [Zn+2].CC([O-])=O.CC([O-])=O.CCC(=O)C1=CC=C(F)C([C@H]2[C@H](C2)NC(=O)NC=2N=CC(=CC=2)C#N)=C1O UHVMMEOXYDMDKI-JKYCWFKZSA-L 0.000 claims description 30
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 235000015278 beef Nutrition 0.000 description 2
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- DMBHHRLKUKUOEG-UHFFFAOYSA-N diphenylamine Chemical compound C=1C=CC=CC=1NC1=CC=CC=C1 DMBHHRLKUKUOEG-UHFFFAOYSA-N 0.000 description 2
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- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
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- QAOWNCQODCNURD-UHFFFAOYSA-L sulfate group Chemical group S(=O)(=O)([O-])[O-] QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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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
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
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- C12P19/00—Preparation of compounds containing saccharide radicals
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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Abstract
The invention discloses a luminous bacillus rufoci and application thereof in preparing carrageenan oligosaccharides, belonging to the technical field of bioengineering. The Lu's luminous bacillus is Lu's luminous bacillusPhotobacterium rosenbergii) GDSX-4, the 16S rDNA sequence of which is shown as SEQ ID NO. 1. The luminous bacillus rupestis GDSX-4 provided by the invention can degrade kappa-carrageenanTo obtain kappa-carrageenan oligosaccharide. Preparing a crude enzyme solution from the luminous bacillus rufin GDSX-4, and reacting for 0.5-72 h at 40 ℃ in a substrate solution containing kappa-carrageenan to prepare kappa-new karaoke, kappa-new karaoke hexaose and kappa-new karaoke disaccharide. The invention provides a method for preparing carrageenan oligosaccharides with mild reaction and uniform products.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and relates to a luminous bacillus rupestris and application thereof in preparation of carrageenan oligosaccharides.
Background
Carrageenan @Carrageenan) Also known as carrageenan and carageenan, is an acidic polysaccharide extracted from red algae and has a repetitive alpha-1, 4-D-galactopyranose-beta-1, 3-D-galactopyranose disaccharide unit framework structure. The use safety of carrageenan is always controversial, on the one hand, the carrageenan is macromolecular sulfated polysaccharide and is not easy to digest and absorb in organisms; on the other hand, studies have shown that carrageenan is prone to cause gastrointestinal inflammation and intestinal immune responses in organisms. In addition, the carrageenan has various biological activities of reducing blood fat, resisting coagulation, resisting thrombus, regulating immunity, stimulating connective tissue growth and the like, and has good application prospect in the field of biological medicines. However, the carrageenan has a relatively large molecular weight, poor solubility and absorbability, and limits the application of the carrageenan.
Compared with carrageenan, the carrageenan oligosaccharide is a degradation product of the carrageenan, has the characteristics of lower molecular weight, higher solubility, easy absorption, better stability and better safety, and simultaneously, the activity of the carrageenan oligosaccharide is obviously improved compared with the carrageenan due to the full exposure of active groups on a molecular chain. The current carrageenan degradation modes comprise chemical degradation methods, such as oxidative degradation, acid degradation and the like; physical degradation methods such as irradiation degradation, microwave degradation, etc.; enzymatic degradation and the like. The chemical hydrolysis reaction process is often too severe to cause structural damage to the product during the hydrolysis process, and thus has a great limitation in its application. The enzyme degradation can protect active groups of the reaction substrate to the greatest extent from being damaged in the degradation process, the degradation products are uniform, the reaction conditions are mild, and the activity of the products is higher, so that the preparation method is considered to be a preparation method with development potential. Therefore, the strain can produce enzymes for degrading carrageenan, enrich microbial resources for preparing carrageenan oligosaccharides by enzyme degradation, and has important significance.
Disclosure of Invention
The invention provides a luminous bacillus rupestis and application thereof in preparing carrageenan oligosaccharides, in order to provide a strain capable of degrading carrageenan to prepare carrageenan oligosaccharides. The luminous bacillus of Ru is specifically luminous bacillus of Ru' sPhotobacterium rosenbergii) GDSX-4 is capable of degrading kappa-carrageenan to yield kappa-neokara disaccharide, kappa-neokara tetrasaccharide and kappa-neokara hexasaccharide. The invention provides a method for preparing carrageenan oligosaccharides with mild reaction and uniform products.
