Beta-glucanase recombinant bacteria, construction and application in preparation of water-soluble beta-glucan

Abstract

This invention discloses a recombinant β-glucanase bacterium, its construction, and its application in the preparation of water-soluble β-glucan. Belonging to the fields of genetic engineering and enzyme engineering, the recombinant plasmid pWB980-g9050 is introduced and expressed in the β-glucanase recombinant bacterium. The plasmid contains the gene sequence shown in SEQ ID NO.1 from *Chaetoceros* JY25 with accession number CGMCC No. 6882. The full gene sequence of the recombinant plasmid is shown in SEQ ID NO.2. This recombinant bacterium can be used in the preparation of water-soluble β-glucan. This invention achieves efficient and targeted biotransformation of yeast β-glucan by constructing a recombinant *Bacillus subtilis* strain that efficiently expresses a specific endoglucanase and employing a simple process based on crude enzyme solution fermentation. This not only stably produces water-soluble products with concentrated molecular weight distribution and well-defined structures, but also utilizes mild and environmentally friendly processes, demonstrating excellent potential for industrial application.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and enzyme engineering technology, and relates to recombinant β-glucanase bacteria, their construction, and their application in the preparation of water-soluble β-glucan. Background Technology

[0002] β-glucan is a natural polysaccharide compound composed of glucose molecules linked by β-1,3-glycosidic bonds to form the backbone and β-1,6-glycosidic bonds to form the branches, meaning that glucose molecules branch out from the backbone via β-1,6-glycosidic bonds. β-glucan is widely found in plants, fungi, and bacteria, and possesses various physiological functions such as immune regulation, anti-tumor activity, lipid-lowering, and blood sugar-lowering effects. It has been widely applied in medicine, health products, and food industries.

[0003] Yeast β-glucan is a major component of yeast cell walls and a rich source of natural glucans. However, its dense triple helix structure and high molecular weight result in extremely poor solubility, limiting its applications. Current physical or chemical methods to improve its water solubility suffer from low conversion rates and severe pollution. In contrast, enzymatic hydrolysis offers milder and more environmentally friendly conditions, making it a more promising direction for modification. Existing technologies have publicly used β-glucanases for treatment, but these methods typically rely on single enzyme preparations, which have limited degradation efficiency for the complex network structure of yeast β-glucan, and commercial enzyme preparations are expensive.

[0004] The inventors discovered in their research that the crude enzyme broth from a specific *Chaetomium* strain (CGMCC No. 6882) exhibits unique advantages in degrading yeast β-glucan to prepare specific low-molecular-weight water-soluble enzymes. However, direct fermentation using natural strains still suffers from drawbacks such as a long production cycle and the need for further improvement in enzymatic hydrolysis rate.

[0005] Therefore, it is urgent to utilize modern genetic engineering technology to identify and clone key gene sequences from the aforementioned specific strains with high degradation capabilities, and to construct recombinant engineered bacteria that can express these genes more efficiently and stably. Summary of the Invention

[0006] The purpose of this invention is to overcome the limitations of existing enzymatic methods that rely on high-cost commercial enzymes and low production efficiency of natural strains, and to provide a method for preparing water-soluble β-glucan by constructing recombinant bacteria for β-glucanase and its application in the preparation of water-soluble β-glucan. By using genetic engineering methods to construct recombinant engineered bacteria that can efficiently express specific β-glucanase, a method for preparing water-soluble β-glucan with higher production efficiency that does not rely on commercial enzymes or natural fungal fermentation is provided. Preservation Information: CGMCC NO. 6882, China General Microbiological Culture Collection Center, Chaetomium sp. 2012-11-26

[0007] The technical solution of the present invention specifically includes: The key to the β-glucanase recombinant bacteria is that the recombinant plasmid pWB980-g9050 is introduced and expressed in the above-mentioned β-glucanase recombinant bacteria. The above plasmid contains the gene sequence shown in SEQ ID NO.1 of Chaetomium JY25 with the preservation number CGMCC No.6882; the full gene sequence of the above-mentioned recombinant plasmid is shown in SEQ ID NO.2.

[0008] Furthermore, the aforementioned recombinant β-glucanase bacteria are Bacillus subtilis Bs168 or Bacillus subtilis WB600; the aforementioned recombinant plasmid contains a secretory signal peptide sequence.

[0009] The key to the above-mentioned method for constructing recombinant β-glucanase bacteria lies in the following steps: Obtain the gene sequence shown in SEQ ID NO.1 from Chaetomium JY25 with accession number CGMCC No.6882; The gene sequence shown in SEQ ID NO.1 was cloned into an expression vector to construct a recombinant plasmid; The complete genome sequence of the above recombinant plasmid is shown in SEQ ID NO.2; The above recombinant plasmid was introduced into the host bacteria to obtain the above β-glucanase recombinant bacteria.

[0010] Furthermore, the gene sequence shown in SEQ ID NO.1 was obtained through whole-gene synthesis, with a tag sequence for protein purification and cleavage added upstream of its coding sequence during synthesis.

[0011] Furthermore, the aforementioned tag sequence includes a 6×His tag, a thrombin recognition cleavage site, and an endothelial kinase recognition cleavage site.

