Bacterial Lipolytic Enzymes Research Articles

Investigation of amino acids related to Staphylococcus saprophyticus AG1 EstAG1 carboxylesterase catalytic function revealed a new family of bacterial lipolytic enzymes

Most of the lipolytic enzymes (carboxylesterases, EC 3.1.1.1 and triacylglycerol acylhydrolases, EC 3.1.1.3) originate from bacteria and form a large group of functionally important enzymes that are also well known for their use in multiple biotechnology sectors. Rapid and increasing amount of bacterial lipolytic enzymes being discovered and characterized led to a necessity to classify them. More than twenty years ago bacterial lipolytic enzymes were originally classified into eight families and six true lipase sub-families based on the differences in their amino acid sequences and biochemical properties. Later, this classification was comprehensively updated to 19 families with eight subfamilies, and more recently, employing deeper comparative analysis methods, classification expanded to 35 families and 11 subfamilies. Bacterial lipolytic enzymes that cannot be classified into currently existing families are still being discovered. This work provides site-directed mutagenesis and differential scanning fluorimetry based investigation of catalytic function-related amino acids of previously discovered and characterized EstAG1 carboxylesterase from Staphylococcus saprophyticus AG1. Experimental results obtained in this work revealed that EstAG1 carboxylesterase can be placed into a new family of bacterial lipolytic enzymes.

Efficient monoacylglycerol synthesis by carboxylesterase EstGtA2 from Geobacillus thermodenitrificans in a solvent-free two-phase system

The present study investigated high-yield monoacylglycerol (MAG) synthesis by bacterial lipolytic enzymes in a solvent-free two-phase system. Esterification by monoacylglycerol lipase from Bacillus sp. H-257 (H257) required a high glycerol/fatty acid molar ratio for efficient MAG synthesis. Screening of H257 homologues revealed that carboxylesterase derived from Geobacillus thermodenitrificans, EstGtA2, exhibited a higher esterification rate than H257. Moreover, neutralizing the pH of the acidic reaction solution by adding potassium hydroxide (KOH) solution further increased the esterification rate. The esterification rate by EstGtA2 reached 75% under conditions of equivalent molar amounts of glycerol and fatty acid, and the MAG rate (MAG/total glyceride) was 97%. The neutralized pH of the reaction solution likely affected the thermal stability of EstGtA2 during the esterification reaction. Screening for thermal-tolerant variants revealed that the EstGtA2S26I variant was stable at 75°C for 30min, a condition under which wild-type EstGtA2 was completely inactivated. The esterification rate by the EstGtA2S26I variant reached 90%, and the MAG rate was 96%. The addition of alkali and the use of a thermal-tolerant enzyme were important for obtaining high-yield MAG in a solvent-free two-phase system utilizing EstGtA2.

Ancestral sequence reconstruction of ancient lipase from family I.3 bacterial lipolytic enzymes

Family I.3 lipase is distinguished from other families by the amino acid sequence and secretion mechanism. Little is known about the evolutionary process driving these differences. This study attempt to understand how the diverse temperature stabilities of bacterial lipases from family I.3 evolved. To achieve that, eighty-three protein sequences sharing a minimum 30% sequence identity with Antarctic Pseudomonas sp. AMS8 lipase were used to infer phylogenetic tree. Using ancestral sequence reconstruction (ASR) technique, the last universal common ancestor (LUCA) sequence of family I.3 was reconstructed. A gene encoding LUCA was synthesized, cloned and expressed as inclusion bodies in E. coli system. Insoluble form of LUCA was refolded using urea dilution method and then purified using affinity chromatography. The purified LUCA exhibited an optimum temperature and pH at 70 ℃ and 10 respectively. Various metal ions increased or retained the activity of LUCA. LUCA also demonstrated tolerance towards various organic solvents in 25% v/v concentration. The finding from this study could support the understanding on temperature and environment during ancient time. In overall, reconstructed ancestral enzymes have improved physicochemical properties that make them suitable for industrial applications and ASR technique can be employed as a general technique for enzyme engineering.

Crystal structures of a novel family IV esterase in free and substrate-bound form.

