JPH0336B2 - - Google Patents

Description

[Detailed description of the invention]

[Field of Industrial Application] The present invention relates to a novel protein kinase and a method for producing the same. Specifically, the present invention relates to a novel protein kinase that is derived from prokaryotes, has excellent stability, is independent of cyclic nucleotides, and is capable of converting rice globulin or swine plasma protein into phosphoric acid. The present invention relates to a protein kinase that can be converted into a protein kinase and a method for producing the enzyme using a microorganism. [Prior art and problems to be solved by the invention] In plants, residues containing large amounts of protein after oil extraction, such as soybeans, sunflowers, and rapeseed, are often used as feed rather than as food resources; In Japan, the blood that is obtained in large quantities when obtaining meat is often thrown away. Effective use of these proteins is also important in avoiding future food shortages due to rapid population growth. However, unused proteins are often difficult to use in their original form, so it is necessary to make some kind of modification to the proteins to make them easier to use. As a modification method, enzymatic methods are attracting attention because they are safe and can be carried out under mild conditions. Protein kinase is one of the enzymes that change the properties of proteins. This enzyme has been found in eukaryotes, and research is progressing particularly in animals.
While there is a lot of research being done on the biochemical side, there is not much research being done on the usage side. Regarding enzymes, there are those that require expensive stimulatory factors such as cyclic nucleotides, hormones, and calmodulin, those that are not suitable for application due to extremely high substrate specificity, and those that do not increase activity with existing stimulatory factors. It has been known. In non-eukaryotes, oncogene products of retroviruses are known to have tyrosine kinase activity. Among prokaryotes, there are reports that serine/threonine kinase activity has been detected in Salmonella typhimurium and tyrosine kinase activity has been detected in Escherichia coli and photosynthetic bacteria, but little else is known. Furthermore, in eukaryotes, protein kinases have been discovered that have the activity of phosphorylating externally added proteins such as casein, but in prokaryotes,
There are only reports that there is evidence that bacterial cell proteins are phosphorylated, and there are no reports that have been found to have the ability to phosphorylate externally applied proteins. So far, there have been reports of reaction systems using immobilized enzymes to phosphorylate proteins. The enzyme used in this reaction system is a partially purified protein kinase from animal internal organs. When considering the use of enzymes, it is more economical to use microorganisms than to use animal cells because the culture method is simpler, mass culture is easier, the culture time is shorter, and microorganisms are more advantageous than animal cells. [Means to Solve the Problem] As a result of repeated studies to obtain protein kinases derived from microorganisms, the present inventors discovered that specific microorganisms produce novel protein kinases with excellent stability. Based on this knowledge, we have completed the present invention. That is, in the present invention, the molecular weight measured by gel filtration of a product solubilized with Triton X-100 is approximately
65,000 and about 16,000, and the optimum PH is 7.0, and the PH
7.0, the optimal temperature for a reaction time of 3 minutes is 65°C, it is independent of cyclic AMP and cyclic GMP, and is a protein kinase that can phosphorylate rice globulin and porcine plasma proteins. The present invention relates to a method for producing protein kinase, which comprises culturing a microorganism capable of producing protein kinase in a medium, collecting microbial cells from the culture, and collecting a membrane fraction. The microorganism used in the present invention may be any microorganism as long as it can produce the above-mentioned enzyme, and specific examples include Micrococcus luteus FK1001 (IAM 11007), which was provided by the Useful Bacterial Strain Preservation Facility of the Institute of Applied Microbiology, the University of Tokyo. In addition to the present strain, mutant strains obtained by applying conventional mutation methods can also be used in the present invention as long as they have the ability to produce the desired enzyme. When culturing this strain, normal aerobic bacterial culture methods can be applied, and the carbon sources of the medium are preferably glucose, fructose, etc., and the nitrogen sources are ammonium chloride, ammonium sulfate, yeast extract, peptone, meat, etc. Extracts, casamino acids, etc. can be used. Others: salt, potassium chloride,
Inorganic salts such as calcium chloride, cobalt chloride, sodium phosphate, potassium phosphate, iron sulfate, etc. can also be added. In addition, the appropriate pH of the medium is 6.8 to 7.8. The appropriate culture method is liquid culture, at 28-37℃.
Shaking culture is carried out at 130-170 rpm, preferably 150 rpm at 30°C. Cultivation may be carried out until the microorganisms proliferate to a sufficient extent to produce the desired culture, but usually cells in the logarithmic growth phase are collected and used. An example of producing the protein kinase of the present invention is shown below. First, after culturing as described above, microbial cells are collected by centrifugation or the like, spheroplasts are formed using achromopeptidase, and spheroplasts are collected by centrifugation or the like. Next, after disrupting the spheroplasts in a hypotonic solution, a membrane fraction is collected by centrifugation or the like, a buffer containing Triton X-100 is added thereto, and the mixture is stirred at low temperature. Thereafter, the supernatant obtained by solid-liquid separation is used as a Triton X-100 soluble membrane fraction and used as a crude enzyme. In addition, to search for strains with protein kinase activity, use the supernatant obtained by harvesting, crushing, and solid-liquid separation, and holding it at 30°C for 1 hour with the substrates histone type-A and [gamma- ^32P ]ATP. Yes, 20%
After stopping the reaction with trichloroacetic acid, the radioactivity of the solid content obtained by filtration was measured using a liquid scintillation counter. Protein kinase activity was determined by adding the above substrate, [gamma ³² P] ATP, and enzyme solution to a potassium phosphate buffer (PH7.0) containing magnesium acetate, keeping the reaction solution at a constant temperature, and carrying out the reaction. After stopping the reaction, the solid content obtained by filtration was washed with trichloroacetic acid, dried, and the radioactivity of the solid content was measured using a liquid scintillation counter, and the amount of phosphoric acid incorporated into the protein was calculated. Ta. The crude enzyme solution prepared by the above method has the following properties. In addition, unless otherwise specified, the reaction is 45
The reaction was carried out at ℃ for 3 minutes, and rice globulin was used as the substrate. (1) Optimal PH As shown in Figure 1, the optimal PH of this enzyme is 7.0.
It's nearby. Therefore, reactions using this enzyme can be carried out under mild conditions. (2) Optimal temperature The optimal temperature at PH7.0 and reaction time of 3 minutes is:
As shown in Figure 2, the temperature is around 65°C. In addition,
In this case, rice globulin was used as the substrate. (3) Requirement for divalent cations As shown in Table 1, the activity of this enzyme increases significantly when magnesium, manganese, etc. are added.

