Influence of Carbon and Nitrogen Sources on the α-amylase Production by a Newly Isolated Thermophilic Streptomyces sp. MSC702 (MTCC 10772)
The present study deals with the standardization of different cultural conditions for extracellular α-amylase production by thermophilic Streptomyces sp. MSC702 in submerged fermentation (SmF). The appropriate incubation period (48 h), temperature (50°C) and pH (7.0) were determined. The effects of derived and natural carbon sources, inorganic and organic nitrogen sources were also examined. Maximum α-amylase production i.e., 435.71 and 373.89 U mL-1 were achieved by employing derived (D-inositol) and natural (rice bran) carbon sources, respectively. Among the tested nitrogen sources, ammonium sulphate and peptone were found the best inorganic and organic sources, respectively. The C:N ratio found to be the optimum was 1:1. The highest α-amylase activity (807.64 U mL-1) was obtained by utilizing rice bran and wheat bran in 1:2 ratio as the substrate with supplements of D-inositol (1% w/v), ammonium sulphate (0.5% w/v) and peptone (1% w/v). By using the optimized cultural conditions with further characterization, this α-amylase may be utilized in wide spread applications like detergent, saccharification and pharmaceutical industry.
Received: May 15, 2011;
Accepted: September 16, 2011;
Published: December 07, 2011
Programs to select new microorganisms for enzyme production are increasing
around the world. Thermophilic actinomycetes are an integral part of the indigenous
soil microflora involved in the decomposition of plant materials where temperature
reaches up to 50-60°C such as hay, bagasse, compost etc. On the basis of
taxonomical position these microorganisms is well defined in the prokaryote
group; they are filamentous, gram-positive, aerobic and chemo-organotrophic
bacteria (Stamford et al., 2001). They are one
of the most investigated groups since they constitute a potential source of
biotechnologically interesting substances (Lealem and Gashe,
1994; Arunachalam et al., 2010).
In the present day scenario, α-amylase (EC 188.8.131.52, 1,4-α-D-glucan
glucanohydrolase) find application in all the industrial processes such as in
food, detergents, textiles, paper industry, pharmaceutical and fine chemical
industries for the hydrolysis of starch (Pandey et al.,
2000; Gupta et al., 2003; Sivaramakrishnan
et al., 2006). Microbial α-amylases have completely replaced
chemical hydrolysis in the starch processing industry (Gupta
et al., 2003). It is desirable that α-amylases should be active
at the high temperatures of gelatinization (100-110°C) and liquefaction
(80-90°C) to economize processes; therefore, there has been a need and continual
search for more thermostable α-amylases (Rasooli et
Contemporary of this, microbial α-amylases can also find applications
in the bioethanol production method to release low molecular weight sugars from
stored starches. Bioethanol (most common biofuel worldwide) is an biologically
produced alcohol by the action of microorganisms and enzymes through fermenting
the sugar components of plant materials and it is made mostly from sugar and
starch crops such as wheat, corn, sugar beets, sugar cane etc. (Hahn-Hagerdal
et al., 2006). However, these raw materials base which also have
to be used for animal feed and human needs, will not be sufficient to meet the
increasing demand for fuel ethanol. Both these factors call for the exploitation
of lignocellulose feedstocks, such as agricultural and forest residues as well
as dedicated crops, for the production of ethanol (Farrell
et al., 2006). Some starchy residues (agricultural residues i.e.,
rice/wheat straw and bran, corn straw and sugarcane bagasse etc.) are easily
obtainable and appear to be the cheapest raw material for ethanol production
(Chum and Overend, 2001; Kim and
Dale, 2004; Demirbas, 2005; Uma
et al., 2010; Roslan et al., 2011).
A high degree of gelatinization and liquefaction processes are occurred in the
initial step of production of ethanol from starch or starchy residues. So, there
is a need of thermostable α-amylases to carry out these processes (Sodhi
et al., 2005). Thermophilic actinomycetes including Thermomonospora
and Thermoactinomyces are versatile producers of the thermostable α-amylases
(Reddy et al., 2003).