In order to achieve the technical aim of the invention, on one hand, the invention provides a strain of luminous bacillus rufimbriae, in particular luminous bacillus rufimbriaePhotobacterium rosenbergii）GDSX-4。
The invention relates to a luminous bacillus rufomiiPhotobacterium rosenbergii) GDSX-4 was collected from powder Fang Jiang, supplied by the university of ocean, guangdong, and the information stored is as follows.
Classification naming:Photobacterium rosenbergii GDSX-4；
preservation time: 2023, 10, 13;
preservation unit: the collection of microorganism strains in Guangdong province;
deposit unit address: guangzhou city first middle road No. 100 college No. 59 building 5;
preservation number: GDMCC NO: 63878.
The luminous bacillus rufimbriae of the invention produces enzyme for degrading carrageenan.
The invention is prepared from Fang Jiang%Gracilaria coronopifolia) The strain GDSX-4 is obtained after medium enrichment culture, preliminary screening and secondary screening, and is obtained through morphological identification, and the strain GDSX-4 is gram-negative bacteria and rod-shaped; the molecular identification shows that the strain GDSX-4 is L.lukei. Lu's luminous bacillusPhotobacterium rosenbergii) The 16S rDNA sequence of GDSX-4 is shown as SEQ ID NO. 1.
Further, the culture conditions of the luminous bacillus rufoci provided by the invention are as follows: enrichment medium is cultured for 24h at 30 ℃ and 150r/min in a shaking table. The enrichment medium comprises: 10g/L beef extract, 20g/L tryptone, 21.120g/L NaCl, 0.5820g/L KCl and CaCl 2 0.820g/L，MgCl 2 ·6H 2 O 3.620g/L，NaHCO 3 0.0820g/L，MgSO 4 ·7H 2 O2.62 g/L, pH 7.3.
Furthermore, the luminous bacillus rufin provided by the invention can degrade carrageenan, wherein the carrageenan is specifically kappa-carrageenan, and the degradation product is kappa-carrageenan oligosaccharide. Kappa-carrageenan oligosaccharides include kappa-neokaraoke disaccharide (DP 2), kappa-neokaraoke tetrasaccharide (DP 4) and kappa-neokaraoke hexasaccharide (DP 6).
On the other hand, the invention claims the application of the luminous bacillus rupestris in preparing carrageenan oligosaccharides. The luminous bacillus rufirluki can degrade kappa-carrageenan to obtain kappa-carrageenan oligosaccharides, wherein the kappa-carrageenan oligosaccharides comprise kappa-neokara disaccharide, kappa-neokara tetrasaccharide and kappa-neokara hexasaccharide.
Further, the invention cultures the luminous bacillus rufimbriae GDSX-4 in a solid enzyme production culture medium for 48 hours at 30 ℃, and the crude enzyme liquid is obtained by ultrasonic crushing and centrifugating the supernatant after the bacterial body is resuspended. And adding the crude enzyme solution into the kappa-carrageenan solution, and degrading for 0.5-72 h at 40 ℃ to obtain kappa-carrageenan oligosaccharide. Preferably, the degradation time is 1-48 hours. Further preferably, the time of degradation is 48 hours.
In another aspect, the invention claims an enzyme that degrades carrageenan, the enzymeIs prepared from luminous bacillus rufomiiPhotobacterium rosenbergii) GDSX-4 was prepared.
On the other hand, the invention claims a microbial agent which contains the luminous bacillus rufimbriaePhotobacterium rosenbergii) GDSX-4, lu's luminous bacillusPhotobacterium rosenbergii) GDSX-4 is used in the preparation of carrageenan oligosaccharides including kappa-neokara disaccharide, kappa-neokara tetrasaccharide and kappa-neokara hexasaccharide.
Furthermore, the invention discloses a preparation method of carrageenan oligosaccharides, which uses luminous bacillus of lushi @ to prepare the carrageenan oligosaccharidesPhotobacterium rosenbergii) GDSX-4 is used for preparing carrageenan oligosaccharides, or enzyme for degrading carrageenan is used for preparing the carrageenan oligosaccharides, or microbial agent is used for preparing the carrageenan oligosaccharides. The temperature for preparing the carrageenan oligosaccharide is 40 ℃ and the time is 0.5-72 h; carrageenan oligosaccharides include kappa-neokara disaccharide, kappa-neokara tetrasaccharide and kappa-neokara hexasaccharide.
