JPH0330689A - Novel production of cellulose using microorganism - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed Description of the Invention] (Industrial Application Field) Bacterial cellulose produced by certain acetic acid bacteria is almost pure cellulose and is formed of single-crystalline microfibrils. Cellulose membranes and cellulose fibers, which are aggregates thereof, are known to have extremely high strength and elastic modulus, and therefore their application as various industrial materials is being studied. For example, it has recently been industrialized and marketed as an acoustic material (headphones). Furthermore, since this cellulose is edible, it is also used in the field of food materials. The present invention provides a highly efficient method for producing cellulose using a new effective culture solution for cellulose synthesis useful in these fields.

(Prior art) Research on the synthesis of cellulose using microorganisms has been carried out for a long time, and the culture solution of acetic acid bacteria used for this purpose mainly contains D-glucose as a carbon source and refined sugars such as sucrose. However, these products are created through processes such as synthesis, washing, and purification, and are currently quite expensive. In addition, as a carbon source, in a method for producing polysaccharide using a culture solution containing partially decomposed cellulose (cellooligosaccharide) as a main component, disclosed in Japanese Patent Application Laid-Open No. 54-37889, Carbohydrate Chemis
try Vol.

IIl, P-134-139 (1 decomposition method),
P-139~143 (enzymatic degradation method), P-143~
Cellulose partial decomposition products prepared by No. 149 (acetolysis method) have been used, but these also require very complicated purification steps before being used, and are also industrially expensive.

In addition, in the use of cellulase preparations in culture and the use of cellulose-producing microorganisms in JP-A No. 63-74490, it is mentioned that cellulose synthesis efficiency is improved by adding a small amount of physiologically active cellulase. There is. However, not only is the synthesis efficiency of cellulose only 1.5 times that of conventional methods, but it is extremely inefficient because cellulose is synthesized by adding new and expensive additives to an already expensive carbon source. This is a manufacturing method.

There are methods in which micronutrients such as inositol and phytic acid are added, such as in the method for producing cellulosic substances using microorganisms disclosed in JP-A No. 61-212295, and the same applies to these methods.

As described above, as long as the culture medium used to date is used, there is no production method that allows the culture medium to be easily prepared, the main component carbon source to be produced at low cost, and at the same time has a high conversion efficiency to cellulose. . Furthermore, there is no precedent for a manufacturing method that actively uses byproducts from the production of culture fluid.

(Problems to be Solved by the Invention) The purpose of the present invention is not only to facilitate the preparation of a culture solution and to produce the main component carbon source at a low cost, but also to use the by-products derived at that time. The objective is to find a production method with high conversion efficiency to cellulose.

(Means for Solving the Problem) As a result of extensive research to achieve the above object, the present inventors have completed the present invention using the following means. That is, the present invention relates to a method for producing cellulose, which is characterized in that a culture solution containing a thermally denatured cellulase is used in a method of biosynthesizing cellulose using microorganisms. The present invention relates to a method for producing cellulose using a culture solution containing a liquid obtained by enzymatically decomposing a cellulosic substance with cellulase and then thermally denaturing it.

In the present invention, cellulase may be any enzyme as long as it has the activity of cleaving β-1,4 glucosidic bonds. The concentration of cellulase used is not particularly limited, but is preferably in the range of 0.05 to 2% solution concentration.

The type of cellulase used is not particularly limited, and preferred examples include Trichoderma cellulase, Driselase cellulase, and the like.

The thermally denatured cellulase in the present invention refers to a substance that has lost the activity of cleaving β-1,4-glucoside bonds by heat-treating cellulase. There are no particular restrictions on the conditions such as temperature and time for deactivation.

Here, if a cellulose material is first enzymatically decomposed with cellulase and then thermally denatured as the cellulase thermally denatured product, it is possible to obtain the carbon source necessary for culture at the same time as the cellulase thermally denatured product. This is more preferable since the efficiency in producing cellulose is improved.

The above-mentioned cellulosic substance may be any substance as long as it has a polysaccharide consisting of β-1,4-glucoside bonds as a main component. For example, the material may be old newspapers, pulp such as magazines, industrial waste such as bagasse, beet dregs, wood flour, etc., and conditions such as shape and composition are not limited.

The microorganism used in the present invention is a bacterium belonging to the genus Acetobacter or Gluconobacter, and any microorganism that can synthesize cellulose can be used. Among these, preferred examples include Acetobacter xylinum ATCC-10245, Same (NB
I-1051 etc. can be mentioned.

