CN113293114B - Alteromonas HT1 and culture method and application thereof - Google Patents
Alteromonas HT1 and culture method and application thereof Download PDFInfo
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Abstract
The invention relates to alteromonas HT1 and a culture method and application thereof. Alteromonas (Alteromonas sp.) HT1 is preserved in China center for type culture collection in 3 months and 9 days in 2021, wherein the preservation address is eight Lopa Alojia mountains in Wuchang district, Wuhan city, Hubei province, and the preservation number is CCTCC NO. M2021217. Alteromonas (alteromonas sp.) HT1 can grow in a culture medium with enteromorpha as a unique carbon source, and the product obtained by degrading enteromorpha polysaccharide is enteromorpha oligosaccharide which mainly exists in the forms of disaccharide, trisaccharide, tetrasaccharide and pentasaccharide. The strain and enteromorpha polysaccharide lyase secreted by the strain can be applied to degradation of marine green algae and preparation of functional oligosaccharide, and have good application potential in the pharmaceutical industry, the cosmetic industry, the food industry and the like.
Description
Technical Field
The invention relates to alteromonas HT1 and a culture method and application thereof, belonging to the technical field of microorganisms.
Background
Enteromorpha prolifera is widely distributed in coastal areas around the world, belongs to algae of Ulvales of Chlorophyceae of Chlorophyta, and is also important algae influencing the coastal environment in summer in recent years. The enteromorpha polysaccharide accounts for 43.4-60.2% of the dry weight of the enteromorpha, is sulfated polysaccharide consisting of 3-sulfated rhamnose, glucuronic acid and xylose, has various biological functions of resisting cancers, resisting viruses, regulating blood fat, reducing blood fat and the like due to unique physicochemical properties, and has good application potential in the fields of food, medicine and the like. However, the characteristics of high viscosity and high molecular weight of the enteromorpha polysaccharide seriously affect the absorption and utilization of the enteromorpha polysaccharide by people, so that the enteromorpha polysaccharide is degraded into the enteromorpha oligosaccharide with low molecular weight and good solubility, and the enteromorpha oligosaccharide becomes an important way for improving the biological function and the added value of the enteromorpha polysaccharide.
The polysaccharide skeleton of the enteromorpha polysaccharide consists of D-GlcUAp-alpha- (1 → 4) -3-sulfuric acid-L-rhamnose p-beta- (1 → 4) -D-xylose p-beta- (14) -3-sulfuric acid-L-rhamnose p units. The acidolysis method is often adopted for the degradation of the low molecular weight algal polysaccharide, but the acidolysis method has poor specificity and wide molecular weight distribution range, and the molecular weight and the sulfation degree of the oligosaccharide are difficult to control. Therefore, the enzymatic degradation with mild reaction conditions, high specificity and high efficiency is a method which is of great interest for processing enteromorpha polysaccharide.
Enteromorpha polysaccharide lyase is mainly derived from marine bacteria, few strains are available at present, and only the strains which can produce the Enteromorpha polysaccharide lyase are reported to be Alteromonas marcedodi B7 and Alteromonas sp.A321, and the strains lack special industrial enzyme strains for degrading Enteromorpha polysaccharide at present. Therefore, the strain capable of producing high-efficiency enteromorpha polysaccharide degradation is obtained through screening, and the method has important significance for development and utilization of enteromorpha resources.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides an alteromonas HT1 and a culture method and application thereof.
The technical scheme of the invention is as follows:
an Alteromonas (Alteromonas sp.) HT1 is preserved in China center for type culture collection in 3 months and 9 days in 2021 with the preservation address of eight Loa Jia mountain in Wuchang district, Wuhan city, Hubei province and the preservation number of CCTCC NO. M2021217.
The Alteromonas (Alteromonas sp.) HT1 is from the surface of enteromorpha, is negative in gram stain, and has circular, white, moist, smooth and non-pigment colony observed on a solid culture medium; the observation of the transmission electron microscope shows that the alteromonas HT1 cells are in a short rod shape, the length is 2.0-2.5 mu m, the width is 1.0-1.5 mu m, and the cells have single flagellum.
The 16S rDNA gene sequence of alteromonas HT1 is shown in SEQ ID NO. 1.
The culture method of the alteromonas HT1 bacterial liquid comprises the following steps:
(1) taking alteromonas HT1, streaking and inoculating in a TYS solid culture medium, and carrying out inverted culture at 20-30 ℃ for 1-3 days;
(2) selecting a single colony of alteromonas HT1 from a TYS solid culture medium, inoculating the single colony into a TYS liquid culture medium, and performing shake culture overnight at the temperature of 20-30 ℃ and the rpm of 160-200 to prepare a seed solution;
(3) and (3) inoculating the seed solution prepared in the step (2) into a liquid culture medium which takes enteromorpha polysaccharide as a unique carbon source according to the volume percentage of 1%, and performing shake cultivation for 2-4 days at the temperature of 20-30 ℃ and the rotating speed of 160-200 rpm to obtain the bacterium solution of alteromonas HT 1.