[0012] A method for preparing water-soluble β-glucan, the key being the use of the above-mentioned recombinant β-glucanase bacteria or the recombinant β-glucanase bacteria obtained by the above-mentioned construction method, specifically including: S1. The above-mentioned recombinant β-glucanase bacteria were subjected to liquid fermentation culture to obtain fermentation broth; S2. The fermentation broth obtained in step S1 is subjected to solid-liquid separation to obtain a crude enzyme solution containing β-glucan degrading enzyme system. S3. The above crude enzyme solution and insoluble yeast β-glucan substrate were subjected to enzymatic hydrolysis at pH 5.5-6.5 and temperature 45℃-65℃ to obtain the enzymatic hydrolysis product. S4. Terminate the reaction and separate and purify the enzymatic hydrolysis products to obtain low molecular weight, water-soluble β-glucan with a weight average molecular weight of 25kDa to 28kDa.

[0013] Furthermore, in step S1, the liquid fermentation culture is carried out using LB liquid medium or corn steep liquor dry powder medium; the corn steep liquor dry powder medium includes: 20 g / L corn steep liquor dry powder, 10 g / L sodium chloride, natural pH value, and water as the solvent.

[0014] Specifically, the conditions for the liquid fermentation culture in step S1 are: temperature 26℃~32℃, shaking speed 160r / min~200r / min, and culture time 14h~22h.

[0015] Specifically, in step S2, the solid-liquid separation is performed by centrifugation at 8000 r / min to 12000 r / min for 5 to 15 minutes. After centrifugation, the supernatant is collected to obtain the crude enzyme solution containing the β-glucan-degrading enzyme system; at the same time, the precipitate (mainly undigested insoluble yeast β-glucan substrate) is collected, and this precipitate can be directly returned to the reaction system in step S3 for recycling.

[0016] More specifically, the enzymatic hydrolysis reaction in step S3 takes 4 to 8 hours; the insoluble yeast β-glucan substrate is added in the form of a suspension with a mass concentration of 1.7% to 2.3%, and the volume ratio of the crude enzyme solution to the suspension is 1:(4 to 9).

[0017] It should be noted that the pH of the above enzymatic hydrolysis reaction is achieved by adjusting the initial pH of the insoluble yeast β-glucan substrate suspension, and the pH of the reaction system is basically maintained at the initial pH of the suspension.

[0018] Compared with the prior art, the present invention has the following advantages: First, this invention constructs a recombinant Bacillus subtilis engineered strain capable of efficiently expressing a specific endoglucanase through genetic engineering. The catalytic functional unit derived from a safe strain is successfully introduced into a host suitable for industrial production, achieving targeted enhancement and stable inheritance of degradation capacity, and providing a reliable core catalyst for efficient and stable biocatalytic processes.

[0019] Secondly, the enzymatic hydrolysis process based on whole-cell fermentation of crude enzyme solution adopted in this invention is simple and highly economical. This invention eliminates the need for separation and purification of the target enzyme, directly utilizing the fermentation broth containing highly active recombinant enzymes for the reaction. This not only avoids complex downstream processing steps, significantly reducing production costs and operational complexity, but also more completely preserves the natural catalytic microenvironment of the enzyme system, which is beneficial for maintaining its efficient and stable degradation performance.

[0020] Third, the molecular weight of the product of this invention is highly controllable. This invention can directionally depolymerize insoluble high molecular weight dextran networks into water-soluble fragments with concentrated molecular weight distribution and well-defined structures, overcoming the technical problem of uneven products from traditional chemical methods or non-specific enzymatic methods.

[0021] Fourth, the overall process conditions of this invention are mild and environmentally friendly, possessing good industrial adaptability. The reaction process takes place in an aqueous phase, requiring a wide range of temperature and pH conditions. Furthermore, incompletely converted substrates can be recycled, achieving reuse and aligning with the concepts of green production and sustainable development. Attached Figure Description

[0022] Figure 1 This is the PCR result of E. coli transformation using the pWB980-g9050 plasmid for identification.

[0023] Figure 2 This is the pWB980-g9050 plasmid map.

[0024] Figure 3 This is the PCR result of Bacillus subtilis transformed with the pWB980-g9050 plasmid.

[0025] Figure 4 This is an analysis of the induced expression of Bacillus subtilis Bs168-pWB980-g9050.

[0026] Figure 5 This is an analysis of the induced expression of Bacillus subtilis WB600-pWB980-g9050.

[0027] Figure 6 The total sugar content standard curve was plotted using the phenol-sulfuric acid method with glucose as the standard.

[0028] Figure 7 This is a photograph of sample 1 prepared in Example 2.

[0029] Figure 8 This is a photo of commercially available water-insoluble yeast beta-glucan.

[0030] Figure 9 This refers to the dissolution effect of sample 1.

[0031] Figure 10 This refers to the solubility effect of commercially available water-insoluble yeast β-glucan.

[0032] Figure 11 This is the gel permeation chromatography (GPC / SEC) chromatogram of sample 1.

[0033] Figure 12 This is a gel permeation chromatography (GPC / SEC) chromatogram of commercially available water-insoluble yeast β-glucan.

[0034] Figure 13 The infrared spectra (IR spectra) are of water-soluble β-glucan, represented by sample 1, and commercially available insoluble yeast β-glucan.

