RESUMO
The marine environment is a rich reservoir of diverse biological entities, many of which possess unique properties that are of immense value to biotechnological applications. One such example is the red fluorescent protein derived from the coral Discosoma sp. This protein, encoded by the DsRed gene, has been the subject of extensive research due to its potential applications in various fields. In the study, a variant of the red fluorescent protein was generated through random mutagenesis using the DsRed2 gene as a template. The process employed error-prone PCR (epPCR) to introduce random mutations, leading to the isolation of twelve gene variants. Among these, one variant stood out due to its unique spectral properties, exhibiting dual fluorescence emission at both 480 nm (green) and 550 nm (red). This novel variant was expressed in both Escherichia coli and zebrafish (Danio rerio) muscle, confirming the dual fluorescence emission in both model systems. One of the immediate applications of this novel protein variant is in ornamental aquaculture. The dual fluorescence can serve as a unique marker or trait, enhancing the aesthetic appeal of aquatic species in ornamental settings.
Assuntos
Antozoários , Proteína Vermelha Fluorescente , Animais , Fluorescência , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Antozoários/genética , Antozoários/metabolismo , Biotecnologia , Proteínas de Fluorescência VerdeRESUMO
This work aimed the application of a new biocatalyst for biodiesel production from residual agro-industrial fatty acids. A recombinant Pichia pastoris displaying lipase from Rhizomucor miehei (RML) on the cell surface, using the PIR-1 anchor system, were prepared using glycerol as the carbon source. The biocatalyst, named RML-PIR1 showed optimum temperature of 45 °C (74.0 U/L). The stability tests resulted in t1/2 of 3.49 and 2.15 h at 40 and 45 °C, respectively. RML-PIR1 was applied in esterification reactions using industrial co-products as substrates, palm fatty acid distillate (PFAD) and soybean fatty acid distillate (SFAD). The highest productivity was observed for SFAD after 48 h presenting 79.1% of conversion using only 10% of biocatalyst and free-solvent system. This is about ca. eight times higher than commercial free RML in the same conditions. The stabilizing agents study revealed that the treatment using glutaraldehyde (GA) and poly(ethylene glycol) (PEG) enabled increased stability and reuse of biocatalyst. It was observed by SEM analysis that the treatment modified the cell morphology. RML-PIR1-GA presented 87.9% of the initial activity after 6 reuses, whilst the activity of unmodified RML-PIR decreased by 40% after the first use. These results were superior to those obtained in the literature, making this new biocatalyst promising for biotechnological applications, such as the production of biofuels on a large scale.
Assuntos
Agricultura , Biocombustíveis/microbiologia , Resíduos Industriais , Lipase/metabolismo , Rhizomucor/enzimologia , Saccharomycetales/metabolismo , Biocatálise , Esterificação , Especificidade por Substrato , TemperaturaRESUMO
BACKGROUND: Insects can be found in numerous diverse environments, being exposed to pathogenic organisms like fungi and bacteria. Once these pathogens cross insect physical barriers, the innate immune system operates through cellular and humoral responses. Antimicrobial peptides are small molecules produced by immune signaling cascades that develop an important and generalist role in insect defenses against a variety of microorganisms. In the present work, a cecropin B-like peptide (AgCecropB) sequence was identified in the velvetbean caterpillar Anticarsia gemmatalis and cloned in a bacterial plasmid vector for further heterologous expression and antimicrobial tests. METHODS: AgCecropB sequence (without the signal peptide) was cloned in the plasmid vector pET-M30-MBP and expressed in the Escherichia coli BL21(DE3) expression host. Expression was induced with IPTG and a recombinant peptide was purified using two affinity chromatography steps with Histrap column. The purified peptide was submitted to high-resolution mass spectrometry (HRMS) and structural analyses. Antimicrobial tests were performed using gram-positive (Bacillus thuringiensis) and gram-negative (Burkholderia kururiensis and E. coli) bacteria. RESULTS: AgCecropB was expressed in E. coli BL21 (DE3) at 28°C with IPTG 0.5 mM. The recombinant peptide was purified and enriched after purification steps. HRMS confirmed AgCrecropB molecular mass (4.6 kDa) and circular dichroism assay showed α-helix structure in the presence of SDS. AgCrecropB inhibited almost 50% of gram-positive B. thuringiensis bacteria growth. CONCLUSIONS: The first cecropin B-like peptide was described in A. gemmatalis and a recombinant peptide was expressed using a bacterial platform. Data confirmed tertiary structure as predicted for the cecropin peptide family. AgCecropB was capable to inhibit B. thuringiensis growth in vitro.
