Yeast engineering strain for producing endo-beta-1,4-glucanase by fermentation and its application in xanthan gum degradation

Abstract

The application discloses a yeast engineering bacterium for fermentative production of endo-beta-1,4-glucanase and application of the yeast engineering bacterium in xanthan gum degradation, and belongs to the technical field of biological enzymolysis.The application adopts recombinant Pichia pastoris to express an endo-beta-1,4-glucanase gene, inoculates the recombinant Pichia pastoris into a fermentation culture medium for culture, inoculates endo-beta-1,4-glucanase fermentation supernatant into a fermentation liquid containing Xanthomonas campestris for 36-144h of co-culture, so that the content of xanthan gum oligosaccharide in the fermentation liquid can be as high as 50% or more, and the molecular weight distribution of the xanthan gum oligosaccharide in the fermentation liquid is between 500Da and 2000Da.The method has the advantages of simple process and uniform product, and has great application potential and advantages.

Description

Technical Field

[0001] This invention relates to engineered yeast strains for the fermentation production of endo-β-1,4-glucanase and their application in xanthan gum degradation, belonging to the field of bioenzymatic hydrolysis technology. Background Technology

[0002] Xanthan gum is an anionic heteropolysaccharide composed of repeating pentasaccharide units. It contains a cellulose-based β-1,4-glucan backbone, with side chains linked by internal α-1,3-mannose residues, β-1,2-glucuronic acid residues, and terminal β-1,4-mannose residues. Xanthan gum's unique side chains, stable secondary structure, and special tertiary structure determine its pseudoplastic rheology, water solubility, thickening properties, suspension properties, emulsifying properties, and a range of other properties. Xanthan gum oligosaccharides are small-molecule oligosaccharides produced after the xanthan gum backbone is cleaved. They possess good antibacterial properties, can scavenge free radicals, exhibit activating factor activity, and possess antitumor and immunomodulatory activities, promoting the growth of beneficial gut microbiota. Low-molecular-weight sugars are mainly prepared through two routes: artificial synthesis and polysaccharide degradation. Artificial synthesis can synthesize both non-natural and natural sugars, but this method has low yields, complex synthesis steps, and high costs, making large-scale production difficult. Therefore, research on the degradation process of low molecular weight oligosaccharides in xanthan gum is of great significance for industrial production and daily life. Currently, there are few reports on enzymatic degradation of xanthan gum, and commercially available cellulases are mainly used to degrade the cellulose-like backbone of xanthan gum, but the results are not ideal. Summary of the Invention

[0003] To address the shortcomings of existing technologies, such as "the enzymatic hydrolysis of xanthan gum is not ideal, and the molecular weight of the product is still relatively large, which seriously affects the application performance of subsequent xanthan gum oligosaccharides," this invention provides an endogenous β-1,4-glucanase and its application in the enzymatic hydrolysis of xanthan gum. Enzymatic hydrolysis can be carried out simultaneously in the initial stage of xanthan gum production by Xanthomonas campestris fermentation, and xanthan gum released into the culture medium by Xanthomonas campestris fermentation can be degraded in an instant, thereby improving the enzymatic hydrolysis efficiency of the endogenous β-1,4-glucanase.

[0004] This invention provides a recombinant Pichia pastoris expressing the β-1,4-glucanase gene shown in SEQ ID NO.1.

[0005] In one embodiment, the recombinant Pichia pastoris uses pPIC9K as the expression vector and Pichia pastoris GS115 as the host.

[0006] The present invention also provides a method for hydrolyzing xanthan gum using β-1,4-glucanase, the encoding gene of which is shown in SEQ ID NO.1.

[0007] In one embodiment, the hydrolysis is carried out at 65–80°C for at least 20 min; or at 28–32°C for at least 4–5 days.

[0008] In one embodiment, the β-1,4-glucanase is produced by fermentation of the recombinant Pichia pastoris.

[0009] In one embodiment, the fermentation production specifically involves: culturing the recombinant Pichia pastoris in BMMY medium at 28–30°C, and adding methanol every 24 hours to induce enzyme production.

[0010] In one embodiment, the fermentation time is 3 to 4 days.

[0011] In one embodiment, the recombinant Pichia pastoris is cultured in BMGY medium to OD. 600 The concentration is 2-6, and then transferred to BMMY medium for fermentation.

[0012] In one embodiment, the xanthan gum is produced by fermentation of Xanthomonas oryzae.

