CN109593813B - Culture medium for fermentation of 2-keto-L-gulonic acid and fermentation production method of 2-keto-L-gulonic acid - Google Patents
Culture medium for fermentation of 2-keto-L-gulonic acid and fermentation production method of 2-keto-L-gulonic acid Download PDFInfo
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Abstract
The invention provides a culture medium for fermentation of 2-keto-L-gulonic acid, which contains 0.007-0.009 g/L of mixed nutrient, wherein the mixed nutrient comprises the following components in percentage by weight: 25-67% of cytochrome C and 33-75% of heme. The invention also provides a fermentation production method of the 2-keto-L-gulonic acid, which is characterized in that the mixed nutrient is supplemented when the concentration of the mixed nutrient in a fermentation culture medium is reduced to 0.001-0.003 g/L so as to maintain the concentration of the mixed nutrient to be 0.001-0.003 g/L until the fermentation is finished, or pyrroloquinoline quinone is supplemented when the online oxygen value is 20-40% so as to ensure that the concentration of the pyrroloquinoline quinone in the culture medium is 0.003-0.005 g/L, so that the fermentation period can be shortened, and the synthesis efficiency of the 2-keto-L-gulonic acid can be improved.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation, and particularly relates to a novel culture medium for fermentation of 2-keto-L-gulonic acid and a fermentation production method of 2-keto-L-gulonic acid, wherein the culture medium and the fermentation production method can improve the synthesis efficiency of the 2-keto-L-gulonic acid and shorten the fermentation period.
Background
Vitamin C (VC for short), L-Ascorbic acid (L-Ascorbic acid), is a water-soluble Vitamin that must be taken in by human body from outside, and is widely used in the fields of medicine, food, cosmetics, feed, etc. 2-keto-L-gulonic acid (2-keto-L-gulonic acid, 2-KGA for short) is an important precursor for synthesizing vitamin C, and at present, a two-step fermentation method is still a main process for industrially producing VC, wherein the second step of mixed fermentation is a key technology of the process, and only under the assistance of nutrition of large bacteria (or associated bacteria), small bacteria can normally grow and metabolize to synthesize related enzymes. Then, the microspecies convert sorbose into sorbosone by using sorbose dehydrogenase on a cell membrane, then convert the sorbosone into 2-KGA under the action of the sorbosone dehydrogenase, simultaneously transfer electrons separated from the sorbose and the sorbosone to oxygen through an electron transfer chain on the cell membrane to form oxygen anions, and the oxygen anions are combined with hydrogen ions derived from the sorbose and the sorbosone to finally generate water. Therefore, in the fermentation process, it is necessary to improve the synthesis efficiency of 2-KGA by improving the efficiency of enzyme activity and electron transfer.
Regarding the improvement of enzyme activity, patent document 1 found Fe3+、Mg2+And Ca2+Effective in increasing the activities of L-sorbose dehydrogenase and L-sorbosone dehydrogenase, but Cu2+Excessive Cu inhibits the activity of L-sorbosone dehydrogenase2+Resulting in autolysis of B.megaterium during fermentation, thereby affecting the yield of 2-KGA, while Mg is added2+And Mn2+This phenomenon can be effectively suppressed. Therefore, patent document 1 aims to improve the 2-KGA production by adding these metal ions to the medium appropriately to effectively control the enzyme activity.
Patent document 2 also optimizes the culture medium, and adds reduced glutathione to make the bacteria resist osmotic pressure and maintain normal in vivo redox environment, thereby promoting the mixed bacteria system to better adapt to fermentation environment and increasing the biomass of ketogulonic acid bacteria. Patent document 3 analyzes sugar metabolism networks of large and small bacteria based on sequencing and functional annotation of whole genomes of the large and small bacteria, and finds and verifies that a batch of cheap carbon sources, such as ethanol, acetaldehyde and acetic acid (salt), D-sorbitol, D-mannitol and D-mannose, can be well utilized by the small bacteria in a mixed fermentation process and have the effect of promoting the growth of bacteria.
Regarding the improvement of electron transfer efficiency, patent document 4 has found that lipoic acid, as a constituent of a coenzyme or prosthetic group, plays an important role in substance metabolism by performing a function of coupling acyl transfer and electron transfer. Thus, patent document 4 improves the growth and acid-producing ability of ketogulonic acid bacteria by supplementing such a nutrient factor to the culture medium.
