JPS5918987B2 - Cellulase production method - Google Patents

Description

[Detailed description of the invention] The present invention relates to a method for producing cellulase.

Cellulosic materials such as paper scraps, wood chips, sawdust, straw, and rice husks amount to enormous amounts, but they are currently largely unused (incinerated).

Recently, due to the strong desire to develop alternative energy to petroleum, it has become possible to saccharify unused cellulose materials and reuse them as an energy resource.

That is, monosaccharides are obtained from cellulose materials, which are converted into ethanol by alcohol fermentation or the like, or converted into other chemical substances by fermentation technology or chemical technology.

The technical and economic problem here is cellulose saccharification technology.

Methods for saccharification of cellulose include chemical methods in which hydrolysis is performed using sulfuric acid, hydrochloric acid, etc., and biological methods in which decomposition is performed using enzymes or microorganisms.

However, although the former has been studied for a long time, it is economically difficult to put it into practical use, and the latter is carried out under mild reaction conditions, so it consumes less energy and requires less chemicals. However, there are still some problems, particularly the establishment of methods for isolating and culturing strains with high cellulose productivity.

Various cellulase-producing bacteria have been searched for, and among them, Trichoderma reese, a wood-decaying fungus, has been investigated.
erma reesei) is the most superior in that it has high C1 activity.

Recently, T, re, which has high C1 activity due to mutation,
esei QM 9414, T, reese, i
MCG 77 has been isolated and is attracting attention.

The production of cellulase by these cellulase-producing bacteria is induced in the presence of cellulose and inhibited by glucose.

Furthermore, when cellulose is used as a substrate, the specific growth rate (μ) is very slow at approximately 0.02/hr, and it takes a long time to induce cellulase, resulting in extremely low cellulase productivity. There was a lot of trouble.

Therefore, efforts are being made to greatly improve productivity through isolation of superior bacteria through mutation, fed batch culture, continuous culture, and the like.

However, the productivity of cellulases currently reported is only about 50 IU/A-hr at the highest, which is not very satisfactory.

In view of these circumstances, the present inventors have conducted intensive research and found that induction time is an important factor in the production of cellulase, and that the enzymatic activity of the produced cellulase is
The present invention was completed based on the knowledge that the effect depends on the bacterial cell concentration.

That is, the present invention relates to the production of cellulase using glucose as a substrate belonging to the genus TrichOderma.

The process is divided into a bacterial cell production process in which cellulase-producing bacteria are grown, and an enzyme production process in which cellulase is produced from the resulting bacterial cells in the presence of cellulose, an inducer. This is a method for producing cellulase, the gist of which is to maintain a high bacterial cell concentration in a fermentation tank by maintaining it at a high concentration through separate tanks of the same type and another.

Hereinafter, the present invention will be specifically explained with reference to the drawings.

First, a glucose source is communicated and supplied from the supply pipe 3 to the first fermentation tank 2 equipped with the stirrer 1 .

Further, a cellulose source to be supplied to a second fermentation tank 5, which will be described later, is also supplied to the first fermentation tank 2 through the supply pipe 3 together with a glucose source.

Of course, the cellulose source may be directly supplied to the second fermenter 5.

Furthermore, nutrient salts are added to the first fermentation tank 2 as necessary.

Then, cellulase-producing bacteria belonging to the genus Trichoderma are inoculated into the first fermenter 2.

As the glucose source, bagasse saccharification liquid and the like are mainly used.

As the cellulose source, delignified bagasse and physically or chemically treated natural cellulose materials are mainly used.

Cellulase-producing bacteria include T, reesei Q
M 9414, T.

reesei MCG 77 is preferably used, but
Not limited to these.

In this way, cellulase-producing bacteria are grown and produced in the first fermenter 2.

The following equation is obtained from the material balance of glucose in the bacterial cell production process.

Xl-Y8 (So-8) ・・・・・・・・・(
1. second
It is continuously sent to the fermenter 5.

The second fermentation tank 5 is also equipped with an agitator 6 and has a separate separation tank 7.

Circulation of the culture solution through the separation tank 7 is performed, for example, as follows.

A portion of the culture solution in the second fermentation tank 5 is taken out into the separation tank 7 via the separation tube 8 at regular intervals and left to stand.

After the required time, the supernatant portion is discharged out of the system via the discharge pipe 9, and the sediment portion is returned to the second fermentation tank 5 via the return pipe 10.

