Cell Surface Expression of Biologically Active Influenza C Virus HEF Glycoprotein Expressed from cDNA

The hemagglutinin, esterase, and fusion (HEF) glycoprotein of influenza C virus possesses receptor binding, receptor destroying, and membrane fusion activities. The HEF cDNAs from influenza C/Ann Arbor/1/50 (HEF-AA) and influenza C/Taylor/1223/47 (HEF-Tay) viruses were cloned and expressed, and transport of HEF to the cell surface was monitored by susceptibility to cleavage by exogenous trypsin, indirect immunofluorescence microscopy, and flow cytometry. Previously it has been found in studies with the C/Johannesburg/1/66 strain of influenza C virus (HEF-JHB) that transport of HEF to the cell surface is severely inhibited, and it is thought that the short cytoplasmic tail, Arg-Thr-Lys, is involved in blocking HEF cell surface expression (F. Oeffner, H.-D. Klenk, and G. Herrler, J. Gen. Virol. 80:363-369, 1999). As the cytoplasmic tail amino acid sequences of HEF-AA and HEF-Tay are identical to that of HEF-JHB, the data indicate that cell surface expression of HEF-AA and HEF-Tay is not inhibited by this amino acid sequence. Furthermore, the abundant cell surface transport of HEF-AA and HEF-Tay indicates that their cell surface expression does not require coexpression of another viral protein. The HEF-AA and HEF-Tay HEF glycoproteins bound human erythrocytes, promoted membrane fusion in a low-pH and trypsin-dependent manner, and displayed esterase activity, indicating that the HEF glycoprotein alone mediates all three known functions at the cell surface.     

A cold-active esterase with a substrate preference for vinyl esters from a psychrotroph, Acinetobacter sp. strain no. 6: gene cloning, purification, and characterization

It has recently been shown that fatty acid vinyl esters serve as effective acylating agents for the synthesis of esters by enzymatic transesterification in high yields. To enhance the usefulness of this system at low temperatures, we have searched for the gene coding for a cold-active lipolytic enzyme with a substrate preference for fatty acid vinyl esters and obtained it from the genomic library of Acinetobacter sp. strain no. 6, a psychrotroph isolated from Siberian soil. The gene (termed aelh, 777 bp) encoded a protein of 258 amino acids, and sequence analysis revealed that the enzyme shows a high sequence similarity to β-ketoadipate enol-lactone hydrolase involved in the β-ketoadipate pathway for the bacterial catabolism of benzoic acid. The aelh gene was expressed in the E. coli C600 cells under the control of lac promoter and the expression product was purified to homogeneity and characterized. It was a monomeric esterase preferentially catalyzing the hydrolysis of enol esters, such as fatty acid vinyl esters with a short-chain acyl group. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride, a specific inhibitor for serine hydrolases. The enzyme could also catalyze transesterification, for example, between vinyl propionate and propanol yielding propyl propionate at 4 °C. These results indicate the usefulness of an esterase (termed AELH) for the enzymatic synthesis of esters by transesterification using vinyl esters as an acyl donor.     

Est10: A Novel Alkaline Esterase Isolated from Bovine Rumen Belonging to the New Family XV of Lipolytic Enzymes

A metagenomic fosmid library from bovine rumen was used to identify clones with lipolytic activity. One positive clone was isolated. The gene responsible for the observed phenotype was identified by in vitro transposon mutagenesis and sequencing and was named est10. The 367 amino acids sequence harbors a signal peptide, the conserved secondary structure arrangement of alpha/beta hydrolases, and a GHSQG pentapeptide which is characteristic of esterases and lipases. Homology based 3D-modelling confirmed the conserved spatial orientation of the serine in a nucleophilic elbow. By sequence comparison, Est10 is related to hydrolases that are grouped into the non-specific Pfam family DUF3089 and to other characterized esterases that were recently classified into the new family XV of lipolytic enzymes. Est10 was heterologously expressed in Escherichia coli as a His-tagged fusion protein, purified and biochemically characterized. Est10 showed maximum activity towards C4 aliphatic chains and undetectable activity towards C10 and longer chains which prompted its classification as an esterase. However, it was able to efficiently catalyze the hydrolysis of aryl esters such as methyl phenylacetate and phenyl acetate. The optimum pH of this enzyme is 9.0, which is uncommon for esterases, and it exhibits an optimal temperature at 40°C. The activity of Est10 was inhibited by metal ions, detergents, chelating agents and additives. We have characterized an alkaline esterase produced by a still unidentified bacterium belonging to a recently proposed new family of esterases.     