Prior art documents:
patent document 1: CN101402988A
Patent document 2: CN102424830A
Patent document 3: CN104357529A
Patent document 4: CN102321698B
Disclosure of Invention
Problems to be solved by the invention
The inventor conducts intensive research on the sorbose metabolic pathway to produce 2-KGA, and tries to find a new key substance for effectively controlling enzyme activity and electron transfer efficiency, optimize a culture medium and improve a fermentation production method by using the substance.
In the sorbose metabolic pathway, sorbose is catalytically converted into sorbosone by sorbose dehydrogenase, and sorbosone is catalytically converted into 2-KGA by sorbosone dehydrogenase. Pyrroloquinoline quinone (PQQ) is a coenzyme of sorbose dehydrogenase and sorbosone dehydrogenase on a cell membrane of a microorganism, in the sorbose catalysis process, after the sorbose is dehydrogenated, the coenzyme PQQ of the sorbosone dehydrogenase transfers electrons to cytochrome C, and then the cytochrome C transfers the electrons to cytochrome terminal oxidase. In the catalytic process of sorbone, the heme and PQQ coenzyme of sorbone dehydrogenase jointly form a quinone-heme protein complex, after sorbone is dehydrogenated, PQQ transfers electrons to the heme, and the heme further transfers the electrons directly to cytochrome terminal oxidase. Cytochrome C and heme are key rate-limiting substances for cell membrane electron transfer, the content of cytochrome C and heme in cells is extremely low, the requirements of normal growth and metabolism of cells can be met only, the electron transfer rate is greatly limited, and the production requirements of rapid cell growth and efficient catalysis cannot be met.
Based on the characteristics of the metabolic pathway, the invention adds the mixed nutrient of the cytochrome C and the heme from an external source to increase the content of the cytochrome C and the heme in a mixed bacteria fermentation system, promotes the cell growth and metabolism, and assists the coenzyme PQQ to enhance the enzyme catalysis efficiency of the sorbosone dehydrogenase, thereby achieving the purpose of improving the synthesis efficiency of the 2-keto-L-gulonic acid.
At present, the prior art does not carry out systematic research on mixed fermentation metabolism change caused by mixed nutrients of coenzyme PQQ, cytochrome C and heme in the actual fermentation process, and does not carry out systematic research on a better and more economic control strategy of the mixed nutrients of coenzyme PQQ, cytochrome C and heme in the large-scale mixed fermentation production.
Means for solving the problems
The optimization of the culture medium of the present invention is mainly achieved by adding a mixed nutrient consisting of cytochrome C and heme at a specific concentration, and the improvement of the fermentation production method of 2-KGA of the present invention is mainly achieved by using the optimized culture medium, in some preferred embodiments, further using the culture medium and timely supplementing the mixed nutrient or coenzyme PQQ.
Specifically, the invention comprises the following technical scheme:
[1] a culture medium for fermentation of 2-keto-L-gulonic acid, said medium comprising a mixed nutrient consisting of cytochrome C and heme.
[2] The culture medium according to [1], wherein the content of the mixed nutrient in the culture medium is 0.007-0.009 g/L, preferably 0.0075-0.0085 g/L.
[3] The culture medium according to [1] or [2], wherein the mixed nutrient consists of, in weight percent: 25-67% of cytochrome C and 33-75% of heme.
[4] The medium according to any one of [1] to [3], wherein the medium has a pH of 6.0 to 8.0, and contains (g/L): 80-120 parts of L-sorbose, 4.0-6.0 parts of corn steep liquor, 11.0-13.0 parts of urea, 0.5-1.5 parts of monopotassium phosphate, 0.1-0.2 part of anhydrous magnesium sulfate, 4.0-6.0 parts of calcium carbonate and 0.007-0.009 part of the mixed nutrient.
[5] A method for producing 2-keto-L-gulonic acid by fermentation, wherein the method comprises inoculating a seed solution into a fermentation medium containing a mixed nutrient consisting of cytochrome C and heme to perform fermentation culture, thereby producing 2-keto-L-gulonic acid.
[6] The fermentation production method according to [5], wherein the content of the mixed nutrient in the culture medium is 0.007-0.009 g/L, preferably 0.0075-0.0085 g/L.
