New thermophilic and thermostable esterase with sequence similarity to the hormone-sensitive lipase family, cloned from a metagenomic library

A gene coding for a thermostable esterase was isolated by functional screening of Escherichia coli cells that had been transformed with fosmid environmental DNA libraries constructed with metagenomes from thermal environmental samples. The gene conferring esterase activity on E. coli grown on tributyrin agar was composed of 936 bp, corresponding to 311 amino acid residues with a molecular mass of 34 kDa. The enzyme showed significant amino acid similarity (64%) to the enzyme from a hyperthermophilic archaeon, Pyrobaculum calidifontis. An amino acid sequence comparison with other esterases and lipases revealed that the enzyme should be classified as a new member of the hormone-sensitive lipase family. The recombinant esterase that was overexpressed and purified from E. coli was active above 30 degrees C up to 95 degrees C and had a high thermal stability. It displayed a high degree of activity in a pH range of 5.5 to 7.5, with an optimal pH of approximately 6.0. The best substrate for the enzyme among the p-nitrophenyl esters (C(4) to C(16)) examined was p-nitrophenyl caproate (C(6)), and no lipolytic activity was observed with esters containing an acyl chain length of longer than 10 carbon atoms, indicating that the enzyme is an esterase and not a lipase.     

A novel lipolytic enzyme located in the outer membrane of Pseudomonas aeruginosa

A lipase-negative deletion mutant of Pseudomonas aeruginosa PAO1 still showed extracellular lipolytic activity toward short-chain p-nitrophenylesters. By screening a genomic DNA library of P. aeruginosa PAO1, an esterase gene, estA, was identified, cloned, and sequenced, revealing an open reading frame of 1,941 bp. The product of estA is a 69.5-kDa protein, which is probably processed by removal of an N-terminal signal peptide to yield a 67-kDa mature protein. A molecular mass of 66 kDa was determined for (35)S-labeled EstA by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The amino acid sequence of EstA indicated that the esterase is a member of a novel GDSL family of lipolytic enzymes. The estA gene showed high similarity to an open reading frame of unknown function located in the trpE-trpG region of P. putida and to a gene encoding an outer membrane esterase of Salmonella typhimurium. Amino acid sequence alignments led us to predict that this esterase is an autotransporter protein which possesses a carboxy-terminal beta-barrel domain, allowing the secretion of the amino-terminal passenger domain harboring the catalytic activity. Expression of estA in P. aeruginosa and Escherichia coli and subsequent cell fractionation revealed that the enzyme was associated with the cellular membranes. Trypsin treatment of whole cells released a significant amount of esterase, indicating that the enzyme was located in the outer membrane with the catalytic domain exposed to the surface. To our knowledge, this esterase is unique in that it exemplifies in P. aeruginosa (i) the first enzyme identified in the outer membrane and (ii) the first example of a type IV secretion mechanism.     

Purification, cloning, and DNA sequence analysis of a chitinase from an overproducing mutant of Streptomyces peucetius defective in daunorubicin biosynthesis

Extracellular chitinases of Streptomyces peucetius and a chitinase overproducing mutant, SPVI, were purified to homogeneity by ion exchange and gel filtration chromatography. The purified enzyme has a molecular mass of 42 kDa on SDS-PAGE, and the N-terminal amino acid sequence of the protein from the wild type showed homology to catalytic domains (Domain IV) of several other Streptomyces chitinases such as S. lividans 66, S. coelicolor A3(2), S. plicatus, and S. thermoviolaceus OPC-520. Purified SPVI chitinase cross-reacted to anti-chitinase antibodies of wild-type S. peucetius chitinase. A genomic library of SPVI constructed in E. coli using lambda DASH II was probed with chiC of S. lividans 66 to screen for the chitinase gene. A 2.7 kb fragment containing the chitinase gene was subcloned from a lambda DASH II clone, and sequenced. The deduced protein had a molecular mass of 68 kDa, and showed domain organization similar to that of S. lividans 66 chiC. The N-terminal amino acid sequence of the purified S. peucetius chitinase matched with the N-terminus of the catalytic domain, indicating the proteolytic processing of 68 kDa chitinase precursor protein to 42 kDa mature chitinase containing the catalytic domain only. A putative chiR sequence of a two-component regulatory system was found upstream of the chiC sequence.     