A novel esterase from Ralstonia sp. M1: gene cloning, sequencing, high-level expression and characterization

A newly isolated gene from Ralstonia sp. M1, encoding an esterase, was cloned in Escherichia coli and its nucleotide sequence determined. The 1.6kb insert revealed one complete open reading frame, predicted to encode an esterase (320 aa, 34.1kDa) with a pI of 9.86. EstR contained a putative oxyanion hole H36G37, a conserved pentapeptide G103HSLG107 and a conserved catalytic His265 and Asp237. The EstR sequence shared 64-70 and 44-48% identity with the hydrolases/acyltransferases from Burkholderia strains and from Ralstonia strains, respectively, 44 and 38% identity with the lactone-specific esterase from Pseudomonas fluorescens and Mesorhizobium loti, respectively. The esterase EstR was expressed with a high level of 41mg/g wet cells. The Ni-NTA-purified esterase EstR showed an optimal activity in the temperature range 60-65 degrees C and pH range 7.5-9.0 towards p-nitrophenyl caproate. The enzyme was found to be highly resistant to many organic solvents especially induced by ethanolamine. Metal ions showed slight effect on esterase activity. The inhibitor phenylmethanesulfonyl fluoride inhibited strongly the esterase. Triton X-45 induced the activation of EstR, but other detergents slightly to strongly decreased or completely inhibited. Among tested p-NP esters, caproate was the most preferential substrate of this esterase.     

O-acetylation and de-O-acetylation of sialic acids. Sialic acid esterases of diverse evolutionary origins have serine active sites and essential arginine residues

We and others have recently described 9-O-acetyl-sialic acid esterase (9-O-Ac-SA esterase) activities that appear to be specific for removal of O-acetyl esters from the 9-position of naturally occurring sialic acids. We have now examined a variety of species for such enzymes and found them in vertebrates and higher invertebrates, but not in plants or in lower invertebrates. This evolutionary distribution correlates well with that of the sialic acids themselves. All of the 9-O-Ac-SA esterase activities tested were inhibited by diisopropyl fluorophosphate (DFP) in a dose-dependent fashion. This indicates that each of these enzymes has a serine active site similar to the well known serine esterases and serine proteases. Methyl esterification of the carboxyl group of 9-O-acetyl-N-acetylneuraminic acid significantly reduced the activity of all of the 9-O-Ac-SA esterases against the O-acetyl group. This indicates that each of these enzymes may recognize the negatively charged carboxyl group of the sialic acid. Enzymes that recognize anionic substrates frequently have an essential arginine residue (Riordan, J. F., McElvany, K. D., and Borders, C. L., Jr. (1977) Science 195, 884-886). We therefore studied the effects of the arginine-specific modifying reagents 2,3-butanedione and phenylglyoxal on 9-O-Ac-SA esterase activities from influenza C virus, human erythrocytes, rat liver, starfish gonads, and sea bass brain. All of these enzymes were inhibited in a dose-dependent fashion by both reagents, under conditions previously known to avoid nonspecific modification. In contrast, the typical serine proteases trypsin and kallikrein and the serine esterase acetylcholinesterase were not significantly affected, even by the highest concentrations of these reagents used. These data indicate that five 9-O-Ac-SA esterase activities from evolutionarily distinct origins all have serine active sites and essential arginine residues. We postulate that the arginine residue is involved in substrate recognition via the negatively charged carboxyl group of the sialic acids. Thus, these 9-O-Ac-SA esterase activities may be members of a previously undescribed class of serine esterase.     

Chlorophyllase as a serine hydrolase: identification of a putative catalytic triad

Chlorophyllases (Chlases), cloned so far, contain a lipase motif with the active serine residue of the catalytic triad of triglyceride lipases. Inhibitors specific for the catalytic serine residue in serine hydrolases, which include lipases effectively inhibited the activity of the recombinant Chenopodium album Chlase (CaCLH). From this evidence we assumed that the catalytic mechanism of hydrolysis by Chlase might be similar to those of serine hydrolases that have a catalytic triad composed of serine, histidine and aspartic acid in their active site. Thus, we introduced mutations into the putative catalytic residue (Ser162) and conserved amino acid residues (histidine, aspartic acid and cysteine) to generate recombinant CaCLH mutants. The three amino acid residues (Ser162, Asp191 and His262) essential for Chlase activity were identified. These results indicate that Chlase is a serine hydrolase and, by analogy with a plausible catalytic mechanism of serine hydrolases, we proposed a mechanism for hydrolysis catalyzed by Chlase.     

