A β-amylase-producing Bacillus subtilis strain and its application

Abstract

The present invention discloses a strain of bacillus subtilis producing beta-amylase and its application, belong to the field of bioengineering technology, the bacillus subtilis is named as bacillus subtilis (Bacillus subtilis) JXJY-0811, has been deposited in the General Microbiology Center of China National Committee for the Collection of Microorganisms on June 7, 2024, and the deposit number is CGMCC NO.30886. The present invention uses the clear circle method to screen from soil and obtain bacterial strains that can secrete beta-amylase and amylase activity extracellularly, and is identified as bacillus subtilis through whole genome sequencing. The extracellular beta-amylase suitable temperature conditions of the strain are 50-75 DEG C, suitable pH value is 3.5-6.5, and extracellular enzyme activity is 9640.10U / mL, and after 60 hours of fermentation, the highest enzyme activity of 5L fermentation tank beta-amylase can reach 51980.36U / mL. The beta-amylase obtained by fermentation is used to process soluble starch solution, and the main product is maltose, which can be used for the production of maltose and high maltose syrup.

Description

Technical Field

[0001] The present invention relates to the technical field of bioengineering, in particular to a beta-amylase-producing Bacillus subtilis and application thereof. Background Art

[0002] β-amylase, also known as α-1,4-glucan-4-maltohydrolase, can cleave the α-1,4-glucosidic bonds between the non-reducing ends of starch and glycogen, producing β-optically active maltose. When β-amylase acts on amylose, the product is primarily maltose, with a small amount of glucose. When hydrolyzing amylopectin, β-amylase is unable to cleave the α-1,6-glucosidic bonds, resulting in the hydrolysis products including limit dextrins in addition to maltose.

[0003] One of the key uses of β-amylase in the food and pharmaceutical industries is as a saccharifying agent in the preparation of maltose syrup, the primary component of which is maltose. β-amylase is one of the primary saccharifying enzymes in starch processing. The reaction requires no coenzymes, operates under mild conditions, and produces few byproducts. It is used as a saccharifying agent in the industrial production of maltose, beer, and beverages, and has significant applications in food processing, beer fermentation, textiles, pharmaceuticals, chemical and medical analysis, and other industries.

[0004] With the continuous development of the enzyme industry and the increasing demand for maltose quality, the use of enzyme preparations to produce maltose syrup has become a research hotspot. The increasing demand for β-amylase has made it one of the most promising industrial enzymes. Bacillus subtilis is one of the main strains producing β-amylase. Under specific culture conditions and fermentation processes, Bacillus subtilis can produce β-amylase. Microbial β-amylase has attracted attention in industrial production due to its low production cost, minimal environmental impact, and high enzyme activity. β-amylase produced by microorganisms such as Bacillus subtilis has been widely used in food processing, beer fermentation, textile processing, and other fields.

[0005] While Bacillus subtilis offers numerous advantages in producing β-amylase, such as rapid growth, low nutritional requirements, and efficient secretion of proteins and metabolites, it also presents some potential drawbacks. Compared to plant-derived β-amylases, microbial β-amylases typically have lower heat tolerance. This means that at higher operating temperatures, the enzyme may lose activity, impacting production efficiency and product quality. The optimal operating temperature for bacterial β-amylase is generally below 50°C, which may limit its application in certain high-temperature industrial processes. Furthermore, the form in which Bacillus subtilis expresses β-amylase can also impact its industrial application. Intracellularly expressed β-amylase increases the difficulty and cost of handling during application. Summary of the Invention

[0006] The present invention discloses a strain of Bacillus subtilis producing β-amylase and its application, which belongs to the field of bioengineering technology. The Bacillus subtilis is named Bacillus subtilis (Bacillus subtilis) JXJY-0811, which has been deposited in the General Microbiology Center of China Microorganism Culture Collection Administration Committee on June 7, 2024, with a deposit number of CGMCC NO.30886. The present invention uses the clear circle method to screen from the soil to obtain a strain that can secrete β-amylase extracellularly and has a high amylase activity, and is identified as Bacillus subtilis by whole genome sequencing. The β-amylase obtained by fermentation is used to treat starch solution, and the main product is maltose, which can be used for the production of maltose and high maltose syrup.

[0007] The first object of the present invention is to provide a Bacillus subtilis strain producing β-amylase, named Bacillus subtilis JXJY-0811, which was deposited in the General Microbiology Center of the China Culture Collection of Microorganisms on June 7, 2024, with a deposit number of CGMCC NO.30886, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences.