Preferably, in step (1), the TYS solid medium is formulated as: 0.5 wt% of peptone, 0.1 wt% of yeast powder, 1.5 wt% of agar powder and 3 wt% of artificial seawater, and the pH value is 7.8.
Preferably, in step (2), the formula of the TYS liquid medium is: 0.5 wt% of peptone, 0.1 wt% of yeast powder and 3 wt% of artificial seawater, and the pH value is 7.8.
According to the preferable formula of the liquid culture medium with the enteromorpha polysaccharide as the only carbon source in the step (3), the formula is as follows: 0.01 wt% of peptone, 0.01 wt% of yeast powder, 3 wt% of NaCl, 0.05 wt% of NH 4 Cl，0.3wt％MgCl 2 ·6H 2 O，0.2wt％K 2 SO 4 ，0.02wt％K 2 HPO 4 ，0.001wt％CaCl 2 ，0.0006wt％FeCl 3 ·6H 2 O，0.0005wt％Na 2 MoO 2 ·7H 2 O，0.0004wt％CuCl 2 ·2H 2 O, 0.6 wt% Tris, 0.2 wt% Enteromorpha polysaccharide, pH 7.8.
The alteromonas HT1 is applied to preparation of enteromorpha polysaccharide lyase and degradation of enteromorpha polysaccharide.
An enteromorpha polysaccharide lyase, the amino acid sequence is shown in SEQ ID NO.3, and the enteromorpha polysaccharide lyase is separated from the alteromonas HT 1.
The nucleotide sequence of the coding gene of the enteromorpha polysaccharide lyase is shown in SEQ ID NO.2, and the coding gene is derived from the alteromonas HT 1.
The enteromorpha polysaccharide lyase is applied to degrading enteromorpha polysaccharide.
Has the advantages that:
1. according to the invention, the screened Alteromonas (Alteromonas sp.) HT1 capable of efficiently degrading enteromorpha polysaccharide can grow in a culture medium taking enteromorpha as a unique carbon source, and a product obtained by degrading the enteromorpha polysaccharide is enteromorpha oligosaccharide which mainly exists in the forms of disaccharide, trisaccharide, tetrasaccharide and pentasaccharide. The strain and enteromorpha polysaccharide lyase secreted by the strain can be applied to degradation of marine green algae and preparation of functional oligosaccharide, and have good application potential in the pharmaceutical industry, the cosmetic industry, the food industry and the like.
2. The invention provides an enteromorpha polysaccharide lyase, which is used for separating self-bred telomonas sp HT1, is easy for heterologous expression and purification, and can efficiently degrade enteromorpha polysaccharide to generate enteromorpha oligosaccharide. The enzymolysis reaction condition is mild, and the method is suitable for high-value processing of enteromorpha and other green algae resources. And the obtained enzymolysis product mainly comprises disaccharide, trisaccharide and pentasaccharide, so the method has obvious technical advantages in the production of enteromorpha prolifera oligosaccharide.
Drawings
FIG. 1 is a Transmission Electron Microscope (TEM) image of Alteromonas (Alteromonas sp.) HT 1.
FIG. 2 is a 16S rDNA phylogenetic tree of Alteromonas (Alteromonas sp.) HT 1.
FIG. 3 is an HPLC chromatogram of a product obtained by degrading Enteromorpha polysaccharide with supernatant of Alteromonas sp HT1 bacterial liquid.
FIG. 4 is an SDS-PAGE electrophoresis picture of heterologously expressed and purified enteromorpha polysaccharase U-3282;
in the figure: m: protein molecular weight standards (marker); u-3282: enteromorpha polysaccharide lyase U-3282;
FIG. 5 is a High Performance Liquid Chromatography (HPLC) analysis chart of Enteromorpha polysaccharide degradation product by Enteromorpha polysaccharide lyase U-3282
FIG. 6 is a mass spectrometry analysis diagram of Enteromorpha polysaccharide lyase U-3282 on the main product peak of the Enteromorpha polysaccharide degradation product.
In a figure: main product peak 1, panel b: main product peak 2, panel c: major product peak 3.
Detailed Description
The invention is further illustrated with reference to the following figures and examples, without however being limited thereto.
The source of the biological material is as follows: alteromonas sp.ht1 was deposited in the chinese culture collection center (CCTCC), address: china center for type culture Collection, with preservation number CCTCC NO. M2021217, of eight Wuhan universities in Wuchang district, Wuhan City, Hubei province.
In the examples, the raw materials for preparing the culture medium are all the raw materials commonly used in the field.
