Maximum enzyme production of 8050 U/g dry substrate was obtained in wheat bran supplemented with polygalacturonic acid PGA; 1%, w/v and neurobion a multivitamin additive; 27 ll/g dry sub
Trang 1Enhanced production of pectinase by Bacillus sp DT7
using solid state fermentation
a Department of Microbiology, Panjab University, Chandigarh 160014, India
b Department of Biotechnology, Panjab University, Chandigarh 160014, India Received 22 May 2002; received in revised form 10 August 2002; accepted 14 August 2002
Abstract
Bacillus sp DT7 produced very high levels of alkaline and thermotolerant pectinase by solid state fermentation Production of this enzyme was affected by nature of solid substrate, level of moisture content, presence or absence of carbon, nitrogen, mineral and vitamin supplements Maximum enzyme production of 8050 U/g dry substrate was obtained in wheat bran supplemented with polygalacturonic acid (PGA; 1%, w/v) and neurobion (a multivitamin additive; 27 ll/g dry substrate) with distilled water at 75% moisture level, after 36 h of incubation at 37°C
Ó 2002 Published by Elsevier Science Ltd
Keywords: Solid state fermentation; Bacillus; Pectinase
1 Introduction
In recent years there has been a renewed interest in
solid-state fermentation (SSF) processes for the
pro-duction of bioactive compounds While efforts continue
largely to exploit filamentous fungi and yeasts for the
production of various enzymes, attempts have also been
made to explore possibilities of using bacterial strains in
SSF systems (Pandey et al., 2000) Enzyme production
by SSF using bacterial spp has been reported for many
enzymes such as xylanase (Gessesse and Mamo, 1999)
and amylase (Babu and Satyanarayana, 1995) but
re-ports on pectinase production by SSF using bacterial
spp are lacking in the literature The use of SSF for the
production of enzymes mainly from fungi may be due to
the general belief that SSF technique is applicable only
to filamentous fungi (Lonesane and Ghidyal, 1992)
Studies on comparative production of pectinase by
As-pergillus sp using submerged fermentation (SmF) and
SSF has shown the latter to be a better option
(Solis-Pereyra et al., 1993; Minjares-Carranco et al., 1997)
Moreover, the use of SSF has been reported to be more
advantageous than SmF as it allows cheaper production
of enzyme having better physiochemical properties than that produced by SmF (Solis-Pereyra et al., 1993)
In this study, we report the production of very high levels of an alkali- and thermotolerant pectinase pro-duced by a mesophilic Bacillus sp DT7 (Kashyap et al., 2000) using SSF conditions
2 Methods 2.1 Solid state fermentation Solid substrates (5.0 g; wheat bran, rice bran, apple pomace) in 250 ml Erlenmeyer flasks were moistened with specified content of distilled water and autoclaved at
15 psi for 30 min 2.0 ml of culture was used as an in-oculum in each flask of solid substrate, which were then incubated at 37 °C for specified time intervals Visual observations regarding growth were made on each day and samples were drawn periodically to assess the en-zyme production Enen-zyme was extracted with 25 ml, Tris–HCl buffer (0.01 M, pH 8.0) and filtered The pooled filtrate of two extractions was centrifuged (10,000g for 15 min; 4°C) and used as the source of enzyme
Pectinase activity in the extracted liquid was assayed
by the calorimetric method described earlier (Kashyap
et al., 2000) Pectinase activity in the supernatant was expressed as U/g of solid substrate used
*
Corresponding author Address: Department of Microbiology,
Panjab University, Chandigarh 160014, India Fax: +91-172-541409.
E-mail address: rooptt1@glide.net.in (R Tewari).
0960-8524/03/$ - see front matter Ó 2002 Published by Elsevier Science Ltd.
