Isolation and analysis of a gene from the marine microalga Isochrysis galbana that encodes a lipase-like protein

Microalgae constitute a novel study area for characterising new lipolytic enzymes of biotechnological interest. A new gene from the microalga Isochrysis galbana has been isolated and a preliminary characterisation performed. The full-length cDNA contains 2,060 base pairs with an open reading frame of 1,374 nucleotides encoding a polypeptide of 457 amino acids with a predicted molecular mass of 49 kDa, and a theoretical pI of 5.65. The deduced protein includes highly conserved motifs found in α/β fold hydrolases, and shares some similarities with putative or known lipases. Sequence comparison indicated that the catalytic triad corresponds to residues Ser¹⁸⁸, Asp³⁰⁶ and His³⁹¹, with the nucleophilic residue Ser¹⁸⁸ positioned within the consensus G-X-S¹⁸⁸-X-G pentapeptide. Phylogenetic analyses established close relationships with fungal lipases and microalgal sequences.     

Cloning and identification of a novel C18-Δ9 polyunsaturated fatty acid specific elongase gene from DHA-producing <Emphasis Type="Italic">Isochrysis galbana</Emphasis> H29

Isochrysis galbana, a marine prymnesiophyte microalga, is able to produce a high level of long chain polyunsaturated fatty acids such as docosahexaenoic acid (DHA, C22:6n-3). In this article, a novel gene (IgASE2) that encoded a C18-Δ9 polyunsaturase fatty acids specific (C18-Δ9-PUFAs-specific) elongase was isolated and characterized from DHA-rich microalga, I. galbana H29. A full-length cDNA of 1653 bp was cloned by rapidamplification of cDNA ends (RACE) PCR techniques. The IgASE2 contained a 786 bp ORF encoding a protein of 261 amino acids that shared 87% identity with the reported Δ9-elongase IgASE1, a 44 bp 5′ untranslated region and an 823 bp 3′ untranslated region. The function of IgASE2 was demonstrated by its heterologous expression in Saccharomyces cerevisiae. In S. cerevisiae, IgASE2 elongated linoleic acid (LA, C18:2n-6), α-linolenic (ALA, C18:3n-3) to eicosadienoic acid (EDA, C20:2n-6) and eicosatrienoic acid (ETrA, C20:3n-3). The conversion ratios of LA to EDA and ALA to ETrA were 60.47 and 58.36%, respectively. However, IgASE2 could not catalyze the elongation reactions of oleic acid (OA, C18:1n-9) and other fatty acids. These results confirmed that IgASE2 had C18-Δ9-PUFAs-specific elongase activity.     

Development of Escherichia coli MG1655 strains to produce long chain fatty acids by engineering fatty acid synthesis (FAS) metabolism

The goal of this research was to develop recombinant Escherichia coli to improve fatty acid synthesis (FAS). Genes encoding acetyl-CoA carboxylase (accA, accB, accC), malonyl-CoA-[acyl-carrier-protein] transacylase (fabD), and acyl-acyl carrier protein thioesterase (EC 3.1.2.14 gene), which are all enzymes that catalyze key steps in the synthesis of fatty acids, were cloned and over-expressed in E. coli MG1655. The acetyl-CoA carboxylase (ACC) enzyme catalyzes the addition of CO₂ to acetyl-CoA to generate malonyl-CoA. The enzyme encoded by the fabD gene converts malonyl-CoA to malonyl-[acp], and the EC 3.1.2.14 gene converts fatty acyl-ACP chains to long chain fatty acids. All the genes except for the EC 3.1.2.14 gene were homologous to E. coli genes and were used to improve the enzymatic activities to over-express components of the FAS pathway through metabolic engineering. All recombinant E. coli MG1655 strains containing various gene combinations were developed using the pTrc99A expression vector. To observe changes in metabolism, the in vitro metabolites and fatty acids produced by the recombinants were analyzed. The fatty acids (C16) from recombinant strains were produced 1.23-2.41 times higher than that from the wild type.     

