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Glycoside hydrolase family 97 (GH97) comprises enzymes like anomer‐inverting α‐glucoside hydrolases (i.e., glucoamylase) and anomer‐retaining α‐galactosidases. In a soil bacterium, Flavobacterium johnsoniae, we previously identified a GH97 enzyme (FjGH97A) within the branched dextran utilization locus. It functions as an α‐glucoside hydrolase, targeting α‐(1→6)‐glucosidic linkages in dextran and isomaltooligosaccharides (i.e., glucodextranase). FjGH97A exhibits a preference for α‐(1→6)‐glucoside linkages over α‐(1→4)‐linkages, while Bacteroides thetaiotaomicron glucoamylase SusB (with 69% sequence identity), which is involved in the starch utilization system, exhibits the highest specificity for α‐(1→4)‐glucosidic linkages. Here, we examined the crystal structures of FjGH97A in complexes with glucose, panose, or isomaltotriose, and analyzed the substrate preferences of its mutants to identify the amino acid residues that determine the substrate specificity for α‐(1→4)‐ and α‐(1→6)‐glucosidic linkages. The overall structure of FjGH97A resembles other GH97 enzymes, with conserved catalytic residues similar to anomer‐inverting GH97 enzymes. A comparison of active sites between FjGH97A and SusB revealed differences in amino acid residues at subsites +1 and +2 (specifically Ala195 and Ile378 in FjGH97A). Among the three mutants (A195S, I378F, and A195S‐I378F), A195S and A195S‐I378F exhibited increased activity toward α‐(1→4)‐glucoside bonds compared to α‐(1→6)‐glucoside bonds. This suggests that Ala195, located on the Gly184‐Thr203 loop (named loop‐N) conserved within the GH97 subgroup, including FjGH97A and SusB, holds significance in determining linkage specificity. The conservation of alanine in the active site of the GH97 enzymes, within the same gene cluster as the putative dextranase, indicates its crucial role in determining the specificity for α‐(1→6)‐glucoside linkage. [ABSTRACT FROM AUTHOR]

In this study, the α-glucosidase (maltase-glucoamylase: MGAM) and α-amylase inhibitory properties elicited by xylooligosaccharides (XOSs) prepared from dulse xylan were analysed as a potential mechanism to control postprandial hyperglycaemia for type-2 diabetes prevention and treatment. Xylan was purified from red alga dulse powder and used for enzymatic hydrolysis using Sucrase X to produce XOSs. Fractionation of XOSs produced xylobiose (X2), β-(1→3)-xylosyl xylobiose (DX3), xylotriose (X3), β-(1→3)-xylosyl-xylotriose (DX4), and a dulse XOS mixture with n ≥ 4 xylose units (DXM). The different fractions exhibited moderate MGAM (IC50 = 11.41–23.44 mg/mL) and α-amylase (IC50 = 18.07–53.04 mg/mL) inhibitory activity, which was lower than that of acarbose. Kinetics studies revealed that XOSs bound to the active site of carbohydrate digestive enzymes, limiting access to the substrate by competitive inhibition. A molecular docking analysis of XOSs with MGAM and α-amylase clearly showed moderate strength of interactions, both hydrogen bonds and non-bonded contacts, at the active site of the enzymes. Overall, XOSs from dulse could prevent postprandial hyperglycaemia as functional food by a usual and continuous consumption. [ABSTRACT FROM AUTHOR]

In this study, microcapsule beads‐0–3‐layers (M‐0–3 indicates microencapsulated beads with 0, 1, 2, and 3 layers) were prepared, their properties were measured and characterized, and the effects of M‐0–3 on solid‐state fermentation were investigated. The results showed that in a liquid environment, the releasing glucoamylase activities of M‐0–3 were 55.77%, 47.67%, 45.85%, and 42.87% in 360 h, respectively. In the solid environment, the reducing sugar production efficiency of M‐0–3 was 29.84%, 27.72%, 19.16%, and 15.93% in 15 days, respectively. Adding M‐0–3 improved the alcohol and reduced sugar content while decreasing the residual starch content of the Jiupei, indicating that adding M‐0–3 was beneficial to the solid‐state fermentation of Baijiu. Solid‐state fermentation simulation experiments illustrated that microcapsule beads play a positive role in the production of Baijiu, enhancing raw material utilization and yield of Baijiu production. [ABSTRACT FROM AUTHOR]

Miniaturized cultivation systems offer the potential to enhance experimental throughput in bioprocess development. However, they usually lack the miniaturized pumps necessary for fed-batch mode, which is commonly employed in industrial bioprocesses. An alternative are enzyme-mediated glucose release systems from starch-derived polymers, facilitating continuous glucose supply. Nevertheless, while the glucose release, and thus the feed rate, is controlled by the enzyme concentration, it also strongly depends on the type of starch derivative, and the culture conditions as well as pH and temperature. So far it was not possible to implement controlled feeding strategies (e.g., exponential feeding). In this context, we propose a model-based approach to achieve precise control over enzyme-mediated glucose release in cultivations. To this aim, an existing mathematical model was integrated into a computational framework to calculate setpoints for enzyme additions. We demonstrate the ability of the tool to maintain different pre-defined exponential growth rates during Escherichia coli cultivations in parallel mini-bioreactors integrated into a robotic facility. Although in this case study, the intermittent additions of enzyme and dextrin were performed by a liquid handler, the approach is adaptable to manual applications. Thus, we present a straightforward and robust approach for implementing defined continuous fed-batch processes in small-scale systems, where continuous feeding was only possible with low accuracy or high technical efforts until now. [ABSTRACT FROM AUTHOR]

Wheat Qu plays the role of saccharification fermentation, providing microorganisms and flavor in the fermentation of huangjiu , and the use of functional microorganisms to fortify wheat Qu is becoming increasingly popular. Yet, the mechanisms promoting microbial successions of wheat Qu remain unclear. In this study, we first correlated microbial community succession with physicochemical factors (moisture, temperature, acidity, glucoamylase and amylase) in inoculated raw wheat Qu (IRWQ) with Saccharopolyspora rosea. The Mantel test was performed to investigate the significance and found that temperature (r = 0.759, P = 0.001), moisture (r = 0.732, P = 0.006), and acidity (r = 0.712, P = 0.017) correlated significantly with the bacterial community in phase 1 (0–40 h). Meanwhile, temperature correlated significantly with the fungal community in phases 1 and 2 (40–120 h). To confirm the effect of temperature on microbial communities, the artificial reduction of bio-heat (37°C) in IRWQ also reduced the relative abundance of heat-resistant microorganisms including Bacillus and Saccharopolyspora. A higher abundance of Saccharopolyspora (87%) in IRWQ was observed following biofortified inoculation of S. rosea , in which glucoamylase activity increased by 40% compared to non-inoculated raw wheat Qu (NIRWQ) (1086 U/g vs 776 U/g). Finally, the IRWQ was employed to mechanized huangjiu fermentation and it was found to reduce the bitter amino acid and higher alcohol content by 27% and 8%, respectively, improving the drinking comfort and quality of huangjiu. [ABSTRACT FROM AUTHOR]

BACKGROUND: Baijiu brewing adopts the solid‐state fermentation method, using starchy raw materials, Jiuqu as saccharifying fermenting agent, and distilled spirits made by digestion, saccharification, fermentation and distillation. In the late stages of solid‐state fermentation of Baijiu, the reduced activity of glucoamylase leads to higher residual starch content in the Jiupei, which affects the liquor yield. The direct addition of exogenous glucoamylase leads to problems such as the temperature of the fermentation environment rising too quickly, seriously affecting the growth of microorganisms. RESULTS: To solve the problem of reduced activity of glucoamylase in the late stage of solid‐state fermentation of Baijiu, microcapsule beads (M‐B) based on microcapsule emulsion were prepared and the effect of M‐B on solid‐state fermentation of Baijiu was investigated. The results showed that the release of M‐B before and after drying was 53.27% and 25.77% in the liquid state (120 h) and 29.84% and 22.62% in the solid state (15 days), respectively. Adding M‐B improved the alcohol by 0.33 %vol and reducing sugar content by 0.51%, reduced the residual starch content by 1.21% of the Jiupei, and had an insignificant effect on the moisture and acidity of the Jiupei. CONCLUSION: M‐B have excellent sustained‐release properties. The addition of M‐B in solid‐state fermentation significantly increased the alcohol content, reduced the residual starch content of Jiupei, ultimately improving the starch utilization rate and liquor yield of Baijiu brewing. The preparation of M‐B provides methods and approaches for applying other active substances and microorganisms in the brewing of Baijiu. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Why was the work done: Diastatic variants of Saccharomyces cerevisiae are unusual in producing an extracellular glucoamylase which enables the breakdown of starch to fermentable sugars. Diastatic S. cerevisiae has long been viewed negatively as a contaminant of especially beer packaged in cans or bottles. However, this view is being reconsidered due to the opportunities that diastatic strains present for niche fermented products and distillation applications. What are the main findings: This review highlights the utilisation of diastatic S. cerevisiae for its flavour potential, and processing applications in the brewing, distilling, and biofuel industries. Further, genetic differences are compared with non-diastatic strains of S. cerevisiae, together with commonly employed and emerging methods of detection. Why is the work important: Diastatic yeast strains can be used to create flavour profiles that resemble traditional beverages and can be used to achieve fermentation with higher attenuation. This offers greater fermentation efficiency in, for example, the development of low-calorie beers. Additionally, the ability of diastatic strains of S. cerevisiae to convert non-fermentable oligosaccharides to fermentable sugars enables applications that range from novel beverages using unusual raw materials to more efficient distillation and biofuel production. The negative attributes that are associated with diastatic S. cerevisiae yeasts can be managed through co-inoculation or hybridisation with standard strains. [ABSTRACT FROM AUTHOR]

