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

Abstract

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 (Topt 50 °C) and moderate alkaliphilic (pHopt 8) enzyme, showing high thermostability at 30 and 40 °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 °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.

Characterization of a novel thermostable alkaline lipase derived from a compost metagenomic library and its potential application in the detergent industry

“…Comparisons of their enzymatic properties with Lip54q are shown in Table 5. The results in Table 5 show that the optimum pH of Lip54q was higher than those for EstCS1 (Park et al, 2020a), Est56 (Lu and Daniel, 2021), Est1 (Lu et al, 2019), and EstCS3 (Park et al, 2020b) and was equal to those for Est2 (Lu et al, 2019) and Est8L (Park et al, 2021), but lower than that of Est13L (Park et al, 2021). In addition, the optimum temperature for Lip54q was similar to those of most esterases but significantly lower than those for Est1 and Est2 (Lu et al, 2019).…”

“…Compost is an ideal habitat in which to explore for new lipid hydrolases. In recent years, several lipid hydrolases with unique enzymatic characteristics have been identified from composting habitats (Lu et al, 2019;Park et al, 2020aPark et al, ,b, 2021Lu and Daniel, 2021). Although these enzymes belong to different ester hydrolase families, including family IV (Park et al, 2020a(Park et al, , 2021Lu and Daniel, 2021), family V (Lu et al, 2019), and family VIII (Lu et al, 2019;Park et al, 2020bPark et al, , 2021, the optimal substrates for these enzymes are p-nitrophenyl ester substrates with short carbon chains.…”

Effect of Xanthan Gum, Kappa–Carrageenan, and Guar Gum on the Functional Characteristics of Egg White Liquid and Intermolecular Interaction Mechanism

“…With the addition of NaCl to the mixture, Cl – competed with the negative polysaccharides for the binding sites of positively charged proteins, and Na + competed with the positively charged proteins for the binding sites of the negative polysaccharides, which shielded the electrostatic attraction between the negative polysaccharides and proteins [ 20 ]. Because of their ability to form strong hydrogen bonds, urea functional groups preferentially adsorb onto protein hydrophilic residues through hydrogen bonds, reducing the intramolecular and intermolecular interactions of proteins and polysaccharides, which leads to decreased solution viscosity [ 38 , 39 ]. After shielding the electrostatic and hydrogen–bond interactions, the initial apparent viscosity decreased to varying degrees because the addition of NaCl or urea blocked the intramolecular and intermolecular interactions of polysaccharides and proteins, and the phenomenon of molecular entanglement was weakened, resulting in decreased apparent viscosity [ 40 ].…”

Molecular Cloning and Characterization of a New Family VI Esterase from an Activated Sludge Metagenome

“…Due to the development of metagenomic libraries from various complex samples such as soils [12][13][14][15][16], compost [17][18][19], activated sludge [20][21][22][23], and sediments [24][25][26], many novel lipolytic enzymes have been discovered. Tremendous genetic resources from a number of unculturable microorganisms might be utilized more effectively and successfully when the metagenomic approach is used in the discovery of novel genes.…”