World Journal
of Microbiology
& Biotechnology
11,695
Short Note: Cellulase production Volvariella diplasia*
by the edible mushroom
U.S. Puntambekar Volvariella diplasia produced cellulolytic enzymes (550 U CM-cellulase and 69 U filter-paper cellulase/l) when grown in shake culture at pH 5.4 and 28°C with 0.5% cellulose powder as carbon source. Alkali-treated as well as untreated cellulosic substrates were hydrolysed by both enzymes (sp. act. 2.75 U/mg protein), with cellobiose and glucose as the end products.
Key words: Cellulase, submerged
fermentation,
Volvariella diplasia.
In a physiological growth study of an edible, paddy-straw mushroom, Volvariella diplasia, Langer et al. (1980) observed that the cellulose and hemicellulose contents of the paddy straw used as substrate fell. Production of cellulolytic enzymes in submerged cultures of this species is reported here for the first time. A IO-ml sample of 7-day-old V. diplasia (NCIM 1126) mycelium in potato/dextrose broth was transferred to 100 ml Mandels & Weber (1969) medium, containing various carbon sources, and incubated on an orbital shaker at 200 rev/min. The broth was then filtered through a glassfibre filter and assayed for various extracellular enzyme activities, defined and measured according to Lachke et al. (1986). Maximum activities of CM-cellulase (552 U/l) and filterpaper cellulase (69.4 U/l) were produced at pH 5.4 and 28°C after 15 days. No cellulase activity was detected in any of the culture filtrates grown on carbon sources other than cellulosic substrates, indicating the induced nature of the enzymes. Among the different cellulosic substrates tested, 0.5% (w/v) cellulose powder (CP 123; Schleicher & Schull, Germany) gave greatest enzyme production. Substrates such as straw, wheat bran, mesta wood and bagasse induced lower enzyme activities, possibly because of the close association between secreted cellulases and unhydrolysed residual substrates which could not be detected. &Glucosidase (pNPGase) activity, however, was so low in the culture filtrate (10 U/l) that it was only detected after lo-fold concentration by ultra-filtration through a UMlO membrane. Saccharification was also carried out with various cellulosic substrates which had been left untreated or treated with 5 M NaOH (Table 1). Pre-treatment and saccharification were performed according to Mandels et al. (1974). Crystalline cellulosic substrates such as Avicel, were hydrolysed to the same extent as cellulose CP 123 (Table I), indicating an exoglucanase-type activity. This activity was The author is with Chemical Laboratory, ‘NCL Communication @ 1995 Rapid
Science
NCIM, Division of Biochemical Sciences, National Pune 411 006, Maharashtra, India: fax: 212 330233. No. 6166. Publishers
also evident from the end-product analysis, in which cellobiose and glucose predominated after 24 h. The cellobiose produced was further broken down to glucose by the traces of P-glucosidase present in the culture filtrate. Volvariella diplasia (NCIM 1126) also produced a significant amount of xylanase (6000 U/l). Studies on the optimization of the enzyme production and its characterization are in progress. Table 1. Enzymatic celluloslc substrates.*
hydrolysis
Substrate
Cellulose powder Cotton (absorbent) Filter paper (Whatman Avicel Rice straw (50 mesh) Bagasse (50 mesh) Mesta wood
No. 1)
01 untreated
and
alkali-treated
Saccharification
(%) alter
Untreated
Alkali-treated
11.6 5.8 7.1 8.9 8.0 4.4 2.2
* The hydrolysis was carried out with 100 mg substrate 0.05 M acetate buffer (pli 4.8) containing 25 U enzyme 2.75 U/mg protein). ND-Not detected.
72 h
18.1 20.0 20.0 ND
xi.8 13.8 14.8 in 10 ml (sp. act.
References Lachke, A.H., Bastawde, K.B., Powar, V.K. & Srinivasan, M.C. 1986 Isolation of a hypercellulolytic mutant (Cu-1) of Penicillium funicuhum. Enzyme and Microbial Technology 8, 105-108. Langer, P.N., Sehgal, J.P. & Garcha, H.S. 1980 Chemical changes in wheat and paddy straw after fungal cultivation. Indian journal of Animal Science50, 942-946. Mandels, M., Hontz, L. & Nystrom, J. 1974 Enzymatic hydrolysis of waste cellulose. Biotechnology and &engineering 16, 14711493. Mandels, M. & Weber, J. 1969 The production of cellulases. Advances in Chemistry 95, 391-414.
