CN118006510A - Micro-bubble bacteria for producing algin lyase and application thereof - Google Patents
Micro-bubble bacteria for producing algin lyase and application thereof Download PDFInfo
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- CN118006510A CN118006510A CN202410410699.2A CN202410410699A CN118006510A CN 118006510 A CN118006510 A CN 118006510A CN 202410410699 A CN202410410699 A CN 202410410699A CN 118006510 A CN118006510 A CN 118006510A
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- algin
- microbubble
- lyase
- algin lyase
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- 235000010443 alginic acid Nutrition 0.000 title claims abstract description 99
- 229920000615 alginic acid Polymers 0.000 title claims abstract description 99
- 108090000856 Lyases Proteins 0.000 title claims abstract description 51
- 102000004317 Lyases Human genes 0.000 title claims abstract description 51
- 241000894006 Bacteria Species 0.000 title abstract description 22
- 241000582681 Microbulbifer donghaiensis Species 0.000 claims abstract description 8
- 244000005700 microbiome Species 0.000 claims abstract description 6
- 238000000034 method Methods 0.000 claims description 38
- 108090000790 Enzymes Proteins 0.000 claims description 27
- 102000004190 Enzymes Human genes 0.000 claims description 27
- 238000000855 fermentation Methods 0.000 claims description 25
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- 230000000694 effects Effects 0.000 abstract description 12
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Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
The invention belongs to the field of microorganisms, and discloses a microbubble strain for producing algin lyase and application thereof; the classification of the micro-vesicular bacteria producing the algin lyase is named as east sea micro-vesicular bacteria (Microbulbifer donghaiensis), and the micro-vesicular bacteria are preserved in the microorganism strain preservation center of Guangdong province at the year 2024, 02, with the preservation number of GDMCC No:64370; the strain can produce algin lyase; the activity of the algin lyase can reach 25.2 U.mL ‑1; the invention also provides the application of the microbubble bacteria and the algin lyase derived from the microbubble bacteria in the production of algin lysate.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to a microbubble strain for producing algin lyase and application thereof.
Background
Sodium alginate, also known as sodium alginate, was originally extracted from algins produced by brown algae organisms, and is commonly polymerized from beta-D-mannuronic acid (beta-D-mannuronic acid, M) and alpha-L-guluronic acid (G) by 1, 4-glycosidic bonds in industry, and has various uses in the food industry, such as thickening, gelling, stabilizing systems. The brown alginate oligosaccharide (Alginate Oligosaccharides, AOS) is a molecule with polymerization degree of 2-25, is prepared by decomposing brown alginate, and has multiple functions of regulating human immunity, resisting oxidation, resisting tumor, protecting nervous system, promoting plant growth, inhibiting bacteria, etc. The AOS has special chemical and biological characteristics, and has potential and wide application value in the fields of agriculture, food, medicine, health care products, cosmetics, metallurgy, chemical industry and the like. In addition, the algin also has the biological activities of reducing blood fat, resisting tumor, resisting bacteria and the like.
The molecular weight of the commercial algin is usually 33000-400000 Da, the dissolution speed is low, the solution viscosity is high, and the algin is difficult to penetrate through biological membranes and is absorbed and utilized by organisms. And brown alginate oligosaccharides with the polymerization degree of 2-25 after degradation often show better physicochemical characteristics and higher biological activity. The biological activity of AOS is affected by the degree of polymerization and the M/G composition ratio. Therefore, how to efficiently and directionally prepare AOS becomes an important problem for research and utilization of AOS, wherein a biodegradation method is one of potential means for solving the problem.
The biodegradation method generally refers to degrading the algin by adding algin lyase or producing the algin lyase by microbial fermentation, so as to obtain the AOS, and the method has the advantages of mild reaction conditions, specific biological enzyme action sites, high yield and the like, and can effectively avoid the problems of high energy consumption, long reaction time, poor repeatability, environmental pollution and the like in the physical and chemical degradation process. The algin-lysing bacteria identified are mainly from the genera Pseudomonas sp, bacillus sp, vibrio sp and Sphingomonas sp. However, most algin-lyase strains have low enzyme production capacity, so industrial mass production of AOS prepared by microbial fermentation is not realized. Based on the method, the screening of the efficient and safe algin cracking bacteria has important significance and practical production value.
