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Maltodextrin glucosidase (MalZ) is a key enzyme in the maltose utilization pathway in Escherichia coli that liberates glucose from the reducing end of the short malto-oligosaccharides. Unlike other enzymes in the GH13_21 subfamily, the hydrolytic activity of MalZ is limited to maltodextrin rather than long starch substrates, forming various transglycosylation products in α-1,3, α-1,4 or α-1,6 linkages. The mechanism for the substrate binding and hydrolysis of this enzyme is not well understood yet. Here, we present the dimeric crystal structure of MalZ, with the N-domain generating a unique substrate binding groove. The N-domain bears CBM34 architecture and forms a part of the active site in the catalytic domain of the adjacent molecule. The groove found between the N-domain and catalytic domain from the adjacent molecule, shapes active sites suitable for short malto-oligosaccharides, but hinders long stretches of oligosaccharides. The conserved residue of E44 protrudes at subsite +2, elucidating the hydrolysis pattern of the substrate by the glucose unit from the reducing end. The structural analysis provides a molecular basis for the substrate specificity and the enzymatic property, and has potential industrial application for protein engineering.

Maltogenic amylase CoMA from Corallococcus sp. strain EGB catalyzes the hydrolysis and transglycosylation of maltooligosaccharides and soluble starch into maltose, the sole hydrolysate. This process yields pure maltose with potentially wide applications. Here, we identified and evaluated the role of Phenylalanine 314 (F314), a key amino acid located near the active center, in the catalytic activities of the CoMA. Site-directed mutagenesis analysis showed that the activity of a F314L mutant on potato starch substrate decreased to 26% of that of wild-type protein. Compared with the wild-type, F314L exhibited similar substrate specificity, hydrolysis pattern, pH, and temperature requirements. Circular dichroism spectrum data showed that the F314L mutation did not affect the structure of the folded protein. In addition, kinetic analysis demonstrated that F314L exhibited an increased Km value with lower substrate affinity. Homology modeling showed that the benzene ring structure of F314L was involved in π-π-conjugation, which might potentially affect the affinity of CoMA towards starch. Taken together, these data demonstrated that F314 is essential for the hydrolytic activity of the CoMA from Corallococcus sp. strain EGB. This article is protected by copyright. All rights reserved.

Abiotic stressors, such as drought and high salinity, seriously affect plant growth, productivity, and quality. Maintaining reactive oxygen species (ROS) homeostasis and osmotic balance plays a crucial role in abiotic stress tolerance. β-amylase (BAM) hydrolyzes α-1,4-glycosidic bonds by releasing maltose from starch in the regulation of soluble sugars. However, the function and mechanism of BAMs related to abiotic stress resistance remain unclear in sweetpotato (Ipomoea batatas (L.) Lam.). In this study, we isolated a novel β-amylase gene IbBAM1.1, which was strongly induced by PEG6000, NaCl, and maltose treatments in sweetpotato variety Yanshu25. Overexpression of IbBAM1.1 conferred enhanced tolerance to the drought and high salinity stressors in Arabidopsis thaliana. The activity of β-amylase and the degradation of starch were promoted under drought or salt stress. Accordingly, the contents of osmoprotectants, including maltose and proline were significantly higher in the transgenic lines than those in wild type (WT) plants. Less ROS, such as H2O2 and O2−, accumulated in the overexpressing lines than in WT plants. Superoxide dismutase activity was strongly enhanced and the level of malondialdehyde was lower under the drought or salt treatment in transgenic plants. Taken together, these results demonstrate that IbBAM1.1 acted as a positive regulator, at least in part, by regulating the level of osmoprotectants to balance the osmotic pressure and activate the scavenging system to maintain ROS homeostasis in the plants.

Trehalose is a non-reducing disaccharide in increasing demand for applications in food, nutraceutical, and pharmaceutical industries. Single-step trehalose production by trehalose synthase (TreS) using maltose as a starting material is a promising alternative process for industrial application due to its simplicity and cost advantage. Pseudomonas monteilii TBRC 1196 was identified using the developed screening method as a potent strain for TreS production. The TreS gene from P. monteilii TBRC 1196 was first cloned and expressed in Escherichia coli. Purified recombinant trehalose synthase (PmTreS) had a molecular weight of 76 kDa and showed optimal pH and temperature at 9.0 and 40°C, respectively. The enzyme exhibited >90% residual activity under mesophilic condition under a broad pH range of 7-10 for 6 h. Maximum trehalose yield by PmTreS was 68.1% with low yield of glucose (4%) as a byproduct under optimal conditions, equivalent to productivity of 4.5 g/l/h using enzyme loading of 2 mg/g substrate and high concentration maltose solution (100 g/l) in a lab-scale bioreactor. The enzyme represents a potent biocatalyst for energy-saving trehalose production with potential for inhibiting microbial contamination by alkaline condition.

Background and objectives Whether iron supplementation in patients on hemodialysis could be delivered by less frequent but higher single doses compared with the currently more common higher-frequency schedules of lower single iron doses is unknown. Design, setting, participants, & measurements We carried out an open-label, randomized, controlled noninferiority trial over 40 weeks in patients on prevalent hemodialysis (n=142). We administered in total 2 g iron as 100 mg iron sucrose biweekly in a continuous (20 × 100 mg) fashion or 500 mg ferric carboxymaltose every 10 weeks in a periodic (4 × 500 mg) fashion. The primary end point was the change in hemoglobin at week 40 from baseline with a noninferiority margin of -0.8 g/dl. Secondary end points were changes in ferritin, transferrin, transferrin saturation, and erythropoiesis-stimulating agent use. Results In total, 108 patients completed the study. At 40 weeks, hemoglobin changed by -0.27 g/dl (95% confidence interval, -0.64 to 0.09) in the iron sucrose arm and by -0.74 g/dl (95% confidence interval, -1.1 to -0.39) in the ferric carboxymaltose arm compared with baseline. Noninferiority was not established in the per-protocol population as hemoglobin changes compared with baseline differed by -0.47 g/dl (95% confidence interval, -0.95 to 0.01) in the ferric carboxymaltose arm compared with the iron sucrose arm. Proportional changes from baseline to week 40 differed by -31% (98.3% confidence interval, -52 to -0.1) for ferritin, by 1% (98.3% confidence interval, -7 to 10) for transferrin, and by -27% (98.3% confidence interval, -39 to -13) for transferrin saturation in the ferric carboxymaltose arm compared with the iron sucrose arm. Erythropoiesis-stimulating agent dosing did not differ between groups. The overall number of adverse events was similar; however, more infections were observed in the iron sucrose arm. Conclusions An equal cumulative dose of ferric carboxymaltose administered less frequently did not meet noninferiority for maintaining hemoglobin levels compared with iron sucrose administered more frequently. Clinical trial registry name and registration number Comparison Study of Two Iron Compounds for Treatment of Anemia in Hemodialysis Patients (COPEFER), NCT02198495 PODCAST: This article contains a podcast athttps://www.asn-online.org/media/podcast/CJASN/2021_09_27_CJN03850321.mp3.

The major activity of β-amylase (BMY) is the production of maltose by the hydrolytic degradation of starch. BMY is found to be produced by some plants and few microorganisms only. The industrial importance of the enzyme warrants its application in a larger scale with the help of genetic engineering, for which the regulatory mechanism is to be clearly understood. In plants, the activities of BMY are regulated by various environmental stimuli including stress of drought, cold and heat. In vascular plant, Arabidopsis sp. the enzyme is coded by nine BAM genes, whereas in most bacteria, BMY enzymes are coded by the spoII gene family. The activities of these genes are in turn controlled by various compounds. Production and inhibition of the microbial BMY is regulated by the activation and inactivation of various BAM genes. Various types of transcriptional regulators associated with the plant- BMYs regulate the production of BMY enzyme. The enhancement in the expression of such genes reflects evolutionary significance. Bacterial genes, on the other hand, as exemplified by Bacillus sp and Clostridium sp, clearly depict the importance of a single regulatory gene, the absence or mutation of which totally abolishes the BMY activity.

