CN113186176B - Method for producing rhizomucor miehei lipase by total synthesis culture medium and fermentation method - Google Patents

Abstract

The application discloses a fully synthetic medium and a method for producing Rhizomucor miehei lipase by fermentation. The molar ratio of carbon to nitrogen in the total synthetic medium is 11-18; the total synthetic medium includes carbon source and nitrogen source, and the carbon source is sucrose, trehalose, lactose or fructose; The nitrogen source is alanine. The fully synthetic culture medium provided by this application for fermenting and producing Rhizomucor miehei lipase by Aspergillus oryzae uses sucrose, trehalose, etc. as carbon source, alanine as nitrogen source, and is used in combination with scientific carbon-nitrogen ratio , and there is an obvious interaction between carbon source and nitrogen source, which can effectively promote the synthesis of Rhizomucor miehei lipase by Aspergillus oryzae. The activity of the Rhizomucor miehei lipase obtained by fermenting and culturing the total synthetic medium in the present application is as high as 60.42 U/ml, and the effect is remarkable.

Description

Method for producing Rhizomucor miehei lipase by a kind of fully synthetic medium and fermentation method

technical field

The application relates to the technical field of industrial microbes, in particular to a fully synthetic medium and a method for producing Rhizomucor miehei lipase by fermentation.

Background technique

Rhizomucor miehei Lipase (RML) is the first enzyme to obtain crystal structure analysis and 3D structure analysis, and it has become 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 hydrolysis, alcoholysis and esterification of esters, and can be widely used in food, chemical, and feed production , detergents and biodiesel. Naturally occurring Rhizomucor miehei lipase is extremely rare and unstable, and cannot be used on a large scale in an industrialized manner. Common lipase exogenous expression hosts include yeast, Escherichia coli and filamentous fungi, and filamentous fungi have a powerful post-protein modification function compared to the former two, which is conducive to the stability and secretion of extracellular proteases. Aspergillus oryzae is one of the commonly used production strains in fermentation production. It is often used in the fermentation production of soy sauce and is a high-quality and high-yielding strain of neutral protease. Aspergillus oryzae is included as a safe strain by GRAS (generally recognized as safe). Aspergillus oryzae has strong protein expression characteristics, and can be used as an expression vector to produce various enzymes, such as lipase, protease, amylase, glucoamylase, etc. It has high expression and strong secretion ability, and is a good producer.

The wild-type R. miehei strain has poor lipase-producing ability and poor enzyme stability, so the natural Rhizomucor miehei lipase (RML) enzyme content is very low, which is not enough for large-scale application in the process, and its development is limited. However, Aspergillus oryzae has good growth characteristics, high-efficiency protein synthesis and secretion ability, and Aspergillus oryzae can be used as a host for stable expression of exogenous proteins. People often use yeast as the host cell for the expression of Rhizomucor miehei lipase. Zhang Yu et al. successfully constructed the pPIC9K-rml inducible expression vector, and after optimizing the fermentation conditions, measured the Rhizomucor miehei in the fermentation supernatant by the p-NP method. The lipase activity reached 116U/mL, which was 3.14 times higher than before optimization.

The medium can provide microorganisms with the nutrients necessary for cell growth and product synthesis, and its composition and concentration play a vital role in the fermentation and cultivation of microorganisms. However, at present, Aspergillus oryzae is mainly cultivated by using compound medium, but its composition is complex, which is not conducive to the quantitative analysis and research of cell metabolism characteristics. Although the conventional synthetic medium for filamentous fungi is basically suitable for Aspergillus oryzae, the lipase activity of Rhizomucor miehei is very low, so it is difficult to be used in the actual fermentation process.

Therefore, it is urgent to provide a fully synthetic medium for Aspergillus oryzae fermentation to produce Rhizomucor miehei lipase and a fermentation method for Rhizomucor miehei lipase, which can obtain Rhizomucor miehei lipase with high enzyme activity.

