D-tagatose-producing strain and application thereof

Abstract

The application discloses a D-tagatose-producing strain and application thereof, the strain is classified and named as endogenous bacillus (Bacillus endophyticus) (Bacillus endophyticus) Z723, and has a preservation number of GDMCC No: 63266. Priestia endophytica The strain utilizes galactitol as a substrate to produce D-tagatose through a whole-cell catalysis method, and the conversion rate can reach 92%. The strain provides a new strain for high-conversion-rate D-tagatose synthesis and related oxidoreductase mining, and has a wide application prospect in the fields of food and medical treatment.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a D-tagatose-producing strain and its applications. Background Technology

[0002] Currently, consumer preferences have undergone a paradigm shift from synthetic compounds to natural bioactive molecules, as these are considered safe and free of side effects. Among potential nutritional supplements, D-tagatose has attracted attention due to its low calorie value, prebiotic potential, anti-diabetic properties, and potential for preventing lifestyle-related diseases; however, D-tagatose exists only in small quantities in nature. As a novel sweetener, D-tagatose offers functional health benefits such as low calories and low absorption, satisfying both the need for sweetness and health requirements, and is widely used in food, pharmaceuticals, cosmetics, and other fields.

[0003] To date, the most frequently reported pathway for D-tagatose production involves isomerizing D-galactose via L-arabinose isomerase. For example, Rao et al. constructed a genetically engineered Bacillus subtilis strain and used arabinose isomerase and galactosidase to convert lactose, adding 3 mM Mn... 2+ Whole-cell catalysis yielded only 57.2% of tagatose conversion. The coexistence of D-galactose and D-tagatose in the reaction solution resulted in low yields, hindering downstream product separation. Furthermore, most L-arabinose isomerases exhibit a strict metal ion dependence (e.g., Mn) on D-tagatose production. 2+ This affects the application of D-tagatose in food and medicine. Redox reactions allow for the almost complete conversion of galactitol to D-tagatose without metal ion dependence, showing potential as an alternative to isomerization pathways for D-tagatose synthesis. However, currently, only a handful of microorganisms have been reported that can catalyze the production of D-tagatose from galactitol. Mycobacterium smegmatis A few species, and relatively few related oxidoreductases. Summary of the Invention

[0004] The primary objective of this invention is to provide a novel D-tagatose-producing strain, classified as *Endophytosporum* (…). Priestia endophytica Z723, accession number GDMCC No: 63266.

[0005] A second objective of this invention is to provide the application of the above-mentioned strain in the fermentation production of D-tagatose.

[0006] As a preferred embodiment, the substrate is converted into D-tagatose using resting cells of the strain via whole-cell catalysis.

[0007] In a preferred embodiment, after the strain is seed cultured, the seed liquid is inoculated into a fermentation medium for fermentation culture. The fermentation broth is centrifuged to collect the cells, which are then resuspended and washed in PBS buffer. After centrifugation again, the precipitate is collected, which is the resting cells.

[0008] As a preferred embodiment, resting cells of the strain are resuspended in PBS buffer containing the substrate and shaken to prepare D-tagatose.

[0009] As a preferred embodiment, the seed culture medium is LB liquid medium.

[0010] In a preferred embodiment, the strain produces D-tagatose using galactitol as a substrate.

[0011] In a preferred embodiment, the substrate concentration in the conversion system is 10-60 g / L.

[0012] As a preferred embodiment, the pH of the conversion system is 6-10; preferably 7-9.

[0013] As a preferred embodiment, the temperature in the conversion system is 25-50℃, preferably 40-50℃.

[0014] As a preferred embodiment, the reaction time of the conversion system is 12-24 h.

[0015] This invention isolates and screens a strain of endophytic Bacillus from saline-alkali soil in Xinjiang, my country, capable of biosynthesizing D-tagatose. This strain, belonging to the endophytic Bacillus family, can synthesize D-tagatose at a concentration of 18.4 g / L using 20 g / L galactitol as a substrate in its resting cells. The conversion rate of D-tagatose production using galactitol can reach 92%, providing a new strain for high-conversion-rate D-tagatose synthesis and the discovery of related oxidoreductases. The rare sugar D-tagatose produced by the strain screened in this invention has broad application prospects in the food and medical fields. Attached Figure Description

[0016] Figure 1 To isolate and screen D-tagatose-producing strains.

[0017] Figure 2 This is a microscopic image of a D-tagatose-producing strain.

[0018] Figure 3 Phylogenetic tree of endophytic spore bacillus Z723.

[0019] Figure 4 The standard curve for the quantitative analysis of D-tagatose, a product of cysteine ​​carbazole, using the colorimetric method.

[0020] Figure 5The growth curve of endophytic spore bacillus Z723 is shown.

[0021] Figure 6 The effect of pH on the conversion of D-tagatose in the reaction system.

