WO2002031119A1

WO2002031119A1 - Mutant resistant to catabolite repression

Info

Publication number: WO2002031119A1
Application number: PCT/KR2000/001147
Authority: WO
Inventors: Bon-Tag Koo; Kee-Don Park; Jin-Man Kim; Jung-Ki Lee; Guk-Jin Kim; Hyun-Kuk Kim
Original assignee: Inbionet Corp
Current assignee: Inbionet Corp
Priority date: 2000-10-13
Filing date: 2000-10-13
Publication date: 2002-04-18
Anticipated expiration: 2003-04-13
Also published as: AU2000279668A1

Abstract

The present invention relates to catabolite repression-resistant thermophilic strains of Bacillus thermoglucosidasius BtgMul (KCCM-10221) producing amylolytic enzyme, and of Bacillus thermoglucosidasius BtgcelMul (KCCM - 10220) producing cellulase. Since the co-culture of the two strains, BtgMul and BtgcelMul, notably increase activiy of amylolytic enzyme and celluase, they might be applied to food garbage volume reduction process by declining the food garbage volume sharply.

Description

MUTANT RESISTANT TO CATABOLITE REPRESSION

TECHNICAL FIELD

The present invention relates to novel Bacillus strains that can produce industrially useful enzymes. More particularly, the present invention relates to the catabolite repression insensitive mutants of thermophihc Bacillus thermoglucosidasius with mutated glucose transport system and the processes for preparation and application thereof.

BACKGROUND ART

Amylase and cellulase have been utilized most frequently for industrial uses. Starch and cellulose can be decomposed into bioavailable sugars by degrading with bacterial enzymes and be utilized by the microorganism.

However, the repression of the' amylase and cellulase, so called catabolite repression, is induced when easily usable carbon sources, such as glucose, exist in bacterial medium or when glucose is accumulated properly within medium through the degradation by the secreted enzymes. Thus far, there remain some difficulties to produce the above enzymes massively by using bacterial fermentation. DISCLOSURE OF INVENTION

The object of the present invention is to provide catabolite repression insensitive Bacillus strains producing amylase and cellulase specifically, which can improve the enzyme productivity as well as save on glucose for secondary fermentation. Particularly, the present invention provides amylase and cellulase producing Bacillus thermoglucosidasius strains, which are made by damaging the repression mechanism of the enzymes and by destroying artificially the main bacterial mechanism of glucose absorption through gene mutation technique.

The present invention provides the mutant strain, Bacillus thermoglucosidasius BtgMul (KCCM-10221) producing amylase, and the mutant strain, Bacillus thermoglucosidasius BtgcelMul (KCCM-10220) producing cellulase which are damaged in the catabolite repression process and are inhibited in glucose uptake process due to the destruction of glucose transport system.

Precisely, the above mutant Bacillus thermoglucosidasius strains can be prepared by the processes comprising steps as follows:

(a) mutating thermophihc Bacillus thermoglucosidasius strains by using N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and ultraviolet ray;

(b) selecting mutant strains by exploiting 2-deoxyglucose;

(c) cultivating the mutants obtained above for 22 to 26 hours at 52 to 58^°C in Nutrient agar medium (beef extract 0.3%; peptone 0.5%>) containing 0.3%> of glucose and 1.0%> of starch or cellulose, which is suitable for the measurement of the enzyme activities; (d) dyeing the agar plate with 0.1 N concentration of iodide solution or Congo-Red reagent;

(e) separating catabolite repression insensitive mutants by collecting colonies forming transparent circles on the above plate; (f) selecting the mutant strain that obtains the catabolite repression insensibility for amylase, or selecting the mutant strain that obtains the catabolite repression insensibility for cellulase.

In addition, the present invention provides a method for producing the amylase or the cellulase by using the Bacillus thermoglucosidasius strains obtained above.

Furthermore, the present invention provides a method for decomposing food waste by using the above Bacillus thermoglucosidasius mutants.

Further features of the present invention will appear hereinafter.

In the present invention, the mutant Bacillus thermoglucosidasius strains are separated according to the methods described below. N-methyl-N'-nitro-N- nitrosoguanidine (NTG) and ultraviolet ray is treated for mutating thermophilic Bacillus thermoglucosidasius strains. Then the mutant strains are selected by using culture medium containing 2-deoxyglucose. The mutant strains obtained above is cultivated again for 22 to 26 hours in Nutrient agar medium (beef extract 0.3%>; peptone 0.5%>), which contains 0.3% of glucose, 1.0% of starch or cellulose. The above agar plate is dyed with 0.1 N of iodide solution or Congo-Red reagent. Finally, catabolite repression insensitive Bacillus mutants are distinguished by clearness, and collected from the colonies forming transparent circles.