Enteromorpha polysaccharide: heyday by China oceanic university
Seawater culture medium: 3% NaCl (w/v), 0.6% Tris (w/v), 0.3% MgCl 2 ·6H 2 O(w/v)、0.2％K 2 SO 4 (w/v)、0.05％NH 4 Cl(w/v)、0.02％K 2 HPO 4 (w/v)、0.001％CaCl 2 (w/v)、0.0006％FeCl 3 ·6H 2 O(w/v)、0.0005％Na 2 MO 4 ％、0.0004％CuCl 2 ·2H 2 O(w/v)。
TYS solid medium: 0.5 wt% of peptone, 0.1 wt% of yeast powder, 1.5 wt% of agar powder and 3 wt% of artificial seawater, and the pH value is 7.8.
TYS liquid medium: 0.5 wt% of peptone, 0.1 wt% of yeast powder and 3 wt% of artificial seawater, and the pH value is 7.8.
Liquid with enteromorpha polysaccharide as unique carbon sourceCulture medium: 3% NaCl (w/v), 0.3% MgCl 2 ·6H 2 O(w/v)、0.2％K 2 SO 4 (w/v)、0.05％NH 4 Cl(w/v)、0.02％K 2 HPO 4 (w/v)、0.001％CaCl 2 (w/v)、0.0006％FeCl 3 ·6H 2 O(w/v)、0.0004％CuCl 2 ·2H 2 Dissolving O (w/v), 0.6% Tris (w/v) and 0.5% Enteromorpha polysaccharide (w/v) in deionized water, and adjusting the pH to 7.5-8.0.
EXAMPLE 1 screening of strains
Adding 2g of enteromorpha sample into the prepared seawater culture medium in an ultraclean workbench, placing the mixture into a shaking table at 25 ℃ for shake culture for one week, and obtaining enteromorpha suspension after the seawater culture medium is turbid. Diluting Enteromorpha prolifera suspension to 10 -1 ～10 -5 And (3) respectively coating the obtained product in a TYS solid culture medium, culturing for 48 hours in a constant-temperature incubator at 25 ℃, then selecting single colonies, and continuously performing streak culture until the single colonies are obtained after purification.
And (3) selecting the purified single colonies on the TYS solid culture medium, respectively inoculating the single colonies in a TYS liquid culture medium, and culturing overnight in a shaking table at 25 ℃ to obtain a seed solution. Inoculating the seed solution into a liquid culture medium which takes enteromorpha polysaccharide as a unique carbon source according to the inoculation amount of 1%, and performing shake culture for 3 days at the temperature of 25 ℃ and the speed of 180rpm to obtain the extracellular fermentation solution supernatant of the strain. Taking 1mL of extracellular fermentation liquid supernatant, and centrifuging at 13000rpm for 5min to obtain the supernatant which is extracellular crude enzyme liquid of the strain. Adding Tris-HCl with the final concentration of 50mM and pH 8.0 and an enteromorpha polysaccharide substrate of 2mg/mL into a reaction system of 200 mu L, placing the reaction system in a metal bath at the temperature of 30 ℃ for heat preservation for 5min, adding 20 mu L of extracellular crude enzyme solution, reacting for 5h, and taking the inactivated crude enzyme solution as a blank control. After the reaction, 100. mu.L of DNS dye solution was added and heated at 100 ℃ for 5min to terminate the reaction. After cooling, the reaction was centrifuged slightly, and 200. mu.L of the resulting solution was taken for OD measurement 540 Absorbance in OD 540 The absorbance was used as an index, and OD was determined 540 The bacterial strain with the value increased over 0.1 is the bacterial strain for producing the enteromorpha polysaccharide lyase, and the strain is recorded as HT 1.
Strain HT1 was stored in glycerol at a final concentration of 18% (v/v) and stored in an ultra low temperature freezer at-80 ℃.
EXAMPLE 2 identification of the strains
(1) Characteristics of the Strain
The strain HT1 is gram-negative bacterium, and its colony is round, white, wet and smooth as shown in FIG. 1. The cells of strain HT1 were rod-shaped and had flagella (length 2.0-2.5 μm, width 1.0-1.5 μm).
(2) Identification of strains
DNA was extracted from the strain selected in example 1 using a BioTeke bacterial genome extraction kit, and the 16S rRNA gene of HT1 strain was PCR-amplified using 27F and 1492R primers according to the instructions of the kit, and the amplified product was ligated to pMD19-T vector (TaKaRa, China) and sequenced on an Applied Biosystems 3730xl DNA sequencer. .
The PCR primers used were universal primers:
27F：5'-AGAGTTTGATCCTGGCTCAG-3'，
1492R：5'-GGTTACCTTGTTACGACTTC-3'，
the PCR reaction conditions are as follows: pre-denaturation at 95 deg.C for 5 min; denaturation at 95 ℃ for 30 s; annealing at 55 ℃ for 30 s; extension at 72 ℃ for 90 s; 30 cycles, extension at 72 ℃ for 5min, storage at 4 ℃.
