JP2844350B2 - Manufacturing methods for substances produced by microorganisms - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial application] The present invention relates to a method for producing a substance produced by a microorganism, more specifically, to reduce the content of contaminants in a culture by a mixed culture of two or more microorganisms. The present invention relates to a method for producing a substance in a high yield.

[Conventional technology]

BACKGROUND ART Conventionally, many methods for producing a substance produced by a microorganism by culturing the microorganism (hereinafter, referred to as a “culture product”) have been reported and put into practical use. Incidentally, the culture contains many contaminants other than the target substance. These contaminants usually consist of nucleic acids, proteins, etc., in addition to the cells used, and these are produced as by-products from the cells used or produced by lysis. When a culture product is collected from a culture, it is usually necessary to remove these contaminants, and such a contaminant removal step is generally called a post-treatment step. The post-processing step is
It usually consists of two unit operations, pre-fractionation (coarse fractionation) and precision fractionation [Bioseparation Engineering Handbook, p30, Science Forum, 1988]. Of these, the prefractionation is extremely important because it has a significant effect on the precision fractionation performed later. That is, as the removal rate of contaminants by the pre-fractionation increases, the burden of precision fractionation (membrane separation, chromatography, etc.) is reduced, and the yield is improved.

Centrifugation and filtration are mainly used as the pre-fractionation. Particularly, nucleic acids, proteins, etc., which lower the purity of the product or lower the efficiency of separation and purification, are removed by a combination of the following methods. Attempts have been made to increase For example, a nucleic acid precipitation method using streptomycin sulfate or manganese chloride, a nucleic acid decomposition method using nuclease, a protein or bacteria precipitation method using ammonium sulfate or phosphate, etc. [Biochemical Engineering Second Edition, p376, Publishing Center, 1973].

[Problems to be solved by the invention]

However, the above-mentioned processing methods combined with these prefractionations have the following problems in industrialization. For example, when ammonium sulfate or the like is used, the load of wastewater treatment increases, and when a flocculant such as streptomycin sulfate or a nuclease is used, the cost in industrial production increases. In general, the cost of the post-treatment process accounts for a large proportion of the entire product, and the cost problem is important especially for products with low market prices (Bioseparation Engineering Handbook, p28, Science Forum, 1988).

Therefore, it has been a major problem in the technical field that the post-treatment is performed efficiently and at low cost without increasing the load such as wastewater treatment in the post-treatment process of the culture.

[Means for solving the problem]

Under these circumstances, the present inventors have conducted intensive studies to solve the above-mentioned problems, and found that the following microorganisms that produce enzymes that degrade or reduce nucleic acids and proteins in the culture step are combined with microorganisms that produce cellulase or amylase. It has been found that the target substance can be obtained in a high yield in the post-treatment step following the culture step if the mixed culture is carried out in the following manner.

That is, the present invention provides a cellulase-producing bacterium or an amylase-producing bacterium belonging to the genus Bacillus (hereinafter, referred to as "main microorganism").
And Bacillus cereus (IFO 3003), Bacillus cereus subspecies mycoides (IFO 3015),
Bacillus mycoides (AHU 1360), Bacillus subtilis (AHU 1033) and Bacillus mycoides (AHU)
1034). A process for producing cellulase or amylase, comprising mixing and culturing a nucleic acid / protease producing bacterium (hereinafter referred to as “sub-microorganism”) selected from 1034).

The sub-microorganism used in the present invention is a nucleic acid,
A microorganism that produces an enzyme that decomposes or reduces impurities such as proteins, and does not inhibit the growth of the main microorganism and the production of the culture product, and is not inhibited from growing by the main microorganism. Microorganisms that produce enzymes that degrade contaminants such as decomposing enzymes.

Representative examples of the main microorganisms include Bacillus sp. KSM635 (Cellulase-producing bacterium No. 533), Bacillus alcalophilus (ATCC2159), an amylase-producing bacterium.
1) can be shown.

The present invention is carried out by mixing and culturing the above-mentioned main microorganisms and sub-microorganisms, and collecting a culture product from the culture by ordinary means.

