Novel Long-Term Phytase from Serratia odorifera: Cloning, Expression, and Characterization.

The appA-So gene, encoding a phytase from Serratia odorifera, was cloned and heterologously expressed in Komagataella phaffii. The open reading frame of appA-So comprised 1281 bp that encoded a 426-amino acid protein, including a 27-amino acid signal peptide. The encoded phytase, AppA-So, showed 52% homology with other histidine acid phosphatases. The purified recombinant phytase showed optimal activity at 55 °C and pH 4.5, exhibiting enzymatic activity between pH 3.7 and 5.8, with a specific activity of 1123 U/mg at pH 4.5 and 37 °C. The AppA-So protein retained more than 85% of its initial activity after incubation in different pH conditions (pH 2.5-6.5) at 37 °C for 3 h. AppA-So activity was maintained over time and displayed a low Michaelis-Menten constant (Km) of 0.093 g/L. To the best of our knowledge, this is the first report of the cloning and characterization of the phytase from S. odorifera. Comparison of AppA-So with other well-known phytases suggests that the S. odorifera phytase has the lowest Km and highest stable activity over time, making it very suitable for use in the animal feed industry

Evaluation of Phosphorus Digestibility from Monocalcium and Dicalcium Phosphate Sources and Comparison between Total Tract and Prececal Digestibility Standard Methods in Broilers

The objective of this study was to compare the total tract (total excreta and marker) and prececal methodologies to determine phosphorus (P) digestibility and to evaluate its variation as a function of the physicochemical characteristics of the inorganic phosphate used (monocalcium, MCP and dicalcium, DCP) from different commercial sources. A total of 176 1-day-old male broilers were used in two digestibility experiments. In Experiment 1, one MCP and one DCP were incorporated in the basal diet at two levels. In Experiment 2, MCP and DCP from three commercial sources were incorporated to the basal diet at one level. Physicochemical characteristics of inorganic phosphates were examined, as well. Additionally, bone mineralization and growth performance traits were investigated in both trials. The digestibility of MCP ranged from 75.2 to 87.4% and from 80.5 to 86.6% for DCP amongst methodologies, but differences between total tract and preceal methodologies were not statistically significant. Particle size, surface area, degree of crystallinity and impurities varied amongst commercial sources. The P digestibility of the three tested commercial sources of MCP was 79.6% (MCP1), 70.2% (MCP2) and 65.6% (MCP3); p > 0.05. The P digestibility of the 3 tested commercial sources of DCP was 80.1% (DCP1), 77.4% (DCP2) and 71.4% (DCP3); p > 0.05

Aspectes moleculars de dues malalties de transport lisosòmic: la cistinosi i la malaltia de Niemann-Pick tipus C