A microalgal strain Micractinium reisseri FM1 which could assimilate organic carbon sources effectively and grow rapidly under mixotrophic condition was screened and identified through morphological and 18S rRNA gene sequence. Addition of glucose, maltose and sodium acetate at 2 and 4 g L−1 contents could promote the biomass production, lipid content and productivity significantly. Compared with batch culture (8 g L−1), fed-batch culture (fed 2 g L−1 4 times) with glucose, maltose and sodium acetate respectively, could not only enhance biomass, but also boost lipid productivity. More importantly, the composition of fatty acid could be enriched and the proportion of saturated to unsaturated fatty acids could be improved on the basis of promoting alga growth and lipid production by feeding organic carbon and regulating culture mode. M. reisseri FM1 has the potential to assimilate organic carbon sources for high-cell density cultivation and production of biodiesel or functional polyunsaturated fatty acids.

Background and Objective: Cardiovascular Diseases (CVDs) remain the main cause of mortality globally. High cholesterol levels (hypercholesterolemia) and high blood glucose (diabetes) are among the factors that increase the risk for CVDs. Application of inhibitors for the digestive enzymes accountable for the macronutrient hydrolysis, such as carbohydrates and fats, is one of the prevalent approaches in the development of medications against CVDs. The present study was performed to examine, in vitro, the lipase and amylase inhibitory potential of phenolic rich extract of leaves of four date palm cultivars. Materials and Methods: In the current study, the research investigated the potentiality of phytochemicals extracted from leaves of four date palm cultivars (Rawthan, Rabeaa, Barny and Ajwa), collected from Al-Madinah Governorate as lipase and amylase inhibitors and as antioxidants. Moreover, the total contents of flavonoids and phenolics were assessed. Results: The results revealed that all the tested cultivars showed promising lipase and amylase inhibition and antioxidants capacities. However, Rawthan and Ajwa were the most powerful cultivars. Conclusion: Therefore, the results presented herein suggest as the earliest report, the potential use of date palm leaves as a potential source for lipase and amylase inhibitors as an approach to decrease the risk for CVDs.

l-Menthyl α-D-glucopyranoside (α-MenG) is a glycoside derivative of l-menthol with improved water-solubility and new flavor property as a food additive. α-MenG can be synthesized through biotransformation, but its scale-up production was rarely reported. In this study, the properties of an α-glucosidase from Xanthomonas campestris pv. campestris 8004 (Agl-2) in catalyzing the glucosylation of menthol was investigated. Agl-2 can almost completely glycosylate l-menthol (> 99%) when using 1.2 M maltose as glycosyl donor. Accumulated glucose resulted from maltose hydrolysis and transglycosylation caused the inhibition of the glucosylation rate (40% reduction of the glucosylation rate in the presence of 1.2 M glucose) which can be avoided through whole-cell catalysis with recombinant E. coli. Interestingly, in spite of the poor solubility of menthol, the productivity of α-MenG reached 24.7 g/(L·h) in a 2 L catalyzing system, indicating industrialization of the reported approach.

The α-amylase paralogue Amyrel present in true flies (Diptera Muscomorpha) has been classified as a glycoside hydrolase in CAZy family GH13 on the basis of its primary structure. Here, we report that, in fact, Amyrel is currently unique among animals as it possesses both the hydrolytic α-amylase activity (EC 3.2.1.1) and a 4-α-glucanotransferase (EC 2.4.1.25) transglycosylation activity. Amyrel reacts specifically on α-(1–4) glycosidic bonds of starch and related polymers but produces a complex mixture of maltooligosaccharides, which is in sharp contrast with canonical animal α-amylases. With model maltooligosaccharides G2 (maltose) to G7, the Amyrel reaction starts by a disproportionation leading to Gn − 1 and Gn + 1 products, which by themselves become substrates for new disproportionation cycles. As a result, all detectable odd- and even-numbered maltooligosaccharides, at least up to G12, were observed. However, hydrolysis of these products proceeds simultaneously, as shown by p-nitrophenyl-tagged oligosaccharides and microcalorimetry, and upon prolonged reaction, glucose is the major end-product followed by maltose. The main structural determinant of these atypical activities was found to be a Gly-His-Gly-Ala deletion in the so-called flexible loop bordering the active site. Indeed, engineering this deletion in porcine pancreatic and Drosophila melanogaster α-amylases results in reaction patterns similar to those of Amyrel. It is proposed that this deletion provides more freedom to the substrate for subsites occupancy and allows a less-constrained action pattern resulting in versatile activities at the active site.

We expressed a bacterial glucan synthase (Agrobacterium GlgA) in the cytosol of developing endosperm cells in wheat grains, to discover whether it could generate a glucan from cytosolic ADP-glucose. Transgenic lines had high glucan synthase activity during grain filling, but did not accumulate glucan. Instead, grains accumulated very high concentrations of maltose. They had large volumes during development due to high water content, and very shrivelled grains at maturity. Starch synthesis was severely reduced. We propose that cytosolic glucan synthesized by the glucan synthase was immediately hydrolysed to maltose by cytosolic β-amylase(s). Maltose accumulation resulted in a high osmotic potential in developing grain, drawing in excess water that stretched the seed coat and pericarp. Loss of water during grain maturation then led to shrinkage when the grains matured. Maltose accumulation is likely to account for the reduced starch synthesis in transgenic grains, through signalling and toxic effects. Using bioinformatics, we identify an isoform of β-amylase likely to be responsible for maltose accumulation. Removal of this isoform through identification of TILLING mutants or genome editing, combined with co-expression of heterologous glucan synthase and a glucan branching enzyme, may in future enable elevated yields of carbohydrate through simultaneous accumulation of starch and cytosolic glucan.

The efficiency of enzymatic cyclodextrin production using cyclodextrin glycosyltransferases (CGTases) is limited by product inhibition. In this study, maltose binding site 2 (MBS2) of the β-CGTase from Bacillus circulans STB01 was modified to decrease product inhibition. First, two point mutants were prepared at position 599 (A599V and A599N). Then, two double mutants incorporating alanine at position 633 (A599N/Y633A and A599V/Y633A) were prepared. Finally, the entire MBS2 region was replaced by that of the α-CGTase from Paenibacillus macerans JFB05-01 to form multipoint mutant MBS2 β → α. All five mutants exhibited mixed-type product inhibition, although both the competitive and uncompetitive components of this inhibition were decreased. The total cyclization activities of A599N, A599V and A599V/Y633A were 15.6%, 76.8% and 70.9% lower than that of the wild-type, respectively, while that of A599N/Y633A was 22.4% higher. Among the mutants, only MBS2 β → α showed catalytic efficiency (kcat/Km) comparable with that of the wild-type. Moreover, A599N, A599N/Y633A and MBS2 β → α produced cyclodextrin yields 13.1%, 15.8% and 19.7% greater than that of the wild-type, respectively. These results suggest that A599N, A599N/Y633A and MBS2 β → α may be more suitable than the wild-type for cyclodextrin production.

Background The level of available carbohydrates in our diet is directly linked to two major diseases: obesity and Type II diabetes. Despite this, to date there is no method available to allow direct and accurate measurement of available carbohydrates in human and animal foods. Objective The aim of this research was to develop a method that would allow simple and accurate measurement of available carbohydrates, defined as non-resistant starch, maltodextrins, maltose, isomaltose, sucrose, lactose, glucose, fructose, and galactose. Method Non-resistant (digestible) starch is hydrolyzed to glucose and maltose by pancreatic α-amylase (PAA) and amyloglucosidase at pH 6.0 with shaking or stirring at 37°C for 4 h. Sucrose, lactose, maltose, and isomaltose are completely hydrolyzed by specific enzymes to their constituent monosaccharides, which are then measured using pure enzymes in a single reaction cuvette. Results A method has been developed that allows the accurate measurement of available carbohydrates in all cereal, vegetable, fruit, food, and feed products, including dairy products. Conclusions A single-laboratory validation was performed on a wide range of food and feed products. The inter-day repeatability (RSDr, %) was Highlights A unique method has been developed for the direct measurement of available carbohydrates, entailing separate measurement of glucose, fructose, and galactose, information of value in determining the glycemic index of foods.

