Polysaccharide coenzyme GumC mutant for controlling xanthan gum chain length and application of polysaccharide coenzyme GumC mutant

Abstract

The invention belongs to the technical field of enzyme engineering, and particularly relates to a polysaccharide coenzyme GumC mutant for controlling the chain length of xanthan gum and application of the polysaccharide coenzyme GumC mutant. The mutant is obtained by mutating at least one corresponding site in a highly conserved domain, a transmembrane domain, a connecting region and a periplasmic domain in polysaccharide coenzyme GumC. The polysaccharide coenzyme GumC mutant capable of remarkably reducing the chain length of the xanthan gum, disclosed by the invention, can be applied to preparation of low molecular weight xanthan gum (Low Molecular Weight Xanthan Gum, LXG).

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering, and in particular relates to a polysaccharide copolymerase GumC mutant for controlling the chain length of xanthan gum and application thereof. Background Art

[0002] Xanthan gum is a functional extracellular polysaccharide isolated from the fermentation broth of Xanthomonas campestris. Its structural characteristics are that it is a polysaccharide polymer compound composed of D-glucose, D-mannose and D-glucuronic acid in a ratio of 2:2:1. It is composed of repeated pentasaccharide units. The main chain is composed of glucose connected by β-1,4 glycosidic bonds. The side chain trisaccharides are connected to the main chain glucose units through α-(1,3)-glycosidic bonds. The side chain trisaccharides are composed of β-mannose-(1,4)β-glucuronic acid-(1,2)α-mannose. Figure 1 As shown;

[0003] Xanthan gum, due to its unique physical and chemical properties, is widely used in a variety of fields, including food, cosmetics, industry, and medicine. Its primary functions include thickening, stabilization, emulsification, suspension, moisture retention, rheology modification, bioactivity, salt tolerance, and high-temperature resistance. These properties make it a versatile additive that can meet diverse application needs. However, the xanthan gum market is currently highly competitive, with significant price fluctuations. Furthermore, due to insufficient technological innovation, the development of alternatives such as guar gum and carrageenan could weaken the market appeal of xanthan gum, significantly impacting the xanthan gum industry.

[0004] In some applications, the high viscosity of xanthan gum can lead to processing difficulties or incompatibility with product performance, so its viscosity needs to be reduced. There is generally a positive correlation between the molecular weight and viscosity of xanthan gum, meaning that the larger the molecular weight, the higher the viscosity. Therefore, by controlling the molecular chain length of xanthan gum, its viscosity can be effectively reduced, resulting in low-viscosity xanthan gum, and thus forming low molecular weight xanthan gum (LXG).

[0005] Low-molecular-weight xanthan gum is obtained by degrading high-molecular-weight xanthan gum. Its molecular weight typically ranges from 1,000 to 10,000 Da, far lower than that of traditional xanthan gum (which typically has a molecular weight in the millions of Da). Low-molecular-weight xanthan gum has stable physical and chemical properties and, compared to high-molecular-weight xanthan gum, offers advantages such as improved solubility, lower viscosity, and higher bioavailability. Furthermore, low-molecular-weight xanthan gum exhibits excellent biological activities, such as antibacterial, antioxidant, and antitumor activity. It can also induce plant disease resistance, regulate plant growth, and promote the growth of beneficial intestinal microorganisms. These properties give low-molecular-weight xanthan gum broad application prospects in various fields.

[0006] Currently, there are three main methods for obtaining low-molecular-weight xanthan gum: physical, chemical, and biological. Physical methods (such as high temperature, radiation, ultrasound, and mechanical force) are costly, have complex reaction conditions that require optimization, and may be accompanied by branching and cross-linking reactions. Chemical methods (such as acids, bases, and oxidants), while effective, are violent and uncontrollable, and the degradation process may lead to the formation of monosaccharides, accompanied by changes in the skeleton structure and the production of by-products. Biological methods (enzymatic hydrolysis), while mild and environmentally friendly, are limited in the types of enzymes currently available for xanthan gum degradation, and the molecular weight and distribution of the products during the degradation process are uneven.

[0007] Therefore, it is necessary to conduct in-depth research on the xanthan gum production process to control the molecular weight of the final product and obtain the ideal low molecular weight xanthan gum. Summary of the Invention

[0008] In view of the limitations of the existing technology for obtaining low molecular weight xanthan gum, the purpose of the present invention is to provide a polysaccharide copolymerase GumC mutant for controlling the chain length of xanthan gum and its application.

