Xanthan gum hydrolase, xanthan gum as well as preparation method and application of xanthan gum hydrolase and xanthan gum

Abstract

The invention discloses xanthan gum hydrolase, xanthan gum as well as a preparation method and application of the xanthan gum hydrolase. According to the invention, a gene of xanthan gum hydrolase is obtained through codon optimization, then a recombinant plasmid pET-15b is transferred into a host cell BL21 (DE3), and xanthan gum hydrolase with enzyme activity of 18.8 * 10 < 3 > U / g is obtained through expression. The enzyme can perform enzymolysis on xanthan gum with high molecular weight to obtain the xanthan gum with the molecular weight of less than or equal to 100 wDa. The low-molecular-weight xanthan gum product is enriched, and a new thought and technical support are provided for application of the xanthan gum in the high-value-added fields such as biological medicine, functional food and fine chemical engineering.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and xanthan gum production, specifically relating to a xanthan gum hydrolase, xanthan gum, its preparation method, and its application. Background Technology

[0002] Xanthan gum is a high-molecular-weight extracellular polysaccharide produced by the fermentation of *Xanthomonas campestris*. Its backbone consists of D-glucose linked by β-1,4-glycosidic bonds, while its side chains contain D-mannose, D-glucuronic acid, and pyruvate groups. This unique structure endows xanthan gum with excellent thickening, suspending, and stability properties, making it widely used in food, pharmaceuticals, cosmetics, and oil extraction. However, high molecular weight xanthan gum is limited in some applications due to its high viscosity. Therefore, the preparation of medium or low molecular weight xanthan gum products, especially xanthan gum with a molecular weight of approximately 100 wDa, has become an important research direction for expanding its functions and applications.

[0003] Recent studies have found that xanthan gum with a molecular weight of around 100 wDa, while maintaining a certain viscosity, exhibits better solubility, dispersibility, and biocompatibility, making it suitable for applications such as drug delivery systems, tissue engineering scaffolds, and high-end personal care products. Xanthan gum with a molecular weight of 100 wDa not only effectively improves product texture but also serves as a carrier for functional ingredients, enhancing the stability and bioavailability of active substances. Furthermore, this type of xanthan gum is more readily degradable in the environment, aligning with the requirements of green and sustainable development.

[0004] Currently, the main methods for degrading xanthan gum include physical, chemical, and enzymatic methods. Physical methods, such as high temperature, high pressure, or ultrasonic treatment, can reduce the molecular weight of xanthan gum, but they often make it difficult to precisely control the molecular weight distribution of the product and easily lead to random breakage of polysaccharide chains, affecting product uniformity and functional properties. Chemical degradation methods, such as acid hydrolysis or oxidative degradation, can achieve molecular weight control to some extent, but the reaction conditions are harsh, potentially introducing chemical residues, causing a decrease in product purity and environmental pollution. In contrast, enzymatic degradation has attracted much attention due to its mild conditions, high specificity, and good product uniformity. Xanthan gum hydrolysase can efficiently and specifically cleave the β-1-4-glycosidic bonds in the xanthan gum molecular backbone. By controlling the enzymatic hydrolysis conditions, xanthan gum with a target molecular weight (e.g., 100 wDa) can be precisely prepared, and the product has a complete structure, stable physicochemical properties, and is easy to purify and apply subsequently.

[0005] Xanthan gum hydrolases are mainly divided into two categories: one is endo-xanthan gum hydrolases, which can randomly cleave the β-1,4-glycosidic bonds of the xanthan gum backbone, rapidly reducing the viscosity of the solution; the other is exo-xanthan gum hydrolases, which stepwise release monosaccharides or oligosaccharides from the non-reducing ends of the polysaccharide chain. These enzymes are mostly derived from certain bacteria and fungi, such as Bacillus, Pseudomonas, and Aspergillus.

[0006] Therefore, developing xanthan gum hydrolases for producing low molecular weight xanthan gum (e.g., 100 wDa) is of significant research value and has broad market prospects for the application of xanthan gum in high-value-added fields such as biomedicine, functional foods, and fine chemicals. Summary of the Invention

[0007] In order to better develop low molecular weight xanthan gum products and improve the quality and application value of xanthan gum, the present invention provides the following technical solutions.

