CN113862165B

CN113862165B - Method for directly producing low-molecular-weight xanthan gum by co-culture fermentation

Info

Publication number: CN113862165B
Application number: CN202111098889.8A
Authority: CN
Inventors: 高敏杰; 詹晓北; 杨国帅; 蒋芸; 李志涛
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2021-09-18
Filing date: 2021-09-18
Publication date: 2023-07-25
Anticipated expiration: 2041-09-18
Also published as: CN113862165A

Abstract

The invention discloses a method for directly producing low-molecular-weight xanthan gum by co-culture fermentation, belonging to the technical field of microorganisms and fermentation. The method comprises the steps of inoculating Xanthomonas campestris capable of producing xanthan gum strain into a fermentation medium for culturing for 24-48 h, inoculating recombinant pichia pastoris capable of producing xanthan gum endonuclease into the fermentation medium fed with yeast extract for co-culturing for 24-120 h, so that the content of low molecular weight xanthan gum in the fermentation liquid is up to 3.71-5.65 g/L, and the molecular weight of the low molecular weight xanthan gum in the fermentation liquid is distributed in 3X 10 ⁴ ～5×10 ⁴ Between Da, therefore, the low molecular weight xanthan gum produced by the method has the advantages of high yield and uniform molecular weight of the prepared low molecular weight xanthan gum; the fermentation medium used in the invention has wide sources and low price, so the method for producing the low molecular weight xanthan gum has the advantage of low cost.

Description

Method for directly producing low-molecular-weight xanthan gum by co-culture fermentation

Technical Field

The invention relates to a method for directly producing low molecular weight xanthan gum by co-culture fermentation, belonging to the technical field of microorganisms and fermentation.

Background

Xanthan gum, commonly referred to as xanthan gum, huang Shengjiao, is a natural anionic polysaccharide of complex structure. Originally, the plant pathogenic bacteria Xanthomonas campestris isolated from the United states department of agriculture Xanthomonas campestris NRRL B-1459 were produced by fermentation. Xanthan gum is composed of a certain amount of "pentasaccharide repeating unit" composed of glucose, mannose and glucuronic acid,the monosaccharide composition and the molar ratio are glucose: mannose: glucuronic acid=2.0: 2.0:1.0, relative molecular weight of 0.2X10 ⁷ ～5.0×10 ⁷ Da, the monosaccharide composition and the molar ratio, and the relative molecular weight are closely related to the production strain, the fermentation condition and the extraction process. Because of the excellent physicochemical properties, the xanthan gum can endow jam food with unique flavor, viscosity, structure, water retention and appearance, can also increase the water retention of baked food and prolong the shelf life of the baked food, has wide application in the food field, and can be used as an unlimited food additive by the U.S. food and drug administration.

The low molecular weight xanthan gum degraded from xanthan gum has higher bioactivity than the high molecular weight xanthan gum. The relative molecular weight is 1X 10 ⁶ ～1.5×10 ⁶ Da xanthan gum can inhibit chondrocyte apoptosis; the relative molecular weight is 1.7X10 ⁶ Da xanthan gum can inhibit cartilage matrix destruction; the relative molecular weight is 1X 10 ⁶ ～1.5×10 ⁶ Da xanthan gum has the efficacy of treating osteoarthritis; the relative molecular weight is 1.3X10 ⁵ Da xanthan gum is capable of scavenging free radicals; the relative molecular weight is 7.5X10 ³ Da xanthan gum has antioxidant activity; the low molecular xanthan gum can also inhibit apoptosis induced by oxidative stress, and has antibacterial, antitumor and immunity activities. Therefore, the research on the production of low molecular weight xanthan gum is of great significance.

Common polysaccharide degradation methods include physical, chemical and biological enzymatic methods. Physical degradation methods include high-temperature degradation methods, electrolytic degradation methods, radiation degradation methods and the like, and the methods degrade polysaccharide rapidly, but have the problems of high cost, low yield, colored impurities and the like. The chemical method generally utilizes strong acid, strong alkali or strong oxidant to degrade polysaccharide, and has high hydrolysis efficiency, but has the problems of uncontrollable degradation, often accompanied generation of toxic substances, environmental pollution and the like. Compared with the physical method and the chemical method, the biological enzyme method has mild reaction conditions, controllable process, and moderate cost, can obtain the product with the expected molecular weight, and is widely applied. The conventional biological enzyme method for degrading polysaccharide generally comprises the steps of respectively producing, extracting, separating and purifying enzyme and sugar, then adding the enzyme into a polysaccharide solution, and reacting for a period of time to obtain the low molecular weight polysaccharide. However, the higher the concentration of xanthan gum, the higher the solution viscosity, the effect of enzyme is affected, and the efficiency is low. It is therefore highly desirable to find a process for producing low molecular weight xanthan gum that can efficiently degrade xanthan gum.

Disclosure of Invention

The invention provides recombinant pichia pastoris, which takes pPIC9K as an expression vector and pichia pastoris GS115 as an expression host, and expresses xanthan gum endonuclease with an amino acid sequence shown as SEQ ID NO. 1.

In one embodiment of the present invention, the AOX1 promoter on the pPIC9K plasmid is replaced with GAP promoter, the nucleotide sequence of which is shown as SEQ ID NO.3, to obtain an expression vector (named pGAP9K vector).

In one embodiment of the invention, the nucleotide sequence of the xanthan endonuclease is shown as SEQ ID NO. 2.

In one embodiment of the invention, the constitutive promoter GAP is used to express the xanthan endonuclease.

