Hello,

It is a complex protocol to identify a compound, following steps should be adopted.

1. broth culture / supernatent of the centrifuged broth to be subjected for chromatography studies using the available reference antibiotics (classes of antibiotics). compare the Rf values to ascertain the compound class.

2. The above samples can be purified using column chromatography, deferentially eluted products are further spotted on TLC and extracted.

3. The purified product then be analysed using HPLC/ GCMS and the compound be determined by X ray chrystallography for characterization and identification.

Thank you 

We worked a bit on purification and characterization of the functionality of an antimicrobial protein. Please refer to the article, if you may wish to at  http://link.springer.com/article/10.1007/s12010-014-1415-z

As for the identification and structural characterization, Dr. Hanumaiah has given a good general start to the topic.

Insilico modelling approaches may be tried, if the sequence is known. Additionally, X-ray crystallography of the crystal, or NMR may be used. Some suggest NMR to be a better technique, as it allows protein structure prediction in its native form without crystallization.

Happy researching....

This is not some thing very difficult , I had been the head of the GSK Fermentation Division in Lahore Pakistan .  Vast literature is available on this subject , both for the Lab & Industrial Scale Cultures . For the Lab scales, you just harvest the cultures , depending upon the lab batch scales ,  by  the Sharples  super centrifuge machine , collect the cell paste from the Rotor ,   the cell paste is frozen  to break the cells by any Cell disruption  Machine at high pressure of 20000 PSI pressure , the cells will rupture , releasing the  cell contents , which is centrifuged again   to remove the debris & the thick liquid / paste . The contents are subjected  to the organic solvent extractions to get the extract,  the extract is purified by the cellulose  or silica gel  flash column  chromatography , identify the fractions , evaporate them to get the purified antibiotic which can then further be purified  by prep HPLC & then analysed by HPLC or  GC-MS . Depending upon the sensitivity  of the antibiotic / its metabolites to aerial oxidation , nitrogen blanketing can be done as well during the purification steps.  The industrial operations are complex  but the pilot scale operations can be developed with  minimum costs.  The commercially , all machines & equipment are available   to achieve any scale of the antibiotic.

You can use simple analytical or prep TLC chromatography techniques as well for the identification of these compounds. Always remember to run the Standards  with these techniques. 

Dr Abid Aziz Ajaz 

Plant Operations Manager 

Stallion  Pharmaceuticals Pvt Limited

Plot No: 581 ,Sundar Industrial Estate

Lahore.Pakistan.

0092-301-4147207

Ema :  [email protected]

______________________________________________________________