[0035] In the attached figures, 1 represents the dissolution effect of sample 1 at a concentration of 5 g / L, 2 represents the dissolution effect of sample 1 at a concentration of 25 g / L, 3 represents the dissolution effect of sample 1 at a concentration of 50 g / L, 4 represents the dissolution effect of sample 1 at a concentration of 75 g / L, 5 represents the dissolution effect of sample 1 at a concentration of 100 g / L, 6 represents the dissolution effect of commercially available water-insoluble yeast β-glucan at a concentration of 5 g / L, 7 represents the dissolution effect of commercially available water-insoluble yeast β-glucan at a concentration of 25 g / L, 8 represents the dissolution effect of commercially available water-insoluble yeast β-glucan at a concentration of 50 g / L, 9 represents the dissolution effect of commercially available water-insoluble yeast β-glucan at a concentration of 75 g / L, and 10 represents the dissolution effect of commercially available water-insoluble yeast β-glucan at a concentration of 100 g / L. Detailed Implementation

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0038] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0039] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0040] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0041] Unless otherwise specified in the examples, the procedures can be followed according to conventional conditions. If the manufacturers of the reagents or instruments used are not specified, such as yeast β-glucan (water insoluble), they are all conventional products that can be purchased commercially.

[0042] The *Chaetoceros* strain JY25 (hereinafter referred to as *Chaetoceros* JY25), with accession number CGMCC No. 6882, used in this study was obtained from Henan University of Technology. After activation of the lyophilized powder, the strain was plate-cultured for 7 days. Mycelia from the colony edges were picked and inoculated into liquid seed culture medium (components included: 10 g / L tryptone; 5 g / L yeast extract; 10 g / L sodium chloride, pH=7.0, sterilized at 115℃ for 30 min, cooled for later use). The medium was then cultured at 28℃ and 180 rpm for 24 h with shaking to obtain a seed culture with good mycelial growth and uniform appearance. The seed culture was aseptically aliquoted into 1 mL sterile centrifuge tubes and stored long-term in an ultra-low temperature freezer at -80℃. To ensure the activity and genetic stability of the strain, the frozen strain was revived and retested every 6 months, and its morphological characteristics were verified by plate culture and microscopic observation to ensure its suitability for subsequent fermentation.

[0043] The LB liquid medium formula is as follows: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH=7.0±0.2, solvent is water; sterilize at 115℃ for 30 min.

[0044] Example 1 This embodiment provides a method for constructing recombinant β-glucanase bacteria, specifically including: 1. Gene alignment analysis and synthesis of pWB980-g9050 plasmid: Using the endonuclease gene sequence (XM_038893329.1) in the NCBI database, the genome sequencing sequence of Chaetomium JY25 was compared with that of Chaetomium to obtain a sequence highly homologous to the reported sequence as shown in SEQ ID NO.1, and the genome sequencing annotation sequence number is g9050; The entire gene was synthesized using the pWB980-ori plasmid, which contains a secretory signal peptide. A 6×His tag, thrombin recognition site, and cleavage site were then added upstream of the gene. thrombin site ), endothelial kinase recognition sites and cleavage sites ( enterokinase site ), used to cut the label sequence.

[0045] 2. pWB980-g9050 plasmid was transformed into E. coli: The synthesized plasmid was transformed into E. coli DH5a competent cells, cultured, and colonies were selected for positive clone identification. PCR results for bacterial culture identification are as follows Figure 1 As shown: M is DM2000, samples 1-8 are bacterial cultures, 9 is a positive plasmid control, and 10 is a negative control; like Figure 1 As shown, the bacterial culture PCR identification result band was consistent with the positive control band, confirming successful transformation. Three more parallel samples were sent for sequencing verification, and the results showed that the vector construction was also successful. It was then named pWB980-g9050. The constructed plasmid map is shown below. Figure 2 The full genome sequence of the recombinant plasmid is shown in SEQ ID NO.2.

[0046] 3. pWB980-g9050 plasmid was transformed into Bacillus subtilis: The successfully constructed pWB980-g9050 plasmid was transformed into Bacillus subtilis competent cells using the heat shock method. Two strains were used: Bacillus subtilis Bs168 and Bacillus subtilis WB600. Then, transformed colonies were selected for colony PCR verification. The colony PCR results are shown below. Figure 3 Wherein: M is DM2000; Sample 1 is a negative control; Sample 2 is a positive plasmid control; Samples 1-4 were transformed with pWB980-g9050 plasmid into Bacillus subtilis 168 bacterial culture PCR, and Samples 5-8 were transformed with pWB980-g9050 plasmid into Bacillus subtilis WB600 bacterial culture PCR. like Figure 3 As shown, both strains amplified bands consistent with the positive control, indicating successful transformation. They were named Bacillus subtilis Bs168-pWB980-g9050 and Bacillus subtilis WB600-pWB980-g9050, respectively.