The invention also provides a method for producing the xanthan gum, which comprises the steps of co-culturing a xanthomonas campestris strain (Xanthomonas campestris) capable of producing the xanthan gum by utilizing glycerol and a pichia pastoris genetically engineered strain capable of expressing the xanthan gum endonuclease to obtain fermentation liquor, and then extracting the fermentation liquor to prepare the xanthan gum with low molecular weight;

the method comprises the following steps:

(1) Inoculating a seed solution of Xanthomonas campestris (Xanthomonas campestris) into a fermentation medium for culturing to obtain a culture solution;

(2) And (3) adding the recombinant pichia pastoris seed solution into the culture solution obtained in the step (1), performing co-culture fermentation to obtain a fermentation solution, and extracting the fermentation solution to obtain the xanthan gum with low molecular weight.

In one embodiment of the invention, the xanthomonas campestris is: campestris cctccc NO: m2015714.

In one embodiment of the invention, the x.campestris cctccc NO: m2015714 is described in the Chinese patent publication No. CN 105505824B.

In one embodiment of the invention, the OD of the recombinant pichia pastoris seed solution ₆₀₀ At least 2.0.

In one embodiment of the invention, the Xanthomonas campestris seed solution has an OD ₆₀₀ At least 0.8.

In one embodiment of the invention, the preparation method of the Xanthomonas campestris seed solution comprises the following steps:

streaking Xanthomonas campestris capable of producing xanthan gum by glycerol in Xanthomonas campestris flat-plate culture medium, culturing at 30deg.C for 3-4 d, selecting single colony, inoculating into Xanthomonas campestris seed culture medium, culturing at 30deg.C and 220rpm for 22-26 hr, at the same time, OD in the culture solution ₆₀₀ Reaching 0.8 to 1.0 to obtain the Xanthomonas campestris seed solution.

In one embodiment of the invention, the Xanthomonas campestris plate medium (g/L): glycerin 50.0, fish meal peptone 5.0, beef extract 3.0, yeast extract 1.0, agar 20.0, pH 7.0-7.2.

In one embodiment of the invention, xanthomonas campestris seed medium (g/L): glycerin 50.0, fish meal peptone 5.0, beef extract 3.0, yeast extract 1.0, pH 7.0-7.2.

In one embodiment of the invention, the preparation method of the recombinant pichia pastoris solution comprises the following steps:

scribing the Pichia pastoris engineering bacteria in a YPD flat-plate culture medium, culturing for 2.5-3.5 d at 30 ℃, picking single bacterial colony, inoculating the single bacterial colony into a YPD liquid culture medium, and culturing for 16-24 h at 30 ℃ and 200rpm, wherein the OD600 in the culture solution reaches 2.0-6.0 at the moment, so as to obtain recombinant Pichia pastoris seed solution.

In one embodiment of the invention, the inoculation amount of the recombinant pichia pastoris seed solution in the fermentation medium is 10-70% (v/v).

In one embodiment of the present invention, the seed solution of Xanthomonas campestris is inoculated in a fermentation medium in an amount of 5 to 20% (v/v).

In one embodiment of the invention, the method is:

(1) Inoculating Xanthomonas campestris (Xanthomonas campestris) seed solution into a fermentation medium, and fermenting at 28-33 ℃ and 180-230 rpm for 24-48 h to obtain a culture solution;

(2) Adding the recombinant pichia pastoris seed solution into the culture solution obtained in the step (1), and performing co-culture fermentation at the temperature of 28-30 ℃ and at the speed of 170-220 rpm.

In one embodiment of the present invention, the fermentation medium is contained in a reaction system in a liquid amount of: 5-35% (v/v).

In one embodiment of the invention, the conditions for co-cultivation fermentation by adding the recombinant pichia pastoris seed solution are as follows: the fermentation temperature is 28-30 ℃, the pH is 5.0-8.0, and the rotating speed is 170-220 rpm.

In one embodiment of the present invention, the method further comprises adding a nitrogen source to the culture broth prepared in step (1) before adding the recombinant pichia pastoris seed broth to the culture broth prepared in step (1).

In one embodiment of the invention, the nitrogen source is ammonium sulfate, KNO ₃ Ammonium dihydrogen phosphate, yeast extract, soybean peptone, fish meal peptone, bean sprout juice, corn steep liquor, malt extract or tryptone.

In one embodiment of the present invention, the nitrogen source is added in an amount of: 5-25 g/L.

In one embodiment of the invention, the nitrogen source is yeast extract.

In one embodiment of the present invention, the yeast extract is added to the culture medium in an amount such that the final concentration is 5 to 25g/L.

In one embodiment of the invention, the fermentation medium (g/L) comprises: 20 to 50 portions of glycerin, 1.5 to 4.5 portions of fish meal peptone, 1.0 to 2.0 portions of yeast extract and NaNO ₃ 0.6～1.0，MgSO ₄ ·7H ₂ O 2.0～3.0，FeSO ₄ ·7H ₂ O 0.005～0.015，K ₂ HPO ₄ ·3H ₂ O 3.0～4.0，KH ₂ PO ₄ 1.5～2.5。

In one embodiment of the invention, the pH regulation strategy is the optimal pH regulation strategy in the co-culture fermentation, wherein the pH regulation in the co-culture fermentation is two-stage pH regulation, the first stage of the pH regulation in the co-culture fermentation is the xanthomonas campestris growth phase, and the second stage of the pH regulation in the co-culture fermentation is the co-culture fermentation phase by adding recombinant pichia pastoris seed liquid into a fermentation medium.

The method comprises the following steps: adjusting the pH value of the first stage to 7.0-7.2; and adjusting the pH value of the second stage to 5.0-8.0.

The invention also provides the xanthan gum prepared by the method.