Contents of the invention

This application provides a fully synthetic medium for fermenting Aspergillus oryzae to produce Rhizomucor oryzae lipase, which can promote the synthesis of Rhizomucor oryzae lipase (RML) by Aspergillus oryzae, and then obtain Rhizomucor oryzae lipase with high enzyme activity .

The application provides a fully synthetic medium, in which the ratio of the molar amount of carbon and nitrogen in the fully synthetic medium (C: N=n _c /n _N , carbon nitrogen Ratio) is 11-18:1; the total synthetic medium includes carbon source and nitrogen source, the carbon source is sucrose, trehalose, lactose or fructose; the nitrogen source is alanine.

The fully synthetic medium is used for fermenting Aspergillus oryzae to produce Rhizomucor miehei lipase.

Optionally, in some embodiments of the present application, the molar ratio 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 synthetic medium is 15-22 g/L; and/or

When the carbon source is trehalose, the concentration of trehalose in the total synthetic medium is 30-40g/L; and/or

When the carbon source is lactose, the concentration of lactose in the total synthetic medium is 25-30 g/L; and/or

When the carbon source is fructose, the concentration of fructose in the total synthetic medium is 26-28 g/L.

Optionally, in some embodiments of the present application, the fully synthetic medium further includes metal ions, and the metal ions include Ca ²⁺ and/or Mn ²⁺ .

Optionally, in some embodiments of the present application, in the total synthetic medium, the amount of Mn ²⁺ added is 0.2%-0.6% of the total mass of the total synthetic medium.

Optionally, in some embodiments of the present application, in the total synthetic medium, the amount of Ca ²⁺ added is 0.02%-0.06% of the total mass of the total synthetic medium.

Optionally, in some embodiments of the present application, the fully synthetic medium includes a carbon source and a nitrogen source, wherein the carbon source is sucrose, and the nitrogen source is alanine;

The fully synthetic medium also includes manganese sulfate, potassium dihydrogen phosphate, sodium chloride, magnesium sulfate heptahydrate, calcium chloride anhydrous, disodium hydrogen phosphate dodecahydrate, zinc sulfate heptahydrate, copper sulfate pentahydrate, hexahydrate Nickel Chloride Hydrate, Ferrous Sulfate Heptahydrate, and Antifoam. The pH value of the fully synthetic medium is 4.5-5.5.

Optionally, in some embodiments of the present application, the fully synthetic medium includes raw materials: sucrose 18-21g/L, alanine 3.5-4.2g/L, manganese sulfate 0.7-1.0g/L, diphosphate Potassium hydrogen 0.65-1.4g/L, sodium chloride 0.7-1.3g/L, magnesium sulfate heptahydrate 0.9-1.1g/L, calcium chloride anhydrous 0.08-0.15g/L, disodium hydrogen phosphate dodecahydrate 9 ～12g/L, zinc sulfate heptahydrate 0.01～0.8g/L, copper sulfate pentahydrate 0.0009～0.15g/L, nickel chloride hexahydrate 0.0002～0.04g/L, ferrous sulfate heptahydrate 0.004～0.007g/L And defoamer 0.5 ~ 1.5g/L. The pH value of the fully synthetic medium is 4.5-5.5.

Optionally, in some embodiments of the present application, the defoamer is polyether foam powder.

Optionally, in some embodiments of the present application, the fully synthetic medium includes raw materials: sucrose 19.60 g/L, alanine 4.02 g/L, manganese sulfate 0.86 g/L, potassium dihydrogen phosphate 1.31 g/L L, sodium chloride 0.75g/L, magnesium sulfate heptahydrate 1g/L, calcium chloride anhydrous 0.13g/L, disodium hydrogen phosphate dodecahydrate 10.5g/L, zinc sulfate heptahydrate 0.0126g/L, five Copper sulfate hydrate 0.000925g/L, nickel chloride hexahydrate 0.000225g/L, ferrous sulfate heptahydrate 0.00517g/L and defoamer 1g/L. The pH value of the fully synthetic medium is about 5.0.