Providing a strain capable of efficiently producing alginate lyase is one of the important problems to be solved in the art.
Disclosure of Invention
The present invention proposes the following technical solution to the above problems existing in the prior art.
In a first aspect, the present invention provides an algin lyase-producing eastern sea microbubble strain (Microbulbifer donghaiensis) deposited on the cantonese microbiological strain collection center located in the building No. 59 in the viet-show martyr of guangdong, guangzhou, 2024, 02, under accession number GDMCC No:64370.
In a second aspect, the present invention provides a method for preparing algin lyase from the strain of microbubble east China sea according to the first aspect of the present invention, wherein the method comprises: (1) Inoculating the seed solution of the Donghai microbubble strain into a liquid culture medium, and performing shake culture at 25-35 ℃ and 180-240 rpm to obtain fermentation liquor containing algin lyase.
In a third aspect, the present invention provides an algin lyase, which is characterized in that the algin lyase is obtained by fermentation of the strain of the micro-vesicular bacteria in the east sea of the first aspect of the present invention.
In a fourth aspect, the invention provides the eastern sea microbubble strain of the first aspect of the invention and the application of the algin lyase of the third aspect of the invention in the preparation of the fucoidin.
In a fifth aspect, the present invention provides a method for preparing brown alginate oligosaccharides, comprising: the algin lyase of the third aspect of the present invention is contacted with a sample containing algin to perform enzymolysis.
The beneficial effects of the invention are as follows:
The invention provides a new east sea microbubble strain which can be used for preparing alginate oligosaccharides, and enriches the strain sources for producing alginate lyase. The preservation number provided by the invention is GDMCC No:64370 of the microbubble fungus SKLFSR 131 has high-efficiency alginate degradation effect, and the strain can degrade the alginate into the alginate oligosaccharides by using the alginate lyase synthesized by the strain under mild external conditions (the temperature is about 35 ℃ and the pH is about 8.0). The east sea microbubble bacteria and the algin lyase derived from the east sea microbubble bacteria can be used for preparing the algin oligosaccharides, and have important significance in improving the utilization efficiency of the algin and promoting the development and preparation of the functional algin oligosaccharides.
Drawings
FIG. 1 is a transparent circle screen of strain SKLFSR;
FIG. 2 is a microscopic morphological observation (5000X) of strain SKLFSR;
FIG. 3 is a graph showing the fermentation enzyme production and bacterial growth curve of strain SKLFSR;
FIG. 4 shows a thin layer chromatography analysis of the enzymatic hydrolysis product.
Detailed Description
The invention discloses an algin-producing lyase strain and application thereof, and a person skilled in the art can use the content of the algin-producing lyase strain to properly improve the process parameters to realize the algin-producing lyase strain. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter described herein belongs. Before describing the present invention in detail, the following definitions are provided to better understand the present invention.
Where a range of values is provided, such as a range of concentrations, a range of percentages, or a range of ratios, it is to be understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of the range, and any other stated or intervening value in that stated range, is encompassed within the subject matter unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and such embodiments are also included in the subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the subject matter.
In the context of the present invention, many embodiments use the expression "comprising", "including" or "consisting essentially/mainly of … …". The terms "comprises," "comprising," or "consists essentially of … …" are generally understood to be open ended terms that include not only the individual elements, components, assemblies, method steps, etc., specifically listed thereafter, but also other elements, components, assemblies, method steps. In addition, the expression "comprising," "including," or "consisting essentially of … …" is also to be understood in this document as a closed-form expression, in certain instances, to mean that only the elements, components, assemblies, method steps specifically listed thereafter are included, and that no other elements, components, assemblies, method steps are included. At this time, the expression is equivalent to the expression "consisting of … …".
As described above, the invention provides a strain of micro-vesicular bacteria producing alginate lyase and application thereof.