The invention also provides the application of the recombinant pichia pastoris or the method in preparing the low molecular weight xanthan gum.

Advantageous effects

(1) The invention provides a method for producing low molecular weight xanthan gum, which comprises the steps of inoculating a strain capable of producing the xanthan gum into a fermentation medium for culturing for 24-48 h, inoculating the strain capable of producing the xanthan gum endonuclease into the fermentation medium fed with yeast extract for co-culturing for 24-120 h, and enabling the content of the low molecular weight xanthan gum in the fermentation liquid to reach 3.71-5.65 g/L.

(2) The invention provides a method for producing low molecular weight xanthan gum, the molecular weight of the low molecular weight xanthan gum produced by the method is 3 multiplied by 10 ⁴ ～5×10 ⁴ Da, so the low molecular weight xanthan gum produced by the method has the advantage of uniform molecular weight of the prepared low molecular weight xanthan gum.

(3) The invention provides a method for producing low-molecular-weight xanthan gum, which only needs to use two bacteria to carry out simple fermentation in a fermentation medium, and the fermentation medium has wide sources and low cost, so the method for producing the low-molecular-weight xanthan gum has the advantage of low cost.

Drawings

Fig. 1: schematic of plasmid pPIC 9K.

Fig. 2: schematic of plasmid pGAP 9K.

Fig. 3: co-cultivation fermentation curves.

Detailed Description

The detection method involved in the following examples is as follows:

detection of xanthan gum yield:

the yield of xanthan gum in the supernatant was determined by the anthrone-sulfuric acid method.

Detection of xanthan gum molecular weight:

the molecular weight of xanthan gum is determined by high performance size exclusion chromatography (high-performance size exclusion chromatograph, HPSEC) by the following specific method: the instrument was a Waters1525 high performance liquid chromatograph equipped with a 2414 differential refractive detector and an Empower3 workstation. The chromatographic conditions were as follows: ultrahydrogel ^TM Linear 300mm x 7.8mm id secondary series column; mobile phase 0.1 mol.L ^-1 NaNO ₃ The method comprises the steps of carrying out a first treatment on the surface of the Flow rate 0.9 mL/min ^-1 The method comprises the steps of carrying out a first treatment on the surface of the Column temperature 45 ℃; sample preparation: the sample is dissolved in 0.1 mol.L ^-1 NaNO ₃ In the above, the sample was filtered through a 0.22 μm microporous membrane and fed to the sample injection volume of 20. Mu.L.

Determination of the enzyme activity of the xanthan gum endoenzyme:

preparing an enzyme reaction system, and measuring the concentration of reducing sugar in the reaction system by using a dinitrosalicylic acid method (DNS) after the reaction is finished. The method for calculating the enzyme activity of the xanthan gum incision enzyme comprises the following steps: the amount of enzyme required to release 1. Mu.M reducing sugar per minute was 1U, enzyme activity unit: U/mL.

The following examples relate to the following media:

LB liquid medium (g/L): tryptone 10, naCl 10, yeast powder 5, pH 7.

LB plate medium (g/L): tryptone 10, naCl 10, yeast powder 5, pH 7 and agar 20.

MD plate media (g/L): YNB 13.4, biotin 4X 10 ^-4 Glucose 20, agar 20.

Xanthomonas campestris plate medium (g/L): glycerin 50.0, fish meal peptone 5.0, beef extract 3.0, yeast extract 1.0, agar 20.0, pH 7.0-7.2.

Xanthomonas campestris seed medium (g/L): glycerin 50.0, fish meal peptone 5.0, beef extract 3.0, yeast extract 1.0, pH 7.0-7.2.

Fermentation medium (g/L): 40.0 portions of glycerin, 3.0 portions of fish meal peptone, 1.5 portions of yeast extract and NaNO ₃ 0.8，MgSO ₄ ·7H ₂ O2.5，FeSO ₄ ·7H ₂ O 0.01，K ₂ HPO ₄ ·3H ₂ O 3.5，KH ₂ PO ₄ 2.0；pH 7.0～7.2。

YPD plate Medium (g/L): yeast extract 10, tryptone 20, glucose 20, agar 20.

YPD liquid Medium (g/L): yeast extract 10, tryptone 20 and glucose 20.

Example 1: construction of recombinant Pichia pastoris

The method comprises the following specific steps:

(1) The sequence of the xanthan endonuclease gene EX (GenBank: ALX 66163.1) was obtained from NCBI (disclosed in paper Novel Endotype Xanthanase from Xanthan-Degrading Microbacterium sp. Strain XT 11), and the xanthan endonuclease EX fragment was synthesized by the whole gene of the division of biological engineering (Shanghai); inserting the recombinant plasmid pPIC9K into the plasmid pPIC9K by a double digestion (EcoR I, not I) method to obtain a recombinant plasmid pPIC9K-EX (shown in figure 1);

(2) Inoculating Escherichia coli stored in a laboratory into an LB culture medium, activating, and preparing Escherichia coli competence;

(3) Transferring the recombinant plasmid pPIC9K-EX obtained in the step (2) into the competence of escherichia coli by heat shock, carrying out water bath at 37 ℃ for 45-60 minutes, coating 50-100 mu L of the mixture on an LB plate added with ampicillin, and culturing for 14-16 hours at 37 ℃;

(4) Performing colony PCR on the positive single bacteria on the LB plate in the step (3), randomly selecting 5-10 positive clones, culturing at 37 ℃ and 200rpm for 14-16 h, and sequencing to obtain positive clones meeting the requirements; extracting plasmid of positive clone meeting requirement, preparing recombinant plasmid pPIC9K-EX, and storing at-20deg.C;

