Purification and characterization of the first recombinant bird pancreatic lipase expressed in Pichia pastoris: The turkey

<p>Abstract</p> <p>Background</p> <p>The turkey pancreatic lipase (TPL) was purified from delipidated pancreases. Some biochemical properties and kinetic studies were determined using emulsified system and monomolecular film techniques. Those studies have shown that despite the accumulation of free fatty acids at the olive oil/water interface, TPL continues to hydrolyse efficiently the olive oil and the TC₄in the absence of colipase and bile salts, contrary to most classical digestive lipases which denaturate rapidly under the same conditions. The aim of the present study was to express TPL in the methylotrophic yeast <it>Pichia pastoris </it>in order to get a large amount of this enzyme exhibiting interesting biochemical properties, to purify and characterize the recombinant enzyme.</p> <p>Results</p> <p>The recombinant TPL was secreted into the culture medium and the expression level reached about 15 mg/l after 4 days of culture. Using Q-PCR, the number of expression cassette integrated on <it>Pichia </it>genomic DNA was estimated to 5. The purified rTPL, with molecular mass of 50 kDa, has a specific activity of 5300 U/mg on emulsified olive oil and 9500 U/mg on tributyrin. The optimal temperature and pH of rTPL were 37°C and pH 8.5. The stability, reaction kinetics and effects of calcium ions and bile salts were also determined.</p> <p>Conclusions</p> <p>Our results show that the expressed TPL have the same properties as the native TPL previously purified. This result allows us the use of the recombinant enzyme to investigate the TPL structure-function relationships.</p

Indigo Formation and Rapid NADPH Consumption Provide Robust Prediction of Raspberry Ketone Synthesis by Engineered Cytochrome P450 BM3

Natural raspberry ketone has a high value in the flavor, fragrance and pharmaceutical industries. Its extraction is costly, justifying the search for biosynthetic routes. We hypothesized that cytochrome P450 BM3 (P450 BM3) could be engineered to catalyze the hydroxylation of 4‐phenyl‐2‐butanone, a naturally sourceable precursor, to raspberry ketone. The synthesis of indigo by variants of P450 BM3 has previously served as a predictor of promiscuous oxidation reactions. To this end, we screened 53 active‐site variants of P450 BM3 using orthogonal high‐throughput workflows to identify the most streamlined route to all indigo‐forming variants. Among the three known and 13 new indigo‐forming variants, eight hydroxylated 4‐phenyl‐2‐butanone to raspberry ketone. Previously unreported variant A82Q displayed the highest initial rates and coupling efficiencies in synthesis of indigo and of raspberry ketone. It produced the highest total concentration of raspberry ketone despite producing less total indigo than previously reported variants. Its productivity, although modest, clearly demonstrates the potential for development of a biocatalytic route to raspberry ketone. In addition to validating indigo as a robust predictor of this promiscuous activity, we demonstrate that monitoring rapid NADPH consumption serves as an alternative predictor of a promiscuous reactivity in P450 BM3

Biología reproductiva del olivo (BRO-EEZ-CSIC)

Trabajo presentado a las III Jornadas Nacionales del grupo de Horticultura de la SECH, celebradas en Sevilla el 6 y 7 de octubre de 2011.Peer reviewe

High Y-chromosomal differentiation among ethnic groups of Dir and Swat districts, Pakistan

The ethnic groups that inhabit the mountainous Dir and Swat districts of northern Pakistan are marked by high levels of cultural and phenotypic diversity. To obtain knowledge of the extent of genetic diversity in this region, we investigated Y‐chromosomal diversity in five population samples representing the three main ethnic groups residing within these districts, including Gujars, Pashtuns and Kohistanis. A total of 27 Y‐chromosomal short tandem repeats (Y‐STRs) and 331 Y‐chromosomal single nucleotide polymorphisms (Y‐SNPs) were investigated. In the Y‐STRs, we observed very high and significant levels of genetic differentiation in nine of the 10 pairwise between‐group comparisons (RST 0.179–0.746), and the differences were mirrored in the Y‐SNP haplogroup frequency distribution. No genetic differences were found between the two Pashtun subethnic groups Tarklanis and Yusafzais (RST = 0.000). Utmankhels, also considered Pashtuns culturally, were not closely related to any of the other population samples (RST 0.451–0.746). Thus, our findings provide examples of both associations and dissociations between cultural and genetic legacies. When analyzed within a larger continental‐scale context, these five ethnic groups fall mostly outside the previously characterized Y‐chromosomal gene pools of the Indo‐Pakistani subcontinent. Male founder effects, coupled with culturally and topographically based constraints upon marriage and movement, are likely responsible for the high degree of genetic structure in this region