Bacterial lipolytic enzymes of family IV are homologs of the mammalian hormone-sensitive lipases (HSL) and have been successfully used for various biotechnological applications. The broad substrate specificity and ability for enantio-, regio-, and stereoselective hydrolysis are remarkable features of enzymes from this class. Many crystal structures are available for esterases and lipases, but structures of enzyme-substrate or enzyme-inhibitor complexes are less frequent although important to understand the molecular basis of enzyme-substrate interaction and to rationalize biochemical enzyme characteristics. Here, we report on the structures of a novel family IV esterase isolated from a metagenomic screen, which shows a broad substrate specificity. We solved the crystal structures in the apo form and with a bound substrate analogue at 1.35 and 1.81Å resolution, respectively. This enzyme named PtEst1 hydrolyzed more than 60 out 96 structurally different ester substrates thus being substrate promiscuous. Its broad substrate specificity is in accord with a large active site cavity, which is covered by an α-helical cap domain. The substrate analogue methyl 4-methylumbelliferyl hexylphosphonate was rapidly hydrolyzed by the enzyme leading to a complete inactivation caused by covalent binding of phosphinic acid to the catalytic serine. Interestingly, the alcohol leaving group 4-methylumbelliferone was found remaining in the active site cavity, and additionally, a complete inhibitor molecule was found at the cap domain next to the entrance of the substrate tunnel. This unique situation allowed gaining valuable insights into the role of the cap domain for enzyme-substrate interaction of esterases belonging to family IV. DATABASE: Structural data of PtEst1 are available in the worldwide protein data bank (https://www.rcsb.org) under the accession codes: 6Z68 (apo-PtEst1) and 6Z69 (PtEst1-inhibitor complex).

Metagenomic Approach to Bacterial Diversity and Lipolytic Enzymes’ Genes from a Steam Soil of Los Humeros Geothermal Field (Puebla, México)

Los Humeros volcanic complex, located at the eastern end of the Trans Mexican Volcanic Belt, possess superficial thermal manifestations (fumaroles, heat steam-soils and alteration zones), with temperatures between 50 °C and 90 °C. This is the first work to explore the bacterial diversity of a steam soil of Los Humeros geothermal field using a metagenomic 16S RNA gene analysis, and the presence of lipolytic enzymes’ genes after an enrichment culture by a whole-genome shotgun approach. High diversity indexes were found in soil samples, with Firmicutes, Chloroflexi, Proteobacteria, Actinobacteria and Acidobacteria as the most prevalent phyla. Alicyclobacillus was the most prevalent genus at 15 cm and Anoxybacillus at 50 cm depth; anaerobic and obligate thermophilic and hyperthermophilic members were found at 50 cm depth. Firmicutes was favored by the oil-rich medium, and the dominant genera were Geobacillus and Caldibacillus. 165 genes related to lipolytic enzymes were found; carboxilesterases and phospholipases C were the most abundant, and 43% were assigned to uncharacterized lipolytic enzymes. The diversity as well as the high percentage of non-characterized lipolytic enzymes found after soil enrichment in an oil-rich medium suggest the presence of biotechnologically promising organisms and enzymes that deserve to be investigated.

Characterization of Novel Salt-Tolerant Esterase Isolated from the Marine Bacterium Alteromonas sp. 39-G1.

An esterase gene, estA1, was cloned from Alteromonas sp. 39-G1 isolated from the Beaufort Sea. The gene is composed of 1,140 nucleotides and codes for a 41,190 Da protein containing 379 amino acids. As a result of a BLAST search, the protein sequence of esterase EstA1 was found to be identical to Alteromonas sp. esterase (GenBank: PHS53692). As far as we know, no research on this enzyme has yet been conducted. Phylogenetic analysis showed that esterase EstA1 was a member of the bacterial lipolytic enzyme family IV (hormone sensitive lipases). Two deletion mutants (Δ20 and Δ54) of the esterase EstA1 were produced in Escherichia coli BL21 (DE3) cells with part of the N-terminal of the protein removed and His-tag attached to the C-terminal. These enzymes exhibited the highest activity toward p-nitrophenyl (pNP) acetate (C2) and had little or no activity towards pNP-esters with acyl chains longer than C6. Their optimum temperature and pH of the catalytic activity were 45°C and pH 8.0, respectively. As the NaCl concentration increased, their enzyme activities continued to increase and the highest enzyme activities were measured in 5 M NaCl. These enzymes were found to be stable for up to 8 h in the concentration of 3-5 M NaCl. Moreover, they have been found to be stable for various metal ions, detergents and organic solvents. These salt-tolerant and chemical-resistant properties suggest that the enzyme esterase EstA1 is both academically and industrially useful.