In a first aspect, the present invention provides an algin lyase-producing strain of microbubble eastern bacteria (Microbulbifer donghaiensis) deposited on 02 month 02 of 2024 at the Guangdong province microorganism strain deposit center of floor No. 100 and 59 in the 3 rd View region martyr of Guangzhou province, with deposit number GDMCC No:64370. the eastern sea microbubble strain is a wild strain screened by the inventor from kelp samples collected from the Wiback adult of Shandong, and is also named SKLFSR and 131 in the invention.
In one embodiment, the Donghai microbubble strain has a 16S rDNA sequence as set forth in SEQ ID NO. 1.
The east sea microbubble SKLFSR of the present invention has the following characteristics:
(1) Sodium alginate can be hydrolyzed in a screening culture medium with sodium alginate as a unique carbon source and a transparent ring can be displayed under the treatment of calcium chloride;
(2) Grows well in marine agar medium (2216);
(3) Can produce algin lyase;
(4) Algin can be hydrolyzed to brown alginate oligosaccharides.
Although research has been carried out at present to show that other strains of the genus microbubble (microbubbler) can produce algin lyase, whether the strains of the genus microbubble (Microbulbifer donghaiensis) in the east China sea can produce algin lyase and how the enzyme activities are not yet reported, and the strains of different species in the same genus have larger differences, so that the strains of one species can produce algin lyase and the strains of the other species can not mean that the strains of the other species can also produce algin lyase. Therefore, compared with the existing research, the invention provides a new strain for preparing the algin oligosaccharide, enriches the strain sources for producing the algin lyase, and has important significance for improving the utilization efficiency of the algin and promoting the development and preparation of the functional algin oligosaccharide.
In a second aspect, the present invention provides a method for preparing algin lyase from the strain of microbubble east China sea according to the first aspect of the present invention, wherein the method comprises: (1) Inoculating the seed solution of the Donghai microbubble strain into a liquid culture medium, and performing shake culture at 25-35 ℃ and 180-240 rpm to obtain fermentation liquor containing algin lyase.
In a preferred embodiment, in step (1), the strain of microbubble east China sea bacteria is shake cultivated at 30℃under 200rpm for a period of time, such as at least 20 hours, at least 30 hours or at least 40 hours.
In one embodiment, the method further comprises: before the step (1), inoculating single colony of the Donghai microbubble strain into a liquid culture medium, and performing shake culture at 25-35 ℃ and 180-240 rpm to prepare seed liquid of the Donghai microbubble strain.
In a preferred embodiment, single colonies of the Donghai microbubble strain are shake cultured at 30℃for a period of time, e.g., 24-48 hours, under 200 rpm conditions.
In one embodiment, the liquid medium is 2216 liquid medium.
2216 Liquid culture medium is a culture medium commonly used for enrichment culture of marine bacteria, and mainly comprises: yeast extract, peptone, ferric citrate, sodium chloride, magnesium chloride, potassium chloride, sodium sulfate, calcium chloride, sodium carbonate, potassium bromide 、SrCl2、H3BO3、NaSiO3、NaF、NH4NO3、Na2HPO4、 distilled water, and the PH is 7.6. Wherein, peptone and yeast extract powder in the culture medium can provide nitrogen source, vitamins and growth factors to meet the requirement of bacterial growth; various inorganic salts provide an environment similar to that of seawater, are suitable for the growth of marine bacteria, and can be added with agar as a coagulant to prepare a corresponding solid culture medium.
In a preferred embodiment, the 2216 liquid medium is formulated as follows: yeast extract 1.0 g, peptone 5.0 g, ferric citrate 0.1 g,NaCl 19.45 g, magnesium chloride 5.9 g, potassium chloride 0.55 g, sodium sulfate 3.24 g, calcium chloride 1.8 g, sodium carbonate 0.16 g, potassium bromide 0.08 g,SrCl2 34.0 mg,H3BO3 22.0 mg,NaSiO3 4.0 mg,NaF 2.4 mg,NH4NO3 1.6 mg,Na2HPO4 8.0 mg, distilled water 1000.0 ml, ph 7.6. The 2216 liquid culture medium can be used after being sterilized at the high temperature of 121 ℃ under high pressure of 20 min.
