WO2022078141A1 - Strain for producing alginate lyase, alginate lyase and use thereof - Google Patents

Abstract

The present invention relates to a strain for producing an alginate lyase, the alginate lyase and use thereof. Provided is strain HB182678 for producing the alginate lyase. The strain is classified into Mangrovicoccus, and the alginate lyase produced by the strain has a relatively wide pH stability. Further provided are an alginate lyase, a preparation method therefor, and use of the strain HB182678 and alginate lyase in degrading brown algae.

Description

Alginate lyase-producing strain, alginate lyase and its application

This application claims the priority of the Chinese patent application filed on October 15, 2020 with the application number 202011104345.3 and the invention titled "alginate lyase-producing strain, alginate lyase and its application", the entire contents of which are Incorporated herein by reference.

technical field

The present invention relates to the technical field of microorganisms, in particular to alginate lyase-producing strains, alginate lyases and applications thereof.

Background technique

Alginic acid is a linear polymer formed by connecting guluronic acid (G) and mannuronic acid (M) through β-1,4 glycosidic bonds. It is the main component of brown algae cell walls and widely exists. In kelp, sargassum, macroalgae and other brown algae cells. The algal oligosaccharides prepared by enzymatic degradation have various physiological activities, such as promoting growth, enhancing immunity, neuroprotection, anti-inflammatory, anti-virus, etc. . The enzymatic hydrolysis method has mild reaction conditions, high efficiency, strong substrate specificity, and will not produce by-products harmful to human body and the environment.

Alginate lyase is a type of polysaccharide lyase that can degrade alginate into unsaturated oligosaccharides with double bonds at the non-reducing end through a β-elimination reaction. According to the substrate specificity, alginate lyase can be divided into G block specific lyase (poly G), M block specific lyase (polyM) and MG block bifunctional algin lyase (polyMG). Algin lyase has a wide range of sources and has been isolated from a variety of marine animals, marine and terrestrial microorganisms. Marine bacteria are an important source of algin lyase, such as Pseudomonas, Flavobacterium, Pseudomonas alternatus, Vibrio , Bacillus, etc.

The reported enzyme-producing bacteria still have defects such as low enzymatic activity and single degradation site, which limit the development of alginate lyase and enzymatic hydrolysis to prepare algal oligosaccharides. Therefore, it is an inevitable requirement for the development of algin resources and the exploration of new ways of high-value utilization to screen new bacterial resources that can efficiently degrade alginate.

SUMMARY OF THE INVENTION

In view of this, the present invention provides alginate lyase-producing strains and applications thereof. The strain HB182678 and the alginate lyase prepared by fermentation can be used to degrade brown algae.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The present invention provides the alginate lyase-producing strain Mangrovicoccus sp., whose deposit number is GDMCCNo.61000.

Based on the above research, the present invention also provides the application of the algin lyase-producing strain Mangrovicoccus sp. in the preparation of algin lyase.

The present invention also provides the application of the alginate lyase-producing strain Mangrovicoccus sp. in degrading brown algae.

The present invention also provides the algin lyase produced by the fermentation of the alginate lyase-producing strain Mangrovicoccus sp.

The present invention also provides the application of the alginate lyase in degrading brown algae.

In addition, the present invention also provides a method for preparing algin lyase, using the algin lyase-producing strain, Mangrovicoccus sp., to obtain algin lyase by fermentation.

In some specific embodiments of the present invention, the following steps are included:

Step 1. Strain activation: inoculate the alginate lyase-producing strain Mangrovicoccus sp. in a solid medium, and cultivate at 28-37°C for 24-48 hours to obtain an activated strain;

Step 2, liquid culture: inoculate the activated strain in a liquid seed medium, and shake and culture at 28-37° C. and 120-200 r/min for 12-24 hours to prepare a seed liquid;

Step 3. Fermentation culture: inoculate the seed liquid into a liquid fermentation medium, shake and culture at 28-37° C. and 150-200 r/min for 24-60 hours, collect the fermentation broth, centrifuge, and collect the supernatant to obtain alginate cracking The crude enzyme solution of the enzyme is purified to obtain alginate lyase.