(Received in revised form 19 June 1995; accepted 21 June 1995]
of Microbiology
& Biotechnology
11,695
Short Note: Cellulase production Volvariella diplasia*
by the edible mushroom
U.S. Puntambekar Volvariella diplasia produced cellulolytic enzymes (550 U CM-cellulase and 69 U filter-paper cellulase/l) when grown in shake culture at pH 5.4 and 28°C with 0.5% cellulose powder as carbon source. Alkali-treated as well as untreated cellulosic substrates were hydrolysed by both enzymes (sp. act. 2.75 U/mg protein), with cellobiose and glucose as the end products.
Key words: Cellulase, submerged
fermentation,
Volvariella diplasia.
In a physiological growth study of an edible, paddy-straw mushroom, Volvariella diplasia, Langer et al. (1980) observed that the cellulose and hemicellulose contents of the paddy straw used as substrate fell. Production of cellulolytic enzymes in submerged cultures of this species is reported here for the first time. A IO-ml sample of 7-day-old V. diplasia (NCIM 1126) mycelium in potato/dextrose broth was transferred to 100 ml Mandels & Weber (1969) medium, containing various carbon sources, and incubated on an orbital shaker at 200 rev/min. The broth was then filtered through a glassfibre filter and assayed for various extracellular enzyme activities, defined and measured according to Lachke et al. (1986). Maximum activities of CM-cellulase (552 U/l) and filterpaper cellulase (69.4 U/l) were produced at pH 5.4 and 28°C after 15 days. No cellulase activity was detected in any of the culture filtrates grown on carbon sources other than cellulosic substrates, indicating the induced nature of the enzymes. Among the different cellulosic substrates tested, 0.5% (w/v) cellulose powder (CP 123; Schleicher & Schull, Germany) gave greatest enzyme production. Substrates such as straw, wheat bran, mesta wood and bagasse induced lower enzyme activities, possibly because of the close association between secreted cellulases and unhydrolysed residual substrates which could not be detected. &Glucosidase (pNPGase) activity, however, was so low in the culture filtrate (10 U/l) that it was only detected after lo-fold concentration by ultra-filtration through a UMlO membrane. Saccharification was also carried out with various cellulosic substrates which had been left untreated or treated with 5 M NaOH (Table 1). Pre-treatment and saccharification were performed according to Mandels et al. (1974). Crystalline cellulosic substrates such as Avicel, were hydrolysed to the same extent as cellulose CP 123 (Table I), indicating an exoglucanase-type activity. This activity was The author is with Chemical Laboratory, ‘NCL Communication @ 1995 Rapid
Science
NCIM, Division of Biochemical Sciences, National Pune 411 006, Maharashtra, India: fax: 212 330233. No. 6166. Publishers
also evident from the end-product analysis, in which cellobiose and glucose predominated after 24 h. The cellobiose produced was further broken down to glucose by the traces of P-glucosidase present in the culture filtrate. Volvariella diplasia (NCIM 1126) also produced a significant amount of xylanase (6000 U/l). Studies on the optimization of the enzyme production and its characterization are in progress. Table 1. Enzymatic celluloslc substrates.*
hydrolysis
Substrate
Cellulose powder Cotton (absorbent) Filter paper (Whatman Avicel Rice straw (50 mesh) Bagasse (50 mesh) Mesta wood
No. 1)
01 untreated
and
alkali-treated
Saccharification
(%) alter
Untreated
Alkali-treated
11.6 5.8 7.1 8.9 8.0 4.4 2.2
* The hydrolysis was carried out with 100 mg substrate 0.05 M acetate buffer (pli 4.8) containing 25 U enzyme 2.75 U/mg protein). ND-Not detected.
72 h
18.1 20.0 20.0 ND
xi.8 13.8 14.8 in 10 ml (sp. act.
References Lachke, A.H., Bastawde, K.B., Powar, V.K. & Srinivasan, M.C. 1986 Isolation of a hypercellulolytic mutant (Cu-1) of Penicillium funicuhum. Enzyme and Microbial Technology 8, 105-108. Langer, P.N., Sehgal, J.P. & Garcha, H.S. 1980 Chemical changes in wheat and paddy straw after fungal cultivation. Indian journal of Animal Science50, 942-946. Mandels, M., Hontz, L. & Nystrom, J. 1974 Enzymatic hydrolysis of waste cellulose. Biotechnology and &engineering 16, 14711493. Mandels, M. & Weber, J. 1969 The production of cellulases. Advances in Chemistry 95, 391-414.
(Received in revised form 19 June 1995; accepted 21 June 1995]
