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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

Altres ajuts: acords transformatius de la UAB We would like to thank Dr. Manel Bosch (Advanced Optical Microscopy Facilities, University of Barcelona) for his invaluable advice and collaboration to obtain microscope images of both Haematoxylin-Eosin-stained samples and PAS ones. No disclosure was applicable on this work. Likewise, there is no conflict of interest related with this manuscript. Furthermore, no specific Funding was afforded for this work. This work analysed the expression of prostate polysaccharides in rats with age-related benign prostatic hyperplasia (BPH) for a better understanding of the possible relationship between prostate polysaccharides secretion and BPH onset. For this, prostatic glands from 1 month-old, 3 months-old, 6 months-old and 12 months-old Sprague-Dawley rats were processed in order to identify their overall polysaccharide content. Additionally, serum testosterone was also determined. One-month old rats showed significantly (P < 0.05) lower testosterone levels (0.77 ng/mL±0.12 ng/mL) compared with the other groups, which showed no significant difference among them. PAS staining showed positive polysaccharides markings in both the prostatic lumen and inside of luminal prostatic cells in all groups. Semiquantitative analysis of intraluminal PAS showed that one month-old rats had significantly (P < 0.005) lower PAS intensity when compared with all other groups (100.0 ± 0.5, arbitrary units vs. 107.3 ± 0.6, arbitrary units in 3 months-old ones), whereas 12 months-old ones showed significantly (P < 0.005) higher values when compared with all other groups (133.6 ± 3.5, arbitrary units in 12 months-old rats vs. 108.6 ± 1.4, arbitrary units in 6 months-old ones). The PAS + content practically disappeared when tissues were pre-incubated with either α-amylase or amyloglucosidase, regardless of a previous incubation with proteinase K. Incubation of prostate extracts from 12 months-old rats for 2 h with α-amylase yielded a significantly higher amount of free glucose (1.47 nmol/mg protein±0.23 nmol/mg protein vs. 0.32 nmol/mg protein±0.01 nmol/mg protein in untreated extracts). Similar results were obtained when extracts were pre-incubated with amyloglucosidase. Contrarily, pre-incubation with N-glycosidase induced a significantly (P < 0.05), much lower increase of free glucose. Pre-treatment with proteinase K did not significantly modify these results, which indicate that BPH is related to an increase in the secretion of low ramified ductal α-glycosydic polysaccharides that were not protected against lysis by any type of protein protective core. These changes seem to not be related with concomitant variations in serum testosterone levels.

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.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 LAn 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.

Basidiomycetous yeasts remain an almost unexplored source of enzymes with great potential in several industries. Tausonia pullulans (Tremellomycetes) is a psychrotolerant yeast with several extracellular enzymatic activities reported, although the responsible genes are not known. We performed the genomic sequencing, assembly and annotation of T. pullulans strain CRUB 1754 (Perito Moreno glacier, Argentina), a gene survey of carbohydrate-active enzymes (CAZymes), and analyzed its secretome by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) after growth in glucose (GLU) or starch (STA) as main carbon sources. T. pullulans has 7210 predicted genes, 3.6% being CAZymes. When compared to other Tremellomycetes, it contains a high number of CAZy domains, and in particular higher quantities of glucoamylases (GH15), pectinolytic enzymes (GH28) and lignocellulose decay enzymes (GH7). When the secretome of T. pullulans was analyzed experimentally after growth in starch or glucose, 98 proteins were identified. The 60% of total spectral counts belonged to GHs, oxidoreductases and to other CAZymes. A 65 kDa glucoamylase of family GH15 (TpGA1) showed the highest fold change (tenfold increase in starch). This enzyme contains a conserved active site and showed extensive N-glycosylation. This study increases the knowledge on the extracellular hydrolytic enzymes of basidiomycetous yeasts and, in particular, establishes T. pullulans as a potential source of carbohydrate-active enzymes. KEY POINTS: • Tausonia pullulans genome harbors a high number of genes coding for CAZymes. • Among CAZy domains/families, the glycoside hydrolases are the most abundant. • Secretome analysis in glucose or starch as main C sources identified 98 proteins. • A 65 kDa GH15 glucoamylase showed the highest fold increase upon culture in starch.

