Assaying Paenibacillus alvei CsaB-Catalysed Ketalpyruvyltransfer to Saccharides by Measurement of Phosphate Release

Ketalpyruvyltransferases belong to a widespread but little investigated class of enzymes, which utilise phosphoenolpyruvate (PEP) for the pyruvylation of saccharides. Pyruvylated saccharides play pivotal biological roles, ranging from protein binding to virulence. Limiting factors for the characterisation of ketalpyruvyltransferases are the availability of cognate acceptor substrates and a straightforward enzyme assay. We report on a fast ketalpyruvyltransferase assay based on the colorimetric detection of phosphate released during pyruvyltransfer from PEP onto the acceptor via complexation with Malachite Green and molybdate. To optimise the assay for the model 4,6-ketalpyruvyl::ManNAc-transferase CsaB from Paenibacillus alvei, a β-d-ManNAc-α-d-GlcNAc-diphosphoryl-11-phenoxyundecyl acceptor mimicking an intermediate of the bacterium’s cell wall glycopolymer biosynthesis pathway, upon which CsaB is naturally active, was produced chemo-enzymatically and used together with recombinant CsaB. Optimal assay conditions were 5 min reaction time at 37 °C and pH 7.5, followed by colour development for 1 h at 37 °C and measurement of absorbance at 620 nm. The structure of the generated pyruvylated product was confirmed by NMR spectroscopy. Using the established assay, the first kinetic constants of a 4,6-ketalpyuvyl::ManNAc-transferase could be determined; upon variation of the acceptor and PEP concentrations, a KM, PEP of 19.50 ± 3.50 µM and kcat, PEP of 0.21 ± 0.01 s−1 as well as a KM, Acceptor of 258 ± 38 µM and a kcat, Acceptor of 0.15 ± 0.01 s−1 were revealed. P. alvei CsaB was inactive on synthetic pNP-β-d-ManNAc and β-d-ManNAc-β-d-GlcNAc-1-OMe, supporting the necessity of a complex acceptor substrate.

Isolation, sequencing of the HvnHID gene and its role in the purple-grain colour development in Tibetan hulless barley

2-hydroxyisoflavanone dehydratase (HID) plays an important role in isoflavone biosynthesis. In this study, HID was isolated from the seeds of the purple-grained Tibetan hulless barley variety Nerumuzha and the white-grained variety Kunlun 10. The HvnHID gene includes the 981 bp open reading frame and encodes a protein of 327 amino acids. It has a typical Abhydrolase_3 domain (78–306) and belongs to the carboxylesterase (CXE) family of the Abhydrolase_3 (α/β hydrolase) superfamily. There are eight nucleotide differences in the HvnHID coding sequence and two amino acid differences (one in the Abhydrolase_3 domain) between Nerumuzha and Kunlun 10. The HvnHID of hulless barley has the closest relationship with the HID in Hordeum vulgare, and the most distant relationship in Panicum hallii. At the early-mid stage of the seed colour development, the HvnHID expression levels in the purple and black seeds were significantly higher than in the white and blue ones (P < 0.01). During the seed colour development of purple-grained hulless barley, the expression of the key genes (HvnF3'H, HvnDRF, HvnANT1, and HvnGT) in the anthocyanidin biosynthetic pathway increased significantly, while the HvnHID expression decreased significantly (P < 0.01). Thus, it is likely that HvnHID negatively regulates the anthocyanidin biosynthesis. This result provides an important basis for further study of the biological functions of HvnHID in the anthocyanidin biosynthetic pathway.

Investigating the Effect of Fruit Size on Ripening Recovery of Banana Treated with 1-Methylcyclopropene