[0047] 4. Induction of expression of Bacillus subtilis Bs168-pWB980-g9050 and WB600-pWB980-g9050: Bacillus subtilis Bs168-pWB980-g9050 and WB600-pWB980-g9050 positive bacteria were used to induce expression; Bacillus subtilis Bs168-pWB980-g9050 and WB600-pWB980-g9050 positive bacteria were inoculated into LB liquid medium at a bacterial culture: medium ratio of 1:100. After culturing at 37°C and 200 rpm for 6 h, 0.5 mM IPTG was added to induce expression. Samples were taken at 20 h, 40 h, and 60 h after induction, and 500 μL of the induced bacterial culture was collected. The culture was centrifuged, and the supernatant was transferred to a new centrifuge tube. 200 μL of 2×SDS PAGE loading buffer was added to the precipitate and the precipitate was mixed. At the same time, 50 μL of the supernatant was taken and 50 μL of 2×SDS PAGE loading buffer was added and mixed. The supernatant and precipitate were boiled together for 10 min and then allowed to cool naturally. Then, 20 μL of liquid was loaded and analyzed by SDS-PAGE. The results are as follows: Figure 4 and Figure 5 As shown.

[0048] Figure 4 The results of induced expression in Bacillus subtilis Bs168-pWB980-g9050 showed that no target protein band was observed in the supernatant or precipitate after 40 hours of induction. After 60 hours of induction, a weak 80.2 kDa band appeared in the precipitate, consistent with the expected size, while the target band was absent in the supernatant. This indicates that the enzyme expressed in Bs168-pWB980-g9050 bacteria is intracellular.

[0049] Figure 5 The results of induced expression in Bacillus subtilis WB600-pWB980-g9050 showed that after 20 h of induction, a weak 80.2 kDa target protein band was present in the precipitate, consistent with the expected size. After 40 h of induction, the target band in the precipitate deepened, while no target band was found in the supernatant. After 60 h of induction, the target band in the precipitate became lighter, suggesting that the target protein may have been degraded by the bacterial proteases with prolonged induction time. These results indicate that the enzyme expressed in WB600-pWB980-g9050 bacteria is also intracellular.

[0050] Example 2 This embodiment provides a microbial method for preparing low molecular weight, water-soluble β-glucan. The specific process is as follows: S1. The Bacillus subtilis WB600-pWB980-g9050 constructed in Example 1 was subjected to liquid fermentation culture to obtain a fermentation broth, specifically including: Bacillus subtilis WB600-pWB980-g9050 was inoculated into LB liquid medium and cultured at 28°C with a shaking speed of 180 r / min for 18 h.

[0051] S2. Centrifuge the fermentation broth obtained in step S1 at 10000 r / min for 10 min to obtain a crude enzyme solution containing β-glucan degrading enzyme system.

[0052] S3, enzymatic hydrolysis reaction, specifically including: The crude enzyme solution was added to a suspension containing 2.0% yeast β-glucan substrate at a volume ratio of 1:6 for enzymatic hydrolysis. The hydrolysis temperature was 55℃, the shaking speed was 180 r / min, and the hydrolysis time was 6 h to obtain the hydrolysate. The preparation process of a suspension containing 2.0% yeast β-glucan substrate is as follows: Take 1L of deionized water and adjust the pH of the solution to 6.0 with 0.5mol / L dilute hydrochloric acid; weigh 4g of yeast β-glucan and add it to a 500mL Erlenmeyer flask, and add 196mL of the above-prepared deionized water to the 500mL Erlenmeyer flask to prepare a suspension containing yeast β-glucan substrate for enzymatic hydrolysis.

[0053] S4. Terminate the reaction and separate and purify to obtain water-soluble β-glucan: Take 1g of enzyme hydrolysate into a 100mL centrifuge tube, add 59g of deionized water for dilution, centrifuge at 25℃ and 10000r / min for 10min, and collect the supernatant. The total sugar content in the supernatant was determined using the phenol-sulfuric acid method, and the enzymatic hydrolysis rate of β-glucan was calculated. Separate the enzymatic hydrolysate according to the above method to obtain the supernatant, add 6 times the volume of the collected supernatant of anhydrous ethanol, and let it stand at 4°C for 12 hours for alcohol precipitation. Centrifuge at 5000 r / min for 30 min, collect the precipitate, and wash the precipitate with anhydrous ethanol and acetone respectively; at the same time, collect the precipitate (mainly undigested insoluble yeast β-glucan substrate), which can be directly returned to the reaction system in step S3 for recycling; The precipitate was placed in a cold trap at a temperature of -40℃, a vacuum degree of 10Pa, and a drying time of 24h to obtain water-soluble β-glucan dry powder, which was designated as sample 1.

[0054] Example 3 This embodiment provides a microbial method for preparing low molecular weight, water-soluble β-glucan. The specific process is as follows: S1. The Bacillus subtilis WB600-pWB980-g9050 constructed in Example 1 was subjected to liquid fermentation culture to obtain a fermentation broth, specifically including: Bacillus subtilis WB600-pWB980-g9050 was inoculated into LB liquid medium and cultured at 32°C with a shaking speed of 160 r / min for 14 h.

[0055] S2. Centrifuge the fermentation broth obtained in step S1 at 12000 r / min for 5 min to obtain a crude enzyme solution containing β-glucan degrading enzyme system.

