The protein profile and the presence of enzyme were confirmed

by SDS PAGE analysis . The SDS-PAGE was performed in 13 %

(w/v) gels and samples were heated for 10 min at 45°C in the sample

buffer before loading on wells. After electrophoresis, gels were soaked

in 2.5% (w/v) Triton X-100 for 30 min, washed in 100 mM glycine

buffer pH 10.0, 1.5 mM CaCl2

for 30 min and overlaid with 1% agarose

gel containing 0.1% polygalacturonic acid in the same buffer as above.

After 30 min of incubation at 45°C, gels were stained with 0.05%

(w/v) Ruthenium red (Sigma) for 10 min and washed with water until

pectatelyase bands became visible 

Hi, you have not mentioned if you washed away the SDS before zymography. If not, you will have to, like Kamlesh mentioned, with triton. Secondly, you may want to try loading a higher amount of protein during electrophoresis. Especially with bacterial sources, the number of bands after resolution will be more. So during zymography, protein in the resolved band might not be sufficient to be visible.

Thank you dear Kamlesh sir & Rashmi mam for your valuable comments and suggestions. . I had tried the zymogram as mentioned by Kamlesh sir. I had washed away SDS  with triton X -100 for 30 min. But heating of sample  was not done. Will heating make any change??

I will try by heating and by adding more protein  sample.  

u have do zymogram in cold condition ie 40 c....fixing also very important.

Thank you Vijay sir... yes i performed it in cold condition. Then also no result. 

Try to concentrate ur protein with PEGin 1:1 ration,and u have to do zymography in native page electrophoresis.....avoid heating and lastly u have to maintain same PH in all conditions....