The investigation of carbohydrate recognition in a natural environment suffers from the complexity of overlapping functional effects such as multivalency and heteromultivalency effects. Another key factor in carbohydrate recognition is the presentation mode of glycoligands in three-dimensional (3D) space. In order to trace out the effect of 3D ligand presentation, we utilized an oligosaccharide model to precisely control the spatial relation between a mannose ligand (Man) and a glucose moiety (Glc). A disaccharide (maltose) served as a scaffold to alternately conjugate Man and Glc at position 6 and 6' of a synthetic maltoside, resulting in a pair of regioisomeric heterobivalent glycoclusters. The biological effect of this specific structural tuning was tested in a native system employing mannose-specific adhesion of live E. coli cells. Indeed, the variable 3D presentation of the Man ligand resulted in a 2-fold difference between the regioisomeric heterobivalent glycoclusters as inhibitors of bacterial adhesion. This can be considered a remarkable effect, which could be interpreted by computer-aided modelling of the complexes between the bacterial lectin and the synthetic regioisomeric glycoligands.

Single site OH → F substitution at the termini of maltotetraose leads to significantly improved hydrolytic stability towards α-amylase and α-glucosidase relative to the natural compound. To explore the effect of molecular editing, selectively modified oligosaccharides were preparedviaa convergent α-selective strategy. Incubation experiments in purified α-amylase and α-glucosidase, and in human and murine blood serum, provide insight into the influence of fluorine on the hydrolytic stability of these clinically important scaffolds. Enhancements ofca. 1 order of magnitude result from these subtle single point mutations. Modification at the monosaccharide furthest from the probable enzymatic cleavage termini leads to the greatest improvement in stability. In the case of α-amylase, docking studies revealed that retentive C2-fluorination at the reducing end inverts the orientation in which the substrate is bound. A co-crystal structure of human α-amylase revealed maltose units bound at the active-site. In view of the evolving popularity of C(sp3)-F bioisosteres in medicinal chemistry, and the importance of maltodextrins in bacterial imaging, this discovery begins to reconcile the information-rich nature of carbohydrates with their intrinsic hydrolytic vulnerabilities. © The Royal Society of Chemistry 2020. Western Washington University

Introduction: Immobilization of the whole cell is a modern approach to fermentative production of different enzymes. The technique has several advantages including easy separation of the fermented product, least chance of contamination and no chance of wash out of cells during product recovery. Materials and Methods: ?-amylase produced from Calcium alginate entrapped cells of Bacillus thuringiensis A5-BRSC was purified by ammonium sulphate precipitation, followed by DE-52 ion exchange column chromatography. The molecular weight of the purified enzyme was determined by SDS-PAGE. Study of enzyme kinetics was performed to determine its enzymatic potency. Results: Immobilization of Bacillus thuringiensis A5-BRSC cells in calcium alginate resulted in more than 3.5 times higher ?-amylase activity than free cells within 48 hours of batch fermentation. The molecular weight of the purified enzyme was estimated to be 52 kDa by SDS-PAGE. The enzyme exhibited pH optima at 6.9 and temperature optima at 500C. KM and Vmax of the purified enzyme was determined to be 0.85 mg/ml and 0.98 mg of liberated maltose/ml of enzyme/minute. Conclusion:?-amylase produced form the immobilized cells found to be more potent than the enzyme produced by the free cells. Keywords:Cell immobilization, Bacillus thuringiensis, ?-amylase, Column chromatography

Alpha-amylase producing strain KB 2216 was identified as Bacillus velezensis. The growth pattern showed that 72 h is the optimum incubation period of amylase production, which is a stationary period for the strain. By the purification process, maximum alpha-amylase activity was achieved up to 418.25 U/mL at 72 h of incubation, which was purified with 4.74 folds, 4230.32 U/mg specific activity, with 31.35% yield. The strain was found to produce an oligomeric alpha-amylase, namely Amy3. Amy3 was a trimeric macromolecule of 195 kDa with 62, 64, and 66 kDa subunits, as revealed by zymogram and SDS PAGE analyses. Amy3 was highly active at 55 °C and pH 5.5. It had shown the highest stability at pH 5.0–5.5 and between 0 C and 4 C. It did not require any metal cofactors, but it was inhibited by Ag2+, Hg2+ and Cd2+ divalent cations. Glucose and maltose were shown to be the end products of soluble starch digestion by Amy3. These interesting properties of Amy3 may be useful for many biotechnological applications in the future.

Background: The reports about valuable oligosaccharides in ginseng are quite limited. There is an urgent need to develop a practical procedure to detect and analyze ginseng oligosaccharides. Methods: The oligosaccharide extracts from ginseng were permethylated by solid-phase methylation method and then were analyzed by ultra-high-performance liquid chromatography-Q-Orbitrap/MS. The sequence, linkage, and configuration information of oligosaccharides were determined by using accurate m/z value and tandem mass information. Several standard references were used to further confirm the identification. The oligosaccharide composition in white ginseng and red ginseng was compared using a multivariate statistical analysis method. Results: The nonreducing oligosaccharide erlose among 12 oligosaccharides identified was reported for the first time in ginseng. In the comparison of the oligosaccharide extracts from white ginseng and red ginseng, a clear separation was observed in the partial least squares-discriminate analysis score plot, indicating the sugar differences in these two kinds of ginseng samples. The glycans with variable importance in the projection value large than 1.0 were considered to contribute most to the classification. The contents of oligosaccharides in red ginseng were lower than those in white ginseng, and the contents of maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, maltooctaose, maltononaose, sucrose, and erlose decreased significantly (p

The catalytic depolymerization of biological enzymes during biological liquefaction of coal is very important. In this study, the effects of medium types, lignite addition, and mixed culture on lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase (Lac) secreted by white-rot fungi are investigated. Fifteen strains of white-rot fungi are used for microbial liquefaction of the Zhaotong lignite in Yunnan. The results show that the activities of LiP, MnP, and Lac are highest in the Czapek-Dox medium (DOX), followed by the Sabouraud Maltose Broth (SMB) medium, and are almost zero in the Potato Dextrose Agar (PDA) medium. In the early stage of fungal growth, the medium contains sufficient nutrients, and lignite addition induces an inhibitory effect on extracellular enzyme secretion, whereas in the late growth stage characterized by nutrient deficiency, it exhibits an enhancing effect. For fungi pair combinations, if only single enzyme activity was considered, the positive synergistic effect of Lac in the mixed culture of s.commune and SL203 would be the highest, with the synergistic coefficient of 233.33. While three enzymes were considered, the integrated effects of ZTW07 and SL203 are the highest, with Lac, MnP, and LiP producing synergistic coefficients of 158.795, 1.217, and 1.006, respectively. It can be seen that the mixed culture can greatly improve the enzyme activity of white rot fungi and is one of the effective methods to improve the liquefaction efficiency.

Objectives The main aim of this study is to isolate and characterize fibrinolytic enzyme from Bacillus flexus. Methods Fish meal of Sardinella longiceps and anchovy was optimized using a two-level full factorial design (25) and response surface methodology. The significant physical factors and nutrient sources (peptone, maltose, and magnesium chloride) were identified by statistical approach. The properties of a purified enzyme including their effect at different temperature, pH and the effect of metal ions were evaluated. Results Enzyme yield was improved 3.5 fold than unoptimized medium. Central composite design optimized culture medium enhanced enzyme yield (4711 ± 29.3 U/g of substrate). The fibrinolytic enzyme was highly active at alkaline pH (8.0), 50 °C and the molecular weight was 32 kDa. Conclusions From these findings, it concludes that this fibrinolytic enzyme could be a novel potent thrombolytic agent.