[0009] To achieve the above purpose, the present invention adopts the following technical solutions:

[0010] A polysaccharide copolymerase GumC mutant for controlling the chain length of xanthan gum is obtained by mutation of at least one corresponding site in a highly conserved region, a transmembrane domain, a connecting region and a periplasmic domain in the polysaccharide copolymerase GumC.

[0011] The mutant is a mutation of one or more amino acid sites among positions 406, 407, 419 and 423 in the polysaccharide copolymerase GumC of Xanthomonas campestris.

[0012] The corresponding amino acids at positions 406 (Y) and 407 (D) can be the same or different and mutated to S, A or G respectively; the amino acid at position 419 (A) can be mutated to G or S; and the amino acid at position 423 (G) can be mutated to A or S.

[0013] The mutant is one or a combination of Y406S, Y406A, Y406G, D407A, A419G, A419S, G423A, and G423S in the amino acid sequence shown in SEQ ID NO.1.

[0014] 5 The mutants are Y406S, Y406A, Y406G, D407A, A419G, A419S, G423A, G423S, Y406A and D407A, Y406S and D407A, Y406G and D407A, A419G and G423A, A419G and G423S, A419S and G423A or A419S and G423S in the amino acid sequence shown in SEQ ID NO.1.

[0015] An application of the mutant, namely, application of the mutant in producing low-viscosity xanthan gum.

[0016] An application of the mutant, namely, application of the mutant in producing low molecular weight xanthan gum.

[0017] A method for producing low-viscosity, low-molecular-weight xanthan gum comprises the steps of: utilizing the mutant according to claim 1 to obtain a plasmid containing the mutant, transfecting the mutant into a host for fermentation and culturing; and centrifuging the fermentation broth to collect the supernatant, thereby obtaining low-viscosity, low-molecular-weight xanthan gum.

[0018] Specifically, the mutant was transfected into the plasmid pBBR1MCS-2-T3 to obtain a recombinant plasmid, and the obtained recombinant plasmid was then introduced into Xanthomonas campestris to obtain a strain containing the mutant, and the obtained strain was activated in NYGB solid medium containing antibiotics to an OD of 600 =0.6, and then inoculated into NYGB solid culture medium containing antibiotics at an inoculum size of 1%-10% and cultured with shaking for 24-72 hours. The culture solution was centrifuged to collect the supernatant, purified, and allowed to stand to obtain low-viscosity, low-molecular-weight xanthan gum.

[0019] Beneficial effects of the present invention:

[0020] The present invention discloses a polysaccharide copolymerase GumC mutant capable of effectively controlling xanthan gum chain length. The mutated enzyme is capable of producing low-molecular-weight xanthan gum. The present invention discloses a polysaccharide copolymerase that significantly controls xanthan gum chain length after mutation. This enzyme can control xanthan gum chain length, enabling the production of low-molecular-weight xanthan gum in Xanthomonas campestris, laying a solid foundation for the industrial application of low-molecular-weight xanthan gum. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the structure of xanthan gum.

[0022] Figure 2 This is a signal peptide analysis diagram of GumC provided in an embodiment of the present invention.

[0023] Figure 3 A predicted diagram of the transmembrane domain of GumC provided in an embodiment of the present invention.

[0024] Figure 4 This is a predicted secondary structure diagram of GumC provided by an embodiment of the present invention.

[0025] Figure 5 The tertiary structure prediction diagram (AlphaFold2) of GumC provided in an embodiment of the present invention; PLDDT=88.1.

[0026] Figure 6This is a diagram showing the relative viscosity of low molecular weight xanthan gum obtained using different mutant strains provided in the embodiments of the present invention.

[0027] Figure 7 The gel permeation chromatography results of low molecular weight xanthan gum products of different mutant strains provided in the examples of the present invention. DETAILED DESCRIPTION

[0028] The following non-limiting embodiments may enable those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way.