Compared with the prior art, the technical scheme provided by the invention has at least the following beneficial effects or advantages:
the invention provides a strain with uniform products for preparing carrageenan oligosaccharides by enzymatic degradation of carrageenan. The invention provides a strain capable of degrading carrageenan, namely, a luminous bacillus rupestis strainPhotobacterium rosenbergii) GDSX-4, which is capable of degrading kappa-carrageenan to yield kappa-carrageenan oligosaccharides including kappa-neokara disaccharide, kappa-neokara tetrasaccharide and kappa-neokara hexasaccharide.
According to the invention, after the degradation reaction exceeds 30min, kappa-new kara disaccharide, kappa-new kara tetrasaccharide and kappa-new kara hexasaccharide are mainly generated in the system, and in addition, the oligosaccharide component with the partial polymerization degree more than six is also provided. With the increase of the degradation time, the oligosaccharide spot concentration is increased, and particularly after 48 hours of reaction, the oligosaccharide with the polymerization degree of more than six is greatly reduced, which is probably that part of the oligosaccharide is degraded into kappa-new kara disaccharide.
According to the invention, the liquid phase mass spectrometry analysis shows that the product is mainly kappa-neokara disaccharide before the degradation reaction is carried out for 45min, obvious detection peaks can be seen, and a small part of kappa-neokara tetrasaccharide and kappa-neokara hexasaccharide are generated. When the degradation reaction is carried out for 1h, the response peaks of three oligosaccharide components can be obviously seen, the generation amount of the three oligosaccharide components is gradually increased along with the increase of the degradation time, and particularly, the generation amount of the product reaches the peak value after the degradation is carried out for 48h. After the degradation reaction is finished, the final products are kappa-neocalichease, kappa-neocalichease and kappa-neocalichease, and 44% of the completely generated degradation products are kappa-neocalichease, 32% of the completely generated degradation products are kappa-neocalichease and 24% of the completely generated degradation products are kappa-neocalichease.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention.
FIG. 1 is a plate culture and microscopic image of L.lukei GDSX-4. A in FIG. 1 is a plate streak culture chart of L.luwensis GDSX-4; b in FIG. 1 is a plate diagram of the Lugol's luminous bacillus GDSX-4 after rescreening by Lugol's iodine solution staining method; c in FIG. 1 is a graph showing the result of gram staining by L.lupehensis GDSX-4; d in FIG. 1 is a scanning electron microscope image of L.luwensis GDSX-4.
FIG. 2 is a phylogenetic tree of strain GDSX-4.
FIG. 3 is a graph showing the reaction results of reducing sugar and DNS developer in a reaction system with different degradation times. Ctrl is the sample degraded for 0 h.
FIG. 4 shows the results of thin layer chromatography analysis of the products of L.luwensis GDSX-4 after degradation of kappa-carrageenan. Ctrl is the sample degraded for 0 h; secondly, a standard product kappa-new karaoke disaccharide; fourthly, a standard product kappa-new karaoke; and six is a standard product kappa-new kara-hexasaccharide.
FIG. 5 is a graph showing the results of mass spectrometry detection of kappa-neokaraoke disaccharide, kappa-neokaraoke tetrasaccharide and kappa-neokaraoke hexaose. DP2 is kappa-neokarabiose; DP4 is kappa-neokaraoke; DP6 is kappa-neokara hexose.
FIG. 6 is a total ion chromatogram of a mass spectrometry detection of a product of a process of degrading kappa-carrageenan by L.lupulus GDSX-4. DP2 is kappa-neokarabiose; DP4 is kappa-neokaraoke; DP6 is kappa-neokara hexose.
FIG. 7 is a statistical chart of peak areas of products of the process of degrading kappa-carrageenan by detecting the luminous bacillus rupestis GDSX-4 by mass spectrometry. DP2 is kappa-neokarabiose; DP4 is kappa-neokaraoke; DP6 is kappa-neokara hexose.
Detailed Description
The following describes the technical aspects of the present invention with reference to examples, but the present invention is not limited to the following examples. The experimental methods and the detection methods in each embodiment are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.
Enrichment medium: 10g/L beef extract, 20g/L tryptone, 21.120g/L NaCl, 0.5820g/L KCl and CaCl 2 0.820g/L，MgCl 2 ·6H 2 O 3.620g/L，NaHCO 3 0.0820g/L，MgSO 4 ·7H 2 O2.62 g/L, pH 7.3.
