A Bacillus strain that rapidly and efficiently produces cellulase

Abstract

This invention belongs to the field of microbial technology and relates to a rapidly and efficiently cellulase-producing Bacillus strain. Bacillus sp RW5-1, preservation number: CCTCC NO: M 20242953, exhibits an enzyme production rate that is more than 60% higher than existing strains, and its crude enzyme solution maintains high activity under high temperature (45-100℃) and alkaline (pH 7-9) conditions. This strain is suitable for use in the bioenergy, textile, and detergent industries, offering significant economic benefits.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and relates to a Bacillus strain that rapidly and efficiently produces cellulase. Background Technology

[0002] Cellulase is a key enzyme in the degradation of cellulose and is widely used in bioenergy, textiles, feed, and food industries. Currently, commonly used industrial cellulase-producing strains, such as *Trichoderma reesei* and *Bacillus subtilis*, suffer from drawbacks such as long enzyme production cycles (typically 24-72 hours), insufficient thermal stability (optimal temperature below 50°C), or narrow pH tolerance (mostly acidic or neutral environments). For example, the peak enzyme production period of *Trichoderma reesei* requires more than 48 hours, and its enzyme activity significantly decreases above 60°C; the cellulase activity of *Bacillus licheniformis* loses more than 50% at pH > 8. Therefore, developing a strain with rapid enzyme production, strong adaptability, and high stability has significant industrial value. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a Bacillus strain that produces cellulase with fast enzyme production rate, high enzyme solution thermal stability and strong alkali tolerance.

[0004] A rapidly and efficiently cellulase-producing Bacillus strain, classified as Bacillus sp.RW5-1, was deposited on December 30, 2024, at the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China), with accession number CCTCC NO: M 20242953.

[0005] This invention isolated a cellulase-producing strain from pharmaceutical factory soil. The 16S rDNA was amplified using 27F / 1492R primers and sequenced (purification and sequencing of the PCR product were performed by Shanghai Sangon Biotech). The sequencing results are shown in SEQ ID NO.1. NCBI sequencing results showed that the isolated strain was Bacillus, and it was named RW5-1.

[0006] This invention identifies a novel Bacillus sp. RW5-1 strain, which has the following significant advantages:

[0007] 1. Rapid enzyme production: It enters the logarithmic growth phase after 4-6 hours of cultivation, and the enzyme activity reaches its peak within 14 hours. Figure 2 This reduces the enzyme production cycle by more than 60% compared to traditional strains (such as Trichoderma reesei).

[0008] 2. High thermal stability: The crude enzyme solution retains more than 80% of its original enzyme activity after treatment at 45-100℃ for 60 minutes, and retains 96% of its original enzyme activity after treatment at 55℃ for 30 minutes. Figure 3 It is significantly superior to most known strains (such as Bacillus subtilis, which loses 40% of its original enzyme activity after being treated at 60°C for 30 minutes).

[0009] 3. Strong alkalinity tolerance: Under pH 7-9 conditions, the crude enzyme solution retains more than 70% of the original enzyme activity. Figure 4 This strain is suitable for alkaline industrial environments (such as detergent production), while similar strains (such as Aspergillus niger) have their activity reduced to less than 50% of the original enzyme activity when pH>8.

[0010] This invention discloses a high-producing cellulase Bacillus sp. RW5-1 strain, whose enzyme production rate is increased by more than 60% compared to existing strains, and which maintains high activity under high temperature (45-100℃) and alkaline (pH 7-9) conditions. This strain is suitable for the bioenergy, textile, and detergent industries, and has significant economic benefits. Attached Figure Description

[0011] Figure 1 : Hydrolysis rings stained with RW5-1 iodine solution;

[0012] Figure 2 RW5-1 growth curve and enzyme activity changes;

[0013] Figure 3 Thermostability data of cellulase produced by RW5-1;

[0014] Figure 4 pH tolerance data of cellulase produced by RW5-1;

[0015] Figure 5 RW5-1 Short-term disintegration process of filter paper strips. Detailed Implementation

[0016] The culture media and their formulations used in the following examples are as follows:

[0017] Enrichment medium: 0.5 g / L yeast extract; 0.5 g / L peptone-D; 0.5 g / L casein amino acids; 0.5 g / L glucose; 0.5 g / L soluble starch; 0.3 g / L dipotassium hydrogen phosphate; 0.5 g / L magnesium sulfate; 0.3 g / L sodium pyruvate; 20.0 g / L agar; pH at rest; autoclaved at 121°C for 15 min.

[0018] LB medium: 10.0 g / L peptone; 5.0 g / L yeast extract; 10.0 g / L sodium chloride; pH at rest; autoclaved at 121°C for 20 min.

[0019] Cellulose agar medium: 5.0 g / L cellulose powder; 1.0 g / L sodium nitrate; 1.0 g / L dipotassium hydrogen phosphate; 1.0 g / L potassium chloride; 0.5 g / L magnesium sulfate; 0.5 g / L yeast extract; 15.0 g / L agar; pH at rest; autoclave at 121°C for 15 min.

[0020] Fermentation enzyme production culture: 10.0 g / L CMC-Na; 4.0 g / L ammonium sulfate; 2.0 g / L dipotassium hydrogen phosphate; 0.5 g / L magnesium sulfate; 5.0 g / L beef extract; natural pH, autoclave at 121℃ for 20 min.

[0021] Filter paper strip culture medium: 4.0 g / L ammonium sulfate; 2.0 g / L dipotassium hydrogen phosphate; 0.5 g / L magnesium sulfate; 5.0 g / L beef extract; 0.6 mL 50 mM glucose solution per liter.