The determined 16S rRNA gene sequence was then compared to sequences in GenBank and EzBioCloud ((R))http://www.ezbiocloud.net) A16S rRNA gene sequence database was established using BLASTN. Pairwise sequence similarity values were obtained by the ezbiocoud server. Sequence alignment is carried out by utilizing a ClustalW program in MEGA-X, and a phylogenetic tree is constructed by utilizing a Neighbor-join method of MEGA-X. Bootstrap analysis was performed based on 1000 replicates to assess the stability of branches in the generated phylogenetic tree.
The length of the 16S rDNA gene sequence of the strain obtained by separation and screening in example 1 was 1494bp, obtained by DNA extraction, PCR amplification and sequencing, and BLAST comparison was performed in GenBank to draw a phylogenetic tree, which is shown in FIG. 2.
Based on the 16S rDNA sequence alignment result and the biological characteristics of the strain, the strain is identified as Alteromonas (Alteromonas sp.) named Alteromonas sp.HT1.
EXAMPLE 3 cultivation of the Strain
The culture method of the Alteromonas (Alteromonas sp.) HT1 bacterial liquid comprises the following steps:
(1) inoculating Alternaria alternata (Alteromonas sp.) HT1 in Glycerin tube into TYS solid culture medium by streaking, and culturing at 25 deg.C for 1 day by inversion;
(2) selecting a single colony of alteromonas HT1 from a TYS solid culture medium, inoculating the single colony into a TYS liquid culture medium, and performing shake culture overnight at 25 ℃ and 180rpm to prepare a seed solution;
(3) and (3) inoculating the seed solution prepared in the step (2) to a liquid fermentation culture medium taking enteromorpha polysaccharide as a unique carbon source according to the volume percentage of 1%, and performing shake culture at the temperature of 25 ℃ and the rotating speed of 200rpm for 3 days to obtain a bacterial solution of alteromonas HT 1.
Example 4 high performance liquid chromatography analysis of alteromonas HT1 degraded Enteromorpha polysaccharide and degradation products thereof
Standard reaction system: a200. mu.L reaction contains 2mg/mL polysaccharide substrate, 50mM Tris-HCl buffer pH 8.0, 0.25M NaCl solution.
And adding 20 mu L of the alteromonas HT1 bacterial liquid obtained in the embodiment 3 into a 200 mu L enteromorpha polysaccharide standard reaction system containing 2mg/mL enteromorpha polysaccharide, 0.25M NaCl and 50mM Tris-HCl (pH 8.0), reacting for 12h at 35 ℃, then carrying out boiling water bath on the reaction liquid for 5min to terminate the reaction, and filtering the reaction liquid by using a 0.22 mu M filter membrane to obtain an enteromorpha polysaccharide degradation product.
And (3) performing High Performance Liquid Chromatography (HPLC) analysis by taking the enteromorpha polysaccharide degradation product as an experimental group and taking an enteromorpha polysaccharide standard reaction system as a control group. The High Performance Liquid Chromatography (HPLC) analytical column is Superdex 30 Incase 10/300GL (available from GE Healthcare), the detection wavelength of the ultraviolet detector is 210nm, and the mobile phase is 0.2M NH 4 HCO 3 The flow rate was 0.3mL/min, and the results are shown in FIG. 3.
As can be seen from fig. 3, the bacterial liquid of Alteromonas (Alteromonas sp.) HT1 can effectively degrade enteromorpha polysaccharide, which indicates that the bacterial strain can secrete enteromorpha lyase to the outside of cells, degrade the enteromorpha polysaccharide, and prepare enteromorpha oligosaccharide, wherein the degradation product enteromorpha oligosaccharide mainly exists in the forms of disaccharide, trisaccharide, tetrasaccharide and pentasaccharide.
Example 5 heterologous expression of Enteromorpha lyase
Alteromonas (Alteromonas sp.) HT1 was inoculated in TYS liquid medium, cultured at 30 ℃ for 24h, 2mL of bacterial solution was taken, and after the cells were collected, genomic DNA was extracted according to the instructions of the genome extraction kit of the company Baitach (conventional extraction procedure). Whole genome sequencing analysis was performed by Shanghai Linn organisms.
The sequencing results were analyzed with software on NCBI (National Center for Biotechnology Information, http:// www.ncb1.nlm.nih.gov /). The NCBI analysis software used was Open Reading Frame Finder (ORF Finder, http:// www.ncb1.nlm.nih.gov/gorf. html) and Basic Localalignment Search Tool (BLAST, http:// BLAST. ncb1.nlm. nih. gov/BLAST. cgi).