Yellow mombin (Spondias mombin) and Brazil plum (Spondias tuberosa) seeds are byproducts of exploiting their pulp and currently have no relevant food or industrial applications. Thus, the present study aimed to evaluate the physicochemical, technological, and functional characteristics of flours obtained from yellow mombin (YMF) and Brazil plum (BPF) residues. The flours presented a high percentage of insoluble fiber (68.8–70.2 g/100 g) and low carbohydrate (2.7–4.0 g/100 g) and caloric (91.9–95.3 kcal) values. The flours showed potential for technological application. In addition, the highest concentration of total phenolic content (31.1–50.2 mg GAE/g) was obtained with 70% acetone, which provided excellent results for antioxidant capacity evaluated by 2,2′‐azinobis (3‐ethylbenzothiazoline‐6‐sulfonic acid) (81.0%–89.7%) and 2,2‐diphenyl‐1‐picrylhydrazyl (60.6%–69.1%) radical scavenging capacity assays. Flour extracts in 70% acetone also exhibited inhibition of α‐amylase (63.3%–78.8%) and amyloglucosidase (63.5%–71.0%). The antibacterial study revealed that extracts inhibited the growth of Escherichia coli, Burkholderia cepacia, and Burkholderia multivorans. Therefore, this study suggests the use of yellow mombin and Brazil plum residues for different food or industrial applications. Practical Application: The knowledge gained from this study will open a new approach to add value to yellow mombin and Brazil plum fruit seeds as sources of fiber and bioactive compounds, with promising application in the formulation of functional and nutraceutical products, benefiting both a sustainable environment and a sustainable industry. [ABSTRACT FROM AUTHOR]

Starry stonewort (Nitellopsis obtusa) (Desvaux in Loiseleur) J. Groves is an invasive macro alga that can take over entire water columns and outcompete native species. Previous research has quantified seasonal life history and phenology but there is no research quantifying carbohydrate allocation. The current study utilized samples harvested from Lake Koronis, Minnesota, USA from 2020 and 2021 to quantify starch allocation patterns in N. obtusa. Starch was quantified using the amylase/amyloglucosidase method. Starch data were compared to light transmittance (%), pH and water temperature (°C) via mixed procedures models in SAS. Seasonal average low and high points of starch storage were observed to be June (3.3%) and April (9.9%) for thalli biomass then June (41.7%) and November (54.6%) for bulbils. In regard to allocation patterns, above ground thalli reallocates carbohydrate stores to the below ground rhizoids in May. Carbohydrates are then transported to bulbils from July through September as rhizoid starch decreases and the bulbil starch increases. Ultimately, the carbohydrates are converted to starch and stored in bulbils (21.0–73.7%). In its invaded range, bulbils are important for energy store, vegetative reproduction and spread, as well as temporal distribution. These bulbils spread easily, spread rapidly, can store a lot of energy (73% starch), and are a major concern for the invasion of N. obtusa. Water temperature and light availability were shown to negatively influence starch content of thalli. These findings suggest that in the invaded range, N. obtusa is capable of accumulating large amounts of starch later into the growing season. The energy reserves may give N. obtusa a competitive advantage over native species by allowing a longer growing season resulting in the production of more vegetative propagules and greater longevity in the environments it invades. [ABSTRACT FROM AUTHOR]

The integrated approach of biocatalytic conversion coupled with purification using selective enzyme was developed to achieve continuous trehalose production. The activity of trehalose synthase (TS) derived from three different bacteria— Deinococcus radiodurans , Pseudomonas stutzeri , and Thermus thermophillus —immobilized with chitin-binding domain (ChBD-DrTS, ChBD-PsTS, and ChBD-TtTS, respectively) was compared for the production of trehalose from maltose syrup. The trehalose production yield using ChBD-PsTS was 2.4 and 1.7-fold higher than those using ChBD-DrTS and ChBD-TtTS, respectively, after 24 h at 30 °C and pH 7.0. In this study, a continuous bioconversion system was developed for trehalose production using ChBD-PsTS in a fixed-bed bioreactor, resulting in an approximate yield of 72%, and a productivity of 4 g/L/h. Additionally, residual maltose was converted into glucose using glucoamylase, and the 37% of bioethanol was produced. The integrated approach demonstrated remarkable biocatalytic performance, biocatalyst reusability, and high production yield. [Display omitted] • Immobilizing trehalose synthase from Pseudomonas stutzeri enhances its activity. • The continuous system yielded 72% trehalose at pH 7.0 and 30 °C. • Residual maltose was converted to glucose for subsequent bioethanol fermentation. [ABSTRACT FROM AUTHOR]

Photo-crosslinkable platelet lysate (PL)-based hydrogels have been proven to support human-derived cell cultures owing to their high content of bioactive molecules, such as cytokines and growth factors. As a unique self-maintained and biocompatible 3D scaffold, the recently reported self-feeding hydrogels with enzyme-empowered degradation capacity have shown high biological performance in vitro and in vivo. To take advantage of all features of both PL and self-feeding hydrogels, here UV responsive laminaran-methacrylate (LamMA) and PL-methacrylate (PLMA) derivatives plus glucoamylase (GA), which significantly improve the overall features of a 3D system, is coupled. This self-sustaining hybrid hydrogel emerges as a unique scaffold due to the sustained delivery of glucose produced via enzymatic degradation of laminaran while granting the release of growth factors through the presence of PL. This biomaterial is applied to fabricate high-throughput freestanding microgels with controlled geometric shapes. Furthermore, this multicomponent hybrid hydrogel is successfully implemented as the first reported glucose supplier bioink to manufacture intricate and precisely defined cell-laden structures using a support matrix. Finally, such hydrogels are utilized as a proof of concept to serve as 3D in vitro cancer models, with the aim of recapitulating the tumor microenvironment. [ABSTRACT FROM AUTHOR]

The filamentous fungus Aspergillus niger is well known for its high protein secretion capacity and a preferred host for homologous and heterologous protein production. To improve the protein production capacity of A. niger even further, a set of dedicated protein production strains was made containing up to 10 glucoamylase landing sites (GLSs) at predetermined sites in the genome. These GLSs replace genes encoding enzymes abundantly present or encoding unwanted functions. Each GLS contains the promotor and terminator region of the glucoamylase gene (glaA), one of the highest expressed genes in A. niger. Integrating multiple gene copies, often realized by random integration, is known to boost protein production yields. In our approach the GLSs allow for rapid targeted gene replacement using CRISPR/Cas9‐mediated genome editing. By introducing the same or different unique DNA sequences (dubbed KORE sequences) in each GLS and designing Cas9‐compatible single guide RNAs, one is able to select at which GLS integration of a target gene occurs. In this way a set of identical strains with different copy numbers of the gene of interest can be easily and rapidly made to compare protein production levels. As an illustration of its potential, we successfully used the expression platform to generate multicopy A. niger strains producing the Penicillium expansum PatE::6xHis protein catalysing the final step in patulin biosynthesis. The A. niger strain expressing 10 copies of the patE::6xHis expression cassette produced about 70 μg·mL−1 PatE protein in the culture medium with a purity just under 90%. [ABSTRACT FROM AUTHOR]

The immobilized glucoamylase kinetic in flow calorimetric nonlinear system is discussed. Approximate closed-form of substrate concentration, effectiveness factor is derived by using AkbariGanji method for all values of parameters. The diffusion associated with three electrodes of various geometries (planar, cylindrical and spherical) is studied using this general reaction diffusion indicator parameter and saturated parameters. The problem is numerically simulated using the Maple / MAT-LAB tool. The outcomes of the analysis are compared to simulated data. There is a good agreement with the simulation results. Also covered were the impacts of various parameters on concentration and effectiveness factor. In addition. the significant contribution of this concentration of substrate and effectiveness factor using experiment values of parameters is represented in graphs and tables. The accuracy errors between numerical simulation with our analytical results are 0.008% and 0.48%. This theoretical investigation may be immediately applied to the field of engineering for the kinetics of immobilised enzymes in a flow calorimeter. [ABSTRACT FROM AUTHOR]

