CN108239613B - Paenibacillus feed, culture medium thereof and application of Paenibacillus feed in preparation of enteromorpha polysaccharide degrading enzyme - Google Patents
Paenibacillus feed, culture medium thereof and application of Paenibacillus feed in preparation of enteromorpha polysaccharide degrading enzyme Download PDFInfo
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Abstract
The invention relates to the technical field of microorganisms, and in particular relates to paenibacillus foraging, a culture medium thereof and application thereof in preparation of enteromorpha polysaccharide degrading enzyme. The invention provides paenibacillus fodders with the preservation number of CGMCC NO.12912, and provides a culture medium for screening strains, a culture medium for activating the strains and a culture medium for fermentation. Also provides a method for preparing the enteromorpha polysaccharide degrading enzyme by fermenting the strain and the enteromorpha polysaccharide degrading enzyme prepared by fermenting. The enteromorpha polysaccharide degrading enzyme produced by the strain and fermentation provided by the invention has good enzyme activity, can degrade enteromorpha polysaccharide, and can prepare enteromorpha polysaccharide oligosaccharides with different polymerization degrees according to different parameters. Experiments show that the activity of the enteromorpha polysaccharide degrading enzyme provided by the invention can reach 1.03U/mL.
Description
Technical Field
The invention relates to the technical field of microorganisms, and in particular relates to paenibacillus foraging, a culture medium thereof and application thereof in preparation of enteromorpha polysaccharide degrading enzyme.
Background
The seaweed sulfated polysaccharide is sulfate-based heteropolysaccharide extracted from seaweed, and has a structure containing sulfate groups, monosaccharide compositions comprise glucose (Glc), rhamnose (Rha), xylose (Xyl), glucuronic acid (GlcA), mannose (Man), galactose (Gal) and the like, sulfate is mainly connected to the rhamnose, but the sulfate is different according to the type and the extraction method of enteromorpha.
The existing research proves that the enteromorpha polysaccharide has various physiological activity functions of immunoregulation, tumor resistance, anticoagulation, virus resistance, oxidation resistance, blood fat reduction and the like. In addition, the enteromorpha polysaccharide has excellent thermal reversible gelation, hydrophilicity, no toxicity and other properties. Therefore, the enteromorpha polysaccharide has the potential of being applied to the fields of food, medicines, cosmetics or chemical engineering. With the deep understanding of the structure and function of Enteromorpha prolifera polysaccharide, the application field of Enteromorpha prolifera polysaccharide is continuously widened.
However, the solubility and the absorbability of the enteromorpha polysaccharide are influenced due to overlarge relative molecular mass of the enteromorpha polysaccharide, so that the application of the enteromorpha polysaccharide in the field of medicines and cosmetics is limited. Compared with the enteromorpha polysaccharide, the enteromorpha polysaccharide oligosaccharide has smaller relative molecular mass, increased solubility, stability and safety, and improved biological activity to a certain extent.
At present, methods for preparing enteromorpha polysaccharide oligosaccharide by degrading enteromorpha polysaccharide comprise a physical method and a chemical method. Acid degradation in chemical processes is the traditional method for polysaccharide hydrolysis, and hydrolysis products with different degradation degrees can be obtained by adjusting acidity, temperature and action time. But the acid hydrolysis action condition is violent, the substrate specificity is poor, the sulfate group of the sulfated polysaccharide is easy to be hydrolyzed and shed in the degradation process, and the reduction of the sulfate group content can cause the reduction of the physiological activity of the polysaccharide. Although the sulfate group shedding of the polysaccharide in the degradation process can be reduced by oxidative degradation, the oxidation product is relatively complex, and the difficulty of separation and purification is increased.
The enzyme method for preparing the oligosaccharide has specificity, can selectively carry out enzymolysis and cut off of a specific site on a sugar chain, and can also avoid the shedding of a sulfate group, thereby preparing the specific oligosaccharide; the method has the advantages of mild reaction conditions, easy control of the degradation process, low cost, simple process, high yield, no pollution and the like. However, enzymes with high hydrolysis efficiency are not easy to obtain, and few reports are reported on enzymatic degradation of enteromorpha polysaccharide at present. Therefore, the method for screening the microorganisms capable of utilizing the enteromorpha polysaccharide and extracting the enteromorpha polysaccharide degrading enzyme from the microorganisms for preparing the low-molecular-weight active fragment of the enteromorpha polysaccharide becomes an important direction for the high-valued research of the enteromorpha polysaccharide industry, and has wide development potential in the aspect of the development of marine medicines.