(5) The extracted Pichia pastoris GS genomic DNA is used as a template, and a promoter GAP sequence is obtained through PCR amplification; the recombinant plasmid pPIC9K-EX extracted in the step (4) is replaced by GAP sequence through a double enzyme digestion (Sac I, bamH I) method to obtain recombinant plasmid pGAP9K-EX (shown in figure 2);

(6) Transferring the recombinant plasmid pGAP9K-EX obtained in the step (5) into the competence of escherichia coli by heat shock, carrying out water bath at 37 ℃ for 45-60 minutes, coating 50-100 mu L of the mixture on an LB plate added with ampicillin, and culturing for 14-16 hours at 37 ℃;

(7) Performing colony PCR on positive single bacteria on the LB plate in the step (6), randomly selecting 5-10 positive clones, culturing at 37 ℃ and 200rpm for 14-16 h, and sequencing to obtain positive clones meeting the requirements; extracting plasmids of positive clones meeting the requirements, preparing recombinant plasmids pGAP9K-EX, and storing at-20 ℃;

(8) Laboratory-preserved Pichia pastoris GS was inoculated in YPD medium, activated, and Pichia pastoris GS competent was prepared;

(9) Linearizing the plasmid extracted in step (7) with Sal I enzyme and purifying with the Mini BEST DNA Fragment Purification Kit kit purchased from TAKARA; the linearized plasmid was electrotransferred into Pichia pastoris GS competence and plated on MD plates;

(10) Selecting the colony growing in the step (9), dibbling on a YPD plate added with geneticin (G418), screening a high-copy strain, extracting a genome, and carrying out PCR verification to obtain the genetically engineered bacterium GS115/pGAP9K-EX of the xanthan gum endonuclease with the amino acid sequence shown in SEQ ID NO. 1.

Example 2: seed liquid obtaining

The method comprises the following specific steps:

(1) Xanthomonas campestris CCTCC NO: m2015714 streak on Xanthomonas campestris flat-plate medium, culturing at 30deg.C for 3-4 d, selecting single colony, inoculating to Xanthomonas campestris seed medium, culturing at 30deg.C and 220rpm for 22-26 h, OD ₆₀₀ Reaching 0.8 to 1.0 to obtain the wildXanthomonas campestris seed solution.

(2) Scribing the Pichia pastoris engineering bacteria prepared in the example 1 in YPD plate culture medium, culturing for 2.5-3.5 d at 30 ℃, picking single colony, inoculating the single colony in YPD liquid culture medium, culturing for 16-24 h at 30 ℃ and 200rpm, and OD ₆₀₀ Reaching 2.0 to 6.0 to obtain recombinant pichia pastoris seed liquid.

Example 3: co-cultivation screening of nitrogen sources

The method comprises the following specific steps:

(1) Preparing a fermentation medium:

40.0g/L glycerol, 3.0g/L fish meal peptone, 1.5g/L yeast extract, naNO ₃ 0.8g/L，MgSO ₄ ·7H ₂ O 2.5g/L，FeSO ₄ ·7H ₂ O 0.01g/L，K ₂ HPO ₄ ·3H ₂ O 3.5g/L，KH ₂ PO4 2.0g/L；pH 7.0～7.2。

(2) Effect of adding different nitrogen sources in the mid-fermentation period on Xanthomonas campestris fermentation

The Xanthomonas campestris seed solution (OD) obtained in step (1) of example 2 ₆₀₀ 0.9) inoculating 10% (v/v) of the inoculum size to the fermentation medium obtained in the step (1), wherein the liquid loading amount of the fermentation medium is: 10% (v/v), and culturing at 30deg.C and 220rpm for 36 hr to obtain fermentation broth;

respectively adding ammonium sulfate and KNO into the fermentation liquor ₃ Ammonium dihydrogen phosphate, yeast extract, soybean peptone, bean sprout juice, corn steep liquor, malt extract or tryptone, with final concentration of 10g/L, and culturing at 30deg.C and 220rpm for 120 hr until fermentation is completed. The xanthan gum concentrations in the fermentation broths under the different nitrogen source action conditions were measured with no nitrogen source added to the fermentation broths as a control, and the results are shown in table 1.

TABLE 1 screening of Co-cultured Nitrogen sources

(2) Screening of recombinant Pichia pastoris nitrogen sources

The recombinant Pichia pastoris seed solution (OD) obtained in step (2) of example 2 ₆₀₀ 3.5) was inoculated with 10% (v/v) of ammonium sulfate at 10g/L and KNO at 10g/L, respectively ₃ 10g/L monoammonium phosphate, 10g/L yeast extract, 10g/L soybean peptone, 10g/L fish meal peptone, 10g/L bean sprout juice, 10g/L corn steep liquor, 10g/L malt extract or 10g/L tryptone in place of the nitrogen source (yeast extract 1.5 g/L) were cultured at 30℃for 120 hours at 220rpm until the fermentation was completed. Respectively measuring the incision enzyme activity of xanthan gum in the fermentation liquor; the results are shown in Table 2.

TABLE 2 screening of Co-cultured Nitrogen sources

The results show that: the addition of different nitrogen sources in the Xanthomonas campestris culture process has no great influence on the molecular weight of the produced xanthan gum, when adding ammonium sulfate and KNO ₃ When the ammonium dihydrogen phosphate, yeast extract and corn steep liquor are used, the yield of the xanthan gum is higher; when soybean peptone is added, the yield of xanthan gum is lower although the enzyme activity of the endo-xanthan enzyme is highest; the enzyme activity of the xanthan endonuclease is higher and the yield of xanthan gum is also higher when the yeast extract is added, so the yeast extract is selected as the nitrogen source added during co-culture.