In one embodiment, the method further comprises: after the step (1), centrifuging the fermentation liquor containing the algin lyase at 6000-8000 r/min for 10-30min, and collecting supernatant to obtain crude enzyme liquor. The crude enzyme solution may be used as it is or after further purification.
In a third aspect, the present invention provides an algin lyase, which is characterized in that the algin lyase is obtained by fermentation of the strain of the micro-vesicular bacteria in the east sea of the first aspect of the present invention.
In one embodiment, the algin lyase is prepared by the method of the second aspect of the invention.
In a fourth aspect, the invention provides the eastern sea microbubble strain of the first aspect of the invention and the application of the algin lyase of the third aspect of the invention in the preparation of the fucoidin.
In a fifth aspect, the present invention provides a method for preparing brown alginate oligosaccharides, comprising: the algin lyase of the third aspect of the present invention is contacted with a sample containing algin to perform enzymolysis.
In one embodiment, the brown alginate oligosaccharides are brown alginate disaccharides, brown alginate trisaccharides and/or brown alginate tetrasaccharides.
The eastern sea microbubble strain provided by the invention can degrade algin into alginate oligosaccharides by using the algin lyase synthesized by the strain under mild external conditions (the temperature is about 35 ℃ and the pH is about 8.0). The east sea microbubble bacteria and the algin lyase derived from the microbubble bacteria can be used for preparing the algin oligosaccharides, and have important significance in improving the utilization efficiency of the algin and promoting the development and preparation of functional algin oligosaccharides.
Embodiments of the present invention will be described in detail below with reference to examples. Those skilled in the art will appreciate that the following examples are illustrative only and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
EXAMPLE 1 isolation and screening of microvesicle fungi SKLFSR 131
The kelp sample is obtained from the mountain-east Winheim, the obtained sample is oscillated in physiological saline added with magnetic beads for 2 hours, kept stand overnight, then inoculated into a liquid culture medium taking sodium alginate as the sole carbon source, cultured at 30 ℃ under the condition of 200 r/min until the blocky kelp is degraded, and then transferred, and continuously passaged for 5 times. And respectively diluting culture solution of 5 generations of continuous domestication by 10 -7、10-8、10-9 times of normal saline, then coating the culture solution on a solid culture medium with sodium alginate as a unique carbon source, culturing for 3-5 days at the temperature of 30 ℃, and randomly picking single colonies with different colony morphologies after single colonies grow on a flat plate, and streaking on the solid culture medium with sodium alginate as the unique carbon source. And (3) inoculating the single bacterial colony obtained after streaking into a 24-hole plate filled with a liquid culture medium taking sodium alginate as a unique carbon source, culturing for 24-30 hours at the temperature of 30 ℃ under the condition of 200 r/min, and preserving and re-screening the bacterial liquid after the bacterial liquid is turbid. The single colony obtained by the primary screening was inoculated on a solid medium with sodium alginate as the only carbon source, and after 3 days of culture, the transparent circle size was observed by CaCl 2 chromogenic method (FIG. 1).
The formula of the liquid culture medium with sodium alginate as the only carbon source is as follows: sodium alginate 5 g/L, ammonium sulfate 5 g/L,K2HPO4 2 g/L,NaCl 5 g/L,MgSO4·7H2O 1 g/L,FeSO4·7H2O 0.01 g/L.
The formula of the solid culture medium with sodium alginate as the sole carbon source is that agar is added on the basis of the formula of the liquid culture medium with sodium alginate as the sole carbon source.
Example 2 identification of microbubble SKLFSR 131
(1) Thallus characteristics under electron microscope
The morphological structure of the microvesicle strain SKLFSR selected in example 1 was observed under scanning electron microscopy. FIG. 2 shows characteristics of micro-bubble fungus SKLFSR 131 amplified 5000 times under a scanning electron microscope, and it can be seen from the figure that the micro-bubble fungus is rod-shaped and a plurality of micro-bubble fungus are arranged in bubbles with a size of 0.3-0.5 μm×1.1-7.5 μm.
