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Apr 2018 DOI 10.14302/issn.2690-4829.jen-18-2025
The goal of this work was to clone, express, characterize and assemble a set of soluble thermostablecellulases capable of significantly degrading cellulose. We successfully cloned, expressed, and purified eleven Clostridium thermocellum (Cthe) cellulases and eight Acidothermuscellulolyticus(Acel) cellulases. The performance of the nineteen enzymes was evaluated on crystalline (filter paper) and amorphous (PASC) cellulose. Hydrolysis products generated from these two substrates were converted to glucose using beta-glucosidase and the glucose formed was determined enzymatically. Ten of the eleven Cthe enzymes were highly active on amorphous cellulose. The individual enzymes all produced <10% reducing sugar equivalents from filter paper. Combinations of Cthe cellulases gave higher conversions, with the combination of CelE, CelI, CelG, and CelK converting 34% of the crystalline cellulose. All eight Acel cellulases showed endo-cellulase activity and were highly active on PASC. Only Acel_0615 produced more than 10% reducing sugar equivalents from filter paper, and a combination of six Acel cellulases produced 32% conversion. Acel_0617, a GH48 exo-cellulase, and Acel_0619, a GH12 endo-cellulase, synergistically stimulated cellulose degradation by the combination of Cthe cellulases to almost 80%. Addition of both Acel enzymes to the Cthe enzyme mix did not further stimulate hydrolysis. Cthe CelG and CelI stimulated cellulose degradation by the combination of Acel cellulases to 66%.
Dec 2019 DOI 10.14302/issn.2690-4829.jen-19-3105
Trichoderma reeseiβ-glucosidase (Bgl1) is one of four enzymes demonstrated to act synergistically to degrade cellulose both in vitro and in vivo. Our work attempted to better understand the substrate specificity and potential biotechnological applications of Bgl1. T. reesei Bgl1H cleaves over 80% of the β-(1-4) and β-(1-3) linkages in β-glucan and 14% of the β-(1-4) linkages in amorphous cellulose, significantly more than any tested bacterial β-glucosidase. Bgl1H cleaves 50% of the β-(1-4) linkages in xyloglucan when supplemented with cellulase and α-xyloside. Approximately 20% conversion to glucose was obtained from insoluble β-(1,3)-linked curdlan using only Bgl1H; addition of a curdlanase resulted in conversion of approximately 70% of the curdlan to glucose. Bgl1H also produces xylose from xylooligosaccharides and debranched xylans. For both glucans and xylans, the relative rates of hydrolysis increase with increasing polysaccharide chain lengths. Bgl1H is able to partially degrade β-glucan in a variety of grain components; addition of endo-acting enzymes improved the enzyme’s performance on these grain components. The ability of this enzyme to produce monosaccharides from undigestible polysaccharides suggest it may have potential in improving utilization of carbohydrates in animal feed, fermentations, and other biotechnological applications.
Mar 2019 DOI 10.14302/issn.2471-2140.jaa-19-2699
Xanthine oxidase is a commercially important enzyme with wide area of medical applications to develop diagnostic kits. Xanthine oxidase was extracted, purified and characterized from sheep liver (SLXO). The purification procedure involved acetone precipitation and chromatography on DEAE-cellulose and Sephacryl S-300 columns. The sheep liver xanthine oxidase was homogeneously purified 31.8 folds with 3.5 U/mg specific activity and 24.1% recovery. SLXO native molecular weight was 150 kDa and on SDS-PAGE appeared as single major band of 75 kDa representing a homodimer protein. Isoelectric focusing of the purified SLXO resolved into two closely related isoforms with pI values of 5.6 and 5.8. The apparent Km for xanthine oxidase at optimum pH 7.6 was found to be 0.9 mM xanthine. FeCl2 and NiCl2 increased the activity of SLXO, while CuCl2 and ZnCl2 were found to be potent inhibitors of the purified enzyme. Allopurinol inhibits SLXO competitively with one binding site on the purified molecule and Ki value of 0.06 mM.