Background: Glucoamylase is an important enzyme for starch saccharification in the food and biofuel industries and mainly produced from mesophilic fungi such as Aspergillus and Rhizopus species. Enzymes produced from thermophilic fungi can save the fermentation energy and reduce costs as compared to the fermentation system using mesophiles. Thermophilic fungus Myceliophthora thermophila is industrially deployed fungus to produce enzymes and biobased chemicals from biomass during optimal growth at 45 °C. This study aimed to construct the M. thermophila platform for glucoamylase hyper-production by broadening genomic targeting range of the AsCas12a variants, identifying key candidate genes and strain engineering. Results: In this study, to increase the genome targeting range, we upgraded the CRISPR-Cas12a-mediated technique by engineering two AsCas12a variants carrying the mutations S542R/K607R and S542R/K548V/N552R. Using the engineered AsCas12a variants, we deleted identified key factors involved in the glucoamylase expression and secretion in M. thermophila, including Mtstk-12, Mtap3m, Mtdsc-1 and Mtsah-2. Deletion of four targets led to more than 1.87- and 1.85-fold higher levels of secretion and glucoamylases activity compared to wild-type strain MtWT. Transcript level of the major amylolytic genes showed significantly increased in deletion mutants. The glucoamylase hyper-production strain MtGM12 was generated from our previously strain MtYM6 via genetically engineering these targets Mtstk-12, Mtap3m, Mtdsc-1 and Mtsah-2 and overexpressing Mtamy1 and Mtpga3. Total secreted protein and activities of amylolytic enzymes in the MtGM12 were about 35.6-fold and 51.9‒55.5-fold higher than in MtWT. Transcriptional profiling analyses revealed that the amylolytic gene expression levels were significantly up-regulated in the MtGM12 than in MtWT. More interestingly, the MtGM12 showed predominantly short and highly bulging hyphae with proliferation of rough ER and abundant mitochondria, secretion vesicles and vacuoles when culturing on starch. Conclusions: Our results showed that these AsCas12a variants worked well for gene deletions in M. thermophila. We successfully constructed the glucoamylase hyper-production strain of M. thermophila by the rational redesigning and engineering the transcriptional regulatory and secretion pathway. This targeted engineering strategy will be very helpful to improve industrial fungal strains and promote the morphology engineering for enhanced enzyme production. [ABSTRACT FROM AUTHOR]

The glucoamylase@ZIF‐8 was prepared using ZIF‐8 material as the carrier in this study. The preparation process was optimized by response surface methodology, and the stability of glucoamylase@ZIF‐8 was determined. The material was characterized by scanning electron microscopy, X‐ray diffraction, and Fourier transform infrared spectroscopy. The results showed that the optimum preparation process of glucoamylase@ZIF‐8 was 1.65 mol 2‐methylimidazole, 5.85 mL glucoamylase, 33°C stirring temperature, 90 min stirring time, and 84.0230% ± 0.6006% embedding rate. At 100°C, the free glucoamylase completely lost its activity, whereas the glucoamylase@ZIF‐8 still had a retained enzyme activity of 12.0123% ± 0.86158%; at pH 3–6, the highest activity of glucoamylase@ZIF‐8 was 95.9531% ± 0.96181%, and about 80% of glucoamylase activity could be retained under alkaline conditions. When the ethanol concentration was 13%, the retained enzyme activity was 7.9316% ± 0.19805%, significantly higher than free enzymes. The Km of glucoamylase@ZIF‐8 and free enzyme were 1235.6825 and 80.317 mg/mL, respectively. Vmax was 0.2453 and 0.149 mg/(mL min), respectively. The appearance, crystal strength, and thermal stability of glucoamylase@ZIF‐8 were improved after optimization, and they had high reusability. [ABSTRACT FROM AUTHOR]

Puccinia triticina (Pt) is an important pathogen of wheat. While breeding programmes develop resistant wheat cultivars to mitigate the effects of such rust-causing pathogens, the emergence of new rust races with wider virulence mandates the implementation of other control strategies. Our study investigated whether acidic pH conditions affected selected Carbohydrate-Active Enzymes (CAZymes) in Pt-inoculated Thatcher + Lr9 (IR) wheat compared to those found in the Thatcher (IS) wheat. The β-glucosidase and amyloglucosidase activity levels significantly increased in IR compared to the control from 1 to 14 days post-inoculation (dpi). In contrast, activity levels of invertase did not change in the IR wheat relative to the control at 1 and 7 dpi, but were significantly reduced in the IR plants at 14 dpi. The IS had higher activity of all three hexose-producing enzymes under acidic conditions. These enzyme activities could be increased in the IS to produce hexose sugars required by Pt to develop and advance infection. The phenotypic analysis supported this view because leaf rust disease symptoms were only visible in the IS plants. For cell wall loosening-related enzymes, the IR displayed higher activity of exoglucanase, xylanase and peroxidase enzymes compared to IS. The liquid chromatography–mass spectrometry analysis showed IR had higher concentrations of complex oligosaccharides compared to the IS. Thus, we concluded that the higher exoglucanase, xylanase and peroxidase activity could be involved in cell wall loosening under acidic conditions, while oligosaccharides could be building-blocks for synthesizing cell wall barriers that apprehend Pt growth in inoculated Thatcher + Lr9. [ABSTRACT FROM AUTHOR]

The preparation of bio-butanol from corn starch requires saccharification and fermentation processes. In view of the fact that the pH value at the later stage of fermentation is applicable to the enzymatic hydrolysis of glucoamylase, the effects of simultaneous saccharification and fermentation (SSF) and separate hydrolysis and fermentation (SHF) were compared in this paper. 11.2 g/L butanol and 21.5 g/L total solvent could be obtained by the SSF process, while the yield was 9.74 g/L butanol and 17.2 g/L total solvent in the SHF process. The SSF process required a shorter overall process time (120 h) than the SHF process (144 h) and resulted in a large increase of 38.9% in butanol productivity (2.25 g/Ld for SSF compared to 1.62 g/Ld for SHF). These results show that the application of SSF can reduce the fermentation overall time, simplify the fermentation process, and reduce equipment investment and operating costs. [ABSTRACT FROM AUTHOR]

Crackers are a popular food with an appreciable share of the consumer market. Fat is essential for the sensory properties of this product; however, a high fat content is associated with health disorders. For this reason, developing low-fat products with the same desirable attributes as the corresponding full-fat ones is a high priority for the food industry. The objective of the study was to evaluate the inclusion of modified cassava starch as a fat substitute in crackers by measuring their physical and sensory properties and behavior during storage. Fat reduction in crackers led to higher fracturability values for all treatment but the pregelatinized (PREGEL) one (11.73 ± 0.39 N). The expansion ratio decreased with fat reduction, and the treatments with the highest expansion ratio and specific volume were the control (CTRL) (10.51 ± 0.21 and 1.97 ± 0.09 mL/g), the one with amyloglucosidase (AMG) (10.24 ± 0.10 and 2.20 ± 0.023 mL/g), and the pregelatinized (PREGEL) one (10.40 ± 0.16 and 1.99 ± 0.15 mL/g). The samples with modified starch showed an average total fat reduction of 49.51% compared with the control treatment. The results of sensory analysis by the acceptability test showed a greater inclination towards the CTRL and AMG treatments, in both color (6.73 ± 1.81 and 6.45 ± 2.08 , respectively) and texture (6.95 ± 1.72 and 6.67 ± 1.69 , respectively) parameters. During storage, the fracture strength decreased from 13.66 ± 0.32 N to 11.39 ± 0.57 N for AMG and from 12.5 ± 0.42 N to 10.77 ± 0.61 N for CTRL treatment, while the moisture content increased for both AMG (from 4.09 ± 0.85 % to 4.72 ± 0.13 %) and CTRL (from 3.87 ± 0.14 % to 4.31 ± 0.43 %) treatments between 0 and 30 days of storage. According to these results, it can be concluded that physically and enzymatically modified cassava starches can work as fat replacers in crackers. [ABSTRACT FROM AUTHOR]

Different starch properties may cause alterations in the foodstuff's external appearance. However, modification processes in starches are usually secretive. The use of chemically modified starches is regulated by international standards, which makes it important to identify its presence and type. Mid-infrared spectroscopy (MIR)-modified starches' identification can be hindered by the presence of excess glucose. This research investigates types of modification in commercial starches and in approaches that circumvent MIR's limitations with exploratory analysis. It also considers that enzymatic hydrolysis (α-amylase and amyloglucosidase) can highlight the points of modification in the structure, which can be detected with infrared assays. To determine if sour cassava starch (FCS) is modified and check its type, six samples were selected: a native one, three of the most common cassava modified starches (etherified, esterified, and FCS), and two laboratory processed samples (Acid, Oxidized). The results showed that the sour cassava starch showed similarities with a commercial ester and an oxidized cassava starch, which may be due to the formation of a graft, corresponding to what the literature has already reported for corn starch treated with lactic acid and gamma radiation. [ABSTRACT FROM AUTHOR]