[0008] The second object of the present invention is to provide the use of the above-mentioned Bacillus subtilis in the preparation of microbial agents.

[0009] The third object of the present invention is to provide a microbial agent containing the above-mentioned Bacillus subtilis.

[0010] Furthermore, the microbial agent is a strain fermentation broth or culture supernatant.

[0011] A fourth object of the present invention is to provide use of the above-mentioned Bacillus subtilis or the above-mentioned microbial agent in the preparation of β-amylase.

[0012] In one embodiment of the present invention, LB medium was used to ferment Bacillus subtilis JXJY-0811 to obtain β-amylase.

[0013] Furthermore, the fermentation temperature is 37° C., and the fermentation time is 60 hours.

[0014] Furthermore, 500 g / L glucose and trace element solution were added at a rate of 20 mL / h from 4 to 20 hours.

[0015] Furthermore, the trace element solution includes 0.5g / L CaCl2, 0.18g / L ZnSO4·7H2O, 0.1g / L MnSO4·H2O, 10.05g / L Na2-EDTA, 8.35g / L FeCl3, 0.16g / L CuSO4·5H2O, and 0.18g / L CoCl2·6H2O.

[0016] A fifth object of the present invention is to provide use of the above-mentioned Bacillus subtilis or the above-mentioned microbial agent in the preparation of maltose.

[0017] A sixth object of the present invention is to provide a method for producing β-amylase by fermentation, using the above-mentioned Bacillus subtilis or the above-mentioned microbial agent.

[0018] A seventh object of the present invention is to provide a method for producing maltose by fermentation, using the above-mentioned Bacillus subtilis or the above-mentioned microbial agent.

[0019] Furthermore, the above-mentioned Bacillus subtilis or the above-mentioned microbial agent is added to the soluble starch solution.

[0020] Furthermore, the content of starch is 1%.

[0021] Furthermore, the reaction time is 30 minutes.

[0022] Furthermore, the fermentation temperature is 50-75°C.

[0023] Furthermore, the fermentation pH is 3.5-6.5.

[0024] Beneficial effects of the present invention:

[0025] The invention discloses a Bacillus subtilis JXJY-0811 strain that produces beta-amylase. The strain has an extracellular beta-amylase with an optimum temperature of 50-75°C and an optimum pH of 3.5-6.5, a wide temperature and pH adaptability range, good acid resistance, an extracellular enzyme activity of 9640.10 U / mL, and a maximum beta-amylase activity of 51980.36 U / mL in a 5L fermentation tank after 60 hours of fermentation. The strain has important value for the large-scale industrial production of beta-amylase.

[0026] Biomaterial Deposit

[0027] Bacillus subtitls JXJY-0811, classified and named Bacillus subtilis, was deposited in the General Microbiology Center of China Culture Collection Administration on June 7, 2024, with the deposit number CGMCCNO.30886, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, Beijing, China. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0029] Figure 1 This is a graph of extracellular enzyme activity of the primary screening strains J1, J2 and J3 in Example 1 of the present invention;

[0030] Figure 2 The protein gel of crude enzyme solution and the enzyme activity inside and outside the vacuole of crude enzyme JXJY-0811 β-amylase of Bacillus subtilis in Example 2 of the present invention are shown in Figure 1, where A is the protein gel of crude enzyme solution and B is the enzyme activity inside and outside the vacuole;

[0031] Figure 3 The optimum temperature and optimum pH curves of the Bacillus subtilis JXJY-0811 β-amylase in Example 2 of the present invention, wherein A is the optimum temperature curve and B is the optimum pH curve;

[0032] Figure 4 The fermentation conditions of Bacillus subtilis JXJY-0811 in a 5L tank were optimized in Example 3 of the present invention;

[0033] Figure 5 This is a liquid phase diagram of the reaction product of β-amylase produced by Bacillus subtilis JXJY-0811 and soluble starch in Example 4 of the present invention. DETAILED DESCRIPTION

[0034] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0035] The culture medium used in the examples is as follows:

[0036] Enrichment medium: 5 g / L NaCl, 1 g / L MgSO4, 2 g / L KH2PO4, 2 g / L CaCl2, 6 g / L yeast extract, 6 g / L tryptone;