[0008] In a first aspect, the present invention provides a nucleic acid molecule that encodes xanthan gum hydrolase.

[0009] Preferably, the sequence of the nucleic acid molecule is shown in SEQ ID NO.2.

[0010] The specific sequence of the nucleic acid molecule is as follows:

[0011] In a second aspect, the present invention provides a recombinant plasmid comprising the nucleic acid molecule described in the first aspect.

[0012] Preferably, the recombinant plasmid is selected from any one of the pET plasmid family, such as pET-15b, pET-3ab, pET-11a, pET-28a or pET-19b.

[0013] Furthermore, the recombinant plasmid is pET-15b.

[0014] Thirdly, the present invention provides a host cell comprising the recombinant plasmid described in the second aspect.

[0015] Preferably, the host cells include, but are not limited to, BL21(DE3), Rosetta(DE3), shuffle T7, and Origami B, and more preferably BL21(DE3).

[0016] Fourthly, the present invention provides a method for preparing xanthan gum hydrolase, the method comprising the following steps: 1) The host cells described in the third aspect are seeded into LB medium and cultured with shaking at 30-40°C; 2) Add isopropyl-β-D-thiogalactoside (IPTG) to the culture medium of step 1) to induce the host cells to express xanthan gum hydrolase; 3) Collect the host cells cultured in step 2), break them, separate and purify them to obtain the xanthan gum hydrolase.

[0017] Preferably, the LB medium is formulated as follows: 10 g / L trypsin, 5 g / L yeast extract and 10 g / L sodium chloride.

[0018] Furthermore, the LB medium contains ampicillin (Amp).

[0019] Furthermore, the concentration of ampicillin added is 80-200 μg / mL, for example: 80 μg / mL, 100 μg / mL, 120 μg / mL, 140 μg / mL, 160 μg / mL, 180 μg / mL, 200 μg / mL.

[0020] Preferably, the oscillation speed in step 1) is 180-300 rpm, for example: 180 rpm, 200 rpm, 220 rpm, 240 rpm, 260 rpm, 280 rpm, 300 rpm.

[0021] Preferably, the concentration of IPTG added in step 2) is 0.1-0.5 mM, for example: 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM.

[0022] Preferably, the induction culture time in step 2) is 16-24 h, for example: 16 h, 18 h, 20 h, 22 h, 24 h.

[0023] Preferably, the host cells in step 3) are collected by centrifugation.

[0024] Furthermore, the centrifugal force is 6000-10000×g, for example: 6000×g, 6500×g, 7000×g, 7500×g, 8000×g, 8500×g, 9000×g, 9500×g, 10000×g.

[0025] Furthermore, the centrifugation time is 5-15 min, for example: 5 min, 6 min, 8 min, 10 min, 13 min, 15 min.

[0026] Preferably, the cell disruption method in step 3) includes, but is not limited to, ultrasound, lysozyme disruption, or freeze-thaw.

[0027] Preferably, step 3) further includes the steps of centrifuging to remove cell debris and collecting the supernatant after cell disruption.

[0028] Fifthly, the present invention provides a xanthan gum hydrolase, which is prepared according to the method described in the fourth aspect.

[0029] Preferably, the xanthan gum hydrolase has an enzyme activity ≥18.8×10⁻⁶. 3 U / g.

[0030] Preferably, the xanthan gum hydrolase can hydrolyze xanthan gum with a molecular weight >100 wDa to obtain xanthan gum with a molecular weight ≤100 wDa.

[0031] In a sixth aspect, the present invention provides xanthan gum, which is obtained by enzymatic hydrolysis of xanthan gum as described in the fifth aspect.

[0032] Preferably, the xanthan gum has a molecular weight ≤100 wDa, for example: 100 wDa, 90 wDa, 80 wDa, 70 wDa, 60 wDa, 50 wDa, 40 wDa, 30 wDa, 20 wDa, 10 wDa, and more preferably 100 wDa.

