CN111100826A

CN111100826A - Lexella capable of producing alginate lyase and application thereof

Info

Publication number: CN111100826A
Application number: CN202010122190.XA
Authority: CN
Inventors: 李莉莉; 秦松; 刘正一; 宋宛霖
Original assignee: Yantai Institute of Coastal Zone Research of CAS
Current assignee: Yantai Institute of Coastal Zone Research of CAS
Priority date: 2020-02-27
Filing date: 2020-02-27
Publication date: 2020-05-05
Anticipated expiration: 2040-02-27
Also published as: CN111100826B

Abstract

The invention discloses a lecanicillium capable of producing alginate lyase and application thereof, wherein the lecanicillium is separated from rotten gulfweed picked from the yellow sea, has short growth cycle, can produce the alginate lyase, has higher enzyme activity, and has been subjected to strain preservation in 2019, 4 and 28 days, and the preservation places are as follows: china center for type culture Collection, the collection numbers are: CCTCC M2019314. The invention has the advantages that: (1) the screened strain AlgM can produce high-activity alginate lyase, the capacity of degrading algin is enhanced, and the yield of alginate oligosaccharide is improved; (2) the growth cycle of the screened strain AlgM is short, the strain AlgM can grow by taking sodium alginate, glucose and peptone as carbon sources, and the enzyme can be efficiently produced; (3) the seed culture medium and the fermentation culture medium do not contain inorganic salt, so that the separation and purification process of the brown algae oligosaccharide is simplified.

Description

Lexella capable of producing alginate lyase and application thereof

Technical Field

The invention relates to a strain and application thereof, in particular to a lecanicillium capable of producing alginate lyase and application thereof, belonging to the technical field of marine organisms.

Background

Algin (alginate) is a linear heteropolysaccharide consisting of β -D-mannuronic acid (M) and α -L-guluronic acid (G) linked by 1, 4 glycosidic bonds with a unique random structure.

Alginate oligosaccharides are small molecular weight fragments obtained by degrading algin with acid or lyase, are functional oligosaccharides, and are widely concerned by people.

The algin degradation method mainly comprises a chemical method and a biological enzymolysis method, at present, the chemical method which is hydrolyzed by dilute acid is generally adopted in industry, although the method is simple and easy to implement, the degradation speed is slow, the yield is low, the molecular weight distribution of products is wide, in comparison, the biological enzymolysis method which is formed by eliminating reaction through β to form unsaturated double bonds between non-reducing tail ends C4 and C5 has the advantages of mild condition, high degradation speed and high substrate specificity.

In the biological enzymolysis method, alginate lyase is an important functional enzyme for preparing alginate oligosaccharide. The existing enzyme-producing bacteria have low enzyme activity and weak algin degrading capability, so that the yield of the alginate oligosaccharide is low.

In addition, in the biological enzymolysis method, the following formula is adopted for the fermentation culture medium: 5g/L of sodium alginate, 5g/L of ammonium sulfate, 1g/L of magnesium sulfate, 2g/L of dipotassium hydrogen phosphate, 0.01g/L of ferrous sulfate and tap water. Inorganic salts are added into a fermentation medium, and the types and the concentrations of the added inorganic salts play an important role in the growth and the enzyme production of strains, but the added inorganic salts also cause the separation and purification process of the brown alga oligosaccharides to become complicated in the later period.

Disclosure of Invention

In order to solve the defects of the prior art, the first purpose of the invention is to provide a lecanicillium which can produce high-activity alginate lyase, has a short growth period and can grow by taking sodium alginate, glucose and peptone as carbon sources, and the second purpose of the invention is to provide a fermentation method which can simplify the separation and purification process of alginate oligosaccharides by taking the lecanicillium as an enzyme-producing bacterium.

In order to achieve the above object, the present invention adopts the following technical solutions:

leckera capable of producing alginate lyase (I)Leclerciasp.), characterized in that the lecanicillium is separated from rotten gulfweed picked from the yellow sea, has short growth cycle, can produce alginate lyase, has higher enzyme activity, has been subjected to strain preservation in 2019, 4, 28 days, and has the preservation places: china center for type culture Collection, the collection numbers are: CCTCC M2019314.

