Applied ..d Microbiology Biotechnology

Appl Microbiol Biotechnol (1986) 24:449--453

© Springer-Verlag 1986

Induction and production of cellulases by L-sorbose in Trichoderma reesei* Mikio Kawamori 1, Yasushi Morikawa 2, and Seigo Takasawa 1 Tokyo Research Laboratories, Kyowa Hakko Kogyo Co. Ltd., Asahimachi, Machida-shi, Tokyo 194, Japan 2 Research and Development Division, Kyowa Hakko Kogyo Co. Ltd., Otemachi, Chiyoda-ku, Tokyo 100, Japan

Summary. Cellulase production in Trichoderma reesei mutants was induced by L - s o r b o s e , known to be an inhibitor offl-l,3-glucan synthesis. In the experiments the washed mycelia were used as resting cells. For CMCase induction over 24 h using T. reesei PC-3-7, the most effective pH, temperature and L-sorbose concentration were 2.8, 28°C and 0.3 mg/ml, respectively. Comparison with other cellulase inducers showed that the inductive level of CMCase by L-sorbose was similar to that by sophorose, known to be the most potent inducer of cellulases. Since the induction of CMCase was inhibited completely by 10 gg of cycloheximide per ml, the induction process was considered to involve de novo synthesis. Although L-sorbose had the effective inducibility of CMCase, the assimilation rate of L-sorbose was very low in T. reesei PC-3-7.

Introduction Cellulases are inducible enzymes which are synthesized and usually excreted into the medium by a number of fungi and bacteria during their growth on cellulosic materials. Their production is also induced by some oligomeric sugars and their derivatives such as lactose, cellobiose and sophorose (2-O-fl-D-glucopyranosyl-D-glucose). The mechanism and conditions of cellulase induction using resing cells of T. reesei have been studied by many researchers (Nisizawa et al. 1971; Sternberg and Mandels 1979; Hohn and *

Production of Ethanol from Biomasses. Part III. This work was supported by the funds of the Research Association for Petroleum Alternative Development. Offprint requests to: M. Kawamori

Sahm 1983; Loewenberg 1984). Among these inducers, sophorose is the most potent for the production of cellulases. Much research has been directed toward the improvement of cellulase productivity in T. reesei (Montenecourt and Eveleigh 1978; Gallo et al. 1978; Morikawa et al. 1985; Kawamori et al. 1985a). Because T. reesei grows rapidly on the plate medium, L-sorbose and Triton X-100 were used to restrict the spread of hyphae of T. reesei in our experiments. Assimilation of L-sorbose affected the morphology of Neurospora crassa (Trinci and Collinge 1973) and Trichoderma pseudokoningii (Kubicek 1982; 1983). In the plate culture with L-sorbose, the branching of hyphae was promoted and the spreading of hyphae was restricted, because fl-l,3-glucan synthesis in the cell wall was inhibited by L-sorbose. It was found by chance that L-sorbose also induces cellulases in T. reesei. In our previous paper (Kawamori et al. 1985b), we reported preliminarily that there was a correlation between the cellulase inducibility by L-sorbose and cellulase productivity from alkalitreated bagasse in T. reesei mutants. Recently, Mishra et al. (1984) reported the properties of the constitutive mutants of T. reesei and the effect of various carbon sources, including L - s o r b o s e , o n cellulase production. In the present paper the inductive mechanism of cellulases by L-sorbose is described.

Materials and methods Microorganisms used. Many mutants of T. reesei obtained previously (Morikawa et al. 1985; Kawamori et al. 1985a) and succeeding mutants derived from them were used for the induction experiments. The genealogy of these mutants is shown in Fig. 1. MCG-77 derived from Natick (Gallo et al. 1978) was used as the control strain with high cellulase productivity.