Nucleotide and Deduced Amino Acid Sequences of a New Subtilisin from an Alkaliphilic <Emphasis Type="Italic">Bacillus</Emphasis> Isolate

The gene for a new subtilisin from the alkaliphilic Bacillus sp. KSM-LD1 was cloned and sequenced. The open reading frame of the gene encoded a 97 amino-acid prepro-peptide plus a 307 amino-acid mature enzyme that contained a possible catalytic triad of residues, Asp³², His⁶⁶, and Ser²²⁴. The deduced amino acid sequence of the mature enzyme (LD1) showed approximately 65% identity to those of subtilisins SprC and SprD from alkaliphilic Bacillus sp. LG12. The amino acid sequence identities of LD1 to those of previously reported true subtilisins and high-alkaline proteases were below 60%. LD1 was characteristically stable during incubation with surfactants and chemical oxidants. Interestingly, an oxidizable Met residue is located next to the catalytic Ser²²⁴ of the enzyme as in the cases of the oxidation-susceptible subtilisins reported to date.Received: 19 November 2002 / Accepted: 19 December 2002     

Under low irradiation,the light regime modifies growth and metabolite production in various species of microalgae

The effects of continuous light exposure (24L:0D) and a 12 h:12 h light/dark regime (12L:12D) were compared on the growth and carotenoid, protein, sugar, lipid, and fatty acid contents in Chlorella vulgaris, Nannochloropsis sp., Isochrysis galbana, and Dunaliella salina cultured in a batchwise facility. These microalgae were grown axenically under a low photon flux density (PFD) of 27 μmol photons m^?2 s^?1. C. vulgaris, Nannochloropsis sp., and I. galbana exhibited the highest cell densities when cultured under 24L:0D, whereas D. salina grew better under the alternating light/dark regime. I. galbana accumulated high levels of proteins, sugars, and lipids and exhibited the highest carotenoid content under 24L:0D. Protein production was enhanced in C. vulgaris under 24L:0D. The highest total lipid content was recorded for D. salina, reaching 74.6 % of total proteins, sugars, and lipids in cells at the stationary phase when grown under 12L:12D. The light/dark regime at low PFD was sufficient to stimulate the accumulation of monounsaturated and polyunsaturated fatty acids in all four algae. Their levels, like those of saturated fatty acids, did not differ significantly under the two light regimes. D. salina was an important source of tetradecenoic acid 14:1(n-5). Nannochloropsis sp. produced a large amount of the essential eicosapentaenoic acid, which reached 20 % of total fatty acids under 12L:12D, while I. galbana exhibited the highest level of docosahexaenoic acid, which reached 21 % under both light regimes. This study demonstrated the feasibility of culturing microalgae under low PFD in order to produce large quantities of valuable metabolites, especially various lipids with neutraceutical value.     

Purification, Characterization, Gene Cloning, Sequencing, and Overexpression of Aminopeptidase N from Streptococcus thermophilus A

The general aminopeptidase PepN from Streptococcus thermophilus A was purified to protein homogeneity by hydroxyapatite, anion-exchange, and gel filtration chromatographies. The PepN enzyme was estimated to be a monomer of 95 kDa, with maximal activity on N-Lys–7-amino-4-methylcoumarin at pH 7 and 37°C. It was strongly inhibited by metal chelating agents, suggesting that it is a metallopeptidase. The activity was greatly restored by the bivalent cations Co²⁺, Zn²⁺, and Mn²⁺. Except for proline, glycine, and acidic amino acid residues, PepN has a broad specificity on the N-terminal amino acid of small peptides, but no significant endopeptidase activity has been detected. The N-terminal and short internal amino acid sequences of purified PepN were determined. By using synthetic primers and a battery of PCR techniques, the pepN gene was amplified, subcloned, and further sequenced, revealing an open reading frame of 2,541 nucleotides encoding a protein of 847 amino acids with a molecular weight of 96,252. Amino acid sequence analysis of the pepN gene translation product shows high homology with other PepN enzymes from lactic acid bacteria and exhibits the signature sequence of the zinc metallopeptidase family. The pepN gene was cloned in a T7 promoter-based expression plasmid and the 452-fold overproduced PepN enzyme was purified to homogeneity from the periplasmic extract of the host Escherichia coli strain. The overproduced enzyme showed the same catalytic characteristics as the wild-type enzyme.     