In some preferred embodiments of the invention, the mixed nutrient concentration in the fermentation medium is adjusted to 0.007 to 0.009g/L, preferably 0.0075 to 0.0085g/L before the inoculation of the seed liquid. In the early preliminary experiment, the invention discovers that the yield and the synthesis efficiency of 2-KGA can be obviously improved when the initial concentration of the mixed nutrient in the fermentation medium is 0.007-0.009 g/L. When the concentration of the mixed nutrient is less than 0.007g/L, the cytochrome C and the heme can only meet the requirement of normal growth and metabolism of cells, and the mass production of 2-KGA requires the vigorous growth of somatic cells and the promotion of metabolism, so that the concentration of the mixed nutrient needs to be improved. However, when the mixed nutrient concentration is higher than 0.009g/L, too high concentrations of cytochrome C and heme result in excessive cell growth and large consumption of raw materials, resulting in a decrease in the yield of 2-KGA and a decrease in the synthesis efficiency. The invention also discovers that when the concentration of the mixed nutrient is 0.0075-0.0085 g/L, the yield and the synthesis efficiency of 2-KGA can be improved, and the fermentation period can be effectively shortened.
[7] The fermentation production method according to [5] or [6], wherein the mixed nutrient consists of, in weight percent: 25-67% of cytochrome C and 33-75% of heme.
[8] The fermentation production method according to any one of [5] to [7], wherein the fermentation medium has a pH of 6.0 to 8.0 and contains (g/L): 80-120 parts of L-sorbose, 4.0-6.0 parts of corn steep liquor, 11.0-13.0 parts of urea, 0.5-1.5 parts of monopotassium phosphate, 0.1-0.2 part of anhydrous magnesium sulfate, 4.0-6.0 parts of calcium carbonate and 0.007-0.009 part of the mixed nutrient.
[9] The fermentation production method according to any one of [5] to [8], wherein the mixed nutrient is additionally added when the concentration of the mixed nutrient in the fermentation medium is reduced to 0.001 to 0.003 g/L.
In some preferred embodiments of the present invention, the mixed nutrient is supplemented when the concentration of the mixed nutrient in the fermentation medium is reduced to 0.001-0.003 g/L. The invention discovers that in the later stage of fermentation, when the concentration of the mixed nutrient in the fermentation medium is lower than 0.001g/L, cytochrome C and heme can not meet the requirement of rapid growth and metabolism of somatic cells, and the synthesis rate of 2-KGA is reduced, so that the fermentation period is prolonged, and therefore, the mixed nutrient needs to be supplemented timely. When the concentration of the mixed nutrient is higher than 0.003g/L, the cell overgrowth is caused if the mixed nutrient is supplemented, and the raw material in the medium is excessively consumed, thereby causing the reduction of the yield of 2-KGA.
[10] The fermentation production method according to [9], wherein the mixed nutrient is supplemented to maintain the concentration of the mixed nutrient in the fermentation medium at 0.001-0.003 g/L until the end of fermentation.
In some preferred embodiments of the present invention, the mixed nutrient is supplemented to maintain the concentration of the mixed nutrient in the fermentation medium at 0.001-0.003 g/L until the end of fermentation. The invention discovers that according to the real-time monitoring result of the acid yield, the mixed nutrient is supplemented in a fed-batch mode until the concentration is in the range of 0.001-0.003 g/L, and the higher yield and synthesis efficiency of 2-KGA can be obtained. Mixed nutrients outside this range can lead to cell growth with either insufficient or excessive metabolism, which is detrimental to 2-KGA production.
[11] The fermentation production process according to [9] or [10], wherein the mixed nutrients are supplementarily added by fed-batch.
[12] The fermentation production process according to any one of [5] to [11], wherein pyrroloquinoline quinone PQQ is additionally added so that the concentration thereof in the medium is 0.003 to 0.005g/L when the online dissolved oxygen value is 20 to 40%, preferably 25 to 35%, during the fermentation culture.
In some preferred embodiments of the present invention, the pyrroloquinoline quinone PQQ is additionally added to make the concentration in the culture medium 0.003-0.005 g/L when the online dissolved oxygen value is 20-40%, preferably 25-35%, during the fermentation culture. The invention discovers that the dissolved oxygen value in the fermentation culture process influences the electron transfer effect, oxygen is used as a final acceptor of electron transfer on a cell respiratory chain, oxygen deficiency influences the electron transfer efficiency, but oxygen is also an oxidant, oxygen excess influences the catalytic activity of cellular enzymes, and the specific expression is that the generation rate of 2-KGA is reduced when the online dissolved oxygen value is lower than 20% or higher than 40% at the later stage of the fermentation stage. The invention also finds that when the online dissolved oxygen value is in the range of 25-35%, the yield of 2-KGA can be obviously improved, the fermentation period is effectively shortened, and the synthesis efficiency of 2-KGA is further improved.
