CN113186176B - Method for producing rhizomucor miehei lipase by total synthesis culture medium and fermentation method - Google Patents
Method for producing rhizomucor miehei lipase by total synthesis culture medium and fermentation method Download PDFInfo
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Abstract
The application discloses a fully synthetic culture medium and a method for producing rhizomucor miehei lipase by a fermentation method. The molar weight ratio of carbon element to nitrogen element in the total synthesis culture medium is 11-18; the total synthesis culture medium comprises a carbon source and a nitrogen source, wherein the carbon source is sucrose, trehalose, lactose or fructose; the nitrogen source is alanine. The application provides a total synthesis culture medium for aspergillus oryzae fermentation production rhizomucor miehei lipase uses sucrose, trehalose etc. as the carbon source, uses alanine as the nitrogen source, and uses scientific carbon-nitrogen ratio cooperation addition use to carbon source and nitrogen source have obvious interact, can effectively promote aspergillus oryzae synthesis rhizopus miehei lipase. The activity of the Mucor miehei lipase obtained by fermenting and culturing the fully synthetic culture medium is as high as 60.42U/ml, and the effect is obvious.
Description
Technical Field
The application relates to the technical field of industrial microorganisms, in particular to a method for producing rhizomucor miehei lipase by a fully synthetic culture medium and a fermentation method.
Background
Rhizomucor Miehei Lipase (RML) is the enzyme which has obtained crystal structure analysis and 3D structure analysis at the earliest time, and is one of the most representative fungal lipases due to its huge application potential in industrialization. Rhizomucor Miehei Lipase (RML) has good stability and stereoselectivity, and can participate in various reactions, such as: the hydrolysis, alcoholysis, esterification and the like of the esters can be widely applied to the aspects of food, chemical industry, feed production, detergents, biodiesel and the like. The naturally existing rhizomucor miehei lipase is few and unstable, and can not be used in large scale in industrialization. Common exogenous lipase expression hosts comprise saccharomycetes, escherichia coli and filamentous fungi, and compared with the filamentous fungi, the filamentous fungi have a strong protein post-modification function and are beneficial to the stability and secretion of extracellular protease. Aspergillus oryzae is one of the commonly used production strains in fermentation production, is commonly used for fermentation production of soy sauce, and is a high-quality and high-yield strain of neutral protease. Aspergillus oryzae was included as a safe strain in GRAS (general certified as safe). Aspergillus oryzae has strong protein expression characteristic, can be used as an expression vector to produce various enzymes such as lipase, protease, amylase, glucoamylase and the like, has high expression level and strong secretion capacity, and is a good producer.
The wild type R.miehei strain has poor lipase production capability and poor enzyme solution stability, so that the enzyme quantity of the natural Rhizomucor Miehei Lipase (RML) is very low, the process scale application is not enough, and the development is limited. However, aspergillus oryzae has good growth characteristics and high-efficiency protein synthesis and secretion capacity, and can be used as a host for stably expressing exogenous protein, so that the rhizomucor miehei lipase synthesized by fermenting aspergillus oryzae has wide application prospect, and people often use yeast as a rhizomucor miehei lipase expression host cell in the past, zhang Yu and the like successfully construct pPIC9K-rml inducible expression vectors, and the highest rhizomucor miehei lipase activity in fermentation supernatant fluid measured by a p-NP method after fermentation conditions are optimized reaches 116U/mL, which is improved by 3.14 times compared with that before the optimization.
The culture medium can provide nutrients necessary for cell growth and product synthesis for the microorganisms, and the components and concentrations of the culture medium play a crucial role in microbial fermentation culture. At present, however, aspergillus oryzae is mainly cultured by using a complex medium, but the components of Aspergillus oryzae are complex, and the quantitative analysis and research on the metabolic characteristics of cells are not facilitated. Although conventional synthetic media for filamentous fungi are basically suitable for Aspergillus oryzae, rhizomucor miehei lipase activity is low and thus difficult to use in practical fermentation processes.
Therefore, it is needed to provide a total synthesis medium for aspergillus oryzae fermentation to produce rhizomucor miehei lipase and a fermentation method of the rhizomucor miehei lipase, which can obtain the rhizomucor miehei lipase with high enzyme activity.