Postharvest application of 1-methylcycloprepene (1-MCP) on banana fruit to extend shelf-life and maintain quality is inconsistent as treated fruit do not ripen uniformly. Banana response to 1-MCP treatment can be variable due to within-bunch variation in fruit size, composition, and maturity. Therefore, the present study investigated whether fruit size variation explains variability in ripening recovery. To investigate this relationship, large, medium, and small fruit were treated with 0 nL L−1 1-MCP (control), 400 nL L−1 1-MCP and 50 µL L−1 ethephon + 400 nL L−1 1-MCP. Fruit were then ripened using 800 µL L−1 ethephon and stored at 23 °C for 30 d. Irrespective of fruit size, treating banana with 1-MCP and ethephon + 1-MCP prolonged shelf-life by 30 d compared to control, which were fully ripe at 15 d for medium and large fruit, and 20 d for small fruit. 1-MCP significantly delayed yellow colour development (colour stage 4), chlorophyll degradation (97.4 µg/g), and sucrose (2.57 mg/g) and glucose (0.86 mg/g) accumulation in small compared to medium and large fruit. However, firmness (56.13 N) and starch (0.68 mg/g) were significantly lower in 1-MCP-treated small-sized fruit compared to medium and large fruit. Moisture loss was also significantly higher (19.49%) in 1-MCP-treated small fruit compared to medium (14.89%) and large (18.11%). Combined ethephon and 1-MCP allowed for an increase in ripening in small, medium, and large fruit. Overall, medium and large fruit treated with 1-MCP and ethephon + 1-MCP recovered their ripening capacity better compared to small fruit. The results demonstrate that 1-MCP efficacy is influenced by fruit size, whereas ethephon + 1-MCP treatment was consistent across small, medium, and large fruit. The effect of fruit size on 1-MCP efficacy might explain the inconsistency of the treatment in the banana fruit. Therefore, it is important to apply 1-MCP on fruit of approximately the same size to achieve the full benefit of the treatment. Moreover, fruit treated with 1-MCP + ethephon recovered their ripening capacity, irrespective of size, suggesting that it is a beneficial treatment.

Kinetics of Colour Development during Frying of Potato Pre-Treated with Pulsed Electric Fields and Blanching: Effect of Cultivar

The current research aimed to investigate the effect of pulsed electric fields (1 kV/cm; 50 and 150 kJ/kg) followed by blanching (3 min., 100 °C) on the colour development of potato slices during frying on a kinetic basis. Four potato cultivars ‘Crop77’, ‘Moonlight’, ‘Nadine’, and ‘Russet Burbank’ with different content of glucose and amino acids were used. Lightness (L* values from colorimeter measurement) was used as a parameter to assess the colour development during frying. The implementation of PEF and blanching as sequential pre-treatment prior to frying for all potato cultivars was found effective in improving their lightness in the fried products. PEF pre-treatment did not change the kinetics of L* reduction during frying (between 150 and 190 °C) which followed first-order reaction kinetics. The estimated reaction rate constant (k) and activation energy (Ea based on Arrhenius equation) for non-PEF and PEF-treated samples were cultivar dependent. The estimated Ea values during the frying of PEF-treated ‘Russet Burbank’ and ‘Crop77’ were significantly (p < 0.05) lower (up to 30%) than their non-PEF counterparts, indicating that the change in k value of L* became less temperature dependence during frying. This kinetic study is valuable to aid the optimisation of frying condition in deep-fried potato industries when PEF technology is implemented.

Low Oxygen Storage Improves Tomato Postharvest Cold Tolerance, Especially for Tomatoes Cultivated with Far-Red LED Light

We investigated the effects of low oxygen storage on chilling injury development, colour development, respiration and H2O2 levels of ‘Merlice’ tomatoes cultivated with and without far red (FR) LED lighting during 20 days of shelf-life. Mature green (MG) and red (R) tomatoes were stored at 2 °C in combination with 0.5, 2.5, 5 and 21 kPa O2 for 15 days (experiment 1). MG tomatoes cultivated under either white LED or white LED light with FR LED light were stored at 2 °C in combination with 1, 5 and 21 O2 kPa for 14 days (experiment 2). Chilled MG and R tomatoes from experiment 1 showed decay, firmness loss and higher weight loss during shelf-life which were reduced under low oxygen conditions. FR during cultivation improved chilling tolerance of MG tomatoes. Fastest colour development and lowest respiration rate during shelf-life were observed for MG fruit cultivated with FR lighting prior to storage at 1 kPa O2/0 kPa CO2. H2O2 levels during the shelf-life were not affected during cold storage. The improved cold tolerance of MG tomatoes cultivated with FR lighting is likely due to lower oxygen uptake that led to both higher lycopene synthesis and less softening.