Recent technical advances regarding filamentous fungi have accelerated the engineering of fungal-based production and benefited basic science. However, challenges still remain and limit the speed of fungal applications. For example, high-throughput technologies tailored to filamentous fungi are not yet commonly available for genetic modification. The currently used fungal genetic manipulations are time-consuming and laborious. Here, we developed a flow cytometry-based plating-free system to directly screen and isolate the transformed protoplasts in industrial fungi Myceliophthora thermophila and Aspergillus niger. This system combines genetic engineering via the 2A peptide and the CRISPR-Cas9 system, strain screening by flow cytometry, and direct sorting of colonies for deep-well-plate incubation and phenotypic analysis while avoiding culturing transformed protoplasts in plates, colony picking, conidiation, and cultivation. As a proof of concept, we successfully applied this system to generate the glucoamylase-hyperproducing strains MtYM6 and AnLM3 in M. thermophila and A. niger, respectively. Notably, the protein secretion level and enzyme activities in MtYM6 were 17.3- and 25.1-fold higher than in the host strain. Overall, these findings suggest that the flow cytometry-based plating-free system can be a convenient and efficient tool for strain engineering in fungal biotechnology. We expect this system to facilitate improvements of filamentous fungal strains for industrial applications. KEY POINTS: • Development of a flow cytometry-based plating-free (FCPF) system is presented. • Application of FCPF system in M. thermophila and A. niger for glucoamylase platform. • Hyper-produced strains MtYM6 and AnLM3 for glucoamylase production are generated.

Pompe disease, an autosomal recessive disorder caused by deficient lysosomal acid α-glucosidase (GAA), is characterized by accumulation of intra-lysosomal glycogen in skeletal and oftentimes cardiac muscle. The c.1935C>A (p.Asp645Glu) variant, the most frequent GAA pathogenic mutation in people of Southern Han Chinese ancestry, causes infantile-onset Pompe disease (IOPD), presenting neonatally with severe hypertrophic cardiomyopathy, profound muscle hypotonia, respiratory failure, and infantile mortality. We applied CRISPR-Cas9 homology-directed repair (HDR) using a novel dual sgRNA approach flanking the target site to generate a Gaaem1935C>A knock-in mouse model and a myoblast cell line carrying the Gaa c.1935C>A mutation. Herein we describe the molecular, biochemical, histological, physiological, and behavioral characterization of 3-month-old homozygous Gaaem1935C>A mice. Homozygous Gaaem1935C>A knock-in mice exhibited normal Gaa mRNA expression levels relative to wild-type mice, had near-abolished GAA enzymatic activity, markedly increased tissue glycogen storage, and concomitantly impaired autophagy. Three-month-old mice demonstrated skeletal muscle weakness and hypertrophic cardiomyopathy but no premature mortality. The Gaaem1935C>A knock-in mouse model recapitulates multiple salient aspects of human IOPD caused by the GAA c.1935C>A pathogenic variant. It is an ideal model to assess innovative therapies to treat IOPD, including personalized therapeutic strategies that correct pathogenic variants, restore GAA activity and produce functional phenotypes.

