WO2022078140A1 - Alginate lyase producing strain and application thereof - Google Patents

Abstract

The present invention relates to the technical field of microorganisms, and provides an alginate lyase producing strain HB171785. The strain belongs to γ-proteobacteria genus Neiella, the accession number is GDMCC No.61001, and alginate lyase produced by the strain has good temperature stability. Also provided are alginate lyase and a preparation method therefor. The strain HB171785 and the alginate lyase have application potential in degradation of algin and preparation of alginate oligosaccharides.

Description

Alginate lyase-producing strain and its application

This application claims the priority of the Chinese patent application with the application number 202011103273.0 and the invention titled "alginate lyase-producing strain and its application" filed with the China Patent Office on October 15, 2020, the entire contents of which are incorporated herein by reference Applying.

technical field

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

Background technique

Alginate is widely present in the cell walls of brown algae such as kelp, sargassum, macroalgae, etc. - A linear high molecular polysaccharide polymer composed of β-D-mannuronate randomly linked by 1,4 glycosidic bonds. The large molecular weight of alginate, complex composition, and not easy to degrade, have created obstacles to the resource utilization of brown algae. In recent years, how to efficiently degrade brown algae to prepare highly active seaweed extracts, seaweed biofeeds or fertilizers has become a hot spot in seaweed utilization research.

Studies have shown that fucoidan oligosaccharides prepared by biodegradation and polymerized from 2-20 monosaccharides have various physiological activities, such as growth promotion, immunity enhancement, neuroprotection, anti-inflammatory, antiviral, antioxidant activities, etc. It has wide application potential in food, medicine, agriculture, health care products, cosmetics and other fields. Compared with the traditional acid hydrolysis method, the enzymatic hydrolysis method has attracted more and more attention due to its advantages of high specificity, mild reaction conditions, and high yield of algal oligosaccharides.

Algin lyase is currently the only effective algin degrading enzyme, which can specifically act on 1,4 glycosidic bonds, cut off the algin sugar chain through β elimination reaction and degrade it into unsaturated oligosaccharides with double bonds at the non-reducing end. There are a wide range of sources of algin lyase, including marine algae, mollusks, echinoderms and a variety of microorganisms. Marine bacteria are an important source of algin lyase, such as Vibrio, Flavobacterium, Alternonas, Alternonas Wait. However, enzyme-producing bacteria generally have defects such as low enzyme activity and single degradation site, which limit the development of alginate lyase and enzymatic hydrolysis to prepare algal oligosaccharides. Therefore, it is one of the efficient ways to develop and utilize algin lyase to find new strains that can efficiently degrade alginate.

SUMMARY OF THE INVENTION

In view of this, the present invention provides a new marine bacterial strain Neiella sp.HB171785, the strain preservation number is GDMCC No.61001. The invention also relates to the alginate lyase produced by the fermentation of the strain and its preparation method and application.

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

The present invention provides alginate lyase-producing strain Neiella sp., whose deposit number is GDMCC No.610001.

On the basis of the above research, the present invention also provides the application of the algin lyase-producing strain Neiella sp. in the preparation of algin lyase.

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

The present invention also provides the algin lyase produced by the fermentation of the algin lyase-producing strain Neiella 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 Neiella 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 Neiella 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 strains in a liquid seed medium, and shake at 28-37° C. and 120-200 r/min to a logarithmic growth phase to prepare a seed liquid;

Step 3. Fermentation culture: transfer the seed liquid into a liquid fermentation medium with an inoculum amount of 2-5%, shake and culture at 28-37° C. and 150-200 r/min for 24-60 hours, collect the fermentation broth, and centrifuge. , collect the supernatant to obtain the crude enzyme liquid of alginate lyase, and purify 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; scrape a ring of the bacterial cells from the plate, inoculate it into a shake flask containing 30 mL of seed medium, and shake at 28-37 °C and 120-200 r/min, so that the OD ₆₀₀ is controlled at 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 HB171785. The strain belongs to the genus Neiella of the genus Gamma, the preservation number is GDMCC No.61001, the preservation date is April 17, 2020, and the preservation unit is Guangdong Province Microorganisms Culture Collection Center, the preservation address is: Guangdong Institute of Microbiology, Building 59, Yard, No. 100, Xianlie Middle Road, Guangzhou. The algin lyase produced by this strain has better temperature stability. The invention also provides an algin lyase and a preparation method thereof. The above-mentioned strain HB171785 and the algin lyase have application potential in the degradation of algin and the preparation of algal oligosaccharides.