Baicalin is a biologically active flavone glucuronide with poor water solubility that can be enhanced via glucosylation. In this study, the transglucosylation of baicalin was successfully achieved with CGTases from Thermoanaerobacter sp. and Bacillus macerans using α-cyclodextrin as a glucosyl donor. The synthesis of baicalin glucosides was optimized with CGTase from Thermoanaerobacter sp. Enzymatically modified baicalin derivatives were α-glucosylated with 1 to 17 glucose moieties. The two main glucosides were identified as Baicalein-7-O-α-D-Glucuronidyl-(1→4′)-O-α-D-Glucopyranoside (BG1) and Baicalein-7-O-α-D-Glucuronidyl-(1→4′)-O-α-D-Maltoside (BG2), thereby confirming recent findings reporting that glucuronyl groups are acceptors of this CGTase. Optimized conditions allowed for the attainment of yields above 85% (with a total glucoside content higher than 30 mM). BG1 and BG2 were purified via centrifugal partition chromatography after an enrichment through deglucosylation with amyloglucosidase. Transglucosylation increased the water solubility of BG1 by a factor of 188 in comparison to that of baicalin (molar concentrations), while the same value for BG2 was increased by a factor of 320. Finally, BG1 and BG2 were evaluated using antioxidant and anti-glycation assays. Both glucosides presented antioxidant and anti-glycation properties in the same order of magnitude as that of baicalin, thereby indicating their potential biological activity. [ABSTRACT FROM AUTHOR]

Glycoside hydrolases (GHs), the enzymes that break glycosidic bonds, are ubiquitous in the ecosystem, where they perform a range of biological functions. As an interesting glycosidase family, Glycoside hydrolase family 97 (GH97) contains α-glucosidase, α-galactosidase, and glucoamylase. Only ten members of GH97 have been characterized so far. It is critical to explore novel members to elucidate the catalytic mechanism and application potential of GH97 family. In this study, a novel glucoamylase QsGH97a from Qipengyuania seohaensis SW-135 was cloned and expressed in E. coli. Sequence analysis and NMR results show that QsGH97a is classified into GH97a, and adopts inverting mechanism. The biochemical characterization indicates that QsGH97a shows the optimal activity at 50 °C and pH 8.0. Ca2+ has little effect on the catalytic activity; however, the activity can be substantially increased by 8–13 folds in the presence of Ba2+ or Sr2+. Additionally, the metal content of QsGH97a assay showed a high proportion of Sr2+. The specific metal activity was initially revealed in glucoamylases, which is not found in other members. These results imply that QsGH97a not only is a new member of GH97, but also has potential for industrial applications. Our study reveals that Ba2+ or Sr2+ may be involved in the catalytic mechanism of glucoamylase, laying the groundwork for a more complete knowledge of GH97 and its possible industrial application. [ABSTRACT FROM AUTHOR]

The kaolinite sheets were scrolled by sonication-induced chemical delamination processes into well-developed nanotubes (KNs) which were used as substrates for microwave-based ZnO nanoparticles (ZnO/KNs). The biological activities of synthetic ZnO/KNs structures, in terms of the antioxidant and antidiabetic properties, were assessed in comparative studies with the separated phases of the synthetic ZnO and KNs as well as the commercially used ZnO. The KNs substrate resulted in a notable enhancement in the antioxidant and antidiabetic properties of ZnO, which was assigned positive influence on the surface area, interactive interfaces, charge separation, and agglomeration properties of ZnO in addition to the detectable bioactive properties of the KNs structure. The ZnO/KNs structure achieved remarkable scavenging efficiencies for 1, 1-diphenyl-2-picrylhydrazil (DPPH) (89.8 ± 1.57%), nitric oxide (90.6 ± 1.63%), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) (86.8 ± 1.28%), and superoxide (43.9 ± 1.72%) radicals. Additionally, it shows high inhibition effects on porcine α-amylase (84.3 ± 1.92%), crude α-amylase (70.6 ± 1.37%), pancreatic α-Glucosidase (94.7 ± 1.54%), crude α-Glucosidase (95.4 ± 1.64%), and amyloglucosidase (95.3 ± 1.32%) enzymes. This antidiabetic activity is significantly higher than the activity of miglitol and close to or slightly higher than acarbose, which leads us to recommend the use of ZnO/KNs when considering the cost and side effects of the commercially used drugs. [ABSTRACT FROM AUTHOR]

Green zinc-metal-pillared bentonite mediated curcumin extract (Zn@CN/BE) was synthesized and characterized as a low-cost and multifunctional (curcumin-based phytochemicals, zinc-capped curcumin, zinc/curcumin complexes, and zinc-pillared bentonite) antioxidant and antidiabetic agent with enhanced activity. The activities of the Zn@CN/BE structure were assessed in comparison with curcumin and ZnO as individual components and in the presence of miglitol and acarbose commercial drugs as controls. The structure validated remarkable antioxidant activities against the common oxidizing radicals (nitric oxide (94.7 ± 1.83%), DPPH (96.4 ± 1.63%), ABTS (92.8 ± 1.33%), and superoxide (62.3 ± 1.63 %)) and inhibition activities against the main oxidizing enzymes (porcine α-amylase (89.3 ± 1.13%), murine α-amylase (70.8 ± 1.54%), pancreatic α-Glucosidase (99.3 ± 1.23%), intestinal α-Glucosidase (97.7 ± 1.24%), and amyloglucosidase (98.4 ± 1.64%)). The reported activities are higher than the activities of individual components and the studied ascorbic acid as well as the commercial drugs. This enhancement effect was assigned to the impact of the zinc pillaring process within the curcumin/bentonite host, which induced the stability, dispersions, and interactive interface of the essential active compounds in addition to the solubility and release rate of the intercalated curcumin extract. This paper recommends the application of the Zn@CN/BE structure as an enhanced, low-cost, biocompatible, safe, and simply produced antioxidant and antidiabetic agent. [ABSTRACT FROM AUTHOR]

This study describes the enzymatic conversion of oat starch to maltose and glucose under laboratory conditions. First, oat starch is isolated by two different extraction methods (alkali and acid‐alkali). Second, enzymatic starch hydrolysis is carried out for 10% and 12.5% starch solutions obtained by both extraction methods. Third, pullulanase usage together with amyloglucosidase is investigated during saccharification. Finally, effects of the extraction method, starch concentration, and use of pullulanase are evaluated. The use of a 12.5% starch solution obtained by the acid‐alkali method results in higher rates of conversion of starch to glucose and maltose, expressing better substrate for syrup production compared to alkali starch. The use of pullulanase increases the rate of starch conversion to glucose and maltose. The highest conversion of starch to glucose is found to be 49.06% when pullulanase (12 U) is used with amyloglucosidase. The highest dextrose equivalence (DE) value is 97.71, and it is obtained by using pullulanase in 10% ​​acid‐alkali starch. The data obtained in the study show that oat starch can be a suitable source for use in sugar syrup production. The 12.5% starch ​​concentration provides high yield, and the use of pullulanase increases both the amount of hydrolysis products and the DE value. [ABSTRACT FROM AUTHOR]

The food enzyme glucan 1,4‐α‐glucosidase (4‐α‐d‐glucan α‐glucohydrolase; EC 3.2.1.3) is produced with the non‐genetically modified Aspergillus niger strain NZYM‐BO by Novozymes A/S. It was considered free from viable cells of the production organism. It is intended to be used in seven food manufacturing processes: baking processes, brewing processes, cereal‐based processes, distilled alcohol production, fruit and vegetable processing for juice production, production of dairy analogues and starch processing for the production of glucose syrups and other starch hydrolysates. Since residual amounts of total organic solids (TOS) are removed by distillation and during starch processing, dietary exposure was not calculated for these two food manufacturing processes. For the remaining five food manufacturing processes, dietary exposure to the food enzyme–TOS was estimated to be up to 2.97 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1,920 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 646. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and one match with a respiratory allergen was found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure to this food enzyme cannot be excluded (except for distilled alcohol production), but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use. [ABSTRACT FROM AUTHOR]

Glycosylation, especially N-glycosylation, is one of the most common protein modifications, with immense importance at the molecular, cellular, and organismal level. Thus, accurate and reliable N-glycan analysis is essential in many areas of pharmaceutical and food industry, medicine, and science. However, due to the complexity of the cellular glycosylation process, in-depth glycoanalysis is still a highly challenging endeavor. Contamination of samples with oligosaccharide impurities (OSIs), typically linear glucose homo-oligomers, can cause further complications. Due to their physicochemical similarity to N-glycans, OSIs produce potentially overlapping signals, which can remain unnoticed. If recognized, suspected OSI signals are usually excluded in data evaluation. However, in both cases, interpretation of results can be impaired. Alternatively, sample preparation can be repeated to include an OSI removal step from samples. However, this significantly increases sample amount, time, and effort necessary. To overcome these issues, we investigated the option to enzymatically degrade and thereby remove interfering OSIs as a final sample preparation step. Therefore, we screened ten commercially available enzymes concerning their potential to efficiently degrade maltodextrins and dextrans as most frequently found OSIs. Of these enzymes, only dextranase from Chaetomium erraticum and glucoamylase P from Hormoconis resinae enabled a degradation of OSIs within only 30 min that is free of side reactions with N-glycans. Finally, we applied the straightforward enzymatic degradation of OSIs to N-glycan samples derived from different standard glycoproteins and various stem cell lysates. [ABSTRACT FROM AUTHOR]