(2) Sequence alignment
The bacterial strain selected in example 1 was used to extract the whole genome of the cells using a bacterial genomic DNA extraction kit (Tiangen Biochemical technologies Co., ltd.), followed by PCR amplification of 16S rDNA using a universal 27F/1492R primer (forward primer 5'-AGAGTTTGATCCTGGCTCAG-3', reverse primer 5'-GGTTACCTTGTTACGACTT-3') under the following conditions: 95 ℃ 5 min;95 ℃ for 30 s,55 ℃ for 30 s,72 ℃ for 60 s,35 cycles; 10min at 72 ℃. The PCR amplified products were sequenced by the attorney docket (Shanghai) Biotechnology Co. The deoxidized nucleic acid sequence of the 16S rDNA of the strain is shown as SEQ ID NO:1 is shown as follows:
CCCGAAGGTTAGACTAGCCACTTCTGGAGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGGCCGGGAACGTATTCACCGTGACATTCTGATTCACGATTACTAGCGATTCCGACTTCACGGAGTCGAGTTGCAGACTCCGATCCGGACTACGATTGGTTTTCTCGGATTAGCTCCACCTCGCGGATTCGCAACCGTCTGTACCAACCATTGTAGCACGTGTGTAGCCCAGGACGTAAGGGCCATGATGACTTGACGTCGTCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCCTTTGAGTTCCCACCATTACGTGTTGGCAACAAAGGACAAGGGTTGCGCTCGTTACGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTCACTCAGTTCCCGAAGGCACCAATCCATCTCTGGAAAGTTCTGAGGATGTCAAGCCCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCTACTTATTGCGTTAGCTGCGTCACAAAGTCCTCAAGGAACCCTACGACTAGTAGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTCAGCGTCAGTATCGAGCCAGGCAGTCGCCTTCGCCACTGATGTTCCTTCCTATATCTACGCATTTCACCGCTACACAGGAAATTCCACTACCCTCTCTCGTACTCTAGCCATCCAGTTCTGAATGCAGTTCCCAGGTTAAGCCCGGGGCTTTCACATCCAGCTTAAATAACCGCCTACGCGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGGTGCTTCTTCTGCAGGTAACGTCAATCTTGCAGAGTATTAATCTACAAGCCTTCCTCCCTGCTGAAAGTGCTTTACAACCCGAAGGCCTTCTTCACACACGCGGCATGGCTGGATCAGGGTTGCCCCCATTGTCCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCTGATCATCCTCTCAGACCAGCTACGGATCGTCGCCTTGGTGAGCCATTACCTCACCAACTAGCTAATCCGACGCGGGCATATCCAATAGCACGAGGTCCGAAGATCCCCCGCTTTCCCCCGTAGGGCGTATGCGGTATTAGCATCCGTTTCCGAATGTTGTCCCCCACTACTGGGCAATTTCCCACGCGTTACTCACCCGTCCGCCGCTCTACTCATTCCCGAAGGAACTTTCGCGCTCGACTTGCA
The 16S rDNA deoxynucleic acid sequence was aligned with the 16S rDNA sequence in the gene library of the national center for Biotechnology (National Center for Biotechnology Information USA, NCBI) to find that the 16S rDNA sequence of this strain has 99% homology with the 16S rDNA sequence of the strain CN85 of Torulopsis glabra (Microbulbifer donghaiensis), and this strain was identified as Torulopsis glabra (Microbulbifer donghaiensis) and designated as SKLFSR 131. The strain is preserved in the Guangdong province microorganism strain preservation center of No. 100 building No. 59 in the Xiuzhou Kouzui martyr in Guangdong province on 02 month 02 of 2024, and the preservation number is GDMCC No:64370.
