Journal of Pure and Applied MicrobiologyVol. 9 No. 4

Fed-Batch Fermentation and Downstream Processing for Large-Scale Production of Recombinant Pf-Lactate Dehydrogenase and Its Application in Malaria Diagnosis and as a Platform for Screening of Antimalarial Chemotherapeutic Agent

Saurabh Shrivastava1, Anil K Singh1, Kshitij chandel2, Ritu Gill3 and N. Gopalan1*

1Bio-process Scale up Facility, Defence Research and Development Establishment, Ministry of Defence (Govt. of India), Gwalior - 474 002, India. 2Vector Management Division, Defence Research and Development Establishment, Ministry of Defence (Govt. of India), Gwalior, - 474 002, India. 3Centre for Biotechnology, MD University, Rohtak-124001, India. 4Madhav Institute of Technology and Science, Gwalior, India.

Received on 10 May 2015 and accepted on 20 July 2015

 

ABSTRACT

The potential of malaria parasite (Plasmodium falciparum) generating resistance to currently used antimalarial drugs is a major curse for malaria patients. The multiple targets on which antimalarial drugs act hamper the occurrence of such resistance. As an essential metabolic enzyme involved in energy production in the parasite, the lactate dehydrogenase of P. falciparum (PfLDH) provides an alternative target for parasite killing. In the present study, we have efficiently optimized the fed-batch fermentation and downstream processing for high yield production of recombinant PfLDH using Escherichia coli. The fed-batch operation gives the freedom of manipulating the process via substrate feed rate. The rPfLDH was produced in previously optimized media (Modified Terrific Broth) by fed-batch fermentation using 5 L bioreactor. Fed-batch fermentation resulted in a wet weight of 98.6 g/L and dry cell biomass 24.2 g/L. With the improved downstream process, purified rPfLDH had a yield of 461.20 mg/L. Expression and purification were optimized and the expressed recombinant PfLDH was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The recombinant PfLDH was purified for uniform measurement of enzymatic activity and immobilized on 96-well plates for detection of PfLDH-targeted inhibitors. Hematin (HT) and Gossypol (GP) three well-known inhibitors of PfLDH, were chosen as positive inhibitors for evaluating the feasibility of the in-vitro colorimetric enzyme assay. The fed-batch fermentation and downstream processing methods optimized in this study have enormous application for high yield production of recombinant pLDH in a cost effective manner using bacterial system. The large scale production of recombinant PfLDH is useful in the diagnosis during the incidence of malarial infection and also it can be used as economic potential target site for the screening of chemotherapeutic agents for the development of novel antimalarial drugs.

Keywords : PfLDH, Fed-batch fermentation, Malaria diagnosis, Antimalarial