In the present invention, the culture medium for culturing microorganisms may be made of only a carbon source obtained from cellulosic materials, but it may contain a nitrogen source, inorganic salts, and growth factors as necessary nutrients necessary for growing microorganisms. , more preferred.

There is no limit to the concentration of the carbon source in the medium, but from the viewpoint of cellulose synthesis rate, it is more preferable to use it in the range of 0.1 to 1% by weight.

Further, as the nitrogen source, urea, amino acids, amide compounds, etc., and organic nitrogen-containing compounds such as ammonium salts, nitrates, etc. can be used, and peptone, yeast extract, malt extract, etc. can also be used. There is no limit to the concentration of the nitrogen source, but from the viewpoint of cellulose synthesis rate, it should be 0.05~
It is more preferable to use it in a range of 3% by weight.

Cellulose synthesized and present in the culture solution is, for example,
Separate it from the culture solution using a method such as centrifugation, immerse it in an aqueous solution of about 1% sodium hydroxide to remove proteins such as microorganisms, and then neutralize it with about 1% acetic acid, wash with water, and dry it. recovered by. Cellulose after washing and drying is
It was confirmed by infrared absorption spectrum and X-ray diffraction that it was pure cellulose.

(Effects of the Invention) According to the present invention, the enzymatic saccharification solution of cellulosic substances can be used as a carbon source without condensing or refining, and by simply adding nutrients as necessary, the microorganism culture solution can be used as it is. Not only is the culture solution very easy to prepare, but the main component carbon source can be produced at low cost. Furthermore, by using the thermally denatured cellulase, which is a byproduct derived at that time, without separation, we have developed a production method that has a conversion efficiency of 2.0 to 2.5 times that of a medium using refined sugar. It became possible to provide. Based on this, the present invention is considered to be useful for greatly reducing the production cost of cellulose by microorganisms and expanding its range of use, and is a useful cellulose material that provides an effective material for various industrial fields. This is a manufacturing method.

(Example) Hereinafter, the present invention will be explained in more detail with reference to Examples.
The present invention is not limited to this.

Example 1 Bagasse, beet dregs, coniferous bleached sulfide valve (N
BSP), bleached softwood kraft pulp (NBKP), and unbleached softwood kraft pulp (NUBKP) were each ground to 40 mesh under using a lysaw mill, and then ground for 30 minutes using a rod mill. These are cellulase 0NO
Enzymatic digestion was performed using ZUKA R-10 for 48 hours at a substrate concentration of 50 μ/d, enzyme concentration of 5 μ/d, pH 5.0 (sodium citrate buffer), shaking rate of 120 strokes/min, and temperature of 50°C. After that, the saccharified liquid was filtered out using a G4 glass filter. Table 1 shows the results of analyzing the ti composition in the filtrate by liquid chromatography.

Table 1 Sugar composition (χ) of enzymatic saccharification solutions of various Seltos substances
NBSP NBKP NUBKP
Beet crumbs bagasse glucose? 5.0
74.6 76.2 61.8
62.4 se0 bios 3.6 2.7
1.6 4.2 4.1 Xylose 6.0 8.4 7.4
5.3 23.4 Galactose
5.9 6.4 5.9
4.1 6.27 Rabinose 0.4
0.5 0 16.4
2.2 Mannose 9.1 7.4
8.9 B, 2 2.0 As shown in Table 1, 90 to 98% of the total sugar content in the raw materials was solubilized, and dehua liquid could be easily produced.

Distilled water was added to dilute the various saccharification solutions to a glucose concentration of 2%, and peptone and yeast extract were added to each concentration of 0.5%.

These 15 culture solutions were sterilized in diluted air for 120 minutes in Cl2O to thermally denature the cellulase, and then
Inoculate 0.5 ml of A. acter
The cells were incubated for hours. After culturing, the cellulose film formed on the surface of the culture solution was collected and immersed in a 1% aqueous sodium hydroxide solution for 2 to 4 days to remove proteins such as bacterial cells, then neutralized with 1% acetic acid, washed with water, After drying, the weight was measured, and this was taken as the amount of cellulose produced. The results are shown in Table 2.

Table 2 '#Amount of cellulose produced from each culture solution with cox concentration 2χ (■) NBSP NBKP LBKP N
UBKP Heat Kasu＾TCC-102451
8,6319,9320,9623,4218,12N
BI-105132, 4325, 7828, 6830,
2113,49 Comparative Example 1 A saccharified liquid prepared in the same manner as in Example 1 was filtered through a G4 glass filter, filtered through a 0.45 μm membrane filter, and further filtered through a 0.1 μm membrane filter to obtain a saccharified liquid. The cellulase inside was removed. Using this, a culture solution was prepared in the same manner as in Example 1, and sterilization, inoculation, and culture were performed. After culturing, the amount of cellulose produced was measured in the same manner as in Example 1, and the results are shown in Table 3.