[0029] The present invention performs site-directed mutation of one or more amino acids in the amino acid sequence of a wild-type polysaccharide copolymerase GumC through enzyme engineering to obtain a polysaccharide copolymerase GumC mutant that significantly reduces the xanthan gum chain length. The polysaccharide copolymerase GumC mutant is used to significantly reduce the xanthan gum chain length, accurately control the molecular weight of the xanthan gum, and thus obtain low-molecular-weight xanthan gum, thereby avoiding the problems of molecular weight heterogeneity or instability in traditional methods and having high industrial application prospects.

[0030] Example 1: Construction of polysaccharide copolymerase GumC mutant plasmid

[0031] 1. Construction of recombinant plasmid pBBR1MCS-2-T3-GumC

[0032] a. Based on the nucleotide sequences of polysaccharide copolymerase GumC and vector plasmid pBBR1MCS-2-T3, primers were designed upstream and downstream, respectively; the specific primer information is shown in Table 1.

[0033] The polysaccharide copolymerase GumC is the amino acid sequence of the wild-type polysaccharide copolymerase GumC of Xanthomonas campestris pv. campestris (Xcc). The gene sequence of the polysaccharide copolymerase GumC is derived from the literature (Stingele, F., Neeser, JR, & Mollet, B. (1996). Identification and characterization of the eps (Exopolysaccharide) gene cluster from Streptococcus thermophilus Sfi6. Journal of bacteriology, 178 (6), 1680-1690). https: / / doi.org / 10.1128 / jb.178.6.1680-1690.1996). Furthermore, the wild-type polysaccharide copolymerase GumC protein sequence obtained from NCBI is SEQ ID NO. 1, as follows:

[0034] MARLLPMSMNSDNRSSSSQRHGHLELADVGLMDYWRALVSQLWLIILIAVGALLLAFGITMLMPEKYRATSTLQIERDSLNVVNVDNLMPVESPQDRDFYQTQYQLLQSRSLARAVIREAK LDQEPAFKEQVDEALAKAAEKNPEAGKSLDSRQAIVERSLTDTLLAGLVVEPILNSRLVYVNYDSPDPVLAAKIANTYPKVFIVSTQERRMKASSFATQFLAERLKQLREKVEDSEKDLV SYSTEEQIVSVGDDKPSLPAQNLTDLNALLASAQDSRIKAESAWRQASSGDGMSLPQVLSSPLIQSLRSEQVRLTSEYQQKLSTFKPDYPEMQRLKAQIEESRRQINGEVINIRQSLKAT YDASVHQEQLLNDRIAGLRTNELDLQSRSIRYNMLKRDVDTNRQLYDALLQRYKEIGVASNVGANNVTIVDTADVPTSKTSPKLKLNLALGLIFGVFLGVAVALVRYFLRGPAPESRLN*

[0035] Table 1 Primers required for constructing recombinant plasmids and their specific information

[0036]

[0037] That is, as can be seen above, the primer pair GumC-F and GumC-R are used to amplify the GumC fragment.

[0038] Primer pair MCS2-F and MCS2-R was used to amplify the pBBR1MCS-2-T3 vector fragment.

[0039] b. The recombinant plasmid pBBR1MCS-2-T3-GumC was constructed by seamless DNA cloning.

[0040] (1) Seamless cloning fragment amplification:

[0041] ① The GumC fragment amplification reaction system is: 2×SuperNova PCR Mix (Dye) (purchased from GenStar) 25 μl, forward primer 2.5 μl, reverse primer 2.5 μl, template DNA (Xcc 8004 whole genome) 200 ng, and Sterile Water to 50 μl.

[0042] The PCR reaction procedure for fragment amplification was as follows: initial denaturation at 98°C for 5 minutes, followed by denaturation at 98°C for 15 seconds, annealing for 15 seconds at 57.5°C (extension time shown in Table 1), 35 cycles, and a final incubation at 72°C for 10 minutes. Amplified fragment bands were recovered.

[0043] ②The pBBR1MCS-2-T3 vector fragment amplification reaction system is: 2×SuperNova PCR Mix (Dye) (purchased from GenStar) 25μl, forward primer 2.5μl, reverse primer 2.5μl, template DNA plasmid (pBBR1MCS-2-T3) 200ng, Sterile Water to 50μl.

[0044] The PCR reaction procedure for fragment amplification was as follows: initial denaturation at 98°C for 5 minutes, followed by denaturation at 98°C for 15 seconds, annealing for 15 seconds at 57.5°C (extension time shown in Table 1), 35 cycles, and a final incubation at 72°C for 10 minutes. Amplified fragment bands were recovered.

