1 Int.J.Curr.Biotechnol. Volume 1; Issue 10; Dec, 2013 Renu Singh and Vishal Kapoor, Biochemical characterization of thermophilic actinomycete (Streptomyces sp. MSC702) from mushroom compost in India, Int.J.Curr.Biotechnol., 2013, 1(10):1-7. International Journal of Current Biotechnology Journal Homepage : http://ijcb.mainspringer.com Biochemical characterization of thermophilic actinomycete (Streptomyces sp. MSC702) from mushroom compost in India Renu Singh 1 and Vishal Kapoor 2* 1 Laboratory of Microbiology, Department of Botany, Christ Church College, Kanpur, 208001, India. 2 Department of Civil Engineering, Indian Institute of Technology, Kanpur, 208016, India. ARTICLE INFO ABSTRACT Article History: Received 24 December 2013 Received in revised form 25 December 2013 Accepted 26 December 2013 Available online 31 December 2013 Key words: Streptomyces sp. MSC702, -Amylase, xylanase, endoglucanase activity. A thermophilic and amylolytic actinomycete was isolated from mushroom compost at 55°C. Identification of the strain MSC702 by morphological and physio-biochemical analysis showed that MSC702 strain belongs to the Streptomyces sp. The strain formed white aerial hyphae that carried straight long chains of cylindrical and smooth spores. It was highly halophilic, alkaliphilic and thermophilic in nature. Strain showed remarkable potential to produce hydrolytic enzymes. The suitability of lignocellu- losic rich agro-residue (rice bran) as a carbon source for the production of endoglucanase activity, -amylase and xylanase was tested. Enzyme production was determined using 1% (w/v) of rice bran in production medium containing (g/100mL): K 2 HPO 4 (0.1), (NH 4 ) 2 SO 4 (0.1), NaCl (0.1), MgSO 4 (0.1) at pH 7.0 after incubation of 48 h at 50°C. Strain MSC702 is very promising for producing -amylase and xylanase with the optimum at 360.75 U/ml and 30.6 U/ml, respectively. Endoglucanase activity was estimated as negligible by the strain. Introduction -Amylases and xylanase constituted a class of the most important industrial enzymes which can be used in a number of industrial processes (Gupta et al., 2003). The advantages of using thermostable enzymes in industrial processes include the decreased risk of contamination, cost of external cooling and increased diffusion rate (Lin et al., 1998). It is universally distributed throughout the animal, plant and microbial kingdoms. However, enzymes from fungal and bacterial sources have dominated applications in industrial sectors (Pandey et al., 2000). Several Bacillus sp. and thermostable actinomycetes including Streptomyces , Thermomonospora and Thermoactinomyces are versatile producers of the enzymes (Ben et al., 1999; Singh et al., 2011). Most of the thermostable -amylases and xylanases reported in the literature contained significant cellulolytic activity, which make them less suitable for industries. In the microbial population, Streptomycetes have been widely recognized for their remarkable capacity in producing bioactive compounds, notably antibiotics, enzymes and other pharmacologically active agents (Berdy, 2005; Baltz, 2007; Promnuan et al., 2009). The thermophilic Streptomycetes grow well at 50°C (Kim et al., 1999) and can be isolated from habitats, where decomposition of organic matter takes place at elevated temperatures and under aerobic conditions such as improperly stored hay, manure, straw and various composts, etc. Composting is an acceleration of the natural decomposition and humification of lignocelluloses and throughout the process a wide variety of microorganisms, including mesophilic and thermophilic bacteria flourish (Femor et al., 1985; Savoie et al., 1996). Thus, all mentioned decompose organic matters provided ample chance to isolate a hyperactive amylolytic and xylolytic strain. Actinomycetes are well known producer of enzymes in lignocellulosic medium (Collins et al., 1993; Kelly et al., 1993, McMahon et al., 1999; Yan et al., 2009; Bajaj et al., 2010). Recently we have reported a Streptomyces sp. MSC702 that produces thermostable, Ca +2 -independent, maltose producing -amylase in submerged fermentation (Singh et al., 2012a). Here, the phenotypic and biochemical characteristics of strain MSC702 and its enzymatic potential are described. Since strain MSC702 is an amylolytic as well as xylolytic strain it can be utilized in industrial applications. Material and Methods Collection of samples and isolation Samples of mushroom compost were collected from different layers of the heap and transferred to a sterile *Corresponding author. Email address: [email protected]Mobile: 91-9450744330 ISSN: 2321 - 8371 Abbreviations: ISP - International Streptomyces Project; CMC - Na-carboxymethyl cellulose; DNS - dinitrosalicylic acid.