[13] The fermentation production method according to any one of [5] to [12], wherein the fermentation culture conditions are: the temperature is 24-34 ℃, the pH is 6.0-8.0, the stirring speed is 200-650 rpm, the ventilation volume is 5-18L/min, the tank pressure is 0.02-0.08 MPa, and the time is 40-60 hours.
[14] The fermentation production process according to any one of [5] to [13], wherein the process further comprises separating 2-keto-L-gulonic acid from the fermentation medium after completion of the fermentation.
[15] The fermentation production method according to any one of [5] to [14], wherein the seed liquid is a mixed strain of a microorganism selected from the group consisting of Bacillus megaterium (Bacillus megaterium), Bacillus cereus (Bacillus cereus), Bacillus subtilis, and an associated bacterium selected from the group consisting of Ketogulonigenium vulgare (Ketogulonigenium vulgare) and Bacillus megaterium (Bacillus cereus).
ADVANTAGEOUS EFFECTS OF INVENTION
The fermentation culture gene contains cytochrome C and heme with specific concentration, so that the enzyme catalysis efficiency and the electron transfer efficiency can be effectively improved, the growth and metabolism of cells are promoted, and the synthesis efficiency of 2-KGA is further improved. In addition, the 2-KGA fermentation production method of the invention uses the cytochrome C, the heme and the PQQ in a combined way, so that the cytochrome C and the heme have good assistance effect on coenzyme PQQ, the enzyme catalysis efficiency of the sorbosone dehydrogenase is effectively enhanced, and the synthesis efficiency of the 2-KGA can be improved while the cell growth and metabolism are promoted through the synergistic effect of the three substances in an electron transfer chain.
The culture medium and the fermentation production method have the following advantages:
1) after the culture medium is optimized, the reasonable addition of the cytochrome C and the heme can shorten the fermentation period by 9.8 percent and improve the synthesis efficiency of 2-KGA by 11.6 percent.
2) The reasonable addition of cytochrome C and heme combined with the timely supplement of PQQ can shorten the fermentation period by 17.6 percent and improve the synthesis efficiency of 2-KGA by 23.1 percent.
Detailed Description
In order to better describe the present invention, the following examples are provided for reference. It should be noted that the following examples are only for describing the present invention, and the present invention is not limited thereto.
The source of the strain used in the present invention, the preparation of the medium, the preparation of the mixed strain, the fermentation culture conditions, and the measurement method of the product can be obtained or carried out in the following manner.
(I) origin of the strains
The mixed strain system including a pediococcus and a pediococcus used in the present invention is not limited as long as 2-KGA can be obtained by the mixed strain system in the case of using the culture medium of the present invention. The micrococcus includes a strain conventionally used in the art, preferably ketogulonogenic bacterium vulgare (Ketogulonigenium vulgare), i.e., ketogulonogenic bacterium. The accompanying bacteria include strains conventionally used in the art, preferably Bacillus megaterium (Bacillus megaterium), Bacillus cereus (Bacillus cereus), Bacillus subtilis (Bacillus subtilis), Bacillus thuringiensis (Bacillus thuringiensis), and the like.
The small bacteria and the accompanying bacteria of the mixed bacterial system used in the following examples are respectively: the small bacteria are ketogenic gulonobacterium vulgare (Ketogulonigenium vulgare) (named Gluconobacter oxydans (CGMCC oxydans) with the preservation number of CGMCC NO.1.110, purchased from China general microbiological culture Collection center, No. 3 of West Lu 1 of the morning area of the Yangyo area in Beijing, and the microbial research institute of Chinese academy of sciences, zip code 100101); the accompanying bacteria is Bacillus megaterium (CGMCC NO.1.432, purchased from China general microbiological culture Collection center, No. 3 of West Lu No.1 of Chaoyang district, Beijing, Japan, and postal code 100101).
(II) preparation of culture Medium
The following media components were prepared using materials commonly used in the art, and were prepared by methods well known in the art, each having the following composition:
1) solid medium (g/L): 60-80 parts of L-sorbose, 4.0-6.0 parts of corn steep liquor, 11.0-13.0 parts of urea, 0.5-1.5 parts of monopotassium phosphate, 0.1-0.2 part of anhydrous magnesium sulfate, 4.0-6.0 parts of calcium carbonate, 0.007-0.009 part of mixed nutrient, 15-20 parts of agar and 6.0-8.0 parts of pH.