450

M. Kawamori et al.: Induction and production of cellulases by L-sorbose

~i

(Natick)

MgSO4.7aq, 0.1% Polypepton, 0.05% yeast extract, 0.1% Tween 80 and 0.1% trace element solution in 50 mM tartarateNa buffer (pH 4.0). Trace element solution contained 6 mg of H3BO3, 26 mg of (NH4)6Mo7024.4aq, 100 mg of FeC13-6aq, 40mg of CuSO4.5aq, 8rag of MnClz.4aq and 200rag of ZnCI2 in 100 ml of water. The mycelia were collected on glassfilter and suspended with saline. The weight of mycelia was determined by the method by Morikawa et al. (1985).

G G ~

G

(Glu) ~

G

(Gly)

Fig. 1. Genealogy of Trichoderrna reesei mutants. The process of selecting KDD-10 and KDD-12 (parent strains of this work) via QM 9414 has been reported previously (Kawamori et al. 1985a) MI, Monocolony isolation; UV, Mutation by uv irradiation; NTG, Mutation by N-methyl-N'-nitro-N-nitrosoguanidine treatment; Gly, Resistance to catabolite repression by glycerol; Glu, Resistance to catabolite repression by.glucose; Sor, Enhanced inducibility of cellulase by L-sorbose

Induction experiment for cellulases. For the induction studies, the modified method by Sternberg et al. (1979) was used. The mycelial suspension was mixed with the inducer solution giving a final mycelial dry weight of 2.0 mg/ml. The inducer solution contained various concentrations of L-sorbose and one fourth concentration of the basal medium without carbon source and organic nitrogen source in 50 mM of buffer solution. The induction was carried out in 25-ml tubes containing 5 ml of the reaction mixture and incubated for 24--72 h at 20°--30°C on a reciprocal shaker at 120 strokes per rain. After removing the mycelia, the supernatant was examined for the cellulase assay. Assay of CMCase. CMCase activities from the induction ex-

Preparation of washed mycelia. The spores of T. reesei obtained by V-8 agar slant culture were used to inoculate each 300-ml Erlenmeyer flask containing 50 ml of the basal medium and it was incubated for 1--2 days at 28°C on a rotary shaker at 220 rpm. The basal medium comprised 0.3% glucose, 0.14% (NH4)2SO4, 0 . 2 % KH2PO4, 0.03% CaClz.2aq, 0.03%

Table 1. Induction of CMCase by L-sorbose in Trichoderma reesei mutants in the induction experiment a and the plate assay b

Group

Strain

CMCase (U/mg of myc.)

Previous mutants

KY 746 MCG-77 KDR-11 KDD-10 KDR-27 KDG-12

0.03 0.03 0.03 0.15 trace 0.02

X-30 X-31 PC-l-4 PC-3-7

0.50 0.60 0.55 0.65

New mutants

Clearing zone

m

m

m

+ + + + + +

CMCase induction was carried out in a medium containing 0.3 mg/ml L-sorbose and 50 mM citrate phosphate buffer (pH 2.8) for 24 h at 28 ° C Plate culture with L-sorbose was carried out for 2 days at 30°C, then it was incubated for 1 day at 45°C after being covered with Walseth's cellulose. The lower agar medium consisted of L-sorbose 5 g/l, yeast extract 1 g/l, Triton X100 1 g/l, inorganic compounds solution 50 ml/1 and agar 15g/1. Inorganic compounds solution was as follows: (NH4)2SO4 40g, KH2PO4 80g, Na2HPOa.12aq 120g, MgSOa-7aq 4g, FeSOa.7aq 20rag, CaC12.2aq 20mg, H3BO3 0.2mg, MnSO4.4aq 0.2rag, CuSOn.5aq l mg, (NH4)6Mo7024.4aq0.2 mg and ZnSO4.7 aq 1.4 mg in 1 1 of water. The upper agar medium consisted of Walseth's cellulose 10 g/l, 50 mM acetate buffer (pH 5.5) and agar 10 g/1

periment and the flask culture experiment were examined by the modified method of Mandels et al. (1976). They were assayed at 45°C in sodium acetate buffer (0.05 M at pH 5.0) for 30 min of incubation. The reducing sugar released was determined by the Somogyi-Nelson method (Somogyi 1952) for the induction experiment and by the dinitrosalicylic acid method (Miller 1969) for the flask culture experiment. A unit of enzyme activity represents 1 ~mol of glucose released per min.