Molecular basis of formaldehyde detoxification. Characterization of two S-formylglutathione hydrolases from Escherichia coli, FrmB and YeiG

The Escherichia coli genes frmB (yaiM) and yeiG encode two uncharacterized proteins that share 54% sequence identity and contain a serine esterase motif. We demonstrated that purified FrmB and YeiG have high carboxylesterase activity against the model substrates, p-nitrophenyl esters of fatty acids (C2-C6) and alpha-naphthyl acetate. However, both proteins had the highest hydrolytic activity toward S-formylglutathione, an intermediate of the glutathione-dependent pathway of formaldehyde detoxification. With this substrate, both proteins had similar affinity (Km = 0.41-0.43 mM), but FrmB was almost 5 times more active. Alanine replacement mutagenesis of YeiG demonstrated that Ser145, Asp233, and His256 are absolutely required for activity, indicating that these residues represent a serine hydrolase catalytic triad in this protein and in other S-formylglutathione hydrolases. This was confirmed by inspecting the crystal structure of the Saccharomyces cerevisiae S-formylglutathione hydrolase YJG8 (Protein Data Bank code 1pv1), which has 45% sequence identity to YeiG. The structure revealed a canonical alpha/beta-hydrolase fold and a classical serine hydrolase catalytic triad (Ser161, His276, Asp241). In E. coli cells, the expression of frmB was stimulated 45-75 times by the addition of formaldehyde to the growth medium, whereas YeiG was found to be a constitutive enzyme. The simultaneous deletion of both frmB and yeiG genes was required to increase the sensitivity of the growth of E. coli cells to formaldehyde, suggesting that both FrmB and YeiG contribute to the detoxification of formaldehyde. Thus, FrmB and YeiG are S-formylglutathione hydrolases with a Ser-His-Asp catalytic triad involved in the detoxification of formaldehyde in E. coli.     

The hemagglutinin-esterase of mouse hepatitis virus strain S is a sialate-4-O-acetylesterase

By comparative analysis of the hemagglutinin-esterase (HE) protein of mouse hepatitis virus strain S (MHV-S) and the HE protein of influenza C virus, we found major differences in substrate specificities. In striking contrast to the influenza C virus enzyme, the MHV-S esterase was unable to release acetate from bovine submandibulary gland mucin. Furthermore, MHV-S could not remove influenza C virus receptors from erythrocytes. Analysis with free sialic acid derivatives revealed that the MHV-S HE protein specifically de-O-acetylates 5-N-acetyl-4-O-acetyl sialic acid (Neu4, 5Ac2) but not 5-N-acetyl-9-O-acetyl sialic acid (Neu5,9Ac2), which is the major substrate for esterases of influenza C virus and bovine coronaviruses. In addition, the MHV-S esterase converted glycosidically bound Neu4,5Ac2 of guinea pig serum glycoproteins to Neu5Ac. By expression of the MHV esterase with recombinant vaccinia virus and incubation with guinea pig serum, we demonstrated that the viral HE possesses sialate-4-O-acetylesterase activity. In addition to observed enzymatic activity, MHV-S exhibited affinity to guinea pig and horse serum glycoproteins. Binding required sialate-4-O-acetyl groups and was abolished by chemical de-O-acetylation. Since Neu4,5Ac2 has not been identified in mice, the nature of potential substrates and/or secondary receptors for MHV-S in the natural host remains to be determined. The esterase of MHV-S is the first example of a viral enzyme with high specificity and affinity toward 4-O-acetylated sialic acids.     

Improvement of model for prediction of hemagglutinin mutations in H5N1 influenza viruses with distinguishing of arginine, leucine and serine

In a continuation of our attempt to predict mutations in proteins from influenza A virus, this study attempted to answer the question of whether distinguishing between arginine, leucine and serine can improve the predictability as these residues are governed by different probabilistic mechanism translating from RNA codons to amino acids. In this study, we made the prediction based on the mutation relation among 299 H5N1 hemagglutinins of influenza A virus. Then, we compared the results based on the distinguishing of arginine, leucine and serine with the results without distinguishing of arginine, leucine and serine. The results show that the prediction together with distinguishing between arginine, leucine and serine is better than prediction without distinguishing between these residues.     