In this way, the bacterial cell concentration in the second fermenter 5 is maintained at a high level, and cellulase is produced from the bacterial cells in the presence of cellulose, which is an inducer.

Then, the culture solution in the second fermentation tank 5 is taken out through the enzyme take-out tube 11.

The following equation is obtained from the mass balance of bacterial cells and cellulose in the enzyme production process.

8”+7・(0°−0′3 ・・・・・・・・・
(2)ゝ・-2゜/F FoYo(COC2) μ2−D2”7°X1+Y.

(..-62)°°°°°゛°° (3) Cellulose concentration Fo: 2nd
Discharge rate from the fermentation tank F: Supply rate to the first and second fermentation tanks μ2: Specific growth rate of bacterial cells in the second fermentation tank D2: Dilution rate in the second fermentation tank Now, the productivity of cellulase is Assuming that it depends on the concentration, the following equation is obtained.

π−εX2 ・・・・・・・・・(4)
π: Cellulase productivity (IU/73/hr) ε: Cellulase specific production rate (IU/P cells/hr) Example.

Using the first fermentation tank 2 of 11 and the second fermentation tank 5 of 101, the bagasse saccharification treatment liquid is added to the first fermentation tank 2 as a glucose source,
Supplied as glucose at 35.3P/A,
Delignified bagasse is also used as a cellulose source.
T is supplied as hekinsan at 10 g/l and further added with an appropriate amount of nutrient salts, and is easily obtained from ATCC as a cellulase-producing bacterium belonging to the genus Trichoderma.

reesei QM 9414 (ATCC preservation number 26921) (this bacterium is described in "Biotechnology and Bioengineering Symposium", vol. 6, pp. 35-53 (1976)).
).

Culture conditions in the first fermenter 2, 20, 2/h
r, sensitivity: 33°C, pH: adjusted to 4.5.

In addition, the culture conditions in the second fermenter 5 were set to D2: 0.02.
/hr, temperature: 30°C, and pH: 3.5.

As mentioned above, the circulation of the bacterial cell-containing solution in the enzyme production process involves taking out a portion of the culture solution in the second fermentation tank 5 into the separation tank T at regular intervals, leaving it there, and discharging the supernatant out of the system. At the same time, the sedimentation section was returned to the second fermentation tank 5.

Cellulase productivity (ε) was determined by varying the discharge rate (Fe) of the culture solution taken out from the second fermenter 5.

The results are shown in the table below.

As can be seen from the table, cellulase productivity (ε) is the second
It is proportional to the bacterial cell concentration (X2) in the fermenter 5 and also depends on the discharge rate (Fe) of the culture solution from the same species 5.

In other words, cellulase productivity (ε) is determined by the specific growth rate (μ
2).

By the way, it is already known that in order to improve the productivity of cellulase, it is desirable that the specific growth rate (μ2) be around O.

Therefore, the discharge rate of the culture from the second fermenter 5 (Fe
), it is possible to bring the specific growth rate (μ2) of bacterial cells closer to around O and improve cellulase productivity.

As described above, according to the present invention, cellulase production can be carried out by
The cell production process and the enzyme production process are separated, and the culture solution is circulated through a separation tank in the latter process, so the cellulase productivity can be greatly increased by maintaining a high cell concentration in the enzyme production fermentation tank. can be improved.

In addition, the specific growth rate of bacterial cells in the enzyme production process can be easily reduced to 0.
can be closely regulated, so that the cellulase specific production rate can be maintained high and the productivity of cellulase can be increased.

In addition, by separating the steps, the culture conditions in the enzyme production process can be easily set to the optimal conditions for enzyme production, and the influence of catabolite repression caused by glucose can be suppressed to a minimum in the enzyme production process. can.

[Brief explanation of drawings]

The drawing is a system diagram showing an embodiment of the present invention. 2...First fermentation tank, 5...Second fermentation tank, T...Separation tank.

Claims (1)

[Claims] 1 Cellulase production is carried out by a bacterial cell production process in which cellulase-producing bacteria belonging to the genus Trichoderma proliferate using glucose as a substrate, and an enzyme production process in which the resulting bacterial cells produce cellulase in the presence of cellulose, which is an inducer. The cellulase production process is carried out in two steps, and the culture solution in the fermentation tank is circulated through a separation tank of the same species and another in the enzyme production process, thereby maintaining a high bacterial concentration in the fermentation tank. Production method.