MICROBIOLOGICAL AND BIOCHEMICAL STUDIES ON SOME BACTERIAL ISOLATES THAT PRODUCE EXTRACELLULAR LIPASE

Abstract

Lipase (triacylglycerol lipase, EC3.1.1.3) is an enzyme that hydrolyzes esters of glycerol with preferably long-chain fatty acids. In addition to hydrolyzing esters, lipase can catalyze transesterification, esterification, and aminolysis or oximolysis under anhydrous conditions. Lipases are the most versatile biocatalyst and bring about an arrange of industrial applications such as detergents and tanning industry. In the present study, one hundred bacterial isolates were isolated from Egyptian soil using a rapid and an efficient technique. These isolates were purified for two rounds of purification, and were examined for their ability to produce extracellular lipase enzyme using agar plate methods and specific lipase substrate p­ nitrophenyl palmitate. These isolates were screened to about 40 and further to 20 isolates. Finally, three lipase-producing isolates were chosen based on their great ability to produce true lipase enzyme. These three isolates abbreviated as Ps-x, Ml and M6.

The isolates Ps-x, Ml and M6 were identified using morphological and biochemical tests as Pseudomonas aeruginosa, Pseudomonas cepacia and Pseudomonas pseudomallei respectively. Further confirmation of the identification was carried out for the i solate Ps-x through the 168 rRNA (DNA) technique and result obtained was deposited in the GenBank data base at http://www.ncbi.nlm.nih.gov/.

The level of lipase production was 34.23 U/ml after 24 hours of growth on the production medium for P. aeruginosa while that of P. cepacia and P. pseudomallei gave 13.4 U/ml and 20.3 U/ml after 48 hours, respectively. Growth and extracellular lipase production was monitored for P. aeruginosa. The production of lipase was started at late log phase and, increased gradually till reached its maximal level of production at stationary phase.

Extracellular P. aeruginosa lipase enzyme was purified to homogenity using ammonium sulfate precipitation at 80% saturation followed by two chromatography columns namely Q­ Sepharose and S-Sepharose (ion exchange chromatography). Purification results indicated that, P. aeruginosa under study prod uced two different extracellular lipase enzymes Lipas I and Lipase II and of molecular weights 15.5 and 54,97 kilodaltons, respectively. It was found that 10 J..LI ofthe purified lipase I or J1 are suitable to be used on the reaction mixture (lml). The optimum temperature for the lipase I and lipase II was 50 • C and 45 • C, respectively. The optimum pH for the lipase I and IT was pH 9.0 and pH 10. After one hour of incubation of lipase I or lipase IT at 60 • C the enzyme was retained 48.31% and 66 % of its activity, respectively. Enzyme stability was enhanced in the presence of calcium chJoride to be 87.82% and 77.77% of its activity, respectively. Na+ 1 and Mg+2 had an enhancing effect while Zn +2 had inhibitory effect. Moreover, EDTA and SDS had no significant effect while DMSO had an enhancing effect. PMSF had an inhibitory effect.

In an attempt to isolate DNA fragment that carries a lipolytic activity, genomic library was constructed using a P. Aeruginosa DNA. The genomic library was constructed on pTrcHis (A,B,C) plasmid expression vector. The recombinant plasmids was transformed into competent E. coli DH5a. and screened for lipase gene utilizing LB agar plates supplemented with ampicillin, IPTG and Tween 20. Results indicated that, a number of recombinant clones was found to produce recombinant lipase enzyme. A pilot expression study was carried out on LB ampicillin medium supplemented with IPTG as an inducer.