Functional Screening of Soil Metagenomic Libraries

Supplementary methods

Functional screening of soil metagenomic libraries

All the functional screening media were supplemented either with 1 µg ml-1ampicillin (Sigma, USA) for screening the plasmid libraries (FS1 and FS2) or with 1 µg ml-1chloramphenicol (Sigma, USA) for screening the BAC library (FS3).

Amylase plate diffusion assay

The presence of amylolytic activity in soil metagenome was screened by following the method described by Cowan (1991) with slight modification. The screening media consist of M9 minimal saltagar (Himedia, India) supplemented with 1 % soluble starch (Himedia, India) as sole carbon source. The clones in the soil metagenomic libraries were grown on the starch agar plates at 37 oC for 48 h. After incubation, the plates were flooded with 0.1 % iodine solution (Himedia, India) andthe positive clones were identified by a clear zone around the colonies.Cellulase plate diffusion assay

The cellulase producing clones were screened with M9 minimal salt agar medium (Himedia, India) supplemented with 1 % carboxymethyl cellulose (CMC) (Himedia, India) as described by Teather and Wood (1982).Afterincubation at 37 oC for 48 h and cellulolytic activity was visualized by staining with 1 % Congo Red (Himedia, India) followed by destaining with 5 % NaCl. Cellulase-producing clones were identified by a clear zone around the bacterial colony in a red background.

DNase plate diffusion assay

DNase activity in the soil metagenome was screened with DNase agar (Himedia, India) containing methyl green indicator. After 48 h incubation at 37 oC, the DNase activity was visualized as clear zone in green background (Schau 1986).

Lipase plate diffusion assay

Lipase producing clones were visualized with M9 minimal salt agar (Himedia, India) containing 0.5 % trioleoylglycerol and 0.1 % rhodamine B dye (Sigma, USA). After incubation the plates were irradiated with UV light at 350 nm. The lipase producing clones showed an orange fluorescence around the colony (Kouker and Jaeger1987).

Pectinase plate diffusion assay

Pectinase activity was screened with M9 minimal salt agar media containing 0.5 % pectin (Himedia, India), 0.5 % yeast extract (Himedia, India) and pH 7. After 48 h incubation at 37 oC the plates were flooded with 1 % cetrimide solution (Sigma, USA). Pectinase producing clones were identified with a clear zone around the colonies (Beg et al., 2000).

Xylanase plate diffusion assay

Metagenomic clones were grown in M9 minimal salt agar media (Himedia, India) containing1 % oatspelt xylan (Sigma, USA) and pH7.Afterincubation at 37 oC for 48 h xylanolytic activity was visualized by staining with 1 % congo red solution (Himedia, India) followed by destaining with 5 % Sodium chloride solution (Sigma, USA). (Teather and Wood1982). Xylanase producing clones were identified by a clear zone around the bacterial colony.

Asparaginase plate diffusion assay

Metagenomic clones were screened for L-Asparaginase productionusing plate diffusion assay as described byGulati et al., (1997) with M9 minimal agar (Himedia, India)supplemented with 1 % L-asparagine (Sigma, USA) as soleenergy source and 0.1 % phenol red (Sigma, USA) as pHindicator. After incubation at 37 oC for 48 h.Asparaginase producing clones were visualized by pink colour formation around the colony.

References

Beg QK, Bhushan B, Kapoor M, Hoondal GS (2000) Production and characterization of thermostable xylanase and pectinase from Streptomyces sp. QG-11-3. J Ind Microbiol Biotechnol 24: 396-402

Cowan DA (1991) Industrial enzymes. In Biotechnology, The Science and The Business eds Moses, V. and Cape, R.E. Reading: Harwood Academic Publishers.311–340

Gulati R, Saxena RK, Gupta R (1997) A rapid plate assay for screening l‐asparaginase producing micro‐organisms. Lett Appl Microbiol 24:23-26

Kouker G, Jaeger KE (1987) Specific and sensitive plate assay for bacterial lipases. Appl Environ Microbiol 53: 211-213

Schau HP (1986) JF MacFaddin, Media for Isolation‐Cultivation‐Identification‐Maintenance of Medical Bacteria, Volume I. XI+ 929 S., 163 Abb., 94 Tab. Baltimore, London 1985. Williams and Wilkins. J Basic Microbiol 26: 240-240

Teather RM, Wood PJ (1982) Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen. ApplEnviron Microbiol 43: 777-780