[0045] (2) Seamless cloning plasmid construction: Recover the fragment samples from the gels obtained in steps ① and ② and measure their concentrations using a NanoVue Plus ultramicro-spectrophotometer. Calculate the volume of different fragment samples added to the seamless reaction system according to the following formula.

[0046] Formula: V = fragment length × 0.02 / C

[0047] V: volume (unit: μl)

[0048] C: concentration (unit: ng / μl)

[0049] Assume that according to the above formula, the volume of the GumC fragment sample added to the reaction system is X μl, and the volume of the pBBR1MCS-2-T3 vector fragment sample added to the reaction system is Y μl.

[0050] Seamless ligation system: 5 μl 2×CE Mix V3 (purchased from Vazyme), X μl GumC fragment sample, Y μl pBBR1MCS-2-T3 vector fragment sample, and sterile water to make up to 10 μl. Mix well and place in a 50°C water bath for 30 min.

[0051] c. Introduce the recombinant plasmid into E. coli

[0052] (1) The above seamless connection system was added to the heat-transformed Escherichia coli DH5α competent cells, and the mixture was gently tapped with a pipette. The cells were placed on ice for 30 min, heat-shocked in a 42°C water bath for 60 s, and quickly transferred to an ice bath for 2 min. Then, 1 ml of LB (mass concentration in water: 1% NaCl, 1% peptone, 0.5% yeast extract) liquid culture medium was added, and the cells were cultured in a shaker at 37°C and 200 rpm for 1 h. After the culture was completed, the cells were centrifuged at 4000 rpm for 2 min, 900 μl of supernatant was aspirated, and the bacterial pellet was resuspended with the remaining supernatant. All the supernatant was aspirated and spread on the surface of LB solid culture medium containing Apra resistance at a final concentration of 50 μg / mL, and cultured in a 37°C incubator for 12 h.

[0053] (2) A single colony grown on the solid culture medium in step (1) was inoculated into 10 mL of LB liquid culture medium containing Apra resistance at a final concentration of 50 μg / mL, and cultured in a shaking incubator at 37° C. and 200 rpm for 12 h.

[0054] Plasmid extraction: ① Centrifuge 8 mL of overnight culture at 8,000 g for 2 min at room temperature and discard the supernatant; ② Add 250 μL of Buffer P1 (from the Plasmid Extraction Kit of Sangon Biotech (Shanghai) Co., Ltd., B518191, 100 times) to the pellet to resuspend the bacteria; ③ Add 250 μL of Buffer P2 (from the Plasmid Extraction Kit of Sangon Biotech (Shanghai) Co., Ltd., 100 times), immediately mix by gently inverting the centrifuge tube, and let it stand at room temperature for 2 min to completely lyse the cells; ④ Add 350 μL of Buffer P3 (Plasmid Extraction Kit from Sangon Biotech (Shanghai) Co., Ltd., B518191, 100 times), gently invert up and down to mix, the liquid becomes white flocculent, 12,000g, 10min centrifugation; ⑤ Use a pipette to aspirate the supernatant into a plasmid extraction column (Plasmid Extraction Kit from Sangon Biotech (Shanghai) Co., Ltd., B518191, 100 times), 8,000g, centrifugation for 30s, and discard the collected liquid; ⑥ Add 500μL Wash buffer (Plasmid Extraction Kit from Sangon Biotech (Shanghai) Co., Ltd., B518191, 100 times) to the plasmid extraction column, 9,000g, centrifugation for 30s, and discard the collected liquid; ⑦ Add 500μL Wash buffer to the plasmid extraction column Buffer, 9,000g, centrifuge for 30s, and discard the collected liquid; ⑧ Transfer the plasmid extraction column from step ⑦ above to a clean 1.5mL centrifuge tube, centrifuge at 9,000g for 1min; ⑨ Open the lid of the centrifuge tube at room temperature and let it stand for 10min to allow the ethanol on the adsorption column to fully evaporate; ⑩ Evenly add 80μL Elution Buffer (from the plasmid extraction kit of Sangon Biotech (Shanghai) Co., Ltd., B518191, 100 times) on the adsorption column membrane, incubate at 55℃ for 5min, centrifuge at 9,000g for 1min, collect the liquid plasmid, and perform double enzyme digestion with AscⅠ and XhoⅠ on the plasmid and identify it by 1.0% agarose gel electrophoresis. The recombinant plasmid pBBR1MCS-2-T3-GumC identified as correct by double enzyme digestion was entrusted to Jilin Kumei Company for sequencing verification, and the verified recombinant plasmid pBBR1MCS-2-T3-GumC was preserved.