Because of the industrial importance of thermostable α-amylases, there
is ongoing interest in the isolation of new amylolytic strains of thermophilic
actinomycetes. In order to develop an efficient production of α-amylases,
knowledge regarding the nutritional factors affecting this process needs to
be well identified. Carbon and nitrogen are essential components of the nutrient
medium for the growth of the microorganisms (Narayana and
Vijayalakshmi, 2008). The use of agricultural residues as carbon source
in culture media leads to reduction in the cost of enzyme production (Tangerdy
and Szakacs, 2003). In the present study, experimental selection for the
screening of best carbon and nitrogen sources were done to achieve cost-effective
optimal α-amylase production by newly isolated Streptomyces sp.
MATERIALS AND METHODS
All the media ingredients and reagent chemicals were of analytical grade procured from E-Merck, Hi-Media and Qualigen Chemicals, India, Ltd. Natural carbon sources (sugarcane bagasse, wheat bran, rice bran, corn cob and wheat straw) used as sole carbon source were obtained from the local market, Kanpur, India and used in production medium without any pretreatment. All experiments were carried out in triplicate and average values are given in presented data. All the experiments were conducted in year 2010.
Microorganism and growth conditions: The microorganism used in this
study was Streptomyces sp. MSC702 originally isolated from the mushroom
compost collected from Mushroom Research and Development Centre, Plant Pathology
Department, Chandra Shekhar Azad University of Agriculture and Technology, Kanpur,
India. The stock culture of Streptomyces sp. MSC702 was maintained on
M medium (Obi and Odibo, 1984) modified with 1% (v/v)
trace element solution (Techapun et al., 2002)
Strain isolation: Thermophilic strains of actinomycetes were isolated
from samples of mushroom compost on petri plates of modified M medium using
dilution plate technique (Wakesman, 1927) at 45°C.
Probable amylase producing strains were then selected by flooding the plates
with 0.001 M iodine solution (Obi and Odibo, 1984).
Subsequently, the amylase producing strain was cultivated in modified M medium
agar slants for 2 days at 50°C and stored at 4°C.
Selection of the working strain: Each of 7 probable amylase producing
strains was separately inoculated into 100 mL of M broth (Obi
and Odibo, 1984) in 250 mL Erlenmeyer flask and allowed to ferment for 48
h at 50°C (pH 7.0). Contents were filtered with Whatman filter papers 1
(qualitative circles, 125 mm diameter) and centrifuged at 5000 g for 20 min
at 4°C and the clear cell-free supernatant was used for α-amylase assay.
Preparation of inoculums: The inoculum was prepared by adding 10 mL sterile distilled water in to 2 days old culture slant and make a cell suspension with the help of sterile loop. The spore suspension was taken and agitated in a vortex-cyclomixer for 5 min in order to disperse the spores evenly. The cells (1.9-2.2x108 CFU mL-1) from these slants were inoculated into production media.
Biomass production: To determine the total amount of growth (biomass), the cells were harvested at the interval of 12 h for 96 h. The mycelial mass was removed by vacuum filtration and dried overnight in oven at 80°C to measure dry biomass weight, expressed in terms of mg dry mass 100 mL-1 of culture medium.
Production of α-amylase at different physiological conditions:
Influence of incubation period (12 to 96 h), temperature (45 to 70°C) and
pH (5.0 to 10.0) on α-amylase production by strain MSC702 was studied in
1% (w/v) starch supplemented production medium. The production medium (100 mL)
was prepared according to Obi and Odibo (1984), were
taken in 250 mL of Erlenmeyer flasks and autoclaved at 121°C (15 lbs) for
20 min and cooled. Production medium then inoculated with 1% (v/v) inoculum.
The pH of the medium was adjusted using 1 N HCl or 1 N NaOH.
Production of α-amylase at different carbon and nitrogen sources:
The influence of different carbon sources on the α-amylase production by
strain MSC702 was studied by replacing 1% (w/v) starch with the other derived
and natural carbon sources in production medium. The influence of the nitrogen
sources on the α-amylase production was studied by replacing the 0.1% (w/v)
(NH4)2SO4 with other nitrogen sources in production
medium containing rice bran and wheat bran in 1:2 ratio as sole carbon source.