Structural and functional insights about unique extremophilic bacterial lipolytic enzyme from metagenome source

In the present investigation, a lipid hydrolyzing gene RPK01 was cloned from metagenome source of hot spring. Expression and purification of recombinant protein revealed single purified protein band of ~24 KDa on 12% SDS-PAGE, and is well corroborated with the deduced molecular weight of protein as calculated from its amino acid sequence. The purified protein displayed high activity towards short chain fatty acids and was found to be completely stable at 30°C till 3h, it further retained ~40% activity at 50°C and 60°C temperature till 3h. Additionally, the pH stability assay showed its functionality in broad range of pH, with maximum stability observed at pH 2.0, it decreases from pH 4.0 to pH 12.0, and nearly showed 40% activity in these pH values. Both circular dichroism and intrinsic Trp fluorescence studies revealed conformational stability of protein structure at wide range of temperature and pH. Enzyme activity enhances in presence of non-ionic surfactants like Tween 20 and TritonX-100. Further, inhibitors of the active site residues including PMSF and DEPC alone were unable to inhibit enzyme activity, while cumulative presence of calcium and inhibitors reduces enzyme activity to 90%, indicating conformational changes in the protein. Molecular simulation dynamics analysis revealed a calcium binding site near the lid helix of this protein(Asn75-Ile80).

Structural insights into the catalytic mechanism of lovastatin hydrolase

Structural insights into the catalytic mechanism of lovastatin hydrolase

A proposed update for the classification and description of bacterial lipolytic enzymes.

Bacterial lipolytic enzymes represent an important class of proteins: they provide their host species with access to additional resources and have multiple applications within the biotechnology sector. Since the formalisation of lipolytic enzymes into families and subfamilies, advances in molecular biology have led to the discovery of lipolytic enzymes unable to be classified via the existing system. Utilising sequence-based comparison methods, we have integrated these novel families within the classification system so that it now consists of 35 families and 11 true lipase subfamilies. Representative sequences for each family and subfamily have been defined as well as methodology for accurate comparison of novel sequences against the reference proteins, facilitating the future assignment of novel proteins. Both the code and protein sequences required for integration of additional families are available at: https://github.com/thh32/Lipase_reclassification.

Application of Bacterial Thermostable Lipolytic Enzymes in the Modern Biotechnological Processes: A Review

Review focuses on the modern applications of bacterial lipolytic enzymes in biotechnology and covers the scope of their properties including their activity and functional stability at different temperatures, pH, substrate specificity, and activity in the presence of different chemicals. The recent data on the production of genetically engineered strains producing the bacterial lipolytic enzymes and approaches to improving their productivity are presented. The applications of bacterial lipases in biotechnological processes used in the production of biofuel, chemicals and detergents, in the food industry, and in wastewater treatment are considered.

Expression and characterization of an esterase belonging to a new family via isolation from a metagenomic library of paper mill sludge

A new bacterial lipolytic enzyme Est903 was obtained from paper mill sludge via metagenomic approach. Est903 displayed moderate similarities to two lipolytic enzymes from Rhodopirellula islandica and contained a distinctive pentapeptide motif (GFSAG) that differed from those of all known fourteen families of bacterial lipolytic enzymes. Est903 was regarded as from a new bacterial lipolytic enzyme family through multiple sequence alignment and phylogenetic analysis.The recombinant Est903 showed the highest activity for ρ-nitrophenol butyrate. The pH optimum and temperature optimum of the recombinant enzyme was 9.0 and 51 °C, respectively. Also, this enzyme displayed moderate thermostability, high activity under alkaline conditions, and good tolerance against several organic solvents. In addition, the compatibility test and washing performance analysis revealed that Est903 had good compatibility with commercial laundry detergent and high cleaning ability of oil stains. These good properties make Est903 a potential candidate in organic synthesis or detergent industry.