According to the above method, the Bacillus thermoglucosidasius strain which loses the cellulase activity of the wild type and shows insensibility to catabolite repression of amylase are selected among the above mutant strains. In addition, Bacillus thermoglucosidasius which loses the amylase activity of the wild type strain and sustains the insensibility to catabolite repression of cellulase are also selected among the above mutant strains.

Especially, the mutant strains of the present invention are observed to increase the activities of amylase and cellulase remarkably when the two mutants are co-cultured in Nutrient medium. The peculiar competition between the mutant Bacillus thermoglucosidasius strains might not exist since the two strains that derived from the same parental strain have similar physiological property.

In the mean time, detailed features of the experimental process adopted in the present invention will be demonstrated hereinafter.

The activity of amylase in the medium plate is measured by the process mentioned below. The sample Bacillus strain is cultivated in Nutrient agar medium containing 1.0% of starch, and is dyed onto medium plate by 0.1 N solution of iodide in order to make transparent circles. Then the radius of the transparent circles is measured.

In addition, the enzyme activity of cellulase in the medium plate is measured by the process described below. The sample Bacillus strain is cultivated in Nutrient agar medium containing 1.0% of cellulose, and is dyed by 0.1%) of Congo-Red reagent. After washing the strain with 1 N solution of sodium chloride, the radius of the transparent circles is determined.

In liquid state, the activities of amylase and cellulase are measured by the process as follows. 100 μl of the bacterial culture supernatant (containing enzyme) is added to 900 μl solution of 0.1 %> starch or 1% cellulose, and is reacted at 55°C for 30 minutes. Then the same volume of dinitrosalicylic acid (DNS) solution is mixed, and the mixture is boiled for 15 minutes. The absorbance of the sample is measured at 570 nm. In the comparative sample, 100 μl of sterilized water is utilized instead of the enzyme supernatant. In the above process, unit is defined as enzyme amount showing 0.001 difference of the absorbance value from that of the comparative sample excluding enzymes

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which;

Fig. 1 represents the activities of the catabolite repression insensitive mutants of the present invention, Bacillus thermoglucosidasius BtgMul (KCCM-

10221; a) and Bacillus thermoglucosidasius BtgcelMul (KCCM- 10220; b) showing specific activities for the amylase and cellulase respectively; in Nutrient agar plate containing 1% of starch (Fig. 1A); in Nutrient agar plate containing 1%> of starch and 0.3% of glucose (Fig. IB); in Nutrient agar plate containing 1%> of CMC (Fig. IC); and in Nutrient agar plate containing 1% of CMC and 0.3% of glucose (Fig. ID).

Fig. 2 represents the mutant Bacillus thermoglucosidasius strain damaged in the group translocation process, main glucose transporter, which is separated by using 2-deoxyglucose.

Fig. 3 represents the growth curves of the wild type Bacillus thermoglucosidasius and catabolite repression insensitive Bacillus thermoglucosidasius BtgMul (KCCM-10221) and Bacillus thermoglucosidasius BtgcelMul (KCCM-10220), which are cultivated in glucose condition.

Fig. 4 represents the amylase activities of the wild type Bacillus thermoglucosidasius and catabolite repression insensitive Bacillus thermoglucosidasius BtgMul (KCCM-10221) and Bacillus thermoglucosidasius BtgcelMul (KCCM-10220), which are cultivated in the Nutrient medium containing starch as a carbon source.

Fig. 5 represents the cellulase activities of the wild type Bacillus thermoglucosidasius and the catabolite repression insentive Bacillus thermoglucosidasius BtgMul (KCCM-10221) and Bacillus thermoglucosidasius BtgcelMul (KCCM-10220), which are cultivated in the Nutrient medium containing cellulose as a carbon source. Fig. 6 represents the concentration variation of glucose in the culture medium of the wild type Bacillus thermoglucosidasius and the catabolite repression insensitive Bacillus thermoglucosidasius BtgMul (KCCM-10221) and Bacillus thermoglucosidasius BtgcelMul (KCCM-10220) according to the incubation time.

Fig. 7 represent the food waste weight reduction ratio of the catabolite repression insensitive Bacillus thermoglucosidasius BtgMul (KCCM-10221) (Fig. 7 A) and the number of catabolite repression insensitive Bacillus thermoglucosidasius BtgMul (KCCM-10221) (Fig. 7B) when applied to food waste volume reduction equipment.