A novel cold-adapted esterase from Salinisphaera sp. P7-4: gene cloning, overproduction, and characterization

Salinisphaera sp. P7-4 was isolated from the intestine of silver whiting, Sillago japonicas caught in the Pacific Ocean, and the esterase gene was cloned using the shotgun method. The amino acid sequence deduced from the nucleotide sequence (951 bp) corresponded to a protein of 316 amino acid residues with a molecular weight of 34,443. The esterase had 46 and 44% identities with the esterase enzymes of Ralstonia eutropha JMP134 and Rhodopseudomonas palustris HaA2, respectively. The primary structure of P7-4 esterase showed the conserved catalytic triad (Ser, Asp, His), consensus pentapeptide GXSXG, and oxyanion hole sequence (HG). The protein P7-4 was successfully expressed in Escherichia coli in a biologically active form. The enzyme showed high catalytic activity at low temperatures (5-25° C) with an activation energy of 2.18 kcal/mol. This result indicated that the esterase from Salinisphaera sp. P7-4 is a new cold-adapted enzyme. The enzyme preferentially hydrolyzed acyl-group chains with short chain lengths of ≤10 carbon. Metal ions such as Cd2(+), Co2(+), Cu2(+), Hg2(+), Ni2(+) and Zn2(+) inhibited enzymatic activity. Additionally, EDTA has no effect on its activity, whereas inhibition was observed with PMSF, a serine hydrolase inhibitor.     

The influenza A virus PB2, PA, NP, and M segments play a pivotal role during genome packaging

The genomes of influenza A viruses consist of eight negative-strand RNA segments. Recent studies suggest that influenza viruses are able to specifically package their segmented genomes into the progeny virions. Segment-specific packaging signals of influenza virus RNAs (vRNAs) are located in the 5' and 3' noncoding regions, as well as in the terminal regions, of the open reading frames. How these packaging signals function during genome packaging remains unclear. Previously, we generated a 7-segmented virus in which the hemagglutinin (HA) and neuraminidase (NA) segments of the influenza A/Puerto Rico/8/34 virus were replaced by a chimeric influenza C virus hemagglutinin/esterase/fusion (HEF) segment carrying the HA packaging sequences. The robust growth of the HEF virus suggested that the NA segment is not required for the packaging of other segments. In this study, in order to determine the roles of the other seven segments during influenza A virus genome assembly, we continued to use this HEF virus as a tool and analyzed the effects of replacing the packaging sequences of other segments with those of the NA segment. Our results showed that deleting the packaging signals of the PB1, HA, or NS segment had no effect on the growth of the HEF virus, while growth was greatly impaired when the packaging sequence of the PB2, PA, nucleoprotein (NP), or matrix (M) segment was removed. These results indicate that the PB2, PA, NP, and M segments play a more important role than the remaining four vRNAs during the genome-packaging process.     

Influenza C virus hemagglutinin: comparison with influenza A and B virus hemagglutinins

The complete nucleotide sequence of the influenza C/California/78 virus RNA 4 was obtained by using cloned cDNA derived from the RNA segment. This gene is 2,071 nucleotides long and can code for a polypeptide of 654 amino acids. Although there are no convincing sequence homologies between RNA 4 and the hemagglutinin genes of influenza A and B viruses, we suggest, on the basis of structural features, that RNA 4 of the influenza C virus codes for the hemagglutinin. The structural features which are common to the hemagglutinins of influenza A, B, and C viruses include (i) a hydrophobic signal peptide, (ii) an arginine cleavage site between the hemagglutinin 1 and 2 subunits, (iii) hydrophobic regions at the amino and carboxyl termini of the hemagglutinin 2 subunit, and (iv) several conserved cysteine residues. Additional evidence that RNA 4 of influenza C virus codes for the hemagglutinin is that the tripeptide Ile-Phe-Gly, known to be present at the amino terminus of the hemagglutinin 2 subunit of influenza C virus, is encoded by RNA 4 at a point immediately adjacent to the presumptive arginine cleavage site. The lack of primary sequence homology between the influenza C virus hemagglutinin and the influenza A or B virus hemagglutinins, which all have similar functions, might be attributed to convergent rather than divergent evolution. However, the structural similarities among the influenza A, B, and C virus hemagglutinins strongly suggest that the three hemagglutinin genes have diverged from a common precursor.     