Renu Singh and Vishal Kapoor, Biochemical characterization of thermophilic actinomycete (Streptomyces sp. MSC702)from mushroom compost in India, Int.J.Curr.Biotechnol., 2013, 1(10):1-7.
International Journal of CurrentBiotechnology
Journal Homepage : http://ijcb.mainspringer.com
Biochemical characterization of thermophilic actinomycete (Streptomyces sp.MSC702) from mushroom compost in India
Renu Singh1 and Vishal Kapoor2*
1 Laboratory of Microbiology, Department of Botany, Christ Church College, Kanpur, 208001, India.2 Department of Civil Engineering, Indian Institute of Technology, Kanpur, 208016, India.
A R T I C L E I N F O A B S T R A C T
Article History:Received 24 December 2013Received in revised form 25 December 2013Accepted 26 December 2013Available online 31 December 2013
A thermophilic and amylolytic actinomycete was isolated from mushroom compost at55°C. Identification of the strain MSC702 by morphological and physio-biochemicalanalysis showed that MSC702 strain belongs to the Streptomyces sp. The strainformed white aerial hyphae that carried straight long chains of cylindrical and smoothspores. It was highly halophilic, alkaliphilic and thermophilic in nature. Strain showedremarkable potential to produce hydrolytic enzymes. The suitability of lignocellu-losic rich agro-residue (rice bran) as a carbon source for the production ofendoglucanase activity, -amylase and xylanase was tested. Enzyme production wasdetermined using 1% (w/v) of rice bran in production medium containing (g/100mL):K2HPO4(0.1), (NH
4)2SO4(0.1), NaCl (0.1), MgSO4(0.1) at pH 7.0 after incubation of 48
h at 50°C. Strain MSC702 is very promising for producing -amylase and xylanasewith the optimum at 360.75 U/ml and 30.6 U/ml, respectively. Endoglucanase activitywas estimated as negligible by the strain.
Introduction-Amylases and xylanase constituted a class of the mostimportant industrial enzymes which can be used in anumber of industrial processes (Gupta et al., 2003). Theadvantages of using thermostable enzymes in industrialprocesses include the decreased risk of contamination,cost of external cooling and increased diffusion rate (Linet al., 1998). It is universally distributed throughout theanimal, plant and microbial kingdoms. However, enzymesfrom fungal and bacterial sources have dominatedapplications in industrial sectors (Pandey et al., 2000).Several Bacillus sp. and thermostable actinomycetesincluding Streptomyces, Thermomonospora andThermoactinomyces are versatile producers of theenzymes (Ben et al., 1999; Singh et al., 2011). Most of thethermostable -amylases and xylanases reported in theliterature contained significant cellulolytic activity, whichmake them less suitable for industries.
In the microbial population, Streptomycetes have beenwidely recognized for their remarkable capacity inproducing bioactive compounds, notably antibiotics,enzymes and other pharmacologically active agents(Berdy, 2005; Baltz, 2007; Promnuan et al., 2009). The
thermophilic Streptomycetes grow well at 50°C (Kim etal., 1999) and can be isolated from habitats, wheredecomposition of organic matter takes place at elevatedtemperatures and under aerobic conditions such asimproperly stored hay, manure, straw and variouscomposts, etc. Composting is an acceleration of thenatural decomposition and humification oflignocelluloses and throughout the process a wide varietyof microorganisms, including mesophilic andthermophilic bacteria flourish (Femor et al., 1985; Savoieet al., 1996). Thus, all mentioned decompose organicmatters provided ample chance to isolate a hyperactiveamylolytic and xylolytic strain.