Disclosure of Invention
In view of the above, the technical problem to be solved by the invention is to provide a Paenibacillus pulus feed, a culture medium thereof and an application thereof in preparing Enteromorpha prolifera polysaccharide-degrading enzyme, wherein the Paenibacillus feed can produce the Enteromorpha prolifera polysaccharide-degrading enzyme, and the enzyme activity of the produced Enteromorpha prolifera polysaccharide-degrading enzyme can reach 1.03U/mL.
In order to screen and obtain the strains producing the enteromorpha polysaccharide degrading enzyme, the invention provides a screening culture medium, which comprises water and the following components in percentage by mass:
the pH of the medium was 7.0.
The MgSO4Is MgSO4·7H2O; the CaCl is2The hydrate of (A) is CaCl2·7H2O; the FePO4The hydrate of (A) is FePO4·7H2O。
In some embodiments, the screening medium comprises water and the following components in mass fractions:
the screening culture medium can also be added with agar to prepare a solid culture medium.
In the solid culture medium, the mass fraction of agar is 1-5%. Preferably, the mass fraction of agar is 2%.
The screening culture medium provided by the invention is applied to screening of strains capable of producing enteromorpha polysaccharide degrading enzyme.
The screening method of the strain capable of producing the enteromorpha polysaccharide-degrading enzyme comprises the following steps:
step 1: dissolving sea mud with sterile seawater, inoculating the sea mud to the screening culture medium provided by the invention, and performing shake-flask culture for 2-3 days;
step 2: coating the culture solution obtained in the step 1 on a solid screening culture medium for culturing for 3-5 days;
and step 3: staining the solid screening culture medium cultured in the step 2 by Lugol iodine solution, and streaking the bacterial colony generating the degradation ring to a new solid screening culture medium for continuous culture;
and 4, step 4: repeating step 3 until a single strain is obtained.
The sea mud adopted by the invention is from the sea area near the first sea water bathing place in Qingdao city, Shandong province.
The temperature of the shake flask culture in the step 1 is 28 ℃, and the rotating speed is 200 rpm.
The temperature for the culture in step 2 was 28 ℃.
After screening according to the method provided by the invention, the bacterial colony which generates the largest degradation ring is picked for biological preservation. The preservation number is CGMCC NO. 12912.
The invention provides paenibacillus fodders with the preservation number of CGMCC NO. 12912.
The biochemical characteristics of the strain comprise:
and (3) colony morphology characteristics: the enteromorpha prolifera polysaccharide-containing solid culture medium has the advantages of good growth, milky white and round bacterial colony, slightly convex surface, smoothness, no reflection, opaqueness, regular edge and diameter of about 3.0 mm;
morphological characteristics of the thallus: the cells are straight rod-shaped, 0.6-0.8 mu m is multiplied by 1.8-3.5 mu m, are arranged singly, are gram-positive and uniformly colored, and can produce capsules and move (peritrichomes); spore is mesogenic or juxtameliogenic, is less than or equal to the width of the cell, and is circular or elliptical.
Physiological and biochemical characteristics: the enteromorpha polysaccharide degrading enzyme can be generated in the enteromorpha polysaccharide liquid culture medium; aerobic treatment; alpha-cyclodextrin, beta-cyclodextrin, dextrin, starch, amygdalin, arbutin, cellobiose, D-fructose, D-galactose, gentiobiose, alpha-D-glucose, alpha-D-lactose, maltose, mannose, D-mannitol, D-mannose, melezitose, melibiose, alpha-methyl-D-galactoside, beta-methyl-D-galactoside, 3-methyl-glucose, alpha-methyl-D-glucoside, beta-methyl-D-glucoside, alpha-methyl-D-mannoside, isomaltulose, D-psicose, D-raffinose, salicin, D-sorbitol, stachyose, D-glucoside, D-galactoside, D-galactos, Sucrose, D-tagatose, D-trehalose, turanose, acetic acid, beta-hydroxybutyric acid, methyl pyruvate, pyruvic acid, monomethyl succinate, glycerol, adenosine, 2 '-deoxyadenosine, inosine, thymidine, uridine, adenosine-5' -phosphate, thymidine-5 '-phosphate, uridine-5' -phosphate, 6-phospho-fructose, 6-phospho-glucose.