Malonyl-acyl carrier protein decarboxylase activity promotes fatty acid and cell envelope biosynthesis in Proteobacteria

Bacterial fatty acid synthesis in Escherichia coli is initiated by the condensation of an acetyl-CoA with a malonyl-acyl carrier protein (ACP) by the β-ketoacyl-ACP synthase III enzyme, FabH. E. coli ΔfabH knockout strains are viable because of the yiiD gene that allows FabH-independent fatty acid synthesis initiation. However, the molecular function of the yiiD gene product is not known. Here, we show the yiiD gene product is a malonyl-ACP decarboxylase (MadA). MadA has two independently folded domains: an amino-terminal N-acetyl transferase (GNAT) domain (MadA^N) and a carboxy-terminal hot dog dimerization domain (MadA^C) that encodes the malonyl-ACP decarboxylase function. Members of the proteobacterial Mad protein family are either two domain MadA (GNAT-hot dog) or standalone MadB (hot dog) decarboxylases. Using structure-guided, site-directed mutagenesis of MadB from Shewanella oneidensis, we identified Asn45 on a conserved catalytic loop as critical for decarboxylase activity. We also found that MadA, MadA^C, or MadB expression all restored normal cell size and growth rates to an E. coli ΔfabH strain, whereas the expression of MadA^N did not. Finally, we verified that GlmU, a bifunctional glucosamine-1-phosphate N-acetyl transferase/N-acetyl-glucosamine-1-phosphate uridylyltransferase that synthesizes the key intermediate UDP-GlcNAc, is an ACP binding protein. Acetyl-ACP is the preferred glucosamine-1-phosphate N-acetyl transferase/N-acetyl-glucosamine-1-phosphate uridylyltransferase substrate, in addition to being the substrate for the elongation-condensing enzymes FabB and FabF. Thus, we conclude that the Mad family of malonyl-ACP decarboxylases supplies acetyl-ACP to support the initiation of fatty acid, lipopolysaccharide, peptidoglycan, and enterobacterial common antigen biosynthesis in Proteobacteria.     

Screening and characterization of Isochrysis strains and optimization of culture conditions for docosahexaenoic acid production

Isochrysis is a genus of marine unicellular microalgae that produces docosahexaenoic acid (DHA, C22:6), a very long chain polyunsaturated fatty acid (PUFA) of significant health and nutritional value. Mass cultivation of Isochrysis for DHA production for human consumption has not been established due to disappointing low DHA productivity obtained from commonly used Isochrysis strains. In this study, 19 natural Isochrysis strains were screened for DHA yields and the results showed that the cellular DHA content ranged from 6.8 to 17.0 % of total fatty acids with the highest DHA content occurring in the exponential growth phase. Isochrysis galbana #153180 exhibited the greatest DHA production potential and was selected for further investigation. The effects of different light intensities, forms, and concentrations of nitrogen, phosphorus, and salinity on growth and DHA production of I. galbana #153180 were studied in a bubble column photobioreactor (PBR). Under favorable culture conditions, I. galbana #153180 contained DHA up to 17.5 % of total fatty acids or 1.7 % of cell dry weight. I. galbana #153180 was further tested in outdoor flat-plate PBRs varying in light path length, starting cell density (SCD), and culture mode (batch versus semicontinuous). When optimized, record high biomass and DHA productivity of I. galbana #153180 of 0.72 g L^?1?day^?1 and 13.6 mg?L^?1?day^?1, or 26.4 g?m^?2?day^?1 and 547.7 mg?m^?2?day^?1, respectively, were obtained, suggesting that I. galbana #153180 may be a desirable strain for commercial production of DHA.     