A novel influenza A virus activating enzyme from porcine lung: purification and characterization

Proteolytic activation of hemagglutinin, an envelope glycoprotein of the influenza virus, by host proteases is essential for infection and proliferation of the virus. However, there is no well-defined, inherent source of host proteases in man or swine, both of which are natural hosts for human influenza viruses. We have recently isolated a 32 kDa protein in a high salt extract from porcine lungs, which possess the hemagglutinin processing activity. In this study, we attempted to purify another hemagglutinin processing enzyme from porcine lung. The purified enzyme, named tryptase TC30, exhibited a molecular mass of about 30 kDa by SDS-PAGE and 28.5 kDa by gel filtration chromatography, suggesting that it is a monomer. Tryptase TC30 cleaved peptide substrates with Arg at the P1 position, and preferentially substrates with the Ser-Ile-Gin-Ser-Arg sequence corresponding to the HA cleavage site sequence of the A/PR/8/34 influenza virus. Among various inhibitors tested, trypsin-type serine protease inhibitors, such as aprotinin, antipain, benzamidine and leupeptin, efficiently inhibited the proteolytic activity of the enzyme. The N-terminal 40 amino acid sequence of tryptase TC30 exhibits more than 60% homology to mast cell tryptases from mice MCP-6 and human tryptase-alpha and -beta. These data indicate that tryptase TC30, the 30 kDa enzyme from porcine lung, is a novel hemagglutinin-cleaving enzyme.     

Factor D of the alternative pathway of human complement. Purification, alignment and N-terminal amino acid sequences of the major cyanogen bromide fragments, and localization of the serine residue at the active site.

The serine esterase factor D of the complement system was purified from outdated human plasma with a yield of 20% of the initial haemolytic activity found in serum. This represented an approx. 60 000-fold purification. The final product was homogeneous as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (with an apparent mol.wt. of 24 000), its migration as a single component in a variety of fractionation procedures based on size and charge, and its N-terminal amino-acid-sequence analysis. The N-terminal amino acid sequence of the first 36 residues of the intact molecule was found to be homologous with the N-terminal amino acid sequences of the catalytic chains of other serine esterases. Factor D showed an especially strong homology (greater than 60% identity) with rat 'group-specific protease' [Woodbury, Katunuma, Kobayashi, Titani, & Neurath (1978) Biochemistry 17, 811-819] over the first 16 amino acid residues. This similarity is of interest since it is considered that both enzymes may be synthesized in their active, rather than zymogen, forms. The three major CNBr fragments of factor D, which had apparent mol.wts. of 15 800, 6600 and 1700, were purified and then aligned by N-terminal amino acid sequence analysis and amino acid analysis. By using factor D labelled with di-[1,3-14C]isopropylphosphofluoridate it was shown that the CNBr fragment of apparent mol.wt. 6600, which is located in the C-terminal region of factor D, contained the active serine residue. The amino acid sequence around this residue was determined.     

Identification of a coronavirus hemagglutinin-esterase with a substrate specificity different from those of influenza C virus and bovine coronavirus

We have characterized the hemagglutinin-esterase (HE) of puffinosis virus (PV), a coronavirus closely related to mouse hepatitis virus (MHV). Analysis of the cloned gene revealed approximately 85% sequence identity to HE proteins of MHV and approximately 60% identity to the corresponding esterase of bovine coronavirus. The HE protein exhibited acetylesterase activity with synthetic substrates p-nitrophenyl acetate, alpha-naphthyl acetate, and 4-methylumbelliferyl acetate. In contrast to other viral esterases, no activity was detectable with natural substrates containing 9-O-acetylated sialic acids. Furthermore, PV esterase was unable to remove influenza C virus receptors from human erythrocytes, indicating a substrate specificity different from HEs of influenza C virus and bovine coronavirus. Solid-phase binding assays revealed that purified PV was unable to bind to sialic acid-containing glycoconjugates like bovine submaxillary mucin, mouse alpha1 macroglobulin or bovine brain extract. Because of the close relationship to MHV, possible implications on the substrate specificity of MHV esterases are suggested.     