The effect of C: N ratio on α-amylase biosynthesis was studied using starch
as a fixed carbon source with varying nitrogen source (peptone) in different
concentration. The initial pH of the production medium was 7.0. Production medium
then inoculated with 1% (v/v) inoculum and incubated at 50°C for 48 h. The
saccharolytic activity of α-amylase was determined by 3, 5-dinitrosalicylic
acid method (Miller, 1959).
Analytical procedure: The reaction mixture contained 0.1 mL of appropriately
diluted supernatant enzyme and 0.9 mL of 0.1 M phosphate buffer (pH 7.0) containing
1% (w/v) of soluble starch. The mixture was incubated at 50°C for 10 min.
The amount of reducing sugar level released in the mixture was determined by
the 3, 5-dinitrosalicylic acid method (Miller, 1959).
The absorbance was measured at 550 nm using UV-visible spectrophotometer (UV-1700
PharmaSpec Shimadzu). One unit of α-amylase activity was defined as the
amount of enzyme that released 1 μmol of reducing sugars (maltose) per
minute under the standard assay conditions.
Production of α-amylase under optimum conditions: On the basis of the results obtained with all the optimum parameters, namely viz., rice bran and wheat bran in 1:2 ratio containing D-inositol (1% w/v) as supplementary carbon source and peptone (1% w/v) and ammonium sulphate (0.5% w/v) as supplementary nitrogen sources, pH of 7.0, incubation temperature of 50°C and incubation period of 48 h, the extent of improvement of α-amylase biosynthesis in the optimized medium was evaluated and compared with 1% (w/v) starch supplemented production medium as a control. The fermentation was carried out as described above.
RESULTS AND DISCUSSION
Selection and identification of working strain: During the screening
process of seven amylolytic strains isolated from mushroom compost, strain MSC702
was chosen as working strain on the basis of the highest α-amylase production
amongst all. The newly isolated strain was spore forming, Gram-positive, highly
thermophilic, halophilic and alkaliphilic in nature (data not shown). The phenotypic
and phylogenetic (data not shown) properties of strain MSC702 are consistent
with its assignments to the genus Streptomyces. The data related to comparison
of 16S rRNA sequence of strain MSC702 with other related Streptomyces spp.
was described in Table 1.
Strain MSC702 was deposited in the Microbial Type Culture Collection and Gene Bank (MTCC), Chandigarh, India with accession number MTCC 10772. An almost complete 16S rRNA sequence of strain MSC702 (1474 nucleotides) has been deposited in the GenBank database with nucleotide sequence Accession No. JF325872.
Effect of incubation period, temperature and pH on α-amylase production:
Strain MSC702 showed maximum α-amylase production (276.34 U mL-1,
48 h) in early log phase with drastic decrease in production of enzyme (232.56
U mL-1, 72 h) at late growth phase or early stationary phase (Fig.
1). The production of α-amylase by strain MSC702 began after 12 h and
reached a peak of 276.34 U mL-1 at 48 h. Result showed that α-amylase
production was independent of growth phase. Similar reports on α-amylase
were reported on Streptomyces albidoflavus (Narayana
and Vijayalakshmi, 2008) and S. erumpens MTCC 7317 (Kar
and Ray, 2008).
The incubation period is governed by characteristics of the culture and is
based on growth rate and enzyme production. In most cases, the period employed
was 30 h, although an average of 48-50 h is common (Lonsane
and Ramesh, 1990). The production of α-amylase by strain MSC702 began
after 12 h and reached a peak of 276.34 U mL-1 at 48 h.
|| Details of Streptomyces spp. closely related to Streptomyces
sp. MSC702 on the basis of 16S rRNA gene sequence similarity
||Growth characteristics with reference to α-amylase production
at the interval of 12 h by the strain MSC702 (pH 7.0, at 50°C)
|Fig. 2 (a-b):
||α-Amylase production by Streptomyces sp. MSC702
at different physical parameters. (a) Effect of different temperature range
(45-70°C) at pH 7.0 and 48 h incubation and (b) Effect of different
pH range (5.0-10.0) for 48 h incubation at 50°C
Similar results were obtained by Baysal et al. (2003)
and Liu and Yan (2008) with Bacillus spp. Yang
and Wang (1999) studied the influence of incubation period on α-amylase
produced by Streptomyces rimosus. The production of α-amylase began
after 24 h of cultivation and reached to peak levels after 48 h and declined
thereafter. This result showed the good agreement with the present study.