In some specific embodiments of the present invention, the solid medium in step 1 comprises sodium alginate 3-12 g/L, peptone 2-10 g/L, yeast powder 0.5-4 g/L, sodium chloride 10-30 g/L L, agar powder 18～20g/L, pH 6.5～8.0.

In some specific embodiments of the present invention, the liquid seed medium in step 2 includes 3-12 g/L of sodium alginate, 2-10 g/L of peptone, 0.5-4 g/L of yeast powder, and 10-30 g of sodium chloride /L, pH 6.5-8.0; the inoculation described in step 2 is as follows: scrape a loop of bacterial cells from the plate, inoculate into a shake flask containing 30 mL of seed medium, 28-37 °C, 120-200 r/min Shake culture to control OD ₆₀₀ between 0.8 and 1.2.

In some specific embodiments of the present invention, the liquid fermentation medium in step 3 comprises sodium alginate 5-12g/L, peptone 2-10g/L, yeast powder 0.5-4g/L, sodium chloride 10-30g /L, dipotassium hydrogen phosphate trihydrate 0.5-2 g/L, magnesium sulfate heptahydrate 0.1-0.4 g/L, pH 6.5-8.0; the seed solution inoculation concentration is OD ₆₀₀ =0.05.

The present invention provides an alginate lyase-producing strain HB182678, the strain is classified as Mangrovicoccus, the preservation number is GDMCC No.61000, the preservation date is April 17, 2020, and the preservation unit is Guangdong Province Microbial Culture Collection Center, the preservation address is: Guangdong Institute of Microbiology, Building 59, Yard, No. 100, Xianlie Middle Road, Guangzhou. The alginate lyase produced by the bacteria has a wide range of pH stability. The invention also provides an alginate lyase and a preparation method thereof, as well as the application of the above-mentioned strain HB182678 and the alginate lyase in degrading brown algae, which have good application prospects.

Biological Preservation Instructions

The biological material Mangrovicoccus sp.HB182678, classified and named: Mangrovicoccus sp. was deposited in the Guangdong Provincial Microbial Culture Collection Center on April 17, 2020. The address is Guangdong Institute of Microbiology, 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou. , the deposit number is GDMCC No.61000.

Description of drawings

In order to illustrate the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that are required in the description of the embodiments or the prior art.

Fig. 1 shows the electron microscope photograph of the strain HB182678 of the present invention;

Fig. 2 shows the phylogenetic tree based on 16S rDNA of strain HB182678 of the present invention;

Figure 3 shows the effect of different pH values on the enzyme activity of the crude enzyme solution produced by the strain HB182678 of the present invention.

Detailed ways

The invention discloses an alginate lyase-producing strain and an application thereof, and those skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention. The method and application of the present invention have been described through the preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes and combinations of the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and Apply the technology of the present invention.

The object of the present invention is to provide a Mangrovicoccus strain HB182678, which can effectively degrade algin, and can also be used as a new algin lyase producing bacteria, and can also be directly used for microorganisms for biodegradation of brown algae resource.

The purpose of the present invention is also to provide an algin lyase and a preparation method thereof, the algin lyase can effectively degrade algin, and the technology is mature.

The third object of the present invention is to provide the application of the above-mentioned Mangrovicoccus strain HB182678 and algin lyase in degrading brown algae.

The above-mentioned first object of the present invention is achieved by the following technical solutions: a kind of alginate lyase producing strain HB182678, the classification of this strain is named Mangrovicoccus sp.HB182678, and the deposit number is GDMCC No.61000 , the preservation date is April 17, 2020, the preservation unit is: Guangdong Provincial Microbial Culture Collection Center (GDMCC), and the preservation address is: Guangdong Institute of Microbiology, Building 59, No. 100, Xianlie Middle Road, Guangzhou City.