The reaction of catalase, beta-galactosidase, arginine double hydrolase and gelatinase of the strain is positive; does not exhibit urease, tryptophan deaminase, ornithine decarboxylase, lysine decarboxylase activities; no indole and hydrogen sulfide are generated; the nitrate reduction is negative.
The 16S rDNA gene sequence of the Paenibacillus fodders with the preservation number of CGMCC NO.12912 has high homology with the 16S rDNA sequence of each strain of the Paenibacillus, the maximum similarity reaches 99 percent, and the gene sequence and the Paenibacillus pabuli (AB073191.1) are positioned in the same cluster and have the closest genetic relationship.
And coating the paenibacillus feed with the preservation number of CGMCC NO.12912 on a solid culture medium plate containing enteromorpha polysaccharide, and culturing for 3-5 days. The lugol iodine solution is used for dyeing to form an obvious degradation ring. Therefore, the temperature of the molten metal is controlled,
the invention also provides application of the paenibacillus feed with the preservation number of CGMCC NO.12912 in preparing the enteromorpha polysaccharide degrading enzyme.
The invention also provides a culture medium suitable for the paenibacillus fodders with the preservation number of CGMCC NO. 12912.
A culture medium comprising:
the carbon source is selected from alpha-cyclodextrin, beta-cyclodextrin, dextrin, starch, amygdalin, arbutin, cellobiose, D-fructose, D-galactose, gentiobiose, alpha-D-glucose, alpha-D-lactose, maltose, mannose, D-mannitol, D-mannose, melezitose, melibiose, alpha-methyl-D-galactoside, beta-methyl-D-galactoside, 3-methyl-glucose, alpha-methyl-D-glucoside, beta-methyl-D-glucoside, alpha-methyl-D-mannoside, isomaltulose, D-psicose, D-raffinose, salicin, D-sorbitol, D-glucoside, D-mannoside, D-, Stachyose, sucrose, D-tagatose, D-trehalose, turanose, acetic acid, beta-hydroxybutyric acid, methyl pyruvate, pyruvic acid, monomethyl succinate, glycerol, adenosine, 2 '-deoxyadenosine, inosine, thymidine, uridine, adenosine-5' -phosphate, thymidine-5 '-phosphate, uridine-5' -phosphate, 6-phospho-fructose or 6-phospho-glucose;
the carbon source in the culture medium is selected from agarose, starch, enteromorpha polysaccharide, sucrose and maltose. In some embodiments, the carbon source of the medium is enteromorpha polysaccharide.
The concentration of the carbon source is 0.4 g/L-4 g/L. Specifically 0.4g/L, 0.6g/L, 0.8g/L, 1g/L, 2g/L, 3g/L or 4 g/L. Experiments show that the concentration of the carbon source influences the growth condition of the thallus and the activity of the produced enzyme, the growth condition of the thallus becomes worse with the increase of the concentration of the carbon source, and the activity of the enzyme shows the trend of increasing first and then decreasing with the increase of the concentration of the carbon source.
The nitrogen source in the culture medium is selected from NaNO3Urea, yeast extract, peptone. In the examples of the present invention, the nitrogen source is peptone and yeast extract. In some embodiments, the yeast extract is a yeast extract powder; the peptone is tryptone or casein peptone. The mass ratio of the peptone to the yeast extract is (4-16): (2-14).
In the embodiment of the invention, the concentration of the peptone is 4 g/L-16 g/L. Specifically 4g/L, 6g/L, 8g/L, 10g/L, 12g/L, 14g/L or 16 g/L. Experiments show that the concentration of the peptone influences the growth condition of thalli and the activity of enzyme production, the growth condition of the thalli shows a trend of increasing firstly and then decreasing with the increase of the concentration of the peptone, the highest value of the peptone is 10g/L, and the enzyme activity also shows a trend of increasing firstly and then decreasing with the increase of the concentration of the peptone, and the highest value is 6 g/L.