Methods have been reported for the preparation of ω-functionalized alkyl maltoside and glycoside detergents via a simple and inexpensive synthetic route. The key step was stannic chloride-mediated glycosylation of long-chain alcohols or thiols with maltose octaacetate at 0 or –10°C, respectively, within a very short time (isolated yield 17–44%), which provided more than 98% β-selectivity.

Pullulanase is a debranching enzyme that cleaves explicitly α-1,6 glycosidic bonds, which is widely used in starch saccharification, production of glucose, maltose, and bioethanol. The thermal-resistant pullulanase is isolated from a variety of microorganisms; however, the lack of industrial production of pullulanase has hindered the transformation of the laboratory to industry. In this study, the expensive maltose syrup and soybean meal powder were replaced with cheap corn starch and corn steep liquor, exhibiting 440 U/mL of pullulanase in shake flasks by changing the C/N value and the total energy of the medium. Subsequently, the cultivation conditions were explored in a 50-L and 50-m3 bioreactor. In batch culture, the pullulanase activity reached 896 U/mL, while it increased to 1743 U/mL in fed-batch culture by controlling the dissolved oxygen, pH, reducing sugar content, and temperature. Remarkably, the cultivation volume was enlarged to 50 m3 based on the technical parameters of fed-batch culture. The industrial production of pullulanase was successful, and the activity achieved 1546 U/mL. When the product was stored at room temperature (25 °C) for 6 months, the pullulanase activity was over 90%. The half-lives at 60 and 80 °C were 119.45 h and 51.18 h, respectively, which satisfied the industrial application requirements of pullulanase.

In this experiment, the conjugation reaction between gluten and maltose via Maillard reaction under dry‐heated condition was studied. The process conditions for the preparation of protein–maltose conjugates with optimum solubility were optimized by using Box‐Behnken model. The conjugation reaction and the structure changes of the protein–maltose conjugates were confirmed by infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The results showed that the process conditions for the preparation of protein–maltose conjugates with optimum solubility were as follows: temperature 50.72°C, time 1.92 days, and gluten/maltose (W/W) 267.36%. The infrared spectroscopy showed that the structure of the modified protein had a very obvious change, including the decrease in β‐fold and β‐turn and the increase in α‐helix at a certain degree. But the conjugation reaction has little effect on the irregular coiled structure. The scanning electron microscopy showed that the microstructure of gluten is small grainy, but gluten–maltose conjugate looks sheet with bigger volume., In this experiment, gluten was modified through by glycosylation. The solubility of modified gluten was improved. The structure of modified gluten has changed that confirmed by infrared spectroscopy and scanning electron microscopy.

In this study, water-soluble polysaccharide and fatty acids of Cordia myxa fruit harvested from the southwest of Iran were isolated and investigated. The extraction yield of water-soluble polysaccharide of C. myxa fruit (WSPCM) was 24.12%, and its experimental purity was 79.43%. The High Performance Liquid Chromatography results indicated that the WSPCM consisted of glucose, fructose, sucrose, maltose, ribose, maltitol, and maltoheptaose. The atomic absorption spectrometer analysis indicated that WSPCM was a rich source of Ca and Na. The dynamic light scattering results illustrated that the average particle size and zeta potential were 1510.7 nm and − 64.96, respectively. Fourier transform infrared spectroscopy revealed the presence of α and β-configurations in WSPCM. The total unsaturated and saturated fatty acids were 60.62% and 39.38%, respectively and oleic, palmitic, and linoleic were the main fatty acids. The WSPCM represented good antibacterial activities against both Gram-positive and Gram-negative pathogenic bacteria. Moreover, it showed a relatively good antioxidant activity. In conclusion, WSPCM could be introduced as a novel natural antioxidant and antibacterial substance in functional food.

1-Deoxynojirimycin (1-DNJ) is the major effective component of mulberry leaves, exhibiting inhibitory activity against α-glucosidase. However, due to the low content of 1-DNJ in mulberry products, its level cannot meet the lowest dose to exhibit its activity. In this study, a combination of dietary 5,6,7-trihydroxy-flavonoid aglycones with 1-DNJ showed synergistic inhibitory activity against maltase of mice α-glucosidase and recombinant C- and N-termini of maltase-glucoamylase (MGAM) and baicalein with 1-DNJ exhibited the strongest synergistic effect. The synergistic effect of the combination was also confirmed by the maltose tolerance test in vivo. Enzyme kinetics, molecular docking, fluorescence spectrum, and circular dichroism spectrometry studies indicated that the major mechanism of the synergism is that baicalein was a positive allosteric inhibitor and bound to the noncompetitive site of MGAM, causing an increase of the binding affinity of 1-DNJ to MGAM. Our results might provide a theoretical basis for the design of dietary supplements containing mulberry products.

© The Author(s) 2020, [Background]: α-Glucosidases are widely distributed enzymes with a varied substrate specificity that are traditionally used in biotechnological industries based on oligo- and polysaccharides as starting materials. According to amino acid sequence homology, α-glucosidases are included into two major families, GH13 and GH31. The members of family GH13 contain several α-glucosidases with confirmed hydrolytic activity on sucrose. Previously, a sucrose splitting activity from the nectar colonizing yeast Metschnikowia reukaufii which produced rare sugars with α-(1→1), α-(1→3) and α-(1→6) glycosidic linkages from sucrose was described., [Results]: In this study, genes codifying for α-glucosidases from the nectaries yeast M. gruessii and M. reukaufii were characterised and heterologously expressed in Escherichia coli for the first time. Recombinant proteins (Mg-αGlu and Mr-αGlu) were purified and biochemically analysed. Both enzymes mainly displayed hydrolytic activity towards sucrose, maltose and p-nitrophenyl-α-d-glucopyranoside. Structural analysis of these proteins allowed the identification of common features from the α-amylase family, in particular from glycoside hydrolases that belong to family GH13. The three acidic residues comprising the catalytic triad were identified and their relevance for the protein hydrolytic mechanism confirmed by site-directed mutagenesis. Recombinant enzymes produced oligosaccharides naturally present in honey employing sucrose as initial substrate and gave rise to mixtures with the same products profile (isomelezitose, trehalulose, erlose, melezitose, theanderose and esculose) previously obtained with M. reukaufii cell extracts. Furthermore, the same enzymatic activity was detected with its orthologous Mg-αGlu from M. gruessii. Interestingly, the isomelezitose amounts obtained in reactions mediated by the recombinant proteins, ~ 170 g/L, were the highest reported so far., [Conclusions]: Mg/Mr-αGlu were heterologously overproduced and their biochemical and structural characteristics analysed. The recombinant α-glucosidases displayed excellent properties in terms of mild reaction conditions, in addition to pH and thermal stability. Besides, the enzymes produced a rare mixture of hetero-gluco-oligosaccharides by transglucosylation, mainly isomelezitose and trehalulose. These compounds are natural constituents of honey which purification from this natural source is quite unviable, what make these enzymes very interesting for the biotechnological industry. Finally, it should be remarked that these sugars have potential applications as food additives due to their suitable sweetness, viscosity and humectant capacity., This work was supported by the Projects BIO2016-76601-C3-2-R/-1-R from the Spanish Ministry of Economy and Competitiveness and Fundación Ramón Areces (XIX Call of Research Grants in Life and Material Sciences). The Spanish Ministry of Science, Innovation and Universities supported MG-G fellowship FPU16/02925. We thank Fundación Ramón Areces for an institutional grant to the Center of Molecular Biology Severo Ochoa.