An rne-1 pnp-7 Double Mutation Suppresses the Temperature-Sensitive Defect of lacZ Gene Expression in a divE Mutant

A divE mutant, which has a temperature-sensitive mutation in the tRNA₁^Ser gene, exhibits differential loss of the synthesis of certain proteins, such as β-galactosidase and succinate dehydrogenase, at nonpermissive temperatures. In Escherichia coli, the UCA codon is recognized only by tRNA₁^Ser. Several genes containing UCA codons are normally expressed after a temperature shift to 42°C in the divE mutant. Therefore, it is unlikely that the defect in protein synthesis at 42°C is simply caused by a defect in the decoding function of the mutant tRNA₁^Ser. In this study, we sought to determine the cause of the defect in lacZ gene expression in the divE mutant. It has also been shown that the defect in lacZ gene expression is accompanied by a decrease in the amount of lacZ mRNA. To examine whether inactivation of mRNA degradation pathways restores the defect in lacZ gene expression, we constructed divE mutants containing rne-1, rnb-500, and pnp-7 mutations in various combinations. We found that the defect was almost completely restored by introducing an rne-1 pnp-7 double mutation into the divE mutant. Northern hybridization analysis showed that the rne-1 mutation stabilized lacZ mRNA, whereas the pnp-7 mutation stabilized mutant tRNA₁^Ser, at 44°C. We present a mechanism that may explain these results.     

Heterologous expression and characterization of a malathion-hydrolyzing carboxylesterase from a thermophilic bacterium, Alicyclobacillus tengchongensis

A carboxylesterase gene from thermophilic bacterium, Alicyclobacillus tengchongensis, was cloned and expressed in Escherichia coli BL21 (DE3). The gene coded for a 513 amino acid protein with a calculated molecular mass of 57.82 kDa. The deduced amino acid sequence had structural features highly conserved among serine hydrolases, including Ser204, Glu325, and His415 as a catalytic triad, as well as type-B carboxylesterase serine active site (FGGDPENITIGGQSAG) and type-B carboxylesterase signature 2 (EDCLYLNIWTP). The purified enzyme exhibited optimum activity with β-naphthyl acetate at 60 °C and pH 7 as well as stability at 25 °C and pH 7. One unit of the enzyme hydrolyzed 5 mg malathion l^?1 by 50 % within 25 min and 89 % within 100 min. The enzyme strongly degraded malathion and has a potential use for the detoxification of malathion residues.     

Isolation and characterization of a thermostable esterase from a metagenomic library

A novel esterase gene was isolated by functional screening of a metagenomic library prepared from an activated sludge sample. The gene (est-XG2) consists of 1,506 bp with GC content of 74.8 %, and encodes a protein of 501 amino acids with a molecular mass of 53 kDa. Sequence alignment revealed that Est-XG2 shows a maximum amino acid identity (47 %) with the carboxylesterase from Thermaerobacter marianensis DSM 12885 (YP_004101478). The catalytic triad of Est-XG2 was predicted to be Ser₁₉₂-Glu₃₁₃-His₄₁₂ with Ser₁₉₂ in a conserved pentapeptide (GXSXG), and further confirmed by site-directed mutagenesis. Phylogenetic analysis suggested Est-XG2 belongs to the bacterial lipase/esterase family VII. The recombinant Est-XG2, expressed and purified from Escherichia coli, preferred to hydrolyze short and medium length p-nitrophenyl esters with the best substrate being p-nitrophenyl acetate (K _m and k _cat of 0.33 mM and 36.21 s^?1, respectively). The purified enzyme also had the ability to cleave sterically hindered esters of tertiary alcohols. Biochemical characterization of Est-XG2 revealed that it is a thermophilic esterase that exhibits optimum activity at pH 8.5 and 70 °C. Est-XG2 had moderate tolerance to organic solvents and surfactants. The unique properties of Est-XG2, high thermostability and stability in the presence of organic solvents, may render it a potential candidate for industrial applications.     