[0056] S3, enzymatic hydrolysis reaction, specifically including: The crude enzyme solution was added to a suspension containing 2.3% yeast β-glucan substrate at a volume ratio of 1:4 for enzymatic hydrolysis. The hydrolysis temperature was 65℃, the shaking speed was 200 r / min, and the hydrolysis time was 4 h to obtain the hydrolysate. The preparation process of a suspension containing 2.3% yeast β-glucan substrate is as follows: Take 1L of deionized water and adjust the pH of the solution to 5.5 with 0.5mol / L dilute hydrochloric acid; weigh 4g of yeast β-glucan and add it to a 500mL Erlenmeyer flask, and add 196mL of the above-prepared deionized water to the 500mL Erlenmeyer flask to prepare a suspension containing yeast β-glucan substrate for enzymatic hydrolysis.

[0057] S4. Terminate the reaction and separate and purify to obtain water-soluble β-glucan: Same as step S4 in Example 2, to obtain water-soluble β-glucan dry powder, denoted as Sample 2.

[0058] Example 4 This embodiment provides a microbial method for preparing low molecular weight, water-soluble β-glucan. The specific process is as follows: S1. The Bacillus subtilis WB600-pWB980-g9050 constructed in Example 1 was subjected to liquid fermentation culture to obtain a fermentation broth, specifically including: Bacillus subtilis WB600-pWB980-g9050 was inoculated into LB liquid medium and cultured at 26°C with a shaking speed of 200 r / min for 22 h.

[0059] S2. Centrifuge the fermentation broth obtained in step S1 at 8000 r / min for 15 min to obtain a crude enzyme solution containing β-glucan degrading enzyme system.

[0060] S3, enzymatic hydrolysis reaction, specifically including: The crude enzyme solution was added to a suspension containing 1.7% yeast β-glucan substrate at a volume ratio of 1:9 for enzymatic hydrolysis. The hydrolysis temperature was 45℃, the shaking speed was 160 r / min, and the hydrolysis time was 8 h to obtain the hydrolysate. The preparation process of a suspension containing 1.7% yeast β-glucan substrate is as follows: Take 1L of deionized water and adjust the pH of the solution to 6.5 with 0.5mol / L dilute hydrochloric acid; weigh 4g of yeast β-glucan and add it to a 500mL Erlenmeyer flask, and add 196mL of the above-prepared deionized water to the 500mL Erlenmeyer flask to prepare a suspension containing yeast β-glucan substrate for enzymatic hydrolysis.

[0061] S4. Terminate the reaction and separate and purify to obtain water-soluble β-glucan: Same as step S4 in Example 2, to obtain water-soluble β-glucan dry powder, denoted as sample 3.

[0062] Example 5 This embodiment provides a microbial preparation method for low molecular weight, water-soluble β-glucan. The specific process is the same as in Embodiment 2, except that the liquid culture medium used to inoculate Bacillus subtilis WB600-pWB980-g9050 in step S1 is corn steep liquor dry powder culture medium. The specific components include: corn steep liquor dry powder (water-soluble) 20 g / L, sodium chloride 10 g / L, natural pH value, and water as the solvent. The subsequent process is the same as in Embodiment 2, and water-soluble β-glucan dry powder is prepared, which is denoted as Sample 4.

[0063] Example 6 This embodiment provides a microbial preparation method for low molecular weight, water-soluble β-glucan. The specific process is the same as in Embodiment 2, except that in step S1, Bacillus subtilis Bs168-pWB980-g9050 is used instead of Bacillus subtilis WB600-pWB980-g9050 for inoculation. The subsequent process is the same as in Embodiment 2, and water-soluble β-glucan dry powder is prepared, which is denoted as sample 5.

[0064] Comparative Example 1 This comparative example utilizes *Chaetoceros* JY25 for the microbial preparation of water-soluble β-glucan. The specific process is as follows: S1. The *Chaetoceros* JY25 is subjected to liquid fermentation to obtain the fermentation broth, which specifically includes: Place the frozen Chaetomium JY25 in a clean bench for 1 hour and wait for it to return to room temperature. Then add one tube of Chaetomium JY25 seed culture to LB liquid medium and incubate at 28°C with a shaking speed of 180 r / min for 24 hours. S2. Centrifuge the fermentation broth obtained in step S1 at 10000 r / min for 10 min to obtain a crude enzyme solution containing β-glucan degrading enzyme system.

[0065] S3, enzymatic hydrolysis reaction, specifically including: The crude enzyme solution was added to a suspension containing 2.0% yeast β-glucan substrate at a volume ratio of 1:9 for enzymatic hydrolysis. The hydrolysis temperature was 60℃, the shaking speed was 180 r / min, and the hydrolysis time was 8 h to obtain the hydrolysate. The preparation process of a suspension containing 2.0% yeast β-glucan substrate is as follows: Take 1L of deionized water and adjust the pH of the solution to 5.0 with 1mol / L dilute hydrochloric acid; weigh 4g of yeast β-glucan and add it to a 500mL Erlenmeyer flask, and add 196mL of the above-prepared deionized water with pH=5.0 to the 500mL Erlenmeyer flask to prepare a suspension containing yeast β-glucan substrate for enzymatic hydrolysis.

[0066] S4. Terminate the reaction and separate and purify to obtain water-soluble β-glucan: Same as step S4 in Example 2, to obtain water-soluble β-glucan dry powder, which is designated as control 1.

