A marine-derived glycomonas K1 and its method and application for producing dextranase

Abstract

This invention provides a marine-derived glycomonad K1 strain, a method for producing dextran anhydrase, and its applications. The strain is a Gram-positive actinomycete. Colonies on a solid culture medium containing blue dextran exhibit the following characteristics: initially smooth surface, turning into a white, dry powder after three days of incubation; dried, with radially arranged edges; white on the front and black and opaque on the back. This invention also provides a method for producing dextran anhydrase using the aforementioned glycomonad K1 strain. The dextran anhydrase prepared using this method exhibits good thermal stability and strong pH tolerance, and can be widely used in the sugar, food, pharmaceutical, health, daily necessities, and chemical industries.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically relating to a marine-derived glycomonad K1 and its method and application for producing dextran anhydrase. Background Technology

[0002] Dextranase (EC3.2.1.11), also known as α-glucanase, is a hydrolase that specifically hydrolyzes the α-1,6 anhydride bonds in dextran. Dextranase has been widely studied and applied in medicine, industry, food, and biotechnology. In medical research, dextranase hydrolyzes the α-1,6 anhydride bonds in water-soluble extracellular polysaccharides produced by bacteria such as Streptococcus mutans and Lactobacillus, clearing biofilms and reducing bacterial adhesion, thereby achieving the purpose of removing dental plaque and preventing tooth decay; it can also be used in the production of blood plasma substitutes. In the sugar industry, dextranase can reduce the relative molecular mass of polysaccharides, thereby reducing sugar viscosity and improving sugar quality; in the food industry, dextranase hydrolyzes high molecular weight dextran to prepare functional isomaltooligosaccharides, producing prebiotics; dextran hydrolyzed to varying degrees can be used as a food additive in the production of health foods. Dextranase hydrolyzes starch to prepare porous starch, which is used for encapsulation and sustained release in pharmaceuticals and health products.

[0003] Dextranase has a very broad application prospect and market demand in food, clinical medicine and dental caries prevention. At present, most natural dextranases have the problem of low thermal stability. This problem has become the rate-limiting step for improving the hydrolysis efficiency of dextran. Therefore, obtaining thermally stable dextranase is the key to improving the yield and quality of dextran. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a novel dextranase-producing glycomonas (Saccharomonospora sp.) K1 derived from marine actinomycetes.

[0005] A strain of saccharomonospora K1 was deposited on March 21, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.4.7891 and classified as Saccharomonospora sp. The address of the depository is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and the contact number is 010-64807355.

[0006] The method for using glycomonas K1 to produce dextranase via fermentation provided by this invention includes the following steps:

[0007] 1) Inoculate strain K1 into seed culture medium and culture to obtain seed solution;

[0008] 2) Inoculate the seed culture into the enzyme-producing medium, and after fermentation, centrifuge the fermentation broth to obtain the supernatant containing dextranase.

[0009] Preferably, the seed culture conditions in step 1) are 180 r / min rotation speed, 25% liquid volume, and 48 h culture.

[0010] Preferably, the seed culture medium in step 1) is prepared with 0.5% peptone, 0.1% yeast powder, and aged seawater, with a pH of 8.5.

[0011] Preferably, the enzyme-producing culture medium in step 2) consists of 0.5% fish meal peptone, 0.1% soluble starch, 1% dextran T20, aged seawater, and pH 8.5.

[0012] Preferably, the NaCl concentration in the enzyme-producing culture medium is 2–10 g / L, and more preferably 6 g / L.

[0013] Preferably, the inoculation amount of seed liquid in step 2) is 10%.

[0014] Preferably, the enzyme production culture conditions in step 2) are 80 r / min and 37℃ for 72 h.

[0015] Preferably, the centrifugation conditions for the fermentation broth in step 2) are 10000 r / min for 2 min.

[0016] The optimal temperature for producing dextranase using the method of this invention is 50°C, the optimal pH is 8.5, and the optimal concentration of Ca2+ is 5–10 mM. 2+ 、Sr 2+ and K + It can increase enzyme activity.

[0017] The dextranase produced by the method of the present invention is used to catalyze the hydrolysis of compounds composed of α-1,6 glycoanhydride bonds.

[0018] The dextranase produced by the method of the present invention is used to catalyze the hydrolysis of isomalt oligosaccharides, wherein the isomalt oligosaccharides are isomalthexasaccharide and isomaltheptasaccharide.

[0019] The dextranase produced by the method of the present invention is used to prepare porous starch from sweet potato starch.