Optimum amylase production at 45-55°C is characteristic of the moderately
thermostable actinomycetes like Thermomonospora fusca (Busch
and Stutzenberger, 1997), Streptomyces sp. IMD 267 (McMahon
et al., 1997), Streptomyces sp. D1 (Chakraborty
et al., 2009) and S. erumpens MTCC 7317 (Kar
and Ray, 2008), beyond 55°C, there was sudden decrease in enzyme production.
Streptomyces sp. MSC702 showed highest (269.78 U mL-1) α-amylase
activity at 50°C (Fig. 2a) with retaining ~72% (195.16
U mL-1) and ~35% (95.29 U mL-1) α-amylase production
at 60°C and 65°C, respectively. Many Bacillus sp. such as Bacillus
amyloliquefaciens, B. subtilis and B. licheniformis
reported for α-amylase production at same temperature range (37-60°C)
(Syu and Chen, 1997; Mishra et
al., 2005; Mendu et al., 2005).
Present study showed the optimum pH range 6.5-7.5 for α-amylase biosynthesis
by strain MSC702 with decrease in production thereafter (Fig.
2b). Similarly, Kuo and Hartman (1966) found that
Thermoactinomyces vulgaris synthesized amylase most rapidly at pH values
ranging from 6.5 and 7.5 and that amylase inactivation occurred rapidly if pH
rose above 7.5. Amylase production by Streptomyces aureofaciens 77 (Shatta
et al., 1990), S. erumpens MTCC 7317 (Kar
and Ray, 2008) and S. rimosus (Yang and Wang,
1999) has been increased gradually as the initial pH values ascend from
5.0 to 7.0. These results agreed with our results where maximum α-amylase
production (278.77 U mL-1) achieved at pH 7.0.
Effect of various carbon sources on α-amylase production: Growth and enzyme production of any organism are greatly influenced by both environmental conditions as well as the nutrients available in the growth medium. To investigate the effects of various carbon sources, Streptomyces sp. MSC702 was inoculated in the production medium containing 1% (w/v) derived [(monosaccharides = arabinose, xylose, glucose, fructose, galactose); (disaccharides = sucrose, maltose, lactose, cellobiose); (trisaccharides = raffinose); (polysaccharides = starch, cellulose); (deoxy sugar = L-rhamnose); (polyhydroxy alcohols = D-sorbitol, D-mannitol, D-inositol)] and natural carbon sources (sugarcane bagasse, wheat bran, rice bran, corn cob and wheat straw) separately for 48 h. Results showed that all the substrates supported α-amylase production by the strain, except arabinose, xylose and galactose. The combination of D-inositol with the mixture of rice bran and wheat bran proved superior to the other composition.
Derived carbon sources: All the tested carbon sources supported the
growth of strain MSC702. In present investigation, there was no correlation
between biomass yield and biosynthesis of α-amylase by the strain (Fig.
3). Glucose, fructose, sucrose, maltose, lactose, cellobiose, raffinose,
starch, cellulose, D-sorbitol, D-mannitol and D-inositol were found to support
α-amylase biosynthesis, whereas arabinose, xylose and galactose showed
a repressive effect on α-amylase production (Fig. 3).
L-rhamnose showed no significant effect on α-amylase biosynthesis. All
the tested polyhydroxy alcohols favored α-amylase production. The maximum
enzyme level was obtained in the medium containing D-inositol (435.71 U mL-1).
The results obtained are in good agreement with the findings of Aiyer
(2004) who also stated that amylase synthesis occurred in all the carbon
sources except arabinose and galactose, both of them were least effective for
amylase production. Srivastava and Baruah (1986) also
found that inositol and sorbitol favors the growth and production of amylase
by Bacillus stearothermophilus.
|| Effect of derived carbon sources (1% w/v) on cell growth
and α-amylase production
Natural carbon sources: To evaluate the effect of natural carbon sources
on the production of α-amylase by Streptomyces sp. MSC702, fermentation
was done with the addition of 1% (w/v) Sugarcane Bagasse (SB), Wheat Bran (WB),
Rice Bran (RB), Corn Cob (CC) and Wheat Straw (WS) separately in the production
medium. The order of substrate suitability was RB>CC>WB > WS>SB
(Fig. 4). The α-amylase activities obtained with these
substrates were: 373.89 U mL-1 with rice bran; 368.87 U mL-1
with corn cob; 261.48 U mL-1 with wheat bran; 234.54 U mL-1
with wheat straw and 201.14 U mL-1 with sugarcane bagasse. The utilization
of agricultural residues for production of enzymes was also studied by Milala
et al. (2009).