RESUMO
Insects can be found in numerous diverse environments, being exposed to pathogenic organisms like fungi and bacteria. Once these pathogens cross insect physical barriers, the innate immune system operates through cellular and humoral responses. Antimicrobial peptides are small molecules produced by immune signaling cascades that develop an important and generalist role in insect defenses against a variety of microorganisms. In the present work, a cecropin B-like peptide (AgCecropB) sequence was identified in the velvetbean caterpillar Anticarsia gemmatalis and cloned in a bacterial plasmid vector for further heterologous expression and antimicrobial tests. Methods AgCecropB sequence (without the signal peptide) was cloned in the plasmid vector pET-M30-MBP and expressed in the Escherichia coli BL21(DE3) expression host. Expression was induced with IPTG and a recombinant peptide was purified using two affinity chromatography steps with Histrap column. The purified peptide was submitted to high-resolution mass spectrometry (HRMS) and structural analyses. Antimicrobial tests were performed using gram-positive (Bacillus thuringiensis) and gram-negative (Burkholderia kururiensis and E. coli) bacteria. Results AgCecropB was expressed in E. coli BL21 (DE3) at 28°C with IPTG 0.5 mM. The recombinant peptide was purified and enriched after purification steps. HRMS confirmed AgCrecropB molecular mass (4.6 kDa) and circular dichroism assay showed α-helix structure in the presence of SDS. AgCrecropB inhibited almost 50% of gram-positive B. thuringiensis bacteria growth. Conclusions The first cecropin B-like peptide was described in A. gemmatalis and a recombinant peptide was expressed using a bacterial platform. Data confirmed tertiary structure as predicted for the cecropin peptide family. AgCecropB was capable to inhibit B. thuringiensis growth in vitro.(AU)
Assuntos
Animais , Peptídeos , Glycine max/microbiologia , Proteínas Citotóxicas Formadoras de Poros/classificação , Cecropinas/administração & dosagem , Sistema ImunitárioRESUMO
Insects can be found in numerous diverse environments, being exposed to pathogenic organisms like fungi and bacteria. Once these pathogens cross insect physical barriers, the innate immune system operates through cellular and humoral responses. Antimicrobial peptides are small molecules produced by immune signaling cascades that develop an important and generalist role in insect defenses against a variety of microorganisms. In the present work, a cecropin B-like peptide (AgCecropB) sequence was identified in the velvetbean caterpillar Anticarsia gemmatalis and cloned in a bacterial plasmid vector for further heterologous expression and antimicrobial tests. Methods AgCecropB sequence (without the signal peptide) was cloned in the plasmid vector pET-M30-MBP and expressed in the Escherichia coli BL21(DE3) expression host. Expression was induced with IPTG and a recombinant peptide was purified using two affinity chromatography steps with Histrap column. The purified peptide was submitted to high-resolution mass spectrometry (HRMS) and structural analyses. Antimicrobial tests were performed using gram-positive (Bacillus thuringiensis) and gram-negative (Burkholderia kururiensis and E. coli) bacteria. Results AgCecropB was expressed in E. coli BL21 (DE3) at 28°C with IPTG 0.5 mM. The recombinant peptide was purified and enriched after purification steps. HRMS confirmed AgCrecropB molecular mass (4.6 kDa) and circular dichroism assay showed α-helix structure in the presence of SDS. AgCrecropB inhibited almost 50% of gram-positive B. thuringiensis bacteria growth. Conclusions The first cecropin B-like peptide was described in A. gemmatalis and a recombinant peptide was expressed using a bacterial platform. Data confirmed tertiary structure as predicted for the cecropin peptide family. AgCecropB was capable to inhibit B. thuringiensis growth in vitro.(AU)
Assuntos
Animais , Peptídeos , Glycine max/microbiologia , Proteínas Citotóxicas Formadoras de Poros/classificação , Cecropinas/administração & dosagem , Sistema ImunitárioRESUMO
Yarrowia lipolytica IMUFRJ 50682 is a Brazilian wild-type strain with potential application in bioconversion processes which can be improved through synthetic biology. In this study, we focused on a combinatorial dual cleavage CRISPR/Cas9-mediated for construction of irreversible auxotrophic mutants IMUFRJ 50682, which genomic information is not available, thought paired sgRNAs targeting upstream and downstream sites of URA3 gene. The disruption efficiency ranged from 5 to 28 % for sgRNAs combinations closer to URA3's start and stop codon and the auxotrophic mutants lost about 970 bp containing all coding sequence, validating this method for genomic edition of wild-type strains. In addition, we introduced a fluorescent phenotype and achieved cloning rates varying from 80 to 100 %. The ura3Δ strains IMUFRJ 50682 were also engineered for ß-carotene synthesis as proof of concept. Carotenoid-producing strains exhibited a similar growth profile compared to the wild-type strain and were able to synthesized 30.54-50.06â¯mg/L (up to 4.8â¯mg/g DCW) of ß-carotene in YPD and YNB flask cultures, indicating a promisor future of the auxotrophic mutants IMUFRJ 50682 as a chassis for production of novel value-added chemicals.