During salinity stress, osmoregulatory processes in euryhaline fish need to modify for their survival, and glucose is the preferred mode of extra energy during such conditions. These organisms must have a proper mechanism to maintain glucose homeostasis during such modified osmoregulatory process across different body fluids. Hence, we studied high salinity effect on regulation of glucose homeostasis in Mozambique tilapia. The fish were induced to 15‰ salinity for 21 days. Glucose, glycogen, ion concentrations, Na+-K+-ATPase, pyruvate kinase, γ-amylase activities and GLUT mRNA expressions were investigated in liver, intestine, gill and white muscle tissues. At the end of experiment, Na+ ion concentrations, glucose content and activity of Na+-K+-ATPase especially in the gill and intestine were increased, while decrease in liver and gill glycogen content was seen. Lower concentration of glycogen decrease was observed in the intestine and white muscle of the treated group. High pyruvate kinase activity was noticed in liver and gill tissues that correlates with high Na+-K+-ATPase activity. Elevated γ-amylase activity was observed in the liver and intestine suggesting breakdown of glycogen; however, gill and white muscle did not show any increased activity. Increase in GLUT1 and GLUT4 mRNA expressions was observed especially in the gill and intestine, while increase in GLUT2 mRNA expressions was observed in the liver. Upregulations of GLUTs suggest higher influx of glucose into the cell for catabolism to provide energy and further to drive the enhanced osmoregulatory process. These findings suggest glucose homeostasis being regulated in Mozambique tilapia during salinity acclimation.

Multiple-enzyme cooperation simultaneously is an effective approach to biomass conversion and biodegradation. The challenge, however, lies in the interference of the involved enzymes with each other, especially when a protease is needed, and thus, the difficulty in reusing the enzymes; while extracting/synthesizing new enzymes costs energy and negative impact on the environment. Here, we present a unique approach to immobilize multiple enzymes, including a protease, on a metal-organic material (MOM) via co-precipitation in order to enhance the reusability and sustainability. We prove our strategy on the degradation of starch-containing polysaccharides (require two enzymes to degrade) and food proteins (require a protease to digest) before the quantification of total dietary fiber. As compared to the widely adopted "official" method, which requires the sequential addition of three enzymes under different conditions (pH/temperature), the three enzymes can be simultaneously immobilized on the surface of our MOM crystals to allow for contact with the large substrates (starch), while MOMs offer sufficient protection to the enzymes so that the reusability and long-term storage are improved. Furthermore, the same biodegradation can be carried out without adjusting the reaction condition, further reducing the reaction time. Remarkably, the simultaneous presence of all enzymes enhances the reaction efficiency by a factor of ∼3 as compared to the official method. To our best knowledge, this is the first experimental demonstration of using aqueous-phase co-precipitation to immobilize multiple enzymes for large-substrate biocatalysis. The significantly enhanced efficiency can potentially impact the food industry by reducing the labor requirement and enhancing enzyme cost efficiency, leading to reduced food cost. The reduced energy cost of extracting enzymes and adjusting reaction conditions minimize the negative impact on the environment. The strategy to prevent protease damage in a multi-enzyme system can be adapted to other biocatalytic reactions involving proteases.

Raw starch glucoamylase (RSGA) can degrade the raw starch below the starch gelatinization temperature. In this study, to improve the catalytic activity of raw corn starch, N-glycosylation was introduced into the RSGA from

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.

The anti-glycation effects of polysaccharides from Ecklonia cava were examined according to extraction method-hot buffer (HP), ultrasound (UP), enzyme (EP), or a combination of ultrasound and enzyme (UEP). The physicochemical properties, monosaccharide compositions, and structural characteristics of the polysaccharides were determined. UP, EP, and UEP had higher fucose and galactose compositions than HP. The FT-IR spectra of samples showed the presence of sulfate esters and 4-sulfate galactose. 1H NMR indicated that alginate was removed by purification. UP, EP, and UEP possessed higher sulfate contents than HP. UEP presented with the highest extraction yield and lowest protein and uronic acid contents. The levels of AGE formation, as well as fructosamine, α-dicarbonyl, and protein carbonyl contents were determined during a 3-week incubation in a BSA/fructose system. UEP and UP effectively inhibited AGE, although the inhibition effect was lower than that of aminoguanidine. However, UP and UEP showed higher inhibition of fructosamine, α-dicarbonyl, and protein carbonyl than aminoguanidine. AGE formation was negatively correlated with sulfate content and some monosaccharide compositions (fucose, galactose, and glucose), but positively correlated with molecular weight. Overall, the present study suggests that UEP is a suitable extraction method for obtaining anti-glycation agents from E. cava.