Therefore, the volumetric activities were quite different in the presence of the various derived and natural carbon sources. Carbohydrate present in the greatest proportion in the natural carbon sources are in the form of lignocellulosics and starch. Rice bran was the best carbon source for α-amylase production, contain 65% starch. On the other hand, corn cob, wheat bran, wheat straw and sugarcane bagasse has lesser percentage of starch. Therefore, better α-amylase production may be correlated with the higher content of starch in agro-residues. Due to the higher volumetric activity obtained with rice bran, it was maintained in the fermentation medium for the remaining experiments but it is important to note that combinations of the rice bran with other natural carbon sources, may be a good alternative for higher α-amylase production and due to the less expensive in nature, natural carbon source, could also be used for industrial scale production.
Combinations of natural carbon sources: Figure 4 and
5, show the effect of different combination (1:1 w/v) of natural
carbon sources on α-amylase production taking rice bran as fixed substrate
in all combinations. In the present study, out of the different combinations
(RB:SB; RB:WB; RB:CC; RB:WS) tested, α-amylase activity reached up to 398.1
U mL-1 in rice bran and wheat bran (1:1 w/v) containing medium. Ten
different combinations (0.5:0.5; 1:1; 1:2; 1:3; 1:4; 1:5; 2:1; 2:1; 4:1 and
5:1) of rice bran and wheat bran were analyzed to maximize α-amylase production.
The highest α-amylase activity was found at the level from mixed substrates
of rice bran: wheat bran in 1:2 ratio. Yousaf et al.
(2008) were also reported similar result (rice bran to wheat bran 1:2; amylase
activity ~180 U mL-1). Jecu (2000) also reported
the importance of mixed substrate (wheat bran and wheat straw) for endoglucanase
production by Aspergillus niger.
||Effect of natural carbon sources (1% w/v) and combinations
of rice bran with other natural carbon sources (1:1 ratio w/v) on α-amylase
|| Effect of different ratios of rice bran and wheat bran on
||Effect of combination of derived and natural carbon sources
(1RB: 2WB) on α-amylase
Combination of derived and natural carbon sources: The combined effect
of different derived carbon sources (1% w/v) with rice bran: wheat bran (1:2)
was studied by their addition to production medium (Fig. 6).
D-inositol gave significantly higher (665.37 U mL-1) yield of α-amylase
than other carbon sources when added to production medium containing rice bran
and wheat bran in 1:2 ratio. Similar results shown by Srivastava
and Baruah (1986), they also observed the effect of inositol on amylase
production by Bacillus stearothermophilus.
Effect of nitrogen sources on α-amylase production: A complex nitrogen
source appears to be essential for the production of high amount of α-amylase
by Streptomyces sp. MSC702. The requirement may be explained by
organisms preference for protein as a nitrogen source for growth as well
as for extracellular enzyme production. The amylase synthesis by several microorganisms
has been correlated to the presence or absence of various amino acids and complex
nitrogen sources in the culture medium (Fogarty and Kelly,
1980; Hillier et al., 1997). In present study,
the tested nitrogen sources of inorganic and organic nitrogen were NaNO3,
(NH4)2SO4, NH4NO3, (NH4)2HPO4,
urea, thiourea, peptone, yeast extract, tryptose, casein and amino acids. Among
the tested nitrogen sources, ammonium sulphate was the best inorganic nitrogen
source followed by ammonium nitrate (Fig. 7). While peptone
was the best organic nitrogen source with 9.0% more yield than ammonium sulphate.