Lipid metabolism and intracellular bacterial virulence: key to next-generation therapeutics.

Lipid metabolism is thought to play a key role in the pathogenicity of several intracellular bacteria. Bacterial lipolytic enzymes hydrolyze lipids from the host cell to release free fatty acids which are used as an energy source and building blocks for the synthesis of cell envelope and also to modulate host immune responses. In this review, we discussed the role of lipid metabolism and lipolytic enzymes in the life cycle and virulence of Mycobacterium tuberculosis and other intracellular bacteria. The lipolytic enzymes appear to be potential candidates for developing novel therapeutics by targeting lipid metabolism for controlling M. tuberculosis and other intracellular pathogenic bacteria. [Formula: see text].

Identification and characterization of a novel esterase from Thauera sp.

A novel esterase gene TLip was identified from the strain Thauera sp. and expressed at high levels in Escherichia coli. The TLip protein shared the highest identity (48%) to esterase TesA from Pseudomonas aeruginosa when compared to enzymes with reported properties. Phylogenetic analysis showed that TLip belongs to the GDSL family of bacterial lipolytic enzymes. TLip was an alkaline esterase with a broad optimal temperature range 37-50 °C and an optimal pH of 8.0. Substrate specificity assays showed that TLip preferred medium chain p-nitrophenyl esters (C6 -C12 ). Besides, the activity of TLip was strongly inhibited by Cu2+ but greatly enhanced by Triton X-100 and Tween 80. Thermostability assay revealed that TLip was stable without loss of activity at 37 °C and still retained 69% activity at 50 °C after 2 H of incubation. Together, these provided a good candidate for further exploration of TLip as a promising biocatalyst in industry.

Two novel deep-sea sediment metagenome-derived esterases: residue 199 is the determinant of substrate specificity and preference

BackgroundThe deep-sea environment harbors a vast pool of novel enzymes. Owing to the limitations of cultivation, cultivation-independent has become an effective method for mining novel enzymes from the environment. Based on a deep-sea sediment metagenomics library, lipolytic-positive clones were obtained by activity-based screening methods.ResultsTwo novel esterases, DMWf18-543 and DMWf18-558, were obtained from a deep-sea metagenomic library through activity-based screening and high-throughput sequencing methods. These esterases shared 80.7% amino acid identity with each other and were determined to be new members of bacterial lipolytic enzyme family IV. The two enzymes showed the highest activities toward p-nitrophenyl (p-NP) butyrate at pH 7.0 and 35–40 °C and were found to be resistant to some metal ions (Ba2+, Mg2+, and Sr2+) and detergents (Triton X-100, Tween 20, and Tween 80). DMWf18-543 and DMWf18-558 exhibited distinct substrate specificities and preferences. DMWf18-543 showed a catalytic range for substrates of C2–C8, whereas DMWf18-558 presented a wider range of C2–C14. Additionally, DMWf18-543 preferred p-NP butyrate, whereas DMWf18-558 preferred both p-NP butyrate and p-NP hexanoate. To investigate the mechanism underlying the phenotypic differences between the esterases, their three-dimensional structures were compared by using homology modeling. The results suggested that residue Leu199 of DMWf18-543 shortens and blocks the substrate-binding pocket. This hypothesis was confirmed by the finding that the DMWf18-558-A199L mutant showed a similar substrate specificity profile to that of DMWf18-543.ConclusionsThis study characterized two novel homologous esterases obtained from a deep-sea sediment metagenomic library. The structural modeling and mutagenesis analysis provided insight into the determinants of their substrate specificity and preference. The characterization and mechanistic analyses of these two novel enzymes should provide a basis for further exploration of their potential biotechnological applications.

A Novel Halotolerant Thermoalkaliphilic Esterase from Marine Bacterium Erythrobacter seohaensis SW-135.