Similarly, the concentration of the yeast extract powder is 3g/L to 15 g/L. Specifically 3g/L, 5g/L, 7g/L, 9g/L, 11g/L, 13g/L or 15 g/L. Experiments show that the concentration of the yeast extract powder affects the growth condition of the thallus and the activity of the produced enzyme, the growth condition of the thallus shows a trend of increasing firstly and then decreasing with the increase of the concentration of the yeast extract powder, the highest value of the growth condition of the thallus shows a trend of 9g/L, and the activity of the enzyme shows a trend of increasing firstly and then decreasing with the increase of the concentration of the yeast extract powder, and the highest value of the activity of the enzyme shows a trend of increasing firstly and then decreasing with the increase of the concentration of the yeast.
In the embodiment of the invention, the concentration of NaCl is 5 g/L-35 g/L; specifically 5g/L, 10g/L, 15g/L, 20g/L, 25g/L, 30g/L or 35 g/L. Experiments show that the concentration of NaCl influences the growth condition of the thallus and the activity of the produced enzyme, and the growth condition of the thallus is worsened along with the increase of the concentration of NaCl, and the activity of the enzyme is also reduced.
In the examples of the present invention, Na2HPO4The concentration of (A) is 1 mmol/L-9 mmol/L; specifically 1mmol/L, 2mmol/L, 3mmol/L, 4mmol/L, 5mmol/L, 6mmol/L, 7mmol/L, 8mmol/L or 9 mmol/L. Experiments show that Na2HPO4The concentration of (A) affects the growth of the cells and the activity of the enzyme produced, and is dependent on Na2HPO4The growth of the cells becomes worse with increasing concentration of Na2HPO4The activity of the enzyme is increased and then decreased at the increased concentration, and the highest value is 6 mmol/L.
Experiments show that the pH value of the culture medium influences the growth condition of thalli and the activity of produced enzyme, and in the embodiment of the invention, the pH value of the culture medium is 6.0-8.0. Specifically 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 or 9.0. The highest value of the thallus growth and the enzyme activity is when the pH value is 7.0.
FePO4The hydrate of (a) is iron phosphate heptahydrate; MgSO (MgSO)4The hydrate of (a) is magnesium sulfate heptahydrate.
Examples of media for verifying the effect include:
proved by verification, the culture medium with better effect comprises:
more preferably, the culture medium comprises:
the culture medium provided by the invention is applied to the culture of paenibacillus foraging with the preservation number of CGMCC NO. 12912.
Agar is added to the medium to prepare a solid medium.
The mass fraction of the added agar is 1 to 5 percent.
In order to activate the strain provided by the invention, the invention also provides a culture medium comprising:
the pH value of the culture medium is 6.0-8.0, and preferably 7.0.
The culture medium is applied to the paenibacillus foraging with the activation preservation number of CGMCC NO. 12912.
In a culture medium for activating the strain provided by the invention, the peptone is tryptone or casein peptone; the yeast extract is yeast extract or yeast extract powder; preferably yeast extract powder.
In some embodiments, the culture medium that activates the bacterial species provided herein comprises
The invention also provides a method for preparing the enteromorpha polysaccharide degrading enzyme, which prepares the enteromorpha polysaccharide degrading enzyme by fermenting the feed bacillus with the preservation number of CGMCC NO. 12912.
Specifically, the preparation of the enteromorpha polysaccharide-degrading enzyme comprises the following steps:
step 1: activating feed Paenibacillus with a preservation number of CGMCC NO.12912 by using a culture medium to prepare a seed solution;
step 2: inoculating the seed liquid to a culture medium, and fermenting to obtain a fermentation product;
and step 3: centrifuging the fermented product, taking supernatant, sterilizing, concentrating, desalting, and drying to obtain Enteromorpha polysaccharide degrading enzyme.
In the embodiment of the invention, the culture medium for activating the paenibacillus foraging with the preservation number of CGMCC NO.12912 in the step 1 comprises:
the culture medium adopted for fermentation in the step 2 is provided by the invention and comprises the following components:
the culture medium used for the pretreatment is a solid culture medium formed by adding agar to a culture medium used for fermentation, and specifically, the solid culture medium comprises:
the activation condition in the step 1 is 24-36 ℃, and the shake culture is carried out for 12-24 h; the rotating speed of the shaking table is 150-210 rpm.