Purification and characterization of a cis-epoxysuccinic acid hydrolase from Nocardia tartaricans CAS-52, and expression in Escherichia coli

A highly enantioselective cis-epoxysuccinic acid hydrolase from Nocardia tartaricans was purified to electrophoretic homogeneity. The enzyme was purified 184-fold with a yield of 18.8 %. The purified cis-epoxysuccinic acid hydrolase had a monomeric molecular weight of 28 kDa, and its optimum conditions were 37 °C and pH 7–9. With sodium cis-epoxysuccinate as the substrate, Michaelis–Menten enzyme kinetics analysis gave a Km value of 35.71 mM and a Vmax of 2.65 mM min^?1. The enzyme was activated by Ni²⁺ and Al³⁺, while strongly inhibited by Fe³⁺, Fe²⁺, Cu²⁺, and Ag⁺. The cis-epoxysuccinic acid hydrolase gene was cloned, and its open reading frame sequence predicted a protein composed of 253 amino acids. A pET11a expression plasmid carrying the gene under the control of the T7 promoter was introduced into Escherichia coli, and the cis-epoxysuccinic acid hydrolase gene was successfully expressed in the recombinant strains.     

Cloning and Functional Expression of the Mucrosobin Protein, a β-Fibrinogenase of Trimeresurus mucrosquamatus (Taiwan Habu)

A venom-specific cDNA encoding for a thrombin-like enzyme designated as mucrosobin has been cloned and sequenced from the cDNA library of the venomous gland of Trimeresurus mucrosquamatus. The full-length cDNA of mucrosobin was assembled by oligonucleotide screening and 5′-rapid amplification of cDNA ends. The amino acid sequence deduced from the cDNA consists of 257 amino acid residues with a putative signal peptide of 24 residues. It is highly homologous to the other thrombin-like enzymes (batroxobin, mucofirase, and calobin), suggesting that it is a serine proteinase with a conserved catalytic triad of His⁴¹, Asp⁸⁴ and Ser¹⁷⁹ in the deduced form of mucrosobin protein. Northern blot analysis revealed that the mucrosobin gene encodes an mRNA of 1.5 kb and suggested a tissue-specific expression in the venomous gland. In an effort to study the biological property of mocrosobin, we have expressed the 28-kDa protein as inclusion bodies in Escherichia coli. For analyzing enzymatic activity, the inclusion bodies were solubilized and the recombinant protein was refolded with a two-step dialysis protocol. The refolded recombinant protein exhibited a specific β-fibrinogenolytic activity. This study offers a possibility of using genetic engineering to acquirie a functional snake venom protein with therapeutic potential.     

Phosphorylation of Maize Eukaryotic Translation Initiation Factor 5A (eIF5A) by Casein Kinase 2: IDENTIFICATION OF PHOSPHORYLATED RESIDUE AND INFLUENCE ON INTRACELLULAR LOCALIZATION OF eIF5A*

Maize eukaryotic translation initiation factor 5A (ZmeIF5A) co-purifies with the catalytic α subunit of protein kinase CK2 and is phosphorylated by this enzyme. Phosphorylated ZmeIF5A was also identified after separation of maize leaf proteins by two-dimensional electrophoresis. Multiple sequence alignment of eIF5A proteins showed that in monocots, in contrast to other eukaryotes, there are two serine/threonine residues that could potentially be phosphorylated by CK2. To identify the phosphorylation site(s) of ZmeIF5A, the serine residues potentially phosphorylated by CK2 were mutated. ZmeIF5A and its mutated variants S2A and S4A were expressed in Escherichia coli and purified. Of these recombinant proteins, only ZmeIF5A-S2A was not phosphorylated by maize CK2. Also, Arabidopsis thaliana and Saccharomyces cerevisiae eIF5A-S2A mutants were not phosphorylated despite effective phosphorylation of wild-type variants. A newly developed method exploiting the specificity of thrombin cleavage was used to confirm that Ser² in ZmeIF5A is indeed phosphorylated. To find a role of the Ser² phosphorylation, ZmeIF5A and its variants mutated at Ser² (S2A and S2D) were transiently expressed in maize protoplasts. The expressed fluorescence labeled proteins were visualized by confocal microscopy. Although wild-type ZmeIF5A and its S2A variant were distributed evenly between the nucleus and cytoplasm, the variant with Ser² replaced by aspartic acid, which mimics a phosphorylated serine, was sequestered in the nucleus. These results suggests that phosphorylation of Ser² plays a role in regulation of nucleocytoplasmic shuttling of eIF5A in plant cells.     