NCBI analysis results show that Alteromonas (Alteromonas sp.) HT1 carries an Enteromorpha polysaccharide lyase U-3282, the gene coding region of U-3282 is 1587bp in length, the nucleotide sequence of the gene coding region is shown as SEQ ID NO.2, and the gene coding region has 88.26% homology with ulva polysaccharide lyase (NCBI sequence number: A0A0X9SHN5.1) in the whole genome of Pseudomonas sp.PLSV.
The enteromorpha polysaccharide lyase coded by the U-3282 gene consists of 528 amino acids, the amino acid sequence of the enteromorpha polysaccharide lyase is shown as SEQ ID NO.3, and the theoretical molecular weight of the protein is 60 kDa.
PCR amplification was performed using Alternaria alternate (Alteromonas sp.) HT1 genome as template, with the following primers:
F:5’-AAGAAGGAGATATACATATGTGTGAATCGAGCGATACTGT-3’
R:5’-TGGTGGTGGTGGTGCTCGAGCTCAGAAACACCTAAATCAA-3’
the primer is synthesized by Huada gene.
The amplification procedure was: pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 20sec, annealing at 55 ℃ for 20sec, extension at 72 ℃ for 45sec, 30 cycles; extension at 72 ℃ for 90 sec.
The PCR amplification system (50. mu.L) was as follows:
the PCR-amplified product was subjected to 1 wt% agarose gel electrophoresis, and then the amplified DNA fragment was recovered using a DNA recovery kit of Omega according to the instructions thereof.
The pET-22b vector (purchased from Novagen) was double-digested with restriction enzymes NdeI and XhoI, and the digested product was subjected to 1 wt% agarose gel electrophoresis, followed by recovery of the linearized pET-22b vector using a DNA recovery kit from Omega, as described therein.
The amplified DNA fragment was ligated to the linearized pET-22b vector using In-Fusion (available from TaKaRa).
The ligation system (2.5. mu.L) was as follows:
DNA fragment 1. mu.L
Linearized pET-22b 1. mu.L
5x In-Fusion 0.5μL
Transforming the expression vector into an escherichia coli DH5 alpha strain, then coating the strain on an LB culture medium solid plate containing 100 mu g/mL ampicillin sodium, culturing for 14h at 37 ℃, and selecting a monoclonal; inoculating the single clone into a liquid LB culture medium containing 100 mu g/mL ampicillin sodium for culture, and extracting plasmids; the recombinant plasmid is sent to Huada gene for sequencing verification, and the sequencing result shows that the U-3282 coding gene fragment is successfully inserted between enzyme cutting sites NdeI and XhoI of pET-22b, the insertion direction is correct, and the conditions of base mutation, deletion and addition do not occur, so that the recombinant plasmid vector is named as pET-22 b-3282.
The verified vector plasmid is transformed into an escherichia coli strain BL21(DE3) (purchased from Nanjing Nuojingzu Biotechnology GmbH), and then heterologous induced expression of enteromorpha lyase U-3282 is performed according to the operation steps provided by the company. The expression condition of the enteromorpha prolifera lyase U-3282 is detected by polyacrylamide gel electrophoresis, and the result is shown in figure 4, the enteromorpha prolifera lyase U-3282 is a single strip on electrophoresis gel, the molecular weight is between 60 and 80kDa, and the position is consistent with the predicted molecular weight.
Example 6 high performance liquid chromatography analysis of Enteromorpha prolifera lyase U-3282 for degrading Enteromorpha prolifera polysaccharide and degradation products thereof
U-3282 inactivated by heating at 100 ℃ for 10min was used as blank control. The increase in absorbance at 235nm (A235) due to the production of unsaturated uronic acid in the mixture was then measured using a UV-Vis absorption spectrometer (Jasco V-550).
One enzyme activity (U) is defined as: the amount of enzyme required to increase the A235 value by 0.1 per minute.
Preparing an enteromorpha polysaccharide standard reaction system and an ulva polysaccharide standard reaction system according to the standard reaction system described in the embodiment 4 by using enteromorpha polysaccharide and ulva polysaccharide as substrates, and measuring the enzyme activity of U-3282 enzyme on the enteromorpha polysaccharide standard reaction system and the ulva polysaccharide standard reaction system at 35 ℃. The results are shown in Table 1.
TABLE 1 specific Activity of U-3282
As can be seen from Table 1, the Enteromorpha polysaccharide lyase U-3282 has strong degradation activity on Enteromorpha polysaccharide and ulva polysaccharide, but has stronger degradation capability on Enteromorpha polysaccharide, and the specific activity reaches 10467U/mg.
Taking 1mL of enteromorpha lyase U-3282 heterologously expressed in the embodiment 5, adding the enteromorpha lyase U-3282 into a 200 mu L enteromorpha polysaccharide standard reaction system containing 2mg/mL of enteromorpha polysaccharide, 0.25M NaCl and 50mM Tris-HCl (pH 8.0), reacting overnight at 35 ℃, terminating the reaction by boiling water bath for 5min, centrifuging at 13000rpm for 5min, taking supernatant, and filtering the reaction solution by using a 0.22 mu M hydrophilic filter membrane to obtain an enteromorpha polysaccharide degradation product.