[0067] Comparative Example 2 This comparative example provides a method for preparing water-soluble β-glucan. The specific steps are the same as those in Comparative Example 1, except that the liquid culture medium used to inoculate Bacillus subtilis WB600-pWB980-g9050 in step S1 is corn steep liquor dry powder culture medium. The specific components include: corn steep liquor dry powder (water-soluble) 20 g / L, sodium chloride 10 g / L, natural pH value, and water as the solvent. The subsequent process is the same as in Comparative Example 1, and water-soluble β-glucan dry powder is prepared, which is designated as control 2.

[0068] Comparative Example 3 This comparative example provides a method for preparing water-soluble β-glucan. The specific steps are the same as in Comparative Example 1, except that commercially available β-glucanase (from Longkote Enzyme Preparation Co., Ltd., enzyme activity 50,000 U / g) is used instead of the crude enzyme solution of Chaetomium JY25 used in Comparative Example 1. The specific steps include: S1. Preparation of commercially available enzyme solutions: Weigh 5g of β-glucanase, dissolve it in disodium hydrogen phosphate-citrate buffer at pH 5.0, and bring the volume to 10mL to prepare a commercially available enzyme solution.

[0069] S2, Enzymatic hydrolysis: Same as step S3 in Comparative Example 1, except that commercially available β-glucanase is used instead of the crude enzyme solution used in Comparative Example 1, and the amount of commercially available β-glucanase added (based on dry powder) is 0.3% of the dry weight of the yeast β-glucan substrate.

[0070] S3. Terminate the reaction and separate and purify to obtain water-soluble β-glucan: Same as step S4 of Comparative Example 1, use the phenol-sulfuric acid method to determine the total sugar content in the supernatant, calculate the enzymatic hydrolysis rate of β-glucan, and prepare β-glucan dry powder, which is designated as reference standard 3.

[0071] Note: The aforementioned commercially available β-glucanases are all commercially available enzyme preparations. Their product names, models, and nominal enzyme activities are all from publicly available information from the manufacturers. None of the manufacturers' instructions or publicly available information explicitly provide the specific enzyme type and proportions. Comparing commercially available enzymes from different sources, models, and activities is a routine and necessary practice in this field when evaluating the advancement of new technological solutions. The core purpose of this comparative example is to objectively compare the effect of the crude enzyme solution prepared by this invention with that of existing technology (i.e., commercially available enzymes) on the same substrate under the same conditions, in order to verify the relative advantages of the technical solution of this invention. The specific enzyme component proportions within the commercially available enzymes are technical details of the manufacturers and do not affect the rationality and legal validity of comparing them as a whole existing technology product in this patent, nor do they affect the ability of those skilled in the art to repeat this comparative experiment.

[0072] It should be noted that all enzymatic hydrolysis performance data (such as hydrolysis rate and product molecular weight) in this comparative example represent only the comparison results between the specific commercial enzyme and the crude enzyme solution produced by this invention under the specific reaction system and process parameters set in this invention. These results serve solely for the purpose of demonstrating patent inventiveness and do not constitute any evaluation of the overall quality of any product from any manufacturer or its performance in other application scenarios.

[0073] Analysis and Testing I. Enzymatic hydrolysis rate analysis of β-glucan: This invention uses the phenol-sulfuric acid method to determine the total sugar content and calculate the enzymatic hydrolysis rate of β-glucan. The specific procedure is as follows: Weigh 5.064g of phenol into a beaker and add about 50mL of deionized water. Heat in a 70℃ water bath until the phenol is completely dissolved. After the phenol solution returns to room temperature, transfer it to a 100mL volumetric flask and dilute to 100mL with deionized water to obtain the phenol solution. Take 100 μL of phenol solution and 100 μL of the supernatant obtained after enzymatic hydrolysis and centrifugation into a glass test tube, add 500 μL of concentrated sulfuric acid, mix well, incubate at 30℃ for 30 min, and measure using an ELISA reader on A480. Calculate the total sugar concentration in the supernatant of the enzymatic hydrolysate according to the standard curve. Simultaneously, plot the total sugar content standard curve using glucose as the standard. See [link to standard curve]. Figure 6 .

[0074] According to the standard curve ( The total sugar content in the supernatant was calculated, and the enzymatic hydrolysis rate of dextran was calculated according to Equation 1. The results are shown in Table 1.

[0075] Formula 1 in, C 0: Total sugar concentration in the reaction system before enzymatic hydrolysis, % V 0: Total volume of the reaction system before enzymatic hydrolysis, mL; C1: The concentration of total sugar remaining in the reaction system after enzymatic hydrolysis, % V 1: The total volume of the reaction system after enzymatic hydrolysis, in mL.

[0076] Table 1: Enzymatic hydrolysis rate of dextran in each example and comparative example As shown in Table 1, the β-glucanase recombinant strain constructed in this invention is significantly superior to the comparative strains in terms of enzymatic hydrolysis efficiency against yeast β-glucan, demonstrating the synergistic value of genetically engineered strain construction and fermentation process optimization.