The mixed culture is not particularly limited as long as both the main microorganisms and the sub-microorganisms can proliferate, but a method of adding both the main microorganisms and the sub-microorganisms to an appropriate medium and culturing the same, and culturing the main microorganisms In a medium, a method of adding and culturing a sub-microorganism or a pre-cultured sub-microorganism culture solution, and culturing by adding a main microorganism or a pre-cultured main microorganism culture solution to a medium in which the sub-microorganism is cultured Method. In particular, the mixed culture method is preferred. The mixing ratio of the main microorganism and the sub-microorganism varies depending on the combination of the main microorganism and the sub-microorganism, but at the end of the culture, the main microorganism proliferates sufficiently to produce a culture product, and the sub-microorganism decomposes contaminants. It is desirable to adjust the amount so that the enzyme can be produced sufficiently. In general, it is preferable that the number of sub-microorganisms is smaller than that of the main micro-organism at the end of culturing, particularly preferably 1/100 or less.

As the culture conditions for culturing the main microorganism or the sub-microorganism alone, conditions suitable for the growth of each microorganism may be selected. The culture conditions after mixing both microorganisms are determined in consideration of not only the productivity of the culture product, but also the growth rate of sub-microorganisms, the production rate of degrading enzymes, and the like.

After completion of the culture, a culture product can be collected by a usual method. In the method of the present invention, since the content of impurities such as proteins and nucleic acids is smaller than when the main microorganism is cultured alone, a special means for removing impurities is not required.

〔The invention's effect〕

According to the present invention, since the content of contaminants in the culture is small, post-treatment is easy, the yield of cellulase or amylase is relatively improved, and as a result, the production cost of cellulase or amylase can be reduced. It became.

〔Example〕

Next, the present invention will be described in detail with reference to examples, but the present invention is not limited thereto.

(1) In the following examples, the production of nucleases and proteases from the used sub-microorganisms was confirmed using the respective detection plates. The composition of the detection plate is shown below.

Nucleolytic enzyme detection plate (% by weight) Tryptone 1.5 Soypeptone 0.5 Sodium chloride 0.5 Deoxyribonucleic acid 0.2 Toluidine blue 0.01 Agar 1.5 Proteolytic enzyme detection plate (% by weight) Nutrient broth skim milk 2.0 (2) In the following examples, The slants storing the main microorganism and the sub-microorganism are the following mediums A to C, respectively.
It is made by adding agar to the product.

(3) In the following examples,% indicates% by weight.

Example 1 (1) A cellulase-producing bacterium, Bacillus sp. One platinum loop from the slant is 50 ml of seed medium shown below (medium A)
(Sakaguchi flask) and cultured with shaking at 34 ° C. for 24 hours. 5 ml of the obtained culture was inoculated into 50 ml of a main medium (Sakaguchi flask) and cultured at 34 ° C with shaking.

Medium A Polypeptone 2.0% Yeast extract 0.1% Disodium hydrogen phosphate 0.1% CMC 1.0% Sodium carbonate ^*) 0.75% ^*) Sodium carbonate is sterilized separately (2) Bacillus which produces nucleolytic enzymes and proteolytic enzymes as secondary microorganisms -Subtilis (AHU 1033) was used. The slant was inoculated into the following medium B, 30 ml (Sakaguchi flask), and cultured at 30 ° C. for 24 hours.

Medium B Nutrient broth glucose 1.0% Yeast extract 1.0% (3) 0.005 ml of the obtained culture of the sub-microorganism was added to the culture of the cellulase producing bacterium (10 hours after the start of culture).
(0.01%) and 0.5 ml (1%) were inoculated respectively, and shaking culture was further performed at 34 ° C. for 38 hours.

After completion of the culture, the permeation flux was measured using a membrane filter to determine the cellulase activity, contaminant content, and difficulty of fractionation of the culture solution.

(A) Cellulase activity The CMCase activity of alkaline cellulase K produced from a cellulase-producing bacterium was measured as follows. CMC (2.5
%), 0.1 ml of an enzyme solution was added to a substrate solution consisting of 0.2 ml of 0.5 M glycine buffer (pH 9.0) and 0.1 ml of deionized water, and reacted at 40 ° C. for 20 minutes. After the reaction, reducing sugars were quantified by 3,5-dinitrosalicylic acid (DNS) method. That is, D
1 ml of NS reagent was added, and color was developed by heating at 100 ° C. for 5 minutes. After cooling, 4.5 ml of deionized water was added for dilution. Wavelength 5
It was colorimetrically determined at 35 nm and expressed as relative activity.