Results and discussion

Isolation of mutants with enhanced inducibility of cellulases by L-sorbose We have isolated many T. reesei mutants as shown in Fig. 1 including mutants with hyper cellulase productivity and resistant mutants to catabolite repression, and KDD-10 and KDG-12 were selected as best cellulase producers. We have mainly researched their cellulase productivity on the Walseth's cellulose agar plates which contained L-sorbose and Triton X-100 in order to prevent spreading hyphae of T. reesei. By chance, we found that cellulases were induced by L-sorbose in T. reesei mutants. These mutants showed only low cellulase inducibility by L-sorbose in the induction experiment (Table 1). Therefore, we tried to obtain new mutants with higher cellulase inducibility by L-sorbose than our previous mutants. By the plate method for detection of cellulase yields by L-sorbose, X-series mutants derived from KDD-10 and PC-series mutants derived from KDG-12 were selected, and they showed large clearing zones on the plates. They restricted the growth of hyphae even in the absence of Lsorbose and Triton X-100 in the plate culture.

M. Kawamori et al.: Induction and production of cellulases by L-sorbose

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Fig. 2a. Effect of pH on the CMCase induction by L-sorbose in T. reesei PC-3-7. The CMCase induction was carried out in a medium containing 0.3 mg/ml L-sorbose and 50 mM HCl-glycine buffer (pH 2.0--2.5) or citrate-phosphate buffer (pH 2.5--7.0) for 24 h at 28°C for using older mycelia (O) or younger rnycelia (©) Fig. 2b. Effect of temperature on the CMCase induction by L-sorbose in T. reesei PC-3-7. The CMCase induction was carried out in a medium containing 0.3 m g / m l L-sorbose and 50 mM citrate-phosphate buffer (pH 2.8) for 24 h at various temperatures Fig. 2c. Effect of L-sorbose concentration on the CMCase induction in T. reesei PC-3-7. The CMCase induction was carried out in a medium containing various concentrations of L-sorbose and 50 mM citrate-phosphate buffer (pH 2.8) for 24 h at 28 o C

They obtained high inducible activity of cellulases by L-sorbose, because their cell walls might be changed by the mutation.

weight) was obtained. Thus 0.3mg/ml was chosen as the experimental concentration of Lsorbose for induction.

Conditions for the induction of CMCase by L-sorbose

Inhibition of CMCase induction by cycloheximide

The following points were studied. (1) Effect of pH. The relationship between CMCase induction and pH of the induction medium was investigated. In this experiment the washed mycelia of T. reesei PC-3-7 were obtained in two cultivation ages at the middle logarithmic phase (younger mycelia) and the stationary phase (older mycelia). As shown in Fig. 2a, CMCase was induced at narrow pH range of 2.5--3.0 in the younger mycelia, but it was induced at broad pH range of 2.5--7.0 in the older mycelia. Because CMCase was effectively induced at pH 2.8 in the both mycelia, subsequent experiments were carried out at pH 2.8. (2) Effect of temperature. The induction of CMCase was carried out at various temperatures. As shown in Fig. 2b, CMCase activity reached a maximum level in the range 25 °--30°C. (3) Effect of the concentration of L-sorbose. The induction of CMCase activity was carried out with various concentrations of L-sorbose. As shown in Fig. 2c, CMCase activity increased with increasing concentration of L-sorbose. At levels of L-sorbose of 0.3 mg/ml and higher, CMCase activity of about 0.7 U/mg of mycelia (at dry cell

Effect of cycloheximide (an inhibitor of protein synthesis) on CMCase induction was investigated. As shown in Fig. 3, CMCase induction by L-sorbose was completely inhibited by cycloheximide at 10 lxg/ml. Therefore it seemed probable that CMCase induction by L-sorbose was involved in the protein synthesis. 0.6 0.5