2) Liquid medium (g/L): 80-120 parts of L-sorbose, 4.0-6.0 parts of corn steep liquor, 11.0-13.0 parts of urea, 0.5-1.5 parts of monopotassium phosphate, 0.1-0.2 part of anhydrous magnesium sulfate, 4.0-6.0 parts of calcium carbonate, 0.007-0.009 parts of mixed nutrient and 6.0-8.0 parts of pH.
3) Fermentation medium (g/L): 80-120 parts of L-sorbose, 4.0-6.0 parts of corn steep liquor, 11.0-13.0 parts of urea, 0.5-1.5 parts of monopotassium phosphate, 0.1-0.2 part of anhydrous magnesium sulfate, 4.0-6.0 parts of calcium carbonate, 0.007-0.009 parts of mixed nutrient and 6.0-8.0 parts of pH.
4) Sterilization conditions: sterilizing at 115 deg.C for 25min with conventional sterilizing equipment, wherein the sorbose alone is sterilized at 115 deg.C for 25 min. The solid medium was sterilized and plated.
5) Preparing a mixed nutrient: the weight percentage composition is as follows: and (3) carrying out filtration sterilization on 25-67% of cytochrome C and 33-75% of heme in an ultra-clean workbench for later use without high-temperature sterilization.
6) Preparation of coenzyme PQQ mother liquor: PQQ preparation mother liquor with PQQ concentration of 0.010g/L is added into purified water, and the solution is filtered and sterilized in a clean bench only for standby.
(III) preparation of mixed strains:
respectively inoculating the small bacteria and the associated bacteria on a solid culture medium, and culturing for 24-36 h at 28 ℃. Then, respectively preparing small bacteria and associated bacteria on a solid culture medium into bacterial suspensions, respectively inoculating 1mL of the small bacteria and the associated bacteria into a seed culture medium, simultaneously respectively inoculating 1mL of the small bacteria and the associated bacteria into the seed culture medium in a mixed manner, wherein the liquid loading amount of a triangular flask is 25mL/250mL, the liquid loading amount of the triangular flask is 28-30 ℃, the shaking table rotation speed is 180-200 rpm, culturing is carried out for 18-20 h, and then inoculating the cultured small bacteria and the associated bacteria into a corresponding fermentation culture medium in an inoculation amount of 5%, wherein the liquid loading amount of the triangular flask is 25mL/250mL, the liquid loading amount of the triangular flask is 28-. And detecting the content of residual sugar and 2-KGA and the pH value in the fermentation process, and performing microscopic examination on the thallus morphology.
(IV) fermentation culture conditions:
the temperature is 24-34 ℃, the pH is 6.0-8.0, the stirring speed is 200-650 rpm, the ventilation volume is 5-18L/min, the tank pressure is 0.02-0.08 MPa, and the time is 40-60 h.
(V) detection method
1) Method for measuring 2-KGA content
The determination principle is as follows:
2-KGA is converted into VC through lactone enolization reaction in strong acid medium, and the VC content is measured and converted into 2-KGA content by VC and iodine redox reaction.
The determination method comprises the following steps: a liquid transfer gun sucks 2mL of a sample into a test tube, 2mL of 7mol/L sulfuric acid is added, the sample is uniformly shaken and heated in a boiling water bath for 25min (2-KGA lactonization generates VC), the sample is taken out and cooled, purified water is used for washing into a 250mL triangular flask for 3-4 times, 3mL of 0.5% starch solution is added, and the solution is titrated by 0.1mol/L iodine solution until the blue color is not faded for 30s (the VC can be oxidized by the iodine, and the iodine turns blue when meeting the starch after the VC is consumed), namely the titration end point.
The calculation method comprises the following steps:
wherein:
c- -concentration of iodometric solution (mol/L)
V- -sample consumption iodine titration volume (mL)
0.9071- -VC molecular weight/2-KGA molecular weight
8.806- -0.05 mol/L iodine standard per 1mL corresponds to 8.806mg of VC
Molar conversion of A- - - -2-KGA to VC (in 63.10%)
B- -accurately draw the volume of fermentation broth (mL)
0.05- -theoretical concentration of iodine Standard solution
2) Method for measuring residual sugar content
Adopting an anthrone method to measure the residual sugar in the fermentation liquor, which comprises the following steps: taking 1mL of fermentation liquid sample, adding purified water into a clean volumetric flask, diluting to a constant volume to a certain multiple, sucking 1mL of the diluted liquid into a clean dry test tube, adding 6mL of anthrone solution, shaking up by using a vortex oscillator, and standing at room temperature for 20 min; the blank control replaces the fermentation liquor sample with purified water to carry out the operation; the absorbance value was measured colorimetrically at a wavelength of 620nm using an ultraviolet spectrophotometer, and then converted into the residual sugar content. When the residual sugar content of the fermentation liquor is less than or equal to 0.5mg/mL, the fermentation end point is determined.