Fig. 8 represent the productivities of the amylase (Fig. 8A) and cellulase (Fig. 8B) when Bacillus thermoglucosidasius BtgMul and Bacillus thermoglucosidasius BtgcelMul strains (KCCM-10221 and KCCM-10220) are co-cultured.

BEST MODE FOR CARRYING OUT THE INVENTION

Practical and presently preferred embodiments of the present invention are illustrative as shown below.

Preferred Embodiment 1: Mutation of microorganism Bacillus thermoglucosidasius (KCTC accession number 3400 BP) was obtained from the gene bank of KRIBB (Korea Research Institute of Bioscience and Biotechnology; #52 Oun-dong, Yusong-ku, Taejon 305-333) and was cultured by using 5 μl of Nutrient medium (beef extract 0.3%; peptone 0.5%o) in 55°C for 24 hours. Then NTG (N-methyl-N'-nitro-N-nitrosoguanidine) was added toward 1 mM, and the above strain was cultivated by shaking for 2 more hours. The culture medium was recovered and smeared onto the Nutrient agar medium containing 1%> of starch or cellulose by using the successive dilution smear method.

In addition, 20 erg amount of ultraviolet ray was irradiated onto the culture plate for 40 seconds and again 100 μl of 30 mM 2-deoxyglucose was added onto the center of the solid medium. Then the plate was maintained at 55°C for 24 hours. After the treatment transparent circles appeared - the circles are formed by diffusing 2-deoxyglucose which inhibiting bacterial proliferation - bacterial colonies growing near the transparent circle region were separated.

Preferred Embodiment 2: Selection of mutant strains

The mutant strains damaged in the processes of the catabolite repression and destructed in group translocation process as a major glucose transport system have been adopted from the above selected colonies as follows. (See Fig. 2)

The mutant strains obtained above were inoculated onto Nutrient agar medium containing 0.3% of glucose, 1% of starch or cellulose, and were cultivated for 24 hours. Then 0.1 N of iodide solution or 0.1 % of Congo-Red solution was treated so as to select the catabolite repression insensitive mutant which existed in the transparent circle -of the largest radius and represented the specific enzyme activity of amylase or cellulase independently.

The mutant strains obtained above were named BtgcelMul and BtgMul respectively. The Bacillus thermoglucosidasius BtgcelMul and Bacillus thermoglucosidasius BtgMul strains have been deposited with Corporation Korean Culture Center of Microorganism, Seoul, Korea, on Apr. 2, 1999 (accession number: KCCM-10220; KCCM-10221 respectively). The enzyme activity variation against glucose was depicted in Table 1.

Table 1

According to existence of glucose and substrate, the enzyme activities of

Bacillus thermoglucosidasius BtgcelMul and BtgMul strains

+++ : very high activity; ++ : high activity; - : no activity

Preferred Embodiment 3: Growth curves of the mutant strains

In order to examine the growth of microorganisms, the wild type Bacillus thermoglucosidasius and the mutant Bacillus thermoglucosidasius BtgcelMul (KCCM-10221) and BtgcelMul (KCCM-10220) were inoculated with 0.1% into Nutrient medium containing O.P/o of starch or cellulose and 0.3% of glucose respectively, and cultured at 55°C by shaking. The growth of the Bacillus thermoglucosidasius strains was observed at 8-hour interval. As a result, the wild type and the mutant Bacillus thermoglucosidasius BtgMul (KCCM-10221) and BtgcelMul (KCCM-10220) passed exponential period, and reached the stationary period of proliferation after 16 hour-culture. At that time, the survival numbers of bacteria were 6.2 x 10⁷, 2.3 x 10⁸ and 6.5 x 10⁷ CFU/ml respectively. The detailed results were depicted in Fig. 3.

Preferred Embodiment 4: Examination of the amylase productivity of the mutant strains

In order to investigate the amylase productivity, the wild type Bacillus thermoglucosidasius and the mutants of the present invention, Bacillus thermoglucosidasius BtgcelMul (KCCM-10221) and BtgcelMul (KCCM- 10220), were inoculated with 0.1 %> into Nutrient medium containing O.P/o of starch, and cultured at 55°C by shaking. The enzyme productivities of the Bacillus thermoglucosidasius strains were observed at 8-hour interval. As a result, the wild type increased the amylase activity for absorbing glucose from culture medium until catabolite repression was worked due to glucose accumulation. However, the mutant Bacillus thermoglucosidasius BtgMul (KCCM-10221) augmented the enzyme productivity consistently although glucose concentration increased. The results were depicted in Fig. 4.