[0020] The beneficial effects of this invention are:

[0021] This invention provides a glycomonad bacterium, K1, which is a Gram-positive actinomycete. Its colony characteristics on a solid culture medium containing blue dextran are: initially smooth surface, turning into a white, dry powder after three days of incubation; dry, with radially arranged edges; white on the front and black and opaque on the back. The growth temperature range of this strain is 15-45℃, with an optimal growth temperature of 37℃; the suitable pH range for growth is 6-10, with an optimal growth pH of 8.5; it can grow at NaCl concentrations of 0%-5%, with an optimal NaCl concentration of 0%-1%.

[0022] The present invention also provides a method for producing dextran anhydrase using the aforementioned glycomonads K1, the steps of which are as follows: inoculating Pseudomonas K1 into 2216E medium, rotating at 180 r / min, with a liquid volume of 25%, and culturing at 37°C for 48 h to obtain seed liquid; inoculating the seed liquid into enzyme-producing medium at an inoculation volume of 10%, culturing at 180 r / min, 37°C for 72 h, centrifuging at 10000 r / min for 2 min, and obtaining the supernatant as crude enzyme solution.

[0023] The dextranase prepared by the method of the present invention has the following characteristics: the suitable operating temperature of the dextranase is 50℃, and it has catalytic activity in the temperature range of 30℃ to 70℃. The produced dextranase has good thermal stability, and its activity can still be maintained at more than 80% after incubation at 50℃ for 5 hours. The enzyme has strong pH tolerance and is stable in the pH range of 5.5 to 8.0.

[0024] The dextranase produced by the method of this invention can be widely used in the sugar industry, food industry, pharmaceutical industry, health industry, daily necessities industry, chemical industry, etc. Attached Figure Description

[0025] Figure 1 Scanning electron microscope image of strain K1 (×15k);

[0026] Figure 2 The transparent zone formed by strain K1 on the primary screening plate;

[0027] Figure 3 Phylogenetic tree of strain K1;

[0028] Figure 4 The effect of carbon source on dextranase production;

[0029] Figure 5 The effect of nitrogen source on dextranase production;

[0030] Figure 6 The effect of inoculum size on dextranase production by strain K1;

[0031] Figure 7 The effect of fermentation temperature on enzyme production;

[0032] Figure 8 The effect of fermentation time on dextranase production;

[0033] Figure 9 The effect of culture medium pH on dextranase production;

[0034] Figure 10 The effect of inducers on dextranase production;

[0035] Figure 11 Effect of NaCl on dextranase production

[0036] Figure 12 This is the optimal operating temperature for dextranase;

[0037] Figure 13 The thermal stability of dextranase;

[0038] Figure 14 The optimal pH for dextranase activity and pH stability;

[0039] Figure 15 High-performance liquid chromatography (HPLC) detection of dextranase hydrolysis products: the left figure is the HPLC chromatogram of the sugar standard, and the right figure is the HPLC chromatogram of the hydrolysis products.

[0040] Figure 16 Scanning electron micrographs of the preparation of porous starch by dextranase hydrolysis of sweet potato starch. A: Sweet potato starch, B: Enzymatic hydrolysis for 3 h, C: Enzymatic hydrolysis for 6 h, D: Enzymatic hydrolysis for 9 h, E: Enzymatic hydrolysis for 12 h, F: Enzymatic hydrolysis for 15 h. Detailed Implementation

[0041] The present invention will be further described below with reference to specific embodiments, so that those skilled in the art can better understand and implement the present invention, but the embodiments are not intended to limit the present invention.

[0042] The culture medium involved in this invention

[0043] Selection medium for bacterial strains: soluble starch: 20g, NaCl: 0.5g, FeSO4: 0.01g, KNO3: 1g, K2HPO4: 0.5g, MgSO4: 0.5g, K2Cr2O7: 1g, aged seawater: 1L, pH 8.0, agar 2%.

[0044] Seed culture medium: 0.5% peptone, 0.1% yeast extract, prepared with aged seawater, pH 8.5.

[0045] Initial screening medium for enzyme-producing strains: 0.5% peptone, 0.1% yeast extract, 0.2% blue dextran 2000, 2% agar, prepared with aged seawater, pH 8.0.

[0046] Enzyme-producing culture medium: 0.5% fish meal peptone, 0.1% soluble starch, 1% dextran T20, aged seawater, pH 8.5.