Disclosure of Invention
The application provides a total synthesis culture medium, which is used for producing Rhizomucor miehei lipase by Aspergillus oryzae fermentation, can promote Aspergillus oryzae to synthesize Rhizomucor Miehei Lipase (RML), and further obtains the Rhizomucor miehei lipase with high enzyme activity.
The present application provides a total synthetic medium in which the molar ratio of carbon to nitrogen in the total synthetic medium (C: N = N) c /n N Carbon to nitrogen ratio) Is 11 to 18:1; the total synthesis culture medium comprises a carbon source and a nitrogen source, wherein the carbon source is sucrose, trehalose, lactose or fructose; the nitrogen source is alanine.
The total synthetic culture medium is used for producing Rhizomucor miehei lipase by Aspergillus oryzae fermentation.
Alternatively, in some embodiments of the present application, the ratio of the molar amount of the carbon element in the carbon source to the nitrogen element in the nitrogen source may be 12: 1. 14:1 or 16:1.
optionally, in some embodiments of the present application, when the carbon source is sucrose, the concentration of sucrose in the total synthesis medium is 15 to 22g/L; and/or
When the carbon source is trehalose, the concentration of the trehalose in the total synthesis culture medium is 30-40 g/L; and/or
When the carbon source is lactose, the concentration of the lactose in the total synthesis culture medium is 25-30 g/L; and/or
When the carbon source is fructose, the concentration of the fructose in the total synthesis culture medium is 26-28 g/L.
Optionally, in some embodiments of the present application, the total synthetic medium further comprises metal ions comprising Ca 2+ And/or Mn 2+ 。
Optionally, in some embodiments of the present application, the total synthetic medium contains Mn 2+ The addition amount of (B) is 0.2-0.6% of the total mass of the total synthesis culture medium.
Optionally, in some embodiments of the present application, the total synthetic medium comprises Ca 2+ The addition amount of (B) is 0.02-0.06% of the total mass of the total synthesis culture medium.
Optionally, in some embodiments of the present application, the total synthesis medium comprises a carbon source and a nitrogen source, wherein the carbon source is sucrose and the nitrogen source is alanine;
the total synthesis culture medium also comprises manganese sulfate, potassium dihydrogen phosphate, sodium chloride, magnesium sulfate heptahydrate, anhydrous calcium chloride, disodium hydrogen phosphate dodecahydrate, zinc sulfate heptahydrate, copper sulfate pentahydrate, nickel chloride hexahydrate, ferrous sulfate heptahydrate and a defoaming agent. The pH value of the total synthetic culture medium is 4.5-5.5.
Optionally, in some embodiments of the present application, the total synthetic medium comprises the raw materials: 18 to 21g/L of cane sugar, 3.5 to 4.2g/L of alanine, 0.7 to 1.0g/L of manganese sulfate, 0.65 to 1.4g/L of monopotassium phosphate, 0.7 to 1.3g/L of sodium chloride, 0.9 to 1.1g/L of magnesium sulfate heptahydrate, 0.08 to 0.15g/L of anhydrous calcium chloride, 9 to 12g/L of disodium hydrogen phosphate dodecahydrate, 0.01 to 0.8g/L of zinc sulfate heptahydrate, 0.0009 to 0.15g/L of copper sulfate pentahydrate, 0.0002 to 0.04g/L of nickel chloride hexahydrate, 0.004 to 0.007g/L of ferrous sulfate heptahydrate and 0.5 to 1.5g/L of defoaming agent. The pH value of the total synthetic culture medium is 4.5-5.5.
Optionally, in some embodiments of the present application, the defoamer is a polyether foam powder.
Optionally, in some embodiments of the present application, the total synthetic medium comprises the raw materials: 19.60g/L of sucrose, 4.02g/L of alanine, 0.86g/L of manganese sulfate, 1.31g/L of potassium dihydrogen phosphate, 0.75g/L of sodium chloride, 1g/L of magnesium sulfate heptahydrate, 0.13g/L of anhydrous calcium chloride, 10.5g/L of disodium hydrogen phosphate dodecahydrate, 0.0126g/L of zinc sulfate heptahydrate, 0.000925g/L of copper sulfate pentahydrate, 0.000225g/L of nickel chloride hexahydrate, 0.00517g/L of ferrous sulfate heptahydrate and 1g/L of defoaming agent. The pH of the total synthetic medium was about 5.0.