[0022] Figure 7 The effect of reaction temperature on the conversion of D-tagatose.

[0023] Figure 8 The effect of galactitol substrate concentration on the conversion efficiency of D-tagatose.

[0024] Figure 9 The time-conversion curve of D-tagatose production from galactitol catalyzed by whole-cell endophytic spore bacillus Z723.

[0025] The biological material described in this invention is classified and named endophytic spore-forming bacteria (Endophytic Bacillus). Priestia endophytica Z723 has been deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), accession number GDMCC No: 63266, deposit date: March 12, 2023, deposit address: Guangdong, China. Detailed Implementation

[0026] Example 1: Isolation, screening, and molecular biological identification of D-tagatose-producing strains I. Isolation and Screening of D-Tagalog Producing Strains 1. Take 5 g of soil sample from saline-alkali land in Xinjiang, China, dilute it in 50 mL of sterile water, filter to remove sample residue, prepare bacterial suspension, transfer to 50 mL LB liquid medium (250 mL shake flask), and incubate at 37 ℃ and 200 rpm for 24 h.

[0027] LB liquid medium formulation (g / L): peptone 10, NaCl 10, yeast extract 5, pH 7.0.

[0028] 2. After completing step 1, take 1 mL of the above culture medium and transfer it to fresh enrichment medium. Then, incubate it at 37 ℃ and 200 rpm for 24 h to obtain the culture medium. After dilution, take 100 μL and spread it on the isolation medium plate and incubate it at 37 ℃. After single colonies grow on the plate, pick a single colony, streak it three times for purification, and inoculate it on slant medium for preservation.

[0029] Enrichment medium formula (g / L): galactitol 10, yeast extract 5, (NH4)2SO4 2.5, MgSO4 7H2O 0.3, KH2PO4 2, K2HPO4 4, pH 7.0.

[0030] Plate isolation medium formula (g / L): galactitol 10, yeast extract 5, (NH4)2SO4 2.5, MgSO4 7H2O 0.3, KH2PO4 2, K2HPO4 4, agar 20, pH 7.0.

[0031] Slant solid culture medium formula (g / L): galactitol 10, yeast extract 5, peptone 5, NaCl 5, agar 20, pH 7.0.

[0032] 3. After completing step 2, pick the strain preserved on the slant culture medium and inoculate it into the fermentation medium. Incubate at 37 ℃ with shaking at 200 rpm for 24 h, then centrifuge to collect the cells. Resuspend and wash twice with PBS buffer, centrifuge again, discard the supernatant, and obtain resting cells. Resuspend a certain amount of the cells in 10 mL PBS buffer containing 20 g / L galactitol and transform for 12 h with shaking. Centrifuge for 10 min to obtain the supernatant. Use the cysteine ​​carbazole colorimetric method to screen for strains that change color to blue-purple, such as... Figure 1 As shown, the readings were measured using a spectrophotometer, and the yield was calculated based on the D-tagatose standard curve. The strain with the highest yield and enzyme activity was selected for further identification and preserved in glycerol at -80 °C.

[0033] Fermentation medium formula (g / L): galactitol 20, peptone 10, yeast extract 5, NaCl 10, KH2PO4 2, K2HPO4 4, MgSO47H2O 0.3, CaCl22H2O 0.02, MnSO4H2O 0.05, FeSO47H2O 0.04, pH 7.0.

[0034] II. Molecular biological identification of D-tagatose-producing strains Observe the morphology of the strain under a microscope, such as Figure 2 As shown, the strain is rod-shaped. The 16S rRNA sequence of the strain was sequenced using BLAST and compared with other existing preserved sequences via the online program NCBI. The alignment results were then used to construct a phylogenetic tree using the Neighbor-Joining method in MEGA software. The results are shown below. Figure 3 As shown, the strain was identified as an endophytic Bacillus (Bacillus). Priestia endophytica The strain was named Z723.

[0035] Example 2: Construction of the standard curve for the quantitative analysis of D-tagatose by cystine carbazole colorimetric method Add 1 mL of D-tagatose standard solution (1–50 μg / mL) to eight 15 mL test tubes, respectively. Then, add 0.2 mL of 15 g / L L-cysteine ​​hydrochloride solution, 6 mL of 70% sulfuric acid solution, and 0.2 mL of 1.2 g / L carbazole ethanol solution in the following order: shake to mix, incubate at 60 °C for 10 min, cool to room temperature for 10 min, and measure the absorbance at 550 nm using a spectrophotometer. Use galactitol standard solution instead of the D-tagatose colorimetric reaction solution as a blank. Plot a D-tagatose standard curve with the concentration of D-tagatose standard solution on the x-axis and the absorbance value on the y-axis as shown below. Figure 4 As shown, the D-tagatose content was calculated using a standard curve.