Functional and structural studies of a novel cold-adapted esterase from an Arctic intertidal metagenomic library

A novel cold-adapted lipolytic enzyme gene, est97, was identified from a high Arctic intertidal zone sediment metagenomic library. The deduced amino acid sequence of Est97 showed low similarity with other lipolytic enzymes, the maximum being 30 % identity with a putative lipase from Vibrio caribbenthicus. Common features of lipolytic enzymes, such as the GXSXG sequence motif, were detected. The gene product was over-expressed in Escherichia coli and purified. The recombinant Est97 (rEst97) hydrolysed various ρ-nitrophenyl esters with the best substrate being ρ-nitrophenyl hexanoate (K _m and k _cat of 39 μM and 25.8 s^?1, respectively). This esterase activity of rEst97 was optimal at 35 °C and pH 7.5 and the enzyme was unstable at temperatures above 25 °C. The apparent melting temperature, as determined by differential scanning calorimetry was 39 °C, substantiating Est97 as a cold-adapted esterase. The crystal structure of rEst97 was determined by the single wavelength anomalous dispersion method to 1.6 Å resolution. The protein was found to have a typical α/β-hydrolase fold with Ser144-His226-Asp197 as the catalytic triad. A suggested, relatively short lid domain of rEst97 is composed of residues 80–114, which form an α-helix and a disordered loop. The cold adaptation features seem primarily related to a high number of methionine and glycine residues and flexible loops in the high-resolution structures.     

Isolation, characterization and molecular cloning of a lipolytic enzyme secreted from Malassezia pachydermatis

Lipophilic Malassezia species may induce catheter-associated sepsis in premature neonates and immunocompromised patients receiving parenteral lipid emulsions. To assess the participation of lipolytic enzymes in the pathogenesis of this yeast, we cloned a gene encoding the enzyme. A lipolytic enzyme in the culture supernatant of Malassezia pachydermatis was purified 210-fold to homogeneity. The enzyme showed high esterase activity toward p-nitrophenyl octanoate. The cDNA encoding the enzyme was cloned using a degenerate oligonucleotide primer constructed from the N-terminal amino acid sequence. The cDNA consisted of 1582 bp, including an open reading frame encoding 470 amino acids. The first 19 amino acids and the following 13 amino-acid sequence were predicted to be the signal peptides for secretion and prosequence, respectively. The predicted molecular mass of the 438-amino acid mature protein was 48 kDa. Analysis of the deduced amino acid sequence revealed that it contains the consensus motif (Gly-X-Ser-X-Gly), which is conserved among lipolytic enzymes. Homology investigations showed that the enzyme has similarities principally with 11 lipases produced by Candida albicans (29-34% identity) and some other yeast lipases.     

A novel esterase from Burkholderia gladioli which shows high deacetylation activity on cephalosporins is related to beta-lactamases and DD-peptidases