The application of the lechi capable of producing the alginate lyase in the preparation of the alginate oligosaccharide is characterized in that the method for preparing the alginate oligosaccharide by using the lechi capable of producing the alginate lyase comprises the following specific steps:

step 1: activating a strain AlgM, inoculating a plurality of strains into a seed culture medium, performing shake culture to obtain a seed solution, inoculating the seed solution into a fermentation culture medium, and performing shake culture to obtain a fermentation broth, wherein:

the formula of the seed culture medium is as follows: 5g/L of glucose, 5g/L of peptone, 10g/L of yeast extract powder and seawater preparation;

the formula of the fermentation medium is as follows: 3g/L of glucose, 3g/L of sodium alginate, 3g/L of peptone, 6g/L of yeast extract powder and tap water;

step 2: centrifuging the fermentation liquor at 4 ℃ to obtain a crude enzyme solution;

and step 3: adding the crude enzyme solution into the algin solution for enzymolysis to obtain an enzymolysis solution;

and 4, step 4: separating and purifying the brown algae oligosaccharide.

The application is characterized in that in the step 1, the strain AlgM is activated on an Alg plate culture medium, and the formula of the Alg culture medium is as follows: 5g/L of sodium alginate, 5g/L of ammonium sulfate, 1g/L of magnesium sulfate, 2g/L of dipotassium phosphate, 0.01g/L of ferrous sulfate, 20g/L of agar and tap water.

The application is characterized in that in the step 1, when the seed culture and the fermentation culture are carried out, the culture temperature is 30 ℃, the rotating speed of a shaking table is 200rpm, and the culture time is 28 h.

The above application is characterized in that, in step 3, the concentration of the alginate solution is 3g/L, and the volume ratio of the crude enzyme solution to the alginate solution is 1: 9.

The application is characterized in that, in step 3, the enzymolysis temperature is as follows: 35 ℃ is carried out.

The application is characterized in that, in the step 4, the steps of separating and purifying the brown algae oligosaccharide sequentially comprise: ceramic membrane filtration, alcohol precipitation and suction filtration, rotary evaporation, ultrafiltration dialysis, ion exchange and nanofiltration concentration.

The invention has the advantages that:

(1) the screened strain AlgM can produce high-activity alginate lyase, the capacity of degrading algin is enhanced, and the yield of alginate oligosaccharide is improved;

(2) the growth cycle of the screened strain AlgM is short, the strain AlgM can grow by taking sodium alginate, glucose and peptone as carbon sources, and the enzyme can be efficiently produced;

(3) the seed culture medium and the fermentation culture medium do not contain inorganic salt, so that the separation and purification process of the brown algae oligosaccharide is simplified.

Drawings

FIG. 1 is the phylogenetic tree we constructed;

FIG. 2 is a growth curve of strain AlgM in seed medium;

FIG. 3 is a graph showing the effect of different carbon sources on the production of enzymes by the strain AlgM;

FIG. 4 is a graph showing the results of gram iodine staining;

FIG. 5 is a view showing the result of TLC detection.

Detailed Description

The invention is described in detail below with reference to the figures and the embodiments.

First part, screening and identification of strains

Through the annual research on the zymogenic strains, a strain which has the advantages of high zymogenic activity and short growth cycle and can produce the algin lyase is separated from rotten gulfweed.

The bacterial strain for producing the alginate lyase with the advantages of high enzyme production activity and short growth cycle is obtained by screening through the following method:

1. obtaining starting strain

In 2018, 6 and 11 months, 4 rotten gulfweed plants are picked from the yellow sea, and then a starting strain is obtained by screening and separating the rotten gulfweed plants, wherein the detailed screening and separating process is as follows:

(1) preliminary screening

Homogenizing 4 rotten gulfweed together, then leaving clear liquid, respectively spreading the clear liquid on 6 MALg plate culture media, wherein the 6 MALg plate culture media are respectively marked as H1, H2, H3, H4, H5 and H6, then culturing the 6 MALg plate culture media at a constant temperature of 30 ℃ for 24 hours, then picking up single colonies on each plate culture medium, which generate hydrolysis loops, onto new MALg plate culture media, in the step, picking up 10 single colonies in total from the plate culture media marked as H3, H4 and H5, and respectively marking the new MALg plate culture media corresponding to the 10 single colonies as AlgI, AlgJ, AlgK, AlgL, AlgM, AlgN, AlgO, AlgP, AlgQ and AlgR.

(2) Double sieve

We incubated the 10 single colonies (picked on new MAlg plate medium) obtained from the primary screening at 30 ℃ for 24h, then picked the single colony with the largest hydrolysis loop, at this step, we picked the single colony with the largest hydrolysis loop from the plate medium marked as AlgM, and we marked the strain corresponding to the single colony as AlgM, and finally picked the single colony on MAlg slant medium and stored at-80 ℃.

Formula of MALg culture medium: 5g/L of sodium alginate, 3g/L of glucose, 3g/L of peptone, 6g/L of yeast extract powder, 20g/L of agar and tap water.