To shorten the preparation time of porous starch while simultaneously ensuring its adsorption performance, we treated corn starch with a combination of microwave and complex enzymatic (α-amylase and amyloglucosidase) hydrolysis. Specifically, we investigated the effect of microwave and enzymatic hydrolysis on the structure, physicochemical properties, and adsorption capability of the prepared porous starch. The results showed that the porous starch prepared by combined treatment had denser pores, no new groups were produced in the porous starch, the relative crystallinity, gelatinization temperature, and solubility increased, while the gelatinization enthalpy and swelling power decreased. Compared to enzymatic hydrolysis, the combined treatment yielded starch samples with a larger specific surface area and pore capacity, and the adsorption performance was significantly improved, with water and oil absorption rates increasing from 110.99 % and 133.11 % to 128.29 % and 143.3 %, respectively. These findings indicate that the synergistic processing of microwave and enzymatic hydrolysis has great potential as a productive and rapid method for the preparation of porous starch.

Glucoamylase has a wide range of applications in the production of glucose, antibiotics, amino acids, and other fermentation industries. Fungal glucoamylase, in particular, has attracted much attention because of its wide application in different industries, among which Aspergillus niger is the most popular strain producing glucoamylase. The low availability of NADPH was found to be one of the limiting factors for the overproduction of glucoamylase. In this study, 3 NADH kinases (AN03, AN14, and AN17) and malic enzyme (maeA) were overexpressed in aconidial A. niger by CRISPR/Cas9 technology, significantly increasing the size of the NADPH pool, resulting in the activity of glucoamylase was improved by about 70%, 50%, 90%, and 70%, respectively; the total secreted protein was increased by about 25%, 22%, 52%, and 26%, respectively. Furthermore, the combination of the mitochondrial NADH kinase (AN17) and the malic enzyme (maeA) increased glucoamylase activity by a further 19%. This study provided an effective strategy for enhancing glucoamylase production of A. niger.

This study describes for the first time the purification and characterization of a glucoamylase from Aspergillus wentii (strain PG18), a species of the Aspergillus genus Cremei section. Maximum enzyme production (∼3.5 U/ml) was obtained in submerged culture (72 h) with starch as the carbon source, at 25°C, and with orbital agitation (100 rpm). The enzyme was purified with one-step molecular exclusion chromatography. The 86 kDa purified enzyme hydrolyzed starch in a zymogram and had activity against p-nitrophenyl α- d-glucopyranoside. The optimal enzyme pH and temperature were 5.0 and 60°C (at pH 5.0), respectively. The Tm of the purified enzyme was 60°C, at pH 7.0. The purified glucoamylase had a KM for starch of 1.4 mg/ml and a Vmax of 0.057 mg/min of hydrolyzed starch. Molybdenum activated the purified enzyme, and sodium dodecyl sulfate inhibited it. A thin layer chromatography analysis revealed glucose as the enzyme's main starch hydrolysis product. An enzyme's peptide sequence was obtained by mass spectrometry and used to retrieve a glucoamylase within the annotated genome of A. wentii v1.0. An in silico structural model revealed a N-terminal glycosyl hydrolases family 15 (GH15) domain, which is ligated by a linker to a C-terminal carbohydrate-binding module (CBM) from the CBM20 family.

Biocatalytic activity of amyloglucosidase (AMG), immobilized on superparamagnetic nanoparticles, is dynamically and reversibly activated or inhibited by applying a magnetic field. The magnetic field triggers aggregation/deaggregation of magnetic particles that are also functionalized with urease or esterase enzymes. These enzymes produce a local pH change in the vicinity of the particles changing the AMG activity.