Example 4: screening of Pichia pastoris inoculum size in Co-culture fermentation

According to the results of example 3, the yeast extract was added as a nitrogen source after 36 hours of fermentation of Xanthomonas campestris in this example, and the specific steps were as follows:

(1) The Xanthomonas campestris seed solution (OD) obtained in step (1) of example 2 ₆₀₀ 0.9) inoculating 10% (v/v) of the inoculum size to the fermentation medium prepared in step (1) of example 3, wherein the liquid loading amount of the fermentation medium is: 10% (v/v), and culturing at 30deg.C and 220rpm for 36 hrTo Xanthomonas campestris fermentation broth.

(2) Adding 10%, 20%, 30%, 40%, 50%, 60% and 70% of inoculum size of the Xanthomonas campestris fermentation broth prepared in step (1) into the recombinant Pichia pastoris seed solution (OD) prepared in step (2) of example 2 ₆₀₀ 3.5), and the yeast extract was added to a final concentration of 10g/L, the pH was adjusted to 6.0, and the culture was continued at 30℃and 220rpm for 120 hours until the fermentation was completed. And measuring the yield of the xanthan gum and the enzyme activity of the incision enzyme of the xanthan gum under the condition of different inoculum sizes respectively. The results are shown in Table 3.

TABLE 3 screening of the inoculum size of Pichia pastoris co-cultures

The results show that: with the increase of the inoculum size of the pichia pastoris, the enzyme activity of the incision enzyme of the xanthan gum is firstly increased and then basically kept unchanged; the yield of the xanthan gum is increased and then reduced, the maximum is reached when the inoculation amount is 30% according to the volume ratio, and excessive yeast inoculation can influence the Xanthomonas campestris to produce the xanthan gum, so that the yield of the xanthan gum is reduced; the molecular weight of the xanthan gum is reduced and then kept unchanged, and the molecular weight is kept basically unchanged when the inoculum size of the pichia pastoris is more than 30% (v/v).

Thus, the optimal inoculum size of pichia pastoris was 30% by volume.

Example 5: pH regulation strategy in co-culture fermentation

The method comprises the following specific steps:

(1) The Xanthomonas campestris seed solution (OD) obtained in step (1) of example 2 ₆₀₀ 0.9) inoculating 10% (v/v) of the inoculum size to the fermentation medium prepared in step (1) of example 3, wherein the liquid loading amount of the fermentation medium is: 10% (v/v), at 30 ℃,220rpm under conditions of culture for 36 hours; obtaining the Xanthomonas campestris fermentation liquor. This stage is the Xanthomonas campestris cell growth stage, and the pH in stage (1) is controlled to 7.0.

(2) Into the Xanthomonas campestris fermentation broth prepared in the step (1)Adding the recombinant Pichia pastoris seed solution (OD) prepared in the step (2) of the example 2 according to the inoculation amount of 30% (v/v) of the volume ratio ₆₀₀ 3.5), and the yeast extract was added to a final concentration of 10g/L, and the pH was adjusted to 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, respectively, and the culture was continued at 30℃and 220rpm for 120 hours until the fermentation was completed. And respectively measuring the yield of the xanthan gum and the enzyme activity of the incision enzyme of the xanthan gum after fermentation. The results are shown in Table 4.

TABLE 4 pH control strategy in Co-culture fermentation

The results show that: when the pH of the co-culture stage is 6.0, the enzyme activity of the xanthan endonuclease is highest but the yield of the xanthan gum is lower, and when the pH of the co-culture stage is 6.5, the yield of the xanthan gum is highest and the molecular weight is lower. So 6.5 is the optimal pH.

Example 6: liquid loading amount screening of co-culture fermentation

The method comprises the following specific steps:

(1) The fermentation medium (prepared in step (1) of example 3) was adjusted to 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% of the shake flask capacity (volume ratio), and Xanthomonas campestris seed solution (OD) obtained in step (1) of example 2 ₆₀₀ 0.9) inoculating 10% (v/v) of the strain to a fermentation medium, and culturing at 30 ℃ and 220rpm for 36h to obtain a fermentation broth; controlling the pH of the step (1) to 7.0.

(2) Adding the recombinant pichia pastoris seed solution (OD) prepared in the step (2) of the example 2 into the Xanthomonas campestris fermentation broth prepared in the step (1) according to the inoculum size of 30% by volume ₆₀₀ 3.5), and the yeast extract was added to a final concentration of 10g/L, the pH was adjusted to 6.5, and the culture was continued at 30℃and 220rpm for 120 hours until the fermentation was completed. And respectively measuring the yield of the xanthan gum and the enzyme activity of the incision enzyme of the xanthan gum after fermentation. The results are shown in Table 5.

TABLE 5 liquid loading screening of Co-culture fermentations

The results show that: when the liquid loading amount is 10%, the xanthan gum yield and the xanthan gum endonuclease reach the maximum and the molecular weight of the xanthan gum is smaller.

Example 7: co-culture fermentation

In combination with the optimal fermentation conditions of examples 2 to 5, xanthomonas campestris and Pichia pastoris were subjected to co-culture fermentation, sampling was performed every 12 hours, and the xanthan gum yield and the xanthan gum endonuclease activity were determined.

The method comprises the following specific steps:

(1) The Xanthomonas campestris seed solution (OD) obtained in step (1) of example 2 ₆₀₀ 0.9) inoculating 10% (v/v) of the inoculum size to the fermentation medium prepared in step (1) of example 3, wherein the liquid loading amount of the fermentation medium is: 10% (v/v), and culturing at 30deg.C and 220rpm for 36 hr to obtain fermentation broth; controlling the pH of the step (1) to 7.0.