Structure and function characterization of the phytoene desaturase related to the lutein biosynthesis in Chlorella protothecoides CS-41

Phytoene desaturase is the key enzyme involved in the biosynthesis pathway of lutein. The unicellular microalga, Chlorella protothecoides CS-41, had been selected for the heterotrophic production of high concentrations of lutein. In this study, a cDNA copy of the pds gene from C. protothecoides was obtained using the rapid amplification of cDNA ends (RACE) technique. Phylogenetic analysis of the deduced amino acid sequence revealed that the phytoene desaturases derived from the algal family. Expression of the pds gene in Escherichia coli produced a single protein of 61 kDa. The PDS activity of the expressed protein was confirmed by the production of ζ-carotene as the result from the action of the enzyme’s desaturation activity, which was identified by high-performance liquid chromatography and heterologous complementation analysis. Using random and site-directed mutagenesis, a single amino acid mutation (N144D) was identified and confirmed. This mutant encodes an inactive enzyme, which implies that amino acid 144 is crutial to the activity of the PDS enzyme. Therefore, by gene cloning and expression in prokaryotic cells, the gene for ζ-carotene production or as part of the biosynthetic pathway of lutein had been characterized from Chlorella protothecoides CS-41.     

Cloning,expression and some properties of α-carbonic anhydrase from Helicobacter pylori

The α-carbonic anhydrase gene from Helicobacter pylori strain 26695 has been cloned and sequenced. The full-length protein appears to be toxic to Escherichia coli, so we prepared a modified form of the gene lacking a part that presumably encodes a cleavable signal peptide. This truncated gene could be expressed in E. coli yielding an active enzyme comprising 229 amino acid residues. The amino acid sequence shows 36% identity with that of the enzyme from Neisseria gonorrhoeae and 28% with that of human carbonic anhydrase II. The H. pylori enzyme was purified by sulfonamide affinity chromatography and its circular dichroism spectrum and denaturation profile in guanidine hydrochloride have been measured. Kinetic parameters for CO₂ hydration catalyzed by the H. pylori enzyme at pH 8.9 and 25°C are k_cat=2.4×10⁵ s⁻¹, K_M=17 mM and k_cat/K_M=1.4×10⁷ M⁻¹ s⁻¹. The pH dependence of k_cat/K_M fits with a simple titration curve with pK_a=7.5. Thiocyanate yields an uncompetitive inhibition pattern at pH 9 indicating that the maximal rate of CO₂ hydration is limited by proton transfer between a zinc-bound water molecule and the reaction medium in analogy to other forms of the enzyme. The 4-nitrophenyl acetate hydrolase activity of the H. pylori enzyme is quite low with an apparent catalytic second-order rate constant, k_enz, of 24 M⁻¹ s⁻¹ at pH 8.8 and 25°C. However, with 2-nitrophenyl acetate as substrate a k_enz value of 665 M⁻¹ s⁻¹ was obtained under similar conditions.     