In the present application, the molar amount of carbon element in the fully synthetic medium (abbreviated as carbon molar amount) is the total molar amount of carbon element from carbon source (such as sucrose, etc.) and nitrogen source (such as alanine, etc.) . The molar amount of nitrogen element in the total synthetic medium (nitrogen molar amount) is the total molar amount of nitrogen element in the nitrogen source (such as alanine, etc.).

For example, the calculation method of the carbon-nitrogen ratio: the carbon-nitrogen ratio refers to the molar ratio of the carbon element in the culture medium to the nitrogen element, that is, C: N=n _C /n _N . The carbon molarity in the medium is the total molarity of carbon elements from sucrose and alanine; the nitrogen molarity is the total molarity of nitrogen elements in alanine.

The molar amounts of carbon sources and nitrogen sources are calculated according to the definition of molar amounts, that is, the ratio of carbon content, nitrogen content and relative molecular mass.

Correspondingly, the present application also provides a method for fermentatively producing Rhizomucor miehei lipase, said method comprising: inoculating the seed liquid of Aspergillus oryzae in the above-mentioned fully synthetic medium for fermentation and culturing to obtain a fermented liquid; Rhizomucor miehei lipase was isolated from the fermentation broth.

Optionally, in some embodiments of the present application, in the method, the seed solution is inoculated into the sterilized total synthetic medium with an inoculum amount of 10%.

Optionally, in some embodiments of the present application, the conditions of the fermentation culture are: the culture temperature is 30±2° C., the rotational speed is 150±50 rpm, and the culture is 120±20 hours.

Optionally, in some embodiments of the present application, the process of the fermentation culture specifically includes: inoculating the seed liquid with an inoculum volume concentration of 10% into the sterilized fully synthetic medium, and fermenting the culture, The culture temperature is 30±2° C., the rotational speed is 150±50 rpm, and the culture is 120±20 hours to obtain a fermentation liquid.

The total synthetic culture is based on sterilization at 121° C. for 30 minutes.

Optionally, in some embodiments of the present application, the preparation of the seed liquid: under aseptic conditions, inoculate the spore suspension into the seed medium, culture on a shaking table, the culture temperature is 30±2°C, and the rotation speed is 150±50rpm, cultivated for 28±2 hours or until the thalli in the shake flask are in a state of clumping and hanging on the wall, and the seed liquid is obtained; wherein, the spore suspension contains 10 ⁷ order of magnitude spores per milliliter.

The seed medium includes: corn dextrin 20g/L, corn steep liquor dry powder 10g/L, yeast powder 10g/L, polyether foam enemy 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 present application also provides an application of the fully synthetic medium in the fermentative production of Rhizomucor miehei lipase by Aspergillus oryzae.

In the present application, the Aspergillus oryzae NCBIO1 is Aspergillus oryzae.

The beneficial effect of this application is:

The fully synthetic culture medium provided by this application for fermenting Aspergillus oryzae to produce Rhizomucor miehei lipase uses sucrose etc. There is an obvious interaction with alanine, so that the fully synthetic medium can effectively promote the synthesis of Rhizomucor miehei lipase by Aspergillus oryzae. In this application, the activity of RML obtained by fermenting and culturing the total synthetic medium is as high as 60.42 U/ml, and the effect is remarkable. The synthetic medium can also lay a solid foundation for the research on the metabolism of RML synthesized by Aspergillus oryzae.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings that need to be used in the description of the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application. For those skilled in the art, other drawings can also be obtained based on these drawings without any creative effort.