The gene (estB) encoding for a novel esterase (EstB) from Burkholderia gladioli (formerly Pseudomonas marginata) NCPPB 1891 was cloned in Escherichia coli. Sequence analysis showed an open reading frame encoding a polypeptide of 392 amino acid residues, with a molecular mass of about 42 kDa. Comparison of the amino acid sequence with those of other homologous enzymes indicated homologies to beta-lactamases, penicillin binding proteins and DD-peptidases. The serine residue (Ser(75)) which is located within a present class A beta-lactamase motif ([F,Y]-X-[L,I,V,M,F,Y]-X-S-[T,V]-X-K-X-X-X-X-[A,G,L]-X-X-[L,C]) was identified by site-directed mutagenesis to represent the active nucleophile. A second serine residue (Ser(149)) which is located within a G-x-S-x-G motif which is typically found in esterases and lipases was demonstrated not to play a significant role in enzyme function. The estB gene was overexpressed in E. coli using a tac promoter-based expression system. Investigation of EstB protein with respect to the ability to hydrolyse beta-lactam substrates clearly demonstrated that this protein has no beta-lactamase activity. The recombinant enzyme is active on triglycerides and on nitrophenyl esters with acyl chain lengths up to C6. The preference for short chain length substrates indicated that EstB is a typical carboxylesterase. As a special feature EstB esterase was found to have high deacetylation activity on cephalosporin derivatives.     

Cloning, purification, and enzymatic properties of dipeptidyl peptidase IV from the swine pathogen Streptococcus suis

In this study, the dipeptidyl peptidase IV (DPP IV) of the swine pathogen Streptococcus suis was cloned, overexpressed in Escherichia coli, and characterized. The coding region comprises 2,268 nucleotides containing an open reading frame that codes for a 755-amino-acid protein with a calculated molecular mass of 85 kDa. The amino acid sequence contained the sequence Gly-X-Ser-X-X-Gly, which is a consensus motif flanking the active-site serine shared by serine proteases. The recombinant DPP IV showed a high affinity for the synthetic peptide glycine-proline-p-nitroanilide and was strongly inhibited by Hg2+ and diprotin A.     

Monoacylglycerol lipase from moderately thermophilic Bacillus sp. strain H-257: molecular cloning, sequencing, and expression in Escherichia coli of the gene

Monoacylglycerol lipase [MGLP, EC 3.1.1.23] is produced intracellularly by the moderately thermophilic Bacillus sp. strain H-257. The gene encoding MGLP was cloned, sequenced, and expressed in Escherichia coli. A genomic library of Bacillus sp. strain H-257, prepared in the plasmid vector pACYC184, was screened with a 0.2-kbp DNA fragment amplified by the polymerase chain reaction (PCR) with oligonucleotide primers designed based on the amino acid sequence of a purified MGLP. The plasmid pMGLP31, identified by hybridization with the amplified DNA fragment, contained a 5.3-kbp insert from Bacillus sp. strain H-257 DNA. Sequence analysis of the MGLP gene revealed an open reading frame encoding MGLP consisting of 250 amino acids, with a calculated molecular mass of 27.4 kDa. The deduced amino acid sequence of MGLP contained the consensus pentapeptide (-Gly-Xaa-Ser-Xaa-Gly-), which is conserved among lipases, esterases, and serine proteases. The MGLP is homologous to a putative esterase/lipase from Streptomyces coelicolor (41.8% homology). When pMGLP31 was introduced into E. coli DH1, the transformants produced MGLP intracellularly as an active form to an approximately 13.8-fold greater extent than Bacillus sp. strain H-257. The purified recombinant MGLP was shown to be identical to the native enzyme in terms of chromatographic behavior, isoelectric point, and physicochemical and catalytic properties.     

A novel thermostable branching enzyme from an extremely thermophilic bacterial species, Rhodothermus obamensis

A branching enzyme (EC 2.4.1.18) gene was isolated from an extremely thermophilic bacterium, Rhodothermus obamensis. The predicted protein encodes a polypeptide of 621 amino acids with a predicted molecular mass of 72 kDa. The deduced amino acid sequence shares 42-50% similarity to known bacterial branching enzyme sequences. Similar to the Bacillus branching enzymes, the predicted protein has a shorter N-terminal amino acid extension than that of the Escherichia coli branching enzyme. The deduced amino acid sequence does not appear to contain a signal sequence, suggesting that it is an intracellular enzyme. The R. obamensis branching enzyme was successfully expressed both in E. coli and a filamentous fungus, Aspergillus oryzae. The enzyme showed optimum catalytic activity at pH 6.0-6.5 and 65 degrees C. The enzyme was stable after 30 min at 80 degrees C and retained 50% of activity at 80 degrees C after 16 h. Branching activity of the enzyme was higher toward amylose than toward amylopectin. This is the first thermostable branching enzyme isolated from an extreme thermophile.     

