Bacillus subtilis for producing beta-amylase and application thereof

Abstract

The invention discloses bacillus subtilis capable of producing beta-amylase and application of the bacillus subtilis, and belongs to the technical field of bioengineering, the bacillus subtilis is named as bacillus subtilis JXJY-0811, the bacillus subtilis is preserved in China General Microbiological Culture Collection Center (CGMCC) on June 7, 2024, and the preservation number is CGMCC NO.30886. The invention further discloses a preparation method of the bacillus subtilis capable of producing beta-amylase and application of the bacillus subtilis capable of producing beta-amylase. A strain which can secrete beta-amylase in an extracellular mode and is high in amylase activity is screened from soil through a transparent ring method, and the strain is identified as bacillus subtilis through whole genome sequencing. The appropriate temperature condition of the extracellular beta-amylase of the strain is 50-75 DEG C, the appropriate pH value is 3.5-6.5, the extracellular enzyme activity is 9640.10 U / mL, and the highest enzyme activity of the beta-amylase in a 5L fermentation tank after fermentation for 60 hours can reach 51980.36 U / mL. The beta-amylase obtained through fermentation is used for treating a soluble starch solution, the main product is maltose, and the beta-amylase can be used for producing maltose and high maltose syrup.

Description

Technical Field

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

[0002] β-amylase, whose full name is α-1,4-glucan-4-maltohydrolase, can cut off the α-1,4-glucosidic bonds between the non-reducing ends of starch and glycogen to produce β-optically active maltose. When β-amylase acts on straight-chain starch, the product is mainly maltose, and a small amount of glucose is also produced; when hydrolyzing amylopectin, since β-amylase cannot cut off the α-1,6-glucosidic bonds, the hydrolysis products include limit dextrins in addition to maltose.

[0003] One of the important uses of β-amylase in the food and pharmaceutical industries is as a saccharifying agent to prepare maltose syrup, the main component of which is maltose. β-amylase is one of the main saccharifying enzymes in the starch processing process. The reaction does not require coenzymes, and the conditions are mild and there are few by-products. It can be used as a saccharifying agent in the industrial production of maltose, beer, beverages, etc. It has important application value in food processing, beer fermentation, textiles, medicine, chemical and medical analysis and other industries.

[0004] With the continuous development of the enzyme industry and people's higher requirements for the quality of maltose, the application of enzyme preparations to produce maltose syrup has become a hot topic of research. The increasing demand for β-amylase makes it one of the most promising industrial enzymes. Bacillus subtilis is one of the main strains for producing β-amylase. After specific culture conditions and fermentation processes, Bacillus subtilis can produce β-amylase. β-amylase from microorganisms has been valued in industrial production due to its low production cost, low environmental harm, and high enzyme activity. β-amylase produced by microorganisms such as Bacillus subtilis has been widely used in many fields such as food processing, beer fermentation, and textile processing.

[0005] Although Bacillus subtilis has many advantages in the production of β-amylase, such as fast growth rate, low nutritional requirements, and efficient secretion of proteins and metabolites, it also has some potential defects. Compared with plant-derived β-amylase, microbial-derived β-amylase generally has lower heat resistance. This means that at higher operating temperatures, the enzyme may lose its activity, affecting production efficiency and product quality. The optimal operating temperature of bacterial β-amylase is generally below 50°C, which may limit its application in certain high-temperature industrial processes. In addition, the form in which Bacillus subtilis expresses β-amylase will also affect its industrial application. Intracellularly expressed β-amylase increases the difficulty and cost of processing 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 Microbiological Center of China Microbiological Culture Collection Administration Committee on June 7, 2024, with a deposit number of CGMCC NO.30886. The present invention uses the transparent circle method to screen from the soil to obtain a strain that can secrete β-amylase extracellularly and has a high amylase activity, which 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 China Microorganism Culture Collection Administration 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 a culture supernatant.

[0011] The fourth object of the present invention is to provide the 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 during 4-20 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 the 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 beta-amylase-producing Bacillus subtilis JXJY-0811. The strain has an extracellular beta-amylase with an appropriate temperature condition of 50-75 DEG C and an appropriate pH value 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 enzyme activity of the beta-amylase in a 5L fermentation tank of 51980.36 U / mL after fermentation for 60 hours. The strain has important value for 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 Microorganism 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 according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

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

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

[0031] Figure 3 is the optimum temperature and optimum pH curve 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 the 5L tank of Bacillus subtilis JXJY-0811 in Example 3 of the present invention are optimized;

[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 is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

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

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

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

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

[0039] LB medium: 10 g / L peptone, 5 g / L yeast powder, 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 / L CoCl2·6H2O.

[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 as U / mL.

[0044] Determination method: Accurately draw 9mL of 1.10% soluble starch solution, place it in a 25mL colorimetric tube, preheat it in a 60℃ water bath for 5min, accurately add 1mL of enzyme solution, shake well, start timing immediately, accurately keep the enzymatic reaction in a 60℃ water bath for 30min, quickly draw 0.5mL of reaction solution into 1.5mL of DNS solution, boil for 15min, cool in ice water, add 10.5mL of distilled water, and measure the absorbance at a wavelength of 540nm. The concentration of the test enzyme solution is obtained based on the absorbance. The reaction system with buffer solution instead of enzyme solution under the same conditions is used 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 0.5mL of reaction solution into 10mL;

[0051] 1.9—Coefficient for converting glucose to maltose.

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

[0053] Ten soil samples with dense greenery and fine soil near the Future Food Center of Jiangnan University were selected and numbered 1-10. Under sterile conditions, 1 g of soil sample was added to 100 mL of enrichment culture medium and cultured at 37°C and 220 rpm / min for 24 h. 1 mL of culture solution was diluted with physiological saline for a concentration gradient (10 -1 , 10 -2 , 10 -3 , 10 -4 and 10 -5 ). The diluted solution was spread on the screening plate, and after culturing at 37°C for 48 hours, 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 respectively.

[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 h. 1 mL of seed solution was transferred to 50 mL of TB medium and cultured at 37°C and 220 rpm / min for 36 h. The fermentation broth was centrifuged at 8000 rpm and 4°C for 5 min, and the supernatant was separated to obtain the extracellular crude enzyme solution. The enzyme activity was determined by 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 the end, 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 67 kDa. 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 the strain was sequenced and identified as Bacillus subtilis by comparison with the database.

[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 a phosphate buffer (pH 5.5) to 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 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 of Bacillus subtilis JXJY-0811 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°C and 220rpm for 8-10h, transfer all the seed liquid to a 5L fermenter (liquid volume 2.5L) and perform batch fed fermentation at 500rpm, 2vvm ventilation, and 37°C. Add 500g / L glucose and trace element solution at a rate of 20mL / h for 4-20h. Figure 4 As shown, after 60h of fermentation, the highest enzyme activity of β-amylase in a 5L tank can reach 51980.36U / mL. At the same time, the protein concentration was measured by BCA method, and the final crude enzyme specific activity was 24000U / mg.

[0063] Example 4: Application of β-amylase produced by Bacillus subtilis JXJY-0811 in producing 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°C water bath for reaction for 30 minutes, and the product was analyzed by HPLC after sampling. The results are shown in FIG. 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 clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A Bacillus subtilis strain producing β-amylase, characterized in that: It was named Bacillus subtilis JXJY-0811 and was deposited in the General Microbiology Center of China Culture Collection Administration on June 7, 2024. The deposit number is CGMCC NO.30886 and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences.

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 liquid or a culture supernatant.

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 described in claim 1 or the microbial agent described in claim 3.

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