Adsorption of raw and treated by membranes fermentation brines from table olives processing for phenolic compounds separation and recovery

BACKGROUND: Fermentation brines from table olive processing are effluents characterized by very high salinity and high organic matter concentration, which includes phenols of high value as hydroxytyrosol that is used by pharmaceutical and cosmetic industries. RESULTS: In thiswork the adsorptionwith a phenols-selective resin (MN200) of fermentation brine (rawand pre-treated by ultrafiltration or by ultrafiltration plus nanofiltration) has been studied. The study included the adsorption and desorption process, and the useful life of the resin was evaluated. Results indicated that resin MN200 above 20 g L&#8722;1 yielded phenols separation efficiencies higher than 90%. However, the adsorption of nanofiltrated effluent separated phenols more selectively. Adsorption kinetics fitted properly to a pseudo-second-order kinetics and the Langmuir isotherm correctly model the adsorption process. Results of the intra-particle diffusion model show that the pore diffusion is not the only rate limiting step. Desorption was carried outwith ethanol. More than85%of phenolic compoundswere recovered. The use of the resin during ten cycles showed that the nanofiltrated effluent increased the useful time of the resin. CONCLUSION: The MN200 resin was a viable alternative to phenols removing and further recovering, in FTOP effluents. The optimal conditions were obtained for nanofiltrated effluents.The authors of this work acknowledge the financial support of CDTI (Centre for Industrial Technological Development) through the Spanish Ministry of Science and Innovation.Ferrer-Polonio, E.; Mendoza Roca, JA.; Iborra Clar, A.; Pastor Alcañiz, L. (2016). Adsorption of raw and treated by membranes fermentation brines from table olives processing for phenolic compounds separation and recovery. Journal of Chemical Technology and Biotechnology. 91(7):2094-2102. doi:10.1002/jctb.4807S2094210291

Effect of <i>Spirulina platensis</i> Biomass with High Polysaccharides Content on Quality Attributes of Common Carp (<i>Cyprinus carpio</i>) and Common Barbel (<i>Barbus barbus</i>) Fish Burgers

Lately, microalgae have been used as natural additives in fish-transformed products to improve their nutritional quality. In this research, the effects of adding Spirulina platensis at concentrations of 0.5, 1 and 1.5% w/v on both the texture and the sensory characteristics of canned burgers were studied. In fact, the addition of Spirulina platensis to fish burgers improves their nutritional composition. Compared to the results of the other fish burger treatments, the treatments that contain 1% of Spirulina platensis had better texture and sensory properties (p &lt; 0.05). Besides, these treatments showed higher swelling ability as well as water and oil holding capacities, due to the important dietary fibers and polysaccharides contents found in Spirulina platensis. No mold or foodborne pathogens were detected in any of the canned burgers up to 8 months of storage at 4 &#176;C. Furthermore, burgers prepared with Spirulina were distinguished by the lowest mean (a* and b*) values (p &lt; 0.05), which shows that the yellow color gradually diminished towards a greenish color. Because of the presence of polysaccharides and pigments (chlorophylls, carotenoids and phycocyanin), Spirulina platensis considerably ameliorates the antioxidant activities of the newly prepared fish burgers. On the whole, we can conclude that Spirulina platensis can be used as a nutritious additive to produce new fish-based products with high alimentary qualities

Quantitative analysis of BCL2 mRNA expression in nasopharyngeal carcinoma: an unfavorable and independent prognostic factor