[Table] ** Glycol ether diamine tetraacetic acid
(4) Cyclic nucleotide dependence As shown in Table 2, no increase in activity of this enzyme is observed even when cyclic AMP and cyclic GMP are added. Therefore, it was found that the present enzyme does not require these expensive stimulatory factors.

[Table] (5) Stability When stored at 4°C, the activity of this enzyme was stable for at least 30 days (see Figure 3). Also,
Regarding thermostability, when the activity was measured after heating the enzyme for 10 minutes, it was found to be almost stable up to 60°C (see Figure 4). (6) Molecular weight Protein kinase solubilized with Triton X-100 was analyzed by HPLC (column: Superose 12HR10/30)
When the molecular weight was measured by gel filtration, the protein kinase was found to have a molecular weight of approximately 65,000 (indicated by X ₁ ) and approximately 16,000 (indicated by X ₂ ), and it was found that this protein kinase is a mixture of X ₁ and X ₂ . (7) Substrate specificity When we investigated what kind of proteins this enzyme effectively acts on, as shown in Table 3, it showed the highest activity on rice globulin, and other proteins such as swine plasma, ovalbumin, and histones. Although it acts on type A and phosphorylates it, it does not act on acidic proteins such as casein. In addition, rice globulin and albumin were determined using the method of Iwasaki et al. (Journal of Japan Food Industry Association,
19, No. 2, pp. 70-75 (1972)), and the porcine plasma protein was obtained from an abattoir and was freeze-dried and used.