Biological Preservation Instructions

The biological material Neiella sp.HB171785, classified and named: Neiella 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.61001.

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 HB171785 of the present invention;

Fig. 2 shows the phylogenetic tree of strain HB171785 of the present invention based on 16S rDNA;

Figure 3 shows the effects of different temperatures on the enzyme activity of the crude enzyme solution produced by the strain HB171785 of the present invention.

Detailed ways

The invention discloses a marine bacterial strain Neiella sp. and its application, and those skilled in the art can learn from the content of this article and appropriately improve 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 purpose of the present invention is to provide a Neiella strain HB171785, which can efficiently produce alginate lyase, can be used as a new alginate lyase-producing bacterium, and can also be directly used as a microbial resource for brown algae biodegradation.

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 Neiella strain HB171785 and algin lyase in degrading brown algae.

The above-mentioned first purpose of the present invention is achieved through the following technical solutions: a kind of alginate lyase producing strain HB171785, the strain is classified as γ-transformed genus Neiella, tentatively named: Neiella sp.HB171785, deposit number It is: GDMCC No.61001, the preservation date is: April 17, 2020, the preservation unit is: Guangdong Provincial Microbial Culture Collection Center (GDMCC), and the preservation address is: Guangdong Province, No. Institute of Microbiology.

The Neiella strain HB171785 of the present invention is isolated and screened from sandy soil samples collected from the sea area of Qishui Bay, Wenchang City, Hainan Province. The strain HB171785 was identified as a new species belonging to the bacterial domain, Proteobacteria, Gamma Proteobacteria, Alteromonas, and the genus Neiella, and was tentatively named Neiella sp.HB171785.

The above-mentioned second purpose of the present invention is achieved by the following technical solutions: a kind of algin lyase, obtained by fermentation of the above-mentioned Neiella sp.HB171785 GDMCC No.61001.

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

(1) Bacterial activation: inoculate the above-mentioned Neiella sp.HB171785 on a solid medium, and cultivate at 28-37°C for 24-48 hours to obtain an activated strain;

(2) Liquid culture: scrape a ring of bacteria from the purified plate, insert it into 30 mL 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. to make seed liquid;

(3) Fermentation culture: inoculate the seed liquid in the liquid fermentation medium at 2-10% by volume, and shake and culture at 28-37°C and 150-200r/min for 24-60h, collect the fermentation broth, 8000r/min min centrifuged for 10 min, and the supernatant was collected to obtain the crude alginate lyase 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 by the following technical solutions: the application of the above-mentioned Neiella sp.HB171785 and alginate lyase in degrading brown algae. Brown algae include kelp, sargassum and other brown algae species.

In the marine bacterial strain Neiella sp. 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 HB171785 GDMCC No.61001

Sand samples were collected in the sea area of Qishui Bay, Wenchang City, Hainan Province. Take 10 g of the sample, 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 to 5 days.

When the colonies grow, according to the phenotypic characteristics such as colony shape, color, edge state, transparency, and surface dry and wet state, select colonies that grow well and have different shapes for streaking and purification.

The alginate lyase activity was tested on the isolated strains, and the strain HB171785 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 HB171785 reached 26mm, the D/d value was 10, and the activity was obvious.

The medium formulation used is as follows:

Alginate lyase isolation medium: sodium alginate 5g, peptone 5g, yeast powder 1g, 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.

Example 2 Identification of strain HB171785 GDMCC No.61001

Strain HB171785 GDMCC No.61001 grows well on 2216E agar medium. After 2 days of culture, clear colonies can be seen. The colonies are round, cream-colored to gray-yellow, with neat edges, smooth and moist surface, and a slightly raised center with a diameter of 2-3 mm. Observed under the electron microscope, the cells were long rod-shaped, 2.2-4.5μm long, 0.4-0.7μm wide, and had 1-2 polar or lateral flagella. Gram stain was negative.

The 1459bp 16S rDNA sequence of HB171785 was obtained by PCR amplification 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 HB171785 had the highest homology (98.2%) with Neiella marina J221 ^T. Select related strains with high homology, and use the software MEGA7.0 to construct a phylogenetic tree using the Neighbor-joining method (Figure 2). It can be seen that strain HB171785 and Neiella marina J221 ^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 83.0%, which is less than the limit of 95% of the species specified by the International Committee for the Taxonomy of Bacteria.

The morphological, physiological, biochemical and molecular characteristics of the two strains were compared, and the strain HB171785 (GDMCC No.61001) in the present invention was identified as a new species of the genus Neiella, tentatively named Neiella sp.HB171785.