Actinomycetes are well known producer of enzymes inlignocellulosic medium (Collins et al., 1993; Kelly et al.,1993, McMahon et al., 1999; Yan et al., 2009; Bajaj et al.,2010). Recently we have reported a Streptomyces sp.MSC702 that produces thermostable, Ca+2-independent,maltose producing -amylase in submerged fermentation(Singh et al., 2012a). Here, the phenotypic andbiochemical characteristics of strain MSC702 and itsenzymatic potential are described. Since strain MSC702is an amylolytic as well as xylolytic strain it can be utilizedin industrial applications.
Material and MethodsCollection of samples and isolationSamples of mushroom compost were collected fromdifferent layers of the heap and transferred to a sterile
polythene bags. Samples were kept at 4ºC. Serial dilutionmethod (Waksman, 1927) was applied for isolation ofthermophilic actinomycetes. Strain MSC702 was isolatedon M medium agar (Obi and Odibo, 1984) (pH 7.0 at 45°C).M medium was modified with 1 % (v/v) trace metal saltsolution (Techapun et al., 2002). The strain wasmaintained on modified M medium agar slants at 4ºC. Allthe culture media were autoclaved at 121°C (15 lbs) for 20min. Strain MSC702 was deposited in the Microbial TypeCulture Collection and Gene Bank (MTCC), Chandigarh,India with accession number MTCC 10772.
Phenotypic characterizationMorphological observations of spores and mycelia with2-5 days old cultures grown at 50°C were made usinglight microscopy (Olympus microscope BX40) andscanning electron- microscopy (FEI model QUANTA200). The test strain was examined for a range ofphenotypic properties using standard procedures(Shirling and Gottlieb, 1966; Williams et al., 1983).Tyrosine agar medium (ISP medium 7; Shirling andGottlieb, 1966) was used to determine whether the strainsproduced dark colored melanin pigments. In addition,acid production from carbohydrates was carried out usingmedia and methods described by Gordon et al., (1974).Tolerance to temperature (20, 25, 30, 35, 40, 45, 50, 55, 60,65 and 70°C), pH (3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5,8.0, 8.5, 9.0, 9.5, 10.0, 10.5 and 11.0) and sodium chloride(0, 2%, 4%, 6%, 8%, 10%, 12% and 14%) was tested usingmodified M broth. The physio-biochemical tests usedwere those of Lechevalier (1972), Gordon et al., (1974),Mishra et al., (1980) and Williams et al., (1983).Carbohydrate utilization was determined by using themedium recommended by Shirling and Gottlieb (1966).Antibiotic resistance of strain MSC702 against thirteenantibiotics was determined by inserting the antibioticsimpregnated disc (Hi-Media antibiotics) on culture grownM medium agar plates at 37°C.
Chemotaxonomy
The cell wall fraction was purified and analyzed by themethod of Lechevalier and Lechevalier (1980). Theprocedure of Becker et al., (1965) and Lechevalier andLechevalier (1980) were used to determine the whole-cellchemical composition.
Inoculum preparationThe inoculum was prepared by adding 10 ml steriledistilled water to a 2 to 3-day-old culture slant and a cellsuspension was made with the help of a sterile loop. Cellsuspension (1.9-2.2×108 CFU/ml) was used as theinoculum for enzyme production.
Enzyme production mediumEnzyme production in submerged fermentation (SmF) wascarried out using basal medium containing (% w/v) ricebran 1.0, K2HPO4 0.1, (NH4)2SO4 0.1, NaCl 0.1 andMgSO4.7H2O 0.1 at pH 7.0. Production medium (100 ml)was placed in an Erlenmeyer flask (250 ml) and autoclavedat 121°C for 20 min and cooled. The medium wasinoculated with 1% inoculum and incubated at 50°C for48 h. Samples were harvested by filtering throughWhatman filter papers 1 (qualitative circles, 125 mmdiameter) and centrifuged at 5,000 g for 20 min at 4°C; thecell-free supernatant (crude enzyme) was used forendoglucanase, -amylase and xylanase assay.