α-Amylase production in presence of peptone as a nitrogen source was 584.38
U mL-1. The effects of different concentrations of peptone and ammonium
sulphate on α-amylase production were also studied.
|| Effect of inorganic and organic nitrogen sources (0.1% w/v)
on α-amylase production
||Effect of combinations of peptone and ammonium sulphate with
different concentration on α-amylase production
Results revealed that 1.0% of peptone with 0.5% ammonium sulphate gave highest
α-amylase yield (Fig. 8). In the present study, ammonium
sulphate stimulate α-amylase synthesis with certain amount, on increasing
the amount, it reduced the α-amylase synthesis. Similar result was reported
by Mai et al. (1992), who also observed stimulation
of amylase activity by ammonium salts. Aiyer (2004)
also reported the enhancement of amylase production by using the combination
of peptone with ammonium hydrogen phosphate. In contrast, Saxena
et al. (2007) reported inhibitory effect of ammonium salts on amylase
Among the seventeen amino acids examined, only seven (Table 2)
have stimulatory effect on α-amylase production. Glycine, DL-aspartic acid
and L-glutamic acid were found to be indispensable for α-amylase production.
Glycine (138.82 U mL-1) was found to be most suitable amino acid
for the production of α-amylase. The lowest value (62.38 U mL-1)
of α-amylase production was obtained in presence of threonine. Similar
results have been obtained by other workers (Chandra et
al., 1980; Srivastava and Baruah, 1986). α-Amylase
production by Bacillus amyloliquefaciens ATCC 23350 increased by a factor
of 300 in the presence of glycine (Zhang et al.,
1983). The effect of glycine was not only as a nitrogen source rather it
affected α-amylase production by controlling pH and subsequently α-amylase
production increased. DL-Alanine, DL-valine and DL-methionine were effective
for the production of alkaline amylase by Bacillus sp. However, the role
of amino compounds was considered to be neither as nitrogen nor as a carbon
source but as stimulators of amylase synthesis and excretion (Ikura
and Horikoshi, 1987).
|| Effect of C: N ratio on α-amylase production
Kundu et al. (1973) reported that only asparagines
gave good enzyme yields while the importance of arginine for α-amylase
production from B. subtilis has also been well documented (Lee
and Parulekar, 1993).
Effect of C:N ratio on α-amylase production: For C:N ratio study,
soluble potato starch and peptone were used as carbon and nitrogen sources,
respectively. 1% (w/v) of potato starch was used while the amount of peptone
was varied (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0% w/v) to attain desired C:N ratio.
The activity of α-amylase in fermented broth was recorded after 48 h of
incubation. The maximum α-amylase yield (605.80 U mL-1) was
obtained when C:N ratio was 1.0 (Table 3). Aiyer
(2004) worked on Bacillus licheniformis SPT 27 showed similar results
and having optimum production of amylase in 1:1 C:N ratio where potato soluble
starch and peptone were used as carbon and nitrogen sources, respectively. McTigue
et al. (1995) reported C:N ratio of 1:1 with starch and yeast extract
as carbon and nitrogen sources, respectively.
Production of α-amylase under optimized conditions: A high level
of α-amylase production (807.64 U mL-1) was achieved by utilizing
rice bran and wheat bran in 1:2 ratio as the substrate with supplements (w/v)
of D-inositol (1%), peptone (1%) and ammonium sulphate (0.5%) for 48 h at 50°C,
pH of 7.0.
In the present investigation, we have isolated thermostable α-amylase producing strain Streptomyces sp. MSC702 from mushroom compost. From the above mentioned results, we concluded that ~3 fold increase in α-amylase production was achieved under the optimized fermentation conditions with (w/v) 1% D-inositol, 1% peptone and 0.5% ammonium sulphate as supplements, as compared with the production medium with 1% starch. The enzyme in this research performed some differences in properties related to the production medium which are valuable for higher industrial α-amylase production. The study also concluded that the suitability of newly isolated strain of thermophilic actinomycete Streptomyces sp. MSC702 in the degradation of agro-residues for the release of sugars in bioethanol production. Application of agro-residues in bioprocesses not only provides alternative substrates but it also helps solving their disposal problem.
The authors are grateful to the University Grant Commission, Selection and Award Bureau, New Delhi, India for providing financial support to carry out this work and awarding a fellowship to Renu Singh under the scheme of Rajiv Gandhi National Fellowship.
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