Example 3 determination of fermentation enzyme production Curve and thallus growth Curve
Strain SKLFSR was cultured using a fermentation medium, and samples were taken every 5 hours to determine the OD600 of the fermentation broth and the enzyme activity of the fermentation broth, and the growth curve and fermentation enzyme production curve of strain SKLFSR 131 were determined as follows:
(1) Inoculating the eastern sea microbubble (Microbulbifer donghaiensis) SKLFSR 131 into 2216 solid culture medium, and culturing at 30deg.C for 24-48 hr until single colony is grown;
(2) Picking single colony in the step (1) to 4 mL of 2216 liquid culture medium, culturing at 30 ℃ and 200 rpm overnight for 12 to 14 hours, and activating the strain;
(3) Inoculating the seed bacterial liquid in the step (2) into 2216 liquid culture medium of 200 mL/500 mL (culture medium volume/conical flask volume) in an inoculum size of 2%, fermenting and culturing at 30 ℃ for 40 hours at 200rpm, and taking samples every 5 hours;
(4) After the culture is finished, centrifuging the fermentation broth in the step (3) at 6000-8000 r/min for 10-30 min, and collecting supernatant to obtain crude enzyme liquid;
(5) The OD600 of the fermentation broth was measured, the enzyme activity of the fermentation broth was measured by the dinitrosalicylic acid (DNS) method, and the growth curve and the fermentation enzyme production curve of strain SKLFSR were plotted (FIG. 3).
2216 Solid culture medium comprises the following formula: yeast extract 1.0 g, peptone 5.0 g, ferric citrate 0.1g,NaCl 19.45 g, magnesium chloride 5.9 g, potassium chloride 0.55 g, sodium sulfate 3.24 g, calcium chloride 1.8 g, sodium carbonate 0.16 g, potassium bromide 0.08 g,SrCl2 34.0 mg,H3BO3 22.0 mg,NaSiO3 4.0 mg,NaF 2.4 mg,NH4NO3 1.6 mg,Na2HPO4 8.0 mg, agar 20 g distilled water 1000.0 ml, ph 7.6, and high temperature and high pressure sterilization at 121 ℃ 20 min.
The fermentation medium is 2216 liquid medium, and the formula is as follows: yeast extract 1.0 g, peptone 5.0 g, ferric citrate 0.1 g,NaCl 19.45 g, magnesium chloride 5.9 g, potassium chloride 0.55 g, sodium sulfate 3.24 g, calcium chloride 1.8 g, sodium carbonate 0.16 g, potassium bromide 0.08 g,SrCl2 34.0 mg,H3BO3 22.0 mg,NaSiO3 4.0 mg,NaF 2.4 mg,NH4NO3 1.6 mg,Na2HPO4 8.0 mg, distilled water 1000.0 ml, ph 7.6, and high temperature and high pressure sterilization at 121 ℃ 20min.
The method for drawing the fermentation enzyme production curve comprises the following steps:
(1) Drawing glucose standard curve
Accurately preparing 1 mg/mL of D-anhydrous glucose standard solution, respectively sucking 40, 80, 120 and 160 mu L of standard solution into a clean centrifuge tube, and supplementing sterile water to a final volume of 200 mu L. Adding 150 mu L of dinitrosalicylic acid (DNS) reagent into each tube, uniformly mixing, boiling for reaction 10min, cooling, adding 1 mL sterile water, uniformly mixing, detecting the light absorption value of OD540, and drawing a standard curve by taking the glucose content (mg) as an abscissa and taking the OD540 as an ordinate.
(2) Detection of enzyme activity of SKLFSR.sup.131 algin lyase by dinitrosalicylic acid (DNS) method
80. Mu.L of the crude enzyme solution was mixed with 920. Mu.L of an algin substrate solution (algin 0.5% (m/v), naH 2PO4,Na2HPO4, mM, sodium chloride 300 mM, pH=7.5) uniformly, and water-bath was performed at 30℃for min; under the condition that other conditions are unchanged, the crude enzyme liquid is boiled for 10min to be inactivated to be used as a control group. After the reaction is finished, 200 mu L of reaction solution obtained by the reaction of the crude enzyme solution and the algin substrate solution is taken and uniformly mixed with 150 mu L of DNS solution, boiled for reaction 10min, cooled and then added with 1mL sterile water for uniform mixing, and the change condition of the OD540 absorbance value is detected.