The aim of this study was to carry out the co-immobilization of α-amylase and glucoamylase in crosslinked gelatin porous supports. For this, two methods of co-immobilization were proposed based on the crosslinking with glutaraldehyde (Ggta) or CaCl2 in presence of alginate (Gcal). The supports characterization revealed a porous microstructure with good interaction between its components according to the FTIR analysis and thermal properties. Optimal pH and temperature of the Gcal co-immobilized enzymes were determined at 60 °C and pH 6.0, present an enzymatic activity of 120 μmol·mL·min−1. Moreover, both supports were reused for up to 8 hydrolysis cycles. In addition, co-immobilized enzymes were more efficient than free enzymes in starch saccharification of starch in the long term. These results reveal that the co-immobilization of amylases in gelatinous supports is a promising approach in enzymatic chain reactions.

Starch is composed of two giant polymers of glucose-linked amylose and amylopectin but is not considered to have a branched structure with adjacent α-1,6-linkages (adjacent double branches). We previously discovered that the oligosaccharide DP6-1 contains adjacent double branches in sake, a Japanese rice wine. In this study, we analyzed the oligosaccharides generated from rice starch following porcine pancreatic α-amylase and glucoamylase digestion by hydrophilic interaction chromatography coupled with time-of-flight/mass spectrometry. We found that their elution times were identical to those of the oligosaccharides containing adjacent double branches observed in sake. Nuclear magnetic resonance (NMR) analysis confirmed that one of the oligosaccharides was identical to DP6-1. This indicates for the first time that adjacent double branches exist in the branching structure of starch. The adjacent double branches were estimated to be present at a ratio of approximately 1:100 of the total branching points in rice starch, suggesting that the contribution of the adjacent double branch to the entire structure of starch may be limited. Nonetheless, we found adjacent double branches in starch from rice, maize, wheat, and potato, which indicates that this novel branching pattern is universally present in starch.

We identified a new glucoamylase (TeGA) from Thermoanaerobacter ethanolicus, a thermophilic anaerobic bacterium. Structural studies suggest that TeGA belongs to the family 15 of glycosylhydrolases (GH15).The expression of this enzyme was optimized in E. coli (BL21) cells in order to have the highest amount of soluble protein (around 3 mg/l of culture medium).TeGA showed a high optimum temperature of 75 °C. It also showed one of the highest specific activities reported for a bacterial glucoamylase (75.3 U/mg) and was also stable in a wide pH range (3.0-10.0). Although the enzyme was preferentially active with maltose, it was also able to hydrolyze different soluble starches such as those from potato, corn or rice. TeGA showed a high thermostability up to around 70 °C, which was increased in the presence of PEG8000, and also showed to be stable in the presence of moderate concentrations of ethanol.We propose that TeGA could be suitable for use in different industrial processes such as biofuel production and food processing.

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.

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.

The requirements for the safety of food products obtained by microbial synthesis are including as obligation for to conduct toxicological studies - the study of various biochemical and immunological markers of toxic effects. The necessity of these studies is explained by a possible change in the structure of food ingredients produced by a microbial cell and, consequently, a change in their biological properties, as well as the possible presence of living forms and/or DNA of producer strains or of their toxic metabolites in these ingredients. At the same time, it is well known that the nutrient composition of foods has a significant impact on the composition and properties of microorganisms that make up the gut microbiome, which, in turn, determines the immune status. The purpose of the research was to justify the analyses of gut microbiocenosis composition for inclusion in the protocol of safety investigation of foods obtained by microbial synthesis [on the example of an enzyme preparation (EP) - a complex of glucoamylase and xylanase from a genetically modified strain of Aspergillus awamori Xyl T-15].

Glucoamylase has an essential role as biocatalyst in various important industries of Pakistan. It is synthesized by using various fungal and bacterial strains, and different ecocultural conditions are applied under solid substrate fermentation method (SSF) to get the highest yield of glucoamylase.

Amyloglucosidase (AMG) and α-amylase (AMY) are both involved in starch digestion in human small intestine, whereas their mutual interactions with starch molecules of distinct structures are still unknown. In the current study, starches with different amylose contents (from waxy to high amylose content) were used to investigate the starch-enzyme interactions in the

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.