The fermentation condition in the step 2 is 24-36 ℃, and the shake culture is carried out for 24-48 h; the rotating speed of the shaking table is 150-210 rpm.
In the examples, the fermentation temperature, the rotation speed, the inoculum size and the fermentation volume were screened.
Wherein the fermentation temperature is 20 deg.C, 24 deg.C, 28 deg.C, 32 deg.C, 36 deg.C. The results show that 28 ℃ is the optimum fermentation temperature.
The rotation speed of the fermentation was 40rpm, 80rpm, 120rpm, 160rpm, 200rpm, and 240 rpm. The results show that 200rpm is the optimum rotation speed.
The inoculation amount is 1-8%, specifically 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%. The results revealed that the optimum inoculum size was 3%.
The fermentation volume is 50 mL-120 mL, specifically 50mL, 60mL, 70mL, 80mL, 90mL, 100mL, 110mL, 120 mL. The results show that the optimal fermentation volume is 70 mL.
The research of the invention shows that the activity and the amount of the enteromorpha polysaccharide degrading enzyme obtained by 24h fermentation can reach higher level under the appropriate condition.
The rotation speed of the centrifugation in the step 3 is 6000rpm, and the time is 20 min.
The centrifugation, sterilization, concentration and desalination in step 3 are all carried out at 4 ℃.
The sterilization adopts filtration sterilization, and the filter diameter is 0.22 mu m.
The concentration is carried out by ultrafiltration.
Desalting by dialysis.
The dialysis aperture is 8000-14000 Da
The components of the dialysate include: 0.5mM PBS buffer
Drying is vacuum freeze drying; the procedure was freezing at-80 deg.C, freeze-drying at-50 deg.C, and freeze-drying for 48 h.
The invention also provides the enteromorpha polysaccharide degrading enzyme prepared by fermenting the paenibacillus feed with the preservation number of CGMCC NO. 12912.
The invention provides paenibacillus fodders with the preservation number of CGMCC NO.12912, and provides a method for screening strainsA culture medium for activating a strain, and a culture medium for fermentation. Also provides a method for preparing the enteromorpha polysaccharide degrading enzyme by fermenting the strain and the enteromorpha polysaccharide degrading enzyme prepared by fermenting. The enteromorpha polysaccharide degrading enzyme produced by the strain and fermentation provided by the invention has good enzyme activity, can degrade enteromorpha polysaccharide, and can prepare enteromorpha polysaccharide oligosaccharides with different polymerization degrees according to different parameters. Experiments show that the activity of the enteromorpha polysaccharide degrading enzyme provided by the invention can reach 1.03U/mL. The prepared enteromorpha polysaccharide oligosaccharide (low molecular weight enteromorpha polysaccharide) has a molecular weight of 4.48 × 104Da; the monosaccharide composition is: rhamnose (Rha): glucose (Glc): galactose (Gal): xylose (Xyl): arabinose (Ara) ═ 1.65: 1.00: 0.09: 0.57: 0.17.
biological preservation Instructions
EP-1, taxonomic nomenclature: paenibacillus subtilis as a feed is preserved in China general microbiological culture Collection center (CGMCC) at 29/08 in 2016, and the addresses are as follows: the institute of microbiology, national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing. The preservation number is CGMCC No. 12912.
Drawings
FIG. 1 is a scanning electron micrograph of strain EP-1 (accession number CGMCC No. 12912);
FIG. 2 phylogenetic tree of strain EP-1 (preservation number CGMCC No.12912) and its close strains;
FIG. 3 shows the superoxide radical scavenging activity of Enteromorpha polysaccharide;
FIG. 4 shows Enteromorpha polysaccharide hydroxyl radical scavenging activity;
FIG. 5 shows Enteromorpha polysaccharide DPPH free radical scavenging activity;
FIG. 6 shows the Enteromorpha polysaccharide reducing capacity;
figure 7 shows the metal chelating ability of enteromorpha polysaccharide.
