RESUMEN
Antimicrobial peptides (AMPs) are short and positively charged peptides with broad-spectrum antimicrobial activities. AMPs have been investigated as potential antibiotic alternatives to improve growth performance and prevent pathogen infection in the poultry industry. The antimicrobial peptide tilapia piscidin 4 (TP4) was derived from Oreochromis niloticus, possesses antimicrobial activities and immunomodulatory properties, promotes intestinal health, and protects against pathogen infection. The codon-optimized sequence of TP4 was introduced into the pPICZαA vector and transformed into Pichia pastoris. Large-scale expression was induced following culture with methanol in a 500-liter fermenter. Freeze drying of fermented rTP4 broth and then rTP4 evaluation as a feed additive for Gallus gallus domesticus were performed. The in vitro antimicrobial activity of recombinant TP4 (rTP4) against gram-positive and gram-negative pathogens was evaluated. Evaluation of the effect of temperature on the antimicrobial activity of rTP4 showed its high stability at high temperatures. rTP4 significantly enhanced the phagocytic activity of macrophage cells, indicating that rTP4 has a remarkable ability to stimulate macrophages. rTP4 was used as a dietary supplement at 0.75, 1.5, 3.0, 6.0 and 12% in G. g. domesticus for five weeks, and growth performance, gut microbiota composition, and histology were assessed. The 3.0% rTP4 supplement group showed a significant increase in weight gain ratio and feed efficiency compared to those of the basal broiler diet group. Crude rTP4 was expressed by yeast to significantly promote growth efficiency and resistance against pathogens in G. g. domesticus, which could indicate its use as a suitable alternative to antibiotics as feed additives in the poultry industry.
Asunto(s)
Alimentación Animal , Péptidos Catiónicos Antimicrobianos/farmacología , Pollos/crecimiento & desarrollo , Suplementos Dietéticos , Proteínas de Peces/farmacología , Tilapia/genética , Animales , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/genética , Femenino , Proteínas de Peces/química , Proteínas de Peces/genética , Masculino , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologíaRESUMEN
Supplementing chicken feed with antibiotics can improve survival and prevent disease outbreaks. However, overuse of antibiotics may promote the development of antibiotic-resistant bacteria. Recently, antimicrobial peptides have been proposed as alternatives to antibiotics in animal husbandry. Here, we evaluate the effects of antimicrobial peptide, Epinephelus lanceolatus piscidin (EP), in Gallus gallus domesticus. The gene encoding EP was isolated, sequenced, codon-optimized and cloned into a Pichia pastoris recombinant protein expression system. The expressed recombinant EP (rEP) was then used as a dietary supplement for G. g. domesticus; overall health, growth performance and immunity were assessed. Supernatant from rEP-expressing yeast showed in vitro antimicrobial activity against Gram-positive and Gram-negative bacteria, according to an inhibition-zone diameter (mm) assay. Moreover, the antimicrobial peptide function of rEP was temperature independent. The fermentation broth yielded a spray-dried powder formulation containing 262.9 µg EP/g powder, and LC-MS/MS (tandem MS) analysis confirmed that rEP had a molecular weight of 4279 Da, as expected for the 34-amino acid peptide; the DNA sequence of the expression vector was also validated. We then evaluated rEP as a feed additive for G. g. domesticus. Treatment groups included control, basal diet and rEP at different doses (0.75, 1.5, 3.0, 6.0 and 12%). Compared to control, rEP supplementation increased G. g. domesticus weight gain, feed efficiency, IL-10 and IFN-γ production. Our results suggest that crude rEP could provide an alternative to traditional antibiotic feed additives for G. g. domesticus, serving to enhance growth and health of the animals.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/metabolismo , Pollos/inmunología , Sistema Inmunológico/metabolismo , Perciformes/metabolismo , Secuencia de Aminoácidos , Animales , Antiinfecciosos/análisis , Antiinfecciosos/metabolismo , Antiinfecciosos/farmacología , Péptidos Catiónicos Antimicrobianos/análisis , Péptidos Catiónicos Antimicrobianos/clasificación , Péptidos Catiónicos Antimicrobianos/genética , Pollos/crecimiento & desarrollo , Cromatografía Líquida de Alta Presión , Clonación Molecular , Suplementos Dietéticos , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Sistema Inmunológico/efectos de los fármacos , Interferón gamma/metabolismo , Interleucina-10/metabolismo , Filogenia , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Alineación de Secuencia , Espectrometría de Masas en Tándem , TemperaturaRESUMEN
To obtain the angiotension-I converting enzyme inhibitor (ACEI), a fusion ACEI polypeptide encoded with 8 DNA sequences of GPL, GPM, IKW, IVY, IRPVQ, IWHHT, IYPRY and IAPG, which were selected and designed and cloned into pGAPZαC and then transformed into Pichia pastoris SMD1168H. After 3 days induction, the fraction with highest ACEI activity was expressed and purified using a Ni Sepharose™ 6 Fast Flow. The IC50 of recombinant ACEI polypeptide was 88.2 µM. A 128-fold increase of ACEI activity (0.69 µM) was obtained after pepsin digestion, which was equivalent to 0.022 µM of captopril. Reverse phase HPLC indicated all the 8 peptides contained in ACEI-hydrolysate after pepsin digestion.
Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/metabolismo , Expresión Génica , Péptidos/genética , Pichia/genética , Secuencia de Aminoácidos , Inhibidores de la Enzima Convertidora de Angiotensina/química , Inhibidores de la Enzima Convertidora de Angiotensina/aislamiento & purificación , Clonación Molecular , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Humanos , Péptidos/química , Péptidos/aislamiento & purificación , Péptidos/metabolismo , Peptidil-Dipeptidasa A/química , Pichia/metabolismo , Transformación GenéticaRESUMEN
To obtain a Pichia pastoris mutant with an Escherichia coli phytase gene, which was synthesized according to P. pastoris codon preference, a mature phytase cDNA of E. coli being altered according to the codons usage preference of P. pastoris was artificially synthesized and cloned into an expression vector of pGAPZαC. The final extracellular phytase activity was 112.5 U/mL after 72 h of cultivation. The phytase, with a molecular mass of 46 kDa, was purified to electrophoretical homogeneity after Ni Sepharose 6 Fast Flow chromatography. The yield, purification fold, and specific activity were 63.97%, 26.17, and 1.57 kU/mg, respectively. It had an optimal pH and temperature of 4.0-6.0 and 50 °C, respectively, and was stable at pH 3.0-8.0 and 25-40 °C. The purified recombinant phytase was resistant to trypsin, highly inhibited by Cu(2+), Zn(2+), Hg(2+), Fe(2+), Fe(3+), phenylmethylsulfonyl fluoride, and N-tosyl-l-lysine chloromethyl ketone, but activated by Mg(2+), Ca(2+), Sr(2+), Ba(2+), glutathione, ethylenediaminetetraacetic acid, and N-ethylmaleimide. It revealed higher affinity to calcium phytate than to other phosphate conjugates.
Asunto(s)
6-Fitasa/química , 6-Fitasa/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Escherichia coli/enzimología , Expresión Génica , Pichia/genética , 6-Fitasa/genética , 6-Fitasa/aislamiento & purificación , Clonación Molecular , Estabilidad de Enzimas , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/aislamiento & purificación , Proteínas de Escherichia coli/metabolismo , Cinética , Pichia/metabolismo , Ingeniería de ProteínasRESUMEN
Locust bean gum (LBG) was employed to screen mannanase-producing bacteria. The bacterium with highest mannanase ability was identified as Paenibacillus cookii. It revealed highest activity (6.67 U/mL) when cultivated in 0.1% LBG with 1.5% soytone and 0.5% tryptone after 4 days incubation at 27 °C. Its mannanase was purified to electrophoretical homogeneity after DEAE-Sepharose and Sephacryl S-100 separation. The purified mannanase, with an N-terminus of GLFGINAY, had pH and temperature optimum at 5.0 and 50 °C, respectively, and was stable at pH 5.0-7.0, ≤ 50 °C. It was strongly activated by ß-mercaptoethanol, dithiothreitol, cysteine, and glutathione, but inhibited by Hg(2+), Cu(2+), Zn(2+), Fe(3+), PMSF, iodoacetic acid, and EDTA. According to substrate specificity study, the purified mannanase had high specificity to LBG and konjac.