In the present application, the molar amount of carbon element (abbreviated as carbon molar amount) in the total synthesis medium is the total molar amount of carbon element in the carbon source (e.g., sucrose, etc.) and the nitrogen source (e.g., alanine, etc.). The molar amount of nitrogen element (nitrogen molar amount) in the total synthesis medium is the total molar amount of nitrogen element in the nitrogen source (e.g., alanine, etc.).
For example, the calculation method of the carbon-nitrogen ratio: the carbon-nitrogen ratio refers to the molar ratio of carbon to nitrogen in the medium components, namely C: n = N C /n N . The molar amount of carbon in the medium is derived from the total molar amount of carbon in sucrose and alanine; the molar amount of nitrogen is the total molar amount of nitrogen element in alanine.
The molar amounts of carbon and nitrogen sources are calculated according to the definition of molar amounts, i.e. the ratio of carbon content, nitrogen content to relative molecular mass.
Accordingly, the present application also provides a method for producing rhizomucor miehei lipase by fermentation, the method comprising: inoculating the seed liquid of aspergillus oryzae into the total synthetic culture medium for fermentation culture to obtain fermentation liquid; and (3) separating the rhizomucor miehei lipase from the fermentation liquor.
Optionally, in some embodiments of the present application, in the method, a seed solution is inoculated into the total synthetic medium after sterilization in an inoculum size of 10%.
Alternatively, in some embodiments of the present application, the conditions of the fermentation culture are: the culture temperature is 30 + -2 deg.C, the rotation speed is 150 + -50 rpm, and the culture time is 120 + -20 hours.
Optionally, in some embodiments of the present application, the fermentation culture process specifically includes: inoculating the seed solution into the sterilized fully synthetic culture medium according to the inoculation amount with the volume concentration of 10%, and performing fermentation culture at the culture temperature of 30 +/-2 ℃ and the rotation speed of 150 +/-50 rpm for 120 +/-20 h to obtain the fermentation liquid.
The total synthesis culture medium is sterilized at 121 ℃ for 30 min.
Optionally, in some embodiments of the present application, the preparation of the seed liquid: inoculating the spore suspension into a seed culture medium under the aseptic condition, carrying out shake culture at the culture temperature of 30 +/-2 ℃ and the rotation speed of 150 +/-50 rpm for 28 +/-2 hours or until thalli in a shake flask are agglomerated and hung on the wall, thus obtaining seed liquid; wherein the spore suspension contains 10 per ml 7 An order of magnitude of spore count.
The seed culture medium comprises: corn dextrin 20g/L, corn steep liquor dry powder 10g/L, yeast powder 10g/L, polyether molinate powder 1g/L, KH 2 PO 4 1.244g/L、MgSO 4 ·7H 2 O 0.227g/L、Na 2 HPO 4 ·12H 2 O 5.33g/L；pH 6.5～7.0。
The application also provides an application of the total synthetic medium in the production of Rhizomucor miehei lipase by Aspergillus oryzae fermentation.
In this application, aspergillus oryzae is Aspergillus oryzae NCBIO1.