The mangrove coccus HB182678 of the present invention is isolated and screened from brown algae samples collected from the offshore sea area of Qionghai, Hainan. The strain HB182678 was identified as a new species belonging to the bacterial domain, Proteobacteria, Alpha Proteobacteria, Rhodobacteria, Rhodobacteriaceae, and Mangrovecoccus, and was tentatively named Mangrovicoccus sp.HB182678.

The above-mentioned second purpose of the present invention is achieved by the following technical solutions: a kind of algin lyase is obtained by fermentation with the above-mentioned mangrove coccus HB182678 GDMCC No.61000.

The preparation method of above-mentioned alginate lyase preferably comprises the following steps:

(1) Strain activation: inoculate the above-mentioned mangrove HB182678 on a solid medium, and cultivate at 28 to 37°C for 24 to 48 hours to obtain an activated strain;

(2) Liquid culture: From the purified plate, pick a ring of bacteria and insert it into the liquid seed medium, and shake at 28 to 37 ° C and 120 to 200 r/min for 12 to 15 hours to logarithmic growth phase. into seed liquid;

(3) Fermentation culture: inoculate the seed liquid into the liquid fermentation medium, shake and culture at 28～37°C and 150～200r/min for 24～60h, collect the fermentation broth, centrifuge at 8000r/min for 10min, collect the supernatant to obtain brown algae Gel lyase crude enzyme solution.

In the preparation method of this alginate lyase:

The solid culture medium formula described in step (1) is: sodium alginate 3～12g/L, peptone 2～10g/L, yeast powder 0.5～4g/L, sodium chloride 10～30g/L, agar powder 18g/L ~20g/L, pH6.5~8.0, prepared with distilled water.

The liquid seed medium described in step (2) is composed of: sodium alginate 3～12g/L, peptone 2～10g/L, yeast powder 0.5～4g/L, sodium chloride 10～30g/L, pH 6.5 ~8.0, prepared with distilled water.

The liquid fermentation medium described in step (3) is composed of: sodium alginate 5-12g/L, peptone 2-10g/L, yeast powder 0.5-4g/L, sodium chloride 10-30g/L, trihydrate Dipotassium hydrogen phosphate 0.5～2g/L, magnesium sulfate heptahydrate 0.1～0.4g/L, pH 6.5～8.0, prepared with distilled water.

The above-mentioned third object of the present invention is achieved through the following technical solutions: the application of the above-mentioned mangrove coccus HB182678 and alginate lyase in degrading brown algae. Brown algae include kelp, sargassum and other brown algae species.

In the alginate lyase-producing strain provided by the present invention and its application, the raw materials and reagents used can be purchased from the market.

Below in conjunction with embodiment, the present invention is further elaborated:

Example 1 Isolation and screening of enzyme-producing strain HB182678 GDMCC No.61000

Brown algae samples were collected in the offshore waters of Qionghai City, Hainan Province. Take 10g of the sample, cut it into pieces, dissolve it, and dilute it into a suspension of 10 ^-3 to 10 ^-6 , draw 0.1 mL of a series of suspensions, spread it on the alginate lyase separation medium, and place it in a 30°C incubator to invert for 2 ~ 5d. When the colonies grow, according to the phenotypic characteristics such as colony shape, color, edge state, transparency, surface dry and wet state, select the colonies that grow well and have different shapes for streak purification.

The alginate lyase activity was tested on the isolated strains, and the strain HB182678 with stronger enzyme activity was screened out.

The specific process is as follows:

The strains to be detected were picked and connected to the alginate lyase activity detection medium, and cultured at 30°C for 2-3 days. After obvious colonies grew on the plate, the colony diameter (d) was measured. Add 1 mol/L CaCl ₂ solution to the plate and let it stand for 30-60 min. After the enzymatic dissolving circle appeared on the plate, the diameter of the enzymatic dissolving circle (D) was measured, and the ratio (D/d) of the enzymatic dissolving circle diameter and the colony diameter was used as the primary screening index.