[0077] Specifically, enzymatic hydrolysis using crude enzyme solutions of recombinant Bacillus subtilis WB600-pWB980-g9050 or Bs168-pWB980-g9050 constructed according to this invention resulted in a stable hydrolysis rate of over 63%. Crucially, the process conditions required to achieve this high efficiency are more moderate and flexible. The process parameters in Examples 2 to 5 show that the fermentation time is shorter than that of the wild-type bacteria in Comparative Example 1; the hydrolysis temperature can be efficiently maintained within a wide range of 45℃ to 65℃, and the optimal temperature (55℃ in Example 2) is lower than the 60℃ in Comparative Example 1; the hydrolysis pH environment (5.5–6.5) is closer to neutral, which is milder than the acidic conditions (pH 5.0) used in Comparative Example 1. Therefore, the recombinant engineered strains of this invention not only have a higher upper limit of catalytic efficiency but also reduce dependence on extreme reaction conditions, making the entire process more controllable and industrially adaptable.

[0078] Of particular note is that in Example 5, after using corn steep liquor powder medium instead of LB medium for fermentation, the enzymatic hydrolysis rate was further increased to 69.7%. This reveals that the optimization of the fermentation medium can effectively promote the metabolism and enzyme production of engineered bacteria, thereby releasing their greater catalytic potential, demonstrating the positive impact of process optimization on the final product yield.

[0079] The enzymatic hydrolysis rate of the original Chaetomium JY25 fermentation crude enzyme solution in Comparative Example 1 was only 42.6%. Even after changing to corn steep liquor medium, the improvement in the enzymatic hydrolysis rate was very limited. It can be seen that the recombinant strain modified by genetic engineering has achieved a qualitative leap in enzyme production capacity and catalytic efficiency compared with wild strain.

[0080] In contrast, Comparative Example 3, which directly used commercially available enzyme preparations, had a hydrolysis rate as low as 28.2%, demonstrating the limitations of commercial enzymes in breaking down the complex and dense structure of yeast β-glucan. In comparison, the crude enzyme solution obtained by the present invention based on the fermentation of recombinant engineered bacteria may have an enzyme system composition that is more compatible with the substrate characteristics, thereby achieving more efficient and thorough degradation.

[0081] II. Test of water solubility of β-glucan samples: Sample 1 (see Example 2) of different masses was prepared at 25°C. Figure 7 ) and yeast β-glucan as a substrate (see Figure 8 Add the mixture to water, stir at 100 rpm for 2 minutes, and then let it stand at 25°C for 24 hours. Observe the dissolution effect and the results are shown below. Figure 9 and Figure 10 .

[0082] Depend on Figure 9 and Figure 10 The results show that the sample 1 prepared by the present invention can still completely dissolve at a concentration as high as 100 g / L, forming a homogeneous and stable solution, and no precipitation occurs after standing for 24 hours; while the insoluble yeast β-glucan used as the substrate already shows a large amount of precipitation at a concentration as low as 5 g / L.

[0083] It is evident that the water solubility of β-glucan prepared by the method of this invention is fundamentally improved.

[0084] III. Molecular weight of β-glucan before and after enzymatic hydrolysis: The weight-average molecular weight of each sample and reference standard was determined by gel permeation chromatography. Commercially available yeast β-glucan before enzymatic hydrolysis was used as the water-insoluble glucan sample. The results are shown in Table 2.

[0085] The GPC / SEC spectrum of sample 1 is shown below. Figure 11 The GPC / SEC spectrum of commercially available water-insoluble yeast β-glucan is shown below. Figure 12 .

[0086] Table 2: Weight-average molecular weight of β-glucan before and after enzymatic hydrolysis As can be seen from the results in Table 2, the weight-average molecular weight of samples 1 to 5 prepared by the present invention was precisely controlled within the range of 25 kDa to 28 kDa, which shows that the preparation method of the present invention has high specificity and repeatability.

[0087] It is particularly noteworthy that the commercially available yeast β-glucan used in this invention has an initial molecular weight of approximately 51 kDa, which, while not extremely high, is completely insoluble in water. This phenomenon stems from the unique high-order structure of yeast β-glucan; it is not a simple linear molecule, but rather a backbone composed of β-1,3 glycosidic bonds with numerous β-1,6 glycosidic bond branches, forming a highly complex and dense three-dimensional network structure. More importantly, these molecular chains are tightly packed together through strong hydrogen bonding, forming a stable triple helix conformation. This dense spatial network and strong intermolecular forces, rather than a single molecular weight, are the fundamental reasons for its extremely poor water solubility.

[0088] The recombinant Bacillus subtilis WB600-pWB980-g9050 and Bs168-pWB980-g9050 constructed in this invention, through high expression of the g9050 gene sequence derived from Chaetomium JY25 in Bacillus subtilis WB600 and Bs168, and subsequent targeted reprogramming, exhibit significantly enhanced catalytic effects. Compared to the natural enzyme system of wild fungi, they demonstrate higher overall enzymatic hydrolysis efficiency for yeast β-glucan substrates, achieving a maximum hydrolysis rate of 69.7%. Simultaneously, the depolymerization process of insoluble substrate network structures is highly controllable, enabling stable and precise control of the product molecular weight within a narrow range of 25kDa to 28kDa, resulting in more homogeneous water-soluble β-glucan.