(2) Adding the recombinant pichia pastoris seed solution (OD) prepared in the step (2) of the example 2 into the Xanthomonas campestris fermentation broth prepared in the step (1) according to the inoculum size of 30% by volume ₆₀₀ 3.5), and the yeast extract was added to a final concentration of 10g/L, the pH was adjusted to 6.5, and the culture was continued at 30℃and 220rpm for 144 hours until the fermentation was completed. The xanthan gum yield and the endoenzyme activity of xanthan gum after fermentation are respectively measured, and the results are respectively shown in fig. 3: 5.32g/L, 395.87U/L.

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> university of Jiangnan

<120> a method for directly producing low molecular weight xanthan gum by co-culture fermentation

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<170> PatentIn version 3.3

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Ala Ala Thr Ile Asp Lys Val Thr Val Ser Gln Ala Gly Tyr Ser Ala

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Ser Gly Tyr Lys Val Gly Phe Ala Val Ala Asp Ser Ala Val Pro Gly

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Ser Thr Ser Cys Arg Leu Leu Gln Gly Glu Thr Val Val Leu Pro Ser

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Cys Thr Leu Leu Asp Arg Gly Thr Thr Trp Gly Asp Arg Val Tyr Gln

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Ile Gly Gly Val Arg Ser Pro Arg Phe Ala Ile Glu Asp Asn Val Trp

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Ser Gly Tyr Leu Asp Glu Met Ile Ala Phe Tyr Arg Leu Gln Arg Ser

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Gly Met Asp Thr Glu Asp Ala Tyr Pro Ala Gly Tyr Ser Ser Ile Ala

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Pro Ser Asp Lys Val Phe His Ala Ala Gly His Leu Asp Asp Ala Ala

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Ser Glu Asp Gly Thr Gln His Tyr Asp Leu Thr Gly Gly Trp Tyr Asp

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Ala Gly Asp Tyr Gly Ile Tyr Gly Gly Asn Gln Trp Val Ala Gly Asn

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Trp Asp Val Lys Gly Ser Gly Gly Phe Arg His Pro Glu Phe His Thr