Fig. 1A is a schematic diagram of the influence of different carbon source substrates provided by Example 4 of the present application on the synthesis of Rhizomucor oryzae lipase by Aspergillus oryzae; Fig. 1B is the effect of different carbon source substrates provided by Example 4 of the present application on the growth of Aspergillus oryzae schematic diagram;

Fig. 2A is the schematic diagram of the influence of different nitrogen source substrates provided by Example 5 of the present application on the synthesis of Rhizomucor oryzae lipase by Aspergillus oryzae; Fig. 2B is the effect of different nitrogen source substrates provided by Example 5 of the present application on the growth of Aspergillus oryzae schematic diagram;

Fig. 3 A is the schematic diagram of the influence of the carbon-nitrogen ratio provided by Example 6 of the present application on the synthesis of Rhizomucor oryzae lipase by Aspergillus oryzae; Fig. 3B is the schematic diagram of the influence of the carbon-nitrogen ratio provided by Example 6 of the present application on the growth of Aspergillus oryzae;

Figure 4A is a schematic diagram of the effect of Ca ²⁺ and Mg ²⁺ provided in Example 7 of the present application on the synthesis of Rhizomucor miehei lipase by Aspergillus oryzae; Figure 4B is the effect of Ca ²⁺ and Mg ²⁺ provided in Example 7 of the present application on rice Schematic diagram of the influence of Aspergillus growth; FIG. 4C is a schematic diagram of the influence of Ca ²⁺ and Mg ²⁺ on the protein content after fermentation provided in Example 7 of the present application.

Detailed ways

The following will clearly and completely describe the technical solutions in the embodiments of the application with reference to the drawings in the embodiments of the application. Apparently, the described embodiments are only some of the embodiments of the application, not all of them. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without making creative efforts belong to the scope of protection of this application.

The embodiment of the present application provides a fully synthetic medium and a method for producing Rhizomucor miehei lipase by fermentation. Each will be described in detail below. It should be noted that the description sequence of the following embodiments is not intended to limit the preferred sequence 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 present invention may exist in a range format; it should be understood that the description in a range format is merely for convenience and brevity, and should not be construed as an inflexible limitation on the scope of the invention; therefore, the stated ranges should be considered The description has specifically disclosed all possible subranges as well as individual values within that range. Additionally, whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range.

Detection of RML enzyme activity, protein, and Aspergillus oryzae body weight in this application:

① Determination of RML enzyme activity by p-nitrophenol method: The enzyme activity of lipase was determined by p-nitrophenol palmitate method.

② Determination of protein content by Coomassie brilliant blue method: the protein concentration was determined by Coomassie brilliant blue method, and the standard protein was bovine serum albumin.

③ Aspergillus oryzae body weight determination:

After the fermentation is finished, accurately weigh 20 g of the fermentation broth on an analytical balance with a beaker to record M ₀ , and perform vacuum filtration with a Buchner funnel. The filter paper should be dried in an oven at 80°C in advance to a constant weight and the weight of the filter paper should be recorded as M ₁ . During the suction filtration process, the beaker should be rinsed three times until there is no obvious bacteria residue and stop the suction filtration when no water drops flow out. After the suction filtration, put the filter cake on the analytical balance to weigh _M2 , and then fix the filter cake in the prepared petri dish to prevent the filter cake from overturning in the oven. After drying for 24 hours, quickly weigh the filter cake on the analytical balance Note M ₃ .

Bacteria fresh weight:

Bacterial dry weight (unit: g/L):

Example 1

Under sterile conditions, pipette 30 μL of Aspergillus oryzae spore suspension onto the solid plate medium, evenly spread it, and incubate it upside down in a constant temperature incubator at 30°C for 168 hours until dark green spores appear. Each plate was washed with 10mL sterile water and the spores were evenly dispersed with glass beads, then the number of spores was calculated with a hemocytometer, and the spore suspension diluted to the number of spores in the order of ¹⁰⁷ per milliliter was set aside;

Under sterile conditions, put the pre-prepared sterilized seed culture medium in the seed shake flask with a liquid content of 20%, inoculate 400 μL of spore suspension in each bottle, and then place it in a shaker for cultivation. The cultivation temperature is 30°C and the rotation speed The temperature is 150 rpm, cultivated for about 28 hours or until the bacteria in the shake flask form agglomerates and hang on the wall to obtain seed liquid, which is set aside.

The plate culture medium adopted in this embodiment is: potato 200g/L, glucose 20g/L, agar 15g/L.

