From fruit growth to ripening in plantain: a careful balance between carbohydrate synthesis and breakdown

We investigated the fruit development in two plantain banana cultivars from two weeks after bunch emergence till twelve weeks through high-throughput proteomics, major metabolite quantification and metabolic flux analyses. We give for the first time an insight at early stages of starch synthesis and breakdown. Starch and sugar synthesis and breakdown are processes that take place simultaneously. During the first eight to ten weeks the balance between synthesis and breakdown is clearly in favour of sugar breakdown and a net starch synthesis occurs. During this period, plantain fruit accumulates up to 48% of starch. The initiation of the ripening process is accompanied with a shift in balance towards net starch breakdown. The key enzymes related to this are phosphoglucan water dikinase (PWD), phosphoglucan phosphatase, α-1,6-glucosidase starch debranching enzyme (DBE), alpha glucan phosphorylase (PHS) and 4-alpha glucanotransferase disproportioning enzyme (DPE). The highest correlations with sucrose have been observed for PHS and DPE. There is also a significant correlation between the enzymes involved in ethylene biosynthesis, starch breakdown, pulp softening and ascorbate biosynthesis. The faster ending of maturation and starting of ripening in the Agbagba cultivar are linked to the key enzymes 1-aminocyclopropane-1-carboxylate oxidase and DPE. This knowledge of the mechanisms that regulate starch and sugar metabolisms during maturation and ripening is fundamental to determine the harvest moment, reduce postharvest losses and improve final product quality of breeding programs.

Enhancing carbohydrate repartitioning into lipid and carotenoid by disruption of microalgae starch debranching enzyme

Light/dark cycling is an inherent condition of outdoor microalgae cultivation, but is often unfavorable for lipid accumulation. This study aims to identify promising targets for metabolic engineering of improved lipid accumulation under outdoor conditions. Consequently, the lipid-rich mutant Chlamydomonas sp. KOR1 was developed through light/dark-conditioned screening. During dark periods with depressed CO2 fixation, KOR1 shows rapid carbohydrate degradation together with increased lipid and carotenoid contents. KOR1 was subsequently characterized with extensive mutation of the ISA1 gene encoding a starch debranching enzyme (DBE). Dynamic time-course profiling and metabolomics reveal dramatic changes in KOR1 metabolism throughout light/dark cycles. During light periods, increased flux from CO2 through glycolytic intermediates is directly observed to accompany enhanced formation of small starch-like particles, which are then efficiently repartitioned in the next dark cycle. This study demonstrates that disruption of DBE can improve biofuel production under light/dark conditions, through accelerated carbohydrate repartitioning into lipid and carotenoid.

Effect of biochar on rice starch properties and starch-related gene expression and enzyme activities

We determined the effects of biochar on starch properties and the activities of enzymes and expression levels of genes related to starch in two Japonica rice cultivars. The two rice varieties were subjected to five biochar treatments (0, control; and 5, 10, 20, and 40 t/hm2). In both rice varieties, the content of apparent amylose and resistant starch were lower in biochar treatments than in the control. The proportion of fa chains was higher and that of fb3 chain was lower in the biochar treatments than in the control. Starch viscosity and cooking taste quality were improved by the biochar treatments. In both rice varieties, the activity of granule-bound starch synthase was significantly decreased by biochar treatments, and the activities of soluble starch synthase, starch branching enzyme, and starch debranching enzyme were significantly increased. The transcript levels of genes encoding starch synthases and starch branching enzymes were significantly increased by biochar treatments. We conclude that biochar at a dose of 5–10 t/hm2 can regulate the activity of starch-related enzymes, and this affects the type, content, and fine structure of starch. Therefore, the addition of biochar to soil can improve the viscosity and taste quality of rice starch.

Structural basis of carbohydrate binding in domain C of a type I pullulanase from Paenibacillus barengoltzii

Pullulanase (EC 3.2.1.41) is a well known starch-debranching enzyme that catalyzes the cleavage of α-1,6-glycosidic linkages in α-glucans such as starch and pullulan. Crystal structures of a type I pullulanase from Paenibacillus barengoltzii (PbPulA) and of PbPulA in complex with maltopentaose (G5), maltohexaose (G6)/α-cyclodextrin (α-CD) and β-cyclodextrin (β-CD) were determined in order to better understand substrate binding to this enzyme. PbPulA belongs to glycoside hydrolase (GH) family 13 subfamily 14 and is composed of three domains (CBM48, A and C). Three carbohydrate-binding sites identified in PbPulA were located in CBM48, near the active site and in domain C, respectively. The binding site in CBM48 was specific for β-CD, while that in domain C has not been reported for other pullulanases. The domain C binding site had higher affinity for α-CD than for G6; a small motif (FGGEH) seemed to be one of the major determinants for carbohydrate binding in this domain. Structure-based mutations of several surface-exposed aromatic residues in CBM48 and domain C had a debilitating effect on the activity of the enzyme. These results suggest that both CBM48 and domain C play a role in binding substrates. The crystal forms described contribute to the understanding of pullulanase domain–carbohydrate interactions.

Colorimetric Determination of the Activity of Starch-Debranching Enzyme via Modified Tollens’ Reaction

Nelson–Somogyi and 3,5-dinitrosalicylic acid (DNS) assays are the classical analytical methods for the determination of activity of starch-debranching enzymes, however, they have a narrow detection range and do not adapt to the quantitative measurement of linear polysaccharides. Herein, we developed a simple and accurate colorimetric assay for determining the activity of starch-debranching pullulanase through the modified Tollens’ reaction in combination with UV irradiation. Silver nanoparticles (AgNPs) were formed by reducing aldehyde groups in short-chain glucans (SCGs) generated by debranching of waxy maize starch using pullulanase through the modified Tollens’ reaction. In addition to providing a reducing moiety to the Tollens’ reaction, the debranching product, SCGs, also enhanced the colloidal stability of synthesized AgNPs, of which the amplitude of its surface plasmon resonance (SPR) absorbance peak was proportional to the concentration of SCGs ranging from 0.01–10 mg/mL. The detection limit of this system was 0.01 mg/mL, which was found to be 100 times higher than that of the conventional DNS assay. The purification of SCGs by recrystallization and gelatinization improved the selectivity of this colorimetric assay for debranching products, which provides a simple and accurate means of monitoring the debranching process and characterizing the activity of starch-debranching enzymes.