[cat] La cistinosi i la malaltia de Niemann-Pick tipus C (NPC) són dues patologies hereditàries monogèniques poc freqüents, per aquest motiu estan classificades dins del grup de malalties anomenades rares. La cistinosi està causada per mutacions al gen CTNS, que codifica per una proteïna transmembrana del lisosoma que rep el nom de cistinosina. En canvi, la malaltia de NPC és deguda a mutacions al gen NPC1 o al gen NPC2, que codifiquen per una proteïna integral de la membrana lisosòmica i una proteïna soluble del lisosoma, respectivament, i aquestes reben el mateix nom que el gen, NPC1 i NPC2. El funcionament incorrecte d’aquestes proteïnes de transport dóna lloc a una acumulació de productes, diferent a ambdós casos, a l’interior del lisosoma, sent classificades com a malalties d’acumulació lisosòmica. Aquesta tesi doctoral s’ha centrat en l’anàlisi molecular de pacients afectes d’alguna d’aquestes dues malalties. S’ha realitzat el primer estudi mutacional de cistinosi a la població espanyola, que ha permès la identificació de 15 mutacions diferents, 7 de les quals no havien estat descrites: tres mutacions de canvi de sentit (p.M1T, p.S270F i p.G309V), tres delecions (c.1-19_61del, c.295_310del i c.320_323delATCA) i una mutació de splicing (c.682-1G>T). La deleció de 57 kb és la mutació més freqüent a Espanya (38% dels al•lels) i conjuntament amb altres 5 mutacions representen el 73% dels al•lels estudiats. S’ha establert que els pacients amb fenotip clínic infantil tenen a ambdós al•lels mutacions que trunquen o que afecten aminoàcids conservats de les regions transmembrana de la proteïna. En canvi, la mutació p.S139F s’ha associat a la forma juvenil de la malaltia. L’estudi mutacional realitzat a la malaltia de Niemann-Pick tipus C ha permès establir el genotip d’un gran nombre de pacients, identificant 74 mutacions diferents, 17 de les quals no havien estat descrites: set mutacions de canvi de sentit (p.P474A, p.G535V, p.F995L, p.F1079S, p.L1106P, p.G1209E i p.S1249G), dues mutacions sense sentit (p.Q775X i p.E1089X), dues insercions (p.L1117PfsX4 i p.I1061NfsX4), una deleció en pauta (p.N916del), quatre mutacions de splicing (c.58-3280C>G, c.882-28A>T, c.2604+5G>A i c.3591+5G>A) i la primera gran deleció que afecta al gen NPC1 i gens flanquejants. També s’han pogut establir correlacions genotip-fenotip per a un conjunt de mutacions. També s’ha demostrat la implicació de diferents mecanismes cel•lulars en la malaltia de Niemann-Pick tipus C, segons els tipus de mutacions causants: el “splicing”, el procés de “nonsense-mediated mRNA decay” i la degradació proteica portada a terme pel proteasoma. S’han identificat mutacions intròniques profundes i s’ha caracteritzat el seu efecte en el mRNA, conjuntament amb el de les mutacions de “splicing” que afecten als llocs canònics. El mecanisme de NMD és el responsable de la degradació del mRNA en tots els al•lels analitzats que codifiquen per un PTC al gen NPC1. La majoria de mutacions de canvi de sentit analitzades condueixen a una reducció significativa o a l’absència de la proteïna NPC1, degut a què la proteïna NPC1 mutada és degradada per la via de la ubiquitina-proteasoma. La proteïna NPC1 mutada recuperada, després del tractament amb els inhibidors del proteasoma (ALLN i MG132), és capaç de disminuir els nivells de colesterol a totes les línies cel•lulars NPC estudiades. Aquesta observació podria obrir la porta a l’ús d’aquests fàrmacs com a futur tractament per a la malaltia de NPC causada per determinades mutacions de canvi de sentit.[eng] “Molecular aspects of both lysosomal transport diseases: cystinosis and Niemann-Pick disease type”. The cystinosis and Niemann-Pick disease type C (NPC) are two rare monogenic hereditary diseases. The cystinosis is caused by mutations in the gene CTNS, which encodes a transmembrane protein of the lysosome that is called cystinosin. NPC disease is caused by mutations in the NPC1 or NPC2 gene, encoding an integral lysosomal membrane protein and a soluble protein of the lysosome, respectively, and these are the same name as the gene, NPC1 and NPC2. The impaired transport leads to an accumulation of products, different in both cases, inside the lysosome, being classified as lysosomal storage disorders. This thesis has focused on the molecular analysis of patients with any of these diseases. Molecular analysis in the Spanish cystinosis patients has allowed the identification of 15 different mutations, 7 of which had not been described. The 57-kb deletion is the most common mutation in Spain (38% of alleles) and together with other 5 mutations accounted for 73% of the studied alleles. The p.S139F mutation has been associated with the juvenile form of the disease. Molecular analysis in NPC disease has established the mutation profile in a large number of patients. 74 different mutations have been identified, 17 of which had not been described previously, including the first large deletion affecting the whole NPC1 gene and flanking genes. Genotype-phenotype correlations have been established for several mutations. Different cellular mechanisms are involving in NPC disease: splicing, nonsense-mediated mRNA decay and proteasomal degradation. Deep intronic mutations have been identified and the effect on the mRNA has been characterized. NMD process is responsible for the mRNA decay for all analyzed NPC1 PTC-encoding mutations. Several missense mutations lead to a significant reduction or absence of NPC1 protein, because the NPC1 mutant protein is degraded by the ubiquitin-proteasome pathway. Treatment with proteasome inhibitors partially reverses the NPC1 decrease and reduces cholesterol levels in all studied NPC cell lines. This observation might represent a therapeutical approach for future treatments of NPC disease caused by specific missense mutations