[0089] IV. Structural confirmation of β-glucan samples: Water-soluble β-glucan, represented by sample 1, and water-insoluble β-glucan, represented by commercially available yeast β-glucan as the substrate, were structurally confirmed using infrared spectroscopy. The results are shown in [Figure number missing]. Figure 13 .

[0090] Figure 13 In the middle, at 3375cm -1 2945cm -1 Both exhibit strong absorption peaks, primarily due to the -OH stretching vibration and the CH stretching vibration; these two absorption peaks are characteristic of polysaccharides. (1622 cm⁻¹) -1 The absorption peak is an aldehyde peak, caused by C=O stretching. 1416 cm⁻¹ -1 The absorption peak is due to the CH bending vibration; 1112 cm⁻¹ -1 The absorption peak is a characteristic absorption peak of the pyran ring, caused by the stretching vibration of the COH side group and the COC glycosidic bond, and is also at 1112 cm⁻¹. -1 The absorption peak is also a characteristic absorption peak of glucose. 615 cm⁻¹ -1 The absorption peak indicates the presence of CH in the polysaccharide. (890-900 cm⁻¹) -1 The absorption peak is a characteristic absorption peak of β-glycosidic bonds.

[0091] Therefore, it is evident that the enzymatic hydrolysis of water-soluble yeast β-glucan using the recombinant bacteria constructed in this invention selectively disrupts the higher-order physical structures that cause insolubility while completely preserving its core chemical structure. Specifically, the basic chemical backbone, glucose units, and glycosidic bond configuration (β-type) of β-glucan remain unchanged. Thus, this invention significantly improves the water solubility of the product while maximally maintaining the natural molecular structure and potential bioactivity of β-glucan.

[0092] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A recombinant β-glucanase bacterium, characterized in that, The recombinant plasmid pWB980-g9050 was introduced and expressed in the β-glucanase recombinant bacteria. The recombinant plasmid contains the gene sequence shown in SEQ ID NO.1 of Chaetomium JY25 with accession number CGMCC No. 6882; the full gene sequence of the recombinant plasmid is shown in SEQ ID NO.

2.

2. The recombinant β-glucanase strain according to claim 1, characterized in that, The β-glucanase recombinant bacteria is Bacillus subtilis Bs168 or Bacillus subtilis WB600; the recombinant plasmid contains a secretory signal peptide sequence.

3. The method for constructing recombinant β-glucanase bacteria according to claim 1, characterized in that, The method includes the following steps: Obtain the gene sequence shown in SEQ ID NO.1 from Chaetomium JY25 with accession number CGMCC No. 6882; The gene sequence shown in SEQ ID NO.1 was cloned into an expression vector to construct a recombinant plasmid; The complete genome sequence of the recombinant plasmid is shown in SEQ ID NO.2; The recombinant plasmid was introduced into the host bacteria to obtain the β-glucanase recombinant bacteria.

4. The construction method according to claim 3, characterized in that, The gene sequence shown in SEQ ID NO.1 was obtained by whole-gene synthesis, with a tag sequence for protein purification and cleavage added upstream of its coding sequence during synthesis.

5. The construction method according to claim 4, characterized in that, The tag sequence includes a 6×His tag, a thrombin recognition cleavage site, and an endothelial kinase recognition cleavage site.

6. A method for preparing water-soluble β-glucan, characterized in that, The specific preparation method using the recombinant β-glucanase bacteria as described in any one of claims 1-2 or the recombinant β-glucanase bacteria obtained using the construction method as described in any one of claims 3-5 includes: S1. The β-glucanase recombinant bacteria are subjected to liquid fermentation culture to obtain fermentation broth; S2. The fermentation broth obtained in step S1 is subjected to solid-liquid separation to obtain a crude enzyme solution containing β-glucan degrading enzyme system. S3. The crude enzyme solution and insoluble yeast β-glucan substrate are subjected to enzymatic hydrolysis at pH 5.5-6.5 and temperature 45℃-65℃ to obtain the enzymatic hydrolysis product. S4. Terminate the reaction and separate and purify the enzymatic hydrolysis products to obtain low molecular weight, water-soluble β-glucan with a weight average molecular weight of 25kDa to 28kDa.

7. The preparation method according to claim 6, characterized in that, The liquid fermentation culture described in step S1 uses LB liquid medium or corn steep liquor dry powder medium; the corn steep liquor dry powder medium includes: 20 g / L corn steep liquor dry powder, 10 g / L sodium chloride, natural pH value, and water as the solvent.

8. The preparation method according to claim 6, characterized in that, The conditions for liquid fermentation culture in step S1 are: temperature 26℃~32℃, shaking speed 160r / min~200r / min, and culture time 14h~22h.

9. The preparation method according to claim 6, characterized in that, The solid-liquid separation described in step S2 is centrifugal separation, with centrifugation conditions of 8000 r / min to 12000 r / min for 5 min to 15 min.

10. The preparation method according to claim 6, characterized in that, The enzymatic hydrolysis reaction in step S3 takes 4 to 8 hours; the insoluble yeast β-glucan substrate is added in the form of a suspension with a mass concentration of 1.7% to 2.3%, and the volume ratio of the crude enzyme solution to the suspension is 1:(4 to 9).

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