Cloning, sequencing, and expression of the gene encoding an intracellular beta-D-xylosidase from Streptomyces thermoviolaceus OPC-520

The intracellular beta-xylosidase was induced when Streptomyces thermoviolaceus OPC-520 was grown at 50 degrees C in a minimal medium containing xylan or xylooligosaccharides. The 82-kDa protein with beta-xylosidase activity was partially purified and its N-terminal amino acid sequence was analyzed. The gene encoding the enzyme was cloned, sequenced, and expressed in Escherichia coli. The bxlA gene consists of a 2,100-bp open reading frame encoding 770 amino acids. The deduced amino acid sequence of the bxlA gene product had significant similarity with beta-xylosidases classified into family 3 of glycosyl hydrolases. The bxlA gene was expressed in E. coli, and the recombinant protein was purified to homogeneity. The enzyme was a monomer with a molecular mass of 82 kDa. The purified enzyme showed hydrolytic activity towards only p-nitrophenyl-beta-D-xylopyranoside among the synthetic glycosides tested. Thin-layer chromatography analysis showed that the enzyme is an exo-type enzyme that hydrolyze xylooligosaccharides, but had no activity toward xylan. High activity against pNPX occurred in the pH range 6.0-7.0 and temperature range 40-50 degrees C.     

Molecular cloning and expression of the gene encoding family 19 chitinase from Streptomyces sp. J-13-3

The gene encoding chitinase from Streptomyces sp. (strain J-13-3) was cloned and its nucleotide structure was analyzed. The chitinase consisted of 298 amino acids containing a signal peptides (29 amino acids) and a mature protein (269 amino acids), and had calculated molecular mass of 31,081 Da. The calculated molecular mass (28,229 Da) of the mature protein was almost same as that of the native chitinase determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometer. Comparison of the encoded amino acid sequences with those of other chitinases showed that J-13-3 chitinase was a member of the glycosyl-hydrolase family 19 chitinases and the mature protein had a chitin binding domain (65 amino acids) containing AKWWTQ motif and a catalytic domain (204 amino acids). The J-13-3 strain had a single chitinase gene. The chitinase (298 amino acids) with C-terminal His tag was overexpressed in Escherichia coli BL21(DE3) cells. The recombinant chitinase purified from the cell extract had identical N-terminal amino acid sequence of the mature protein in spite of confirmation of the nucleotide sequence, suggesting that the signal peptide sequence is successfully cut off at the predicted site by signal peptidase from E. coli and will be a useful genetic tool in protein engineering for production of soluble recombinant protein. The optimum temperature and pH ranges of the purified chitinase were at 35-40 degrees C and 5.5-6.0, respectively. The purified chitinase hydrolyzed colloidal chitin and trimer to hexamer of N-acetylglucosamine and also inhibited the hyphal extension of Tricoderma reesei.     

Characterization and heterologous expression of a novel lysophospholipase gene from Antrodia cinnamomea

Aims: A novel lysophospholipase (LysoPL) from the basidiomycetous fungi Antrodia cinnamomea named ACLysoPL was cloned, heteroexpressed in Escherichia coli and characterized. Methods and Results: The gene encoding ACLysoPL was obtained from expressed sequence tags from A. cinnamomea. The full length of this gene has a 945 ‐bp open reading frame encoding 314 amino acids with a molecular weight of 35·5 kDa. ACLysoPL contains a lipase consensus sequence (GXSXG) motif and a Ser–His–Asp catalytic triad. A putative peroxisomal targeting signal type 1 was found in the C‐terminal. Heterologous expression of ACLysoPL in E. coli showed that the enzyme preferentially hydrolyses long‐chain acyl esterases at pH 7 and 30°C. ACLysoPL is a psychrophilic enzyme about 40% of whose maximum activity remained at 4°C. The LysoPL activities with lysophospholipids as substrate were analysed by gas chromatography/mass spectrometry. Conclusion: We have identified and characterized a gene named ACLysoPL encoding a protein performing LysoPL and esterase activities. Significance and Impact of the Study: This is the first LysoPL of A. cinnamomea identified and characterized at the molecular level.     