[0013] In one embodiment, the method includes the following steps:

[0014] (1) Xanthan gum was produced by fermentation using Xanthomonas oryzae as the fermenting microorganism;

[0015] (2) Apply the β-1,4-glucanase to hydrolyze xanthan gum.

[0016] In one embodiment, the Xanthomonas campestris species has the accession number CCTCC M 2015714 and has been disclosed in Chinese invention patent with authorization announcement number CN105505824B.

[0017] In one embodiment, Xanthomonas campestris (X. campestris) CCTCC M2015714 seed culture was inoculated into a fermentation medium and cultured for 36 hours.

[0018] In one embodiment, step (2) involves adding the supernatant of the recombinant Pichia pastoris fermentation broth containing β-1,4-glucanase to the fermentation broth of step (1). While Xanthomonas rapa enters the gum-producing stage to produce xanthan gum, the endo-β-1,4-glucanase immediately degrades the generated xanthan gum to obtain xanthan gum oligosaccharide.

[0019] In one embodiment, the amount of supernatant added to the recombinant Pichia pastoris fermentation broth is 10-30% of the volume of Xanthomonas chinensis fermentation broth.

[0020] In one embodiment, the method for preparing the supernatant of the recombinant Pichia pastoris fermentation broth is as follows: Recombinant Pichia pastoris is inoculated into YPD solid medium and cultured at 30°C for 2.5–3 days; a single colony is picked and inoculated into BMGY medium and cultured at 30°C and 200 r / min until OD... 600 The seed culture was obtained by centrifuging at 4°C to collect the bacterial cells. The cells were then inoculated into BMMY medium and cultured at 30°C and 200 r / min. Methanol was added every 24 h to 1.0% (v / v) of the fermentation broth volume. The culture was carried out for 4–5 days, and the supernatant of the fermentation broth was collected by centrifugation.

[0021] In one embodiment, the amount of recombinant Pichia pastoris fermentation supernatant added is 10-30% (v / v).

[0022] In one embodiment, the method further includes rotary evaporation to concentrate, separate and purify, and freeze-drying the enzymatic hydrolysate of the β-1,4-glucanase to obtain xanthan gum oligosaccharides with a molecular weight of 500-2000 Da.

[0023] The present invention also provides a xanthan gum oligosaccharide mixture prepared by the method described herein.

[0024] This invention provides the application of the recombinant Pichia pastoris or the method described herein in the preparation of xanthan gum oligosaccharides.

[0025] In one embodiment, the xanthan gum oligosaccharide is a xanthan gum oligosaccharide with a molecular weight of 1000-2000 Da.

[0026] Beneficial effects:

[0027] (1) This invention provides an enzymatic method for preparing xanthan gum oligosaccharides. Using the method of this invention, a xanthan gum-producing strain is first inoculated into a fermentation medium and cultured for 36 h. Then, the supernatant of the fermentation broth of recombinant Pichia pastoris expressing endo-β-1,4-glucanase is added to the fermentation broth of Xanthomonas chinensis and co-cultured for 36-144 h. During the fermentation process, the polysaccharides are degraded in real time, which can reduce the viscosity of the system, relieve the polysaccharide encapsulation effect and the limitation of dissolved oxygen supply, thereby improving the fermentation performance. The proportion of xanthan gum oligosaccharides in the final fermentation product reaches more than 50%. This method is simple and controllable, has high preparation efficiency, and has little impact on the structure and biological activity of xanthan gum oligosaccharides, and can achieve efficient separation of xanthan gum oligosaccharides.

[0028] (2) The present invention provides a method for producing xanthan gum oligosaccharides. The xanthan gum oligosaccharides produced by the method of the present invention have a molecular weight between 1000 and 2000 Da, and have the advantage of uniform molecular weight of the product. Attached Figure Description

[0029] Figure 1Schematic diagram of recombinant plasmid pPIC9K-persiCel4.

[0030] Figure 2 The relative enzyme activity and stability of endoβ-1,4-glucanase at different pH and temperature.

[0031] Figure 3 The effect of enzymatic hydrolysis reaction under different enzyme solution addition amounts.

[0032] Figure 4 : Molecular weight distribution of hydrolysis products from different samples. Detailed Implementation

[0033] The following strains and plasmids were used in the embodiments of the present invention:

[0034] Escherichia coli JM109: purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0035] Pichia pastoris GS115, purchased from Invitrogen;

[0036] pPIC9K plasmid: Expression plasmid with AOX promoter, genimycin (G418) resistance, purchased from Invitrogen.

