A Novel Cold-Active Lipase from Candida albicans: Cloning, Expression and Characterization of the Recombinant Enzyme

A novel lipase gene lip5 from the yeast Candida albicans was cloned and sequenced. Alignment of amino acid sequences revealed that 86-34% identity exists with lipases from other Candida species. The lipase and its mutants were expressed in the yeast Pichia pastoris, where alternative codon usage caused the mistranslation of 154-Ser and 293-Ser as leucine. 154-Ser to leucine resulted in loss of expression of Lip5, and 293-Ser to leucine caused a marked reduction in the lipase activity. Lip5-DM, which has double mutations that revert 154 and 293 to serine residues, showed good lipase activity, and was overexpressed and purified by (NH(4))(2)SO(4) precipitation and ion-exchange chromatography. The pure Lip5-DM was stable at low temperatures ranging from 15-35 °C and pH 5-9, with the optimal conditions being 15-25 °C and pH 5-6. The activation energy of recombinant lipase was 8.5 Kcal/mol between 5 and 25 °C, suggesting that Lip5-DM was a cold-active lipase. Its activity was found to increase in the presence of Zn(2+), but it was strongly inhibited by Fe(2+), Fe(3+), Hg(2+) and some surfactants. In addition, the Lip5-DM could not tolerate water-miscible organic solvents. Lip5-DM exhibited a preference for the short-and medium-chain length p-nitrophenyl (C4 and C8 acyl group) esters rather than the long chain length p-nitrophenyl esters (C12, C16 and C18 acyl group) with highest activity observed with the C8 derivatives. The recombinant enzyme displayed activity toward triacylglycerols, such as olive oil and safflower oil.     

Cloning, Expression, and Characterization of Thermotolerant Manganese Superoxide Dismutase from Bacillus sp. MHS47

A superoxide dismutase gene from thermotolerant Bacillus sp. MHS47 (MnSOD47) was cloned, sequenced, and expressed. The gene has an open reading frame of 612 bp, corresponding to 203 deduced amino acids, with high homology to the amino acid sequences of B. thuringiensis (accession no. EEN01322), B. anthracis (accession no. NP_846724), B. cereus (accession no. ZP_04187911), B. weihenstephanensis (accession no. YP_001646918), and B. pseudomycoides. The conserved manganese-binding sites (H28, H83, D165, and H169) show that MnSOD47 has the specific characteristics of the manganese superoxide dismutase (MnSOD) enzymes. MnSOD47 expressed an enzyme with a molecular weight of approximately 22.65 kDa and a specific activity of 3537.75 U/mg. The enzyme is active in the pH range 7-8.5, with an optimum pH of 7.5, and at temperatures in the range 30-45 °C, with an optimum temperature of 37 °C. Tests of inhibitors and metal ions indicated that the enzyme activity is inhibited by sodium azide, but not by hydrogen peroxide or potassium cyanide. These data should benefit future studies of MnSODs in other microorganisms and the biotechnological production of MnSOD47, and could also be used to develop a biosensor for the detection of antioxidants and free radical activity. In the future, this basic knowledge could be applicable to the detection of cancer risks in humans and therapeutic treatments.     

Molecular Cloning and Functional Characterization of Mannose Receptor in Zebra Fish (Danio rerio) during Infection with Aeromonas sobria

Mannose receptor (MR) is a member of pattern-recognition receptors (PRRs), which plays a significant role in immunity responses. Much work on MR has been done in mammals and birds while little in fish. In this report, a MR gene (designated as zfMR) was cloned from zebra fish (Danio rerio), which is an attractive model for the studies of animal diseases. The full-length cDNA of zfMR contains 6248 bp encoding a putative protein of 1428 amino acids. The predicted amino acid sequences showed that zfMR contained a cysteine-rich domain, a single fibronectin type II (FN II) domain, eight C-type lectin-like domains (CTLDs), a transmembrane domain and a short C-terminal cytoplasmic domain, sharing highly conserved structures with MRs from the other species. The MR mRNA could be detected in all examined tissues with highest level in kidney. The temporal expression patterns of MR, IL-1β and TNF-α mRNAs were analyzed in the liver, spleen, kidney and intestine post of infection with Aeromonas sobria. By immunohistochemistry assay, slight enhancement of MR protein was also observed in the spleen and intestine of the infected zebra fish. The established zebra fish-A. sobria infection model will be valuable for elucidating the role of MR in fish immune responses to infection.     

