Biochemical characterization of phosphatase, -galactosidase and -mannosidase activities of seeds of an oleaginous cucurbit: Lagenaria siceraria (Molina) Standl blocky-fruited cultivar

Seeds extract of Lagenaria siceraria (Molina) Standl (blocky-fruited cultivar) was screened for enzymatic hydrolytic activities over synthetic variety and natural substrates. The best hydrolytic activities mainly consisted of phosphatase (0.68 ± 0.02 UI/mg), β-galactosidase (0.26 ± 0.03 UI/mg) and α -mannosidase (0.17 ± 0.02 UI/mg). Physicochemicalcharacterization showed that these enzymatic activities were maximal at 55°C in sodium acetate buffer (pHs 4.6 and 5.6). They showed pH and temperature stability and appeared to be resistant in the presence of 5 mM cations (Na+, K+, Ca2+, Ba2+ and Mg2+) concentration and 1% (w/v) detergents (cationic, non-ionic and anionic). The phosphatase activity on different phosphorylated substrates showed it ability to hydrolyze greatly para-nitrophenylphosphate (100 ± 2.3%) and ATP (95.3 ± 2.6%) and in lesser extent sodium phytate (15.2 ± 1.8%). As for natural substrates as lactose and the three different mannobioses linked (α -1,2; α -1,3 α -1,6), that were significantly hydrolyzed by β-galactosidase and α -mannosidase activity respectively. These interesting characteristics deserved to be deeply investigated for the valorisation of Lagenaria siceraria seeds phosphatase, β -galactosidase and α -mannosidase in potential biotechnological applications.Key words: Oleaginous cucurbit, Lagenaria siceraria, blocky-fruited cultivar, seeds, phosphatase activity, β -galactosidase activity, α -mannosidase activity

High throughput automated microbial bioreactor system used for clone selection and rapid scale-down process optimization

High throughput automated fermentation systems have become a useful tool in early bioprocess development. In this study, we investigated a 24 x 15 mL single use microbioreactor system, ambr 15f, designed for microbial culture. We compared the fed-batch growth and production capabilities of this system for two Escherichia coli strains, BL21 (DE3) and MC4100, and two industrially relevant molecules, hGH and scFv. In addition, different carbon sources were tested using bolus, linear or exponential feeding strategies, showing the capacity of the ambr 15f system to handle automated feeding. We used power per unit volume (P/V) as a scale criterion to compare the ambr 15f with 1 L stirred bioreactors which were previously scaled-up to 20 L with a different biological system, thus showing a potential 1,300 fold scale comparability in terms of both growth and product yield. By exposing the cells grown in the ambr 15f system to a level of shear expected in an industrial centrifuge, we determined that the cells are as robust as those from a bench scale bioreactor. These results provide evidence that the ambr 15f system is an efficient high throughput microbial system that can be used for strain and molecule selection as well as rapid scale-up. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 2017

Design and development of a new ambr250® bioreactor vessel for improved cell and gene therapy applications

The emergence of cell and gene therapies has generated significant interest in their clinical and commercial potential. However, these therapies are prohibitively expensive to manufacture and can require extensive time for development due to our limited process knowledge and understanding. The automated ambr250® stirred-tank bioreactor platform provides an effective platform for high-throughput process development. However, the original dual pitched-blade 20 mm impeller and baffles proved sub-optimal for cell therapy candidates that require suspension of microcarriers (e.g. for the culture of adherent human mesenchymal stem cells) or other particles such as activating Dynabeads® (e.g. for the culture of human T-cells). We demonstrate the development of a new ambr250® stirred-tank bioreactor vessel which has been designed specifically to improve the suspension of microcarriers/beads and thereby improve the culture of such cellular systems. The new design is unbaffled and has a single, larger elephant ear impeller. We undertook a range of engineering and physical characterizations to determine which vessel and impeller configuration would be most suitable for suspension based on the minimum agitation speed (NJS) and associated specific power input (P/V)JS. A vessel (diameter, T, = 60 mm) without baffles and incorporating a single elephant ear impeller (diameter 30 mm and 45° pitch-blade angle) was selected as it had the lowest (P/V)JS and therefore potentially, based on Kolmogorov concepts, was the most flexible system. These experimentally-based conclusions were further validated firstly with computational fluid dynamic (CFD) simulations and secondly experimental studies involving the culture of both T-cells with Dynabeads® and hMSCs on microcarriers. The new ambr250® stirred-tank bioreactor successfully supported the culture of both cell types, with the T-cell culture demonstrating significant improvements compared to the original ambr250® and the hMSC-microcarrier culture gave significantly higher yields compared with spinner flask cultures. The new ambr250® bioreactor vessel design is an effective process development tool for cell and gene therapy candidates and potentially for autologous manufacture too