Enzyme assayEndoglucanase, -amylase and xylanase activities wereestimated by analyses of reducing sugar released duringhydrolysis of 1.0% (w/v) Na-carboxymethyl cellulose(CMC), starch and oat-spelt xylan, respectively in 0.1 Mphosphate buffer (pH 7.0) by enzyme (cell-freesupernatant) incubated at 50°C for 10 min. The amountof reducing sugar level released in the mixture wasdetermined by the dinitrosalicylic acid (DNS) method(Miller, 1959). Absorbance at 550 nm (for -amylase) and540 nm (for endoglucanase and xylanase) was recordedby using UV-visible spectrophotometer (UV-1700Pharmaspec Shimadzu) and activity was calculated from
Agar m ediu m Su bstrate myceliu m
A erial myceliu m
D iffu sible pigments
Spore for mation
Yeast ex tract-malt extract (ISP 2)
Colorless, Abundant
W hite, Abundant
None Formed
Oatmeal (IS P 3) Colorless, Abundant
W hite, Abundant
None Formed
Inorganic salt -starch (ISP 4)
Light brown,
Abundant
W hite, Abundant
None Formed
Glycerol-asparagine (IS P 5)
Colorless, Abundant
W hite, Abundant
None Formed
Tyrosine (ISP 7) Light brown,
Abundant
W hite, Abundant
None Formed
Starch-Casein Colorless, Abundant
W hite, moderate
None Formed
N utrien t agar Colorless, Moderate
Not formed
None Not formed
Modified Bennett’s Colorless, Moderate
Not formed
None Not formed
Czapek Dox Colorless, Abundant
W hite, poor
None Formed
Modified M Colorless, Abundant
W hite, Abundant
None Formed
Table 1 Growth and some phenotypic characteristics of strain MSC702 on different media (5 days growth).
Table 2 Chemotaxonomical, biochemical and physiological characteristics of Streptomyces sp. MSC702.
a standard curve using glucose, maltose and xylose asthe standard. One unit (U) of enzyme activity was definedas the amount of enzyme required for the liberation of1mol reducing sugar as glucose, maltose and xylose,respectively, per minute under standard assay conditions.All experiments were carried out in triplicate and averagevalues were given in presented data.
ResultsThe microorganism was isolated from mushroom compostcollected from Mushroom Research and DevelopmentCentre, Department of Plant Pathology, Chandra ShekharAzad University of Agriculture and Technology, Kanpur,India. The type strain is MSC702 (= MTCC 10772).
Morphological and chemical identification
The strain MSC702 is Gram-positive, aerobic, non-motile,halotolerant, alkalitolerant and thermotolerantactinomycete, which forms a highly branched substratemycelium and aerial hyphae which differentiate into longchains of spores (up to 20 spores). Spores are cylindricalshaped and have smooth surfaces (1.1-1.48 m in size)(Fig. 1 A-C). Colonies of Streptomyces MSC702 are white,smooth and powdery on all tested media. Diffusiblepigments are not formed (Table 1). Strain MSC702contains LL-diaminopimelic acid and glycine with nodiagnostic sugars in the whole cell hydrolysates.
Temperature range for growth is 25-70°C with an optimumat 45-55°C. Strain MSC702 is able to grow at pH 5-10 withan optimum at pH 7. It is able to grow in the presence of14% (w/v) NaCl. Growth occurs in presence ofsurfactants such as Tween 80 (1% v/v), Triton X 100 (1%v/v) and Sodium lauryl sulphate (0.1% w/v). The strainhydrolyzed starch, xylan, urea and L-tyrosine. Casein,gelatin, pectin, phenylalanine and carboxy-methyl-cellulose (CMC) are not hydrolyzed. Strain was positivefor nitrate reduction and negative for H2S and catalaseproduction. Good growth is occurred on almost allcarbon sources tested including L-arabinose, xylose,glucose, fructose, galactose, sucrose, maltose, lactose,
cellobiose, raffinose, starch, cellulose, L-rhamnose, D-sorbitol, D-mannitol and meso-inositol. Acid is producedfrom carbon sources L-arabinose, glucose, fructose,galactose, cellobiose, sucrose, maltose, starch and D-mannitol. Acid is not produced from carbon sourcesxylose, raffinose, L-rhamnose, cellulose, D-sorbitol andmeso-inositol. Gas is produced only from the galactose.Growth is recorded in the presence of ampicillin (10 g),ciprofloxacin (5 g), gentamicin (10 g), linezolid (30 g),streptomycin (10 g), vancomycin (30 g), erythromycin(5 g), methicillin (10 g) and penicillin-G (1unit). Growthis inhibited by chloramphenicol (25 g), fusidic acid (10
Characteristics Streptomyces sp. MSC702 S. thermocoprophilus B19T *S. mexicanus CH-
M-1035T
S. thermoviolaceus subsp. apingens DSM
41392T Color of aerial spore mass White Grey Grey Grey
Color of substrate mycelium ND ND Beige Yellow Spore chain Straight Rectiflexibiles Rectiflexibiles Spirales
Spore surface Smooth Smooth Smooth Tuberculate Melanin production - + - - Nitrate reduction + NA NA -
Table 3 Phenotypic properties useful in distinguishing strain MSC702 from other closest phylogenetic thermophilicneighbors.