Molecular Cloning and Characterization of the Calcineurin Subunit A from Plutella xylostella

Calcineurin (or PP2B) has been reported to be involved in an array of physiological process in insects, and the calcineurin subunit A (CNA) plays a central role in calcineurin activity. We cloned the CNA gene from Plutella xylostella (PxCNA). This gene contains an ORF of 1488 bp that encodes a 495 amino acid protein, showing 98%, and 80% identities to the CNA of Bombyx mori, and humans respectively. The full-length of PxCNA and its catalytic domain (CNA_1–341, defined as PxCNα) were both expressed in Escherichia coli. Purified recombinant PxCNA displayed no phosphatase activity, whereas recombinant PxCNα showed high phosphatase activity with a Km of 4.6 mM and a kcat of 0.66 S⁻¹ against pNPP. It could be activated at different degrees by Mn²⁺, Ni²⁺, Mg²⁺, and Ca²⁺. The optimum reaction pH was about 7.5 and the optimum reaction temperature was around 45 °C. An in vitro inhibition assay showed that okadaic acid (OA) and cantharidin (CTD) competitively inhibited recombinant PxCNα activity with the IC₅₀ values of 8.95 μM and 77.64 μM, respectively. However, unlike previous reports, pyrethroid insecticides were unable to inhibit recombinant PxCNα, indicating that the P. xylostella calcineurin appears not to be sensitive to class II pyrethroid insecticides.     

Molecular Cloning and Characterization of DXS and DXR Genes in the Terpenoid Biosynthetic Pathway of Tripterygium wilfordii

1-Deoxy-d-xylulose-5-phosphate synthase (DXS) and 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) genes are the key enzyme genes of terpenoid biosynthesis but still unknown in Tripterygium wilfordii Hook. f. Here, three full-length cDNA encoding DXS1, DXS2 and DXR were cloned from suspension cells of T. wilfordii with ORF sizes of 2154 bp (TwDXS1, GenBank accession no.{"type":"entrez-nucleotide","attrs":{"text":"KM879187","term_id":"871331037","term_text":"KM879187"}}KM879187), 2148 bp (TwDXS2, GenBank accession no.{"type":"entrez-nucleotide","attrs":{"text":"KM879186","term_id":"871331021","term_text":"KM879186"}}KM879186), 1410 bp (TwDXR, GenBank accession no.{"type":"entrez-nucleotide","attrs":{"text":"KM879185","term_id":"871330999","term_text":"KM879185"}}KM879185). And, the TwDXS1, TwDXS2 and TwDXR were characterized by color complementation in lycopene accumulating strains of Escherichia coli, which indicated that they encoded functional proteins and promoted lycopene pathway flux. TwDXS1 and TwDXS2 are constitutively expressed in the roots, stems and leaves and the expression level showed an order of roots > stems > leaves. After the suspension cells were induced by methyl jasmonate, the mRNA expression level of TwDXS1, TwDXS2, and TwDXR increased, and triptophenolide was rapidly accumulated to 149.52 µg·g⁻¹, a 5.88-fold increase compared with the control. So the TwDXS1, TwDXS2, and TwDXR could be important genes involved in terpenoid biosynthesis in Tripterygium wilfordii Hook. f.     

High-Level Expression of Pro-Form Lipase from Rhizopus oryzae in Pichia pastoris and Its Purification and Characterization

A gene encoding Rhizopus oryzae lipase containing prosequence (ProROL) was cloned into the pPICZαA and electrotransformed into the Pichia pastoris X-33 strain. The lipase was functionally expressed and secreted in Pichia pastoris with a molecular weight of 35 kDa. The maximum lipase activity of recombinant lipase (rProROL) was 21,000 U/mL, which was obtained in a fed-batch cultivation after 168 h induction with methanol in a 50-L bioreactor. After fermentation, the supernatant was concentrated by ultrafiltration with a 10 kDa cut off membrane and purified with ion exchange chromatography using SP Sepharose Fast Flow chromatography. The optimum pH and temperature of the rProROL were pH 9.0 and 40 °C, respectively. The lipase was stable from pH 4.0 to 9.0 and from 25 to 55 °C. The enzyme activity was enhanced by Ca²⁺ and inhibited by Hg²⁺ and Ag⁺. The lipase showed high activity toward triglyceride-Tripalmitin (C16:0) and triglyceride-Trilaurin (C12:0).     

