This study investigated the biodegradation of the antifungal drug griseofulvin by Bacillus subtilis isolated from expired pharmaceutical raw materials. Key findings include: 1) B. subtilis was able to degrade griseofulvin as detected by TLC, IR and HPLC analysis of degradation products but not physical/chemical tests. 2) The degradation enzyme was purified using column chromatography and identified to have a molecular weight of 5.3439 kDa. 3) Gas chromatography-mass spectrometry identified diethyl phthalate and carbon dioxide as biodegradation products, indicating B. subtilis' ability to metabolize griseofulvin. 4

1. By
Dr. Yahia Ahmed Maher
Assistant professor of Microbiology
Biodegradationof Griseofulvinby Bacillussubtilis
isolatedfromexpiredpharmaceuticalsraw materials
Qura University-Faculty of Dentistry Umm Al

3. Aim of the Work
2 Purification of the biodegradation enzymes.
3 Identification of the biodegradation products.
Study the biodegradation process of
pharmaceutical raw materials.1

5. • The pharmaceutical industry products are
present in everyday life, for a long time the
production of chemicals and pharmaceuticals
as well as their usage and application caused
heavy pollution of the environment and
serious health effects (Sheldon, 2007).
Introduction

6. • Biodegradation is a very important process in
the transformation of organic pollutants as
pharmaceuticals by bacterial growth as energy
source during metabolically processes that
helps to maintain ecosystem and human
health (Yu and Wu, 2012).
Introduction

7. • Some microorganisms may cause drug
contaminant and unable to replicate but could
remain viable and cause lowering of the
quantity of the therapeutic agent in the dosage
form and decrease in its bioavailability or
released a toxic product into environment such
as phenol and acids (United States
Pharmacopoeia 2004).
Introduction

8. • Griseofulvin is antifungal produced by growth
of certain strains of Penicillium griseofulvum
and used against dermatophytic infection,
young actively metabolized cells and inactive
against Actinomyces, Nocardia, and other
bacteria (United States Pharmacopoeia 2004).
Introduction

9. Structure formula of Griseofulvin.
Introduction

11. 1
• Sample collection.
2
• Culture medium and growth conditions.
3
• Isolation, purification and identification.
4
• Studying the optimum condition for enzyme
production.
5
• Detection of degrading Griseofulvin enzyme.
1- Study the biodegradation processes

12. 2- Griseofulvin identification method
A- Physical test B- Chemical reaction C- Spectroscopical analysis
a- UV spectroscopy
detection
c- IR detectionb- TLC detection d- HPLC detection

13. 1
• Filtration the broth and precipitatedby using ammonium sulfate.
2
• Dialysis.
3
• Purifiedby using DEAE-Cellulose, sephadex G-200 column
chromatography and SDS-polyacrylamide gel electrophoresis.
4
• Identificationof the Griseofulvinbiodegradationproducts by
using GC-Mass Spectrometry.
3- Extraction and purificationof
Griseofulvin biodegradation

15. Sample collection
• Eleven bacterial isolates from six expired
pharmaceutical raw materials on Tryptic Soy Agar
(TSA) and Nutrient Agar (NA) medium from total
different seventy-eight samples of expired
pharmaceutical raw materials which collected
from the storage room of Memphis Company for
pharmaceutical and chemical industry.

16. Contaminated and non-contaminated
expired pharmaceutical raw materials

17. Microphotograph of Bacillus subtilis

18. Detection of degrading Griseofulvin enzyme

19. Griseofulvin cup plate clearing zone (CZ)
technique of Griseofulvin degrading enzyme

20. 2- Griseofulvin identification method
• The physical, chemical and UV spectroscopy don’t
detect any degradation for Griseofulvin while it is
detected by TLC, IR and HPLC.

21. TLC recording visible estimation of
standard Griseofulvin
ST T

22. IR spectrum analysis of standard and
biodegraded Griseofulvin

23. HPLC analyses of standard and
biodegraded Griseofulvin
92.85%

24. Standard marker and purified
Griseofulvin degrading enzyme
A, standard marker lane.
B, Griseofulvin degrading
enzyme lane (5.3439 KDa).

25. Relativeabundance
Retention time (Min)
GC-MS detection of standard Griseofulvin
Griseofulvin

26. Relativeabundance
Retention time (Min)
GC-MS detection of biodegradation
Griseofulvin
Diethyl phtalatate
CO2C2H5
CO2C2H5
Griseofulvin

27. Conclusion
The biodegradation processes require present adapted isolate
to synthesize appropriate enzyme.
The biodegradation products were extracted and identified by
GC-Mass Spectrometry.
Remediation of the pharmaceutical raw materials wastes by
using biodegradation technique.