[0055] 2. Construction of polysaccharide copolymerase GumC mutant plasmid

[0056] a. The present invention uses structure prediction, homology comparison and rational analysis to obtain the mutation site of polysaccharide copolymerase that controls the xanthan gum chain length. The secondary and tertiary structure prediction diagrams of GumC are shown in FIG. Figures 2 to 5As shown. Downstream primers were designed based on the position of the mutation site in the polysaccharide copolymerase GumC amino acid sequence and the corresponding nucleotide sequence (see the table below). Using the pBBR1MCS-2-T3-GumC recombinant plasmid as a template, upstream universal primers for the mutation site were set upstream of the polysaccharide copolymerase GumC nucleotide sequence.

[0057]

[0058] b. Construct the mutant plasmid by RF cloning.

[0059] The PCR reaction system for RFⅠ was: 1 μL of PrimeSTAR (purchased from Takara), 4 μL of the forward primers with different mutations described above, 4 μL of the reverse primers with different mutations described above, 100 ng of template DNA plasmid (pBBR1MCS-2-T3-GumC), 8 μL of dNTPs, 20 μL of 5× Primer STAR Buffer, and 60 μL of sterile water.

[0060] The PCR reaction procedure for RFⅠ was as follows: initial denaturation at 98°C for 3 minutes, followed by denaturation at 98°C for 30 seconds, annealing for 30 seconds (annealing temperature see Table 2), extension at 72°C (extension time see Table 2), 35 cycles, and a final incubation at 72°C for 10 minutes. Amplified fragments were recovered.

[0061] The PCR reaction system for RFⅡ was: 0.25 μL PrimeSTAR (purchased from Takara), 100 ng of RFⅠ recovered product, 100 ng of template DNA plasmid (pBBR1MCS-2-T3-GumC), 2 μL dNTP, 5 μL 5× Primer STAR Buffer, and 16 μL sterile water.

[0062] The PCR reaction program for RFⅡ was as follows: denaturation at 98°C for 10 s, annealing for 15 s (annealing temperature see Table 1), extension at 72°C (extension time see Table 1), 30 cycles, and finally incubation at 16°C for 10 min.

[0063] The result was a plasmid containing different mutation sites (i.e., pBBR1MCS-2-T3-GumC Y406S 、pB BR1MCS-2-T3-GumC Y406A 、pBBR1MCS-2-T3-GumC Y406G 、pBBR1MCS-2-T3-GumC D407A 、pBBR1MCS-2-T3-GumC Y406A / D407A 、pBBR1MCS-2-T3-GumC Y406S / D407A 、pBBR1MCS-2-T3-GumC Y406G / D407A 、pBBR1MCS-2-T3-GumC A419G 、pBBR1MCS-2-T3-GumC A419S 、pBBR1MCS-2-T3-GumC G423A 、pBBR1MCS-2-T3-GumC G423S 、pBBR1MCS-2-T3-GumC A419G / G423A 、pBBR1MCS-2-T3-Gum C A419G / G423S 、pBBR1MCS-2-T3-GumC A419S / G423A 、pBBR1MCS-2-T3-GumC A419S / G423S ), and entrusted the obtained plasmid to Jilin Kumei Company for sequencing verification, and saved the verified correct mutant plasmid.

[0064] Example 2: Polysaccharide copolymerase GumC mutant plasmid transfection and low-viscosity xanthan gum extraction

[0065] 1. Transfection of polysaccharide copolymerase GumC mutant plasmid

[0066] a. Preparation of competent cells for electroporation transformation of Xanthomonas campestris:

[0067] (1) Streak a plate of Xanthomonas campestris on a solid medium containing NYGB (mass concentration in water: 2% glycerol, 0.3% yeast extract, 0.5% tryptone, 1.5% Agar) containing a final concentration of 25 μg / mL Rif resistance and culture in a 30°C incubator for 36 h.