Table 3 Amount of cellulose produced from each culture solution with a glucose concentration of 2χ (mg) NBKP NUBKP 8TCC-1024512, 8121, 17NBI-1
05116, 3020, 14 Comparative Example 2 A solution having the same composition as the saccharified solution prepared in Example 1 was prepared by dissolving purified sugar, a commercially available special reagent, in ion exchange distilled water. This was diluted and a nitrogen source was added in the same manner as in Example 1, and the pH was adjusted to be the same as the culture solution used in Example 1. Using this, sterilization, inoculation, and culture were performed in the same manner as in Example 1. After culturing, the amount of cellulose produced was measured in the same manner as in Example 1, and the results are shown in Table 4.

Table 4 Amount of cellulose produced from each culture solution at concentration 2χ (■) NBSP NBKP NUBKP
Beat Kasu ATCC-1024510,359,45
8,419,58 Example 2 A culture solution using refined sugar was prepared in the same manner as in Comparative Example 2. Cellulase 0 NOZUKA R-10 dissolved at 0.5% i? ! After steam sterilizing at 120°C for 20 minutes to thermally denature the cellulase, this culture solution was sterilized and inoculated (Aceto) in the same manner as in Example 1.
bacter xylinum ATCC40245)
, cultured. After culturing, the amount of cellulose produced was measured in the same manner as in Example 1, and the results are shown in Table 5.

Example 3 A solution having the same composition as the saccharification solution prepared in Example 1 was prepared by dissolving purified sugar, a commercially available special reagent, in a citric acid buffer. This was diluted with a citrate buffer in the same manner as in Example 1, a nitrogen source was added, and the pH was adjusted to be the same as the culture solution used in Example 1. Add 0.0% of cellulase NOZUKA R-10 to this solution. 5
% solution concentration, steam sterilized at 120°C for 20 minutes to thermally denature the cellulase, and then sterilized and inoculated (Ace) in the same manner as in Example 1.
tobacterxylinum ATCC-1024
5) Culture was performed. After culturing, the amount of cellulose produced was measured in the same manner as in Example 1, and the results are shown in Table 6.

Table 5 Amount of cellulose produced from each culture solution with a glucose concentration of 2χ (mg) NBSP NBKP NLIBKP8TC
To C-0245 20.57 19.80
21.22 Table 6 Amount of cellulose produced from each culture solution with glucose concentration 2z (■) NBSP NBKP NLIBKPΔTC
C-1024530,0233,2627,95 Comparative Example 3 H3 medium (D-glucose; 2.0%, peptone: 0.
5%, yeast extract: 0.5%, citric acid, 0.115%
, disodium hydrogen phosphate; 0°27%) were prepared, sterilized and inoculated (Acetobacter) in the same manner as in Example 1.
er xylinum ATCC-10245) was cultured. After culturing, the amount of cellulose produced was measured in the same manner as in Example 1, and the result was 18.20.

Example 4 Driselase-based cellulase was added to the H3 medium used in Comparative Example 3 to a 0.5% solution dust, and then
After steam sterilizing at °C for 20 minutes to thermally denature the cellulase, this culture solution was sterilized in the same manner as in Comparative Example 3.
Inoculation and culture were performed. After culturing, the amount of cellulose produced was measured in the same manner as in Example 1, and the result was 32.34.

Example 5 Cellulase 0NOZUKA R-10, which is a Trichoderma cellulase, was added to the H5 medium used in Comparative Example 3.
After adding 5 to 0.5% solution dust, 120
After performing steam sterilization at °C for 20 minutes to thermally denature the cellulase, this culture solution was sterilized, inoculated, and cultured in the same manner as in Comparative Example 3. After culturing, the amount of cellulose produced was measured in the same manner as in Example 1. As a result, the cellulase concentration was 0.
05% is 23.84mg, 0.1% is 30
．． 94mg, 27.66+ng for 0°3%,
In the case of 0.5%, it was 23.11■.

(1 idiot)

Claims (2)

[Claims] (1) A method for producing cellulose, which comprises using a culture solution containing a thermally denatured cellulase in a method for biosynthesizing cellulose using microorganisms. (2) The method for producing cellulose according to claim 1, wherein the culture solution containing the cellulase thermally denatured product is a culture solution containing a solution obtained by enzymatically decomposing a cellulose substance with cellulase and then thermally denaturing it.