Characterization of a novel cold-active esterase isolated from swamp sediment metagenome

A functional screen of a metagenomic library from “Upo” swamp sediment in Korea identified a gene EstL28, the product of which displayed lipolytic properties on a tributyrin-supplemented medium. The EstL28 sequence encodes a 290 amino acid protein (designated as EstL28), with a predicted molecular weight of 31.3 kDa. The encoded EstL28 protein exhibited the highest sequence similarity (45 %) to a hydrolase found in Streptococcus sanguinis. Phylogenetic analysis indicated that EstL28 belongs to a currently uncharacterized family of esterases. Within the conserved α/β-hydrolase 6 domain, the EstL28 retains the catalytic triad Ser₁₀₃–Asp₂₄₈–His₂₆₈ that is typical of esterases. The Ser₁₀₃ residue in the catalytic triad is located in the consensus pentapeptide motif GXSXG. The purified EstL28 enzyme worked optimally at 35 °C and pH 8.5 and remained stable at temperatures lower than 20 °C. The catalytic activity of EstL28 was maximal with p-nitrophenyl butyrate, indicating that it was an esterase. This enzyme also exhibited stable activity in the presence of methanol, ethanol, isopropanol, and dimethyl sulfoxide. Therefore, the level of stability in organic solvents and cold temperature suggests that EstL28 has potential for many biotechnological applications.     

Effects of green LED light and three stresses on biomass and lipid accumulation with two-phase culture of microalgae

The effects of wavelengths of light-emitting diode (LED), nitrate concentration, and salt concentration were evaluated for the two-phase culture of the microalgal species Phaeodactylum tricornutum, Dunaliella tertiolecta, and Isochrysis galbana on cell growth and lipid production. Blue LEDs produced the highest biomass of P. tricornutum at a nitrate concentration of 8 mg/L, reaching 0.97 g dcw/L with a specific growth rate (μ) of 0.047 h⁻¹, followed by I. galbana with 0.79 g dcw/L and μ = 0.040 h⁻¹ and D. tertiolecta with 0.55 g dcw/L and μ = 0.028 h⁻¹. Of the three microalgae, P. tricornutum had the highest specific growth rate of μ_max = 0.070 h⁻¹ and lowest saturation constant of K_s = 4.18 mg/L, resulting in fast cell growth. The highest lipid production was obtained under green LED wavelength stress on day 14, reaching 60.6% (w/w) of the dry cell weight among the three microalgae. The main fatty acids produced by the three microalgae were myristic acid (C14:0), palmitic acid (C16:0), oleic acid (C18:1), and arachidic acid (C20:0), which comprised 72.68%–84.16% (w/w) of the total fatty acids content under three stresses.     

Characterization of an acid inducible lipase Rv3203 from Mycobacterium tuberculosis H37Rv

The Rv3203 (LipV) of Mycobacterium tuberculosis (Mtb) H37Rv, is annotated as a member of Lip family based on the presence of characteristic consensus esterase motif ‘GXSXG’. In vitro culture studies of Mtb H37Ra indicated that expression of Rv3203 gene was up-regulated during acidic stress as compared to normal whereas no expression was observed under nutrient and oxidative stress conditions. Therefore, detailed characterization of Rv3203 was done by gene cloning and its further expression and purification as his-tagged protein in microbial expression system. The enzyme was purified to homogeneity by affinity chromatography. It demonstrated broad substrate specificity and preferentially hydrolyzed p-nitrophenyl myristate. The purified enzyme demonstrated an optimum activity at pH 8.0 and temperature 50 °C. The specific activity, K _m and V _max of enzyme was determined to be 21.29 U mg^?1 protein, 714.28 μM and 62.5 μmol ml^?1 min^?1, respectively. The pH stability assay and circular dichroism spectroscopic analysis revealed that Rv3203 protein is more stable in acidic condition. Tetrahydrolipstatin, a specific lipase inhibitor and RHC80267, a diacylglycerol lipase inhibitor abolished the activity of this enzyme. The catalytic triad residues were determined to be Ser⁵⁰, Asp¹⁸⁰ and His²⁰³ residues by site-directed mutagenesis.