A broad range of enzymes are used to modify starch for various applications. Here, a thermophilic 4-α-glucanotransferase from Thermoproteus uzoniensis (TuαGT) is engineered by N-terminal fusion of the starch binding domains (SBDs) of carbohydrate binding module family 20 (CBM20) to enhance its affinity for granular starch. The SBDs are N-terminal tandem domains (SBDSt1 and SBDSt2) from Solanum tuberosum disproportionating enzyme 2 (StDPE2) and the C-terminal domain (SBDGA) of glucoamylase from Aspergillus niger (AnGA). In silico analysis of CBM20s revealed that SBDGA and copies one and two of GH77 DPE2s belong to well separated clusters in the evolutionary tree; the second copies being more closely related to non-CAZyme CBM20s. The activity of SBD-TuαGT fusions increased 1.2–2.4-fold on amylose and decreased 3–9 fold on maltotriose compared with TuαGT. The fusions showed similar disproportionation activity on gelatinised normal maize starch (NMS). Notably, hydrolytic activity was 1.3–1.7-fold elevated for the fusions leading to a reduced molecule weight and higher α-1,6/α-1,4-linkage ratio of the modified starch. Notably, SBDGA-TuαGT and-SBDSt2-TuαGT showed Kd of 0.7 and 1.5 mg/mL for waxy maize starch (WMS) granules, whereas TuαGT and SBDSt1-TuαGT had 3–5-fold lower affinity. SBDSt2 contributed more than SBDSt1 to activity, substrate binding, and the stability of TuαGT fusions. [ABSTRACT FROM AUTHOR]

Glucoamylase was often used in the brewing industry but was unstable to several environmental factors and reacted quickly to produce fermentable sugar, which limited its applications. Microencapsulation could effectively overcome the drawbacks. This study evaluated the feasibility of the preparation of glucoamylase microcapsules (GM) using W/O/W complex coacervation‐freeze‐drying method. The parameters of the microcapsules were optimized by the response surface optimization design: core‐wall ratio at 1:1, wall‐material concentration at 8%, and coagulation time for 20 min. Under current condition, the final microencapsulation efficiency reached 85.64 ± 1.60%. Glucoamylase could be slowly released for more than 96 h in the liquid state, and could react slowly for more than 336 h in the solid state. The optimized GM were incubated for 1 h, and the relative enzyme activity was better than that of free glucoamylase under high temperature conditions. The water capacity, solubility, morphology, differential scanning calorimetry, and Fourier transform infrared spectroscopy were conducted. Glucoamylase exhibited good sustained release characteristics. The microcapsules were more resistant to environmental stimuli and showed stronger robustness after optimization. Practical Application: Saccharification enzymes are often used in the winemaking industry, and direct addition will cause the fermentation process to heat up too quickly, resulting in the inactivation of microorganisms and saccharification enzymes, affecting the efficiency of saccharification enzymes. Therefore, microcapsules are used to encapsulate the saccharification enzyme, and its efficacy is slowly released for a long time during the fermentation process. [ABSTRACT FROM AUTHOR]

The food enzyme glucan‐1,4‐α‐glucosidase (4‐α‐d‐glucan glucohydrolase; EC 3.1.2.3) is produced with the non‐genetically modified Rhizopus delemar strain CU634‐1775 by Shin Nihon Chemical Co., Ltd. The food enzyme is free from viable cells of the production strain. It is intended to be used in six food manufacturing processes: baking processes, starch processing for production of glucose syrups and other starch hydrolysates, fruit and vegetable processing for juice production, fruit and vegetable processing for products other than juices, brewing processes and distilled alcohol production. As residual amounts of total organic solids (TOS) are removed by distillation and by the purification steps applied during the production of glucose syrups, dietary exposure was not calculated for these two food processes. For the remaining four food processes, dietary exposure to the food enzyme‐total organic solids was estimated to be up to 1.238 mg TOS/kg body weight (bw) per day. Genotoxicity tests did not raise a safety concern. Systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1,735 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 1,401. In a search for the similarity of the amino acid sequence of the food enzyme to known allergens, a single match with a respiratory allergen was found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use. [ABSTRACT FROM AUTHOR]

Summary: Glucose‐rich supernatants were developed based on the enzymatic hydrolysis of raw and extruded chickpea flours first treated with alcalase® and then with a combination of alpha‐amylase and amyloglucosidase or only amyloglucosidase. The optimum treatments were the extruded hydrolysates further treated with amyloglucosidase 'EF(AMG)', or the raw chickpea flour treated with a combination of alpha‐amylase and amyloglucosidase 'RF (Amyl+AMG)'. The resulting supernatants were further fermented with five different strains of Lactobacillus and monitored for 24 h in terms of pH, titratable acidity, and microbial counts. Also, the glucose content and oligosaccharides, total protein, and viability were determined. Both supernatants presented a successful fermentation as estimated by the pH drop (<4.2), lactic acid produced (>0.7%), and microbial counts 9 log (CFU mL−1) which increased up to 5 log during the 24 h fermentation. However, the EF(AMG) supernatant induced fewer reductions in glucose and protein contents in comparison to the RF (Amyl+AMG) counterpart. Among bacteria, the strain Lp 29 in EF(AMG) showed the fastest growth rate (K) and the highest residual protein. In all cases, the proposed technology enhanced the growth of five Lactobacillus strains by taking advantage of the hydrolyzed chickpea chemical composition. [ABSTRACT FROM AUTHOR]

The food enzyme glucan 1,4‐α‐glucosidase (4‐α‐d‐glucan glucohydrolase; EC 3.2.1.3) is produced with the non‐genetically modified Rhizopus arrhizus strain AE‐G by Amano Enzyme Inc. The food enzyme is free from viable cells of the production organism. The applicant proposed the use of the food enzyme in baking processes, coffee processing and manufacture of enzymatically modified dairy ingredients (EMDI). The Panel considered only the baking processes as the relevant intended use of this food enzyme. Dietary exposure to the food enzyme–total organic solids (TOS) was estimated to be up to 0.94 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not raise a safety concern. The systemic toxicity was assessed by a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1,868 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 1,987. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and one match was found. The Panel considered that under the intended conditions of use the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns when used in baking processes under the intended conditions of use. [ABSTRACT FROM AUTHOR]

Background: Raw starch-degrading enzyme (RSDE) is applied in biorefining of starch to produce biofuels efficiently and economically. At present, RSDE is obtained via secretion by filamentous fungi such as Penicillium oxalicum. However, high production cost is a barrier to large-scale industrial application. Genetic engineering is a potentially efficient approach for improving production of RSDE. In this study, we combined genetic engineering and random mutagenesis of P. oxalicum to enhance RSDE production. Results: A total of 3619 mutated P. oxalicum colonies were isolated after six rounds of ethyl methanesulfonate and Co60-γ-ray mutagenesis with the strain A2-13 as the parent strain. Mutant TE4-10 achieved the highest RSDE production of 218.6 ± 3.8 U/mL with raw cassava flour as substrate, a 23.2% compared with A2-13. Simultaneous deletion of transcription repressor gene PoxCxrC and overexpression of activator gene PoxAmyR in TE4-10 resulted in engineered strain GXUR001 with an RSDE yield of 252.6 U/mL, an increase of 15.6% relative to TE4-10. Comparative transcriptomics and real-time quantitative reverse transcription PCR revealed that transcriptional levels of major amylase genes, including raw starch-degrading glucoamylase gene PoxGA15A, were markedly increased in GXUR001. The hydrolysis efficiency of raw flour from cassava and corn by crude RSDE of GXUR001 reached 93.0% and 100%, respectively, after 120 h and 84 h with loading of 150 g/L of corresponding substrate. Conclusions: Combining genetic engineering and random mutagenesis efficiently enhanced production of RSDE by P. oxalicum. The RSDE-hyperproducing mutant GXUR001 was generated, and its crude RSDE could efficiently degrade raw starch. This strain has great potential for enzyme preparation and further genetic engineering. [ABSTRACT FROM AUTHOR]

