A Novel Carboxylesterase Derived from a Compost Metagenome Exhibiting High Stability and Activity towards High Salinity

Halotolerant lipolytic enzymes have gained growing interest, due to potential applications under harsh conditions, such as hypersalinity and presence of organic solvents. In this study, a lipolytic gene, est56, encoding 287 amino acids was identified by functional screening of a compost metagenome. Subsequently, the gene was heterologously expressed, and the recombinant protein (Est56) was purified and characterized. Est56 is a mesophilic (T-opt 50 degrees C) and moderate alkaliphilic (pH(opt) 8) enzyme, showing high thermostability at 30 and 40 degrees C. Strikingly, Est56 is halotolerant as it exhibited high activity and stability in the presence of up to 4 M NaCl or KCl. Est56 also displayed enhanced stability against high temperatures (50 and 60 degrees C) and urea (2, 4, and 6 M) in the presence of NaCl. In addition, the recently reported halotolerant lipolytic enzymes were summarized. Phylogenetic analysis grouped these enzymes into 13 lipolytic protein families. The majority (45%) including Est56 belonged to family IV. To explore the haloadaptation of halotolerant enzymes, the amino acid composition between halotolerant and halophilic enzymes was statistically compared. The most distinctive feature of halophilic from non-halophilic enzymes are the higher content of acidic residues (Asp and Glu), and a lower content of lysine, aliphatic hydrophobic (Leu, Met and Ile) and polar (Asn) residues. The amino acid composition and 3-D structure analysis suggested that the high content of acidic residues (Asp and Glu, 12.2%) and low content of lysine residues (0.7%), as well as the excess of surface-exposed acidic residues might be responsible for the haloadaptation of Est56.

Automated summary

Halotolerant lipolytic enzyme Est56, identified from a compost metagenome, exhibited high thermal stability, pH optima at 50 degrees C and pH 8, and tolerance to high salt concentrations up to 4 M NaCl or KCl. Phylogenetic analysis grouped Est56 and other similar enzymes into 13 lipolytic protein families, with the majority (45%) belonging to family IV. Amino acid composition comparison between halotolerant and halophilic enzymes revealed higher acidic residues and lower lysine content in halophilic enzymes, suggesting a key role in haloadaptation for Est56.