Feedstuffs potential of harvest by-products from two oleaginous curcurbits

Among the generated crop residues and by-products in tropical agriculture, those of cucurbits represent great opportunities for animal nutrition. Nutritive profile of harvest by-products (dried leaves, fermented fruits, non fermented fruits and seeds shells) of two oleaginous cucurbits (Citrullus lanatus and Lagenaria siceraria) were investigated in order to explore their potential use as feedstuffs. The moisture, ash, and crude fibres contents were 4.81 to 12.87, 9.93 to 18.29, and 2.18 to 16.35%, respectively. Shells of L. siceraria seeds yielded the highest carbohydrate content (84.80 ± 2.78 %) while the highest calorific value (380.92 ± 11.40 kcal/100 g) was obtained in C. lanatus bebu. The contents of threonine (Thr), lysine (Lys) and methionine (Met) in dried leaves of C. lanatus bebu were 4.16, 6.86 and 6.89 g/100 g proteins, respectively. The content of methionine (Met) was 5.81 g/100 g proteins in fermented fruits of C. lanatus (wlêwlê). The harvest by-products analyzed in this study contained remarkably high amounts of potassium (671.78 – 4738.79 mg/100 g) and calcium (342.08 – 2963.95 mg/100 g) with highest value (4738.79 ± 230.10; 2963.95 ± 135.74 mg/100 g) for non-fermented fruits of L. siceraria and dried leaves of C. lanatus wlêwlê, respectively. The analyzed plants parts were also notable sources of magnesium, ranging from 221.45 ± 1.96 mg/100 g (non-fermented fruits of L. siceraria) to 872.10 ± 48.49 mg/100 g (dried leaves of C. lanatus wlêwlê). All these results suggest that the studied by-products could be used as valuable feedstuffs. Key words: Harvest by-products, nutritive value, cucurbits, feedstuff

Molecular confirmation of varietal status in bottle gourd (Lagenaria siceraria) using genotyping-by-sequencing

Bottle gourd (Lagenaria siceraria) is an important food, medicinal, and utilitarian crop with a large pan-tropical distribution. Two morphologically different types in the siceraria subspecies are sufficiently different to be considered as varieties, but they are assigned into different taxonomic ranks. Genotyping-by-sequencing (GBS) of 95 different accessions from the Nangui Abrogoua University collection was used to confirm the varietal status in bottle gourd. This analysis produced 22 575 single-nucleotide polymorphisms (SNPs). Cluster analyses conducted with 2250 (9.96%) SNPs distinctly separated hard-shelled from soft-shelled types. Analysis of 23 SNPs located in 11 genes coding for traits that differentiate the two types of gourds revealed that genes in the soft-shelled types had about 21% fewer SNPs than genes within hard-shelled gourds, but the latter had more non-synonymous SNPs. Cluster analyses conducted with the 23 SNPs fitted well with the structure defined by the 2250 SNPs, suggesting the implication of these SNPs in the varietal differentiation of bottle gourd. These nucleotide changes along with the genetic relationships between the accessions provide molecular proof supporting the status of two varieties. To prevent the confusion inherent in the use of synonyms and homonyms in bottle gourd, we suggest the terms hard-shelled and soft-shelled to designate, respectively, the varieties used as utensils and those grown for their edible seeds

On the dispersal of bottle gourd [Lagenaria siceraria (Mol.) Standl.] out of Africa : a contribution from the analysis of nuclear ribosomal DNA haplotypes, divergent paralogs and variants of 5.8S protein sequences

Bottle gourd (Lagenaria siceraria), a multipurpose crop, is among the first domesticates of humans. This study analyses nuclear ribosomal DNA (nrDNA) of the two cultivated subspecies to improve our understanding on the African origin and the dispersal to Asia. A total of 146 nrDNA sequences representing 79 individuals from African cultivars and 67 individuals from Asian cultivars were compared; the resulting nrDNA sequences were composed of 35 and 16 haplotypes specific to Africa and Asia, respectively, and two additional haplotypes shared by both continents. When all the rDNA haplotypes were bulked, the genetic differentiation (F (ST) ) was significant between the subspecies (P < 0.001), within Africa (P < 0.001) and within Asia (P < 0.05), and the nucleotide diversity was 2.5-fold higher in Africa. Sorting the haplotypes by classes of paralogs revealed more classes in Africa, and in classes where African and Asian cultivars were represented, the diversity was higher in Africa, in general. The 5.8S-coding regions showed two to four amino acid differences resulting to nine protein sequence variants, one of which encompassed all the Asian cultivars. The nucleotide diversity at that shared variant was 1.43-fold higher in Africa than in Asia. Analyses of phylogenetic networks revealed major shared haplotypes containing 23.91 % of the cultivars and having founder locations. We suggest that African cultivars reached Asia. The study tags for the first time nrDNA haplotypes capable of discriminating between and within the subspecies. Thirty single nucleotide polymorphisms (SNPs) and five insertion-deletions (Indels) derived from the haplotypes and registered in GenBank are provided