Novel cold-adapted alkaline lipase from an intertidal flat metagenome and proposal for a new family of bacterial lipases

Abstract

A new lipase, LipEH166, isolated from an intertidal flat metagenome, showed no amino acid similarity to any known lipolytic enzyme except in the consensus region. This suggested that LipEH166 and its homologues belong to a new family of lipolytic enzymes. Partial characterization indicated that LipEH166 is a novel cold-adapted alkaline lipase.

FIG. 1.

Phylogenetic tree of LipEH166 and closely related proteins. Phylogenetic analysis was performed using Megalign in the Lasergene program (version 6.0; DNAStar Inc.). Amino acid sequences were aligned by Clustal V. Except for LipEH166 and related proteins, the protein sequences for families of bacterial lipolytic enzymes previously classified by Arpigny and Jaeger (1) and for the LipG family (11) were retrieved from GenBank (http://www.ncbi.nlm.nih.gov).

FIG. 2.

Substrate specificity of LipEH166 for pNPEs. The lipase activity of the purified LipEH166 enzyme toward various pNPEs was assayed at 25°C and pH 8.0. The chain length of pNPEs is expressed as the carbon number of esters. Data are averages from duplicate experiments.

FIG. 3.

Effect of temperature on LipEH166. (A) Enzyme activity was analyzed at each temperature under standard assay conditions. (B) The proportion of unfolded LipEH166 is displayed as a function of temperature. Each percentage was calculated from the change in ellipticity at 222 nm, obtained by circular dichroism using a spectropolarimeter. Data are averages from triplicate experiments.

FIG. 4.

Effect of pH on LipEH166. Enzyme activity was measured at each pH under standard assay conditions (▪). In addition, the enzyme was preincubated at the indicated pH, and the remaining activity was determined (•). Data are averages from triplicate experiments.