[0033] In a seventh aspect, the present invention provides a method for preparing xanthan gum as described in the sixth aspect, the method comprising the following steps: (1) Prepare xanthan gum solution; (2) Add the xanthan gum hydrolase described in the fifth aspect to the xanthan gum solution, and carry out an enzymatic hydrolysis reaction; (3) Inactivate the enzymatic hydrolysis reactants, determine the molecular weight of the reaction products, and obtain the xanthan gum.

[0034] Preferably, the xanthan gum in the xanthan gum solution in step (1) has a molecular weight > 100 wDa.

[0035] Preferably, the enzymatic hydrolysis reaction temperature in step (2) is 35-40℃, for example: 35℃, 36℃, 37℃, 38℃, 39℃, 40℃.

[0036] Preferably, the concentration of xanthan gum hydrolase used in step (2) is 0.5~5 mg / mL, for example: 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL, 5 mg / mL.

[0037] Preferably, the inactivation temperature in step (3) is ≥95℃, for example: 95℃, 96℃, 97℃, 98℃, 99℃, 100℃.

[0038] Preferably, the inactivation time in step (3) is 5-15 min, for example: 5 min, 7 min, 10 min, 12 min, 15 min.

[0039] Eighthly, the present invention provides the use of the nucleic acid molecule described in the first aspect, the recombinant plasmid described in the second aspect, or the host cell described in the third aspect in the preparation of xanthan gum.

[0040] Preferably, the xanthan gum has a molecular weight ≤100 wDa, for example: 100 wDa, 90 wDa, 80 wDa, 70 wDa, 60 wDa, 50 wDa, 40 wDa, 30 wDa, 20 wDa, 10 wDa, and more preferably 100 wDa.

[0041] The beneficial effects of this invention are: This invention obtains the gene for xanthan gum hydrolase through codon optimization, and then transforms it into host cells BL21(DE3) using the recombinant plasmid pET-15b, resulting in an enzyme activity of 18.8 × 10⁻⁶. 3This invention provides a xanthan gum hydrolase at a concentration of U / g. This enzyme can hydrolyze high molecular weight xanthan gum to obtain xanthan gum with a molecular weight ≤100 wDa. This invention enriches the product range of low molecular weight xanthan gum and provides new ideas and technical support for the application of xanthan gum in high-value-added fields such as biomedicine, functional foods, and fine chemicals. Attached Figure Description

[0042] Figure 1 The image shown is an SDS-PAGE gel image of xanthan gum hydrolase. 1 is the marker; 2 is the bacterial precipitate; 3 is the supernatant; 4 is the flow-through buffer; 5 is the 25 mM imidazole elution buffer; 6 is the enzyme solution. Figure 2 The image shows a TLC diagram of xanthan gum degradation by xanthan gum hydrolase. 1 is the enzymatic hydrolysis solution; 2 is the control. Figure 3 The results of the optimal temperature determination for xanthan gum hydrolase are shown below. Figure 4 The results show the optimal pH determination of xanthan gum hydrolase. Detailed Implementation

[0043] The technical solution of the present invention will be further described below with reference to embodiments and accompanying drawings. The advantages and features of the present invention will become clearer as the description unfolds. However, it should be understood that the embodiments are merely exemplary and do not constitute a limitation on the scope of the present invention.

[0044] It should be noted that, unless otherwise specified, the experimental methods used in the following embodiments are conventional methods in the art. Unless otherwise defined, all scientific and technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art.