[0068] (2) A single colony was picked from the plate and inoculated into 10 mL of NYGB liquid medium (mass concentration in water: 2% glycerol, 0.3% yeast extract powder, 0.5% tryptone) containing a final concentration of 25 μg / mL Rif resistance, and cultured at 30°C, 200 rpm in a shaking incubator until the OD 600 =0.6.

[0069] (3) The seed solution was expanded to 50 mL NYGB liquid medium containing a final concentration of 25 μg / mL Rif resistance at a volume ratio of 1:50, and cultured in a shaking incubator at 30°C and 200 rpm until the OD 600 =0.6.

[0070] (4) Pre-cool the bacterial solution on ice for 30 minutes.

[0071] (5) Place the bacterial suspension in a 250 mL centrifuge cup and centrifuge at 1200 g for 3 min at 4°C using a Xiangyi GL21-M centrifuge.

[0072] (6) Discard the supernatant and resuspend the bacterial pellet in 100 mL of pre-cooled 300 mM sucrose. Centrifuge at 1200 g for 3 min at 4°C using a Xiangyi GL21-M centrifuge.

[0073] (7) Discard the supernatant and resuspend the bacterial pellet in 50 mL of pre-cooled 300 mM sucrose. Centrifuge at 1200 g for 3 min at 4°C using a Xiangyi GL21-M centrifuge.

[0074] (8) Discard the supernatant and resuspend the bacterial pellet with 1 mL of pre-chilled 300 mM sucrose. Aliquot 100 μL into sterile 1.5 mL EP tubes and store at -80°C until use.

[0075] b. Transfection of polysaccharide copolymerase GumC mutant plasmid

[0076] (1) Preheat the electroporation instrument for 20 minutes and place the electroporation cup in a -20°C refrigerator for 20 minutes.

[0077] (2) The prepared Xanthomonas campestris competent cells were electroporated and taken out from a -80°C refrigerator and thawed on ice. 300 ng of the different GumC mutant plasmids obtained above were added, the mixture was gently tapped to mix, and the cells were incubated on ice for 10 min.

[0078] (3) The competent cells were aspirated and placed in the slit of the electroporation cup, and the electroporation cup was placed in the electroporation fusion instrument. The electric shock was performed at 2500V for 5ms. After the end, the electroporation cup was taken out and 1mL of SOC liquid culture medium (2% trypsin Chen, 0.5% yeast extract, 0.05% NaCl, 2.5mM KCl, 10mM MgCl2, 10mM MgSO4, 20mM D-glucose pH7.5) was added to the electroporation cup. The cells were resuspended, aspirated, and placed in a 1.5mL EP tube. The cells were cultured at 30℃ and 200rpm on a shaking table for 2h.

[0079] (4) After the culture was completed, the cells were centrifuged at 4000 rpm for 2 min, 900 μl of supernatant was aspirated, and the bacterial pellet was resuspended with the remaining supernatant. All the supernatant was aspirated and spread on the surface of NYGB solid culture medium containing Rif resistance at a final concentration of 25 μg / mL and Apra resistance at a final concentration of 50 μg / mL, and cultured in a 30°C incubator for 48 h.

[0080] (5) The single colony grown on the solid culture medium in step (4) was inoculated into 10 mL of NYGB liquid culture medium containing Rif resistance at a final concentration of 25 μg / mL and Apra resistance at a final concentration of 50 μg / mL, and cultured in a shaking incubator at 30°C and 200 rpm for 36 h.

[0081] (6) After the culture was completed, the plasmid was extracted and double-digested with AscⅠ and XhoⅠ and identified by 1.0% agarose gel electrophoresis. The mutant plasmid identified as correct by double-digestion was entrusted to Jilin Kumei Company for sequencing verification to ensure that the mutant plasmid was successfully transfected.

[0082] (7) Preserve the successfully transfected bacteria: Mix 700 μL of bacterial solution and 300 μL of 60% glycerol in a sterilized cryovial and store in a -80°C refrigerator.

[0083] 2. Low viscosity xanthan gum extraction:

[0084] a. The above-mentioned different frozen tube bacteria containing GumC mutant plasmids were streaked onto the surface of NYGB solid medium containing a final concentration of 25 μg / mL R-Fif resistance and a final concentration of 50 μg / mL Apra resistance, and cultured in a 30°C incubator for 36 h.