(B) Contaminant content For the contaminants, the culture solution was subjected to centrifugation (3000 rpm, 20 min.), And the obtained supernatant fraction was examined by high performance liquid chromatography. The high molecular weight (MW 1,000,000 or more) fraction was used as the contaminant fraction. The column used was TSKgel GW4000S
WXL and 0.2 M phosphate buffer, pH 7.2 were used as elution buffers, and detection was performed with UV (260 nm). The contaminants were represented by relative amounts using DNA as a standard.

(C) Permeation flux As an example where impurities impose a load on the post-treatment process,
Using a microfiltration membrane (olefin-based polymer, 1.33 cm ² ), the permeation flux was examined (2.0 kg / cm ² , 10 ml) and expressed as a relative value.

 Table 1 shows the obtained results.

Example 2 In Example 1, Bacillus cereus (IFO 30) producing nucleases and proteases as sub-microorganisms was used.
03), and culturing was performed under the same conditions as in Example 1 except for the above. Table 2 shows the obtained results.

Example 3 In Example 1, culturing was carried out under the same conditions as in Example 1 except that Bacillus cereus subspecies mycoides (IFO 3015), which produces a nuclease and a proteolytic enzyme, was used as a sub-microorganism. Table 3 shows the obtained results.

Example 4 In Example 1, Bacillus mycoides (AH) which produces nucleases and proteases as sub-microorganisms
U1360), and culturing was carried out under the same conditions as in Example 1 except for the above. Table 4 shows the obtained results.

Example 5 (1) Bacillus alcalophilus (ATCC 21591) was used as an amylase-producing bacterium as a main microorganism. One platinum loop was inoculated from the slant into 30 ml (Sakaguchi flask) of the following medium (C) and cultured at 30 ° C. for 24 hours. 1 ml of the obtained culture solution was further inoculated into 30 ml of the same medium (Sakaguchi flask) and cultured at 34 ° C with shaking.

Medium C Peptone 1.0% Yeast extract 1.0% Dipotassium hydrogen phosphate 0.1% Magnesium sulfate 0.02% Soluble starch 1.0% Sodium carbonate ^*] (anhydrous) 1.0% Glucose ^*] 1.0% Fructose ^*] 1.5% ^{*] With} glucose and fructose Separate sterilization of sodium carbonate (2) Bacillus mycoides (AHU 1034) which produces nucleases and proteolytic enzymes was used as secondary microorganisms. 30 ml of medium B (Sakaguchi flask) was inoculated from the slant and cultured at 30 ° C. for 24 hours. 0.3 ml (1%) of the obtained culture solution of the heterologous microorganism was inoculated into the culture solution of the amylase-producing bacterium (10 hours after the start of the culture) and cultured at 34 ° C. for 38 hours.

After the completion of the culture, the amylase activity, contaminant content and permeation of a membrane filter in the culture solution were measured.

(A) Amylase activity The activity of amylase produced from an amylase-producing bacterium was measured as follows. Soluble starch (2.5%) 0.2m
l, 0.1 ml of enzyme solution was added to a substrate solution consisting of 0.1 ml of 0.5 M glycine buffer (pH 9.0) and 0.1 ml of deionized water, and the mixture was heated at 40 ° C.
Reacted for 20 minutes. After the reaction, reducing sugars were quantified by 3,5-dinitrosalicylic acid (DNS) method. That is, 1 ml of DNS reagent is added to 0.5 ml of the reaction solution.
l, add color by heating at 100 ° C for 5 minutes,
Was added and diluted. This was colorimetrically determined at a wavelength of 535 nm and represented by relative activity.

The contaminant content and permeate flux were measured as in Example 1. Table 5 shows the obtained results.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12R 1: 085) (72) Inventor Tomokazu Sato 1-8762 Doi Honcho, Hazaki-cho, Kashima-gun, Ibaraki Pref. 62-44172 (JP, A) (58) Fields investigated (Int. Cl. ⁶ , DB name) C12N 9/00-9/99 C12P 1/00-1/06

Claims (1)

(57) [Claims] 1. A cellulase-producing or amylase-producing bacterium belonging to the genus Bacillus and Bacillus cereus (IFO 300).
3), Bacillus cereus subspecies mycoides (IFO 3015), Bacillus mycoides (AHU 13)
60), a method for producing cellulase or amylase, comprising co-cultivating a nuclease / protease producing bacterium selected from Bacillus subtilis (AHU 1033) and Bacillus mycoides (AHU 1034).