A novel flavin-containing monooxygenase from Methylophaga sp strain SK1 and its indigo synthesis in Escherichia coli

We cloned a gene from Methylophaga sp. strain SK1. This gene was responsible for producing, the blue pigment, indigo. The complete open reading frame was 1371 bp long, which encodes a protein of 456 amino acids. The molecular mass of the encoded protein was 105 kDa, consisting of homodimer of 54 kDa with an isoelectric point of 5.14. The deduced amino acid sequence from the gene showed approximately 30% identities with flavin-containing monooxygenases (FMOs) of human (FMO1-FMO5), Arabidopsis, and yeast. It contained three characteristic sequence motifs of FMOs: FAD binding domain, FMO-identifying sequence motif, and NADPH binding domain. In addition, the biochemical properties such as substrate specificities and absorption spectra were similar to the eukaryotic FMO families. Thus, we assigned the enzyme to be a bacterial FMO. The recombinant Escherichia coli expressing the bacterial FMO produced up to 160 mg of indigo per liter in the tryptophan medium after 12h cultivation. This suggests that the recombinant strain has a potential to be applied in microbial indigo production.     

A novel streptomycete lipase: cloning,sequencing and high-level expression of the Streptomyces rimosus GDS(L)-lipase gene

An extracellular lipase from Streptomyces rimosus R6-554W has been recently purified and biochemically characterized. In this report the cloning, sequencing, and high-level expression of its gene is described. The cloned DNA contained an ORF of 804 bp encoding a 268-amino-acid polypeptide with 34 amino acid residues at the amino terminus of the sequence that were not found in the mature protein. The theoretical molecular mass (24.172 kDa) deduced from the amino acid sequence of the mature enzyme was experimentally confirmed. This lipase showed no overall amino acid sequence similarity to other lipases in the databases. However, two hypothetical proteins, i. e. putative hydrolases, derived from the genome sequencing data of Streptomyces coelicolor A3(2), showed 66% and 33% identity. In addition, a significant similarity to esterases from Streptomyces diastatochromogenes and Aspergillus terreus was found. Sequence analysis revealed that our novel S. rimosus lipase containing a GDS(L)-like consensus motif belongs to family II of lipolytic enzymes, previously unrecognized in Streptomyces. When the lipase gene was expressed in a S. rimosus lipase-deficient strain harboring the lipase gene on a high-copy-number vector, lipase activity was 22-fold higher than in the original strain.     

Cloning and sequence analysis of cDNA encoding Rhizopus niveus lipase.

Complementary DNA encoding Rhizopus niveus lipase (RNL) was isolated from the R. niveus IF04759 cDNA library using a synthetic oligonucleotide corresponding to the amino acid sequence of the enzyme. A clone, which had an insert of 1.0 kilobase pairs, was found to contain the coding region of the enzyme. The lipase gene was expressed in Escherichia coli as a lacZ fusion protein. The mature RNL consisted of 297 amino acid residues with a molecular mass of 32 kDa. The RNL sequence showed significant overall homology to Rhizomucor miehei lipase and the putative active site residues were strictly conserved.     