【table】

〔Example〕

Next, the present invention will be explained in detail with reference to examples. Example 1 Micrococcus luteus FK1001 strain (IAM11007) was inoculated into a medium with the following composition and incubated at 30°C.
Rotary shaking culture was performed for 16 hours. Medium composition: Polypeptone 10g/Meat extract 10g/Salt 5g/PH7.0 Bacterial cells were collected from the culture solution by centrifugation, spheroplasts were formed using achromopeptidase, and the cells were collected. Next, the spheroplasts were disrupted in a hypotonic solution and centrifuged to collect membrane fractions. A buffer containing Triton X-100 was added to this fraction, and the mixture was stirred at 4°C. Next, a supernatant was obtained by centrifugation, and this was used as a crude enzyme solution. When the activity of this crude enzyme solution was measured,
Approximately 300 picomole of phosphoric acid was transferred to rice globulin protein in 1 minute at 45°C per 1 mg of crude enzyme protein.
If the enzyme that phosphorylates 1 picomole per minute is 1 unit, then 1200 to 1500 units of the enzyme can be obtained from the second culture solution. Example 2 As mentioned above, the enzyme of the present invention phosphorylates rice globulin well, but when the globulin was separated by gel filtration, it was investigated which fraction phosphorylated well. Rice globulin was gel-filtered using Sephadex G-100, crude enzyme was added to the eluted fraction as a substrate, and protein kinase activity was measured. As a result, it was found that a relatively high molecular weight fraction was phosphorylated (see Figure 5). Next, fraction number 1
When No. 23 was collected and concentrated, gel filtration was performed using Sephacryl S-200, and the same experiment was carried out, two phosphorylated fractions appeared as shown in FIG. The maximum activity around fraction number 10 was calculated to have a molecular weight of approximately 123,000, and the maximum activity around fraction number 15 was calculated to have a molecular weight of 70,000. The molecular weight was calculated from the elution positions of the standard proteins B to F shown in FIG. 6 using a Lineweber bulk plot. [Effects of the Invention] According to the present invention, a protein kinase with excellent stability can be produced in large quantities by a simple method. By using this enzyme, it becomes possible to use unused proteins as food.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the PH dependence of the enzyme of the present invention, FIG. 2 is a graph showing the temperature dependence of the enzyme of the present invention, FIG. 3 is a graph showing the storage stability of the enzyme of the present invention, and FIG. 4 is a graph showing the storage stability of the enzyme of the present invention. The figure is a graph showing the thermostability of the enzyme of the present invention, and Figures 5 and 6 are graphs showing the phosphorylation of the eluted fraction after gel filtration of rice globulin.

Claims (1)

[Claims] 1. The molecular weights measured by gel filtration of the solubilized product with Triton The optimum temperature is around 65℃, and it is independent of cyclic AMP and cyclic GMP.
A protein kinase capable of phosphorylating rice globulin and porcine plasma proteins. 2 Belongs to the genus Micrococcus, Triton X-100
The molecular weights of the solubilized products measured by gel filtration are approximately 65,000 and approximately 16,000, and the optimal pH is 7.0, and the optimal temperature at pH 7.0 and reaction time of 3 minutes is
A microorganism that can produce a protein kinase that is around 65°C, is independent of cyclic AMP and cyclic GMP, and can phosphorylate rice globulin and swine plasma protein is cultured in a medium, and microbial cells are collected from the culture. A method for producing protein kinase, which comprises collecting a membrane fraction. 3. The method according to claim 2, wherein the protein kinase-producing bacterium belonging to the genus Micrococcus is Micrococcus luteus FK1001 strain (IAM 11007). 4. The method according to claim 2, wherein the membrane fraction is a Triton X-100 soluble membrane fraction.