[0085] b. Pick a single colony from the plate and inoculate it into 10 mL NYGB liquid medium containing a final concentration of 25 μg / mL Rif resistance and a final concentration of 50 μg / mL Apra resistance, and culture at 30°C, 200 rpm in a shaker until OD 600 =0.6.

[0086] c. Expand the seed solution at a ratio of 1:100 to 500 mL of NYGB liquid medium containing Rif resistance at a final concentration of 25 μg / mL and Apra resistance at a final concentration of 50 μg / mL, and culture at 30°C, 200 rpm in a shaker for 72 h.

[0087] d. Remove the cultured fermentation broth from the incubator, pour the fermentation broth into a 250mL centrifuge cup, and place it in a GL-21M centrifuge at 10,000g×30min to remove the bacteria in the fermentation broth.

[0088] e. After centrifugation, pour the supernatant into a beaker, and then pour three times the volume of anhydrous ethanol into the beaker. Let it stand for 5-10 minutes to allow the low-viscosity xanthan gum to precipitate and float to the surface.

[0089] f. After the low-viscosity xanthan gum has completely floated and aggregated, use a glass rod to pick out the low-molecular-weight xanthan gum and place it in a clean 50-mL centrifuge tube. Open the tube lid and allow the remaining ethanol to evaporate at room temperature. After evaporation, place the tube in an 80°C oven to dry the extracted low-viscosity xanthan gum.

[0090] Example 4: Viscosity and molecular weight determination of low-viscosity rubber

[0091] The viscosity of the low-viscosity xanthan gum samples obtained using different plasmids containing GumC mutants in Example 2 above was measured:

[0092] 1. Determination of colloid viscosity by titration time:

[0093] a. Prepare 10 mg / mL xanthan gum control sample: Place 10 mg of each of the above-extracted xanthan gums in a 2 mL EP tube and add ddH2O to the volume to 1 mL to completely dissolve it.

[0094] b. Prepare 10 mg / mL low molecular weight xanthan gum sample: Take 10 mg of each extracted low molecular weight xanthan gum in a 2 mL EP tube, add ddH2O to make the volume 1 mL, and dissolve it completely.

[0095] c. Titration operation: (Use a 200μL burette for titration)

[0096] (1) Insert the 200μL burette into the sample and use the ear bulb to aspirate the sample to the 200μL mark.

[0097] Open the burette and allow the titrant to begin dripping into the sample. Simultaneously, start a stopwatch and record the time it takes for the titrant to drip into the sample. When 100 μL of titrant has dripped into the sample, stop the stopwatch and record the titration time.

[0098] (2) To ensure the accuracy of the results, repeat step ① at least three times for each sample group and take the average value as the final result. The wild type was used as the control.

[0099] The results are as follows Figure 5 As shown in the figure, it can be seen that the viscosity of xanthan gum produced by the mutant strains obtained in the embodiment of the present invention is lower than that of the wild type. G423A and GumC Y406G The viscosity is significantly reduced.

[0100] 2. Use gel permeation chromatography (GPC) to detect the distribution of relative molecular mass of low molecular weight xanthan gum.

[0101] In this study, gel permeation chromatography (GPC) was used to detect the viscosity of GumC G423A and GumC Y406G The distribution of relative molecular weight of low molecular weight xanthan gum produced by the mutant strain was determined:

[0102] a. Rinse the syringe three times with methanol and H2O. Use a pipette to draw up 200 μL of a 1 mg / mL low-molecular-weight xanthan gum sample. Pass it through a 0.22 μm filter to remove impurities. Use the xanthan gum sample obtained from the mutant strain described above to rinse the syringe three times. Draw up 50 μL of the sample and add it to the injection loop. Repeat this process twice to clean the injection loop. Rotate the wrench to inject the sample.

[0103] b. An Agilent 1260 Infinity system was used, with three columns connected in series (PL aquagel-OH 60, PLaquagel-OH MIXED-M, and PL aquagel-OH30 separation columns).

[0104] c. Set the column oven temperature to 40°C and the volume flow rate to 0.8 mL / min. Before testing the sample, flush the column and system with 0.1 mol / L NaNO3 until the baseline fluctuation is within 0.5 VW. After the baseline is stable, analyze the low molecular weight xanthan gum sample.