Identification and characterization of a novel salt‐tolerant esterase from a Tibetan glacier metagenomic library

A salt‐tolerant esterase, designated H9Est, was identified from a metagenomic library of the Karuola glacier. H9Est gene comprised 1071 bp and encoded a polypeptide of 357 amino acids with a molecular mass of 40 kDa. Sequence analysis revealed that H9Est belonged to the family IV of bacterial lypolitic enzyme. H9Est was overexpressed in Escherichia coli and the purified enzyme showed hydrolytic activity towards p‐nitrophenyl esters with carbon chain from 2 to 8. The optimal esterase activity was at 40°C and pH 8.0 and the enzyme retained its activity towards some miscible organic solvents such as polyethylene glycol. A three‐dimensional model of H9Est revealed that S200, D294, and H324 formed the H9Est catalytic triad. Circular Dichroism spectra and molecular dynamic simulation indicated that the esterase had a wide denaturation temperature range and flexible loops that would be beneficial for H9Est performance at low temperatures while retaining heat‐resistant features. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:890–899, 2015     

Intra- and extracellular beta-galactosidases from Bifidobacterium bifidum and B. infantis: molecular cloning, heterologous expression, and comparative characterization

Three beta-galactosidase genes from Bifidobacterium bifidum DSM20215 and one beta-galactosidase gene from Bifidobacterium infantis DSM20088 were isolated and characterized. The three B. bifidum beta-galactosidases exhibited a low degree of amino acid sequence similarity to each other and to previously published beta-galactosidases classified as family 2 glycosyl hydrolases. Likewise, the B. infantis beta-galactosidase was distantly related to enzymes classified as family 42 glycosyl hydrolases. One of the enzymes from B. bifidum, termed BIF3, is most probably an extracellular enzyme, since it contained a signal sequence which was cleaved off during heterologous expression of the enzyme in Escherichia coli. Other exceptional features of the BIF3 beta-galactosidase were (i) the monomeric structure of the active enzyme, comprising 1,752 amino acid residues (188 kDa) and (ii) the molecular organization into an N-terminal beta-galactosidase domain and a C-terminal galactose binding domain. The other two B. bifidum beta-galactosidases and the enzyme from B. infantis were multimeric, intracellular enzymes with molecular masses similar to typical family 2 and family 42 glycosyl hydrolases, respectively. Despite the differences in size, molecular composition, and amino acid sequence, all four beta-galactosidases were highly specific for hydrolysis of beta-D-galactosidic linkages, and all four enzymes were able to transgalactosylate with lactose as a substrate.     

Polysaccharide lyase: molecular cloning, sequencing, and overexpression of the xanthan lyase gene of Bacillus sp. strain GL1

When grown on xanthan as a carbon source, the bacterium Bacillus sp. strain GL1 produces extracellular xanthan lyase (75 kDa), catalyzing the first step of xanthan depolymerization (H. Nankai, W. Hashimoto, H. Miki, S. Kawai, and K. Murata, Appl. Environ. Microbiol. 65:2520-2526, 1999). A gene for the lyase was cloned, and its nucleotide sequence was determined. The gene contained an open reading frame consisting of 2,793 bp coding for a polypeptide with a molecular weight of 99,308. The polypeptide had a signal peptide (2 kDa) consisting of 25 amino acid residues preceding the N-terminal amino acid sequence of the enzyme and exhibited significant homology with hyaluronidase of Streptomyces griseus (identity score, 37.7%). Escherichia coli transformed with the gene without the signal peptide sequence showed a xanthan lyase activity and produced intracellularly a large amount of the enzyme (400 mg/liter of culture) with a molecular mass of 97 kDa. During storage at 4 degrees C, the purified enzyme (97 kDa) from E. coli was converted to a low-molecular-mass (75-kDa) enzyme with properties closely similar to those of the enzyme (75 kDa) from Bacillus sp. strain GL1, specifically in optimum pH and temperature for activity, substrate specificity, and mode of action. Logarithmically growing cells of Bacillus sp. strain GL1 on the medium with xanthan were also found to secrete not only xanthan lyase (75 kDa) but also a 97-kDa protein with the same N-terminal amino acid sequence as that of xanthan lyase (75 kDa). These results suggest that, in Bacillus sp. strain GL1, xanthan lyase is first synthesized as a preproform (99 kDa), secreted as a precursor (97 kDa) by a signal peptide-dependent mechanism, and then processed into a mature form (75 kDa) through excision of a C-terminal protein fragment with a molecular mass of 22 kDa.     