Data for strain MSC702 are from this study; data for * strain from Petrosyan et al., (2003); data for remaining strainsfrom Kim et al., (2000).Abbreviations: ND, Not distinctive; NA. Not analyzed. Symbols: +, positive; -, negative
g), novobiocin (5 g) and tetracyclin (25 g).Chemotaxonomical, biochemical and physiologicalcharacteristics of strain MSC702 are presented in Table2.
Effect of various mediums on the growth of strainMSC702Growth of aerial mycelium was found on all media testedexcept Nutrient agar and modified Bennett’s agar medium.Morphological characteristics on different media areshown in Table 1.
Chemotaxonomical identificationCell wall composition analysis revealed the presence ofLL-DAP and glycine and absence of characteristicssugars in the peptidoglycan that’s why it belonged tocell wall chemotype I (Lechevalier and Lechevalier, 1970).
Enzyme productionIn the presence of rice bran as a substrate, the -amylaseand xylanase production by strain MSC702 wasnoticeable. The enzyme activity was reached at maximum360.75 U/ml and 30.6 U/ml for amylase and xylanase,respectively, on rice bran as sole carbon source.Endoglucanase activity was almost negligible in themedium (Fig. 2).
DiscussionThe morphological and chemical properties of strainMSC702 are consistent with its assignments to the genusStreptomyces (Williams et al., 1989; Manfio et al., 1995).The aerial mycelium of strain MSC702 was white on alltested media. The substrate mycelium was differed fromcolorless to brown on ISP 4 and ISP 7 media. Diffusiblepigments were not observed on any tested media andmelanin was not produced (Table 1).
A comparative study between strain MSC702 and closelyrelated thermophilic species of the genus Streptomycesrevealed that it differed from S. thermocoprophilus B19T,S. mexicanus CH-M-1035T and S. thermoviolaceus subsp.apingens DSM 41392T in morphological, cultural, andphysiological characteristics as summarized in Table 3.In addition, the aerial mycelium of strain MSC702 waswhite with straight spore chain and diffusible pigmentswere not produced. In contrast, the aerial mycelium of S.
Fig. 1 Streptomyces sp. MSC702 images. A. Culture char-acteristics; B. Microphotograph under light microscopeC. Scanning electron micrograph of the spore chain ofStreptomyces sp. MSC702 grown on modified M agarmedium at 50°C for 3 days.
Fig. 2 Enzyme activity profile. Enzyme activity at different incubation period by strain MSC702 taking rice bran as asole carbon source (pH 7.0, at 50°C).
thermocoprophilus B19T is grey with rectiflexibiles sporechain and produced brown diffusible pigments. Amolecular identification of 16S rRNA and completeanalysis of sequence data of strain MSC702 has beendone (Singh et al., 2012b).
It is apparent from the phenotypic, genotypic andbiochemical data that strain MSC702 belong to genusStreptomyces and showed significant enzymatic potential.Though several enzyme producing microbial strains havebeen isolated and characterized, limited studies areavailable in the literature on the production of -amylaseand xylanase simultaneously, using lignocellulosics as asubstrate. The strain MSC702 is a better producer of -amylase (360.75 U/ml) and xylanase (30.6 U/ml) incomparison to endoglucanase enzyme (0.01 U/ml), in ricebran containing medium. Pulp and paper industry requiredcellulase free -amylase and xylanase for modification ofstarch of coated paper i.e. for the production of low-viscosity, high molecular weight starch (Gupta et al., 2003)and to avoid hydrolysis of the cellulose fibres (Pathaniaet al., 2012), respectively utilizes the potentiality of thestrain MSC702.