Human sweet taste receptor (hSTR) recognizes a wide array of sweeteners, resulting in sweet taste perception. Maltitol and lactitol have been extensively used in place of sucrose due to their capability to prevent dental caries. Herein, several molecular modeling approaches were applied to investigate the structural and energetic properties of these two polyols/hSTR complexes. Triplicate 500 ns molecular dynamics (MD) simulations and molecular mechanics/generalized Born surface area (MM/GBSA)-based free energy calculations revealed that the TAS1R2 monomer is the preferential binding site for maltitol and lactitol rather than the TAS1R3 region. Several polar residues (D142, S144, Y215, D278, E302, R383, and especially N143) were involved in polyols binding through electrostatic attractions and H-bond formations. The molecular complexation process not only induced the stable form of ligands but also stimulated the conformational adaptation of the TAS1R2 monomer to become a close-packed structure through an induced-fit mechanism. Notably, the binding affinity of the maltitol/TAS1R2 complex (ΔGbind of -17.93 ± 1.49 kcal/mol) was significantly higher than that of the lactitol/TAS1R2 system (-8.53 ± 1.78 kcal/mol), in line with the experimental relative sweetness. These findings provide an in-depth understanding of the differences in the sweetness response between maltitol and lactitol, which could be helpful to design novel polyol derivatives with higher sweet taste perception.

The quality of Indonesia tuber flour can be improved by α-amylases which hydrolyzes the flour amylose to glucose and maltose. These monosaccharides causes the flour to have better homogeniety similar to wheat flour and easier to digest. This research aimed at investigating carbohydrate degradation of tuber paste flour by the addition of α-amylase from two Lactobacillus species. Lactobacillus species used were Lactobacillus bulgaricus and L. plantarum B110, while the flour types were made of local taro (Colocasia esculenta), gadung (Dioscorea hispida) and sweet potato (Ipomoea batatas), as well as wheat (Triticum) as a reference. Crude α-amylase activity and reducing sugars were detected by the Dinitrosalycylic acid (DNS) method. Data were statistically analyzed with ANOVA. Research results indicated that α-amylase from L. bulgaricus and L. plantarum B110 have been characterized for their optimum activity and stabilitiy. The reducing sugar content in taro, gadung, sweet potato paste flour and wheat paste flour added with α-amylase of L. bulgaricus increased by 0.008, 0.006, 0.004 and 0.001%, respectively. Meanwhile, the reducing sugars of the above flours added with amylase from L. plantarum B110, increased by 0.008, 0.008, 0.008, and 0.003%, respectively. Increase in reducing sugar contents in carbohydrate degradation of local tuber paste flour added with L. bulgaricus α-amylases was higher than that in wheat paste flour with a 0.001% increase. Similarly, the 0.008% increase of sugar content in tuber paste added with L. plantarum B110 α-amylase was also higher than that in wheat flour with 0.003% increase. Therefore, local tuber paste flour can be used as an alternative to wheat paste flour.

According to whole-genome sequencing, Aspergillus niger produces multiple enzymes of glycoside hydrolases (GH) 31. Here we focus on a GH31 α-glucosidase, AgdB, from A. niger . AgdB has also previously been reported as being expressed in the yeast species, Pichia pastoris ; while the recombinant enzyme (rAgdB) has been shown to catalyze tranglycosylation via a complex mechanism. We constructed an expression system for A. niger AgdB using Aspergillus nidulans . To better elucidate the complicated mechanism employed by AgdB for transglucosylation, we also established a method to quantify glucosidic linkages in the transglucosylation products using 2D NMR spectroscopy. Results from the enzyme activity analysis indicated that the optimum temperature was 65 °C and optimum pH range was 6.0-7.0. Further, the NMR results showed that when maltose or maltopentaose served as the substrate, α-1,2-, α-1,3-, and small amount of α-1,1-β-linked oligosaccharides are present throughout the transglucosylation products of AgdB. These results suggest that AgdB is an α-glucosidase that serves as a transglucosylase capable of effectively producing oligosaccharides with α-1,2-, α-1,3-glucosidic linkages.

Shoot tips of Potato (Solanum phureja) were cryopreserved with the encapsulation-dehydration method, including: coating of shoot tips in calcium alginate beads, pre-culture in a solution containing 0.75 M of sugar (0.1 M of sucrose +0.65 M of another cryoprotectant sugar), and dehydration (for 4.5 h) on silica gel to reduce water content to 21–27%. Regrowth of shoot tips was assessed after freezing in liquid nitrogen. After cryopreservation, coated shoot tips were cultivated on a medium enriched with growth substances to measure concentrations of soluble carbohydrates. Maltose and trehalose ensured a better cryopreservation (88% and 91% of regrowth, respectively). Regrowth rates of potato shoot tips in the presence of polyols were very low and no recovery was obtained after freezing of shoot tips that were pre-cultivated in monosaccharides. Concentration of soluble carbohydrates increased in shoot tips pre-cultivated in sucrose, trehalose or glucose, especially when sucrose was applied as the cryoprotectant. The application of sucrose helped potato shoot-tips to acquire a certain tolerance to dehydration and cryofreezing. The analysis of soluble carbohydrates in potato shoot tips confirmed the accumulation of sugars during the pre-culture process, principally in the form of sucrose. The statistical analysis showed that soluble carbohydrate contents in shoot tips increased when total carbohydrates increased in the cultivation medium. The modelling approach indicated that the contents of soluble carbohydrates increased when pre-cultivation of potato shoot-tips was conducted in sucrose media compared to the control and other sugars. Keywords: Cryopreservation, Pre-culture, Soluble carbohydrates, Liquid nitrogen, Coated shoot tips, Encapsulation-dehydration, Potato

BACKGROUND The carbohydrates in beer play an important role as they are essential for fermentation. Any change in their composition may influence the sensory characteristics of the beer and so their determination is of great interest. This study compares the carbohydrates in three types of commercial beer - barley malt beer, wheat beer, and barley malt beer with adjuncts - and examines their influence on beer quality, which is important for selecting raw ingredients and production conditions, and for quality control. RESULTS Among the oligosaccharides in three types of beer, raffinose was the most, followed by maltotetraose, maltotriose and maltose. Monosaccharides were only present in small amounts. Dextrin, oligosaccharides with 2-6 polymerization degree and non-starch polysaccharides (NSP) make up 15.90-34.83%, 17.59-38.63%, and 2.33-7.47% of the total carbohydrates in beer, respectively. The dextrin content and NSP content were significantly (P

Natural compounds are a major source for pharmaceutical industries. Cell suspension culture provides a reliable method to enhance the productivity of compounds exhibiting antioxidant properties. In the present study, cell cultures of date palm (Phoenix dactylifera L.), a desert fruit species rich in nutrients and medicinal compounds, were grown in different culture parameters to determine the optimum conditions for culture growth and accumulation of active compounds. The tested parameters included MS strength of culture medium, sucrose concentration (1–8%), pH (4–6.5), inoculum density (1–20 g/l) carbohydrates (3%) and their combinations (sucrose, glucose, fructose and maltose) and different media (MS, B5, N6 and NN). The response to these parameters was assessed in terms of packed cell volume (PCV), total phenolic, flavonoid and antioxidant activity. The results showed the MS medium containing 3% sucrose at pH 5.8 enhanced the PCV production. Inoculum density of 5 g/l induced maximum flavonoid content in cell culture. The highest phenolic accumulation and antioxidant activity were observed in cell cultures grown in double-strength MS medium, 20 g/l inoculum density and 4% sucrose at pH 6. A positive correlation was detected between phenolic content and antioxidant activity. These findings can facilitate the scale-up production of pharmaceutically important compounds from date palm cell culture.