Detailed Description
The invention provides paenibacillus foddei, a culture medium thereof and application thereof in preparing enteromorpha polysaccharide degrading enzyme, and technical personnel in the field can realize the purpose by appropriately improving process parameters by referring to the contents. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The instruments adopted by the invention are all common commercial products and can be purchased in the market.
The invention is further illustrated by the following examples:
example 1
Separation and purification of Enteromorpha polysaccharide degrading bacteria (Paenibacillus pabuli) EP-1.
Collecting sea mud in sea area near the first sea water bath of Qingdao city, Shandong province, adding sterile seawater for dissolving, transferring into primary screening culture medium, and shake-flask enrichment culturing for 2-3 days. And coating the culture solution on a solid culture medium plate containing enteromorpha polysaccharide, and culturing for 3-5 days. And (3) dyeing by Lugol iodine solution, transferring the bacterial colony with an obvious degradation ring to a new enteromorpha polysaccharide solid culture medium plate for culture, and repeatedly scribing until a pure culture is obtained. Primary screening of culture medium components (mass-volume ratio): enteromorpha polysaccharide 4%, NaCl1.5%, NaNO30.5％，MgSO4·7H2O0.05％，CaCl20.01 percent, 0.005 percent of ferric phosphate and 7.0 of pH value.
Inoculating the pure culture obtained by primary screening into a fermentation culture medium, and performing shake culture. And then, scribing the culture solution on an enteromorpha polysaccharide solid culture medium, and picking out the bacterial colony which generates the largest degradation ring for the next experiment. All the screened strains are separated and purified and then preserved.
Morphological and physiological characteristics identification of strain (Paenibacillus pabuli. sp) EP-1 the strain EP-1 for identification is inoculated in an enteromorpha polysaccharide culture medium, 15g/l of agar can be added when preparing a solid culture medium, and sterilization is carried out for 30min at 121 ℃.
Obtaining a single colony by plate streak culture, wherein the strain has the following morphological characteristics:
(1) colony morphology: the enteromorpha prolifera polysaccharide solid culture medium has the advantages of good growth, milky white and round colony, slightly raised surface, smoothness, no reflection, opaqueness, neat edge and diameter of about 3.0 mm;
(2) cell morphology: the cells are in straight rod shape, 0.6-0.8 μm multiplied by 1.8-3.5 μm, are arranged singly, are gram-positive and uniformly colored, can produce capsules and can move (peritrichomes); spore is mesogenic or juxtameliogenic, is less than or equal to the width of the cell, and is circular or elliptical.
Amplifying 16S rRNA gene of strain (Paenibacillus pabuli.sp) EP-1, sequencing and analyzing strain EP-1, streaking on the inclined plane of enteromorpha polysaccharide solid culture medium, and culturing at 28 ℃ until forming bacterial lawn. Under the aseptic condition, a ring of thalli is scraped by an inoculating loop and is placed in a 1.5mL aseptic centrifuge tube, and a bacterial genome rapid extraction kit is adopted to extract genome DNA which is used as a template for PCR directly. The universal primers for amplifying the 16S rRNA gene were as follows:
a forward primer: 5'-AGAGTTTGATCCTGGCTCAG-3', respectively;
reverse primer: 5'-ACGGTTACCTTGTTACGACTT-3', respectively;
the primers correspond to bases 8 to 27 and 1510 to 1492 of 16S rRNA gene of Escherichia coli, respectively. The 50 μ L PCR reaction system was: 10 XBuffer 5. mu.L, 10mM dNTPs 1. mu.L, 4. mu.M primers 1. mu.L each, ddH2O42. mu.L, Taq DNA polymerase 0.5. mu.L (2.5U), DNA template 0.5. mu.L (10-100 ng). The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 75 s; after 33 cycles, extension was carried out at 72 ℃ for 10min, and sequencing of the obtained PCR product was carried out by Biotechnology engineering (Shanghai) Co., Ltd. The sequence length of the 16S rRNA gene of the obtained strain EP-1 was 1372 bp.