[0022] 1. Strain isolation and identification:

[0023] 1g of soil from Wudang Pharmaceutical Factory in Hubei Province was suspended in sterile PBS by shaking. A 10-fold serial dilution was prepared and spread onto an enrichment medium. After incubation at 30°C for 24 hours, single colonies were obtained through repeated streak purification. The bacterial culture was inoculated onto cellulose agar medium and incubated at 30°C for 48 hours. Iodine staining was performed; strains showing a clear zone were identified as cellulase-positive bacteria and preserved in glycerol. The iodine hydrolysis zone of positive bacteria is shown in the image. Figure 1 .

[0024] Positive strains were inoculated into enzyme-producing culture medium and fermented for 4 days. The crude enzyme solution was obtained by centrifugation. Enzyme activity was determined by the filter paper enzyme activity method (DNS method to determine the amount of reducing sugar produced after 60 minutes of reaction at 50°C). The unit enzyme activity was defined as the amount of enzyme that produces 1 μmol of reducing sugar per minute.

[0025] 16S rDNA was amplified using primers 27F / 1492R and sequenced (purification and sequencing of the PCR product were performed by Shanghai Sangon Biotech). The sequencing results are shown in SEQ ID NO.1. Sequencing results from NCBI confirmed that the isolated strain was Bacillus sp., and it was named RW5-1. This strain was deposited on December 30, 2024, at the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China), with the taxonomic name Bacillus sp. RW5-1 and accession number CCTCCNO: M 20242953.

[0026] 2. Growth and enzyme production of RW5-1 over 24 hours:

[0027] RW5-1 was inoculated into fermentation medium and cultured at 30℃ and 150 rpm for 24 hours. Enzyme activity and growth concentration were measured at 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h, and 24h. Figure 2 The results showed that the strain reached the logarithmic growth phase in 4-6 hours, and the enzyme activity reached its peak in 14 hours (more than 10 hours earlier than the traditional strain).

[0028] Thermostatic stability of cellulase produced by RW5-1:

[0029] After fermentation of RW5-1 for 14 hours to produce enzyme, the mixture was centrifuged (5000 rpm, 10 min) to obtain crude enzyme solution. The enzyme solution was then incubated at 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, 80℃, 90℃, and 100℃ for 30 min and 60 min, respectively. The residual enzyme activity was measured, with the initial enzyme activity taken as 100%. The relative enzyme activity of cellulase at different temperatures was calculated as follows: Figure 3 .

[0030] The results showed that the cellulase exhibited good stability between 45 and 100°C, retaining over 80% of its activity within 60 minutes of incubation. After 30 minutes of incubation at 55°C, 96% of the maximum enzyme activity was retained. After 60 minutes of incubation at 45°C, 94% of the maximum enzyme activity was retained, and at temperatures above 80°C, the activity retention reached up to 90%.

[0031] pH tolerance of cellulase produced by RW5-1:

[0032] After fermenting RW5-1 for 14 hours to produce enzyme, the mixture was centrifuged (5000 rpm, 10 min) to obtain crude enzyme solution. The crude enzyme solution was then placed in buffer solutions of different pH values. Citrate buffer was used to prepare solutions with pH 3–6, and phosphate buffer was used to prepare solutions with pH 7–8. After 2 hours of incubation, residual enzyme activity was measured. The initial enzyme activity was taken as 100%, and the relative enzyme activity of cellulase at different pH values ​​was calculated.

[0033] Figure 4 The results showed that cellulase activity was relatively stable at pH 7–9, and the relative enzyme activity was higher when incubated at 45°C than when incubated at 55°C, with the enzyme activity remaining above 70%.

[0034] RW5-1 Filter Paper Disintegration Test

[0035] RW5-1 in the logarithmic growth phase was inoculated into filter paper strip culture medium at a 10% inoculation rate. Three filter paper strips (1×6 cm dried filter paper strips) were placed in each Erlenmeyer flask and incubated in a constant temperature shaker at 30℃ and 150 r / min. The disintegration of the filter paper strips was observed periodically during the incubation period. The results are as follows: Figure 5 As shown

[0036] Five hours after inoculation, the filter paper strip broke into three segments; after 15 hours, it broke into many small segments and became fibrous. Figure 5 This indicates its ability to rapidly degrade cellulose.

[0037] SEQ ID NO.1:

[0038]

Claims

1. A rapidly and efficiently cellulase-producing Bacillus sp. strain, named Bacillus RW5-1, which was deposited at the China Center for Type Culture Collection (CCTCC) on December 30, 2024, with accession number CCTCC NO: M20242953, is characterized by, It enters the logarithmic growth phase and begins enzyme production within 4-6 hours, and the enzyme activity reaches its peak after 14 hours.

2. The use of the Bacillus strain of claim 1 in the production of cellulase, characterized in that, The crude enzyme solution is suitable for industrial environments with high temperatures of 45-100℃ and / or pH of 7-9.

3. The use according to claim 2, characterized in that, The crude enzyme solution retains more than 80% of the original enzyme activity when treated at 45-100℃ for 60 minutes, and retains 96% of the original enzyme activity when treated at 55℃ for 30 minutes.

4. The use according to claim 2, characterized in that, The crude enzyme solution retains more than 70% of the original enzyme activity under pH 7-9 conditions.

5. Use of the Bacillus of claim 1 in the degradation of cellulose.

6. The use according to claim 5, characterized in that, The strain and filter paper strips were cultured at 35°C for 5-15 hours, which effectively and rapidly decomposed the filter paper strips.

Images