2. Culturing strain AlgM to obtain seed liquid

(1) Culture medium

The formula of the Alg culture medium is as follows: 5g/L of sodium alginate, 5g/L of ammonium sulfate, 1g/L of magnesium sulfate, 2g/L of dipotassium phosphate, 0.01g/L of ferrous sulfate, 20g/L of agar and tap water.

The seed culture medium formula comprises: 5g/L of glucose, 5g/L of peptone, 10g/L of yeast extract powder and seawater.

(2) Seed culture

Activating the strain AlgM on an Alg plate culture medium, then inoculating the activated strain into a seed culture medium, and culturing at 30 ℃ and 200rpm for 24h to obtain a seed solution.

3. Fermentation culture

(1) Fermentation medium

The fermentation medium formula comprises: 3g/L of glucose, 3g/L of sodium alginate, 3g/L of peptone, 6g/L of yeast extract powder and tap water.

(2) Fermentation culture conditions

A300 mL Erlenmeyer flask was filled with 100mL of fermentation medium, and the seed solution was inoculated into the fermentation medium at an inoculum size of 2% (volume fraction) for each experimental group, and cultured at 30 ℃ and 200rpm for 24 hours.

4. Activity measurement of alginate lyase

Centrifuging the fermentation liquor at 4 ℃ and 8000g for 10min to obtain a crude enzyme solution, placing the crude enzyme solution in an ice bath, slowly adding ammonium sulfate into the supernatant, keeping the saturation of the ammonium sulfate at 20% under a magnetic stirrer, standing overnight at 4 ℃, then centrifuging at 4 ℃ and 8000g for 10min to remove impure proteins (slowly adding to prevent protein denaturation), continuously adding the ammonium sulfate into the supernatant until the saturation is 80%, standing overnight at 4 ℃, then centrifuging at 10000g for 30min, taking precipitates, and dissolving the precipitates with a small amount of PBS buffer solution to obtain a concentrated enzyme solution.

The algin lyase is characterized by that it utilizes β elimination mechanism to form unsaturated double bond between non-reducing end C4 and C5, said unsaturated double bond has strong absorption at 235nm, and the light absorption value and quantity of formed unsaturated double bond are linearly related, and 0.9mL of algin solution (pH 7.0) whose concentration is 3g/L is taken, 0.1mL of concentrated enzyme solution is added, and heat-insulating treatment is carried out at 35 deg.C for 10min, so that the light absorption value of system at 235nm can be measured.

The unit of enzyme activity is defined as: the area of the transparent ring is increased by 0.1mm every 1min²The amount of enzyme required is one enzyme activity unit (EU).

The enzyme activity of the concentrated enzyme solution is 2.60EU by experiment.

5. Identification of the Strain AlgM

Extracting DNA of the strain AlgM by using a kit extraction method, obtaining 16S rDNA of the strain through PCR amplification, performing similarity comparison on the 16S rDNA sequence of the strain and a bacterial 16S rDNA sequence in a database by using a Blast program in NCBI, constructing a phylogenetic tree (shown in figure 1) by using MEGA 5.1 software (Neighbor-Joining), analyzing the evolutionary relationship of each strain, and determining the bacterial species.

By comparison, we determined that the strain AlgM is Klebsiella (R) ((R))Leclerciasp.）。

Second part, deposit of the Strain AlgM

Because the strain AlgM has higher enzyme activity, the strain AlgM is preserved in China center for type culture Collection with the preservation addresses as follows: wuhan, China, the preservation date is: 28 th 4 month in 2019, the preservation number is: CCTCC M2019314, categorically named: lerlichia sp.

Third part, application of strain AlgM

The strain AlgM obtained by screening and separating can produce alginate lyase, and the produced alginate lyase has high enzyme activity, so that the strain can be applied to the preparation of alginate oligosaccharides.

We will now describe in detail the method of obtaining fucoidan by degrading algin using the strain AlgM.

1. Drawing a growth curve of the strain AlgM

And (2) selecting the strain AlgM on an Alg plate culture medium for activation, then selecting a ring of the activated strain AlgM by using an inoculating ring, inoculating the activated strain AlgM into a seed culture medium, carrying out shake flask culture at the temperature of 30 ℃, sampling at the rotating speed of a shaking table of 200rpm every 3h, diluting to a proper concentration, measuring the light absorption value at 600nm, and drawing a growth curve of the strain AlgM.

The growth curve of the resulting strain AlgM is plotted in FIG. 2.