This strain has been deposited in the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on April 17, 2020, and its survival has been proved, and its deposit registration number is GDMCC No.61001. 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 alginate lyase in the present invention is prepared by fermentation of Neiella sp.HB171785 whose deposit number is GDMCC No.61001 in Example 1, and the specific method comprises the following steps:

(1) Strain activation: The strain HB171785 in Example 1 was inoculated into a solid medium, and cultured 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 15h to make seed liquid; the OD ₆₀₀ is controlled between 0.8 and 1.2;

(3) Fermentation culture: inoculate the seed liquid into the liquid fermentation medium, shake and culture at 30°C and 180r/min for 40h, collect the fermentation liquid, centrifuge, and collect the supernatant to obtain the crude algin lyase enzyme liquid; seed liquid inoculation The concentration was _OD600 = 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 effect of different fermentation time on the enzyme production activity of strain HB171785 is shown in Table 1. The alginate lyase enzyme activity of strain HB171785 was the largest at 148.6U/mL when it was fermented for 36h.

Table 1 Effects of different fermentation time on the enzyme production activity of strain HB171785

Example 5 Optimum temperature and temperature stability of alginate lyase

Experiment 1: After keeping the crude enzyme solution at different temperatures (4, 20, 30, 40, 50, 60, 70, 80°C) for 1 h, the residual activity of alginate lyase was measured respectively to determine the stability of the enzyme. The enzyme activity of the crude enzyme solution stored at 4°C was defined as 100%.

Experiment 2: Determine the enzyme activity of the crude enzyme solution in 50mM pH7.0 phosphate buffer containing 3g/L sodium alginate at different temperatures to determine the optimum reaction temperature. The relative enzyme activity measured at the optimum reaction temperature was defined as 100%.

The results showed that when the crude enzyme solution was kept below 40 ℃ for 1 hour, the remaining enzyme activity reached more than 90%. When kept at 60℃ for 1h, the enzyme activity still retains 52.4%, which shows that the enzyme has good temperature stability. The enzyme activity of the crude enzyme solution was the highest at 50°C, and the enzyme activity was above 80% in the range of 40-60°C. When the temperature is lower than 40℃ or higher than 60℃, the enzyme activity decreases sharply.

Table 2 The influence of pH value on the enzyme activity of crude enzyme liquid produced by strain HB171785 of the present invention

Example 6 Degradation of brown algae by Neiella sp.HB171785

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 Neiella sp. HB171785 prepared according to Example 3 was inoculated, the inoculation concentration was OD ₆₀₀ =0.1, and the culture was carried out at 180 rpm and 30° C. The turbidity of the medium and the change of the shape of the seaweed were observed.

It was observed that with the extension of culture time, the seaweed gradually dissolved at 8h, 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 85.6% and 90.3%, respectively; at 20h, the seaweed block It became smaller, the debris increased, the turbidity of the solution deepened and became opaque, and the solid weight of kelp and sargassum remained 54.1% and 62.7% respectively; after 40 hours, the kelp and sargassum were completely degraded, the solution became turbid, and the solid seaweed block was completely disappear. Strain HB171785 can efficiently degrade kelp and sargassum in a short time, and can be used for brown algae resource utilization.

The alginate lyase-producing strains provided by the present invention and their applications 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 Neiella sp. is characterized in that its deposit number is GDMCCNo.610001.
2. The application of the algin lyase-producing strain Neiella sp. as claimed in claim 1 in the preparation of algin lyase.
3. Application of the alginate lyase-producing strain Neiella sp. as claimed in claim 1 in degrading brown algae.
4. The algin lyase produced by fermentation of the alginate lyase strain Neiella 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 Neiella sp. as claimed in claim 1, to obtain algin lyase by fermentation.
7. The method of claim 6, comprising the steps of:

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

   Step 2, liquid culture: inoculate the activated strains in a liquid seed medium, and shake at 28-37° C. and 120-200 r/min to a logarithmic growth phase to prepare a seed liquid;

   Step 3. Fermentation culture: transfer the seed liquid into a liquid fermentation medium with an inoculum amount of 2-5%, shake and culture at 28-37° C. and 150-200 r/min for 24-60 hours, collect the fermentation broth, and centrifuge. , collect the supernatant to obtain the crude enzyme liquid of alginate lyase, and purify 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; scrape a ring of bacteria from the plate, inoculate it into a shake flask with 30mL seed medium, 28～37℃, 120～200r/min shaking culture, make OD ₆₀₀ is controlled between 0.8 and 1.
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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