(3) Definition of enzyme activity unit: under the above reaction conditions, the content of alginate lyase required for producing 1. Mu.g of reducing sugar in1 min was one enzyme activity unit (U.mL -1). The specific calculation formula is as follows:
enzyme Activity (U.mL -1) = (m.times.N.times.1000)/(T.times.V)
M-the content (mg) of reducing sugar produced by the enzymatic reaction of the crude enzyme solution, calculated from the glucose standard curve and OD 540;
N—dilution of fermentation broth (undiluted to 1);
T-enzymatic reaction time (min);
v-enzyme solution (fermentation broth) volume (mL).
According to the formula, the enzyme activity of the algin lyase produced by the strain SKLFSR 131 cultured for different time under the culture condition is calculated, and a fermentation enzyme production curve can be drawn.
It was found that SKLFSR.about.5 hours after inoculation in 2216 liquid medium, the growth phase was continued, 20 hours later, the slow phase was continued, and the culture was continued until the period reached 35 hours. The cell growth was maximized at 25 hours and the enzyme activity was maximized at 30 hours, and the enzyme activity measured by the DNS method was 25.2.+ -. 0.34U/mL (FIG. 3).
Example 4 preparation of alginate oligosaccharides
(1) Thin layer chromatography analysis of enzymatic hydrolysis products
The crude enzyme solution of algin lyase prepared in example 3 is used for enzymolysis of algin, 0.5U algin lyase is added into 500 mu L of sodium alginate solution (pH 8.0) containing 50 mmol/L phosphate and 300 mmol/L NaCl (mass volume ratio), the reaction is carried out for 72 hours at 35 ℃, and the reaction is stopped by boiling for 10 min, thus obtaining the final product.
(2) Carrying out thin layer chromatography identification on the enzymolysis product
Taking 1 mu L of the final product, spotting on a TLC silica gel plate, placing in a chromatographic cylinder for unfolding, taking out and airing when the developer approaches to the position 1 cm above the silica gel plate, adding the developer, taking out and airing after 15 seconds, and displaying at 150 ℃. The developing agent is n-butanol, acetic acid and H 2 O (3:2:3, volume ratio), the color-developing agent is sulfuric acid, ethanol (1:9, volume ratio).
Experimental results show that the product of SKLFSR.sup.131 enzymatic hydrolysis is selected from the group consisting of fucoidan disaccharide, fucoidan trisaccharide and fucoidan tetrasaccharide (FIG. 4).
Claims (10)
1. An eastern sea microbubble strain (Microbulbifer donghaiensis) for producing algin lyase, which is deposited on the Guangdong province microorganism strain collection of No. 100 building No. 59 in Xiuzhou martyr of Guangzhou province in 2024, 02 month 02, with deposit number of GDMCC No:64370.
2. A method of preparing algin lyase from the eastern sea microbubble strain of claim 1, comprising: (1) Inoculating the seed solution of the Donghai microbubble strain into a liquid culture medium, and performing shake culture at 25-35 ℃ and 180-240 rpm to obtain fermentation liquor containing algin lyase.
3. The method according to claim 2, wherein the method further comprises: before the step (1), inoculating single colony of the Donghai microbubble strain into a liquid culture medium, and performing shake culture at 25-35 ℃ and 180-240 rpm to prepare seed liquid of the Donghai microbubble strain.
4. A method according to claim 2 or 3, wherein the liquid medium is 2216 liquid medium.
5. A method according to claim 2 or 3, characterized in that the method further comprises: after the step (1), centrifuging the fermentation liquor containing the algin lyase at 6000-8000 r/min for 10-30 min, and collecting supernatant to obtain crude enzyme liquor.
6. An algin lyase obtained by fermentation of the eastern sea microbubble strain of claim 1.
7. The algin lyase according to claim 6, wherein the algin lyase is prepared by the method of claim 2.
8. Use of the eastern sea microbubble strain of claim 1 or the algin lyase of claim 6 or 7 in the preparation of alginate oligosaccharides.
9. A method for preparing brown alginate oligosaccharides, said method comprising: the algin lyase of claim 6 or 7 is contacted with an algin-containing sample for enzymatic hydrolysis.
10. The method of claim 9, wherein the alginate oligosaccharides are alginate disaccharides, alginate trisaccharides and/or alginate tetrasaccharides.
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