Performing High Performance Liquid Chromatography (HPLC) analysis by using the enteromorpha polysaccharide degradation product as an experimental group and an enteromorpha polysaccharide standard reaction system as a control group, wherein the HPLC analysis column is Superdex 30 Incase 10/300GL (purchased from GE Healthcare), the flow rate is 0.3mL/min, the detection wavelength of an ultraviolet detector is 210nm, and the mobile phase is 0.2M NH 4 HCO 3 The results are shown in FIG. 5. Collecting main product peak of experimental group, freeze-drying, adding 100 μ L of triple distilled water, freeze-drying again, and removing NH 4 HCO 3 Dissolving in distilled water, addingAnalysis by electrospray ionization mass spectrometry (ESI-MS) gave the results shown in FIG. 6.
As can be seen from FIG. 5, HPLC analysis of the degradation product of Enteromorpha prolifera polysaccharide by U-3282 mainly generates three main peaks (1, 2, 3), while the control group does not have the main product peak, which indicates that Enteromorpha prolifera polysaccharide lyase U-3282 can effectively degrade Enteromorpha prolifera polysaccharide. From FIG. 6, it can be seen that U-3282 mainly contains disaccharide GlcA as degradation product of Enteromorpha polysaccharide 1 Rha 1 (SO 3 H) 1 Trisaccharide GlcA 1 Rha 2 (SO 3 H) 2 And pentasaccharide GlcA 1 Xyl 1 Rha 3 (SO 3 H) 2 Therefore, the enteromorpha polysaccharide lyase U-3282 has obvious technical advantages in the production of enteromorpha oligosaccharide.
SEQUENCE LISTING
<110> Shandong university
<120> alteromonas strain HT1 and culture method and application thereof
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 1493
<212> DNA
<213> Alternamonas (Alteromonas sp.) HT1
<400> 1
agagtttgat catggctcag attgaacgct ggcggcaggc ctaacacatg caagtcgaac 60
ggtaacagga tgtgcttgca catcgctgac gagtggcgga cgggtgagta atgcttggga 120
acttgccttt gcgaggggga taacagttgg aaacgactgc taataccgca taatgtcttc 180
ggaccaaacg gggcttaggc tccggcgcaa agagaggccc aagtgagatt agctagttgg 240
taaggtaacg gcttaccaag gcgacgatct ctagctgttc tgagaggaag atcagccaca 300
ctgggactga gacacggccc agactcctac gggaggcagc agtggggaat attgcacaat 360
gggggaaacc ctgatgcagc catgccgcgt gtgtgaagaa ggccttcggg ttgtaaagca 420
ctttcagttg tgaggaaaag ttagtagtta atacctgcta gccgtgacgt taacaacaga 480
agaagcaccg gctaactccg tgccagcagc cgcggtaata cggagggtgc gagcgttaat 540
cggaattact gggcgtaaag cgcacgcagg cggtttgtta agctagatgt gaaagccccg 600
ggctcaacct gggatggtca tttagaactg gcagactaga gtcttggaga ggggagtgga 660
attccaggtg tagcggtgaa atgcgtagat atctggagga acatcagtgg cgaaggcgac 720
tccctggcca aagactgacg ctcatgtgcg aaagtgtggg tagcgaacag gattagatac 780
cctggtagtc cacaccgtaa acgctgtcta ctagctgtgt gtgcctttaa ggcgtgcgta 840
gcgaagctaa cgcgctaagt agaccgcctg gggagtacgg ccgcaaggtt aaaactcaaa 900
tgaattgacg ggggcccgca caagcggtgg agcatgtggt taattcgatg caacgcgaag 960
aaccttacct acacttgaca tgctgagaag ttactagaga tagtttcgtg ccttcgggaa 1020
ctcagacaca ggtgctgcat ggctgtcgtc agctcgtgtc gtgagatgtt gggttaagtc 1080
ccgcaacgag cgcaaccctt gtccttagtt gccagcctta agttgggcac tctaaggaga 1140
ctgccggtga caaaccggag gaaggtgggg acgacgtcaa gtcatcatgg cccttacgtg 1200
tagggctaca cacgtgctac aatggcattt acagagggaa gcgagacagt gatgtggagc 1260