3) Method for measuring online dissolved oxygen value
Installing a METTLER dissolved oxygen electrode (electrode model: InPro 6800, Metlerthelo Tooliduo instruments (Shanghai) Co., Ltd.), adjusting each process parameter to a preset value before starting fermentation, correcting the online dissolved oxygen parameter value by adopting a two-point method (respectively correcting the online dissolved oxygen value of 0% and the online dissolved oxygen value of 100%), and then carrying out process operation according to the online dissolved oxygen value.
Example 1
1) Preparing a mixed nutrient, wherein the mixed nutrient comprises the following components in percentage by weight: 33% of cytochrome C, 67% of heme. After the preparation is finished, the mixture is filtered and sterilized in a super clean bench for standby.
2) Preparation of fermentation Medium (g/L): 120.0 parts of L-sorbose, 5.0 parts of corn steep liquor, 12.0 parts of urea, 1.0 part of monopotassium phosphate, 0.1 part of anhydrous magnesium sulfate, 5.0 parts of calcium carbonate, 0.008 part of mixed nutrient, pH value adjustment of 7.0 part, and sterilization at 115 ℃ for 25min, wherein the sorbose is sterilized at 115 ℃ for 25min alone.
3) The seed solution of the expanded culture was inoculated to a fermentation medium in an inoculum size of 20%, and the mixture was fermented in a 15L fermentor (70% liquid loading), at a temperature of 32 ℃ and a pH of 6.8, at a stirring speed of 600rpm, at a ventilation rate of 15L/min, and under a tank pressure of 0.03MPa, to synthesize 2-KGA. And when the concentration of the mixed nutrient is reduced to 0.002g/L in the middle and later stages of fermentation, maintaining the concentration of the mixed nutrient in the fermentation liquor to be 0.002g/L to the end point of fermentation by adopting a fed-batch mode. The fermentation period is 46h, the end acid amount is 114.62mg/mL, the residual sugar is 0.32mg/mL, and the synthesis efficiency of 2-KGA is 2.492 mg/mL-h-1。
Examples 2 to 8 and comparative examples 1 to 3
The contents of cytochrome C and hemoglobin in the mixed nutrient, the initial addition concentration of the mixed nutrient, the fed-batch concentration of the mixed nutrient, and the like were adjusted according to the method of example 1, and the other conditions were the same as in example 1, and the results are shown in table 1 below:
TABLE 1
As is clear from the data in Table 1, the media of comparative examples 1 to 3 were not supplemented with cytochrome and/or hemoglobin, and compared to the media supplemented with cytochrome and/or hemoglobin, examples 1 to 8 were shorter in fermentation cycle and higher in 2-KGA synthesis efficiency.
Wherein, the culture medium of the embodiment 1-5 is added with the cytochrome C with the content of 25-67% and the heme with the content of 33-75%, the concentration of the mixed nutrient in the fermentation culture medium before inoculation is kept in the range of 0.007-0.009 g/L, and the mixed nutrient is further added in the middle and later stages of fermentation to keep the concentration in the range of 0.001-0.003 g/L, so that the cytochrome C and the heme are sufficiently supplied in the whole fermentation process of the embodiment 1-5, the growth and metabolism of cells are favorably promoted, and the higher 2-KGA yield and the synthesis efficiency are realized in a shorter fermentation period than those of the embodiment 1-3. Wherein, the effect of the embodiment 1 is best, compared with the comparative example 1 without adding the mixed nutrient, the fermentation period can be shortened by 9.8 percent, and the synthesis efficiency of the 2-KGA is improved by 11.6 percent.
However, when the content of cytochrome C in the mixed nutrient is not in the range of 25 to 67% and the content of hemoglobin is not in the range of 33 to 75% (comparative examples 1 to 3), the fermentation period is long, and the yield and synthesis efficiency of the product 2-KGA are low. In addition, when the concentration of the mixed nutrient in the fermentation medium before inoculation is not in the range of 0.007-0.009 g/L (example 8), or the mixed nutrient is not supplemented at a proper time in the middle and later stages of fermentation to maintain the concentration in the range of 0.001-0.003 g/L (examples 6-7), the fermentation period is long or the synthesis efficiency of the product 2-KGA is slightly low, but is still shorter than that of comparative examples 1-3 and the synthesis efficiency of 2-KGA is high, compared with examples 1-5.