The beneficial effect of this application lies in:
the application provides a total synthesis culture medium for aspergillus oryzae fermentation production rhizomucor miehei lipase to sucrose etc. are the carbon source, use alanine as the nitrogen source, and use with scientific carbon nitrogen ratio cooperation addition, and there is obvious interact in sucrose and alanine, make total synthesis culture medium can effectively promote aspergillus oryzae synthesis rhizomucor miehei lipase. The activity of RML obtained by fermenting and culturing the fully synthetic culture medium is as high as 60.42U/ml, and the effect is obvious. The synthetic medium can also lay a solid foundation for researching the metabolism of the Aspergillus oryzae for synthesizing the RML.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1A is a schematic illustration of the effect of different carbon source substrates provided in example 4 of the present application on Aspergillus oryzae synthesis of Rhizomucor miehei lipase; FIG. 1B is a schematic illustration of the effect of different carbon source substrates on Aspergillus oryzae growth as provided in example 4 herein;
FIG. 2A is a schematic representation of the effect of different nitrogen source substrates provided in example 5 of the present application on Aspergillus oryzae synthesis of Rhizomucor miehei lipase; FIG. 2B is a schematic representation of the effect of different nitrogen source substrates provided in example 5 of the present application on the growth of Aspergillus oryzae;
FIG. 3A is a schematic representation of the effect of carbon to nitrogen ratio on Aspergillus oryzae to synthesize Rhizomucor miehei lipase as provided in example 6 of the present application; FIG. 3B is a schematic representation of the effect of carbon to nitrogen ratio on the growth of Aspergillus oryzae as provided in example 6 herein;
FIG. 4A shows Ca provided in example 7 of the present application 2+ 、Mg 2+ Schematic diagram of the influence of Aspergillus oryzae on the synthesis of Rhizomucor miehei lipase; FIG. 4B shows Ca provided in example 7 of the present application 2+ 、Mg 2+ For riceSchematic representation of the effect of aspergillus growth; FIG. 4C shows Ca provided in example 7 of the present application 2+ 、Mg 2+ Schematic representation of the effect on protein content after fermentation.
Detailed Description
The technical solutions in the embodiments of the present application will be described clearly and completely with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only some embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
The embodiment of the application provides a fully synthetic culture medium and a method for producing rhizomucor miehei lipase by a fermentation method. The following are detailed descriptions. It should be noted that the following description of the embodiments is not intended to limit the preferred order of the embodiments. In addition, in the description of the present application, the term "including" means "including but not limited to". Various embodiments of the invention may exist in a range of versions; it is to be understood that the description in the form of a range is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention; accordingly, the described range descriptions should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. In addition, whenever a numerical range is indicated herein, it is meant to include any number (fractional or integer) recited within the range so indicated.
In the application, the RML enzyme activity, the protein and the aspergillus oryzae bacterium amount are detected as follows:
(1) determination of RML enzyme activity by the p-nitrophenol method: the lipase activity is measured by a p-nitrophenol palmitate method.
(2) Protein content determination by coomassie brilliant blue method: the protein concentration was determined by Coomassie brilliant blue method, the standard protein being bovine serum albumin.
(3) Determination of Aspergillus oryzae quantity:
after fermentation is finished, accurately weighing 20g of fermentation liquor by a beaker on an analytical balance, and recording M 0 Vacuum filtration was performed using a buchner funnel. The filter paper is advanced to 80 DEG COven-drying to constant weight and recording the weight of the filter paper to be recorded M 1 And in the suction filtration process, repeatedly washing the beaker for three times until no obvious thallus remains and no water drops flow out, and stopping suction filtration. After the filtration, the filter cake is placed on an analytical balance and weighed to obtain M 2 Then fixing the filter cake in a culture dish prepared in advance, preventing the filter cake in an oven from overturning, quickly weighing the dry weight of the filter cake on an analytical balance after drying for 24 hours, and recording M 3 。
Fresh weight of the thallus:
dry cell weight (unit: g/L):
example 1
And (3) sucking 30 mu L of aspergillus oryzae spore suspension on a solid plate culture medium by a liquid transfer gun under an aseptic condition, uniformly coating, and inversely culturing for 168h at the temperature of 30 ℃ in a constant-temperature incubator until dark green spores appear. Spores were washed with 10mL of sterile water and broken down evenly with glass beads for each plate, and then diluted to 10mL using a hemocytometer to count the number of spores 7 Spore suspension with spore number of magnitude order for standby;
under the aseptic condition, putting a seed culture medium prepared and sterilized in advance into a seed shake flask with the liquid loading capacity of 20%, inoculating 400 mu L of spore suspension into each flask, then putting the bottles into a shaking table for culturing at the culture temperature of 30 ℃ and the rotation speed of 150rpm for about 28h or until thalli in the shake flask are agglomerated and hung on the wall to obtain seed liquid for later use.
The plate medium used in this example was: 200g/L of potato, 20g/L of glucose and 15g/L of agar.