Among them, the diameter of enzymolysis circle of strain HB182678 reached 25mm, the D/d value was 9, and the activity was obvious.

The medium formulation used is as follows:

Alginate lyase separation medium: sodium alginate 5g, yeast powder 1g, peptone 5g, ferric phosphate 0.01g, NaCl 20g, agar 18g, dilute to 1L with distilled water, pH 7.6.

Alginate lyase activity detection medium: sodium alginate 5g, (NH ₄ ) ₂ SO ₄ 5g, K ₂ HPO ₄ 2g, NaCl 20g, MgSO ₄ ·7H ₂ O 1g, FeSO ₄ ·7H ₂ O 0.01g, agar 18g, make up to 1L with distilled water, pH 7.6.

Embodiment 2 Identification of Mangrove HB182678 GDMCC No.61000

Strain HB182678 GDMCC No.61000 grows well on 2216E agar medium, and clear colonies can be seen after 2 days of culture. Observed under the electron microscope, the cells were ellipsoid, about 1.3-2.0μm×1.2-1.5μm in length and width, and without flagella. Gram stain was negative.

The 1425bp 16S rDNA sequence of HB182678 was obtained by PCR amplification, transformation and sequencing, and its nucleotide sequence is shown in SEQ ID No.1. The sequence was aligned with that in the EzBioCloud database, and it was found that strain HB182678 had the highest homology (96.3%) with Mangrovicoccus ximenensis Tlg56 ^T. Select related strains with high homology, and use the software MEGA7.0 to construct a phylogenetic tree by Neighbor-joining method (Figure 2). It can be seen that strain HB182678 and Mangrovicoccus ximenensis Tlg56 ^T are on the same branch, and the two are closely related. . Comparing the average nucleotide similarity ANI value of the two genomes, it is only 83.5%, which is less than the limit of 95% of species stipulated by the International Committee on the Taxonomy of Bacteria.

The morphological, physiological, biochemical and molecular characteristics of the two strains were compared, and the strain HB182678 (GDMCC No. 61000) in the present invention was identified as a new species of the genus Mangrovicoccus (Mangrovicoccus), and was tentatively named as Mangrovicoccus sp.HB182678.

This strain has been deposited in the Guangdong Provincial Microorganism Culture Collection Center (GDMCC) on April 17, 2020, and its survival has been proved, and its deposit registration number is GDMCC No.61000. The preservation address is Guangdong Institute of Microbiology, Building 59, Yard, No. 100, Xianlie Middle Road, Guangzhou.

Embodiment 3 Alginate lyase and preparation method thereof

The algin lyase in the present invention is prepared by fermentation of Mangrovicoccus sp.HB182678 whose deposit number is GDMCC No.61000 in Example 1, and the specific method comprises the following steps:

(1) Strain activation: inoculate the mangrove HB182678 in Example 1 on a solid medium, and cultivate at 30° C. for 48 hours to obtain an activated strain;

(2) Liquid culture: insert the activated strain into the liquid seed medium, and shake it at 30°C and 180r/min for 12 to 18 hours to prepare seed liquid; the OD ₆₀₀ is controlled between 0.8 and 1.2;

(3) Fermentation culture: inoculate the seed liquid in the liquid fermentation medium, shake and culture at 30° C. and 180 r/min for 40 hours, collect the fermentation liquid, centrifuge, and collect the supernatant to obtain the crude algin lyase enzyme liquid; the seeds The inoculum concentration was OD ₆₀₀ =0.05.

The solid medium in step (1) consists of: sodium alginate 5g/L, peptone 5g/L, yeast powder 1g/L, sodium chloride 20g/L, agar powder 18g/L, pH 7.0, prepared with distilled water.