[0047] Preparation of trace mineral salt solutions (per liter):

[0048] CuSO4·5H2O, 0.01g; ZnSO4·7H2O, 0.1g; CoCl2·6H2O, 0.005g; MnCl2·4H2O, 0.2g; Na2MoO4·2H2O, 0.1g; KBr, 0.05g; KI, 0.05g; H3BO 3, 0.1g; NaF, 0.05g; LiCl, 0.05g; Al2(SO4) 3, 0.05g; NiCl2·6H2O, 0.01g; VoSO4·2H2O, 0.005g; H2WO4·2H2O, 0.002g; Na2SeO 4, 0.005g; SrCl·6H2O, 0.005g; BaCl2, 0.005g.

[0049] Example 1: Screening method for strains

[0050] Marine mud samples from the waters off Gaogong Island in Haizhou Bay, Lianyungang City, Jiangsu Province, China, were serially diluted 10-fold with sterile water and then spread onto a primary screening medium for enzyme-producing strains. The samples were incubated at 30°C for 48-72 hours, and the appearance of a clear zone around the colonies was observed. The size of the clear zone and the colony size were also measured. Colonies producing clear zones were picked and further purified by streak plating. The purified strains were inoculated into enzyme-producing medium and incubated at 30°C and 180 rpm for 2 days. The supernatant was then centrifuged at 10,000 rpm for 2 minutes, and the enzyme activity was determined. The strain with the highest enzyme activity was selected and named K1.

[0051] Example 2: Morphological characteristics and molecular biological identification of strain K1

[0052] 2.1 Morphological characteristics

[0053] After culturing for 72 hours in the initial screening medium for enzyme-producing strains, the colonies exhibited radial edges, were white on the front and black on the back, and were powdery. Strain K1 was identified as a Gram-positive actinomycete (see...). Figure 1 In solid culture media containing blue dextran, a clear zone can be produced (see...). Figure 2 ).

[0054] 2.2 Molecular biological identification of strain K1

[0055] The genome of strain K1 was extracted using the TIANamp Bacteria DNAKit. Universal primer pairs for amplifying prokaryotic 16S rDNA sequences (27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-GGTTACCTTGTTACGCTT-3') were selected. PCR reaction system and procedure: PCR reaction system (50 μL): 10 μL 5x reaction buffer, 1 μL 10 mM dNTPS, 2.5 μL 10 μM primer F, 2.5 μL 10 μM primer R, 2 μL template DNA, 0.5 μL DNA polymerase, and ultrapure water to a final volume of 50 μL. Reaction procedure: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, 30 cycles; 72℃ for 2 min; storage at 4℃. The PCR products were sequenced by Parasitic Biotechnology (Shanghai) Co., Ltd., to obtain the 16S rDNA gene sequence of the strain. The 16S rDNA gene sequence of strain K1 was submitted to the NCBI database, and homology comparison of the 16S rDNA sequence identified the strain as *Saccharomonosporasp.*. Multiple comparisons were performed using MEGA software, and a phylogenetic tree was constructed using the Neibor-joing method. The results showed that strain K1 is most closely related to *Saccharomonosporaspora*. See also... Figure 3 .

[0056] Saccharomonosporasp. strain K1 was deposited on March 21, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.4.7891 and classified as Saccharomonosporasp. The address of the depository is No. 3, No. 1 Courtyard, Beichen West Road, Chaoyang District, Beijing, and the contact number is 010-64807355.

[0057] Example 3: Method for producing dextranase by strain K1

[0058] 3.1 Effects of carbon and nitrogen sources on enzyme production by strain K1

[0059] Carbon source: 0.5% carbon source (soluble starch, dextrin, glucose, D-fructose, yeast extract, lactose, maltose) and 0.1% nitrogen source (tryptone, fish meal peptone, soybean meal, ammonium chloride, sodium nitrate, ammonium sulfate, potassium nitrate) were used to replace the yeast extract and peptone in the original enzyme-producing medium. After inoculation, the fermentation broth was cultured in a shaker at 37℃ for 72 h, and then the enzyme activity was detected. The results showed that soluble starch had the highest dextranase activity when used as the carbon source. Figure 4 Fish meal peptone promotes enzyme production ( ), Figure 5 Subsequently, 0.5% soluble starch and 0.1% fish meal peptone were selected as the carbon and nitrogen sources for the enzyme-producing culture medium.

[0060] 3.2 Effects of inoculum size and fermentation temperature on enzyme production by strain K1

[0061] Seed culture medium containing strain K1 for 48 hours was inoculated into enzyme-producing medium at inoculation rates of 5% and 10%. The 10% inoculation rate resulted in faster and higher enzyme production. Figure 6 10% was inoculated into enzyme-producing medium and cultured at 15-45℃ for 72 h. The enzyme activity of the fermentation broth was then measured. Results are shown below. Figure 7 The optimal temperature for enzyme production by strain K1 is 37℃. Below 20℃ or above 40℃, the enzyme production decreases significantly.