[0037] Screening medium: 2 g / L soluble starch, 1 g / L peptone, 5 g / L NaCl, 3 g / L beef extract, 20 g / L agar;

[0038] Lugol's iodine solution: 3g / L I2, 6g / L KI;

[0039] LB medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, 20 g / L agar;

[0040] TB fermentation medium: 24 g / L yeast powder, 12 g / L peptone, 5 g / L glycerol, 16.43 g / L K2HPO4·3H2O, 2.32 g / L KH2PO4;

[0041] Trace element solution: CaCl2 0.5g / L, 0.18g / L ZnSO4·7H2O, 0.1g / L MnSO4·H2O, 10.05g / LNa2-EDTA, 8.35g / L FeCl3, 0.16g / L CuSO4·5H2O, 0.18g / LCoCl2·6H2O.

[0042] The β-amylase activity detection method used in the examples is as follows:

[0043] The number of mg of maltose formed by 1 mL of enzyme solution hydrolyzing 1.10% starch solution per hour at pH 5.50 and 60°C is one enzyme activity unit, expressed in U / mL.

[0044] Assay method: Accurately pipette 9 mL of 1.10% soluble starch solution into a 25 mL colorimetric tube. Preheat in a 60°C water bath for 5 minutes. Accurately add 1 mL of enzyme solution, shake well, and immediately start the timer. Incubate the enzymatic reaction in a 60°C water bath for 30 minutes. Quickly pipette 0.5 mL of the reaction solution into 1.5 mL of DNS solution, boil for 15 minutes, cool in ice water, add 10.5 mL of distilled water, and measure the absorbance at 540 nm. Calculate the concentration of the test enzyme solution based on the absorbance. A control reaction using buffer instead of enzyme solution under the same conditions serves as a control.

[0045] β-amylase activity calculation formula: Enzyme activity (U / mL) = OD × 2 × 20 × 1.9 × K × n

[0046] Where:

[0047] K—standard curve constant;

[0048] n—dilution multiple;

[0049] 2—30 min reaction time converted to 60 min;

[0050] 20—Convert 0.5 mL of reaction solution into 10 mL;

[0051] 1.9—Conversion coefficient of glucose to maltose.

[0052] Example 1: Screening of wild-type Bacillus subtilis strains

[0053] Ten soil samples were collected from the Future Food Center of Jiangnan University, with dense greenery and fine soil, and numbered 1-10. Under sterile conditions, 1g of soil sample was added to 100mL of enrichment culture medium and cultured at 37℃ and 220rpm / min for 24h. 1mL of culture medium was diluted with physiological saline to make a concentration gradient (10 -1 , 10 -2 , 10 -3 , 10 -4 and 10 -5 The diluted solution was spread on a screening plate and incubated at 37°C for 48 h. Lugol's iodine solution was sprayed on the plate surface to observe the colony morphology. Three single colonies with obvious hydrolysis zones were selected and named J1, J2, and J3.

[0054] For rescreening, J1, J2, and J3 were first inoculated into 3-4 mL of LB medium and cultured at 37°C and 220 rpm / min for 10-12 hours. 1 mL of the seed solution was then transferred to 50 mL of TB medium and cultured at 37°C and 220 rpm / min for 36 hours. The resulting fermentation broth was centrifuged at 8000 rpm and 4°C for 5 minutes. The supernatant was separated to obtain the extracellular crude enzyme solution, and the enzyme activity was determined using the DNS method. The results were as follows: Figure 1 As shown, the strain J2 with the highest enzyme activity was named Bacillus subtilis JXJY-0811; the bacteria were resuspended in PBS buffer (pH = 7.2-7.4) and disrupted using an ultrasonic disruptor. After completion, they were centrifuged at 8000 rpm and 4°C for 10 min, and the supernatant was collected as the intracellular crude enzyme solution, and the intracellular precipitate was resuspended in PBS buffer.

[0055] The extracellular crude enzyme solution, intracellular crude enzyme solution and intracellular precipitation were subjected to SDS-PAGE gel electrophoresis experiment. The results are as follows: Figure 2 As shown in A, the molecular weight of the enzyme is about 67kDa. The intracellular and extracellular enzyme activities were measured, and the results were as follows Figure 2 As shown in Figure B, Bacillus subtilis JXJY-0811 has an extracellular β-amylase activity of 9640.10 U / mL and an intracellular β-amylase activity of 3708.19 U / mL. The whole genome of this strain was sequenced and identified as Bacillus subtilis through database comparison.