Programmed cell death plays a vital role in a wide variety of physiological processes. Defects in apoptotic cell death contribute to neoplastic diseases by preventing or delaying normal cell death. BCL2 (Bcl-2) is an anti-apoptotic gene with marked up-regulation in various malignancies, such as breast cancer, in which expression of the BCL2 protein has been proposed as a prognostic tumor biomarker. The purpose of the current study was to investigate mRNA expression of the BCL2 gene in nasopharyngeal carcinoma (NPC) biopsies and assess its prognostic value. For this purpose, total RNA was isolated from 89 malignant and hyperplastic nasopharyngeal biopsies from Tunisian patients. After testing the quality of the extracted RNA, cDNA was prepared by reverse transcription. A highly sensitive real-time PCR methodology for BCL2 mRNA quantification was developed using SYBRA (R) Green chemistry. GAPDH served as an endogenous control gene. Relative quantification analysis was performed using the comparative C (T) (2(-a dagger a dagger CT)) method. High BCL2 mRNA levels were detected in advanced-stage nasopharyngeal tumors (p = 0.030). Furthermore, BCL2 mRNA expression was strongly associated with lymph node involvement (p = 0.009) and presence of distal metastases (p = 0.013). Survival analysis demonstrated that patients with BCL2-positive nasopharyngeal tumors have significantly shorter disease-free and overall survival (p = 0.011 and p = 0.028, respectively). The major contribution of the current study is the quantification and evaluation, for the first time, of the prognostic significance of the BCL2 mRNA expression in nasopharyngeal carcinoma (NPC) patients. Our results suggest that mRNA expression levels of BCL2 may represent a novel unfavorable and independent tumor biomarker for nasopharyngeal carcinoma

Effect of Microalgae Incorporation on Quality Characteristics and Functional and Antioxidant Capacities of Ready-to-Eat Fish Burgers Made from Common Carp (<i>Cyprinus carpio</i>)

Microalgae have been used as natural ingredients to produce functional and nutritional food products. The impact of the addition of Chlorella minutissima, Isochrysis galbana, and Picochlorum sp. at concentrations of 0.5, 1, and 1.5% w/v on the texture and sensory attributes of canned burgers were investigated. The results show that carp formulations containing 1% microalgae show significantly better classification performance for many textural and sensory parameters compared to the rest of the formulations. Also, these treatments had higher swelling ability as well as water and oil holding capacities, thanks to the important dietary fiber and polysaccharide contents found in microalgae. Moreover, microalgae-supplemented burgers were characterized as having low a* and b* values, which made the color appear to be pale orange. Additionally, thanks to its richness in pigments and polysaccharides, microalgae considerably ameliorated the antioxidant activities of the new prepared fish burgers. Thus, microalgae could be used as natural and nutritious ingredient to develop new fish-based products

Effect of zeolite (clinoptilolite) as feed additive in Tunisian broilers on the total flora, meat texture and the production of omega 3 polyunsaturated fatty acid

Abstract Background Increasing consumer demand for healthier food products has led to the development of governmental policies regarding health claims in many developed countries. In this context, contamination of poultry by food-borne pathogens is considered one of the major problems facing the progress of the poultry industry in Tunisia. Result Zeolite (Clinoptilolites) was added to chicken feed at concentrations 0,5% or 1% and was evaluated for its effectiveness to reduce total flora in chickens and its effects on performance of the production. The broilers were given free and continuous access to a nutritionally non-limiting diet (in meal form)that was either a basal diet or a' zeolite diet' (the basal diet supplemented with clinoptilolite at a level of 0,5% or 1%). It was found that adding zeolite in the broiler diet significantly (p Conclusion This study showed the significance of using zeolite, as a feed additive for broilers, as part of a comprehensive program to control total flora at the broiler farm and to increase level of Omega 3 fatty acid on the chicken body.</p

Quality Characteristics and Functional and Antioxidant Capacities of Algae-Fortified Fish Burgers Prepared from Common Barbel ( Barbus barbus )

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