[0045] Example 1: Preparation of xanthan gum hydrolase 1.1 Obtaining the target gene 1.1.1 Design the protein sequence of xanthan gum hydrolase. The specific amino acid sequence is as follows: MEEAAADAQNAEINYNRSVPLEVKGNKIVKQGTDEMVVLRGVNVPSMDWGMAEHLFESMTMVYDSWGANLIRLPINPKYWKNGSVWDEKNLTKEQYQKYIDDMVKAAQARGKYIILDCHRYVMPQQDDLDMWKELAVKYGNNSAVLFGLLNEPHDIKPVGVEKPTTVEQWDVWYNGGQIIVGGEEVTAIGHQQLLNEIRKQGANNICIAGGLNWAFDISGFADGYNERPNGYRLIDTAEGHGVMYDSHAYPVKGAKTAWDTIIGPVRRVAPVIIGEWGWDSSDKNISGGDCTSDIWMNQIMNWMDDTDNQYDGIPVNWTAWNLHMSSSPKMLYSWDYKTTAYNGTHIKNRLLSYNTAPEKLDGVYSTDFSTDDVFRSYTAPSGKASIKYSDESGNVAITPAAANWYATLNFPFDWDLNGIQTITMDISAATAGSVNIGLYGSDMEVWTKAVDVNTEVQTVTIGINELVKQGNPQTDGKLDAALSGIYFGAATADTGSITIDNVKIVKLATPVYTANTYPHKDMGEESYIDIDTTGFKKQTTAWNSKFTGTTMQITDANVLNINGETTKTKCVTYTRDATDTEGCRAKFDLNTVPSMDAKYFTIDIKGNGIAQKLTVSLSGLAYITVNMAEGDTDWHQYIYSLEGNVEYPEDITYVQISADTRTTAEFYIDNIGFSNTKSERLIPYPEKTFVYDFATYNKNTTKYEAAISTESGSEGDTIVATKEEGGLGFDSKALEVKYSRNGNTPSKAKVVYSPNDFFKGNVNDDERTANRATLKADMEYMTDFVFYGKSTSGKNEKINVGVIDTASAMTTYTDTKEFTLTTEWKQFRVPFDEFKILDGGSNLDCARVRGFIFSSAENSGEGSFMIDNITHTSIKGDIEWGLPGGGGSFEEQKLISEEDL (SEQ ID NO.1).

[0046] 1.1.2 Based on the amino acid sequence designed in 1.1.1, obtain the sequence of the target gene, and then synthesize its sequence (SEQ ID NO.2).

[0047]

[0048] 1.2 Preparation and transformation of recombinant plasmids NcoI and BamHI were selected as cloning sites. The xanthan gum hydrolase gene synthesized in 1.1 was cloned into the vector pET-15b. The sequencing was correct, and the recombinant plasmid was successfully constructed. It was then transformed into Escherichia coli BL21 (DE3).

[0049] 1.3 Expression and purification of xanthan gum hydrolase protein 1.3.1 Add 50 μL of 100 μg / mL ampicillin (Amp) to 50 mL of LB medium (10 g / L trypsin, 5 g / L yeast extract, and 10 g / L sodium chloride), and then inoculate with the BL21 (DE3) bacterial suspension obtained in 1.2. Incubate overnight with vigorous shaking at 37°C and 220 rpm.

[0050] 1.3.2 The overnight culture was inoculated into 1 L of LB medium containing 100 μg / mL Amp and cultured at 37°C and 220 rpm until OD was reached. 600 When the pH value reaches 0.6-0.8, IPTG (0.4 mM) is added to induce the expression of xanthan gum hydrolase. The culture is then incubated at 16℃ and 180 rpm for 16-20 hours. The culture is then centrifuged at 4℃ and 8000 ×g for 10 min to collect the bacterial cells.

[0051] 1.3.3 The bacterial cells collected in 1.3.2 were sonicated and centrifuged at 4℃, 24000×g for 30 min to remove cell debris. The supernatant was collected and purified using a Ni column, followed by SDS-PAGE analysis. Figure 1 As shown, the molecular weight of xanthan gum hydrolase protein is 100 kDa.

[0052] 1.3.4 The concentration of the purified protein was determined on a nanodrop. Based on the volume of the purified enzyme, the amount of enzyme expressed in the E. coli shake flask was calculated to be 63 mg / L.

[0053] Example 2: Detection of the degradation activity of xanthan gum hydrolase A 5 mg / mL xanthan gum solution (molecular weight 388 wDa, purchased from Fufeng Biotechnology) was prepared. No metal ions or buffer solutions were added, and pH adjustment was unnecessary. 1 mg / mL of the xanthan gum hydrolase solution prepared in Example 1 was added directly. Thin-layer chromatography (TLC) was used to characterize the reaction progress. It was found that xanthan gum pentasaccharides and decasaccharides were generated after 3 minutes of reaction (see...). Figure 2 ).