Solid screening medium: 12g/L carrageenan, 15g/L NaCl and NaNO 3 20g/L，CaCl 2 0.1,g/L，MgSO 4 ·7H 2 O 0.5g/L，FeSO 4 0.004g/L，K 2 HPO 4 1g/L。
Solid enzyme-producing medium: 15g/L carrageenan, 5g/L peptone, 1g/L yeast powder and 1L filter sterilized aged seawater are used for dissolution.
Powder Fang Jiang is supplied by university of ocean, guangdong.
Kappa-carrageenan was purchased from Qingdao sea marine oligosaccharide technologies Inc.
Example 1
This example provides the screening and identification of L.luwensis GDSX-4.
1. Screening of strains
Fang Jiang of the powderGracilaria coronopifolia) Crushing and homogenizing, and shake culturing for 24h at the temperature of 30 ℃ and 150r/min in an enrichment medium. 100 mu L of enrichment culture solution is coated on a solid screening culture medium, the culture medium is inverted at 30 ℃ for 48 hours, the growth state of colonies is observed, colonies with obvious pits are picked, and plate streaking is repeated until a single colony is obtained (a in FIG. 1). Picking single colony and inoculating to solid enzyme-producing culture medium 3Culturing at 0deg.C for 48 hr to obtain strain after primary screening. mu.L of the initially screened bacterial cells were dropped on a solid screening medium containing carrageenan as a sole carbon source, and after culturing for 48 hours at 30℃in an inverted manner, the cells were stained with 5mL of Lugol's iodine solution (b in FIG. 1), and the diameter of the transparent ring was measured with a vernier caliper.
As can be seen from a in FIG. 1 and b in FIG. 1, the strain GDSX-4 was able to grow in a carrageenan-containing solid screening medium, stained with Lugol's iodine solution, and a hydrolytic transparent circle with a diameter of 3.0cm was evident. Observing colony morphology, wherein single colony is round, has neat edge, wet and glossy surface, is milky white, has moderate size and is easy to pick up.
2. Morphological identification of strains
Gram staining was performed on the strain obtained by screening, the morphology of the strain was observed by a microscope (c in FIG. 1), and the gram staining result of the somatic cells was observed, wherein purple was gram-positive bacteria and red was gram-negative bacteria. The cell shape was observed by a scanning electron microscope (d in FIG. 1).
As can be seen from c in FIG. 1 and d in FIG. 1, the cells were red, gram-negative and rod-shaped.
3. Molecular characterization of strains
The single colony pure culture obtained is extracted with Ezup column type bacterial genome DNA extraction kit (purchased from Shanghai Limited of bioengineering), and the obtained DNA is subjected to 16S rRNA full-length amplification under the following experimental conditions and primer sequences:
universal primer sequence: 27F (5 '-AGTTTGATCMTGGCTCAG-3') and 1492R (5 '-GGTTACCTTGTTACGACTT 3').
Amplification system: 25. Mu.L of the reaction system, 1. Mu.L of each of the DNA template, 2.5. Mu.L of the 10 XPCR Buffer, 0.5. Mu.L of dNTP Mix (10 mM), 0.2. Mu.L of Taq enzyme (5U/L), and 25. Mu.L of deionized water were used as the primers. Taq enzyme was purchased from MBI (Fermentas).
Amplification conditions: pre-denaturation at 95 ℃ for 5min; denaturation at 94℃for 30s, annealing at 57℃for 30s, extension at 72℃for 90s,30 cycles; and at 72℃for 10min. The PCR product is purified and sent to a biological engineering (Shanghai) Co., ltd for DNA sequencing, and the 16S rDNA sequence of the strain GDSX-4 is shown as SEQ ID NO. 1.
And submitting the sequencing result to an EZbio database for Blast comparison. Downloading the sequences with higher homology, performing multiple sequence alignment by using ClustalX, selecting the closest 8 mode strains, and constructing a phylogenetic tree by using MEGA, as shown in FIG. 2.
As can be seen from FIG. 2, the strain GDSX-4 and L.lushiPhotobacterium rosenbergii) The homology is 99.93%, and according to the comparison of 16S rDNA, the strain GDSX-4 is identified as the luminous bacillus of Ru, named as the luminous bacillus of RuPhotobacterium rosenbergii）GDSX-4。
Example 2
The embodiment provides application of the luminous bacillus rupestis GDSX-4 in carrageenan oligosaccharide preparation.