Therefore, cytochrome C and heme are used as key rate-limiting substances for cell membrane electron transfer, and the content of cytochrome C and heme in the initial medium and the middle and later stages of fermentation is properly adjusted to promote the growth and metabolism of cells and improve the enzyme activity (examples 1-5). However, too high concentrations of cytochrome C and heme result in excessive cell growth and excessive consumption of raw materials, resulting in a decrease in 2-KGA production and a decrease in synthesis efficiency (examples 7 to 8).
Example 9
1) Preparing a mixed nutrient, wherein the mixed nutrient comprises the following components in percentage by weight: 33% of cytochrome C, 67% of heme. A coenzyme PQQ mother liquor was prepared, wherein the concentration of coenzyme PQQ was 0.10 g/L. Respectively filtering and sterilizing in a superclean bench for later use.
2) Preparation of fermentation Medium (g/L): 120.0 parts of L-sorbose, 5.0 parts of corn steep liquor, 12.0 parts of urea, 1.0 part of monopotassium phosphate, 0.1 part of anhydrous magnesium sulfate, 5.0 parts of calcium carbonate, 0.008 part of mixed nutrient, pH value adjustment of 7.0 part, and sterilization at 115 ℃ for 25min, wherein the sorbose is sterilized at 115 ℃ for 25min alone.
3) Inoculating the liquid of the expanded culture with 20% of inoculum size into fermentation medium containing mixed nutrient, fermenting with 15L fermentation tank (liquid content 70%), at 32 deg.C, pH6.8, stirring speed 600rpm, ventilation 12L/min, and tank pressure 0.03MPa, and maintaining mixed nutrient in the fermentation liquid by feeding when the concentration of mixed nutrient is reduced to 0.002g/LThe concentration of the nutrient is 0.002g/L to the end point of fermentation. And when the online dissolved oxygen value is reduced to 30% in the fermentation process, supplementing PQQ, and supplementing coenzyme PQQ by adopting a fed-batch and material-supplementing mode to maintain the concentration of PQQ in the fermentation liquor at 0.004g/L to the fermentation end point. The fermentation period is 42h, the end acid amount is 115.02mg/mL, the residual sugar is 0.37mg/mL, and the 2-KGA synthesis efficiency is 2.748 mg/mL-h-1。
Examples 10 to 13 and reference examples 1 to 3
The method of example 9 was followed to adjust parameters such as PQQ concentration after feeding and on-line dissolved oxygen value during feeding, and the other conditions were the same as in example 9, and the results are shown in table 2 below:
TABLE 2
As can be seen from the data in Table 2, in comparison with reference example 1 in which PQQ was not fed, in examples 9 to 13, PQQ was fed when the on-line dissolved oxygen value of the fermentation culture was 20% to 40% and the concentration thereof in the medium was 0.003 to 0.005g/L, thereby effectively shortening the fermentation period and improving the yield of 2-KGA and the synthesis efficiency. Among them, example 9 is the most effective, and compared with reference example 1, the cycle is shortened by 8.7%, and the 2-KGA synthesis efficiency is improved by 10.3%, and compared with comparative example 1 in which neither mixed nutrients nor PQQ are added, the cycle is shortened by 17.6%, and the 2-KGA synthesis efficiency is improved by 23.1%.
In contrast, in reference examples 2 to 3, PQQ was not fed at an appropriate oxygen-dissolving time or the concentration of PQQ fed was too high, and thus the fermentation period was long, and the 2-KGA yield and synthesis efficiency were low.
The present inventors have found that, as a result of analysis of the above results, PQQ as a coenzyme, in combination with sorbose dehydrogenase and sorbosone dehydrogenase can improve the enzymatic catalytic efficiency of the sorbose dehydrogenase and sorbosone dehydrogenase, and therefore, when cells enter the stationary phase, the catalytic efficiency of the enzymes can be effectively improved by adding a proper amount of PQQ after the sorbose dehydrogenase and sorbosone dehydrogenase reach a saturated concentration, but PQQ having an excessively high concentration has a certain toxic effect on the growth and metabolism of the cells, and is not favorable for the synthesis of 2-KGA.