[0054] Example 3: Determination of the enzymatic properties of xanthan gum hydrolase 3.1 Determination of the optimal temperature for xanthan gum hydrolase Prepare a 5 mg / mL xanthan gum solution without pH adjustment. Mix 500 μL of the prepared solution with a 1 mg / mL xanthan gum hydrolase solution and react for 10 min in a metal bath at 31, 34, 37, 40, 43, 46, 49, 52, 55, and 58 °C. After the reaction, inactivate the enzyme solution at high temperature and centrifuge. Dilute the reaction solution to 2 mg / mL and set up a blank control (mix the pre-inactivated enzyme solution with 500 μL of xanthan gum solution and react for 10 min in a metal bath at 31, 34, 37, 40, 43, 46, 49, 52, 55, and 58 °C, centrifuge, inactivate at high temperature, and centrifuge). Set up three parallel experiments for each group. Mix the reaction solution and DNS at a ratio of 1:2, boil in boiling water at 100 °C for 5 min, and immediately cool. Measure the absorbance (OD) at 540 nm and plot the OD results.

[0055] like Figure 3 As shown, the optimal temperature for xanthan gum hydrolase is 49℃.

[0056] 3.2 Determination of the optimal pH for xanthan gum hydrolase Prepare buffer solutions with different pH values: 50 mM sodium acetate solution at pH 4.5, 5, 5.5, 6, and 6.5; 1×PBS solution at pH 6, 6.5, 7, and 7.5; and 50 mM Tris-HCl at pH 7, 7.5, 8, 8.5, and 9. Weigh xanthan gum and dissolve it in the above buffer solutions to a concentration of 5 mg / mL. Mix 500 μL of the prepared solution with 1 mg / mL xanthan gum hydrolase solution and react in a metal bath at 49°C for 10 min. After the reaction, inactivate the enzyme solution by high temperature and centrifuge. Dilute the reaction solution to 2 mg / mL and set up a blank control (mix the pre-inactivated enzyme solution with 500 μL of xanthan gum solution at different pH values, react in a metal bath at 49°C for 10 min, centrifuge, and then inactivate by high temperature and centrifuge). Set up three parallel experiments for each group. Mix the reaction solution and DNS at a ratio of 1:2 until homogeneous, boil in 100℃ boiling water for 5 min, and immediately cool. Measure the absorbance (OD) at 540 nm and plot the optimal pH curve based on the OD results.

[0057] like Figure 4 As shown, the optimal pH for xanthan gum hydrolase is 6.

[0058] 3.3 Determination of xanthan gum hydrolase activity under optimal conditions 3.3.1 Prepare a 5 mg / mL xanthan gum solution without pH adjustment. Mix 500 μL of the prepared solution with a 1 mg / mL xanthan gum hydrolase solution and react in a metal bath at 37°C for 10 min. After the reaction, inactivate the enzyme at high temperature and centrifuge. Dilute the reaction solution to 2 mg / mL and set up a blank control (mix the pre-inactivated enzyme solution with 500 μL of xanthan gum solution, react in a metal bath at 37°C for 10 min, centrifuge, inactivate at high temperature, and centrifuge). Set up 3 parallel experiments for each group. Mix the reaction solution and DNS at a ratio of 1:2, boil in boiling water at 100°C for 5 min, and immediately cool. Measure the absorbance (OD) at 540 nm using a microplate reader.

[0059] 3.3.2 Calculate the absorbance difference y between the average value of the control group and the average value of the experimental group. Substitute y into the OD540 standard curve equation y=2.5533x-0.0948 to obtain the glucose concentration corresponding to the standard curve. Then calculate the xanthan gum hydrolase activity according to the following formula.

[0060] Enzyme activity units (U / g) = (r × 0.3 × K × 10⁻⁶) 6 ×60) / (T×m) In the formula: r is the glucose concentration (mg / mL), K is the dilution factor, T is the enzymatic hydrolysis time (min), and m is the amount of enzyme added (mg).

