Pustaka Ilmiah Universitas Padjadjaran

Abstrak

Transformation of Puljl43 Plasmid into Monascus Purpureus Albino Mutant

Monascus purpureus, a red angkak mould, produces various secondary metabolites, such as pigments, monacolin K, and citrinin. The pigments are used as food and cosmetic colorants, Monacolin K has anti hypercholesterolemia activity , whereas its citrinin, eventhough has antimicrobial activity against gram positive bacteria, unfortunately showed nephrotoxic properties. Many researches have been done in investigating the way to eliminate this toxic metabolite. Since citrinin’s precursor is as same as its pigments, their biosynthesis can be manipulated by genetic engineering method, however it needs comprehensive information about genetic transformation because there are still very limited information on Monascus transformations.

This research aimed to study the method of inserting pULJL43 plasmid containing ble gene into genome of M. purpureus KM2 (albino mutant), to obtain stable transformants. This was a preliminary work, as part of our major research for further genetic engineering application to eliminate citrinin.

The wild type of the fungus was firstly mutated to be albino mutant. The research was initiated by the determination of minimum inhibitory concentration (MIC) of phleomycin from the albino’s. The plasmid pULJL43 from E. coli was isolated, analysed its purity by electrophoresis and the concentration of the plasmid was determined by spectrophotometric method. Then, the protoplasts of albino mutant were produced. The plasmid was transform to this protoplast by protoplast-polyethylene glycol (PEG) method. Plasmid-containing transformants were produced and then grown on phleomycin-containing YMP solid medium. The transformation efficiency (transformant colonies per g pULJL43 plasmid) was determined, and the transformants stability was observed for 5th generations. The 5th phleomycin MIC concentration in YMP solid medium. Detection insertion gene (ble gene) was characterized by PCR. Electrophoresis of PCR product of transformant’s DNA and pULJL43 plasmid as positive control, showed no difference.

The research results showed that the transformation of pULJL43 plasmid into protoplast of Monascus albino mutant was succeeded and it was proved by PCR. The minimum inhibitory concentration of phleomycin for albino mutant was 1 g/mL, whereas the transformants were grown on YMP solid medium containing 5 g/mL phleomycin, and 31 to 45 transformants were yielded per g plasmid. Stable transformants were proved stable for 5 generations and the 5 generation still grown in YMP solid medium containing 10 g/mL phleomycin.