Partial Purification and Characterization of Molybdenum Reducing Enzyme from Klebsiella Oxytoca Hkeem

• Main Author: Lim, Hui Keem
• Format: Thesis
• Language: English; English
• Published: 2011
• Online Access: http://psasir.upm.edu.my/id/eprint/20548/1/FBSB_2011_20_IR.pdf; http://psasir.upm.edu.my/id/eprint/20548/

As a result of widespread application in numerous industrial processes, heavy metals have become a contaminant of many environmental systems. Molybdenum’s ubiquitous application in many industrial products makes it a silent pollutant with levels as high as several hundreds of ppm. Thus in this study, the isolate molybdenum reducing bacterium, which showed the highest molybdenum reducing activity, were isolated from a soil sample collected near a steel factory in Selangor. This isolate was later identified as Klebsiella oxytoca strain hkeem through molecular phylogenetic analysis of sequenced 16S rRNA gene sequence. Klebsiella oxytoca strain hkeem reduced the heavy metal molybdenum to molybdenum blue optimally in selective low phosphate medium agar, at pH 7.3 and 30ºC, after 24 hours of incubation. In specific, Klebsiella oxytoca strain hkeem could reduce molybdenum to molybdenum blue under aerobic conditions in the medium with fructose as electron donor, yeast extract, phosphate ion (4.5 mM) and molybdate ion (80 mM). Partial purification and characterization were conducted on the molybdenum reducing enzyme, with anion exchange on Q-sepharose and gel filtration on Zorbax GFX-250. Based on the SDS polyacrylamide-gel electrophoresis (SDS-PAGE), two bands were observed on the gel filtration fraction at 90 and 38 kDa, respectively. Meanwhile, the enzyme showed an optimum activity at substrate pH 5.5 and 25ºC. The kinetics of electron donor (NADH) and electron acceptor (LPPM) for the enzyme were followed the classical Michaelis-Menten rectangular hyperbolic curve. Km and Vmax for the electron donor substrate, NADH was 2.83 mM and 12.23 nmole molybdenum blue produced/min/mg/protein, respectively. However the Km and Vmax for the electron acceptor substrate phosphomolybdate (50 mM) were 1.66 mM and 32.06 nmole molybdenum blue/min/mg/protein respectively. Although many molybdenum-reducing bacteria have been isolated, molybdenum-reducing activity of Klebsiella oxytoca strain hkeem up to 80 mM of molybdate was reported as the most potent molybdenum-reducing isolate to date. Based on the results obtained, Klebsiella oxytoca strain hkeem was proven as a more powerful molybdenum reducer to develop a cost-effective bioremediation work, especially since bacterial molybdenum reduction has been suggested as an important remediation tool for cleaning up molybdenum pollutant in the environment.