Chlorella vulgaris, like a wide range of other microalgae, are able to grow mixotrophically. This maximizes its growth and production of polysaccharides (PS). The extracted polysaccharides have a complex monosaccharide composition (fructose, maltose, lactose and glucose), sulphate (210.65 ± 10.5 mg g−1 PS), uronic acids (171.97 ± 5.7 mg g−1 PS), total protein content (32.99 ± 2.1 mg g−1 PS), and total carbohydrate (495.44 ± 8.4 mg g−1 PS). Fourier Transform infrared spectroscopy (FT-IR) analysis of the extracted polysaccharides showed the presence of N–H, O–H, C–H, –CH3, >CH2, COO−1, S=O and the C=O functional groups. UV–Visible spectral analysis shows the presence of proteins, nucleic acids and chemical groups (ester, carbonyl, carboxyl and amine). Purified polysaccharides were light green in color and in a form of odorless powder. It was soluble in water but insoluble in other organic solvents. Thermogravimetric analysis demonstrates that Chlorella vulgaris soluble polysaccharide is thermostable until 240°C and degradation occurs in three distinct phases. Differential scanning calorimetry (DSC) analysis showed the characteristic exothermic transition of Chlorella vulgaris soluble polysaccharides with crystallization temperature peaks at 144.1°C, 162.3°C and 227.7°C. The X–ray diffractogram illustrated the semicrystalline nature of these polysaccharides. Silver nanoparticles (AgNPs) had been biosynthesized using a solution of Chlorella vulgaris soluble polysaccharides. The pale green color solution of soluble polysaccharides was turned brown when it was incubated for 24 hours with 100 mM silver nitrate in the dark, it showed peak maximum located at 430 nm. FT-IR analysis for the biosynthesized AgNPs reported the presence of carbonyl, –CH3, >CH2, C–H,–OH and –NH functional groups. Scanning and transmission electron microscopy show that AgNPs have spherical shape with an average particle size of 5.76. Energy-dispersive X-ray (EDX) analysis showed the dominance of silver. The biosynthesized silver nanoparticles were tested for its antimicrobial activity and have positive effects against Bacillus sp., Erwinia sp., Candida sp. Priming seeds of Triticum vulgare and Phaseolus vulgaris with polysaccharides solutions (3 and 5 mg mL−1) resulted in significant enhancement of seedling growth. Increased root length, leaf area, shoot length, photosynthetic pigments, protein content, carbohydrate content, fresh and dry biomass were observed, in addition these growth increments may be attributed to the increase of antioxidant activities.

Mechanocatalysis is a promising method for depolymerization of lignocellulosic biomass. Microbial utilization of the resulting oligosaccharides is one potential route of adding value to the depolymerized biomass. However, it is unclear how readily these oligosaccharides are utilized by standard cell factories. Here, we investigate utilization of cellulose subjected to mechanocatalytic depolymerization, using ethanologenic Escherichia coli as a model fermentation organism. The mechanocatalytic oligosaccharides supported ethanol titers similar to those observed when glucose was provided at comparable concentrations. Tracking of the various oligomers, using maltose (alpha-1,4) and cellobiose (beta-1,4) oligomers as representative standards of the orientation, but not linkage, of the glycosidic bond, suggests that the malto-like-oligomers are more readily utilized than cello-like-oligomers, consistent with poor growth with cellotetraose or cellopentaose as sole carbon source. Thus, mechanocatalytic oligosaccharides are a promising substrate for cell factories, and microbial utilization of these sugars could possibly be improved by addressing utilization of cello-like oligomers.

In this study, Streptomyces sp. Al-Dhabi-49 was isolated from the soil sample of Saudi Arabian environment for the simultaneous production of lipase and protease in submerged fermentation. The process parameters were optimized to enhance enzymes production. The production of protease and lipase was found to be maximum after 5 days of incubation (139.2 ± 2.1 U/ml, 253 ± 4.4 U/ml). Proteolytic enzyme increases with the increase in pH up to 9.0 (147.2 ± 3.6 U/ml) and enzyme production depleted significantly at higher pH values. In the case of lipase, production was maximum in the culture medium containing pH 8.0 (166 ± 1.3 U/ml). The maximum production of protease was observed at 40 °C (174 ± 12.1 U/ml) by Streptomyces sp. Lipase activity was found to be optimum at the range of temperatures (30–50 °C) and maximum production was achieved at 35 °C (168 ± 7.8 U/ml). Among the evaluated carbon sources, maltose significantly influenced on protease production (218 ± 12.8 U/ml). Lipase production was maximum when Streptomyces sp. was cultured in the presence of glucose (162 ± 10.8U/ml). Among various concentrations of peptone, 1.0% (w/v) significantly enhanced protease production. The lipase production was very high in the culture medium containing malt extract as nitrogen source (86 ± 10.2 U/ml). Protease production was maximum in the presence of Ca2+ as ionic source (212 ± 3.8 U/ml) and lipase production was enhanced by the addition of Mg2+ with the fermentation medium (163.7 ± 6.2 U/ml). Keywords: Streptomyces sp., Identification, Protease, Lipase, Optimization, Enzyme characterization

Mono-, di- and triacetin are three glycerol esters which are usually synthesized via costly and environmentally unfriendly chemical synthesis methods. Here, Escherichia coli is metabolically engineered for the production of mono-, di- and triacetin using glycerol as a substrate. First, a novel biosynthetic route of mono- and diacetin is established by overexpression of a native enzyme, maltose O-acetyltransferase (MAA). Next, the biosynthetic pathway is extended to produce a mixture of mono-, di- and triacetin by overexpression of chloramphenicol-O-acetyltransferase (CAT). By successive strategies, including heterologous gene expression, metabolic engineering, and culture optimization, a recombinant E. coli is enabled to produce more than 27 g L−1 of a mixture of mono-, di- and triacetin in shake flask cultures, which is a >650-fold increase over the initial production of 0.04 g L−1. In vitro studies confirm the acetylation of glycerol to mono- and diacetin by MAA, and the additional acetylation to triacetin by CAT. When crude glycerol is used as a substrate, the engineered strain produced a total of 25.9 g L−1 of the acetin mixture, about the same as that achieved from pure glycerol. To our knowledge, this is the first successful report of microbial production of the artificial chemicals, acetins.

In this study a novel thermo stable alkaline protease producing Streptomyces sp. Al-Dhabi-82 was isolated from the soil. The effect of fermentation period on enzyme production by Streptomyces sp. Al-Dhabi-82 was optimized in submerged fermentation. Protease activity was found to be maximum after 5 days of incubation (129.5 ± 7.1 U/ml) and depleted after 6 days of incubation (113.8 ± 4.1 U/ml). Enzyme production increases with the increase in pH up to 9.0 (136.2 ± 3.6 U/ml) and enzyme production depleted significantly at pH 11.0 (67 ± 2.9 U/ml). Maximum production of protease was observed at 40 °C (164 ± 11.1 U/ml). Among the evaluated carbon sources, maltose significantly influenced on protease production (212 ± 14.8 U/ml). The optimum amount of protease (269 ± 10.4 U/ml) produced by Streptomyces sp. Al-Dhabi-82 was observed in the production medium containing yeast extract. Enzyme production was maximum in the presence of 0.15% Ca2+. The specific activity of crude enzyme was 26 U/ mg protein and it increased as 276 U/mg protein after chromatography separation. The molecular weight of purified protease obtained from sephadex G-75 gel filtration chromatography was estimated to be 37 kDa using SDS-PAGE. This enzyme showed high activity at pH 9.0, and lost about 16% activity at pH 10. The optimum temperature for Streptomyces sp. Al-Dhabi-82 protease was 40 °C. The extracellular alkaline protease from Streptomyces sp. Al-Dhabi-82 hydrolyzed chicken feather completely. Keywords: Actinomycetes, Streptomyces sp., Alkaline protease, Optimization

Trichoderma viride AUMC 13021 isolated from Mangrove soil of Ras Mohammed protected area at Sharm El-Sheikh, Egypt, was optimized to promote chitinase activity under submerged fermentation. The maximum enzyme yield (38.33 U/mg protein) was obtained at 1.4% of colloidal chitin, 96 h of incubation, 35°C, pH 6.5 and 125, rpm and using maltose (1%) and yeast extract (1%) as supplementation of salt basal medium. The enzyme has been purified with an overall yield of 73.1% and 5.48 purification fold, and a specific activity of 210.16 U/mg protein. The molecular mass of the purified chitinase was 62 kDa. Maximal activity of chitinase was recorded at pH 6.5 and 40°C. The highest activity was recorded in the case of colloidal chitin, with an apparent Km value of 6.66 mg/ml and Vmax of 90.8 U/ml. The purified chitinase was activated by Ca2+ and Mn2+ while the activity was inhibited by Hg2+, Zn2+, Cu2+, Co2+, dodecyl sulphate and EDTA. In vivo, the median lethal dose (LD50) was approximately 18.43 mg/kg body weight of Sprague Dawley rats. MTT assay showed that the purified chitinase has a toxic effect to MCF7 with an IC50 value 20 μg/ml, and HCT-116 cell lines with an IC50 value 44 μg/ml. Moreover, the purified enzyme showed significant antifungal activity against Fusarium oxysporum f. sp. lycopersici race 3 the causal agent of tomato wilt.