AcknowledgementsThe authors acknowledge Dr. Ved Ratan (MushroomResearch and Development Centre, Department of PlantPathology, Chandra Shekhar Azad University ofAgriculture and Technology, Kanpur, India) for supportin collection of the samples and to Dr. Jitendra Kumarand Mr. Shivendra Kumar Jaiswal (Advanced Centre forMaterial Science, Indian Institute of Technology, Kanpur)for providing the facility of scanning electron microscopy.
ReferencesBajaj B.K., Razdan K. and Sharma A., 2010. Thermoactivealkali-stable xylanase production from a newly isolatedStreptomyces sp. SU 9. Indian J. Chem. Technol. 17: 375-380.Baltz R.H., 2007. Antimicrobials from actinomycetes: backto the future. Microbe. 2: 125-131.Becker B., Lechevalier M.P. and Lechevalier H.A., 1965.Chemical composition of cell-wall preparations fromstrains of various form-genera of aerobic actinomycetes.Applied Microbiol. 13: 236-243.Ben A.M., Mezghani M. and Bejar S., 1999. Athermostable -amylase producing maltohexose from anew isolated Bacillus sp. US100: Study of activity andmolecular cloning of corresponding agent. Enzym.Microb. Technol. 24: 548-549.Berdy J., 2005. Bioactive microbial metabolites. J. Antibiot.58: 1-26.Collins B.S., Kelly C.T., Fogarty W.M. and Doyle E.M.,1993. The high maltose producing -amylase of thethermophilic actinomycete, Thermomonospora curvata.Appl. Microbiol. Biotechnol. 39: 31-35.Femor T.R., Randle P.E. and Smith J.F., 1985. Compost asa substrate and its preparation. In: Flegg P, Spencer DM,Wood DA (ed) The biology and technology of thecultivated mushroom. Wiley, Chichester, UK, pp 81-110.Gordon R.E., Barnett D.A., Handerhan, J.E., Pang Hor-Nay C., 1974. Nocardia coeliaca, Nocardiaautotrophica, and the nocardin strain. Int. J. Syst.Bacteriol. 24: 54-63.Gupta R., Gigras P., Mohapatra H., Goswami V.K. andChauhan B., 2003. Microbial -amylases: abiotechnological perspective. Process Biochem. 38: 1599-1616.
Kelly T.T., Collins B.S., Fogarty W.M. and Doyle E.M.,1993. Mechanisms of action of the -amylase ofMicromonospora melanosporea. Appl. Microbiol.Biotechnol. 39: 599-603. Kim B., Al-Tai A.M., Kim S.B., Somasundaram P. andGoodfellow M., 2000. Streptomyces thermocoprophilussp nov, a cellulase-free endo-xylanase-producingstreptomycete. Int. J. Syst. Evol. Microbiol. 50: 505-509.Kim B., Sahin N., Minnikin D.E., Zakrzewska-CzerwinskaJ., Mordarski M. and Goodfellow M., 1999. Classificationof thermophilic streptomycetes including the descriptionof Streptomyces thermoalcalitolerans. Int. J. Syst.Bacteriol. 49: 7-17.Lechevalier M.P. and Lechevalier H.A., 1970. Chemicalcomposition as a criterion in the classification of aerobicactinomycetes. Int. J. Syst. Bacteriol. 20: 435-443.Lechevalier M.P. and Lechevalier H.A., 1980. Thechemotaxonomy of actinomycetes. In: Dietz X, Thayer Y(ed) Actinomycete taxonomy. Society for industrialmicrobiology, Arlington, VA, pp 227-291Lechevalier M.P., 1972. Description of a new species,Oerskovia xanthineolytica , and emendation ofOerskovia Prauser et al. Int. J. Syst. Bacteriol. 22: 260-264.Lin L.L., Chyau C.C. and Hsu W.H., 1998. Production andproperties of a raw starch-degrading amylase from thethermophilic and alkalophilic Bacillus sp. TS-23.Biotechnol. Appl. Biochem. 