The resulting sequences were compared for similarity using the Blast program. Phylogenetic tree construction (FIG. 2) and homology comparison were performed on each species of Paenibacillus and EP-1 using the neighbor-joining method using the MEGA (molecular evolution Genetics analysis)6 software package. It was found that the EP-1 strain 16SrRNA gene sequence has high homology with the 16S rRNA sequence of each species of Paenibacillus, and the maximum similarity reaches 99%. The evolutionary tree shows that EP-1 and Paenibacillus pabuli (AB073191.1) are located in the same cluster, the genetic relationship is recent, and the EP-1 strain is preliminarily identified as Paenibacillus fodders.
The strain EP-1 is preserved in CGMCC with the preservation number of CGMCC NO. 12912.
Example 2
Respectively preparing culture media shown in the table 1, activating the feed paenibacillus with the preservation number of CGMCC NO.12912, and then inoculating the feed paenibacillus to the culture media shown in the table 1. Inoculating 5%, culturing at 28 deg.C and 200rpm for 24 hr, and determining OD of bacterial liquid600The value is obtained.
Centrifuging the obtained bacterial liquid at 6000rpm for 20min to obtain fermentation supernatant; and (3) filtering the obtained fermented supernatant with a 0.22-micron filter membrane for sterilization, performing ultrafiltration concentration, dialyzing for desalting, and performing vacuum freeze drying at the temperature of 4 ℃ to obtain the enteromorpha polysaccharide degrading enzyme. And (4) measuring the enzyme activity. The detection method of the enzyme activity is a DNS method, and the enzyme activity is defined as follows: 1mL of enzyme solution produced 1. mu.g of reducing sugars as one activity unit for 1 min.
TABLE 1 culture Medium formulation
According to table 1, the optimal carbon source is enteromorpha polysaccharide, and the optimal nitrogen source is a composition of peptone and yeast powder. Under proper conditions, the enzyme activity of the enteromorpha polysaccharide degrading enzyme can reach 1.03U/mL.
Example 3
Adopting the culture medium with the best effect in Table 1 (the formula is 0.2 percent of Enteromorpha polysaccharide, 0.6 percent of peptone, 0.7 percent of yeast extract powder, 0.5 percent of NaCl, and MgSO4·7H2O 0.1％,Na2HP046mmol/L) to prepare the enteromorpha polysaccharide degrading enzyme by fermentation. Pre-treating and activating before fermentation.
1) And pretreatment: marking the feed Paenibacillus with the preservation number of CGMCC NO.12912 on an enteromorpha polysaccharide solid culture medium, and culturing for 5-7 d at 25-35 ℃;
2) and activating: inoculating 1-3 rings of the solid culture medium strain into a test tube of a seed culture solution, and culturing at the temperature of 25-35 ℃ for 12-24 h at 150-210 rpm.
3) And fermenting: and (3) inoculating the seed culture medium into a fermentation culture medium, and performing shake cultivation for 24-48 h to obtain a fermentation product.
Step 3, fermentation tests with different parameters are carried out, and the parameters are shown in the table 2:
after the fermentation was completed, the OD of the bacterial liquid was measured600The value is obtained.
Centrifuging the obtained bacterial liquid at 6000rpm for 20min to obtain fermentation supernatant; and (3) filtering the obtained fermented supernatant with a 0.22-micron filter membrane for sterilization, performing ultrafiltration concentration, dialyzing for desalting, and performing vacuum freeze drying at the temperature of 4 ℃ to obtain the enteromorpha polysaccharide degrading enzyme. And (4) measuring the enzyme activity. The detection method of the enzyme activity is a DNS method, and the enzyme activity is defined as follows: 1mL of enzyme solution produced 1. mu.g of reducing sugars as one activity unit for 1 min.
TABLE 2 fermentation conditions
The result shows that the inoculation amount is 3%, the culture temperature is 28 ℃, the rotating speed of a shaking table is 200rpm, the culture is carried out for 24 hours, the fermentation volume is 70mL, and the generated enzyme activity is highest.