[0105] d. Use a differential refractive index detector (RI) and pullulan as the molecular weight standard. Integrate the refractive index (RI) of each group to obtain the final peak area. Divide the peak area by the retention time range corresponding to each peak to calculate the total RI peak area. Calculate the relative molecular mass distribution of the low molecular weight xanthan gum sample. Use the wild-type xanthan gum as a control.

[0106] The results are as follows Figure 6 As shown, GumC G423A and GumC Y406G The low molecular weight xanthan gum produced by the mutant strain contains two main components: one is the original chain length xanthan gum with an average relative molecular weight of 2619 kDa, and the other is GumC G423A The low molecular weight xanthan gum produced by the mutant strain has an average relative molecular weight of 4542Da; the other is GumC Y406G The low molecular weight xanthan gum produced by the mutant strain has an average relative molecular weight of 3958 Da.

[0107] In summary, the present invention uses enzyme engineering technology to perform amino acid site-directed mutagenesis on the polysaccharide copolymerase GumC from Xanthomonas campestris pv. campestris (Xcc) to obtain a series of mutants (such as GumC G423A 、GumC Y406G These mutants can significantly reduce the viscosity of xanthan gum and thus reduce its chain length. In particular, GumC G423A and GumC Y406GThe two mutants are particularly effective in reducing xanthan gum chain length. This invention provides a method for precisely controlling the molecular weight of xanthan gum, enabling the preparation of low molecular weight xanthan gum (LXG), and providing a feasible technical approach for its industrial application.

Claims

1. A polysaccharide copolymerase GumC mutant for controlling xanthan gum chain length, characterized in that: The mutant is obtained by mutation of at least one corresponding site in the highly conserved region, transmembrane domain, connecting region and periplasmic domain of the polysaccharide copolymerase GumC.

2. The polysaccharide copolymerase GumC mutant for controlling xanthan gum chain length according to claim 1, characterized in that: The mutant is a mutation of one or more amino acid sites among positions 406, 407, 419 and 423 in the polysaccharide copolymerase GumC of Xanthomonas campestris.

3. The polysaccharide copolymerase GumC mutant for controlling xanthan gum chain length according to claim 2, characterized in that: The corresponding amino acids at positions 406 (Y) and 407 (D) can be the same or different and mutated to S, A or G respectively; the amino acid at position 419 (A) can be mutated to G or S; and the amino acid at position 423 (G) can be mutated to A or S.

4. The polysaccharide copolymerase GumC mutant for controlling xanthan gum chain length according to claim 3, characterized in that: The mutant is one or a combination of Y406S, Y406A, Y406G, D407A, A419G, A419S, G423A, and G423S in the amino acid sequence shown in SEQ ID NO.

1.

5. The polysaccharide copolymerase GumC mutant for controlling xanthan gum chain length according to claim 4, characterized in that: The mutants are Y406S, Y406A, Y406G, D407A, A419G, A419S, G423A, G423S, Y406A and D407A, Y406S and D407A, Y406G and D407A, A419G and G423A, A419G and G423S, A419S and G423A or A419S and G423S in the amino acid sequence shown in SEQ ID NO.

1.

6. A use of the mutant according to claim 1, characterized in that: The mutant is used in producing low-viscosity xanthan gum.

7. A use of the mutant according to claim 1, characterized in that: The mutant is used in producing low molecular weight xanthan gum.

8. A method for producing low-viscosity, low-molecular-weight xanthan gum, characterized in that: The mutant plasmid obtained by using the mutant according to claim 1 is transfected into a host for fermentation and culture, and the fermentation broth is centrifuged to collect the supernatant to obtain low-viscosity, low-molecular-weight xanthan gum.

9. The method according to claim 8, characterized in that: The mutant was transfected into plasmid pBBR1MCS-2-T3 to obtain a recombinant plasmid, which was then introduced into Xanthomonas campestris to obtain a strain containing the mutant. The resulting strain was activated in NYGB solid medium containing antibiotics to an OD of 600 =0.6, and then inoculated into NYGB solid culture medium containing antibiotics at an inoculum size of 1%-10% and cultured with shaking for 24-72 hours. The culture solution was centrifuged to collect the supernatant, purified, and allowed to stand to obtain low-viscosity, low-molecular-weight xanthan gum.

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