As can be seen from fig. 2, the growth of strain AlgM has a distinct 3 phases: a delay period (0-9 h), a logarithmic period (9-28 h) and a stationary period (28-33 h), wherein the intracellular enzyme activity of the strain AlgM is increased along with the increase of time in the first 28h of the whole culture period, and the intracellular enzyme activity of the strain AlgM reaches the maximum and then is more obviously reduced when the strain AlgM is cultured to the 28h, and the presumed: this change is associated with the consumption of nutrients in the medium and the aging of the strain itself.

Based on FIG. 2, we determined the seed culture time to be 28 h.

2. Screening of carbon sources in fermentation Medium

The main functions of the carbon source include: provides carbon skeleton for the synthesis of macromolecules in cells and provides energy for the metabolism of cells.

In this embodiment, we mainly studied the effect of different carbon sources on the production of enzyme by the strain AlgM when starch, sodium alginate and glucose are used as carbon sources and the carbon content is the same, and the experimental results are shown in fig. 3.

As can be seen from FIG. 3, when the strain AlgM was cultured in a medium containing glucose as a carbon source, the produced alginate lyase had the highest enzyme activity, which reached 79%.

Based on FIG. 3, we replaced a portion of the sodium alginate in the original fermentation medium formulation with glucose.

3. Inorganic salts in fermentation media

The formula of the fermentation medium containing inorganic salt comprises the following components: 3g/L of glucose, 3g/L of sodium alginate, 5g/L of ammonium sulfate, 1g/L of magnesium sulfate, 2g/L of dipotassium hydrogen phosphate, 0.01g/L of ferrous sulfate and tap water.

The formula of the fermentation medium without inorganic salt comprises the following components: 3g/L of glucose, 3g/L of sodium alginate, 3g/L of peptone, 6g/L of yeast extract powder and tap water.

Selecting a strain AlgM on an Alg plate culture medium for activation, then selecting a ring of activated strain AlgM by using an inoculating ring, inoculating the activated strain AlgM into a seed culture medium, carrying out shake flask culture at the temperature of 30 ℃, carrying out shaking table rotation speed of 200rpm, respectively inoculating seed liquid into a fermentation culture medium containing inorganic salt and a fermentation culture medium without the inorganic salt according to an inoculation amount of 2% (volume fraction) after culturing for 28h, culturing for 24h at the temperature of 30 ℃ and 200rpm, and then centrifuging the fermentation liquid for 10min at the temperature of 4 ℃ and 8000g to obtain a crude enzyme liquid.

Under the aseptic condition, vertically placing an oxford cup on a MALg plate culture medium, adding 200 mu l of crude enzyme solution into the oxford cup, then placing the oxford cup in an incubator at 30 ℃ for culturing for 24h, pouring gram iodine solution on the MALg plate culture medium after the culture is finished, measuring the diameter of a transparent ring around the oxford cup, and calculating the area of the transparent ring.

The relative enzyme activity can be indirectly reflected by comparing the area of the transparent ring, and the larger the area of the transparent ring is, the larger the enzyme activity is, and the smaller the enzyme activity is otherwise.

The results of gram-iodine staining are shown in FIG. 4.

As can be seen from FIG. 4, the enzyme activity of alginate lyase produced by the strain AlgM was higher when the strain AlgM was cultured in the fermentation medium containing no inorganic salts than when the strain AlgM was cultured in the fermentation medium containing no inorganic salts.

Based on FIG. 4, during the fermentation phase, we chose the aforementioned inorganic salt-free fermentation medium to fermentatively culture the strain AlgM.

4. Fermentation culture

Activating the strain AlgM on an Alg plate culture medium, inoculating the activated strain AlgM into a seed culture medium, culturing at 30 ℃ and 200rpm for 28h to obtain a seed solution, inoculating the seed solution into a fermentation culture medium according to the inoculation amount of 2% (volume fraction), and culturing at 30 ℃ and 200rpm for 28h to obtain a fermentation liquid. Then, the fermentation broth was centrifuged at 8000g for 10min at 4 ℃ to obtain a crude enzyme solution.

5. Enzymolysis

Adding the crude enzyme solution into the alginate solution with the concentration of 3g/L, wherein the volume ratio of the crude enzyme solution to the alginate solution is 1:9, and performing enzymolysis for 22h at 35 ℃ to obtain an enzymolysis solution.