[0036] Example 3: Growth curve determination of Bacillus Z723 D-tagatin-producing endophytic Bacillus Z723 was inoculated into 5 mL of LB liquid medium and activated at 37 ℃ and 200 rpm for 12 h. Then, an inoculation rate of 2% was applied. v / v The culture medium was transferred and incubated at 37 °C and 200 rpm. Absorbance values ​​were measured at 2-hour intervals, with a total growth period of 24 hours. Growth curves were plotted as shown below. Figure 5 As shown.

[0037] Example 4: Effects of pH, reaction temperature, and concentration of galactitol substrate added to the reaction system on the conversion of D-tagatose. I. Effect of pH of the reaction system on the conversion of D-tagatose Endophytic Bacillus Z723, which produces D-tagatose, was inoculated into fermentation medium and cultured at 37 °C with shaking at 200 rpm for 24 h. The cells were then collected by centrifugation, resuspended twice in PBS buffer, and centrifuged again, discarding the supernatant to obtain resting cells. A certain amount of the cells was resuspended in 1 mL of PBS buffer (pH 3.0–10.0) containing 20 g / L galactitol, and reacted at 37 °C with shaking for 2 h. The supernatant was obtained by centrifugation for 10 min. The concentration of the D-tagatose product was determined using the cysteine ​​carbazole colorimetric method at different pH systems to determine the optimal pH. Figure 6 As shown, the optimal pH is 8.0.

[0038] II. Effect of reaction temperature on the conversion efficiency of D-tagatose The resting cells were resuspended in 1 mL of PBS buffer (pH 8.0) containing 20 g / L galactitol, and transformed by incubation at 25–50 °C with shaking for 2 h. The supernatant was obtained by centrifugation for 10 min. The concentration of the transformation product D-tagatose at different reaction temperatures was determined using the cysteine ​​carbazole colorimetric method to identify the optimal reaction temperature. Figure 7 As shown, 45 ℃ is the optimal reaction temperature, and the yield decreases when the temperature is higher or lower than 45 ℃.

[0039] III. Effect of galactitol substrate concentration on D-tagatose conversion efficiency The resting cells were resuspended in 1 mL of PBS buffer (pH 8.0) containing 10, 20, 30, 40, 50, and 60 g / L galactitol. The cells were transformed by incubation at 37 °C with shaking for 2 h, followed by centrifugation for 10 min to obtain the supernatant. The concentration of the transformation product D-tagatose at different galactitol substrate concentrations was determined using the cysteine ​​carbazole colorimetric method to identify the optimal substrate concentration. Figure 8 As shown, 20 g / L is the optimal substrate concentration. When the substrate concentration is higher than 20 g / L, the concentration of D-tagatose product increases slightly, the binding of cells to the substrate tends to be saturated, and the conversion rate decreases.

[0040] Example 5: Under optimal conversion conditions Priestia endophytica Z723 Synthesizes D-Tragose Will Priestia endophytica Z723 resting cells were transformed under the optimized reaction conditions described in Example 4 using PBS buffer (pH 8.0) containing 20 g / L galactitol at a constant temperature of 45 °C with shaking for 24 h. The concentration of the transformation product D-tagatose at different time points was determined using the cysteine ​​carbazole colorimetric method. Figure 9 As shown, it can be converted to produce 18.4 g / L D-tagatose after 24 h, with a conversion rate of 92%.

Claims

1. A D-tagatose-producing strain, characterized in that, Its classification name is Endophytic Bacillus ( Priestia endophytica Z723, accession number GDMCC No: 63266.

2. The application of the strain described in claim 1 in the fermentation production of D-tagatose, characterized in that, The strain produces D-tagatose using galactitol as a substrate.

3. The application according to claim 2, characterized in that, The substrate was converted into D-tagatose using resting cells of the strain via whole-cell catalysis.

4. The application according to claim 3, characterized in that, After seed culture of the strain, the seed culture is inoculated into fermentation medium for fermentation culture. The fermentation broth is centrifuged to collect the cells, which are then resuspended and washed in PBS buffer. After centrifugation again, the precipitate is collected, which is the resting cells.

5. The application according to claim 3, characterized in that, D-tagatose was prepared by resuspending the resting cells of the strain in PBS buffer containing the substrate and shaking.

6. The application according to claim 4, characterized in that, The medium used for seed culture was LB liquid medium.

7. The application according to claim 3, characterized in that, The substrate concentration in the conversion system is 10-60 g / L.

8. The application according to claim 5, characterized in that, The pH in the conversion system is 3-10.

9. The application according to claim 8, characterized in that, The pH in the conversion system is 7-9.

10. The application according to claim 5, characterized in that, The temperature in the conversion system is 25-50℃.

11. The application according to claim 10, characterized in that, The temperature in the conversion system is 40-50℃.

Images