Preferred Embodiment 5: Examination of the cellulase productivity of the mutant strains

In order to examine the cellulase productivity, the wild type Bacillus thermoglucosidasius and the mutants of the present invention, Bacillus thermoglucosidasius BtgMul (KCCM-10221) and BtgcelMul (KCCM-10220), were inoculated with O.P/o into Nutrient medium containing O.P/o of cellulose and cultured at 55°C by shaking. The enzyme productivities of the Bacillus thermoglucosidasius strains were measured at 8-hour interval. As a result, the wild type reduced the enzyme productivity through catabolite repression since glucose was accumulated. However, the mutant Bacillus thermoglucosidasius BtgcelMul (KCCM-10220) increased the enzyme productivity consistently according to time. The results were depicted in Fig. 5.

Preferred Embodiment 6: Concentration variation of glucose in the medium of the mutant strains during fermentation

In order to investigate the concentration variation of glucose, the wild type Bacillus thermoglucosidasius and the mutants of the present invention, Bacillus thermoglucosidasius BtgMul (KCCM-10221) and BtgcelMul (KCCM- 10220), were inoculated with O.P/o into Nutrient medium containing O.P/o of starch or cellulose and cultured at 55°C by shaking. The concentration variation of the Bacillus thermoglucosidasius strains was observed at 8-hour interval. As a result, the wild type strain has consumed glucose rapidly in liquid medium. However both the mutant Bacillus thermoglucosidasius accumulated glucose. The data was depicted in Fig. 6.

Preferred Embodiment 7: Application of the mutant strains for developing equipment of food waste volume reduction

Food waste collected from mass feeding facilities, restaurants and so on was mixed well with sterilized wood chip, and was agitated in a reactor inoculating 1,000 ppm amount of the mutant strain BtgMul (KCCM-10221). In order to investigate the weight reduction ratios of total mass, 1 kg of new organic waste matter was admixed at 1-day interval. As a result, the total ratio of mass reduction was 140%) approximately after 6 day of stabilization period (see Fig. 7A).

Furthermore, the each cell number of the mutant strains within the reactor was measured by using Nutrient agar medium containing final 30 mM concentration of 2-deoxyglucose and 1%> of starch or cellulose. Thus, the mutant Bacillus thermoglucosidasius BtgMul (KCCM-10221) was identified to be a major population. The detailed data was shown in Fig. 7B. Preferred Embodiment 8: Increase of ÷he enzyme activity in co-culture of the mutant Bacillus thermoglucosidasius strains

In order to measure enzyme activities, the two mutant Bacillus thermoglucosidasius strains were co-cultured in Nutrient medium, which autoclaved for 20 minutes, containing 0.1% of starch and cellulose. As comparative samples, the same medium not inoculating bacteria and the medium inoculating the above mutant strains respectively were utilized. The detailed results were shown in Fig. 8 A and Fig. 8B.

However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the scope of the present invention.

INDUSTRIAL APPLICABILITY

As demonstrated in Preferred Embodiments 1 and 2, the mutant Bacillus thermoglucosidasius BtgMul shows catabolite repression insensibility specific for amylase, and is damaged in group translocation mechanism. Hence, the mutant represented high amylase activity in Nutrient agar medium containing 0.3% of glucose. On the other hand, Bacillus thermoglucosidasius BtgcelMul showing the catabolite repression insensibility specific for cellulase represented high cellulase activity in Nutrient agar medium containing 0.3%) of glucose. The mutant Bacillus thermoglucosidasius BtgMul (KCCM-10221) and BtgcelMul (KCCM-10220) were multiplied to 2.3 x 10⁸ and 6.5 x 10⁷ CFU/ml respectively in Nutrient medium containing O.P/o of starch or cellulose and 0.3 % of glucose, which was the higher growth rate than 6.2 x 10⁷ CFU/ml of the wild type. In detail, the amylase activity of Bacillus thermoglucosidasius BtgMul increased continuously during 48 to 56 hours culture. On the other hand, the enzyme activity of the wild type Bacillus thermoglucosidasius reduced on account of glucose effect. Approximately, the enzyme activity of the mutant Bacillus thermoglucosidasius BtgMul was more than six times of the highest activity of the wild type.

The cellulase activity of the mutant Bacillus thermoglucosidasius BtgMul (KCCM-10221) was approximately more than twice as high as that of the wild type strain. The glucose concentration of medium was decreased consistently in the wild type consuming glucose, but the glucose concentration increased to 4.5 g/1 of maximum value in the mutant Bacillus thermoglucosidasius BtgMul (KCCM-10221) and BtgcelMul (KCCM-10220) culture, which described the glucose accumulation toward 1.5 times of the initial concentration.