ggacccctta aagaatgtcg tagtccggat tggagtctgc aactcgactc catgaagtcg 1320
gaatcgctag taatcgcagg tcagaatact gcggtgaata cgttcccggg ccttgtacac 1380
accgcccgtc acaccatggg agtgggatgc aaaagaagta gttagtctaa ccttcgggag 1440
gacgattacc actttgtgtt tcatgactgg ggtgaagtcg taacaaggta acc 1493
<210> 2
<211> 1587
<212> DNA
<213> Alternamonas (Alteromonas sp.) HT1
<400> 2
atgaaattaa aactaaaaat gcgtggtcta gcgctgcaaa ttagcttacc tgttaaagtt 60
ttggccgtgc tttcaatgtt aacagcctgt gaatcgagcg atactgtagg tgtatcttca 120
agcgcaaaca aagcaagtgg ggtatttcta gagagcagta caaaaataac aaatggcgcg 180
cttcattttg atggtaaaaa gctcaatcac aaaacctttc aaaacccttc aacaggaccc 240
gagtacgact acttttttgg aagaaatatt tcggctcatg gcgatgcggt caaaccttat 300
aagcactacg ttttcatgac ctggtacaaa ggcggcaagg aagaacgcaa tgttatgctt 360
tctaggttta actccaaaac tggcgttgtt aaaaccattc agtttcctca tcgacacaca 420
gggtttcgag gaaatccact ggtaggggag tcacacaaca ccataggttt agccgtaagc 480
ccgataaatg gcaccattca tatggtttac gacatgcacg cttatgtcga cgatggtgaa 540
aacggtagat tcaagggacg attcgttaac gactttttcc gttattctta ttcagtggta 600
ggcgcagcag atgtgccaga tgatgaattt acattaaagc agtttgtaaa agataccagt 660
gaagtgagtc agggggcaga cgactacaaa catctgacca tgacaggtga cttacaagac 720
aaagaaaatt tttctgcgct cacatacccc aaattctata caagtaaaaa tggcgaactc 780
ttgcattaca tgcgatgggg cggcaacaac aatggtgctt attatttcaa taaatacgac 840
gccgaaaaac aagtgtggac tcgttttata cctttcaatc ataaagacca aaaaacgcat 900
ggcaatgctt acaactgggg cttatatggt cagatgaaat atataaacgg taaattacgc 960
gtaggctttc agcaacgtag tgcgaataat aatgaccgct ataagtatca aaatggcgtt 1020
tactatgcgt attctgacca ccctgatggc ttaggagaat ggaaaaatgt agatggcaag 1080
gacatgactt ggcctttggt aaactcagat gaaataaaaa tctttgagcc tggcgattat 1140
atcgatcatc aagaacctaa ctctgtgcac attgtaacgg gttttgactg gacagttacc 1200
gaaaacgaag atattcactt tattacccac gttcgctcta ccgatactaa acgaagtgat 1260
tacaaagagg tgtccataca tgcgtttaaa caggctaacg ccaatgactt tacagttacg 1320
actgacttta ctggtgccga ttctatttat acgtcgggtg acagcatttt cattataggg 1380
ctcaaaaacg gttacccatt tgttgaaaaa gcgaaaggcg gtacgaatga ttttgaagta 1440
gtgtaccagc aaaccacagg aacaaaattt gaccacggca ctattcatat tgaaaacggc 1500
aaagcttact actatttgat ggagaaaggg gccggtaacg cgctaccgct tcatctacaa 1560
attattgatt taggtgtttc tgagtaa 1587
<210> 3
<211> 528
<212> PRT
<213> Alternamonas (Alteromonas sp.) HT1
<400> 3
Met Lys Leu Lys Leu Lys Met Arg Gly Leu Ala Leu Gln Ile Ser Leu
1 5 10 15
Pro Val Lys Val Leu Ala Val Leu Ser Met Leu Thr Ala Cys Glu Ser
20 25 30
Ser Asp Thr Val Gly Val Ser Ser Ser Ala Asn Lys Ala Ser Gly Val
35 40 45
Phe Leu Glu Ser Ser Thr Lys Ile Thr Asn Gly Ala Leu His Phe Asp
50 55 60
Gly Lys Lys Leu Asn His Lys Thr Phe Gln Asn Pro Ser Thr Gly Pro
65 70 75 80
Glu Tyr Asp Tyr Phe Phe Gly Arg Asn Ile Ser Ala His Gly Asp Ala
85 90 95
Val Lys Pro Tyr Lys His Tyr Val Phe Met Thr Trp Tyr Lys Gly Gly
100 105 110
Lys Glu Glu Arg Asn Val Met Leu Ser Arg Phe Asn Ser Lys Thr Gly
115 120 125
Val Val Lys Thr Ile Gln Phe Pro His Arg His Thr Gly Phe Arg Gly
130 135 140
Asn Pro Leu Val Gly Glu Ser His Asn Thr Ile Gly Leu Ala Val Ser
145 150 155 160
Pro Ile Asn Gly Thr Ile His Met Val Tyr Asp Met His Ala Tyr Val
165 170 175
Asp Asp Gly Glu Asn Gly Arg Phe Lys Gly Arg Phe Val Asn Asp Phe
180 185 190
Phe Arg Tyr Ser Tyr Ser Val Val Gly Ala Ala Asp Val Pro Asp Asp