Example 4
Preparation of Low molecular weight Enteromorpha polysaccharide (Enteromorpha polysaccharide oligosaccharide) Using Enteromorpha polysaccharide degrading enzymes prepared in example 2 or example 3
Cleaning the dry Enteromorpha prolifera powder with tap water (removing salt and other impurities), and drying at 60 deg.C for use. Extracting polysaccharide by a hot water extraction method, according to the ratio of enteromorpha: adding distilled water into water (w/v): 1:30, heating and stirring at 100 ℃ for extracting for 2h, filtering by using 200-mesh bolting silk, repeatedly extracting filter residue once, combining two crude filtrates, and measuring the volume. Standing the coarse filtrate, cooling to gel, cutting into strips, and freezing in a refrigerator at-20 deg.C. And taking out the rubber strips after the rubber strips are completely frozen, breaking the rubber strips, adding 95% alcohol with the volume 1.5 times that of the crude filtrate, and standing the mixture at room temperature to freeze and dehydrate ice blocks. Filtering with 200 mesh silk, squeezing out water and alcohol by hand. And further dehydrating the filter residue by using a certain amount of 95% alcohol, and then drying at 60 ℃ to obtain the enteromorpha crude polysaccharide.
Separating and purifying the crude polysaccharide by using a DEAE Sepharose Fast Flow weak anion exchange chromatographic column, collecting fractions by using a partial collector, and detecting the total sugar content by using a sulfuric acid-phenol method. According to the linear elution result, respectively eluting 2 column volumes with distilled water, 2 column volumes with 0.5mol/L NaCl, 3 column volumes with 1.07mol/L NaCl, 2 column volumes with 4mol/L NaCl, and washing 6 column volumes with deionized water (in order to remove redundant salt solution); column volume: 60 mL; sample loading amount: 30 mg; flow rate: 3.5 mL/min. Collecting part of the sample by a collector, detecting the total sugar content by a sulfuric acid-phenol method, and plotting the elution volume, the absorbance and the salt concentration. Collecting distilled water eluate, dialyzing to remove salt, concentrating, lyophilizing, and storing in a drier.
The polysaccharide fraction obtained by anion exchange chromatography on DEAE Sepharose Fast Flow was further purified by Sepharose 6B gel column chromatography (2.6X 80 cm). The eluent was water at a flow rate of 1 mL/min. Fractions were collected using a fraction collector (10 mL/tube); the total sugar content was determined by the sulfuric acid-phenol method, and the absorbance was measured at 490nm and plotted as absorbance-elution time. Mixing the same components, dialyzing, desalting, lyophilizing, and storing in a desiccator.
The preparation of the enteromorpha polysaccharide with low molecular weight is carried out by an enzyme hydrolysis method. Reacting Enteromorpha polysaccharide degrading enzyme and Enteromorpha polysaccharide (the amount of enzyme is 2% of polysaccharide mass) at 45 deg.C for 72h, boiling with boiling water for 10min to inactivate protein, centrifuging at 8000rpm for 10min, collecting supernatant, adding 95% ethanol, and precipitating to remove unreacted polysaccharide. And (3) concentrating the supernatant through rotary evaporation, dialyzing (less than or equal to 2000Da), and freeze-drying to obtain the enteromorpha polysaccharide with low molecular weight.
Detected as lowThe Enteromorpha polysaccharide with molecular weight is white and easily soluble in water, the total sugar content is 56.08 +/-1.19%, no uronic acid is seen, the sulfate group content is 17.934 +/-0.12%, and the protein content is 0.64 +/-0.14; molecular weight of 4.48X 104Da; the monosaccharide composition is: rhamnose (Rha): glucose (Glc): galactose (Gal): xylose (Xyl): arabinose (Ara) ═ 1.65: 1.00: 0.09: 0.57: 0.17.
the biological activity of the low molecular weight polysaccharide was further measured, and the results showed that it was strong in superoxide radical scavenging activity (FIG. 3), hydroxyl radical scavenging ability (FIG. 4), DPPH radical scavenging ability (FIG. 5), reducing ability (FIG. 6), and metal chelating ability (FIG. 7).
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.
Claims (3)
1. Paenibacillus foeniculaceus with preservation number of CGMCC NO.12912 (Paenibacillus pabuli ） EP-1。
2. Application of paenibacillus feed with the preservation number of CGMCC NO.12912 in preparing enteromorpha polysaccharide degrading enzyme.
3. A method for preparing enteromorpha polysaccharide degrading enzyme is characterized in that the enteromorpha polysaccharide degrading enzyme is prepared by fermenting feed paenibacillus with the preservation number of CGMCC NO. 12912.
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