A novel esterase gene, e69, was cloned from Erythrobacter seohaensis SW-135, which was isolated from a tidal flat sediment of the Yellow Sea in Korea. This gene is 825 bp in length and codes for a 29.54 kDa protein containing 274 amino acids. Phylogenetic analysis showed that E69 is a new member of the bacterial lipolytic enzyme family IV. This enzyme exhibited the highest level of activity toward p-nitrophenyl (NP) butyrate but little or no activity toward the other p-NP esters tested. The optimum temperature and pH of the catalytic activity of E69 were 60°C and pH 10.5, respectively. The enzyme exhibited stable activity over a wide range of alkaline pH values (7.5–9.5). In addition, E69 was found to be a halotolerant esterase as it exhibited the highest hydrolytic activity in the presence of 0.5 M NaCl and was still active in the presence of 3 M NaCl. Moreover, it possessed some degree of tolerance to Triton X-100 and several organic solvents. Through homology modeling and comparison with other esterases, it was suggested that the absence of the cap domain and its narrow substrate-binding pocket might be responsible for its narrow substrate specificity. Sequence and structural analysis results suggested that its high ratio of negatively to positively charged residues, large hydrophobic surface area, and negative electrostatic potential on the surface may be responsible for its alkaline adaptation. The results of this study provide insight into marine alkaliphilic esterases, and the unique properties of E69 make it a promising candidate as a biocatalyst for industrial applications.

Characterization of a novel thermo-stable lipase from endophyte Pseudomonas putida in Pistacia chinensis Bunge

A novel lipolytic enzyme-producing endophytic strain PC2 was successfully isolated from the seeds of an ideal bioenergy plant Pistacia chinensis Bunge. Based on the analysis of morphology and 16S rRNA sequence, bacterial strain PC2 was identified as a subspecies of Pseudomonas putida, therefore named as P. putida PC2. Whole-genome sequencing showed PC2 contained a 1224-nucleotide lipase gene (named lip-PC2) predicted to encode a 407-amino-acid protein. Purified lipases from both the original PC2 strain and heterologously expressed Escherichia coli were nearly 50 kD with specific activity of 9.48 U/mL. LIP-PC2 displayed the maximal activity at 50°C or pH 8.0, and maintained above 80% relative activity in the range of from 40 to 60°C or pH in the range of from 6.0 to 8.0, indicating thermostable and alkaline properties. Enzyme activity was enhanced by Mg2+, Na+ and Mn2+, but strongly inhibited by Cu2+, Zn2+ Co2+, EDTA as well as organic solvents and surfactants. Additionally, the analysis of amino acid sequence and structure indicated that LIP-PC2 was a novel member belonging to family I.3 of bacterial lipolytic enzymes and its catalytic triad was consisted of Ser-200, Asp-342 and His-374.

Biochemical characterization of an enantioselective esterase from Brevundimonas sp. LY-2

BackgroundLactofen, a member of the diphenylether herbicides, has high activity and is commonly used to control broadleaf weeds. As a post-emergent herbicide, it is directly released to the environment, and easily caused the pollution. This herbicide is degraded in soil mainly by microbial activity, but the functional enzyme involved in the biodegradation of lactofen is still not clear now.ResultsA novel esterase gene lacH, involved in the degradation of lactofen, was cloned from the strain Brevundimonas sp. LY-2. The gene contained an open reading frame of 921 bp, and a putative signal peptide at the N-terminal was identified with the most likely cleavage site between Ala 28 and Ala 29. The encoded protein, LacH, could catalyze the hydrolysis of lactofen to form acifluorfen. Phylogenetic analysis showed that LacH belong to family V of bacterial lipolytic enzymes. Biochemical characterization analysis showed that LacH was a neutral esterase with an optimal pH of 7.0 and an optimal temperature of 40 °C toward lactofen. Besides, the activity of LacH was strongly inhibited by Hg2+ and Zn2+. LacH preferred short chain p-nitrophenyl esters (C2–C6), exhibited maximum activity toward p-nitrophenyl acetate. Furthermore, the enantioselectivity of LacH during lactofen hydrolysis was also studied, and the results show that R-(−)-lactofen was degraded faster than S-(+)-lactofen, indicating the occurrence of enantioselectivity in the enzymatic reaction.ConclusionsOur studies characterized a novel esterase involved in the biodegradation of diphenylether herbicide lactofen. The esterase showed enantioselectivity during lactofen degradation, which revealed the occurrence of enzyme-mediated enantioselective degradation of chiral herbicides.