[0037] Determination of recombinant endoβ-1,4-glucanase activity: The activity of endoβ-1,4-glucanase was determined using the 3,5-dinitrosalicylic acid (DNS) method. 20 μL of enzyme solution was added to 60 μL of 1% xanthan gum solution, reacted at 50 °C for 20 min, followed by the addition of 120 μL of DNS reagent and boiling for 5 min. The absorbance of the sample was measured at 540 nm. One unit of enzyme activity (U / mL) was defined as the amount of endoβ-1,4-glucanase required to release 1 μM of reducing sugar per minute.

[0038] Xanthan gum yield determination: Xanthan gum yield in the supernatant was determined by the anthrone-sulfuric acid method.

[0039] Reducing sugar content determination: The reducing sugar content in the co-culture system was determined using the DNS method.

[0040] Determination of molecular weight of xanthan gum and oligosaccharides: Their molecular weight distribution was determined by high-performance gel filtration chromatography (HPGFC). The instrument used was a Waters 1525 high-performance liquid chromatograph, and the chromatographic conditions were as follows: Ultrahydrogel... TMLinear 300mm×7.8mm id; mobile phase 0.1mol / L NaNO3; flow rate 0.5mL / min; column temperature 45℃. Molecular weight calibration curves were plotted using dextran standards with weight-average molecular weights of 180, 2700, 9750, 135030, and 300600 Da. The molecular weight of the polysaccharide was calculated based on the sample retention time and the calibration curves.

[0041] The culture media involved in the following examples are as follows:

[0042] LB medium (g / L): yeast extract 5g, tryptone 10g, NaCl 5g, (agar powder 20g).

[0043] YPD medium (g / L): yeast extract 10, peptone 20, glucose 20, (agar powder 20).

[0044] MD medium (g / L): glucose 20, YNB 13.4, biotin 4×10⁻⁶ -4 , 15g agar powder.

[0045] BMGY medium (g / L): yeast extract 10, peptone 20, YNB 13.4, biotin 4×10⁻⁶ -4 10,100mM potassium phosphate buffer (pH 6.0).

[0046] BMMY medium (g / L): yeast extract 10, peptone 20, YNB 13.4, biotin 4×10⁻⁶ -4 5% methanol, 100mM potassium phosphate buffer (pH 6.0).

[0047] Seed culture medium for Xanthomonas oryzae (g / L): glycerol 50, fish meal peptone 5, beef extract 3, yeast extract 1, (agar 20), pH 7.0–7.2.

[0048] Fermentation medium for Xanthomonas brassicae (g / L): 40.0 g / L glycerol, 1.5 g / L fish meal peptone, 0.5 g / L yeast extract, 2.0 g / L NaNO3, 2.5 g / L MgSO4·7H2O, 0.01 g / L FeSO4·7H2O, 3.5 g / L K2HPO4·3H2O, 2.0 g / L KH2PO4; pH 7.0–7.2.

[0049] Example 1: Construction of recombinant Pichia pastoris GS115-persiCel4 expressing the endogenous β-1,4-glucanase persiCel4

[0050] (1) Construct expression plasmid pPIC9K-persiCel4

[0051] The persiCel4 gene sequence shown in SEQ ID NO.1 was synthesized. The persiCel4 gene and the Pichia pastoris methanol-inducible expression vector pPIC9K were double-digested with the DNA restriction endonucleases SnaBI and NotI. Then, the persiCel4 gene was ligated to the vector pPIC9K using T4 DNA ligase to form the expression plasmid pPIC9K-persiCel4. Figure 1 The recombinant plasmid was transformed into Escherichia coli JM109, then plated on LB plates containing the corresponding antibiotics and cultured overnight. Colony PCR was performed, and five positive clones were randomly selected and cultured at 37°C and 200 rpm for 14–16 h. The clones were then sent to the company for sequencing analysis. If the sequencing results showed no frameshift or other base mutations, the vector was successfully constructed.

[0052] (2) Constructing a Pichia pastoris engineered strain that expresses persiCel4 in a methanol-induced manner