doi:10.1016/S0960-8524(02)00206-7
Bioresource Technology 88 (2003) 251–254
Trang 22.2 Optimization studies for pectinase production
The effect of environmental parameters in SSF was
studied using wheat bran, unless otherwise stated, by
altering physiochemical and culture conditions
Effect of different solid substrates: The effect of
dif-ferent solid substrates on pectinase production was
studied by using a variety of solid substrates (wheat
bran, rice bran, apple pomace) The original moisture
content of the apple pomace was 80% Effect of moisture
contents on pectinase production using wheat bran and
rice bran was studied by adding different amount (50%,
60%, 66.7%, 75%, 77.8%, and 80%) of distilled water as
the moistening agent to these solid substrates
Effect of salts: Different salts: CaCl2 2H2O,
MgSO4 7H2O, CoCl2 2H2O, MnSO4 4H2O, H3BO3,
ZnCl2, KCl and NaCl (1 mM, final concentration) were
dissolved in the distilled water used to adjust moisture
level in the solid substrate
Effect of carbon sources: Different carbon sources:
glucose, mannitol, pectin, polygalacturonic acid (PGA),
galactose, sucrose, lactose, maltose, sodium acetate (SA)
and xylose were supplemented separately to a final
concentration of 1% (w/v) in solid media They were
dissolved to required concentrations in 15 ml of distilled
water, which was used as moistening agent per 5 g of
solid substrate
Effect of nitrogen sources: Different organic and
in-organic nitrogen sources: yeast extract, peptone,
tryp-tone, glycine, urea, ammonium chloride, ammonium
nitrate, ammonium sulphate and ammonium citrate
were supplemented separately to a final concentration of
1% (w/v) in solid media after dissolving them in distilled
water used for adjusting the moisture content
Effect of vitamin supplementation: Effect of neurobion
(Nb) (a multivitamin solution of vitamin B1, B6and B12;
E-Merck, India Ltd.) on pectinase production was
studied by supplementing different concentrations (9–54
ll/g dry substrate) of Nb into the solid medium For this
5.0 g wheat bran and 15 ml of distilled water were
au-toclaved (15 psi, 30 min) separately Nb was then added
to the distilled water, which was subsequently used as
moistening agent
Effect of optimized components: Effect of optimized
components on pectinase production in optimized
me-dium (wheat bran) was studied by supplementing these
components alone or in combination in distilled water
used as moistening agent
3 Results and discussion
Research on the selection of suitable substrates for
pectinase production has mainly centered on tropical
agro-industrial crops and residues Of the various
sub-strates reported in the literature, wheat bran has been
the prime among all (Pandey et al., 2000) In the present study, out of three substrates (wheat bran, rice bran and apple pomace) used wheat bran yielded 4600 U of pec-tinase/g dry substrate at 75% moisture level after 36 h of incubation at 37°C When rice bran and apple pomace were used separately as prime substrates, maximum pectinase yields obtained were 3265.25 and 78.0 U/g dry substrate at 66.7 and 80% moisture contents respectively after 48 h of incubation at 37°C A decline in pectinase activity in wheat bran (26%) and rice bran (8.2%) was observed after 72 h of incubation Moreover, the pro-duction of this enzyme using SSF process was much higher than the SmF process reported previously from the same bacteria (Kashyap et al., 2000) Higher pro-duction of pectinase in SSF process may be due to the reason that solid substrate not only supplies the nutrient
to the microbial cultures growing in it, but also serves as anchorage for the cells (Pandey et al., 2000) allowing them to utilize the substrate effectively However, some
of the nutrients in the solid substrate may be available in suboptimal concentrations, or even not present in the substrates In such cases, it would be necessary to sup-plement them externally Therefore, wheat bran was supplemented with different salts (final concentration, 1 mM), carbon (final concentration, 1% w/v) and nitrogen (1%, w/v) sources and Nb (27 ll/g dry substrate) to evaluate their effect on pectinase production at 75% moisture level
Addition of CaCl2 2H2O and MgSO4 7H2O en-hanced pectinase production by 28.0% and 11.6% re-spectively, whereas CoCl2 2H2O and MnSO4 4H2O did not have any effect on pectinase production (Table 1) Salts such as H3BO3, ZnCl2, KCl and NaCl inhibited the pectinase production up to 12% Carbon sources such as PGA, SA, pectin and lactose were found to enhance pectinase production from 11% to 44% (Table 2)
When various nitrogen sources were supplemented in wheat bran medium, yeast extract (YE), peptone and ammonium chloride were found to enhance pectinase production up to 24% (Table 3) Addition of glycine,
Table 1 Production of pectinase from Bacillus sp DT7 in wheat bran using various salts
Salt (1 mM) Relative activity (%)
CaCl 2 2H 2 O 128.0 MgSO 4 2H 2 O 11.5 CoCl 2 2H 2 O 102.0 MnSO 4 4H 2 O 104.8
Note: Incubated at 37 °C for 36 h incubation 100% activity is equiv-alent to 4600 U/g dry substrate.