The proteasome inhibitor bortezomib reduced cholesterol accumulation in fibroblasts from Niemann-Pick type C patients carrying missense mutations

© 2014 FEBS. Niemann-Pick disease type C (NPC) is a lipid storage disorder mainly caused by mutations in the NPC1 gene. Approximately 60% of these mutations are missense changes that may induce reduced NPC1 protein levels by increased degradation via ubiquitin-proteasome. This is the case for the most prevalent worldwide mutation, p.Ile1061Thr, as well as for other three missense changes. In the present study, we analyzed the NPC1 levels in fibroblasts from eighteen NPC patients presenting missense mutations. We found that fourteen of these cells lines showed decreased levels of NPC1. Six of these cell lines were homozygous, whereas the other eight were associated with a frame shifting mutation. We focused our attention in the NPC homozygous samples and demonstrated that, in most of the cases, NPC1 reduction was a consequence of a decrease of its half-life. NPC cells were treated not only with the proteasome inhibitors carbobenzoxy-L-leucyl-L-leucyl-L-leucinal or N-acetyl-leucyl-leucylnorleucinal, both widely used as a research tools, but also with bortezomib, the first proteasome inhibitor to reach clinical applications, although it has never been used in NPC disease. We observed that, after treatment, the mutant NPC1 protein levels were partially recovered in most of the cell lines. Importantly, these mutant proteins partially recovered their activity and substantially reduced free cholesterol levels. These results suggest that by enhancing the NPC1 protein stability with the use of proteasome inhibitors, their functionality might be recovered and this might represent a therapeutical approach for future treatments of NPC disease resulting from specific missense mutations.The Spanish Ministerio de Ciencia e Innovación. Grant Numbers: PI10/0936, SAF2012-38078, RTICC RD12/0036/0054Peer Reviewe

Effect of Dietary Mineral Content and Phytase Dose on Nutrient Utilization, Performance, Egg Traits and Bone Mineralization in Laying Hens from 22 to 31 Weeks of Age

A total of 192 laying hens were used to evaluate the effect of dietary mineral content and phytase dose on nutrient utilization, egg production and quality and bone mineralization of young laying hens. Four dietary treatments were studied: PC, positive control with no added phytase, 4.07% Ca and 0.61% P; NC, negative control with no added phytase, 2.97% Ca and 0.37% P; and P500 and P1000, where NC diet was supplemented with phytase at 500 and 1000 FTU/kg, respectively. Hens’ performance and egg traits were controlled from 22 to 31 weeks of age. Coefficients of total tract apparent digestibility (CTTAD) of nutrients were determined at 25 and 31 weeks of age. Apparent ileal digestibility (AID) and blood content of Ca and P, as well as bone traits, were determined at 31 weeks of age. Ca and P retention was higher in birds on PC diet at 25 weeks, but not at 31 weeks of age compared to those on NC diet (p < 0.05). P1000 birds had the highest CTTAD values for dry and organic matter at both ages (p < 0.001). CTTAD of Ca was significantly higher in P1000 diet than in NC diet at 31 weeks of age (p < 0.001). Birds fed with P500 diet at 25 weeks of age and P1000 at 31 weeks of age showed higher CTTAD and retention of P, but lower excretion of P than those fed NC diet (p < 0.05). Phytase inclusion linearly increased AID of dry matter and P (p < 0.001). P500 hens fed had the greatest body weight at the end of the trial (p < 0.05) and P1000 birds had the best feed conversion ratio (p < 0.05). Fowl fed a PC diet produced eggs with higher shell thickness and yolk color than those fed on NC diet (p < 0.05). Phytase inclusion linearly increased the yolk color (p < 0.05). Tibia of laying hens fed with PC had significantly higher ash content than those on NC diet (p < 0.05), and birds fed with P1000 presented intermediate values. It can be concluded that it would be advisable to increase the dose of phytase in the feed of laying hens to obtain long-term benefits