Sake, the Japanese rice wine, contains a variety of oligosaccharides and glucosides produced by fungal enzymes during the brewing process. This study investigates the effect of knocking out the Aspergillus oryzae α-glucosidase (agdA) gene on the transglycosylation products in brewed sake. In addition to α-ethyl glucoside and α-glyceryl glucoside, the amount of two compounds that have molecular mass values similar to that of ethyl maltose decreased by agdA gene knockout. Both compounds were synthesized, in vitro, from maltose and ethanol with purified agdA. Nuclear magnetic resonance analysis identified the two compounds as ethyl α-maltoside and ethyl α-isomaltoside, respectively, which are novel compounds in sake as well as in the natural environment. Quantitative analysis of 111 commercially available types of sake showed that these novel compounds were widely present at concentrations of several hundred mg/L, suggesting that both of them are ones of the common glycosides in sake.

The β-amylase was encapsulated in emulsion liquid membrane (ELM), which acted as a reactor for conversion of starch to maltose. The membrane phase was consisted of surfactant (span 80), stabilizer (polystyrene), carrier for maltose transport (methyl cholate) and solvent (xylene). The substrate starch in feed phase entered into the internal phase by the process of diffusion and hydrolyzed to maltose by encapsulated β-amylase. Methyl cholate present in the membrane acts as a carrier for the product maltose, which helps in transport of maltose to feed phase from internal aqueous phase. The residual activity of β-amylase after the five-reaction cycle was found to decrease to ∼70%, which indicated possibility to recycle the components of the emulsion and enzyme. The pH and temperature of the encapsulated enzyme were found to be optimum at 5.5 and 60 °C, respectively. The novelty of the present work lies in the development of Enzyme Emulsion Liquid Membranes (EELM) bioreactor for the hydrolysis of starch into maltose mediated by encapsulated β-amylase. The attempt has been made for the first time for the successful encapsulation of β-amylase into EELM. The best results gave the highest residual enzyme activity (94.1%) and maltose production (29.13 mg/mL).

Rapid pollen tube growth requires uptake of Suc or its hydrolytic products, hexoses, from the apoplast of surrounding tissues in the style. Due to species-specific sugar requirements, reliance of pollen germination and tube growth on cell wall invertase and Suc or hexose transporters varies between species, but it is not known if plants have a sugar transporter that mediates the uptake of both hexose and Suc for pollen tube growth. Here, we show that a sugar transporter protein in apple (Malus domestica), MdSTP13a, takes up both hexose and Suc when expressed in yeast, and is essential for pollen tube growth on Glc and Suc but not on maltose. MdSTP13a-mediated direct uptake of Suc is primarily responsible for apple pollen tube growth on Suc medium. Sorbitol, a major photosynthate and transport carbohydrate in apple, modulates pollen tube growth via the MYB transcription factor MdMYB39L, which binds to the promoter of MdSTP13a to activate its expression. Antisense repression of MdSTP13a blocks sorbitol-modulated pollen tube growth. These findings demonstrate that MdSTP13a takes up both hexose and Suc for sorbitol-modulated pollen tube growth in apple, revealing a situation where acquisition of sugars for pollen tube growth is regulated by a sugar alcohol.

The compositional and quality characteristics of two wheat varieties Triticum dicoccum (Triticum dicoccum var. dicoccum, Triticum dicoccum var. rufum) produced in the Republic of Azerbaijan have been tested and are relatively useful in assessing their applicability to bread production. The wheat species studied, Triticum dicoccum, were found to have a higher protein and cell content, as well as essential proteins of lysine, phenylalanine, leucine and isoleucine, methionine and valine, relative to Gorbustan wheat varieties. The chromatographic method was used to determine the carbohydrate composition of the Triticum dicoccum grain. The following redistribution of low molecular weight carbohydrate fractions is noted: the maltose content is higher, and galactose, glucose and fructose are much lower than those of the modern wheat variety Gorbustan. Such a distribution of carbohydrates can reduce the formation of toxic products when baking bread. In addition, the wheat grain Triticum dicoccum is characterized by a higher content of sterols, in particular β-sitosterol. The antioxidant activity expressed as percentage inhibition of DPPG free radicals in the Triticum dicoccum grain is twice as high as this indicator for wheat of the commercial variety Gorbustan. By scanning electron microscopy, it has been established that the microstructure of the grain surface and the cross section has varietal characteristics. Grain Triticum dicoccum var. rufum has a thicker shell, tighter and tighter, unlike the grain of Triticum dicoccum var. dicoccum. With all the benefits of the wheat grain Triticum dicoccum, its technological properties were even worse. But the use of technological methods to boost gluten will ensure the production of high-quality healthy bread from old wheat grain.

α-Glucan is a major cell wall component and a virulence and adhesion factor for fungal cells. However, the biosynthetic pathway of α-glucan was still unclear. α-Glucan was shown to be composed mainly of 1,3-glycosidically linked glucose, with trace amounts of 1,4-glycosidically linked glucose. Besides the α-glucan synthetases, amylase-like proteins were also important for α-glucan synthesis. In our previous work, we showed that Aspergillus nidulans AmyG was an intracellular protein and was crucial for the proper formation of α-glucan. In the present study, we expressed and purified AmyG in an Escherichia coli system. Enzymatic characterization found that AmyG mainly functioned as an α-amylase that degraded starch into maltose. AmyG also showed weak glucanotransferase activity. Most intriguingly, supplementation with maltose in shaken liquid medium could restore the α-glucan content and the phenotypic defect of a ΔamyG strain. These data suggested that AmyG functions mainly as an intracellular α-amylase to provide maltose during α-glucan synthesis in A. nidulans. IMPORTANCE Short α-1,4-glucan was suggested as the primer structure for α-glucan synthesis. However, the exact structure and its source remain elusive. AmyG was essential to promote α-glucan synthesis and had a major impact on the structure of α-glucan in the cell wall. Data presented here revealed that AmyG belongs to the GH13_5 family and showed strong amylase function, digesting starch into maltose. Supplementation with maltose efficiently rescued the phenotypic defect and α-glucan deficiency in an ΔamyG strain but not in an ΔagsB strain. These results provide the first piece of evidence for the primer structure of α-glucan in fungal cells, although it might be specific to A. nidulans.