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Fig. 3. Effect of cycloheximide on the CMCase induction by L-sorbose in T. reesei PC-3-7. The CMCase induction was carried out in a medium containing 0.3 mg/rnl L-sorbose and 50 rnM citrate-phosphate buffer (pH 2.8) for 24 h at 28° C

452

M. Kawamori et al.: Induction and production of cellulases by L-sorbose

Effect o f various compounds on CMCase induction

2.5

Various c o m p o u n d s were tested for their i n d u c tive ability o f C M C a s e as well as L-sorbose in T. reesei K Y 746, M C G - 7 7 a n d PC-3-7. The results are s u m m a r i z e d in Table 2. A m o n g s t these c o m p o u n d s , s o p h o r o s e was the most effective i n d u c e r o f C M C a s e p r o d u c t i o n . Cellulose a n d its derivatives such as Avicel (crystalline cellulose), Walseth's cellulose (acid swollen cellulose) a n d alkalitreated bagasse s h o w e d greater or lesser C M C a s e i n d u c t i o n activity. Lactose h a d a small effect on C M C a s e i n d u c t i o n , but cellobiose a n d glucose h a d no effect. These results c o r r e s p o n d e d to the results o f Sternberg et al. (1979). C o m p o u n d s related to L-sorbose (D-sorbose, D-tagatose a n d sorbitol) did n o t i n d u c e C M C a s e p r o d u c t i o n . In T. reesei PC-3-7, the effects o f L-sorbose on C M C a s e i n d u c t i o n were similar to those o f s o p h o r o s e a n d alkali-treated bagasse.

Time course o f induction o f CMCase and consumption o f inducers A typical time course o f C M C a s e i n d u c t i o n a n d c o n s u m p t i o n o f L-sorbose, s o p h o r o s e a n d glucose in T. reesei PC-3-7 is s h o w n in Fig. 4. A l t h o u g h the C M C a s e i n d u c t i o n by L-sorbose h a d a lag time, the rate o f C M C a s e i n d u c t i o n by L-sorbose was almost the same as that by s o p h o r o s e . The rate o f c o n s u m p t i o n o f L-sorbose was very slow

Table 2. CMCase induction by various compounds in T. reesei mutants a Compound

Avicel Walseth's cellulose Alkali-treated bagasse b Sophorose Lactose Cellobiose Glucose L-Sorbose D-Sorbose D-Tagatose Sorbitol

CMCase (U/mg of myc.) KY 746

MCG-77

PC-3-7

0.23 0.18 0.48 1.1 trace 0 0 0.03 0 0 0

0.17 0.29 0.40 1.1 0.26 0 0 0.03 0 0 0

0.22 0.39 0.81 1.2 trace 0 0 0.65 0 0 0

The CMCase induction was carried out in a medium containing 0.3 mg/ml various inducer and 50 mM citrate phosphate buffer (pH 2.8) for 24 h at 28 ° C b Bagasse was treated with 0.3N NaOH for 15 min at 120°C

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Fig. 4. Time course of CMCase induction and consumption of inducers by T. reesei PC-3-7. The CMCase induction was carried out at 28°C in a medium containing 2 mg/ml L-sorbose (@) or sophorose (O) or glucose (Q), 50 mM citrate-phosphate buffer (pH 2.8) and 2 mg/ml mycelia (at dry cell weight). The quantity of sugar was determined with Nihon Bunko Trirotor III HPLC apparatus equipped with refractive index detector. For L-sorbose, Shodex NH pak column (Showa Denko Co. Ltd.) and 85% acetonitrile as an eluent were used. For sophorose and glucose, Shodex Ionpak C-811 column (Showa Denko Co. Ltd.) and 0.1% H3PO4 as an eluent were used Table 3. CMCase production from L-sorbose by T. reesei PC3-7 in flask culture" Carbon source

Max of myc. (mg/ml)

CMCase (U/ml)

L-Sorbose 0.3% Glucose 1.0%+L-Sorbose 0.03% Glucose 1.0%+L-Sorbose 0.3% Avicel 1.0% Avicel 1.0%+ L-Sorbose 0.03% Avicel 1.0%+L-Sorbose 0.3%

0.2 4.5 4.1 1.0 1.0 1.0

5 trace 22 38 42 68

Flask culture was carried out for 7 days at 28 ° C in the medium containing various carbon source and the others which were the same as those used for the collection of washed mycelia

c o m p a r e d with that o f s o p h o r o s e a n d glucose, and r e a c h e d only 0.012 m g / m l , h. I n d u c t i o n by Lsorbose o f cellulase activity other than C M C a s e will be discussed elsewhere.