[0062] 3.3 Effect of fermentation time on enzyme production by strain K1

[0063] Strain K1 was fermented for 108 hours, and enzyme activity was measured every 12 hours. The results showed that enzyme production peaked at 72 hours. Before 72 hours, enzyme production gradually increased with prolonged fermentation time, but continued monitoring of enzyme activity revealed no significant trend. Figure 8 As shown.

[0064] 3.3 Effect of culture medium pH on enzyme production by strain K1

[0065] The enzyme was inoculated at a 10% inoculum into an enzyme-producing medium with an initial pH range of 5-11 and cultured at 37°C for 72 hours. Enzyme activity was then assessed. The optimal initial pH for enzyme production by this strain was 8.5. Enzyme production was significantly affected by both increasing and decreasing pH; when the pH was below 7.0, enzyme activity was undetectable in the fermentation broth of strain K1. (See attached table). Figure 9 .

[0066] 3.4 Effects of inducer concentration and type on enzyme production

[0067] Dextran T20, T40, T70, and T500 were used as enzyme inducers. Different concentrations of inducers were added to the enzyme-producing medium, and enzyme activity was detected after inoculation and culture. Figure 10 As shown, 1% dextran T20 is the optimal dextran enzyme inducer, followed by 1% dextran T40. Insufficient or excessive dextran T20 content is detrimental to enzyme production, and enzyme activity is undetectable without the addition of dextran.

[0068] 3.5 Effect of NaCl concentration on enzyme production

[0069] The enzyme activity was measured after inoculating a fermentation medium with a salinity range of 0-14 g / L at a 10% inoculum size and culturing at 37°C for 72 h. The optimal salinity for enzyme production by this strain was 6 g / L. No enzyme was produced without salt. (See attached results). Figure 11 .

[0070] Example 4: Method for producing dextranase by strain K1 and determination of enzyme properties.

[0071] 4.1 Method for producing dextranase by strain K1

[0072] Strains K1 were inoculated into seed culture medium at 180 rpm and 25% of the volume, and cultured for 48 h to obtain seed culture. The seed culture was then inoculated into enzyme production medium at 10% of the volume and cultured at 37°C at 180 rpm for 72 h. The enzyme solution was then centrifuged at 10,000 rpm for 2 min, and the supernatant was collected and stored at 4°C for later use.

[0073] 4.2 Determination of dextranase activity

[0074] ① Dextran enzyme activity assay: Add 50 μL of enzyme solution to 150 μL of 3% dextran T20 Tris-HCl buffer (0.1 M, pH 8.5), react in a 50℃ water bath for 15 min, add 200 μL of DNS, boil in a boiling water bath for 5 min to stop the reaction and develop color, add 3 mL of deionized water and shake to mix, take 200 μL and add to a 96-well plate, and measure the absorbance at 540 nm.

[0075] ② Definition of enzyme activity unit (U / mL): The amount of enzyme that catalyzes the production of 1 μmol of reducing sugar per minute at a certain temperature and pH is defined as one activity unit.

[0076] 4.3 Optimal temperature for enzyme activity

[0077] Dextranase was reacted with substrates at different temperatures, and enzyme activity was measured. The results are shown below. Figure 12 The optimal temperature for enzyme activity is 50℃, and it exhibits high catalytic activity in the temperature range of 40℃-50℃.

[0078] 4.4 Thermostability of Enzymes

[0079] The enzyme solution was incubated at different temperatures (45℃, 50℃, 55℃, 60℃) for 5 hours. A sample was taken every hour, rapidly cooled, and stored at 4℃. After the incubation period, residual enzyme activity was measured under standardized conditions, with the enzyme activity of the untreated solution set as 100%. The results are shown in [Figure number missing]. Figure 13 It still retains more than 80% of its enzyme activity after being kept at 50℃ for 5 hours.

[0080] 4.5 Optimal pH for enzyme activity

[0081] The enzyme solution was mixed with 3% dextran T20 solutions prepared with different pH buffers, and the reaction was carried out at 50℃. Enzyme activity was then detected. The different pH buffers were: 50 mM sodium phosphate buffer (pH 6.0-7.5) and 50 mM Tris-HCl buffer (pH 7.5-11.0). Results are shown below. Figure 14 The optimal pH for the enzyme solution is 8.5.