Industrial applicability
Cytochrome C, heme and coenzyme PQQ are key factors for improving related enzyme activity and electron transfer efficiency in a sorbose metabolic pathway, and can effectively promote growth and metabolism of cells and improve the yield and synthesis efficiency of 2-KGA. By using the optimized culture medium and timely supplementing the mixed nutrient consisting of cytochrome C and heme, the fermentation period can be shortened by 9.8%, and the synthesis efficiency of 2-KGA can be improved by 11.6%; on the basis, the fermentation production method is further improved, namely PQQ is supplemented timely at a specific stage of fermentation, so that the fermentation period is shortened by 17.6%, and the synthesis efficiency of 2-KGA is improved by 23.1%. Therefore, the culture medium and the fermentation method have important significance for industrial large-scale production of 2-KGA.
Claims (7)
1. A fermentation production method of 2-keto-L-gulonic acid, wherein the method comprises inoculating seed liquid into a fermentation culture medium containing mixed nutrients consisting of cytochrome C and heme to perform fermentation culture so as to produce the 2-keto-L-gulonic acid,
the pH value of the culture medium is 6.0-8.0, and the culture medium contains the following components in g/L: 80-120 parts of L-sorbose, 4.0-6.0 parts of corn steep liquor, 11.0-13.0 parts of urea, 0.5-1.5 parts of monopotassium phosphate, 0.1-0.2 part of anhydrous magnesium sulfate, 4.0-6.0 parts of calcium carbonate, 0.007-0.009 part of mixed nutrient,
wherein the mixed nutrient comprises the following components in percentage by weight: 25-67% of cytochrome C and 33-75% of heme,
sterilizing the medium at 115 ℃ for 25min, wherein the sorbose is sterilized alone at 115 ℃ for 25min,
when the concentration of the mixed nutrient in the fermentation medium is reduced to 0.001-0.003 g/L, the mixed nutrient is supplemented to maintain the concentration of the mixed nutrient in the fermentation medium to be 0.001-0.003 g/L until the fermentation is finished, and in the fermentation culture process, when the online dissolved oxygen value is 20-40%, pyrroloquinoline quinone is supplemented to ensure that the concentration of pyrroloquinoline quinone in the medium is 0.003-0.005 g/L.
2. The fermentation production method of claim 1, wherein the content of the mixed nutrient in the culture medium is 0.0075-0.0085 g/L.
3. The fermentative production method of claim 1, wherein the mixed nutrient is supplemented by fedbatch.
4. The fermentation production method of claim 1, wherein pyrroloquinoline quinone is added to the culture medium at a concentration of 0.003-0.005 g/L when the online dissolved oxygen value is 25-35%.
5. The fermentative production method of claim 1 or 2, wherein the fermentation culture conditions are: the temperature is 24-34 ℃, the pH is 6.0-8.0, the stirring speed is 200-650 rpm, the ventilation volume is 5-18L/min, the tank pressure is 0.02-0.08 MPa, and the time is 40-60 hours.
6. The fermentative production method according to claim 1 or 2, wherein the method further comprises isolating 2-keto-L-gulonic acid from the fermentation medium after fermentation is complete.
7. The fermentative production method according to claim 1 or 2, wherein the seed solution is a mixed strain of a microorganism selected from the group consisting of Bacillus megaterium, Bacillus cereus, Bacillus subtilis and Bacillus thuringiensis and an associated microorganism selected from the group consisting of Ketogulonigenium vulgare and Bacillus subtilis.
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| CN1685050A (en) * | 2002-09-27 | 2005-10-19 | 帝斯曼知识产权资产管理有限公司 | Process for producing vitamin C |
| CN102321698A (en) * | 2011-08-16 | 2012-01-18 | 中国人民解放军军事医学科学院生物工程研究所 | Method for promoting growth and acid production of Ketogulonigenium vulgare |
| CN106434830A (en) * | 2016-12-20 | 2017-02-22 | 宁夏启元药业有限公司 | Method for improving 2-keto-L-gluconic acid fermentation efficiency |
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| CN1685050A (en) * | 2002-09-27 | 2005-10-19 | 帝斯曼知识产权资产管理有限公司 | Process for producing vitamin C |
| CN102321698A (en) * | 2011-08-16 | 2012-01-18 | 中国人民解放军军事医学科学院生物工程研究所 | Method for promoting growth and acid production of Ketogulonigenium vulgare |
| CN106434830A (en) * | 2016-12-20 | 2017-02-22 | 宁夏启元药业有限公司 | Method for improving 2-keto-L-gluconic acid fermentation efficiency |
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| 酮古龙酸菌与呼吸链偶联的2-KGA代谢途径研究进展;李野等;《微生物学报》;20141004;第54卷(第10期);1101-1108 * |
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