6. Separation and purification of brown algae oligosaccharide

Because the seed culture medium and the fermentation culture medium do not contain inorganic salt, the separation and purification process of the brown alga oligosaccharide can be simplified, and the steps of separating and purifying the brown alga oligosaccharide sequentially comprise: ceramic membrane filtration, alcohol precipitation and suction filtration, rotary evaporation, ultrafiltration dialysis, ion exchange and nanofiltration concentration, specifically:

(1) ceramic membrane filtration: and filtering thalli and residues in the enzymolysis liquid by using a ceramic membrane pilot plant, and collecting the dialysate.

(2) Alcohol precipitation and suction filtration: adding ethanol with the volume concentration of 95 percent of 3 times into the dialysate filtered by the ceramic membrane to ensure that the alcohol concentration in the solution is about 75 percent, standing and precipitating for 5 hours, and then carrying out suction filtration in a suction flask to remove residues.

(3) Rotary evaporation: and (3) placing the filtered supernatant into a rotary evaporator, setting the temperature at 50 ℃, carrying out rotary concentration, and carrying out rotary evaporation for 20min per 200mL of the supernatant to evaporate most of ethanol.

(4) Ultrafiltration and dialysis: using roll-type membrane multifunctional small-scale test equipment, pouring the solution subjected to rotary evaporation into a material tank, passing through an ultrafiltration membrane FMV-102 (2500 Da), adding deionized water with the volume of 3-6 times that of the solution in the process of membrane passing, removing macromolecular substances, and collecting ultrafiltration dialysate.

(5) Ion exchange: and (3) using ion exchange experimental equipment filled with anion and cation resins, and pumping the ultrafiltration dialysate into the ion exchange experimental equipment for decolorization and desalination.

(6) Nanofiltration and concentration: and (3) using roll-type membrane multifunctional small-scale test equipment, pouring the solution after ion exchange into a charging tank, filtering the solution by using a nanofiltration membrane FMV-102 (300 Da), adding 1 time of deionized water, concentrating the solution to 1/6 with the original volume, and collecting nanofiltration concentrated solution.

7. TLC detection

Taking a proper amount of nanofiltration concentrated solution, spotting the solution on a TLC plate, and adding n-butyl alcohol: formic acid: water =4:5:1 as a developing agent, the developing agent is developed for 1.5h, the drying is carried out after the completion, the drying is carried out again after a color developing agent (10% sulfuric acid-ethanol) is sprayed, the drying is carried out again, the drying box is placed in a drying box at the temperature of 110 ℃ for 30min, and the detection result is shown in figure 5.

As can be seen from FIG. 5, the standard sample was a 2-6 sugar mixed solution, the sample was a nanofiltration concentrate, and the nanofiltration concentrate contained 3, 4, and 6 sugars, and the degree of polymerization was low, when the bands obtained by the sample were compared with the standard sample.

It should be noted that the above-mentioned embodiments do not limit the present invention in any way, and all technical solutions obtained by using equivalent alternatives or equivalent variations fall within the protection scope of the present invention.

Claims

1. Leckera capable of producing alginate lyase (I)Leclerciasp.), characterized in that the lecanicillium is separated from rotten gulfweed picked from the yellow sea, has short growth cycle, can produce alginate lyase, has higher enzyme activity, has been subjected to strain preservation in 2019, 4, 28 days, and has the preservation places: china center for type culture Collection, the collection numbers are: CCTCC M2019314.

2. The alginate lyase producing bacterium of claim 1, whereinLeclerciasp.) in the preparation of brown algae oligosaccharides.

3. The use according to claim 2, wherein the alginate lyase-producing bacterium of claim 1 is used (i) (a)Leclerciasp.) the method for preparing the brown algae oligosaccharide comprises the following steps:

and 4, step 4: separating and purifying the brown algae oligosaccharide.

4. The use according to claim 3, wherein in step 1, the strain AlgM is activated on Alg plate medium, the formulation of Alg medium being: 5g/L of sodium alginate, 5g/L of ammonium sulfate, 1g/L of magnesium sulfate, 2g/L of dipotassium phosphate, 0.01g/L of ferrous sulfate, 20g/L of agar and tap water.

5. The use according to claim 3, wherein in step 1, the seed culture and the fermentation culture are carried out at 30 ℃ and at 200rpm for 28 h.

6. The use of claim 3, wherein in step 3, the concentration of the alginate solution is 3g/L, and the volume ratio of the crude enzyme solution to the alginate solution is 1: 9.

7. The use according to claim 3, wherein in step 3, the enzymolysis temperature is: 35 ℃ is carried out.

8. The use of claim 3, wherein in step 4, the step of isolating and purifying brown algae oligosaccharide comprises the following steps: ceramic membrane filtration, alcohol precipitation and suction filtration, rotary evaporation, ultrafiltration dialysis, ion exchange and nanofiltration concentration.