The liquid seed medium in step (2) is: sodium alginate 3g/L, peptone 8g/L, yeast powder 1.5g/L, sodium chloride 20g/L, pH 7.0, prepared with distilled water.

The liquid fermentation medium in step (3) is: sodium alginate 7g/L, peptone 8g/L, yeast powder 1.5g/L, sodium chloride 20g/L, dipotassium hydrogen phosphate trihydrate 1g/L, heptahydrate Magnesium sulfate 0.2g/L, pH 7.0, prepared with distilled water.

Embodiment 4 Determination of alginate lyase enzyme activity

The enzymatic activity of alginate lyase was determined by UV absorption method. The process is as follows:

The fermentation broth was collected, centrifuged, and the fermentation supernatant in Example 3 was used as the crude enzyme liquid.

Take 1.8 mL of substrate (3.0 g of sodium alginate dissolved in 1 L of 50 mM pH7.0 phosphate buffer), preheat at 40 °C for 5 min, add 0.2 mL of the crude enzyme solution to be tested, and incubate at 40 °C for 10 min. It is a blank control, and the UV absorption value of the reaction system at OD ₂₃₅ is determined. One unit of enzyme activity (1U) was defined as the amount of enzyme whose OD ₂₃₅ UV absorption value increased by 0.01 per minute under the above-mentioned enzyme activity assay method.

The results showed that the alginate lyase activity of strain HB182678 was the largest at 132.2 U/mL when it was fermented for 40 h.

Table 1 Effects of different fermentation time on the enzyme production activity of strain HB182678

Example 5 Optimum pH and pH Stability of Alginate Lyase

Experiment 1: Prepare 50mM buffers with different pH values [[Na ₂ HPO ₄ -citric acid buffer (pH 3～7), Tris-HCl buffer (pH 8～9), NaHCO ₃ -NaOH (pH 10)]. At 4°C, the crude enzyme solution collected in Example 4 was kept at different pH values (3, 4, 5, 6, 7, 8, 9, 10) for 24 hours, and the residual algin lyase was determined respectively. activity to determine the stability of the enzyme. The enzyme activity measured at the optimum pH was defined as 100%.

Experiment 2: Determine the enzyme activity of the crude enzyme solution in different buffers containing 3 g/L of sodium alginate and 50 mM, and determine the optimal reaction pH value of the enzyme. The enzyme activity measured at the optimal pH was defined as 100%.

The results showed that when the crude enzyme solution was kept at pH 5-8 for 24 hours at 4°C, the remaining enzyme activity reached more than 85%, and the enzyme activity still reached more than 70% at pH 4 and pH 9. It can be seen that the enzyme has a wide range of pH stability. .

The enzyme activity of the crude enzyme solution was the highest at pH 8, the enzyme reaction activity was above 80% in the range of pH 6-8, and the remaining enzyme activity was the highest at pH 7.

Table 2 The influence of pH value on the enzyme activity of crude enzyme liquid produced by strain HB182678 of the present invention

Example 6 Degradation of brown algae by Mangrovicoccus sp.HB182678

Soak kelp (Laminaria japonica) and Sargassum oligocystum for 4 to 5 hours, clean them, cut them into small pieces of 1cm×1cm, add them to 250mL conical flasks, and add an appropriate amount of inorganic salt solution. The ingredients are: ( NH ₄ ) ₂ SO ₄ 5 g, K ₂ HPO ₄ 2 g, NaCl 20 g, MgSO ₄ ·7H ₂ O 1 g, FeSO ₄ ·7H ₂ O 0.01 g, distilled water 1 L, pH 7.5. A culture medium containing different species of brown algae was obtained.

The seed solution of Mangrovicoccus sp. HB182678 prepared according to Example 3 was inoculated, and the inoculation concentration was OD ₆₀₀ =0.1. It was cultured at 180 rpm and 30° C., and the turbidity of the medium and the change of the shape of the seaweed were observed.