1. Crude enzyme liquid collection of luminous bacillus rufomii GDSX-4
Culturing the strain in solid enzyme-producing culture medium at 30deg.C for 48 hr, re-suspending with Tris-HCl buffer solution of pH8.0, ultrasonic crushing for 20min, centrifuging the crushed solution at 12000rpm/min for 15min at 4deg.C, and collecting supernatant to obtain crude enzyme solution.
2. Preparation of carrageenan oligosaccharides
A0.5 wt% kappa-carrageenan substrate solution is prepared by Tris-HCl buffer solution (pH 8.0), 10% crude enzyme solution is added according to the total volume of the reaction, 0min,5min,10min,15min,30min,45min,1h,2h,3h,6h,12h,24h,27h,30h,48h and 72h are respectively reacted at 40 ℃, the reaction is stopped by heating and boiling for 5min, and the reaction is cooled to room temperature.
3. Analysis of kappa-carrageenan oligosaccharides by thin layer chromatography
Activating a silica gel thin layer plate for 1h at a drying oven at 100 ℃, taking 3 mu L of carrageenan oligosaccharide sample obtained by the reaction, spotting the sample on the silica gel plate, taking the sample for 0h as a reference (Ctrl), taking the standard products of kappa-new kara-disaccharide (DP 2), kappa-new kara-tetrasaccharide (DP 4) and kappa-new kara-hexa-saccharide (DP 6) as a reference, placing the sample in a developing agent of n-butanol-ethanol-water (3:2:2), drying after developing, soaking the sample in a developing agent (diphenylamine 2g, aniline 2mL,85% phosphoric acid 10mL, concentrated hydrochloric acid 1mL and acetone 100 mL), drying the sample, heating the sample for 10min at 110 ℃ for developing (FIG. 3), and detecting the result by thin layer chromatography is shown in FIG. 4.
As can be seen from fig. 3, the color of the reaction system gradually became darker with the increase of the degradation time, indicating that the content of reducing sugar in the reaction system gradually increased with the change of time.
As can be seen from FIG. 4, only a small amount of oligosaccharides are formed in the system before 30min of degradation reaction of the luminous bacillus rupestis GDSX-4, and after more than 30min of the degradation reaction, kappa-neokara disaccharide, kappa-neokara tetrasaccharide and kappa-neokara hexasaccharide are mainly formed in the system, and in addition, oligosaccharide components with partial polymerization degree of more than six are also formed. With the increase of the degradation time, the oligosaccharide spot concentration is increased, and particularly after 48 hours of reaction, the oligosaccharide with the polymerization degree of more than six is greatly reduced, which is probably that part of the oligosaccharide is degraded into kappa-new kara disaccharide.
4. Analysis of degradation product structure by liquid phase mass spectrometry (Q-TOF-MS)
Diluting the reaction solution after thin layer chromatography analysis to 50 mug/mL, filtering by a 0.22 mu m microporous filter membrane, and injecting into a TOF-MS system for analysis, wherein the chromatographic conditions are as follows: the sample injection amount is 5 mu L; mobile phase: acetonitrile 1mM formic acid (1:1); the flow rate is 0.2mL/min; column temperature was 35 ℃. The mass spectrum adopts an ESI point spray ion source and a negative ion ionization mode; mass scan range: m/z is 50 to 2000Da.
Firstly, the kappa-new kara-disaccharide, kappa-new kara-tetrasaccharide and kappa-new kara-hexasaccharide mixed standard is separated and subjected to primary mass spectrum identification (figure 5), and the change of the oligosaccharide production amount of the lupulus luminous bacillus GDSX-4 in the degradation reaction for 0.5-72 h is further detected (figures 6 and 7).
As can be seen from FIG. 5, the peak with mass-to-charge ratio m/z of 403.05 is An ion peak in which disaccharide carries a sulfate group, the peak is a characteristic ion peak of kappa-neokaraoke disaccharide [ An-G4S ] -and the peak with mass-to-charge ratio m/z of 394.05 is a characteristic ion peak of kappa-neokaraoke [ (An-G4S) 2] -and the peak with mass-to-charge ratio m/z of 391.05 is a characteristic ion peak of kappa-neokaraoke [ (An-G4S) 3] -respectively.