260 265 270

Asp Gly Val Ile Gly Thr Ala Asp Asp Arg Arg Val Ser Gly Met Gly

275 280 285

Val Gly Gly Ser Ala Lys Ala Ser Gly Ser Leu Ala Ala Thr Ala Arg

290 295 300

Ala Ile Arg Ala Ala Ile Asp Ser Gly Asp Ile Asp Ala Gly Ala Ala

305 310 315 320

Ala Ser Trp Glu Thr Arg Ala Ala Glu Ala Glu Glu Ala Ala Val Ala

325 330 335

Phe Tyr Glu Tyr Ala Asp Thr His Arg Gly Asp Pro Leu Gly Gly Tyr

340 345 350

Ser Thr Thr Arg Gly Gly Ile Ala Asn Ser Leu Leu Phe Ala Glu Val

355 360 365

Gln Leu Tyr Leu Leu Ser Gly Asp Ala Ala Tyr Arg Thr Ser Ala Glu

370 375 380

Ala Thr Ile Ala Ala Thr Pro Phe Thr Ile Leu Ser Ser Thr Asn Tyr

385 390 395 400

Trp Asp Met Ala Pro Leu Ser Met Ala Glu Leu Tyr Pro Ala Ala Thr

405 410 415

Ala Thr Gly Lys Ile Asn Ile Gln Arg Tyr Leu Lys Lys Gln Leu Asp

420 425 430

Tyr Val Leu Ser Ser Thr Asp Asp Thr Pro Tyr Gly Val Ile Asn Gln

435 440 445

Phe Lys Asn Phe Gly Val Asn Glu Pro His Val Ser Tyr Met Ala Asp

450 455 460

Ala Leu Arg Tyr Trp Glu Leu Phe Gly Asp Gln Arg Ala Leu Arg Ala

465 470 475 480

Val Gln Lys Gly Leu Tyr Trp Val Phe Gly Asn Asn Pro Trp Gly Thr

485 490 495

Ser Trp Val Ser Gly Val Gly Glu Lys His Thr Met Phe Leu His Thr

500 505 510

Arg Leu Asp Glu Gln Ala Gln Thr Gln Gly Gly Thr Gly Ile Ile Leu

515 520 525

Pro Gly Ala Leu Val Ser Gly Pro Asn Ala Lys Asp Pro Leu Asp Ala

530 535 540

Thr Ser Ala Ser Pro Trp Tyr Glu Asp Arg Pro Gly Ser Ala Asp Val

545 550 555 560

Gly Gln Gln Trp Arg Tyr Asn Glu Tyr Ser Val Ser Ile Gln Ala Gly

565 570 575

Leu Phe Ser Ser Val Phe Gly Leu Ala Ala Ala Gly Asp Ala Pro Trp

580 585 590

Ser Thr Gly Thr Pro Pro Thr Ala Leu Thr Ile Gly Ser Pro Lys Ile

595 600 605

Gly Gln Tyr Val Thr Gly Glu Val Thr Val Phe Ala Gln Ser Gly Ser

610 615 620

Ser Leu Thr Ala His Ala Leu Gly Pro Asp Trp Thr Pro Met Thr Ser

625 630 635 640

Ser Ala Gly Val Ser Thr Gly Val Val Asp Val Ser Gly Leu Ala Pro

645 650 655

Tyr Thr Asn Ala Arg Ile Asp Val Arg Gly Thr Gln Ala Ser Gly Ala

660 665 670

His Ser Tyr Ser Ser Thr His Tyr Thr Val Ala Pro Pro Leu Pro Ala

675 680 685

Pro Asp Ala Pro Leu Leu Tyr Asp Gly Phe Gly Arg Asp Gly Leu Phe

690 695 700

Gly Val Gln Gly Tyr Thr Trp Ala Asn Trp Tyr Asn Asn His Ala Gly

705 710 715 720

Val Gly Gln Val Thr Asn Thr Ile Val Asp Gly Arg Thr Val Gly Arg

725 730 735

Phe Phe Gln Asn Pro Ala Thr Glu Gln Ser Gln Ala Lys Phe Gln Pro

740 745 750

Trp His His Ser Val Asp Ala Asp Gly Tyr Arg Tyr Leu Ser Val Thr

755 760 765

Met Arg Ser Pro Ser Pro Asn Leu Arg Leu Arg Ile Glu Val Ser Asp

770 775 780

Ala Asp Ser Asn His Arg Val Thr Gly Thr Thr Pro Ile Ala Ile Ser

785 790 795 800

Asn Asp Trp Met Thr Tyr Asp Phe Asp Leu Ala Ala Phe Pro Gly Leu

805 810 815

Asp Arg Ser Gln Ala Lys Leu Val Phe Trp Leu Gln Gln Thr Ala Asp

820 825 830

Thr Asp Gly Glu Leu Phe Val Asp Ser Val Glu Phe Thr Asn Glu Glu

835 840 845

Ala Gly Thr Pro Pro Val Leu Ser Asp Val Ser His Thr Ala Gly Pro

850 855 860

Leu Thr Pro Ser Thr Pro Val Thr Val Gln Ala Thr Tyr Thr Asp Ala

865 870 875 880

Asp Gly Val Ala Pro Tyr Ala Val Glu Leu Val Val Asp Gly Val Ile

885 890 895

His Arg Met Ser Pro Val Asp Pro Gly Asp Thr Asp Val Thr Asp Gly

900 905 910

Ala Gln Tyr Ser Val Ala Val Ser Leu Val Lys Gly Val His Ser Tyr

915 920 925

Phe Val Arg Thr Thr Asp Thr Thr Ser Ala Val Val Glu Thr Pro Leu

930 935 940

Val Thr Gly Val Ala Val Gly

945 950

<210> 2

<211> 2856

<212> DNA

<213> artificial sequence

<400> 2

atgtcccgac gacgagcgag ttcgatgtgg agaggtgcgg cagtcgtcac ggcggtcgtg 60

ctgggcgggg cggtgatcgc tgcgccgccc gccgcggccg ccaccatcga caaggtcacg 120

gtcagccagg cgggctacag cgccagcggt tacaaggtcg gcttcgccgt cgccgacagc 180

gcagttccgg gttcgaccag ctgtcgcctg ctccagggcg agacggtcgt gctgccgtcc 240

tgcactcttc tggatcgcgg cacgacctgg ggcgaccgcg tgtatcaggt cgacttcagc 300

gcgttcgacg acgtcggcac cgacttcgcc ctcgagatcg ggggtgtgcg ctccccgcgc 360

ttcgcgatcg aagacaacgt ctggtccggc tacctcgatg agatgatcgc gttctacagg 420

ttgcaacgct cgggaatgga caccgaggat gcataccccg cgggctacag cagcatcgcc 480

ccgtccgaca aggtcttcca cgccgccggt catctcgacg atgccgcgtc cgaggacggc 540

acgcagcact acgacctcac gggtggctgg tacgacgccg gcgactacgg catctacggt 600

gggaaccagt gggtcgcggg gaacatcgcc atctcgtatc tgcgatacgg cgacacaccc 660

gcggtcgggt tcgacggcga ctcgaacggc gtgcccgatc tcgtcgacga ggcctggttc 720

ggcagcgagt acctgctccg gatgctggac gctttcgggg gcccgttctg ggatgtcaag 780

ggcagcggcg gcttccggca tcccgagttc cacaccgacg gcgtgatcgg aacggctgac 840

gaccgacgtg tctccggcat gggtgtgggc ggctcggcca aggcgtcggg ttcgctcgcc 900

gccacagcga gagcgatccg cgccgccatc gacagcggag acatcgacgc cggagccgcc 960

gcgtcctggg agacccgggc cgccgaggcc gaggaagcag cggtcgcgtt ctacgagtac 1020

gccgacacgc accgcggaga tccgctcggc gggtactcga ccacgcgcgg cggcatcgcc 1080

aactccctgc tgttcgccga agtgcagctc taccttctgt cgggcgacgc ggcgtatcgc 1140

acgtcggccg aagcgacgat cgccgcgacc ccgttcacga tcctgtccag tacgaactac 1200