Identification and characterization of a novel cold-adapted esterase from a metagenomic library of mountain soil

A novel lipolytic enzyme was isolated from a metagenomic library after demonstration of lipolytic activity on an LB agar plate containing 1% (w/v) tributyrin. A novel esterase gene (estIM1), encoding a lipolytic enzyme (EstIM1), was cloned using a shotgun method from a pFosEstIM1 clone of the metagenomic library, and the enzyme was characterized. The estIM1 gene had an open reading frame (ORF) of 936 base pairs and encoded a protein of 311 amino acids with a molecular mass 34 kDa and a pI value of 4.32. The deduced amino acid sequence was 62% identical to that of an esterase from an uncultured bacterium (ABQ11271). The amino acid sequence indicated that EstIM1 was a member of the family IV of lipolytic enzymes, all of which contain a GDSAG motif shared with similar enzymes of lactic acid microorganisms. EstIM1 was active over a temperature range of 1–50°C, at alkaline pH. The activation energy for hydrolysis of p-nitrophenyl propionate was 1.04 kcal/mol, within a temperature range of 1–40°C. The activity of EstIM1 was about 60% of maximal even at 1°C, suggesting that EstIM1 is efficiently cold-adapted. Further characterization of this cold-adapted enzyme indicated that the esterase may be very valuable in industrial applications.     

Genetic and biochemical characterization of a new extracellular lipase from Streptomyces cinnamomeus.

Streptomyces cinnamomeus Tü89 secretes a 30-kDa esterase and a 50-kDa lipase. The lipase-encoding gene, lipA, was cloned from genomic DNA into Streptomyces lividans TK23 with plasmid vector pIJ702. Two lipase-positive clones were identified; each recombinant plasmid had a 5.2-kb MboI insert that contained the complete lipA gene. The two plasmids differed in the orientation of the insert and the degree of lipolytic activity produced. The lipA gene was sequenced; lipA encodes a proprotein of 275 amino acids (29,213 Da) with a pI of 5.35. The LipA signal peptide is 30 amino acids long, and the mature lipase sequence is 245 amino acids long (26.2 kDa) and contains six cysteine residues. The conserved catalytic serine residue of LipA is in position 125. Sequence similarity of the mature lipases (29% identity, 60% similarity) was observed mainly in the N-terminal 104 amino acids with the group II Pseudomonas lipases; no similarity to the two Streptomyces lipase sequences was found. lipA was also expressed in Escherichia coli under the control of lacZ promoter. In the presence of the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG), growth of the E. coli clone was severely affected, and the cells lysed in liquid medium. Lipase activity in the E. coli clone was found mainly in the pellet fraction. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, three additional protein bands of 50, 29, and 27 kDa were visible. The 27-kDa protein showed lipolytic activity and represents the mature lipase; the 29- and 50-kDa forms showed no activity and very probably represent the unprocessed form and a dimeric misfolded form, respectively. For higher expression of lipA in S. lividans, the gene was cloned next to the strong aphII promoter. In contrast to the lipA-expressing E. coli clone, S. cinnamomeus and the corresponding S. lividans clone secreted only an active protein of 50 kDa. The lipase showed highest activity with C6 and C18 triglycerides; no activity was observed with phospholipids, Tween 20, or p-nitrophenylesters. Upstream of lipA and in the same orientation, an open reading frame, orfA, is found whose deduced protein sequence (519 amino acids) shows similarity to various membrane-localized transporters. Downstream of lipA and in the opposite orientation, an open reading frame, orfB (encoding a 199-amino-acid protein) is found, which shows no conspicuous sequence similarity to known proteins, other than an NAD and flavin adenine dinucleotide binding-site sequence.