28: 61-68.Manfio G.P., Zakrzewska-Czerwinska J., Atalan E. andGoodfellow M., 1995. Towards minimal standards for thedescription of Streptomyces species. Biotechnologia. 7-8: 242-253.McMahon H.E.M., Kelly C.T. and Fogarty W.M., 1999.Thermostability of three -amylases of Streptomyces sp.IMD 2679. J. Ind. Microbiol. Biotechnol. 22: 96-99.Miller G.L., 1959. Use of dinitrosalicylic acid reagent fordetermination of reducing sugar. Anal. Chem. 31: 426-428.Mishra S.K., Gordon R.E. and Barnett D.A., 1980.Identification of nocardiae and streptomycetes of medicalimportance. J. Clin. Microbiol. 11: 728-736.Obi S.K.C. and Odibo F.J.C., 1984. Partial purification andcharacterization of a thermostable actinomycete -amylase. Appl. Environ. Microbiol. 47(3): 571-575.Pandey A., Nigam P., Soccol C.R., Soccol V.T., Singh D.and Mohan R., 2000. Advances in microbial amylases.Biotechnol. Appl. Biochem. 31: 135-152.Pathania S., Sharma N. and Verma S.K., 2012. Optimizationof cellulase-free xylanase produced by a potentialthermoalkalophilic Paenibacillus sp. N1 isolated fromhot springs of northern Himalayas in India. J. Microbiol.Biotechnol. Food Sci. 2(1): 1-24.Petrosyan P., Varela M.G., Madrigal A.L., Huitron C. andFlores M.E., 2003. Streptomyces mexicanus sp. nov., axylanolytic micro-organism isolated from soil. Int. J. Syst.Evol. Microbiol. 53: 269-273.Promnuan Y., Kudo T. and Chantawannakul P., 2009.Actinomycetes isolated from bee hives in Thialand. WorldJ. Microbiol. Biotechnol. 25: 1685-1689.Savoie J.M., Bruneau D. and Mamoun M., 1996. Resourceallocation ability of wild isolates of Agaricus bisporuson conventional mushroom compost. FEMS Microbiol.Ecol. 21: 285-92.Shirling E.B., Gottlieb D., 1966. Methods forcharacterization of Streptomyces species. Int. J. Syst.Bacteriol. 16: 313-340.Singh R., Kapoor V. and Kumar V., 2011. Influence ofcarbon and nitrogen sources on the -amylase productionby a newly isolated thermotolerant Streptomyces sp.
MSC702 (MTCC 10772). Asian. J Biotechnol. 3(6): 540-553.Singh R., Kapoor V. and Kumar V., 2012a. Production ofthermostable, Ca+2-independent, maltose producing -amylase by Streptomyces sp. MSC702 (MTCC 10772) insubmerged fermentation using agro-residues as solecarbon source. Ann. Microbiol. 62: 1003-1012.Singh R., Kapoor V. and Kumar V., 2012b. Utilization ofagro-residues for the simultaneous production of amylaseand xylanase by thermophilic actinomycetes. Braz. J.Microbiol. 43(4): 1545-1552.Techapun C., Sinsuwongwat S., Poosaran N., WatanabeM. and Sasaki K., 2002. Thermostable and alkaline-tolerant cellulase-free xylanase produced bythermotolerant Streptomyces sp. Ab106. J. Biosci. Bioeng.93(4): 431-433.Waksman S.A., 1927. Principles of soil microbiology.Williams and Willikins Co., Baltimore, pp 897.Williams S.T., Goodfellow M. and Alderson G., 1989. GenusStreptomyces Waksman and Henrici, 1943, 339AL. In:Williams ST, Sharpe ME, Holt JG (ed) Bergey’s Manualof Systematic Bacteriology (Vol IV). Williams and Wilkins,Baltimore, pp 2452-2492.Williams S.T., Goodfellow M., Alderson G., WellingtonE.M.H., Sneath P.H.A. and Sackin M.J. 1983. Numericalclassification of Streptomyces and related genera. J. Gen.Microbiol. 129: 1743-1813.Yan Q., Hao S., Jiang Z., Zhai Q. and Chen W., 2009.Properties of a xylanase from Streptomyces matensis beingsuitable for xylooligosaccharides production. J. Mol.Catal. B: Enzym. 58 (1-4): 72-77.