The seed medium used in this example is: corn dextrin 20g/L, corn steep liquor dry powder 10g/L, yeast powder 10g/L, polyether foam enemy 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 Aspergillus oryzae strain cells in this example were provided by Yihai Kerry Biotechnology R&D Center Co., Ltd. (Shanghai).

Example 2:

This embodiment provides a fully synthetic medium, including raw materials: sucrose 19.60g/L, alanine 4.02g/L, manganese sulfate 0.86g/L, potassium dihydrogen phosphate 1.31g/L, sodium chloride 0.75g/L L. Magnesium sulfate heptahydrate 1g/L, calcium chloride anhydrous 0.13g/L, disodium hydrogen phosphate dodecahydrate 10.5g/L, zinc sulfate heptahydrate 0.0126g/L, copper sulfate pentahydrate 0.000925g/L, Nickel chloride hexahydrate 0.000225g/L, ferrous sulfate heptahydrate 0.00517g/L and defoamer 1g/L. The pH value of the fully synthetic medium is about 5.

The seed solution in Example 1 was inoculated into the fully synthetic medium in this example with a 10% inoculation amount, and fermented and cultured in a shaker at 30°C and 150 rpm, and the amount of liquid in the shaker was 20%. After 120 hours, the RML activity of the fermentation supernatant was measured to reach 60.42 U/ml, which was 9.76 times higher than that of Comparative Example 1.

Example 3:

The present embodiment provides a kind of method that fermentation produces Rhizomucor miehei lipase, comprises the steps:

The seed solution in Example 1 was inoculated in the described total synthetic medium with an inoculum amount of 10%, and the liquid loading amount was 20%, and the shaker fermentation culture was carried out at 30 ± 2° C., 150 ± 50 rpm, and the fermentation time was 120 ±20 hours.

Comparative Example 1:

This comparative example provides a fermentation synthetic medium, including: 4g/L glucose, 2.5g/L (NH ₄ ) ₂ SO ₄ , 0.75g/L KH ₂ PO ₄ , 1g/L NaCl, 1g /L of MgSO ₄ ·7H ₂ O, 0.1g/L of CaCl ₂ , 0.72g/L of ZnSO ₄ ·7H ₂ O, 0.13g/L of CuSO ₄ ·5H ₂ O, 0.03g/L of NiCl ₂ · 6H ₂ O, 0.00517g/L FeSO ₄ ·7H ₂ O, 6g/L Na ₂ HPO ₄ ·12H ₂ O, 1g/L polyether foam enemy powder; pH 5.0.

The seed solution in Example 1 was inoculated into the fermentation synthesis medium in this example with a 10% inoculum amount, and the liquid content was 20%, and the shaker fermentation culture was carried out at 30° C. and 150 rpm. After 120h, the RML activity of the fermentation supernatant was measured to be about 6.19U/ml.

Embodiment 4: (single carbon source comparative experiment)

This example is based on the fully synthetic medium in Example 2, and the composition and content of nitrogen source and inorganic salt remain unchanged. Carbon source experiments. In the fully synthetic medium of this test, the carbon sources and the addition amounts were: glucose (30g/L, 54g/L, 80g/L), sucrose (15g/L, 27g/L, 40g/L), wood Sugar (15g/L, 27g/L, 40g/L), trehalose (15g/L, 27g/L, 40g/L), lactose (15g/L, 27g/L, 40g/L) and fructose (15g/L L, 27g/L, 40g/L). The seed solution in Example 1 was inoculated into the culture medium in this example at an inoculum volume of 10%, and the liquid volume was 20%, and the shaker fermentation culture was carried out at 30° C. and 150 rpm. RML enzyme activity and Aspergillus oryzae body weight were detected for the fermented broth after fermentation. The experimental results are shown in Figure 1A and Figure 1B in detail.