Gene cloning and nucleotide sequencing and properties of a cocaine esterase from Rhodococcus sp. strain MB1

A strain of Rhodococcus designated MB1, which was capable of utilizing cocaine as a sole source of carbon and nitrogen for growth, was isolated from rhizosphere soil of the tropane alkaloid-producing plant Erythroxylum coca. A cocaine esterase was found to initiate degradation of cocaine, which was hydrolyzed to ecgonine methyl ester and benzoate; both of these esterolytic products were further metabolized by Rhodococcus sp. strain MB1. The structural gene encoding a cocaine esterase, designated cocE, was cloned from Rhodococcus sp. strain MB1 genomic libraries by screening recombinant strains of Rhodococcus erythropolis CW25 for growth on cocaine. The nucleotide sequence of cocE corresponded to an open reading frame of 1,724 bp that codes for a protein of 574 amino acids. The amino acid sequence of cocaine esterase has a region of similarity with the active serine consensus of X-prolyl dipeptidyl aminopeptidases, suggesting that the cocaine esterase is a serine esterase. The cocE coding sequence was subcloned into the pCFX1 expression plasmid and expressed in Escherichia coli. The recombinant cocaine esterase was purified to apparent homogeneity and was found to be monomeric, with an M(r) of approximately 65,000. The apparent K(m) of the enzyme (mean +/- standard deviation) for cocaine was measured as 1.33 +/- 0.085 mM. These findings are of potential use in the development of a linked assay for the detection of illicit cocaine.     

A new esterase, belonging to hormone-sensitive lipase family, cloned from Rheinheimera sp. isolated from industrial effluent

The gene for esterase (rEst1) was isolated from a new species of genus Rheinheimera by functional screening of E. coli cells transformed with the pSMART/HaeIII genomic library. E. coli cells harboring the esterase gene insert could grow and produce clear halo zones on tributyrin agar. The rEst1 ORF consisted of 1,029 bp, corresponding to 342 amino acid residues with a molecular mass of 37 kDa. The signal P program 3.0 revealed the presence of a signal peptide of 25 amino acids. Esterase activity, however, was associated with a homotrimeric form of molecular mass 95 kDa and not with the monomeric form. The deduced amino acid sequence showed only 54% sequence identity with the closest lipase from Cellvibrio japonicus strain Ueda 107. Conserved domain search and multiple sequence alignment revealed the presence of an esterase/ lipase conserved domain consisting of a GXSXG motif, HGGG motif (oxyanion hole) and HGF motif, typical of the class IV hormone sensitive lipase family. On the basis of the sequence comparison with known esterases/ lipases, REst1 represents a new esterase belonging to class IV family. The purified enzyme worked optimally at 50 degrees C and pH 8, utilized pNP esters of short chain lengths, and showed best catalytic activity with p-nitrophenyl butyrate (C?), indicating that it was an esterase. The enzyme was completely inhibited by PMSF and DEPC and showed moderate organotolerance.     

ISOLATION AND SEQUENCE ANALYSIS OF A cDNA ENCODING A NOVEL PUTATIVE ESTERASE FROM THE MARINE MICROALGA ISOCHRYSIS GALBANA (PRYMNESIOPHYCEAE,HAPTOPHYTA)1

Microalgae constitute an interesting novel study area for characterizing new esterases, and so we decided to isolate a complete cDNA encoding a new putative microalgal esterase from the haptophyte Isochrysis galbana Parke. Rapid amplifications of both the 5′ and 3′ cDNA ends (RACE) were performed with specific primers, designed using an incomplete candidate gene from the I. galbana expressed sequence tag (EST) database. The full‐length cDNA obtained was designated IgEst1. The coding sequence was 828 bp long, and the deduced amino acid sequence revealed a polypeptide of 275 amino acids with a predicted signal peptide of 23 residues in the N‐terminal region. The following 252 amino acids formed, after in silico analysis, a mature protein with a molecular mass of ～26.92 kDa and had a theoretical pI of 5.87. Alignment analyses revealed slight but significant identity and similarity with carboxylesterases, phospholipases, and lysophospholipases from various organisms including fungi, plants, and animals. The new sequence IgEst1 enclosed the catalytic triad Ser/Asp/His and the consensus pentapeptide Gly‐X‐Ser‐X‐Gly, two highly conserved patterns found in serine hydrolases. Phylogenetic analyses established a close relationship with putative esterases identified in microalgae genomes.