[0082] 4.6 pH stability of enzymes

[0083] The enzyme solution was mixed with buffer solutions of different pH values ​​(according to the buffer solutions in section 4.5), incubated at 25°C for 1 hour, and then the enzyme activity was measured. The enzyme activity of the untreated enzyme solution was set as 100%. Results are shown below. Figure 14 The results showed that after incubation at 30℃ for 1 hour, the enzyme activity of dextran was stable in the pH range of 7.5-9.0, and the residual enzyme activity remained above 60%.

[0084] 4.7 Effects of metal ions and chemical reagents on enzymes

[0085] The enzyme solution was mixed with metal ion solutions of different concentrations to achieve final metal ion concentrations of 1.0 mM, 5 mM, and 10 mM. Enzyme activity was then measured at the enzyme's optimum temperature of 50 °C. Relative enzyme activity was calculated using an enzyme solution without chemical reagents as a control. The results are shown in Table 1. 2+ 、Sr 2+ K + It can increase enzyme activity, Co 2+ Zn 2+ It reduces enzyme activity. Adding 5mM or higher Fe... 3+ This completely destroys enzyme activity. Other metal ions at the above concentrations showed little effect on enzyme activity.

[0086] Table 1. Effects of metal ions on dextranase activity

[0087]

[0088]

[0089] 4.8 Substrate specificity of dextranase in strain K1

[0090] The enzyme solution was applied to various substrates (dextran T20, dextran T40, dextran T70, dextran T500, dextran T2000, soluble starch, and chitosan), and enzyme activity was measured under standard conditions. The dextranase from strain K1 specifically catalyzes compounds composed of α-1,6 glycosidic bonds—dextrans of different molecular weights. It exhibits nearly 32% catalytic activity against soluble starch composed of α-1,4 and α-1,6 glycosidic bonds, but cannot hydrolyze chitosan composed of β-1,4 glycosidic bonds, indicating that this enzyme can only specifically hydrolyze α-1,6 glycosidic bonds.

[0091] Table 2. Substrate specificity of dextranase in strain K1

[0092]

[0093] Example 5: Application of dextranase from strain K1

[0094] 5.1 Preparation of oligosaccharides from dextran hydrolysis by dextranase from strain K1

[0095] The dextranase produced by the method of this invention is used to prepare isomalt oligosaccharides. The dextranase was used to hydrolyze dextran, and the products from hydrolysis at 0.5, 2, and 6 hours were analyzed by high-performance liquid chromatography (HPLC). The results are shown below. Figure 15 The sugar standards are glucose, isomaltose, isomalttriose, isomalttetraose, isomaltpentose, isomalthexaose, and isomaltheptaose, with isomalthexaose and isomaltheptaose being the main products.

[0096] 5.2 Preparation of porous starch by dextranase hydrolysis of sweet potato starch by strain K1

[0097] The dextranase produced by the method of this invention is used to prepare porous sweet potato starch. The products of dextranase hydrolysis at different times were observed by scanning electron microscopy, and the results are shown in the figure. Figure 16 As shown in the figure, the surface of sweet potato starch is porous, and after 15 hours of enzymatic hydrolysis, pores that extend deep into the interior can be observed.

Claims

1. A type of glycomonad ( Saccharomonospora sp.) K1, this glycomonad K1 was deposited on March 21, 2023 at the China General Microbiological Culture Collection Center, with the accession number CGMCC NO.4.7891.

2. The method for using glycomonas K1 as described in claim 1 for fermentation to produce dextranase, comprising the following steps: 1) Inoculate strain K1 into seed culture medium and culture to obtain seed solution; 2) Inoculate the seed culture into the enzyme-producing medium, and after fermentation, centrifuge the fermentation broth to obtain the supernatant containing dextranase.

3. The method according to claim 2, characterized in that, The seed culture conditions in step 1) are 180 r / min rotation speed, 25% liquid volume, and 48 h culture time; the seed culture medium in step 1) is prepared with 0.5% peptone, 0.1% yeast powder, and aged seawater, pH 8.

5.

4. The method according to claim 2, characterized in that, The enzyme-producing culture medium in step 2) consists of 0.5% fish meal peptone, 0.1% soluble starch, 1% dextran T20, aged seawater, and pH 8.5; the NaCl concentration in the enzyme-producing culture medium is 2-10 g / L.

5. The method according to claim 2, characterized in that, The concentration of NaCl in the enzyme-producing culture medium is 6 g / L.

6. The method according to claim 2, characterized in that, In step 2), the inoculation amount of seed solution is 10%.

7. The method according to claim 2, characterized in that, In step 2), the enzyme production culture conditions are 80 r / min and 37℃ for 72 h; the centrifugation conditions for the fermentation broth in step 2) are 10000 r / min for 2 min.

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