[0053] Following the Invitrogen yeast transformation method, the plasmid pPIC9K-persiCel4 was linearized by digestion with the DNA restriction endonuclease Sal I, and the DNA was recovered using a DNA purification kit and stored for later use. 80 μL of Pichia pastoris GS115 competent cells and 5–20 μL of the linearized plasmid were mixed and transferred to a pre-chilled electroporation cuvette for yeast electroporation. The electroporation buffer was plated on MD plates and incubated at 30°C for 3 days for further high-copy screening. The genome of the selected high-copy strains was extracted and verified by PCR. The correctly verified positive transformants were named recombinant Pichia pastoris GS115-persiCel4. Single colonies were inoculated into 25 mL of BMGY medium and cultured at 200 rpm at 30°C for 16–18 h (OD200). 600 =2~6), under aseptic conditions, Pichia pastoris seed culture was taken, centrifuged at 5000 r / min for 5 min at 4℃, the supernatant was discarded, and the cells were added to 100 mL of BMMY medium. The culture was carried out in a shake flask at 30℃ with a rotation speed of 200 r / min. 1.0% of the fermentation broth volume of methanol was added every 24 h. After 72 h of culture, the supernatant of the fermentation broth was collected, and the extracellular endo-β-1,4-glucanase activity was measured. The results showed that the extracellular enzyme activity was 6.224 U / mL.

[0054] Example 2: Determination of the optimal pH and optimal enzyme activity of endoβ-1,4-glucanase

[0055] To determine the optimal pH for endo-β-1,4-glucanase, 50 mM buffer solutions with different pH values ​​were prepared, including: citrate-citrate buffer (pH 3.5–6.0), phosphate buffer (pH 6.5–8.0), Tris-HCl buffer (pH 8.5, 9.0), and Gly-NaOH buffer (pH 9.5, 10.0). The enzyme was added to these buffer solutions, and the relative enzyme activity at each pH was measured. Enzyme activity was measured at 50°C for 3 hours (with 20-minute intervals) to assess pH stability. Relative enzyme activity was calculated with 0 h enzyme activity defined as 100%. To determine the optimal temperature for the enzyme, the enzyme solution was placed in a suitable buffer solution and reacted with the substrate at different temperatures (40–95°C) for 20 min, and the relative enzyme activity was measured. To analyze the thermostability of the enzyme, reactions were performed at different temperatures (65, 75, 85, 95°C) for 3 hours. Relative enzyme activity at different temperature conditions and reaction times was calculated, defined as 0 h enzyme activity as 100%. Results are shown below. Figure 2 As shown, it exhibits high stability within the pH range of 6.0–8.0 and the temperature range of 65–80℃, with the optimal operating conditions being pH 7.0 and temperature 75℃.

[0056] Example 3: Enzymatic hydrolysis of xanthan gum

[0057] (1) Preparation of culture medium:

[0058] BMGY medium: 100 mM potassium phosphate buffer (pH 6.0), 13.4 g / L YNB, 10.0 g / L yeast extract, 20.0 g / L tryptone, 4.0 × 10⁻⁶ mg / L HCl. -4 g / L biotin, 10.0 g / L glycerol.

[0059] BMMY medium: Replace 10.0 g / L glycerol in BMGY medium with 5.0 g / L methanol.

[0060] Xanthomonas aeruginosa solid culture medium (g / L): glycerol 50, fish meal peptone 5, beef extract 3, yeast powder 1, agar 20, pH 7.0~7.2.

[0061] Seed culture medium for Xanthomonas aeruginosa (g / L): glycerol 50, fish meal peptone 5, beef extract 3, yeast extract 1, pH 7.0–7.2.

[0062] Fermentation medium for Xanthomonas brassicae: glycerol 40.0 g / L, fish meal peptone 3.0 g / L, yeast extract 1.5 g / L, NaNO3 0.8 g / L, MgSO4·7H2O 2.5 g / L, FeSO4·7H2O 0.01 g / L, K2HPO4·3H2O 3.5 g / L, KH2PO4 2.0 g / L; pH 7.0–7.2.

[0063] (2) The Pichia pastoris GS115-persiCel4 constructed in Example 1 was inoculated into YPD solid medium and cultured at 30℃ for 2.5–3 days. Single colonies were picked and inoculated into BMGY medium (10% volume, v / v) and cultured at 30℃ and 200 rpm until OD. 600 Seed culture was obtained by centrifuging at 4°C and collecting the cells. The cells were then inoculated into BMMY medium (20% volume, v / v) and cultured at 30°C and 200 rpm. Methanol was added every 24 hours to 1.0% (v / v) of the fermentation broth volume. Fermentation was completed after 3–4 days. The fermentation broth was centrifuged at 8000 rpm for 5 minutes, and the supernatant was collected. The enzyme activity in the supernatant was found to be 6.395 U / mL.