It can be concluded from the figure that the enzyme activities under the conditions of 40g/L trehalose and 15g/L sucrose respectively reach 19.3U/ml and 23.9U/ml, which are respectively 80g/L glucose (1.5U/ml) 12.8 and 15.9 times. It can also be found from Figure 1 that increasing the carbon source within a certain range will promote the growth of Aspergillus oryzae, and when the carbon source is added too much, the cells may be in a relatively hypertonic state, thereby inhibiting the growth of Aspergillus oryzae.

According to the experimental results, it can be known that under the same culture conditions, the RML enzyme activity obtained by cultivating sucrose, trehalose, lactose or fructose in the fully synthetic medium of the present application as a carbon source is higher than that of RML enzyme activities using other types of carbon sources. .

Embodiment 5: (single nitrogen source comparative experiment)

This embodiment is based on the following fermentation medium, carbon source, inorganic salt and other components and content remain unchanged, respectively add valine, threonine, leucine, alanine, serine, sodium nitrate, urea and Ammonium sulfate and other single-factor nitrogen source experiments. In this test fully synthetic medium, the nitrogen source and its addition amount are respectively: valine (15.95g/L), threonine (16.23g/L), leucine (17.86g/L), Alanine (12.14g/L), serine (14.32g/L), sodium nitrate (11.59g/L), urea (8.18g/L), ammonium sulfate (18g/L). The seed solution in Example 1 was inoculated into the culture medium in this example at an inoculum volume of 10%, and the liquid volume was 20%, and shake-crank fermentation culture was carried out at 30° C. and 150 rpm. RML enzyme activity and Aspergillus oryzae body weight were detected for the fermented broth after fermentation. The experimental results are shown in Figure 2A and Figure 2B in detail.

In this embodiment, the composition of the fermentation medium includes: 15 g/L of sucrose, 0.75 g/L of potassium dihydrogen phosphate, 1 g/L of sodium chloride, 1 g/L of magnesium sulfate heptahydrate, 0.1 g/L of anhydrous calcium chloride L, disodium hydrogen phosphate dodecahydrate 6g/L, zinc sulfate heptahydrate 0.72g/L, copper sulfate pentahydrate 0.13g/L, nickel chloride hexahydrate 0.03g/L, ferrous sulfate heptahydrate 0.00517g/L And polyether foam enemy powder 1g/L.

It can be seen from the figure that when different types of nitrogen sources are used, the enzyme activity of RML is significantly different. When alanine was used as a nitrogen source, the enzyme activity was the highest after 120 hours of fermentation, reaching 42.78U/ml, which was 6.8 times higher than that of ammonium sulfate (6.27U/ml). It can be seen that under the same culture conditions, the enzyme activity of RML cultured with alanine as nitrogen source is higher than that of other nitrogen sources.

Embodiment 6: (carbon nitrogen ratio experiment)

This example is based on the following fermentation medium, and the composition and content of inorganic salts remain unchanged, wherein 15 g/L sucrose is used as the carbon source and alanine is used as the nitrogen source. In the fully synthetic medium of this experiment, different concentrations of alanine were added with different carbon-nitrogen ratios (C:N) of 0.5, 1, 4, 6, 8, 10, 12, 16, 20, etc. for the experiment. The seed solution in Example 1 was inoculated into the culture medium in this example at an inoculum volume of 10%, and the liquid volume was 20%, and shake-crank fermentation culture was carried out at 30° C. and 150 rpm. RML enzyme activity and Aspergillus oryzae body weight were detected for the fermented broth after fermentation. The experimental results are shown in Figure 3A and Figure 3B in detail.

The composition of the fermentation medium described in this example includes: sucrose 15g/L, alanine, potassium dihydrogen phosphate 0.75g/L, sodium chloride 1g/L, magnesium sulfate heptahydrate 1g/L, anhydrous calcium chloride 0.1g/L, disodium hydrogen phosphate dodecahydrate 6g/L, zinc sulfate heptahydrate 0.72g/L, copper sulfate pentahydrate 0.13g/L, nickel chloride hexahydrate 0.03g/L, ferrous sulfate heptahydrate 0.00517 g/L and polyether foam enemy powder 1g/L.