[0056] Example 2: Characterization of Enzymatic Properties of Bacillus subtilis JXJY-0811 β-amylase

[0057] (1) Optimal temperature curve of Bacillus subtilis JXJY-0811 β-amylase

[0058] The crude enzyme solution of Bacillus subtilis JXJY-0811 β-amylase prepared according to the method of Example 1 was diluted with phosphate buffer (pH 5.5) in a certain gradient, and the enzyme activity was measured at 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C and 80°C, respectively. The relative enzyme activity was calculated with the highest detected enzyme activity as 100%, and the temperature-relative enzyme activity curve was obtained. Figure 3 As shown in A, the relative enzyme activity of β-amylase produced by Bacillus subtilis JXJY-0811 can reach more than 80% between 50-75°C. The enzyme has a wide temperature range and its optimal action temperature is 60°C.

[0059] (2) Optimal pH curve of Bacillus subtilis JXJY-0811 β-amylase

[0060] The crude enzyme solution of Bacillus subtilis JXJY-0811 prepared according to the method of Example 1 was diluted with buffer solutions of pH 3.0, 3.5, 4.0, 4.5, 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0 in a certain gradient, and the enzyme activity was measured at 60°C. The relative enzyme activity was calculated with the highest detected enzyme activity as 100%. Figure 3 As shown in Figure B, the relative enzyme activity of β-amylase produced by Bacillus subtilis JXJY-0811 can be maintained above 50% between pH 3.5 and 6.5, and the highest enzyme activity is achieved at pH 5.0.

[0061] Example 3: Optimization of fermentation conditions for Bacillus subtilis JXJY-0811 in a 5L tank

[0062] Pick a single colony of Bacillus subtilis and inoculate it into a 250mL shake flask with 25mL LB culture medium. After activation culture at 37℃ and 220rpm for 8-10h, transfer all the seed liquid to a 5L fermenter (with 2.5L liquid volume) and carry out batch fermentation at 500rpm, 2vvm ventilation and 37℃. In 4-20h, add 500g / L glucose and trace element solution at a rate of 20mL / h, such as Figure 4 As shown, after 60 hours of fermentation, the maximum enzyme activity of β-amylase in a 5L tank reached 51980.36 U / mL. The protein concentration was also measured by BCA assay, and the final crude enzyme activity was 24000 U / mg.

[0063] Example 4: Application of β-amylase produced by Bacillus subtilis JXJY-0811 in the production of maltose

[0064] 1% soluble starch solution was used as substrate, and 1.5U of fermentation supernatant of β-amylase prepared in the above example was added. The mixture was placed in a 60℃ water bath for 30min, and the product was analyzed by HPLC after sampling. The results are shown in Figure 2. Figure 5 As shown, the maltose peak time is about 13.3min, and the results show that the main product is maltose, and no maltotriose and glucose peaks are detected. This shows that the β-amylase produced by the strain of the present invention can be used for the production of high maltose syrup, etc.

[0065] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A β-amylase-producing Bacillus subtilis strain, characterized by: Bacillus subtilis ( Bacillus subtilis ) JXJY-0811 was deposited in the General Microbiology Center of China Culture Collection Administration on June 7, 2024, with the deposit number CGMCC NO.30886, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences. The optimum temperature of the β-amylase is 50-75°C, and the optimum pH value is 3.5-6.

5.

2. Use of the Bacillus subtilis according to claim 1 in the preparation of microbial agents.

3. A microbial agent containing the Bacillus subtilis according to claim 1.

4. The microbial agent according to claim 3, characterized in that: The microbial agent is a strain fermentation broth.

5. Use of the Bacillus subtilis according to claim 1 or the microbial agent according to claim 3 in the preparation of β-amylase.

6. Use of the Bacillus subtilis according to claim 1 or the microbial agent according to claim 3 in the preparation of maltose.

7. A method for producing β-amylase by fermentation, characterized in that: Use the Bacillus subtilis according to claim 1 or the microbial agent according to claim 3.

8. A method for producing maltose by fermentation, characterized in that: Use the Bacillus subtilis according to claim 1 or the microbial agent according to claim 3.

9. The method according to claim 8, characterized in that: The fermentation temperature is 50-75℃.

10. The method according to claim 8, characterized in that: The fermentation pH is 3.5-6.5.

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