Pullulanase, a starch debranching enzyme, is required for the preparation of high glucose/maltose syrup from starch. In order to expand its narrow reaction conditions and improve its application value, Bacillus naganoensis pullulanase (PulA) was mutated by site-directed mutagenesis and the biochemical characteristics of the mutants were studied. The mutant PulA-N3 with mutations at asparagine 467, 492 and 709 residues was obtained. It displayed the activity maximum at 60 °C and pH 4.5 and exceeded 90% activities between 45 and 60 °C and from pH 4.0 to pH 5.5, which was improved greatly compared with wild-type PulA. Its thermostability and acidic pH stability were also remarkably improved. Its catalytic rate (kcat/Vmax) was 2.76 times that of PulA. In the preparation of high glucose syrup, the DX (glucose content, %) values of glucose mediated by PulA-N3 and glucoamylase reached 96.08%, which were 0.82% higher than that of PulA. In conclusion, a new pullulanase mutant PulA-N3 was successfully developed, which has high debranching activity in a wide range of temperature and pH, thereby paving the way for highly efficient starch saccharification. [ABSTRACT FROM AUTHOR]

Dark fermentation (DF) is a promising biological process for hydrogen production from biomass. However, low hydrogen yield (HY) is a major hurdle impeding its use at large-scale operation. A potential way to mitigate this problem is to increase the concentration of substrate in the process to increase hydrogen production. The present study investigated the possibility of using high-solid DF to produce hydrogen from cassava processing wastes, i.e. , cassava pulp (CP) and cassava processing wastewater (CPW). CP was suspended in CPW and hydrolyzed enzymatically under optimum conditions of 150 g-CP/L, 29 U/g of α-amylase, 47 U/g of glucoamylase, and 60 FPU/g of cellulase. The hydrolysis performed at 50 °C for 24 h yielded a reducing sugar concentration of 117.7 ± 1.8 g/L, equivalent to 0.78 g-reducing-sugar/g-CP. Subsequent DF of CP-CPW enzymatic slurry, which contained 12.1% water insoluble solids, resulted in a cumulative production of 13.72 ± 0.22 L-H 2 , equivalent to 225.2 ± 3.7 mL-H 2 /g-VS. This was 83.1% of a maximum stoichiometric HY, based on carbohydrate content of CP and soluble metabolites production. The present study shows clearly the applicability of high-solid DF in the production of hydrogen from cassava processing wastes. • Hydrogen production from cassava pulp (CP) and cassava processing wastewater (CPW). • Enzymatic hydrolysis of CP-CPW suspension was statistically optimized. • Proper mixing improved hydrogen production in high-solid dark fermentation. • Hydrogen yield (HY) equivalent to 83.1% of a stoichiometric maximum HY was attained. [ABSTRACT FROM AUTHOR]

BACKGROUND: Mechanized Huangjiu is a stable product, is not subject to seasonal production restrictions, and markedly reduces labor intensity compared to traditional manual Huangjiu. However, the bitterness of mechanized Huangjiu impedes its further development. RESULTS: Based on process optimization, when the fermentation temperature was 45 °C and the fermentation time was 122 h, the inoculation amount of Saccharopolyspora was 5%, the amount of added water was 26%, and the glucoamylase and amylase activities of wheat Qu increased by 27% and 40% respectively, compared with those before optimization. Huangjiu fermented by raw wheat Qu inoculated with Saccharopolyspora rosea F2014 showed a significant (P < 0.05) decrease in bitter amino acid content (1.24 vs. 2.86 g L−1, a decrease of 56%), which attenuated its bitterness. CONCLUSION: An innovative fermentation process of inoculating Saccharopolyspora into raw wheat Qu was developed for the first time. Such a process could be used to control bitterness based on raw wheat Qu inoculated with Saccharopolyspora rosea F2014, instead of traditional wheat Qu in Huangjiu fermentation. © 2022 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

This study evaluated the effects of amylolytic enzymes on the nutritive value, fermentation losses, fermentation profile, and amylolytic activity of rehydrated corn that had been stored for 30, 60, 90, 120, 150, 180, 210, or 240 d. Corn kernels were harvested, ground in a knife mill using a 4-mm screen, rehydrated to 625 g/kg of dry matter (DM). These experimental silos were then randomly assigned to the following treatments (n = 40 each): 1) CON (control) – rehydrated corn silage with no exogenous enzymes incorporated during the ensiling process; 2) AMY – rehydrated corn silage with exogenous alpha-amylase incorporated at 90 U/kg of fresh matter; and 3) GLU – rehydrated corn silage with exogenous glucoamylase incorporated at 90 U/kg of fresh matter. It was found that enzyme incorporation decreased the DM, starch, and non-fibre carbohydrate and increased the total losses and contents of crude protein, ether extract. Enzyme incorporation also reduced the silage pH, while increasing the lactate concentration. silages in the GLU treatment had higher aerobic bacteria counts and acetate and propionate concentrations. Amylolytic enzymes increased fermentation losses and the lactate and ethanol contents and decreased the nutritive value of rehydrated corn silage, reducing the starch content and DM degradation. [ABSTRACT FROM AUTHOR]

The starch in waste bread (WB) from industrial sandwich production was directly converted to ethanol by an amylolytic, ethanologenic thermophile (Parageobacillus thermoglucosidasius strain TM333) under 5 different simultaneous saccharification and fermentation (SSF) regimes. Crude α-amylase from TM333 was used alone or in the presence of amyloglucosidase (AMG), a starch monomerizing enzyme used in industry, with/without prior gelatinisation/liquefaction treatments and P. thermoglucosidasius TM333 fermentation compared with Saccharomyces cerevisiae as a control. Results suggest that TM333 can ferment WB using SSF with yields of 94–100% of theoretical (based on all sugars in WB) in 48 h without the need for AMG addition or any form of heat pre-treatment. This indicates that TM333 can transport and ferment all of the malto-oligosaccharides generated by its α-amylase. In the yeast control experiments, addition of AMG together with the crude α-amylase was necessary for full fermentation over the same time period. This suggests that industrial fermentation of WB starch to bio-ethanol or other products using an enhanced amylolytic P. thermoglucosidasius strain could offer significant cost savings compared to alternatives requiring enzyme supplementation. [ABSTRACT FROM AUTHOR]

Background: Our recent multi-omics analyses of glucoamylase biosynthesis in Aspergillus niger (A. niger) suggested that lipid catabolism was significantly up-regulated during high-yield period under oxygen limitation. Since the catabolism of fatty acids can provide energy compounds such as ATP and important precursors such as acetyl-CoA, we speculated that enhancement of this pathway might be beneficial to glucoamylase overproduction. Results: Based on previous transcriptome data, we selected and individually overexpressed five candidate genes involved in fatty acid degradation under the control of the Tet-on gene switch in A. niger. Overexpression of the fadE, fadA and cyp genes increased the final specific enzyme activity and total secreted protein on shake flask by 21.3 ~ 31.3% and 16.0 ~ 24.2%, respectively. And a better inducible effect by doxycycline was obtained from early logarithmic growth phase (18 h) than stationary phase (42 h). Similar with flask-level results, the glucoamylase content and total extracellular protein in engineered strains OE-fadE (overexpressing fadE) and OE-fadA (overexpressing fadA) on maltose-limited chemostat cultivation were improved by 31.2 ~ 34.1% and 35.1 ~ 38.8% compared to parental strain B36. Meanwhile, intracellular free fatty acids were correspondingly decreased by 41.6 ~ 44.6%. The metabolomic analysis demonstrated intracellular amino acids pools increased 24.86% and 18.49% in two engineered strains OE-fadE and OE-fadA compared to B36. Flux simulation revealed that increased ATP, acetyl-CoA and NADH was supplied into TCA cycle to improve amino acids synthesis for glucoamylase overproduction. Conclusion: This study suggested for the first time that glucoamylase production was significantly improved in A. niger by overexpression of genes fadE and fadA involved in fatty acids degradation pathway. Harnessing the intracellular fatty acids could be a strategy to improve enzyme production in Aspergillus niger cell factory. [ABSTRACT FROM AUTHOR]

Background: There are currently three known congenital disaccharidase deficiencies: congenital lactase deficiency (CLD), congenital sucrase-isomaltase deficiency (CSD), and congenital trehalase deficiency (CTD). No congenital deficiency has been described for maltase-glucoamylase (MGAM). Methods: A literature search was performed in PubMed for the pathogenic variants CLD, CSD, and CTD and the articles retrieved were analyzed to estimate the prevalence of congenital disaccharidase deficiencies. Results: Based on reported variants, the estimated prevalence was 1.3 per 106 births (95% CI: 1.1–1.7) for CLD, and 31.4 per 106 births (95% CI: 28.3–34.8) for CSD. Using data on previously reported variants and variants predicted to be loss-of-function in gnomAD, the overall estimated prevalence was 2.3 per 106 births (95% CI: 1.9–2.9) for CLD, 57.6 per 106 births (95% CI:52.5–63.2) for CSD, and 9.2 per 106 births (95% CI: 2.5–3.7) for CTD. Conclusion: The prevalence of CSD was found to be relatively high, while for other congenital disaccharidase deficiencies, the estimated prevalence was very low. [ABSTRACT FROM AUTHOR]