Construction of a novel lipolytic fusion biocatalyst GDEst-lip for industrial application.

The gene encoding esterase (GDEst-95) from Geobacillus sp. 95 was cloned and sequenced. The resulting open reading frame of 1497 nucleotides encoded a protein with calculated molecular weight of 54.7kDa, which was classified as a carboxylesterase with an identity of 93-97% to carboxylesterases from Geobacillus bacteria. This esterase can be grouped into family VII of bacterial lipolytic enzymes, was active at broad pH (7-12) and temperature (5-85°C) range and displayed maximum activity toward short acyl chain p-nitrophenyl (p-NP) esters. Together with GD-95 lipase from Geobacillus sp. strain 95, GDEst-95 esterase was used for construction of fused chimeric biocatalyst GDEst-lip. GDEst-lip esterase/lipase possessed high lipolytic activity (600U/mg), a broad pH range of 6-12, thermoactivity (5-85°C), thermostability and resistance to various organic solvents or detergents. For these features GDEst-lip biocatalyst has high potential for applications in various industrial areas. In this work the effect of additional homodomains on monomeric GDEst-95 esterase and GD-95 lipase activity, thermostability, substrate specificity and catalytic properties was also investigated. Altogether, this article shows that domain fusing strategies can modulate the activity and physicochemical characteristics of target enzymes for industrial applications.

Identification and Characterization of a Novel Salt-Tolerant Esterase from the Deep-Sea Sediment of the South China Sea.

Marine esterases play an important role in marine organic carbon degradation and cycling. Halotolerant esterases from the sea may have good potentials in industrial processes requiring high salts. Although a large number of marine esterases have been characterized, reports on halotolerant esterases are only a few. Here, a fosmid library containing 7,200 clones was constructed from a deep-sea sediment sample from the South China Sea. A gene H8 encoding an esterase was identified from this library by functional screening and expressed in Escherichia coli. Phylogenetic analysis showed that H8 is a new member of family V of bacterial lipolytic enzymes. H8 could effectively hydrolyze short-chain monoesters (C4–C10), with the highest activity toward p-nitrophenyl hexanoate. The optimal temperature and pH for H8 activity were 35°C and pH 10.0, respectively. H8 had high salt tolerance, remaining stable in 4.5 M NaCl, which suggests that H8 is well adapted to the marine saline environment and that H8 may have industrial potentials. Unlike reported halophilic/halotolerant enzymes with high acidic/basic residue ratios and low pI values, H8 contains a large number of basic residues, leading to its high basic/acidic residue ratio and high predicted pI (9.09). Moreover, more than 10 homologous sequences with similar basic/acidic residue ratios and predicted pI values were found in database, suggesting that H8 and its homologs represent a new group of halotolerant esterases. We also investigated the role of basic residues in H8 halotolerance by site-directed mutation. Mutation of Arg195, Arg203 or Arg236 to acidic Glu significantly decreased the activity and/or stability of H8 under high salts, suggesting that these basic residues play a role in the salt tolerance of H8. These results shed light on marine bacterial esterases and halotolerant enzymes.

Discovery of Polyesterases from Moss-Associated Microorganisms.

In this study, we describe the discovery and analysis of new enzymes from microbial communities associated with plants (moss). The recovered enzymes show the ability to hydrolyze not only common esterase substrates but also the synthetic polyester poly(butylene adipate-co-butylene terephthalate), which is a common material employed in biodegradable plastics. The widespread use of such synthetic polyesters in industry and society requires the development of new sustainable technological solutions for their recycling. The discovered enzymes have the potential to be used as catalysts for selective recovery of valuable building blocks from this material.