[0064] (3) Xanthomonas brasiliensis CCTCC M 2015714 (disclosed in Chinese invention patent with authorization announcement number CN105505824B) was cultured on solid culture medium at 30℃ for 3.5-4 days. Then, a single colony was picked and inoculated into Xanthomonas brasiliensis seed culture medium and cultured at 30℃ and 200r / min for 24 hours to obtain Xanthomonas brasiliensis seed liquid. Xanthomonas brasiliensis seed liquid was inoculated into Xanthomonas brasiliensis fermentation culture medium at an inoculation amount of 10% (v / v) and cultured at 30℃ and 220r / min for 36 hours. Then, 10-30% (v / v) of the recombinant Pichia pastoris fermentation supernatant prepared in step (2) was added to the Xanthomonas brasiliensis fermentation broth according to the final concentration, and cultured at 30℃ and 220r / min for 4-5 days. The total sugar and reducing sugar contents in the fermentation broth were determined at the end of fermentation with Xanthomonas brassicae alone and at the end of fermentation after the addition of recombinant Pichia pastoris supernatant.

[0065] The results are as follows Figure 3 The results showed that when the enzyme solution was added at a concentration of 10-20% (v / v), the total sugar content reached over 6.5 g / L and the reducing sugar content reached over 1.1 g / L; when the addition concentration was 20%, the total sugar content reached 8.46 g / L and the reducing sugar content reached 2.86 g / L.

[0066] Example 4: Preparation of xanthan gum oligosaccharide products

[0067] The fermentation broth obtained in Example 3 was centrifuged to remove the bacterial cells. The supernatant was fractionated and precipitated with alcohol. Finally, 10 times the volume of ethanol was added, and after centrifugation to reconstitute, most of the protein was removed using the Sevage method. The lyophilized sugar powder was collected, and the crude low molecular weight xanthan gum was reconstituted with an appropriate amount of deionized water. Then, the protein was removed using Sep-Pak C18, and the salt ions and other impurities were removed by dialysis using a 500 Da dialysis bag. Finally, the resulting low molecular weight xanthan gum solution was freeze-dried.

[0068] The molecular weight of xanthan gum in different samples was determined, and the results showed that the molecular weight of xanthan gum secreted by Xanthomonas oryzae during fermentation alone was 10922881 Da. Figure 4 a) The molecular weight of the enzymatic hydrolysis products was determined using high performance size exclusion chromatography (HPLC), and the results are as follows: Figure 4 As shown in b, the hydrolysis products include three sugars with molecular weights of 8731711, 38450, and 1808 Da, accounting for 44.83%, 3.67%, and 51.50%, respectively.

[0069] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A recombinant Pichia pastoris, characterized in that, The beta-1,4-glucanase gene shown in SEQ ID NO. 1 is expressed by using pPIC9K as an expression vector and using Pichia pastoris GS115 as a host.

2. Use of a β-1,4-glucanase in hydrolysis of xanthan gum, characterised in that, The coding gene of the beta-1,4-glucanase is shown in SEQ ID NO.

1.

3. Use according to claim 2, characterized in that, The hydrolysis is reacted for at least 20 min at 65-80 DEG C or reacted for 4-5 days at 28-32 DEG C.

4. Use according to claim 2 or 3, characterized in that, The beta-1,4-glucanase is fermented by the recombinant Pichia pastoris of claim 1.

5. Use according to claim 4, characterized in that, The fermentation production is specifically that the recombinant Pichia pastoris is cultured in BMMY medium at 28-30 DEG C, and methanol is added every time to induce enzyme production.

6. A method of producing low molecular weight xanthan oligosaccharides, characterized in that, Xanthomonas campestris 7. The method of claim 6, wherein, The xanthan gum is produced by Xanthomonas aeruginosa (…). The method comprises the following steps: The strain was produced by fermentation; the preservation number of Xanthomonas oryzae is CCTCC M2015714.

8. The method of claim 7, wherein, (1) Xanthomonas campestris is used as a fermentation microorganism to ferment xanthan gum; (2) the supernatant of the fermentation liquor containing beta-1,4-glucanase fermented by the recombinant Pichia pastoris of claim 1 is added into the fermentation liquor of step (1), and the fermentation is continued for 4-5 days. The preparation method of the supernatant of the fermentation liquor containing beta-1,4-glucanase is that the recombinant Pichia pastoris is cultured in BMMY medium at 28-30 DEG C, methanol is added every 24 h to induce enzyme production, and the culture is continued for 4-5 days, and the supernatant of the fermentation liquor is collected by centrifugation.

9. The method of claim 8, wherein, The low molecular weight xanthan oligosaccharide has a molecular weight of 1000-2000 Da.

10. The method of any one of claims 6-9, wherein, ​

Images