252 D Raj Kashyap et al / Bioresource Technology 88 (2003) 251–254
Trang 3urea and ammonium nitrate inhibited pectinase
pro-duction, which may be due to poor growth of Bacillus in
the medium containing these nitrogen sources whereas,
tryptone had no effect on pectinase production in solid
medium
Effect of Nb on pectinase production in solid medium
was studied by supplementing different concentrations
(9–54 ll/g dry substrate) of this multivitamin solution in
wheat bran Pectinase production was enhanced by
65.8% when 27 ll/g dry substrate of multivitamin
solu-tion was added to wheat bran medium Increasing the
concentration of Nb above 27 ll/g dry substrate had no
effect on pectinase production
The effect of optimized components alone or in
combinations on pectinase production by Bacillus sp
DT7, was studied in wheat bran medium at 37°C for 36
h Of the different components supplemented
combina-tion of Nb (27 ll/g dry weight) and PGA (3%, w/w),
enhanced pectinase production by 75% (Table 4),
whereas combination of Nb with other components such
increased pectinase production from 43% to 70% de-pending on the combinations used
From the present study, it is apparent that SSF process for pectinase production from a bacterial source
is a better option than SmF Moreover, as wheat bran is
a cheap and readily available byproduct, the production
of pectinase using SSF may be a cost-effective affair As this enzyme has been successfully used for degumming
of fibre crops in our previous study (Kashyap et al., 2001), the low cost of its production may further broaden the scopes for its use in industries involved in treatment of fibre crops
References
Babu, K.R., Satyanarayana, T., 1995 a-amylase production by the thermophilic Bacillus coagulans in solid state fermentation Process Biochem 30, 305–309.
Gessesse, A., Mamo, G., 1999 High level xylanase production by an alkalophilic Bacillus sp by using solid state fermentation Enzyme Microb Technol 25, 68–72.
Kashyap, D.R., Chandra, S., Kaul, A., Tewari, R., 2000 Production, purification and characterization of pectinase from a Bacillus sp DT7 World J Micobiol Biotechnol 16, 277–282.
Kashyap, D.R., Vohra, P.K., Soni, S.K., Tewari, R., 2001 Degum-ming of buel (Grewia optiva) bast fibres by pectinolytic enzyme from Bacillus sp DT7 Biotechnol Lett 23, 1297–1301.
Table 4 Production of pectinase from Bacillus sp DT7 in wheat bran supple-mented with optimized components
Component Relative activity (%)
CaCl 2 2H 2 O 129.0
PGA þ CaCl 2 2H 2 O 146.5
SA þ CaCl 2 2H 2 O 144.0
YE þ CaCl 2 2H 2 O 130.8 PGA þ SA þ YE 149.0 PGA þ SA þ Nb 169.8 PGA þ CaCl 2 2H 2 O 149.0
SA þ YE þ Nb 169.8 PGA þ YE þ CaCl 2 2H 2 O 145.2
SA þ YE þ Nb 164.6
SA þ YE þ CaCl 2 2H 2 O 163.0
YE þ Nb þ CaCl 2 2H 2 O 167.5 PGA þ SA þ YE þ Nb 169.0 PGA þ SA þ YE þ Nb þ CaCl 2 2H 2 O 170.0 Note: Incubated at 37 °C for 36 h 100% pectinase activity is equivalent
to 4600 U/g dry substrate.
Table 2
Production of pectinase from Bacillus sp DT7 in wheat bran using
various carbon sources
Carbon source
(final concentration, 1% w/v)
Relative activity (%)
Note: Incubated at 37 °C for 36 h 100% activity is equivalent to 4600
U/g dry substrate.
Table 3
Production of pectinase from Bacillus sp DT7 in wheat bran using
various nitrogen sources
Nitrogen source
(1%, w/v, final concentration)
Relative activity (%)
Tryptone 102.0
Ammonium chloride 110.7
Ammonium nitrate 0
Ammonium sulphate 76.42
Note: Incubated at 37 °C for 36 h 100% pectinase activity is equivalent
to 4600 U/g dry substrate.
D Raj Kashyap et al / Bioresource Technology 88 (2003) 251–254 253
Trang 4Lonesane, B.K., Ghidyal, N.P., 1992 Solid substrate cultivation In:
Doelle, H.W., Mitchell, D.A., Rolz, C.E (Eds.), Exoenzyme.
Elsvier Science Publishers Ltd., London, pp 191–209.
Minjares-Carranco, A., Trejo-Aguilar, B.A., Aguilar, G.,
Vniegra-Gonzalez, G., 1997 Physiochemical comparison between pectinase
producing mutants of Aspergillus niger adapted either to solid-state
fermentation or submerged fermentation Enzyme Microb
Tech-nol 21, 25–31.
Pandey, A., Soccol, C.R., Mitchell, D., 2000 New developments in solid state fermentation I Bioprocesses and products Process Biochem 35, 1153–1169.
Solis-Pereyra, S., Favela-Torre, E., Viniegra-Gonzalez, G., Gutierraez-Rojas, M., 1993 Effect of different carbon sources on the synthesis of pectinase by Aspergillus niger in submerged and solid state fermentations Appl Microbiol Biotechnol 39, 36– 41.
254 D Raj Kashyap et al / Bioresource Technology 88 (2003) 251–254