tgggacatgg cgccgctgtc gatggctgag ctgtatcccg ctgcgacggc gaccggaaag 1260

atcaacatcc agcgttacct caagaagcag ctcgactacg tcctctcctc gaccgacgac 1320

accccctacg gcgtgatcaa ccagttcaag aacttcggtg tcaacgagcc gcacgtctcc 1380

tacatggccg acgcgctgcg ctactgggag ctgttcggag accagcgtgc cctgcgagcg 1440

gtgcagaagg gcctgtactg ggtgttcggc aacaacccgt gggggacgag ctgggtctcc 1500

ggcgtgggtg agaagcacac gatgttcctg cacacgcggc tcgacgagca ggcgcagacc 1560

cagggcggca cggggatcat cctgccgggg gcgctcgtct cgggaccgaa tgcgaaggac 1620

ccgctcgacg cgaccagtgc gagcccctgg tacgaagatc gtccaggctc cgccgatgtc 1680

ggtcagcagt ggcgatacaa cgagtacagc gtgagcatcc aggctgggct gttctcgtcc 1740

gtgttcgggc tcgccgccgc cggcgacgcg ccgtggtcca ccgggacacc gccgacggcg 1800

ctgaccatcg gatccccgaa gatcggccag tacgtcaccg gcgaggtgac cgtgttcgcg 1860

cagagcggct cctcgctcac ggcgcacgcg ctcggtccgg attggacgcc gatgacctcg 1920

tcggccggag tctcgaccgg tgtggtcgat gtgagcggcc ttgcgccgta cacgaacgcc 1980

cgtatcgacg tgcgcggcac gcaggcgagc ggtgcccaca gctacagctc tacgcactac 2040

accgtcgcgc cacccctgcc ggctcccgac gcgccgctgc tgtacgacgg cttcggtcgc 2100

gacggcctgt tcggcgtgca ggggtacacc tgggcgaatt ggtacaacaa ccatgccggc 2160

gtcgggcagg tgaccaacac gatcgtggat ggacgtaccg tcggtcgctt cttccagaac 2220

cccgccaccg agcagtcgca ggcgaagttc cagccgtggc atcactcggt ggacgccgac 2280

gggtatcgct atctgtcggt gaccatgcgc agcccatcgc cgaatctccg cttgcgcatc 2340

gaggtctcgg acgcggactc gaaccaccgg gtgaccggga ccacgccgat cgcgatctcg 2400

aacgactgga tgacgtacga cttcgatctt gcggcgttcc ccggcctcga tcggtcgcag 2460

gcgaagctcg tgttctggct gcagcagacc gccgacaccg acggggagct cttcgtcgat 2520

tcggtggagt tcacgaacga ggaggccggc accccgccgg ttctttcgga cgtcagccat 2580

acggccggcc cgctgacacc atcgaccccg gtcacggtcc aggcgaccta caccgacgcc 2640

gacggagtcg ccccctatgc cgtcgagctc gtcgtcgacg gtgtgatcca tcggatgtcg 2700

cccgtcgatc ccggtgatac cgacgtgacc gatggcgcgc agtactccgt cgccgtgtcg 2760

ctcgtgaagg gcgtgcacag ctacttcgtg cgcacgaccg acacgacatc cgcggtggtc 2820

gagacgccgt tggtgacggg agtcgctgtg ggctga 2856

<210> 3

<211> 427

<212> DNA

<213> artificial sequence

<400> 3

aatgtcttgg tgtcctcgtc caatcaggta gccatctctg aaatatctgg ctccgttgca 60

actccgaacg acctgctggc aacgtaaaat tctccggggt aaaacttaaa tgtggagtaa 120

tggaaccaga aacgtctctt cccttctctc tccttccacc gcccgttacc gtccctagga 180

aattttactc tgctggagag cttcttctac ggcccccttg cagcaatgct cttcccagca 240

ttacgttgcg ggtaaaacgg aagtcgtgta cccgacctag cagcccaggg atggaaaagt 300

cccggccgtc gctggcaata atagcgggcg gacgcatgtc atgagattat tggaaaccac 360

cagaatcgaa tataaaaggc gaacaccttt cccaattttg gtttctcctg acccaaagac 420

tttaaat 427

Claims

1. The recombinant pichia pastoris is characterized in that the recombinant pichia pastoris takes pPIC9K as an expression vector, pichia pastoris GS115 as an expression host, and the xanthan gum endonuclease with the amino acid sequence shown as SEQ ID NO.1 is expressed.

2. The recombinant pichia pastoris of claim 1, wherein the nucleotide sequence of the xanthan endonuclease is shown in SEQ ID No. 2.

3. A method of producing xanthan gum, comprising:

(1) The Xanthomonas campestris is treatedXanthomonas campestris）X. campestris Cctccc NO: inoculating M2015714 seed liquid into a fermentation medium for culturing to obtain a culture liquid;

(2) Adding the recombinant pichia pastoris seed solution according to claim 1 or 2 into the culture solution obtained in the step (1), performing co-culture fermentation to obtain a fermentation solution, and extracting the fermentation solution to obtain the xanthan gum with low molecular weight.

4. The method of claim 3, wherein the inoculation amount of the recombinant pichia pastoris seed solution in the fermentation medium is 10-70% according to the volume ratio; the inoculation amount of the Xanthomonas campestris seed solution in the fermentation medium is 5-20% according to the volume ratio.

5. The method of claim 4, wherein the recombinant pichia pastoris OD seed solution ₆₀₀ At least 2.0; OD of the Xanthomonas campestris seed solution ₆₀₀ At least 0.8.

6. The method according to claim 5, wherein the method is:

(1) Inoculating the Xanthomonas campestris seed solution into a fermentation medium, and fermenting for 24-48 hours at 28-33 ℃ and 180-230 rpm to obtain a culture solution;

(2) And (3) adding the recombinant pichia pastoris seed solution into the culture solution obtained in the step (1), and performing co-culture fermentation under the conditions of 28-30 ℃ and 170-220 rpm.

7. The method of claim 6, wherein the fermentation medium comprises: 20-50 g/L glycerol, 1.5-4.5 g/L fish meal peptone, 1.0-2.0 g/L yeast extract and NaNO ₃ 0.6~1.0 g/L，MgSO ₄ ·7H ₂ O 2.0~3.0 g/L，FeSO ₄ ·7H ₂ O 0.005~0.015 g/L，K ₂ HPO ₄ ·3H ₂ O 3.0~4.0 g/L，KH ₂ PO ₄ 1.5~2.5 g/L。

8. Use of the recombinant pichia pastoris according to claim 1 or 2, or the method according to any one of claims 3 to 7, for the preparation of low molecular weight xanthan gum.