In order to investigate the practical application of Bacillus thermoglucosidasius BtgMul, the mutant strain was inoculated with 1,000 ppm amount of the total food waste weight into food waste volume reduction equipment. Then 1 kg of new food waste was added daily and was adjusted for about 1 week. Resultantly, the number of live bacteria increased toward more than 10 CFU/ml and the total mass reduction ratio reached 140%> approximately.

Especially, the mutant Bacillus thermoglucosidasius BtgMul and BtgcelMul co-cultured in the same medium shows about 1.6 times and 1.4 times activities of amylase and cellulase than those cultured independently.

Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the scope of the invention as set forth in the appended claims.

INDICATIONS RELATING TO DEPOSITED MICROORGANISM OR OTHER BIOLOGICAL MATERIAL

(PCT Rule 13W-

The indications made below relate to the deposited microorganism or other biological material referred to in the description on page ? , line 6 ,

B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet | |

Name of depositary institution

Korean Culture Center of icroorganism(KCCM)

Address of depositary institution (including postal code and country)

361-221 , Yurim B/D, Hongje-1- ong, Seodaemun-gu, Seoul 120-091, Republic of Korea

Date of deposit Accession Number

/-.pi II Z, I iW5 KCCM-10220

C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet |_J

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated States)

E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)

The indications listed below will be submitted to the International Bureau later (specify the general nature of the indications e.g., "Accession Number of Deposit")

INDICATIONS RELATING TO DEPOSITED MICROORGANISM OR OTHER BIOLOGICAL MATERIAL

(PCT Rule I3bis)

The indications made below relate to the deposited microorganism or other biological material referred to in the description on page 9 , line I .

B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet | I

Name of depositary institution

Korean Culture Center of Microorganism(KCCM)

Address of depositary institution (including postal code and country)

361-221 , Yurim B/D, Hongje-1-dong, Seodaemun-gu, Seoul 120-091, Republic of Korea

Date of deposit Accession Number

April 2, 1999 KCCM-10221

C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet Q

E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)

For receiving Office use only For International Bureau use only

| | This sheet was received with the international application [ I This sheet was received by the International Bureau on:

Authorized officer Authorized officer

Form PCT RO/134 (Julyl998) n

Claims

What is claimed is:

1. The amylase producing strain, Bacillus thermoglucosidasius BtgMul (accession number: KCCM-10221) that is damaged in catabolite repression process against glucose, and is inhibited in glucose uptake process due to the destruction of glucose transport system.

2. The cellulase producing strain, Bacillus thermoglucosidasius BtgcelMul (accession number: KCCM-10220) that is damaged in catabolite repression against glucose and is inhibited in glucose uptake due to the destruction of glucose transport system.

3. A process for preparing the Bacillus thermoglucosidasius strain of claim 1, comprising steps as follows:

(a) mutating thermophilic Bacillus thermoglucosidasius strains by using N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and ultraviolet ray; (b) selecting mutant strains by using 2-deoxyglucose;

(c) cultivating the mutants obtained above for 22 to 26 hours at 52 to 58^°C in Nutrient agar medium (beef extract 0.3%>; peptone

0.5%)) containing 0.3% of glucose, 1.0% of starch or cellulose which is suitable for measurement of the enzyme activity; (d) dyeing the agar plate with 0.1 N concentration of iodide solution or Congo-Red reagent;

(e) separating catabolite repression insensitive mutants by collecting colony forming transparent circle;

(f) selecting the mutant strain that obtains the catabolite repression insensibility for amylase, and loses the cellulase activity of wild type strain.

4. The process for preparing the Bacillus thermoglucosidasius strain of claim 2, comprising steps as follows: (a) mutating thermophilic Bacillus thermoglucosidasius strains by using N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and ultraviolet ray;

(b) selecting mutant strains by using 2-deoxyglucose;

(c) cultivating the mutants obtained above for 22 ~ 26 hours at 52 to 58 ^°C in Nutrient agar medium (beef extract 0.3%>; peptone 0.5%) containing 03% of glucose, 1.0% of starch or cellulose which is suitable for measurement of the enzyme activity;

(d) dyeing the agar plate with 0.1 N concentration of iodide solution or Congo-Red reagent; (e) separating catabolite repression insensitive mutants by collecting colony forming transparent circle; (f) selecting the mutant strain that obtains the catabolite repression insensibility for cellulase, and loses the amylase activity of wild type strain. A method for producing amylase or cellulase by using the Bacillus thermoglucosidasius strain of claim 1 or claim 2; or a method for decomposing food waste by co-culturing the Bacillus thermoglucosidasius strains of claim 1 and claim 2.