Cloning,Expression and Characterization of a Novel Thermophilic Polygalacturonase from Caldicellulosiruptor bescii DSM 6725

We cloned the gene {"type":"entrez-protein","attrs":{"text":"ACM61449","term_id":"222457187","term_text":"ACM61449"}}ACM61449 from anaerobic, thermophilic Caldicellulosiruptor bescii, and expressed it in Escherichia coli origami (DE3). After purification through thermal treatment and Ni-NTA agarose column extraction, we characterized the properties of the recombinant protein (CbPelA). The optimal temperature and pH of the protein were 72 °C and 5.2, respectively. CbPelA demonstrated high thermal-stability, with a half-life of 14 h at 70 °C. CbPelA also showed very high activity for polygalacturonic acid (PGA), and released monogalacturonic acid as its sole product. The V_max and K_m of CbPelA were 384.6 U·mg⁻¹ and 0.31 mg·mL⁻¹, respectively. CbPelA was also able to hydrolyze methylated pectin (48% and 10% relative activity on 20%–34% and 85% methylated pectin, respectively). The high thermo-activity and methylated pectin hydrolization activity of CbPelA suggest that it has potential applications in the food and textile industry.     

Cloning and Expression of Aspergillus tamarii FS132 Lipase Gene in Pichia pastoris

A lipase gene (atl) was cloned from Aspergillus tamarii FS132 for the first time. The gene was found to have an open reading frame of 1024 base pairs (bp), and the coding region of the gene contained two introns (51 bp and 52 bp). Multi-alignment analysis of the deduced amino acid sequence indicated high homology between the enzyme and mono-and diacylglycerol lipases from fungi Aspergillus. The recombinant lipase was expressed in Pichia pastoris GS115 cells. The recombinant lipase was found to have a molecular mass of 36.7 kDa, and it exhibited lipase activity of 20 U/mL in culture supernatant when tributyrin was used as the substrate.     

Molecular Cloning and Expression of CYP9A61: A Chlorpyrifos-Ethyl and Lambda-Cyhalothrin-Inducible Cytochrome P450 cDNA from Cydia pomonella

Cytochrome P450 monooxygenases (CYPs or P450s) play paramount roles in detoxification of insecticides in a number of insect pests. However, little is known about the roles of P450s and their responses to insecticide exposure in the codling moth Cydia pomonella (L.), an economically important fruit pest. Here we report the characterization and expression analysis of the first P450 gene, designated as CYP9A61, from this pest. The full-length cDNA sequence of CYP9A61 is 2071 bp long and its open reading frame (ORF) encodes 538 amino acids. Sequence analysis shows that CYP9A61 shares 51%–60% identity with other known CYP9s and contains the highly conserved substrate recognition site SRS1, SRS4 and SRS5. Quantitative real-time PCR showed that CYP9A61 were 67-fold higher in the fifth instar larvae than in the first instar, and more abundant in the silk gland and fat body than other tissues. Exposure of the 3rd instar larvae to 12.5 mg L⁻¹ of chlorpyrifos-ethyl for 60 h and 0.19 mg L⁻¹ of lambda-cyhalothrin for 36 h resulted in 2.20-and 3.47-fold induction of CYP9A61, respectively. Exposure of the 3rd instar larvae to these two insecticides also significantly enhanced the total P450 activity. The results suggested that CYP9A61 is an insecticide-detoxifying P450.     

Clinical and Molecular Characterization of Patients with Mucopolysaccharidosis Type I in an Algerian Series

Mucopolysaccharidoses (MPS’s) represent a subgroup of lysosomal storage diseases related to a deficiency of enzymes that catalyze glycosaminoglycans degradation. Mucopolysaccharidosis type I (MPS I) is a rare autosomal recessive disorder caused by a deficiency of α-l-iduronidase encoded by the IDUA gene. Partially degraded heparan sulfate and dermatan sulfate accumulate progressively and lead to multiorgan dysfunction and damage. The aim of this study is to describe the clinical, biochemical, and molecular characteristics of 13 Algerian patients from 11 distinct families. MPS I diagnosis was confirmed by molecular study of the patients’ IDUA gene. Clinical features at the diagnosis and during the follow-up are reported. Eighty-four percent of the studied patients presented with a mild clinical phenotype. Molecular study of the IDUA gene allowed the characterization of four pathological variations at the homozygous or compound heterozygote status: IDUA NM_00203.4:c.1598C>G-p.(Pro533Arg) in 21/26 alleles, IDUA NM_00203.4:c.532G>A-p.(Glu178Lys) in 2/26 alleles, IDUA NM_00203.4:c.501C>G-p.(Tyr167*) in 2/26 alleles, and IDUA NM_00203. 4: c.1743C>G-p.(Tyr581*) in 1/26 alleles. This molecular study unveils the predominance of p.(Pro533Arg) variation in our MPS I patients. In this series, the occurrence of some clinical features linked to the Scheie syndrome is consistent with the literature, such as systematic valvulopathies, corneal opacity, and umbilical hernia; however, storage signs, facial dysmorphic features, and hepatomegaly were more frequent in our series. Screening measures for these debilitating diseases in highly consanguineous at-risk populations must be considered a priority health problem.