It can be found from the figure that as the carbon-nitrogen ratio increases, the synthesis of RML first increases and then decreases. When the carbon-nitrogen ratio is 12, the RML activity of Aspergillus oryzae is the highest, reaching 45.22U/ml, which is higher than that of the control group ( The carbon-nitrogen ratio is 8) which has increased by 2.49 times.

During the fermentation process of Aspergillus oryzae, when the concentration of nitrogen source is too high, it may promote the synthesis of some by-products in the bacteria, resulting in metabolic imbalance; if the concentration of nitrogen source is too low, it may not be able to meet the needs of its own protein synthesis, which will affect the bacteria. growth and metabolism.

Analyzing the experimental results, it can be seen that the scientific carbon and nitrogen used in this application can well guarantee the RML enzyme activity, and the effect is remarkable.

Embodiment 7:

The present embodiment is based on the following fermentation medium to study the influence of metal ions (such as Mn ²⁺ , Ca ²⁺ ) and their different additions on the activity of RML; wherein, the fermentation medium includes: sucrose 15g/L , alanine 12.14g/L, potassium dihydrogen phosphate 0.75g/L, sodium chloride 1g/L, magnesium sulfate heptahydrate 1g/L, disodium hydrogen phosphate dodecahydrate 6g/L, zinc sulfate heptahydrate 0.72g /L, copper sulfate pentahydrate 0.13g/L, nickel chloride hexahydrate 0.03g/L, ferrous sulfate heptahydrate 0.00517g/L and polyether foam enemy powder 1g/L.

On the basis of the above-mentioned fully synthetic medium in this example, different amounts of Mn ²⁺ and Ca ²⁺ were added, such as anhydrous calcium chloride (0.02%, 0.04%, 0.06%), manganese sulfate monohydrate (0.2 %, 0.4%, 0.6%), and without adding Mn ²⁺ , Ca ²⁺ as the blank control group experiment, please refer to Figure 4A, Figure 4B or Figure 4C for details.

The seed solution in Example 1 was inoculated into the culture medium in this example at an inoculum volume of 10%, and the liquid volume was 20%, and shake-crank fermentation culture was carried out at 30° C. and 150 rpm. The fermented fermentation liquid was tested for RML enzyme activity, protein and Aspergillus oryzae body weight respectively. The experimental results are shown in Figure 4A, Figure 4B and Figure 4C in detail.

It can be seen from the figure that after 120 hours of culture in the group of experiments in which 0.2% Mn ²⁺ was added to the culture medium, the RML activity was 51.5 U/ml, compared with 45.22 U/ml in the control group (only Mn ²⁺ was not added, other conditions were the same). U/ml increased by 13.9%.

Obviously, according to the analysis, it can be known that the fully synthetic medium of the present application can effectively improve the activity of the fermentation product RML of Aspergillus oryzae, and has a good application prospect.

The fully synthetic medium for the production of Rhizomucor miehei lipase by Aspergillus oryzae fermented by this application uses sucrose as the carbon source, alanine as the nitrogen source, and is used in combination with a scientific carbon-to-nitrogen ratio, and sucrose There is an obvious interaction with alanine, and the activity of RML obtained by fermenting and cultivating the total synthetic medium is as high as 60.42U/ml, and the effect is remarkable. The synthetic medium can also lay a solid foundation for the research on the metabolism of RML synthesized by Aspergillus oryzae.

A kind of fully synthetic culture medium and fermentation method for producing Rhizomucor oryzae lipase provided by Aspergillus oryzae fermentation provided in the examples of the present application have been introduced in detail above. Specific examples are used in this paper to carry out the principles and implementation methods of the present application. To clarify, the description of the above embodiments is only used to help understand the method of the present application and its core idea; at the same time, for those skilled in the art, according to the idea of the present application, there will be changes in the specific implementation and application scope In summary, the contents of this specification should not be construed as limiting the 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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