Assuntos
Sistemas CRISPR-Cas , Engenharia Metabólica/métodos , Yarrowia/genética , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Meios de Cultura/metabolismo , Fluorescência , Proteínas Fúngicas/genética , Marcação de Genes , Mutação , RNA Guia de Cinetoplastídeos/genética , Uracila/metabolismo , Yarrowia/crescimento & desenvolvimento , Yarrowia/metabolismo , beta Caroteno/biossíntese , beta Caroteno/genéticaRESUMO
l-asparaginase catalyzes the conversion of l-asparagine to l-aspartate and ammonium. This protein is an important therapeutic enzyme used for the treatment of acute lymphoblastic leukemia. In this study, the asparaginase II-encoding gene ASP3 from Saccharomyces cerevisiae was cloned into the expression vector pET28a in-fusion with a 6x histidine tag and was expressed in Escherichia coli BL21 (DE3) cells. The protein was expressed at a high level (225.6 IU/g cells) as an intracellular and soluble molecule and was purified from the supernatant by nickel affinity chromatography. The enzyme showed very low activity against l-glutamine. The denaturing electrophoresis analysis indicated that the recombinant protein had a molecular mass of â¼38â¯kDa. The native enzyme was a tetramer with a molecular mass of approximately 178â¯kDa. The enzyme preparation showed antitumor activity against the K562 and Jurkat cell lines comparable or even superior to the E. coli commercial asparaginase.
Assuntos
Antineoplásicos/metabolismo , Asparaginase/genética , Proteínas de Bactérias/genética , Escherichia coli/metabolismo , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/genética , Antineoplásicos/química , Asparaginase/química , Asparaginase/metabolismo , Asparagina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Linhagem Celular Tumoral , Clonagem Molecular , Expressão Gênica , Glutamina/metabolismo , Humanos , Peso Molecular , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
The effect of gene dosage on the production of Candida antarctica lipase B (CalB) in the methylotrophic yeast Komagataella phaffii, at high densities in a simple medium containing crude glycerin as the sole carbon source, is described. The use of crude glycerin, the main by-product of biodiesel production from vegetable oils, will reduce the production cost of the bioprocess. Two K. phaffii strains were constructed with one or three copies of LipB, an optimized version of the gene encoding CalB under the control of the constitutive PPGK1 promoter. These two constructs were tested and compared on batches using minimal-salts medium with crude glycerin. The strain with three copies achieved a higher enzyme yield (48,760 U/L, 2.3-fold higher than the one-copy strain), with 42 g/L biomass, with no effects on growth.
Assuntos
Candida/enzimologia , Candida/genética , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Lipase/biossíntese , Lipase/genética , Pichia/genética , Saccharomycetales/metabolismo , Candida/metabolismo , Dosagem de Genes/genética , Glicerol/metabolismo , Regiões Promotoras Genéticas/genética , Saccharomycetales/genéticaRESUMO
A modified Pseudomonas aeruginosa strain capable of overexpressing the estA gene, an encoding gene for a membrane-bound esterase, was constructed and its rhamnolipid (RML) production was studied. Fermentations using wild-type (WT) and modified P. aeruginosa strains were conducted until exhaustion of glycerol in Medium Salt Production, using two different C/N ratios. At a C/N of 83.2, the modified strain produced up to 3.9 times more RMLs than the WT, yielding a maximum concentration of 14.62 g/L RML when measured by HPLC and 22 g/L by the orcinol assay. Cell-free supernatant from the modified strain reduced surface tension to 29.4 mN/m and had a CMC of 240 mg/L and CMD of 56.05. This is the first report on the construction of an estA-based recombinant strain for RML production.
Assuntos
Genes Bacterianos , Glicolipídeos/biossíntese , Pseudomonas aeruginosa/metabolismo , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Meios de Cultura , Fermentação , Pseudomonas aeruginosa/genética , Tensão Superficial , Tensoativos/farmacologiaRESUMO
Snakebite envenomation is a neglected condition that constitutes a public health problem in tropical and subtropical countries, including Brazil. Interestingly, some animals are resistant to snake envenomation due to the presence of inhibitory glycoproteins in their serum that target toxic venom components. DM64 is an acidic glycoprotein isolated from Didelphis aurita (opossum) serum that has been characterized as an inhibitor of the myotoxicity induced by bothropic toxins bearing phospholipase A2 (PLA2) structures. This antitoxic protein can serve as an excellent starting template for the design of novel therapeutics against snakebite envenomation, particularly venom-induced local tissue damage. Therefore, the aim of this work was to produce a recombinant DM64 (rDM64) in the methylotrophic yeast Pichia pastoris and to compare its biological properties with those of native DM64. Yeast fermentation in the presence of Pefabloc, a serine protease inhibitor, stimulated cell growth (~1.5-fold), increased the rDM64 production yield approximately 10-fold and significantly reduced the susceptibility of rDM64 to proteolytic degradation. P. pastoris fermentation products were identified by mass spectrometry and Western blotting. The heterologous protein was efficiently purified from the culture medium by affinity chromatography (with immobilized PLA2 myotoxin) and/or an ion exchange column. Although both native and recombinant DM64 exhibit different glycosylation patterns, they show very similar electrophoretic mobilities after PNGase F treatment. rDM64 formed a noncovalent complex with myotoxin II (Lys49-PLA2) from Bothrops asper and displayed biological activity that was similar to that of native DM64, inhibiting the cytotoxicity of myotoxin II by 92% at a 1:1 molar ratio.