In this work, a positively charged chitosan-grafted-polyarginine (CS-N-PArg) as the macro-molecular NO donor, and a negatively charged acetalated starch (AcSt-O-PAsp) as a glucose donor, have been synthesized. To achieve the multi-enzymatic cascade system for local generation of self-supply glucose to increase the H 2 O 2 concentration for the subsequent oxidization of L-Arg into NO, the designed positively charged CS-N-PArg, negatively charged AcSt-O-PAsp, glucoamylase (GA) and glucose oxidase (GOx) are absorbed and assembled in the pore of the gelatin sponge via electrostatic interaction to establish a smart antibacterial dressings (CS/St + GOx/GA). Once stimulated by Escherichia coli (E. coli)-infected wounds (a slightly acidic environment), the cascade reaction system can sequentially induce to generate glucose, H 2 O 2 and NO, which exhibits a meaningful alternative idea for a high-performance antibacterial therapy. • The dressings could release NO gas specifically in bacterial infected skin lesions. • Acetalated starch (AcSt-O-PAsp) could release starch under weakly-acidic environment. • Chitosan–grafted–polyarginine (CS-N-PArg) has been synthesized as NO donor. • The presented exogenous H 2 O 2 way is based on the self-supply glucose. [ABSTRACT FROM AUTHOR]

Cellulose is a major component of dietary fiber and it is proved to influence starch digestibility. The effects of native cellulose (NC), microcrystalline cellulose (MC), soluble cellodextrin (SC) on starch digestion have not been clearly elucidated. In this study, three types of cellulose with representative molecular weights (NC, 422500 Da; MC, 27750 Da; SC, 2202 Da) were prepared and their effects on starch digestion, glucose diffusion, α-amylase and amyloglucosidase activity were compared. The results suggested SC inhibited starch digestibility to a greater degree than those of NC and MC. When addition of SC reached 3 %, rapidly digestible starch proportion decreased from 31.2 % to 11.3 % and resistant starch proportion increased from 15.0 % to 58.0 %. Notably, hindrance effects of SC on glucose diffusion were higher than those of NC and MC. Moreover, SC reduced activity of α-amylase and amyloglucosidase to a larger extent than those of MC and NC. With the effect of starch digestion inhibition, NC, MC and SC could be utilized as functional food ingredients. Especially, the soluble property and the highest starch digestion inhibition ability of SC favors its application in food industry. • SC showed a stronger restraint effect on starch digestibility than either MC and NC • With the addition of 3 % SC, the content of RS increased from 15.0 % to 58.0 %. • SC presented a greater effect on the diffusion of glucose • SC reduced α-amylase and amyloglucosidase activity to a larger extent [ABSTRACT FROM AUTHOR]

Simple Summary: Glucoamylase is one of the most needed industrial enzymes in the food and biofuel industries. Aspergillus niger is a commonly used cell factory for the production of commercial glucoamylase. For decades, genetic manipulation has promoted significant progress in industrial fungi for strain engineering and in obtaining deep insights into their genetic features. However, genetic engineering is more laborious in the glucoamylase-producing industrial strains A. niger N1 and O1 because their fungal features of having few conidia (N1) or of being aconidial (O1) make them difficult to perform transformation on. In this study, we targeted A. niger N1 and O1 and successfully developed high-efficiency transformation tools. We also constructed a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 editing marker-free system using an autonomously replicating plasmid to express Cas9 protein and to guide RNA and the selectable marker. By using the genetic tools developed here, we generated nine albino deletion mutants. After three rounds of sub-culturing under nonselective conditions, the albino deletions lost the autonomously replicating plasmid. Together, the tools and optimization process above provided a good reference to manipulate the tough working industrial strain, not only for the further engineering these two glucoamylase-hyperproducing strains, but also for other industrial strains. The filamentous fungus Aspergillus niger is widely exploited by the fermentation industry for the production of enzymes, particularly glucoamylase. Although a variety of genetic techniques have been successfully used in wild-type A. niger, the transformation of industrially used strains with few conidia (e.g., A. niger N1) or that are even aconidial (e.g., A. niger O1) remains laborious. Herein, we developed genetic tools, including the protoplast-mediated transformation and Agrobacterium tumefaciens-mediated transformation of the A. niger strains N1 and O1 using green fluorescent protein as a reporter marker. Following the optimization of various factors for protoplast release from mycelium, the protoplast-mediated transformation efficiency reached 89.3% (25/28) for N1 and 82.1% (32/39) for O1. The A. tumefaciens-mediated transformation efficiency was 98.2% (55/56) for N1 and 43.8% (28/64) for O1. We also developed a marker-free CRISPR/Cas9 genome editing system using an AMA1-based plasmid to express the Cas9 protein and sgRNA. Out of 22 transformants, 9 albA deletion mutants were constructed in the A. niger N1 background using the protoplast-mediated transformation method and the marker-free CRISPR/Cas9 system developed here. The genome editing methods improved here will accelerate the elucidation of the mechanism of glucoamylase hyperproduction in these industrial fungi and will contribute to the use of efficient targeted mutation in other industrial strains of A. niger. [ABSTRACT FROM AUTHOR]

Simple Summary: As an important energy feed material for livestock and poultry, corn is often in short supply and used immediately after harvest. However, newly harvested corn can have anti-nutritional factors (ANFs) and cause animal diarrhea. Feed-grade enzymes and probiotics can help to alleviate ANFs in feedstuffs. Therefore, in this study, several enzymes and a laboratory Pediococcus acidilactici BCC-1, isolated from chick ceca, were added into the newly harvested corn diet for a 21-day trial in broilers. Here, we found that the newly harvested corn diets resulted in shorter digesta emptying time, increased visual fecal water content, and decreased trypsin activity, which may lead to the occurrence of diarrhea in broilers. The supplementation of Pediococcus acidilactici BCC-1 to newly harvested corn diets could improve the activity of trypsin and decrease the FCR (F/G) of broilers. Moreover, supplemental protease could significantly increase the digesta emptying time, reduce the visual fecal water content, and increase trypsin activity, which may relieve the diarrhea of broilers. In conclusion, Pediococcus acidilactici BCC-1 or protease individually, as well as in combination with glucoamylase are recommended in newly harvested corn diets for broilers. A new grain phenomenon happens in newly harvested corn because of its high content of anti-nutritional factors (ANFs), which can cause low nutrient digestibility and diarrhea in animals. Enzymes and probiotics have been shown to relieve the negative effect of ANFs for animals. The purpose of this study was to investigate the effect of enzymes and probiotics on the performance and nutrient digestibility of broilers, fed with newly harvested corn diets. A total of 624 Arbor Acres Plus male broiler chickens were randomly divided into eight treatment groups (A: normal corn diet, CT: newly harvested corn diet, DE: newly harvested corn diet + glucoamylase, PT: newly harvested corn diet + protease, XL: newly harvested corn diet + xylanase, BCC: newly harvested corn diet + Pediococcus acidilactici BCC-1, DE + PT: newly harvested corn diet + glucoamylase + protease, XL + BCC: newly harvested corn diet + xylanase + Pediococcus acidilactici BCC-1). Each group was divided into six replicates, with 13 birds each. On day 21, growth performance, nutrient digestibility, and digestive enzyme activity were measured. Compared with the normal corn diet (PC), the newly harvested corn diet (NC) produced shorter digesta emptying time (p = 0.015) and increased visual fecal water content (p = 0.002) of broilers, however, there was no effect on performance. Compared to the newly harvested corn diet (NC), supplemental enzyme of DE increased the activity of chymotrypsin (p = 0.016), however, no differences in the digestibility of three kinds of organic matter, digesta emptying time, visual fecal water content, or performance were found. Supplemental protease (PT) significantly increased digesta emptying time (p = 0.004) and decreased the activity of maltase (p = 0.007). However, it had no effect on the digestibility of three kinds of organic matter or the performance of broilers. Supplemental xylanase (XL) decreased the activity of amylase (p = 0.006) and maltase (p < 0.001); however, it had no effect on digesta emptying time, visual fecal water content, the digestibility of three kinds of organic matter, or performance of broilers. Supplemental DE, combined with PT (DE + PT), increased the digesta emptying time (p = 0.016) while decreasing the visual fecal water content (p = 0.011), and the activity of amylase (p = 0.011), lipase (p = 0.021), and maltase (p < 0.001), however, there was no effect on performance. Supplemental BCC individually decreased the activity of amylase (p = 0.024) and maltase (p < 0.001), however, it increased the activity of trypsin (p < 0.001) and tended to improve feed conversion ratio (FCR) (p = 0.081). Supplemental BCC-1, combined with XL (XL + BCC), increased the activity of trypsin (p = 0.001) but decreased the activity of amylase (p = 0.013), lipase (p = 0.019), and maltase (p < 0.001). Pediococcus acidilactici BCC-1 (109 cfu/kg), protease (800,000 U/g) individually, or protease (800,000 U/g) in combination with glucoamylase (800,000 U/g) were supplemented in newly harvested corn diets for growing broilers. Hence, this study mainly explores the alleviation effect of enzyme and probiotics on the negative phenomenon caused by the utilization of newly harvested corn in broilers and provides a better solution for the utilization of newly harvested corn in production practice. [ABSTRACT FROM AUTHOR]