The seed culture media used in this example were: corn dextrin 20g/L, corn steep liquor dry powder 10g/L, yeast powder 10g/L, polyether molinate powder 1g/L, KH 2 PO 4 1.244g/L、MgSO 4 ·7H 2 O 0.227g/L、Na 2 HPO 4 ·12H 2 O 5.33g/L，pH 6.5～7.0。
The cells of the Aspergillus oryzae strain in this example were provided by Haemarginia galanga Biotechnology research and development center, inc. (Shanghai).
Example 2:
this example provides a fully synthetic medium comprising the following raw materials: 19.60g/L of sucrose, 4.02g/L of alanine, 0.86g/L of manganese sulfate, 1.31g/L of potassium dihydrogen phosphate, 0.75g/L of sodium chloride, 1g/L of magnesium sulfate heptahydrate, 0.13g/L of anhydrous calcium chloride, 10.5g/L of disodium hydrogen phosphate dodecahydrate, 0.0126g/L of zinc sulfate heptahydrate, 0.000925g/L of copper sulfate pentahydrate, 0.000225g/L of nickel chloride hexahydrate, 0.00517g/L of ferrous sulfate heptahydrate and 1g/L of antifoaming agent. The pH of the total synthetic medium is about 5.
The seed solution in example 1 was inoculated into the total synthesis medium in this example at an inoculum size of 10%, and the shake flask was cultured by fermentation at 30 ℃ and 150rpm, with the shake flask liquid content being 20%. The RML activity of the fermentation supernatant can reach 60.42U/ml after 120h, which is 9.76 times higher than that of comparative example 1.
Example 3:
the embodiment provides a method for producing rhizomucor miehei lipase by a fermentation method, which comprises the following steps:
the seed solution in example 1 was inoculated into the total synthesis medium at an inoculum size of 10%, the liquid loading was 20%, and the flask was shake-cultured at 30 ± 2 ℃ and 150 ± 50rpm for 120 ± 20 hours.
Comparative example 1:
this comparative example provides a fermentative synthetic medium comprising: 4g/L glucose, 2.5g/L (NH) 4 ) 2 SO 4 0.75g/L KH 2 PO 4 1g/L NaCl, 1g/L MgSO 4 ·7H 2 O, 0.1g/L CaCl 2 0.72g/L of ZnSO 4 ·7H 2 O, 0.13g/L CuSO 4 ·5H 2 O, 0.03g/L NiCl 2 ·6H 2 O, 0.00517g/L FeSO 4 ·7H 2 O, 6g/L of Na 2 HPO 4 ·12H 2 O, 1g/L polyether foam enemy powder; the pH was 5.0.
The seed solution obtained in example 1 was inoculated into the fermentation synthesis medium of this example at an inoculum size of 10%, and the liquid content was 20%, followed by shake flask fermentation culture at 30 ℃ and 150 rpm. After 120h the RML activity of the fermentation supernatant was determined to be about 6.19U/ml.
Example 4: (carbon Source Single comparison experiment)
In this example, based on the total synthesis medium in example 2, the contents and components of nitrogen source and inorganic salts were kept constant, and carbon sources such as glucose, sucrose, xylose, trehalose, lactose and fructose were added to perform a single-factor carbon source experiment. In the total synthesis culture medium of the test, the carbon source and the addition amount are respectively as follows: glucose (30 g/L, 54g/L, 80 g/L), sucrose (15 g/L, 27g/L, 40 g/L), xylose (15 g/L, 27g/L, 40 g/L), trehalose (15 g/L, 27g/L, 40 g/L), lactose (15 g/L, 27g/L, 40 g/L) and fructose (15 g/L, 27g/L, 40 g/L). The seed solutions in example 1 were inoculated into the culture media in this example at an inoculum size of 10%, respectively, and the liquid contents were 20%, followed by shake flask fermentation culture at 30 ℃ and 150 rpm. And (4) respectively detecting the RML enzyme activity and the aspergillus oryzae bacterium amount of the fermented fermentation liquor. The experimental results are shown in detail in FIGS. 1A and 1B.
As can be seen from the figure, the enzyme activity under the conditions of 40g/L trehalose and 15g/L sucrose respectively reaches 19.3U/ml and 23.9U/ml, which are respectively 12.8 times and 15.9 times of the enzyme activity (1.5U/ml) when 80g/L glucose is adopted. It can also be seen from FIG. 1 that an increase in the carbon source within a certain range promotes the growth of Aspergillus oryzae cells, whereas an excessive addition of carbon source may cause the cells to be relatively hypertonic, thereby inhibiting the growth of Aspergillus oryzae.