Dec 2018 DOI 10.14302/issn.2694-2275.jzr-18-2474
Guinea pig diet is essentially based on the use of grasses associated with protein sources. Then, in to improve the herbivores nutrition, the evaluation of intake and In vivo digestibility of Pennisetum purpureum or Panicum maximum in guinea pigs (Cavia porcellus) was carried out in January 2017 at the University of Dschang research and experimental farm and animal production and nutrition laboratory. 20 adult animals of local breed on average weight 450 ± 50g and aged about 5 months were used. They were organized into two batches of 10 animals each (5 males and 5 females). The first batch received 250g of fresh matter of Pennisetum purpureum + 60g concentrate/animal/day, while the second received the same treatment with Panicum maximum as grasses. Leftover and animals were weighed every morning before food distributions, and drinking water containing vitamin C was served ad libitum. According to the results, the highest ingestion was obtained with Panicum maximum. However, the digestibility different nutrients was comparable between treatments; nevertheless, males fed on P. maximum digested crude cellulose (63.78%) better than males fed on P. purpureum (51.17%). With regard to the variation of the bacterial rate of the caecal flora, enterobacteria of animals fed P. maximum was high (11.20 CFU/ml) compared to those of the animals receiving P. purpureum (7.27 CFU/ml), although regardless of the treatment, the level of lactobacilli was higher than that of enterobacteria. In view of the results obtained, these grasses can be alternatively used in feeding guinea pigs.
Aug 2018 DOI 10.14302/issn.2639-3166.jar-18-2264
The management of symbiotic Microbial Biota (MB) in the soil as agents that promote the yield and health of crops, is aimed at inducing modifications of the phenotype of plants, both over and under the ground. It is here shown, in Sorghumsudanensis plants, that: i) a simple response to MB inoculation is the result of the fall out of the raw pH; ii) the simple NIR scans of leaves can be considered to rapidly classify the outcomes; iii) the raw pH can be considered a key-variable of leaf modifications. An experiment was carried out on Sorghumsudanensis. The plants were seeded in pots and grown for 66 d, and then a control non-inoculated group (C) was compared with thirteen Arbuscular Mycorrhizae (AM) Glomus inoculated groups and with two commercial MB products. A total of 374 raw pH measurements conducted on the leaves showed that the 5.18 pH units in the C group were scaled by -1.9% (P<0.0336) in the MB group and by -3.4% in the AM group (P<0.0001), with a relevant diversity between groups. Direct discrimination of these three groups, by means of smart NIR-SCIO, showed a % reclassification of the C, MB and AM groups of 74%, 59% and 96% in the fresh leaves and of 65%, 51% and 94% in the dried ground leaves, respectively. The composition of the dried leaves, based on a set of 14 variables predicted via NIRS models, plus the total foliar dry weight and percentage, showed a typical increase in protein, ash and hemicellulose, and a typical decrease in the cellulose, dry matter, crude fiber and crop maturity index. These variables were related to the foliar pH, as a key-variable, by means of a PLS standard model (R2 0.81) in which a low pH steadily favored the dry mass weight and, to a lesser extent, the hemicellulose and the digestible NDF contents; on the other hand, a high pH increased the dry matter percentage and the cellulose content of the leaves. As expected, the leaves of the inoculated plants showed a more juvenile ontogenic status. The epigean botanical modifications can be considered harmonic expressions of a luxuriant symbiosis, as testified by the homologous NIR categorization. The outlook for a symbiotic agriculture, with mycorrhizal plants, should consider the raw pH as a multifaceted variable.
Feb 2014 DOI 10.14302/issn.2328-0182.japst-13-206
The purpose of this study was to investigate the effect of cremophor RH-40 and polysorbate 80 with hydroxypropyl methylcellulose (HPMC) F4M on the development of formulations of intranasal erythropoietin with low sialic acid content (Neuro-EPO) as a neuroprotective agent. Parameters such as pH, osmolality, apparent viscosity, and protein concentration were controlled for minimizing the differences between formulations. All Neuro-EPO formulations showed similar behaviour in the physicochemistry quality control. However significant differences between formulations were observed in the permanent unilateral ischemia model. The formulations and the vehicles containing cremophor RH-40 showed higher neurotoxicity levels than those containing polysorbate 80 as a nonionic surfactant. Formulations containing HPMC F4M at 0.6% as a bioadhesive polymer showed higher levels of survival and better neurological status than those without the polymer. The formulations with polysorbate 80 and HPMC F4M showed a higher index of survival, smaller incidence of clinical signs of stroke, and similar behavior in the learning and the memory to the false injured animals used as control. These findings suggest that the intranasal pathway constitutes a safe and alternative route of access of the Neuro-EPO to the brain.