Preparation of porous starch (PS) from corn starch (NS) using enzymes amylase (AM) and amyloglucosidase (AMG) was standardized using two factorial experimental design in terms of surface area and pore size distribution. SEM micrographs confirmed the formation of porous structures in the granules. Based on surface area, pore size and statistical analysis 300 U AM and 250 U AMG for 6 h incubation were suggested for porous starch preparation. Physicochemical characteristics of NS and PS were compared using zeta potential, contact angle, rheology, FTIR, XRD, and DSC which suggested the potential of PS as a stabilizer for O/W emulsions. Further studies confirmed emulsion stabilizing efficacy of PS with creaming index of 5.0% against 16.6% for NS. The fluorescence microscopy images of the emulsion after staining with specific dyes revealed that PS acts as a Pickering particle. Furthermore, studies using curcumin as model system indicated that PS acts as better bioactive carrier as compared to NS. The curcumin holding capacities of PS and NS were 82.24 ± 1.07 and 61.03 ± 1.43%, respectively. The study suggested that PS can be effectively used as Pickering particle and bioactive carrier in various food, nutraceutical, and pharmaceutical applications. [ABSTRACT FROM AUTHOR]

A preferable pullulanase with high thermostability and catalytic activity at pH 4.5–5 is desired to match with glucoamylase in the starch-saccharification process. However, most of them exhibit low activity under such low pH conditions. Here, the optimal pH of the hyperthermostable pullulanase from Pyrococcus yayanosii (Pul PY2) was successfully shifted from 6.4 to 5 with a 2-fold increase in the specific activity based on synergistic engineering of the active center and surface. Synergistic engineering was performed by introducing histidine within 6 Å of the active sites, and by enhancing negative charges on the enzymatic surface. Two single-site mutants of Pul PY2 -Q13H and Pul PY2 -I25E with higher hydrolytic activity were obtained, the optimal pH of which was shifted to pH 5 and 5.4, respectively; the combined mutant Pul PY2 -Q13H/I25E exhibited the optimal pH of 5, 3.2-fold increasing catalytic efficiency at pH 5, and high thermostability compared to Pul PY2. These results not only obtained an applicable pullulanase for industrial application, but also provided a strategy for shifting the optimal pH of the enzyme based on synergistic engineering of the active center and surface. [ABSTRACT FROM AUTHOR]

Summary: Daqu provides hydrolytic enzymes that saccharifying the macromolecular carbohydrate in fermenting grains. The process of saccharification is the premise of Baijiu production. But little is known about the protein composition associated with the activity of saccharifying enzymes in daqu. This study investigated related proteins between nongxiangxing daqu and jiangxiangxing daqu by metaproteomics. We present that the amylase system (α‐amylase, glucoamylase and α‐glucosidase) and cellulase system (cellobiohydrolases, endoglucanase and β‐glucosidase) are regarded as the main saccharifying enzymes. Combined with protein‐based taxonomic annotation, the key proteins involving in amylase system were mainly from Kroppenstedtia, Lichtheimia, Byssochlamys and Thermomyces, while Thermoascu contributed the most to cellulase system. Moreover, it was found that the up‐regulated amylase proteins at thermophilic fermentation period in jiangxiangxing daqu in comparison with nongxiangxing daqu were affiliated to Aspergillus, Rhizomucor, Byssochlamys and Thermomyces, along with Thermoascu contributing to cellulase system. These findings will provide clues for improving saccharification efficiency in Baijiu production. [ABSTRACT FROM AUTHOR]

Alpha-glucosidase (maltase, sucrase, isomaltase and glucoamylase) activities which are involved in carbohydrate metabolism are present in human intestinal maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI). Hence, these proteins are important targets to identify drugs against postprandial hyperglycemia thereby for diabetes. To find natural-based drugs against MGAM and SI, Artocarpus heterophyllus leaf was explored for MGAM and SI inhibition in in vitro and in silico. A. heterophyllus leaf aqueous active fraction (AHL-AAF) was prepared using Soxhlet extraction followed by silica column chromatography. The phytoconstituents of AHL-AAF were determined using LC-ESI-MS/MS. AHL-AAF showed dose-dependent and mixed inhibition against maltase (IC50 = 460 µg/ml; Ki = 300 µg/ml), glucoamylase (IC50 = 780 µg/ml; Ki = 480 µg/ml), sucrase (IC50 = 900 µg/ml, Ki = 504 µg/ml) and isomaltase (IC50 = 860 µg/ml, Ki = 400 µg/ml). AHL-AAF phytoconstituents interaction with N-terminal (Nt) and C-terminal (Ct) subunits of human MGAM and SI was analyzed using induced-fit docking, molecular dynamics (MD), and binding free energy calculation. In docking studies, rhamnosyl hexosyl methyl quercetin (RHMQ), P-coumaryl-O-16-hydroxy palmitic acid (PCHP), and spirostanol interacted with active site amino acids of human MGAM and SI. Among these RHMQ stably interacted with all the subunits (Nt-MGAM, Ct-MGAM, Nt-SI and Ct-SI) whereas PCHP with Ct-MGAM and Nt-SI during MD analysis. In molecular docking, the docking score of RHMQ with NtMGAM, CtMGAM, NtSI and CtSI was −8.48, −12.88, −11.98 and −11.37 kcal/mol. The docking score of PCHP for CtMGAM and NtSI was −8.59 and −8.4 kcal/mol, respectively. After MD simulation, the root mean square deviation (RMSD) and root mean square fluctuation (RMSF) values further confirmed the stable protein-ligand interaction. The RMSD value of all the complexes were around 2.5 Å and the corresponding RMSF values were also quite low. In MM/GBSA analysis, the involvement of Van der Waals and lipophilic energy in the protein/ligand interactions are understood. Further binding free energy for Nt-MGAM-PCHP, Nt-MGAM-RHMQ, Nt-SI-PCHP, Nt-SI-RHMQ, Ct-MGAM-PCHP, Ct-MGAM-RHMQ and Ct-SI-RHMQ complexes was found to be −24.94, −46.60, −46.56, −44.48, −40.3, −41.86 and −19.39 kcal/mol, respectively. Altogether, AHL-AAF showed inhibition of α-glucosidase activities of MGAM and SI. AHL-AAF could be further studied for its effect on diabetes in in vivo. [Display omitted] • Artocarpus heterophyllus leaf aqueous active fraction (AHL-AAF) subjected to LC-ESI-MS/MS. • AHL-AAF inhibited mammalian glucosidases activities. • AHL-AAF active molecules interacted with human glucosidase in silico. • MD simulation described the stability of protein-ligand complexes. [ABSTRACT FROM AUTHOR]

In multinuclear and multicellular filamentous fungi little is known about how mRNAs encoding secreted enzymes are transcribed and localized spatiotemporally. To better understand this process we analyzed mRNA encoding GlaA, a glucoamylase secreted in large amounts by the industrial filamentous fungus Aspergillus oryzae , by the MS2 system, in which mRNA can be visualized in living cells. We found that glaA mRNA was significantly transcribed and localized near the hyphal tip and septum, which are the sites of protein secretion, in polarity-dependent expression and localization manners. We also revealed that glaA mRNA exhibits long-range dynamics in the vicinity of the endoplasmic reticulum (ER) in a manner that is dependent on the microtubule motor proteins kinesin-1 and kinesin-3, but independent of early endosomes. Moreover, we elucidated that although glaA mRNA localized to stress granules (SGs) and processing bodies (PBs) under high temperature, glaA mRNA was not seen under ER stress, suggesting that there are different regulatory mechanisms of glaA mRNA by SG and PB under high temperature and ER stress. Collectively, this study uncovers a dynamic regulatory mechanism of mRNA encoding a secretory enzyme in filamentous fungi. [ABSTRACT FROM AUTHOR]

Growing interest among consumers regarding sustainable food production has led to innovations in the use of cleaner additives and manufacturing methods for the food industry, such as the enzymatic modification of starches. The effect of enzymatic modification (pullulanase and amyloglucosidase) on the pasting and thermal properties of cassava starch and the quality properties of fermented dairy beverages are analyzed at the beginning and end of a 20‐day storage period and compared to beverages with chemically modified starch, native cassava starch (NAT), without the addition of starch (control), and a commercial beverage. Amyloglucosidase‐modified starch shows the highest degree of long‐term retrogradation, which can also be perceived in the quality of beverages during storage. Beverages prepared with pullulanase‐modified starch have the highest viscosity (503.56 mPa s) among the evaluated beverages and a lower syneresis (29.18 g/100 g) than control and NAT beverages. The addition of enzymatically modified cassava starch led to a significant increase in the general acceptability of the beverages compared to the control treatment. The results show that pullulanase‐modified starches have the potential to replace chemically modified starches in the production of fermented dairy beverages, enabling the development of products that meet the requirements of the natural foods market. [ABSTRACT FROM AUTHOR]