According to the experimental results, under the same culture conditions, the enzyme activity of the RML cultured by taking sucrose, trehalose, lactose or fructose as carbon sources in the total synthesis culture medium is higher than that of the RML cultured by adopting other carbon sources.
Example 5: (Single Nitrogen source comparative experiment)
In this example, a single-factor nitrogen source experiment was conducted by adding valine, threonine, leucine, alanine, serine, sodium nitrate, urea, ammonium sulfate, and the like, respectively, while maintaining the components and contents of carbon source, inorganic salts, and the like, on the basis of the fermentation medium described below. In the test total synthesis culture medium, the nitrogen source and the addition amount thereof are respectively as follows: valine (15.95 g/L), threonine (16.23 g/L), leucine (17.86 g/L), alanine (12.14 g/L), serine (14.32 g/L), sodium nitrate (11.59 g/L), urea (8.18 g/L), ammonium sulfate (18 g/L). The seed solution in example 1 was inoculated into the culture medium in this example at an inoculum size of 10%, the liquid loading was 20%, and the culture was performed by shake flask fermentation at 30 ℃ and 150 rpm. And (4) respectively detecting the RML enzyme activity and the aspergillus oryzae bacterium amount of the fermented fermentation liquor. The experimental results are shown in detail in fig. 2A and 2B.
In this embodiment, the fermentation medium comprises: 15g/L of sucrose, 0.75g/L of potassium dihydrogen phosphate, 1g/L of sodium chloride, 1g/L of magnesium sulfate heptahydrate, 0.1g/L of anhydrous calcium chloride, 6g/L of disodium hydrogen phosphate dodecahydrate, 0.72g/L of zinc sulfate heptahydrate, 0.13g/L of copper sulfate pentahydrate, 0.03g/L of nickel chloride hexahydrate, 0.00517g/L of ferrous sulfate heptahydrate and 1g/L of polyether foam powder.
As can be seen from the figure, the RML enzyme activities were significantly different when different kinds of nitrogen sources were used. When alanine is used as a nitrogen source, the enzyme activity is maximum after fermentation for 120 hours, can reach 42.78U/ml, and is improved by 6.8 times compared with ammonium sulfate (6.27U/ml). As can be seen, the enzyme activity of RML obtained by culturing with alanine as a nitrogen source under the same culture conditions was higher than that obtained by culturing with other nitrogen sources.
Example 6: (carbon to nitrogen ratio test)
This example was based on a fermentation medium containing 15g/L of sucrose as a carbon source and alanine as a nitrogen source, and the contents and components of inorganic salts were kept constant. In the total synthetic medium of this experiment, alanine was added at different concentrations at different carbon-to-nitrogen ratios (C: N) of 0.5, 1, 4, 6, 8, 10, 12, 16, 20, etc., respectively. The seed solution in example 1 was inoculated into the culture medium in this example at an inoculum size of 10%, the liquid loading was 20%, and the culture was performed by shake flask fermentation at 30 ℃ and 150 rpm. And (4) respectively detecting the RML enzyme activity and the aspergillus oryzae bacterial amount of the fermented liquid. The experimental results are shown in detail in fig. 3A and 3B.
The composition of the fermentation medium in this example includes: 15g/L of sucrose, 0.75g/L of alanine, 0.75g/L of potassium dihydrogen phosphate, 1g/L of sodium chloride, 1g/L of magnesium sulfate heptahydrate, 0.1g/L of anhydrous calcium chloride, 6g/L of disodium hydrogen phosphate dodecahydrate, 0.72g/L of zinc sulfate heptahydrate, 0.13g/L of copper sulfate pentahydrate, 0.03g/L of nickel chloride hexahydrate, 0.00517g/L of ferrous sulfate heptahydrate and 1g/L of polyether foam killer powder.
From the figure, it can be seen that the RML synthesis is increased and then decreased with the increase of the carbon-nitrogen ratio, and at the carbon-nitrogen ratio of 12, the RML synthesis activity of Aspergillus oryzae is the highest, reaching 45.22U/ml, which is 2.49 times higher than that of the control group (the carbon-nitrogen ratio is 8).