It was observed that with the extension of the culture time, the seaweed gradually dissolved at 12h, the color of the colorless culture solution gradually deepened and turned brown, the solution began to become turbid, and the solid weight of kelp and sargassum remained 83.7% and 91.6%, respectively; at 24h, the seaweed The block became smaller, the debris increased, the turbidity of the solution deepened and became opaque, and the solid weight of kelp and sargassum remained 55.9% and 62.3% respectively; after 48 hours, the algae was completely degraded, the solution became turbid, and the solid algae of kelp and sargassum remained. Blocks disappear completely. This indicates that the strain HB182678 can efficiently degrade kelp and Sargasso in a short time, and is the dominant strain for the utilization of brown algae resources.

The alginate lyase-producing strains, alginate lyases and their applications provided by the present invention have been introduced in detail above. The principles and implementations of the present invention are described herein by using specific examples, and the descriptions of the above embodiments are only used to help understand the method and the core idea of the present invention. It should be pointed out that for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can also be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims (10)

1. The alginate lyase-producing strain Mangrovicoccus sp. is characterized in that its deposit number is GDMCCNo.61000.
2. Application of the algin lyase-producing strain Mangrovicoccus sp. as claimed in claim 1 in the preparation of algin lyase.
3. Application of the alginate lyase-producing strain Mangrovicoccus sp. as claimed in claim 1 in degrading brown algae.
4. The algin lyase produced by fermentation of the alginate lyase strain Mangrovicoccus sp. as claimed in claim 1.
5. The application of the alginate lyase as claimed in claim 4 in degrading brown algae.
6. The method for preparing algin lyase is characterized in that, adopting the algin lyase-producing strain Mangrovicoccus sp. as claimed in claim 1, and fermenting to obtain algin lyase.
7. The method of claim 6, comprising the steps of:

   Step 1. Strain activation: inoculate the alginate lyase-producing strain Mangrovicoccus sp. as claimed in claim 1 on a solid medium, and cultivate at 28-37° C. for 24-48 hours to obtain an activated strain;

   Step 2, liquid culture: inoculate the activated strain in a liquid seed medium, and shake and culture at 28-37° C. and 120-200 r/min for 12-24 hours to prepare a seed liquid;

   Step 3. Fermentation culture: inoculate the seed liquid into a liquid fermentation medium, shake and culture at 28-37° C. and 150-200 r/min for 24-60 hours, collect the fermentation broth, centrifuge, and collect the supernatant to obtain alginate cracking The crude enzyme solution of the enzyme is purified to obtain alginate lyase.
8. The method of claim 7, wherein the solid medium in step 1 comprises sodium alginate 3-12 g/L, peptone 2-10 g/L, yeast powder 0.5-4 g/L, sodium chloride 10 g/L ～30g/L, agar powder 18～20g/L, pH 6.5～8.0.
9. The method according to claim 8, wherein the liquid seed medium in step 2 comprises sodium alginate 3-12g/L, peptone 2-10g/L, yeast powder 0.5-4g/L, sodium chloride 10～30g/L, pH 6.5～8.0; the inoculation described in step 2 is as follows: scrape a ring of bacterial cells from the plate, inoculate into a shake flask containing 30mL seed medium, 28～37℃, 150～ Shake culture at 200 r/min to control the OD ₆₀₀ between 0.8 and 1.2.
10. The method of claim 9, wherein the liquid fermentation medium in step 3 comprises sodium alginate 5-12 g/L, peptone 2-10 g/L, yeast powder 0.5-4 g/L, sodium chloride 10-30 g/L, dipotassium hydrogen phosphate trihydrate 0.5-2 g/L, magnesium sulfate heptahydrate 0.1-0.4 g/L, pH 6.5-8.0; the seed solution inoculation concentration is OD ₆₀₀ =0.05.

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