[0061] The calculation results show that the activity of xanthan gum hydrolase is 18.8 × 10⁻⁶. 3 The activity of Xanthanase is approximately 1000 times higher than that of Mixen, which has an enzyme activity of 15.70 ± 0.98 U / g. Mixen is cited in the literature (F Yang. et al. Novel Endotype Xanthanase from Xanthan-Degrading Microbacterium sp. Strain XT11. Applied and Environmental Microbiology. November 9, 2018:1-47).

[0062] Example 4 Preparation of low molecular weight xanthan gum The specific process is as follows: (1) Preparation of xanthan gum solution: Add 30 mL of distilled water to 150 mg of xanthan gum (molecular weight 388 wDa, purchased from Fufeng Biotechnology) and place it in a 50 mL centrifuge tube.

[0063] (2) Enzymatic hydrolysis: The prepared solution in step (1) is placed in a water bath at 37°C for incubation. Xanthan gum hydrolase with a concentration of 1 mg / mL is added to the solution, mixed evenly, and the reaction is carried out at 37°C. Samples are taken at regular intervals.

[0064] (3) Inactivation: Keep the enzymatic reaction solution in step (2) at 100℃ for 5-10 min, then centrifuge at 20℃ and 1000 rpm for 15 min, collect the supernatant, and obtain the reaction product.

[0065] (4) Determine the molecular weight of the reaction product: Dilute the concentration of the reaction solution to 2 mg / mL and determine the molecular weight of the product in a multi-angle laser scattering instrument.

[0066] Table 1. Molecular weight of the reaction products

[0067] As shown in Table 1, xanthan gum hydrolase can degrade high molecular weight xanthan gum (388 w Da) into xanthan gum with a molecular weight of 100 w Da, and the homogeneity of the 100 w Da xanthan gum is best when the reaction proceeds for 3 min.

[0068] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes xanthan gum hydrolase, and the sequence of the nucleic acid molecule is shown in SEQ ID NO.

2.

2. A recombinant plasmid, characterized in that, The recombinant plasmid comprises the nucleic acid molecule of claim 1.

3. A host cell, characterized in that, The host cell contains the recombinant plasmid as described in claim 2.

4. A xanthan gum hydrolase, characterized in that, The preparation method of the xanthan gum hydrolase includes the following steps: 1) The host cells described in claim 3 are seeded into LB medium and cultured with shaking at 30-40°C; 2) Add isopropyl-β-D-thiogalactoside to the culture medium of step 1) to induce the host cells to express xanthan gum hydrolase; 3) Collect the host cells cultured in step 2), break them, separate and purify them to obtain the xanthan gum hydrolase.

5. A type of xanthan gum, characterized in that, The xanthan gum is obtained by enzymatic hydrolysis of the xanthan gum hydrolysate according to claim 4.

6. The xanthan gum according to claim 5, characterized in that, The xanthan gum has a molecular weight ≤100 wDa.

7. The method for preparing xanthan gum according to any one of claims 5-6, characterized in that, The preparation method includes the following steps: (1) Prepare xanthan gum solution; (2) Add the xanthan gum hydrolase of claim 4 to the xanthan gum solution and carry out an enzymatic hydrolysis reaction; (3) Inactivate the enzymatic hydrolysis reactants, determine the molecular weight of the reaction products, and obtain the xanthan gum.

8. The preparation method according to claim 7, characterized in that, The xanthan gum solution in step (1) has a xanthan gum molecular weight > 100 wDa; and / or The enzymatic hydrolysis reaction temperature in step (2) is 35-40℃, and the concentration of the xanthan gum hydrolase used is 0.5~5 mg / mL; and / or The inactivation temperature in step (3) is ≥95℃ and the inactivation time is 5-15 min.

9. The use of the nucleic acid molecule of claim 1, the recombinant plasmid of claim 2, or the host cell of claim 3 in the preparation of xanthan gum.

10. The application according to claim 9, characterized in that, The xanthan gum has a molecular weight ≤100 wDa.

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