CMCase production from L-sorbose in flask culture

a

The flask culture o f T. reesei PC-3-7 was carried out using L-sorbose as a c a r b o n source (Table 3). Because the assimilation rate o f L-sorbose was

M. Kawamori et al.: Induction and production of cellulases by L-sorbose

low, as shown in Fig. 4, flask culture with L-sorbose as a sole carbon source yielded a small quantity of mycelia and reached a low level of CMCase. The addition of L-sorbose to the culture, using glucose or Avicel as a main carbon source, led to the production of a larger amount of mycelia than the culture with L-sorbose as a sole carbon source. Addition of a small quantity of L-sorbose thus brought about the most effective CMCase production in flask culture. Acknowledgement. We gratefully acknowledge the skillful technical assistance of Miss Hiroko Nodake.

References Gallo BJ, Andreotti R, Roche C, Ryu D, Mandels M (1978) Cellulase production by a new mutant strain of Triehoderma reesei MCG-77. Biotechnol Bioeng Symp No. 8:89--101 Hohn H-P, Sahm H (1983) Induction of cellulases in Trichoderma reesei. Enzyme Technology 55--68 Kawamori M, Morikawa Y, Shinsha Y, Takayama K, Takasawa S (1985a) Preparation of mutants resistant to catabolite repression of Trichoderma reesei. Agric Biol Chem 49:2875--2879 Kawamori M, Morikawa Y, Takasawa S (1985b) Inductive formation of cellulases by L-sorbose in Trichoderma reesei. Appl Microbiol Biotechnol 22:235--236 Kubicek CP (1982) fl-Glucosidase excretion by Trichoderma pseudokoningii: Correlation with cell wall bound fl-l,3-glucanase activities. Arch Microbiol 132:349--354

453

Kubicek CP (1983) Influence of L-sorbose on the location of fl-glucosidase in Triehoderma pseudokoningii. FEMS Microbiol Letters 20:285--288 Loewenberg JR (1984) Sophorose induction of an intracellular fl-glucosidase in Trichoderma. Arch Microbiol 137:53--57 Mandels M, Andreotti R, Rhoche C (1976) Measurement of saccharifying cellulase. Biotechnol Bioeng Syrup No. 6:21--33 Miller GL (1969) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426--428 Mishra S, Gopalkrishnan KS (1984) New method for isolation of cellulase constitutive mutants in Trichoderma reesei and partial characterization of one. J Ferment Technol 62:495--500 Morikawa Y, Kawamori M, Ado Y, Shinsha Y, Oda F, Takasawa S (1985) Improvement of cellulase production in Trichoderma reesei. Agric Biol Chem 49:1869--1871 Montenecourt BS, Eveleigh DE (1978) Hypercellulolytic mutants and their role in saccharification. Proc 2nd Ann Symp on Fuels from Biomass 613--625 Nisizawa T, Suzuki H, Nakayama M, Nisizawa K (1971) Inductive formation of cellulase by sophorose in Triehoderma viride. J Biochem 70:375--385 Somogyi M (1952) Notes on sugar determination. J Biol Chem 195:19--23 Sternberg D, Mandels GR (1979) Induction of cellulolytic enzymes in Triehoderma reesei by sophorose. J Bacteriol 139:761--769 Trinci APJ, Collinge A (1973) Influence of L-sorbose on the growth and morphology of Neurospora erassa. J Gen Microbiol 78:179--192

Received September 19, 1985/Revised January 18, 1986