During the fermentation process of aspergillus oryzae, when the concentration of a nitrogen source is too high, thalli can be promoted to synthesize some byproducts, so that the metabolism is unbalanced; the concentration of the nitrogen source is too low, and the requirement of self-synthesized protein can not be met, thereby influencing the growth and metabolism of the thalli.
The analysis of the experimental results shows that the scientific carbon and nitrogen adopted by the method can well ensure the RML enzyme activity, and the effect is obvious.
Example 7:
this example is based on the following fermentation Medium and studies of metal ions (e.g., mn) 2+ 、Ca 2+ ) And the effect of different addition levels thereof on the activity of RML; wherein the fermentation medium comprises: 15g/L of sucrose, 12.14g/L of alanine, 0.75g/L of potassium dihydrogen phosphate, 1g/L of sodium chloride, 1g/L of magnesium sulfate heptahydrate, 6g/L of disodium hydrogen phosphate dodecahydrate, 0.72g/L of zinc sulfate heptahydrate, 0.13g/L of copper sulfate pentahydrate, 0.03g/L of nickel chloride hexahydrate, 0.00517g/L of ferrous sulfate heptahydrate and 1g/L of polyether sodium hypochlorite powder.
In this example, mn was added in different amounts based on the total synthesis medium 2+ 、Ca 2+ For example, anhydrous calcium chloride (0.02%, 0.04%, 0.06%), manganese sulfate monohydrate (0.2%, 0.4%, 0.6%), and so on as not to add Mn 2+ 、Ca 2+ As a blank control experiment, please see FIG. 4A, FIG. 4B or FIG. 4C in detail.
The seed solution in example 1 was inoculated into the culture medium in this example at an inoculum size of 10%, the liquid loading was 20%, and the culture was performed by shake flask fermentation at 30 ℃ and 150 rpm. And (4) respectively detecting the RML enzyme activity, the protein and the aspergillus oryzae bacterial amount of the fermented liquid. The experimental results are shown in fig. 4A, 4B and 4C.
As can be seen from the figure, 0.2% Mn was added to the medium 2+ After 120h incubation, the RML activity of this experiment was 51.5U/ml, compared to the control (Mn alone) 2+ Other conditions were the same) improved by 13.9% from 45.22U/ml.
Obviously, according to analysis, the fully synthetic culture medium can effectively improve the activity of the Aspergillus oryzae fermentation product RML, and has a good application prospect.
The totally synthetic medium for aspergillus oryzae fermentation production of Rhizomucor miehei lipase provided by the application takes sucrose and the like as carbon sources, takes alanine as nitrogen sources, and is matched with and added to use according to a scientific carbon-nitrogen ratio, and sucrose and alanine have obvious interaction, the activity of RML obtained by utilizing the totally synthetic medium for fermentation culture is as high as 60.42U/ml, and the effect is obvious. The synthetic culture medium can also lay a solid foundation for researching the metabolism of Aspergillus oryzae for synthesizing RML.
The fully synthetic medium and the fermentation method for aspergillus oryzae to ferment and produce rhizomucor miehei lipase provided by the embodiments of the present application are described in detail above, and the principles and embodiments of the present application are explained in the present application by applying specific embodiments, and the description of the above embodiments is only used to help understanding the method and the core concept of the present application; meanwhile, for those skilled in the art, according to the idea of the present application, there may be variations in the specific embodiments and the application scope, and in summary, the content of the present specification should not be construed as a limitation to the present application.
