Purification and characterization of 60 kD lipase linked with chaperonin from Pseudomonas aeruginosa BN-1

Abstract

An extracellular lipase from Pseudomonas aeruginosa BN-1 was purified to 42.99 fold. N-terminal sequence of purified enzyme was AAKEVKFGDS identical to sequence of a chaperonin and enzyme may be linked to it. It has an estimated molecular weight of 60 kD, while temperature and pH optima were 37°C and 8.0,  respectively. The enzyme obtained has considerable thermostability retaining 70% of activity at 50°C for 1 h. The enzyme was stable at pH 9.5 for 1 h having 70% of the residual activity. Long acyl chains were preferred as substrate and highest hydrolytic activity was observed against C-16 and C-18 4-nitrophenyl esters. Mustard oil was found to be the preferred substrate as lipolytic activity was 2.75 fold higher when compared with the activity with olive oil as the substrate. The enzymatic activity declined in the presence of Al³⁺, Hg²⁺, Co²⁺ and Mn²⁺, while Ca²⁺ and Ba²⁺ ions enhanced the activity. Non ionic detergents, Tween 80 and sodium deoxycholate, increased the activity by 1.2 and 2.5 fold, respectively. Ethylenediaminetetraacetic acid (EDTA), 2-mercaptoethanol and 1,4-dithio-DL-threitol (DTT) had no effect on lipolytic activity. Sodium dodecyl sulfate (SDS) and phenylmethylsulfonyl fluoride (PMSF) inhibited the enzyme activity by 90 and 98%, respectively. The lipase showed stability in organic solvents.

Key words: Pseudomonas aeruginosa BN^-1, chaperonin, mustard oil, organic solvent, phenyl methyl sulfonyl
fluoride, sodium deoxycholate, Swiss Prot Accession # P 30718.