195 200 205
Glu Phe Thr Leu Lys Gln Phe Val Lys Asp Thr Ser Glu Val Ser Gln
210 215 220
Gly Ala Asp Asp Tyr Lys His Leu Thr Met Thr Gly Asp Leu Gln Asp
225 230 235 240
Lys Glu Asn Phe Ser Ala Leu Thr Tyr Pro Lys Phe Tyr Thr Ser Lys
245 250 255
Asn Gly Glu Leu Leu His Tyr Met Arg Trp Gly Gly Asn Asn Asn Gly
260 265 270
Ala Tyr Tyr Phe Asn Lys Tyr Asp Ala Glu Lys Gln Val Trp Thr Arg
275 280 285
Phe Ile Pro Phe Asn His Lys Asp Gln Lys Thr His Gly Asn Ala Tyr
290 295 300
Asn Trp Gly Leu Tyr Gly Gln Met Lys Tyr Ile Asn Gly Lys Leu Arg
305 310 315 320
Val Gly Phe Gln Gln Arg Ser Ala Asn Asn Asn Asp Arg Tyr Lys Tyr
325 330 335
Gln Asn Gly Val Tyr Tyr Ala Tyr Ser Asp His Pro Asp Gly Leu Gly
340 345 350
Glu Trp Lys Asn Val Asp Gly Lys Asp Met Thr Trp Pro Leu Val Asn
355 360 365
Ser Asp Glu Ile Lys Ile Phe Glu Pro Gly Asp Tyr Ile Asp His Gln
370 375 380
Glu Pro Asn Ser Val His Ile Val Thr Gly Phe Asp Trp Thr Val Thr
385 390 395 400
Glu Asn Glu Asp Ile His Phe Ile Thr His Val Arg Ser Thr Asp Thr
405 410 415
Lys Arg Ser Asp Tyr Lys Glu Val Ser Ile His Ala Phe Lys Gln Ala
420 425 430
Asn Ala Asn Asp Phe Thr Val Thr Thr Asp Phe Thr Gly Ala Asp Ser
435 440 445
Ile Tyr Thr Ser Gly Asp Ser Ile Phe Ile Ile Gly Leu Lys Asn Gly
450 455 460
Tyr Pro Phe Val Glu Lys Ala Lys Gly Gly Thr Asn Asp Phe Glu Val
465 470 475 480
Val Tyr Gln Gln Thr Thr Gly Thr Lys Phe Asp His Gly Thr Ile His
485 490 495
Ile Glu Asn Gly Lys Ala Tyr Tyr Tyr Leu Met Glu Lys Gly Ala Gly
500 505 510
Asn Ala Leu Pro Leu His Leu Gln Ile Ile Asp Leu Gly Val Ser Glu
515 520 525
Claims (3)
1. An enteromorpha polysaccharide lyase is characterized in that the amino acid sequence of the enteromorpha polysaccharide lyase is shown as SEQ ID NO. 3.
2. The coding gene of the enteromorpha polysaccharide lyase in claim 1, wherein the nucleotide sequence of the coding gene of the enteromorpha polysaccharide lyase is shown as SEQ ID No. 2.
3. The use of the Enteromorpha polysaccharide lyase of claim 1 in degrading Enteromorpha polysaccharide.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112029752A (en) * | 2019-06-03 | 2020-12-04 | 中国海洋大学 | Ulva lactuca polysaccharide lyase as well as coding gene and application thereof |
| CN112592914A (en) * | 2020-12-31 | 2021-04-02 | 青岛海大生物集团有限公司 | Special green alga polysaccharide lyase and production process thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112029752A (en) * | 2019-06-03 | 2020-12-04 | 中国海洋大学 | Ulva lactuca polysaccharide lyase as well as coding gene and application thereof |
| CN112592914A (en) * | 2020-12-31 | 2021-04-02 | 青岛海大生物集团有限公司 | Special green alga polysaccharide lyase and production process thereof |
Non-Patent Citations (3)
| Title |
|---|
| 《New Family of Ulvan Lyases Identified in Three Isolates from the Alteromonadales Order》;Moran Kopel et al;《JOURNAL OF BIOLOGICAL CHEMISTRY》;20160311;第291卷(第11期);第5871-5878页 * |
| Alteromonas sp.A321产浒苔多糖降解酶及酶学性质研究;李银平;《万方数据》;20150401;摘要，第23页2.2-第24页2.3，第63页4.5 * |
| Purification and characterization of polysaccharides degradasesproduced by Alteromonas sp. A321;Yinping Li et al;《International Journal of Biological Macromolecules》;20160113;第86卷;摘要 * |
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