As can be seen from FIGS. 6 and 7, the products were mainly kappa-neokara-disaccharide (DP 2) before 45min of the degradation reaction, and distinct detection peaks were seen, while a small portion of kappa-neokara-tetrasaccharide (DP 4) and kappa-neokara-hexasaccharide (DP 6) were produced, but the response values were extremely low, and integration of peak areas was not possible. When the degradation reaction is carried out for 1h, the response peaks of three oligosaccharide components can be obviously seen, the generation amount of the three oligosaccharide components is gradually increased along with the increase of the degradation time, particularly after the degradation is carried out for 48h, the generation amount of the product reaches the peak value, and no more product is generated even if the degradation time is further increased. After the degradation reaction is finished, the final products are kappa-neocalichease, kappa-neocalichease and kappa-neocalichease, and 44% of the completely generated degradation products are kappa-neocalichease, 32% of the completely generated degradation products are kappa-neocalichease and 24% of the completely generated degradation products are kappa-neocalichease.
The embodiments described above are some, but not all, embodiments of the invention. The detailed description of the embodiments of the invention is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments obtained without inventive effort by a person skilled in the art, which are related deductions and substitutions made by the person skilled in the art under the condition of the inventive concept, are within the scope of protection of the present invention.
Claims (10)
1. The luminous bacillus of the strain is characterized in that the luminous bacillus of the strain is luminous bacillus of the strainPhotobacterium rosenbergii) GDSX-4 was deposited at the Cantonese microorganism strain collection at 10 and 13 of 2023 under the accession number: GDMCC NO: 63878;
the luminous bacillus rufimbriae produces enzyme for degrading carrageenan.
2. The luminous bacillus ruffii as claimed in claim 1, wherein the 16S rDNA sequence of the luminous bacillus ruffii is shown in SEQ ID No. 1.
3. The luminous bacillus rufimbriae of claim 1, wherein the carrageenan is kappa-carrageenan;
the degradation product of the carrageenan is kappa-carrageenan oligosaccharide.
4. A luminous bacillus as claimed in claim 3, characterized in that said kappa-carrageenan oligosaccharides comprise kappa-neokara disaccharide, kappa-neokara tetrasaccharide and kappa-neokara hexasaccharide.
5. The use of the lupulus of any one of claims 1-4 in the preparation of carrageenan oligosaccharides.
6. The use according to claim 5, wherein said luminous bacillus rufin degrades kappa-carrageenan to kappa-carrageenan oligosaccharides;
the carrageenan oligosaccharides include kappa-neokara disaccharide, kappa-neokara tetrasaccharide and kappa-neokara hexasaccharide.
7. The use according to claim 6, wherein the degradation temperature is 40 ℃ and the degradation time is 0.5-72 h.
8. An enzyme degrading carrageenan, characterized in that said enzyme is prepared from the luminous bacillus rufii of claim 1.
9. A microbial agent comprising the luminous bacillus rufii of claim 1;
the luminous bacillus rufimbriae is used for preparing carrageenan oligosaccharides;
the carrageenan oligosaccharides include kappa-neokara disaccharide, kappa-neokara tetrasaccharide and kappa-neokara hexasaccharide.
10. A method for preparing carrageenan oligosaccharides, characterized in that the luminous bacillus rufimbriae of claims 1-4 is used for preparing the carrageenan oligosaccharides, or the enzyme of claim 8 is used for preparing the carrageenan oligosaccharides, or the microbial agent of claim 9 is used for preparing the carrageenan oligosaccharides;
the temperature for preparing the carrageenan oligosaccharides is 40 ℃, and the time for preparing the carrageenan oligosaccharides is 0.5-72 h;
the carrageenan oligosaccharides include kappa-neokara disaccharide, kappa-neokara tetrasaccharide and kappa-neokara hexasaccharide.
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| CN109486712A (en) * | 2018-12-05 | 2019-03-19 | 浙江大学 | The Roche pseudomonad PYQ4 and its polyoses producing method of a kind of high-yield extracellular polysaccharide and application |
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| KR20110048746A (en) * | 2009-11-03 | 2011-05-12 | 대한민국(관리부서:국립수산과학원) | Novel photobacterium gaetbulicola gung47 strain for producing lipase |
| CN109486712A (en) * | 2018-12-05 | 2019-03-19 | 浙江大学 | The Roche pseudomonad PYQ4 and its polyoses producing method of a kind of high-yield extracellular polysaccharide and application |
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