Claims (7)
1. A total synthesis culture medium, wherein the molar ratio of carbon to nitrogen in the total synthesis culture medium is 11 to 18:1;
the total synthesis culture medium comprises a carbon source and a nitrogen source, wherein the carbon source is sucrose, trehalose, lactose or fructose; the nitrogen source is alanine;
wherein, when the carbon source is sucrose, the concentration of the sucrose in the total synthesis culture medium is 15-22 g/L; when the carbon source is trehalose, the concentration of the trehalose in the total synthesis culture medium is 30-40 g/L; when the carbon source is lactose, the concentration of the lactose in the total synthesis culture medium is 25-30 g/L; when the carbon source is fructose, the concentration of the fructose in the total synthesis culture medium is 26-28 g/L;
the total synthesis culture medium also comprises metal ions, and the metal ions comprise Mn 2+ ；Mn 2+ The addition amount of (A) is 0.2-0.6% of the total mass of the total synthesis culture medium;
the total synthetic culture medium is used for producing Rhizomucor miehei lipase by Aspergillus oryzae fermentation;
the total synthetic culture medium also comprises the following raw materials: 0.7 to 1.0g/L of manganese sulfate, 0.65 to 1.4g/L of monopotassium phosphate, 0.7 to 1.3g/L of sodium chloride, 0.9 to 1.1g/L of magnesium sulfate heptahydrate, 0.08 to 0.15g/L of anhydrous calcium chloride, 9 to 12g/L of disodium hydrogen phosphate dodecahydrate, 0.01 to 0.8g/L of zinc sulfate heptahydrate, 0.0009 to 0.15g/L of copper sulfate pentahydrate, 0.0002 to 0.04g/L of nickel chloride hexahydrate, 0.004 to 0.007g/L of ferrous sulfate heptahydrate and 0.5 to 1.5g/L of defoaming agent.
2. The fully synthetic media according to claim 1, wherein the fully synthetic media comprises the raw materials: 18 to 21g/L of cane sugar, 3.5 to 4.2g/L of alanine, 0.7 to 1.0g/L of manganese sulfate, 0.65 to 1.4g/L of monopotassium phosphate, 0.7 to 1.3g/L of sodium chloride, 0.9 to 1.1g/L of magnesium sulfate heptahydrate, 0.08 to 0.15g/L of anhydrous calcium chloride, 9 to 12g/L of disodium hydrogen phosphate dodecahydrate, 0.01 to 0.8g/L of zinc sulfate heptahydrate, 0.0009 to 0.15g/L of copper sulfate pentahydrate, 0.0002 to 0.04g/L of nickel chloride hexahydrate, 0.004 to 0.007g/L of ferrous sulfate heptahydrate and 0.5 to 1.5g/L of defoaming agent; the pH value of the total synthetic culture medium is 4.5-5.5.
3. The fully synthetic medium of claim 1, wherein the fully synthetic medium comprises the raw materials: 19.60g/L of sucrose, 4.02g/L of alanine, 0.86g/L of manganese sulfate, 1.31g/L of potassium dihydrogen phosphate, 0.75g/L of sodium chloride, 1g/L of magnesium sulfate heptahydrate, 0.13g/L of anhydrous calcium chloride, 10.5g/L of disodium hydrogen phosphate dodecahydrate, 0.0126g/L of zinc sulfate heptahydrate, 0.000925g/L of copper sulfate pentahydrate, 0.000225g/L of nickel chloride hexahydrate, 0.00517g/L of ferrous sulfate heptahydrate and 1g/L of defoaming agent.
4. A method for producing Rhizomucor miehei lipase by fermentation, which comprises the following steps: inoculating a seed solution of aspergillus oryzae into a total synthesis culture medium of any one of claims 1 to 3 for fermentation culture to obtain a fermentation solution; and separating the rhizomucor miehei lipase from the fermentation liquor.
5. The method for producing Rhizomucor miehei lipase by fermentation according to claim 4, wherein in said method, said seed solution is inoculated into said total synthetic medium after sterilization at an inoculation amount of 10% by volume concentration; and/or the presence of a gas in the atmosphere,
the conditions of the fermentation culture are as follows: the culture temperature is 30 + -2 deg.C, the rotation speed is 150 + -50 rpm, and the culture time is 120 + -20 hr.
6. The method for producing Rhizomucor miehei lipase by fermentation according to claim 5, characterized in that the preparation of the seed liquid: inoculating the spore suspension into a seed culture medium under an aseptic condition, and performing shake culture at the culture temperature of 30 +/-2 ℃ and the rotation speed of 150rpm for 28 +/-2 hours to obtain a seed solution; wherein the spore suspension contains 10 per ml 7 An order of magnitude of spore count.
7. Use of a fully synthetic medium according to any one of claims 1 to 3 in the fermentative production of Rhizomucor miehei lipase by Aspergillus oryzae.
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