A novel putative trehalose synthase gene (treM) was identified from an extreme temperature thermal spring. The gene was expressed in Escherichia coli followed by purification of the protein (TreM). TreM exhibited the pH optima of 7.0 for trehalose and trehalulose production, although it was functional and stable in the pH range of 5.0 to 8.0. Temperature activity profiling revealed that TreM can catalyze trehalose biosynthesis in a wide range of temperatures, from 5°C to 80°C. The optimum activity for trehalose and trehalulose biosynthesis was observed at 45°C and 50°C, respectively. A catalytic reaction performed at the low temperature of 5°C yielded trehalose with significantly reduced by-product (glucose) production in the reaction. TreM displayed remarkable thermal stability at optimum temperatures, with only about 20% loss in the activity after heat (50°C) exposure for 24 h. The maximum bioconversion yield of 74% trehalose (at 5°C) and 90% trehalulose (at 50°C) was obtained from 100 mM maltose and 70 mM sucrose, respectively. TreM was demonstrated to catalyze trehalulose biosynthesis utilizing the low-cost feedstock jaggery, cane molasses, muscovado, and table sugar. IMPORTANCE Trehalose is a rare sugar of high importance in biological research, with its property to stabilize cell membrane and proteins and protect the organism from drought. It is instrumental in the cryopreservation of human cells, e.g., sperm and blood stem cells. It is also very useful in the food industry, especially in the preparation of frozen food products. Trehalose synthase is a glycosyl hydrolase 13 (GH13) family enzyme that has been reported from about 22 bacterial species so far. Of these enzymes, to date, only two have been demonstrated to catalyze the biosynthesis of both trehalose and trehalulose. We have investigated the metagenomic data of an extreme temperature thermal spring to discover a novel gene that encodes a trehalose synthase (TreM) with higher stability and dual transglycosylation activities of trehalose and trehalulose biosynthesis. This enzyme is capable of catalyzing the transformation of maltose to trehalose and sucrose to trehalulose in a wide pH and temperature range. The present investigation endorses the thermal aquatic habitat as a promising genetic resource for the biocatalysts with high potential in producing high-value rare sugars.

With the recent explosion in high-resolution protein structures, one of the next frontiers in biology is elucidating the mechanisms by which conformational rearrangements in proteins are regulated to meet the needs of cells under changing conditions. Rigorously measuring protein energetics and dynamics requires the development of new methods that can resolve structural heterogeneity and conformational distributions. We have previously developed steady-state transition metal ion fluorescence resonance energy transfer (tmFRET) approaches using a fluorescent noncanonical amino acid donor (Anap) and transition metal ion acceptor, to probe conformational rearrangements in soluble and membrane proteins. Here, we show that the fluorescent noncanonical amino acid Acd has superior photophysical properties that extend its utility as a donor for tmFRET. Using maltose binding protein (MBP) expressed in mammalian cells as a model system, we show that Acd is comparable to Anap in steady-state tmFRET experiments and that its long, single-exponential lifetime is better suited for probing conformational distributions using time-resolved FRET. These experiments reveal differences in heterogeneity in the apo and holo conformational states of MBP and produce accurate quantification of the distributions among apo and holo conformational states at subsaturating maltose concentrations. Our new approach using Acd for time-resolved tmFRET sets the stage for measuring the energetics of conformational rearrangements in soluble and membrane proteins in near-native conditions.

β-amylase-rich preparation produced by the soil fungal strain Penicillium citrinum AS-9 was completely purified within the succeeding steps; ultrafiltration, acetone fractionation and gel filtration on Sephadex G-150 column. Acetone fractionation recovered the highest β-amylase activity and the 85% acetone fraction exhibited 3.5-fold activity that of the crude enzyme. The column affected 8.6-fold purification for β-amylase, which produced maltose as the major product of starch hydrolysis. The pure enzyme showed a Km value of 17.5 mM and Vmax of 17.5 Umg-1 protein, applying Woolf plot and exhibited its maximum velocity at pH 7.1 and 50°C. In absence of substrate, thermal treatments of the enzyme solution at pH 5.2 and 5.5 had the most adverse effects on the enzyme activity. CaCl2 (1mM) activated the enzyme, while each of Ca2+, Fe3+, cysteine, cystine, I2 and p-chloromercuribenzoate (PCMB) had different inhibitory effects. Maltose as the enzyme product at final concentration of 80 mM strongly inhibited the enzyme.

Background β-amylase (EC 3.2.1.2) is an exo-enzyme that shows high specificity for cleaving the α-1,4-glucosidic linkage of starch from the non-reducing end, thereby liberating maltose. In this study, we heterologously expressed and characterized a novel β-amylase from Bacillus aryabhattai. Results The amino acid-sequence alignment showed that the enzyme shared the highest sequence identity with β-amylase from Bacillus flexus (80.73%) followed by Bacillus cereus (71.38%). Structural comparison revealed the existence of an additional starch-binding domain (SBD) at the C-terminus of B. aryabhattai β-amylase, which is notably different from plant β-amylases. The recombinant enzyme purified 4.7-fold to homogeneity, with a molecular weight of ~ 57.6 kDa and maximal activity at pH 6.5 and 50 °C. Notably, the enzyme exhibited the highest specific activity (3798.9 U/mg) among reported mesothermal microbial β-amylases and the highest specificity for soluble starch, followed by corn starch. Kinetic analysis showed that the Km and kcat values were 9.9 mg/mL and 116961.1 s− 1, respectively. The optimal reaction conditions to produce maltose from starch resulted in a maximal yield of 87.0%. Moreover, molecular docking suggested that B. aryabhattai β-amylase could efficiently recognize and hydrolyze maltotetraose substrate. Conclusions These results suggested that B. aryabhattai β-amylase could be a potential candidate for use in the industrial production of maltose from starch.

Although Fraxinellone (Frax) isolated from Dictamnus albus L. possessed excellent anti-hepatic fibrosis activity, oral administration of Frax suffered from the inefficient therapeutic outcome in vivo due to negligible oral absorption. At present, the oral formulation of Frax is rarely exploited. For rational formulation design, we evaluated preabsorption risks of Frax and found that Frax was rather stable while poorly dissolved in the gastrointestinal tract (78.88 μg/mL), which predominantly limited its oral absorption. Further solubility test revealed the outstanding capacity of cyclodextrin derivatives (CDs) to solubilize Frax (6.8–12.8 mg/mL). This led us to study the inclusion complexes of Frax with a series of CDs and holistically explore their drug delivery performance. Characterization techniques involving 1H-NMR, FT-IR, DSC, PXRD, and molecular docking confirmed the most stable binding interactions when Frax complexed with 6-O-α-D-maltosyl-β-cyclodextrin (G2-β-CD-Frax). Notably, G2-β-CD-Frax exhibited the highest solubilizing capacity, fast dissolution rate, and superior Caco-2 cell internalization with no obvious toxicity. Pharmacokinetic studies demonstrated markedly higher oral bioavailability of G2-β-CD-Frax (5.8-fold that of free drug) than other Frax-CDs. Further, long-term administration of G2-β-CD-Frax (5 mg/kg) efficiently inhibited CCl4-induced hepatic fibrosis in the mouse without inducing any toxicity. Our results will inspire the continued advancement of optimal oral Frax formulations for anti-fibrotic therapy.

Starch-acting α-glucanotransferase enzymes are of great interest for applications in the food industry. In previous work, we have characterized various 4,6- and 4,3-α-glucanotransferases of the glycosyl hydrolase (GH) family 70 (subfamily GtfB), synthesizing linear or branched α-glucans. Thus far, GtfB enzymes have only been identified in mesophilic Lactobacilli. Database searches showed that related GtfC enzymes occur in Gram-positive bacteria of the genera Exiguobacterium, Bacillus, and Geobacillus, adapted to growth at more extreme temperatures. Here, we report characteristics of the Geobacillus sp. 12AMOR1 GtfC enzyme, with an optimal reaction temperature of 60 °C and a melting temperature of 68 °C, allowing starch conversions at relatively high temperatures. This thermostable 4,6-α-glucanotransferase has a novel product specificity, cleaving off predominantly maltose units from amylose, attaching them with an (α1 → 6)-linkage to acceptor substrates. In fact, this GtfC represents a novel maltogenic α-amylase. Detailed structural characterization of its starch-derived α-glucan products revealed that it yielded a unique polymer with alternating (α1 → 6)/(α1 → 4)-linked glucose units but without branches. Notably, this Geobacillus sp. 12AMOR1 GtfC enzyme showed clear antistaling effects in bread bakery products.