New Smartphone Based Colorimetric Method Development and Validation of Emtricitabine in Bulk and Tablet Dosage Form

The novel smartphone based colorimetric application called PhotoMetrix-PRO has been used for quantitative analysis of Emtricitabine in bulk and tablet dosage form and it compared with conventional UV –visible spectrophtotometry. Smartphone based colorimetric method and UV method was based on detection of intensity of color with increasing concentration Ammonium metavanadate (inorganic oxidizing agent) which was used for preparation Mandeline reagent they used as coloring agent for colour development .The reagent is orange red colour but when it react with Emtricitabine it convert into green color complex. The developed methods have good linearity 50-250μg/ml. The color intensity of API increase with increasing concentration. All images were captured through mobile phone and analysed in PhotoMetrix PRO application. This application convert image into Red, Green and Blue(RGB)histogram and regression models were built into PhotoMetrix-PRO Application both the method have correlation coefficient R2>0.995.The LOD and LOQ for PhotoMetrix PRO 4.77μg/ml and14.92μg/ml and UV-visible spectrophotometry method 4.99 μg/ml and 14.96 μg/ml respectively. The % RSD of Emtricitabine by PhotoMetrix PRO and UV method was 99% (%RSD <2). Appling statistical tool i.e paired two test on both the methods result indicate that both methods are equally significant. Keywords: Smartphone, PhotoMetrix PRO, UV Spectrophotometry, RGB

Analysis of Bacterial Community level Physiological Profiling on the Fermentation of Traditional Pliek u using BIOLOG EcoPlates

Pliek u is an Acehnese traditional condiment made from fermented coconut (Cocos nucifera) endosperm. The traditional pliek u fermentation process typically involves a diverse bacterial community. This research aimed to discover the physiological profile of the bacterial community diversity in pliek u fermentation. BIOLOGTM EcoPlates was used to obtain the physiological functions of the bacterial community during the pliek u fermentation process. The bacteria were then isolated from EcoPlate substrate to determine the predominant microorganism. Results from the analysis showed that the value of the Average Well Colour Development (AWCD) increased during the EcoPlates incubation period. The AWCD values in sample IV were higher than the AWCD values in samples I, II, and III. PCA analysis showed that the use of EcoPlate substrate by the bacterial community at the beginning of the fermentation was correlated with the substrate groups of carbohydrate and polymer, and with the substrate groups of carbohydrate and the amino acid at the end of the fermentation. The phylogenetic analysis showed that EC1 had a close relation with Pseudomonas azotoformans strain NBRC, while EC3 had a close relation with Psedomonas lundensis strain ATCC 49968. In conclusion, there were changes in the use of EcoPlate substrate and the activities of the bacterial community during the pliek u fermentation process.

The Biolog EcoPlate™ Technique for Assessing the Effect of Metal Oxide Nanoparticles on Freshwater Microbial Communities

The application of Biolog EcoPlate™ for community-level physiological profiling of soils is well documented; however, the functional diversity of aquatic bacterial communities has been hardly studied. The objective of this study was to investigate the applicability of the Biolog EcoPlate™ technique and evaluate comparatively the applied endpoints, for the characterisation of the effects of metal oxide nanoparticles (MONPs) on freshwater microbial communities. Microcosm experiments were run to assess the effect of nano ZnO and nano TiO2 in freshwater at 0.8–100 mg/L concentration range. The average well colour development, substrate average well colour development, substrate richness, Shannon index and evenness, Simpson index, McIntosh index and Gini coefficient were determined to quantify the metabolic capabilities and functional diversity. Comprehensive analysis of the experimental data demonstrated that short-term exposure to TiO2 and ZnO NPs affected the metabolic activity at different extent and through different mechanisms of action. TiO2 NPs displayed lower impact on the metabolic profile showing up to 30% inhibition. However, the inhibitory effect of ZnO NPs reached 99% with clearly concentration-dependent responses. This study demonstrated that the McIntosh and Gini coefficients were well applicable and sensitive diversity indices. The parallel use of general metabolic capabilities and functional diversity indices may improve the output information of the ecological studies on microbial communities.

Advance in mechanism of plant leaf colour mutation

As a common mutation trait in plants, leaf colour mutation is related to the degree of chlorophyll and anthocyanin changes and the destruction of chloroplast structure. This study summarizes the latest research progress in leaf colour mutation mechanism, including the metabolic basis of plant leaf colour mutation, leaf colour mutation caused by gene mutation in the chlorophyll metabolism pathway, leaf colour mutation caused by blocked chloroplast development, leaf colour mutation controlled by key transcription factors and non-coding RNAs, leaf colour mutation caused by environmental factors, and leaf colour mutation due to the involvement of the mevalonate pathway. These results will lay a theoretical foundation for leaf colour development, leaf colour improvement, and molecular breeding for leaf colour among tree species.