To obtain novel glucoamylase from Daqu microbe. A dominant strain known as LZ2 with high activity of hydrolyzing starch was isolated from Luzhou Daqu, a Chinese traditional fermentation starter. The LZ2 was identified as Aspergillus oryzae by 18S rDNA sequence analysis. Glucoamylase from LZ2, named as GA-LZ2, was purified to homogeneity and showed a single band with expected molecular mass of 60 kD. The GA-LZ2 effectively degraded amylose, rice starch and wheat starch. Optimal temperature and pH value of enzyme were 60 °C and pH 4.0 respectively. The GA-LZ2 displayed significant thermal stability and pH stability at moderate temperature and low pH. Intriguingly, the thermostability was enhanced in the presence of starch. In addition, GA-LZ2 exhibited insensitivity to glucose, independence of metal ions and tolerance to organic solvents. The GA-LZ2 retained complete activity in the presence of 100 mM glucose and 5% ethanol and methanol. Glucoamylase GA-LZ2 displayed broad substrate specificity, strong stability and tolerance, suggesting that GA-LZ2 carry potential for industrial application in bioethanol production.

The pharmaceutical industry has a high demand for glycoprotein production. The glycoform of glycoproteins is crucial for pharmacological activity. However, in general, cells produce glycoproteins with a heterologous glycoform, which is unfavorable for making uniform, efficacious therapeutic proteins. Here, to produce more glycoproteins with N-glycan uniformity, we applied the GlycoDelete strategy, in which endo-β-N-acetylglucosaminidase (ENGase) from the fungus Hypocrea jecorina (EndoT) is expressed at the Golgi membrane to cleave N-glycan from secretory glycoproteins, to Aspergillus oryzae cells. First, we selected candidate transmembrane domains to target EndoT to the Golgi membrane in A. oryzae cells, generated constructs for expressing the transmembrane-fused EndoT proteins and produced four potential AoGlycoDelete strains. We then confirmed that these strains produced α-amylase with a molecular weight lower than that of native α-amylase without an effect on growth. To test whether the A. oryzae α-amylase proteins had been cleaved by EndoT, we expressed and purified HA-tagged α-amylase AmyB and glucoamylase GlaA proteins from the AoGlycoDelete strain. MS and N-glycan analyses of the intact proteins confirmed neither AmyB-HA nor GlaA-HA produced from the AoGlycoDelete strain contained N-glycan. Lastly, we determined the enzymatic activities of the amylases produced by the AoGlycoDelete strain, which showed that the lack of N-glycan did not affect their activity under the conditions tested. Collectively, our findings demonstrate successful generation of an AoGlycoDelete strain that might be a good candidate for producing pharmaceutical glycoproteins with a uniform N-glycan structure.

BACKGROUND The general enzymatic method for producing reducing sugar is liquefaction followed by saccharification of starch. This method results in lower yields, consuming high energy and time. Therefore, the present study evaluated a new approach for producing reducing sugar from sweet potato starch (SPS), including simultaneous liquefaction (by α-amylase) and saccharification (by glucoamylase) of SPS pretreated with high power ultrasound. The effects of ultrasound parameters on the conversion rate of SPS and mechanism were investigated. RESULTS The optimum ultrasound pretreatment conditions were a frequency of 20 kHz, SPS concentration of 125 g L-1 , temperature of 30 °C, pulsed on-time of 3 s, pulsed off-time of 5 s, power density of 8 W mL-1 and sonication time of 15 min. The ultrasound assisted enzymolysis resulted in a SPS conversion rate of 59.10%, which was improved by 56.35% compared to the control. The results of pasting properties and thermal analysis showed that ultrasound pretreatment decreased the peak viscosity, breakdown temperature, setback viscosity, gelatinization range (TC - TO ) and enthalpy of gelatinization (ΔH) of SPS significantly (P

Enzymatic hydrolysis of starch is an important process in the food industry. In the present work, ultrasound was introduced in glucoamylase pretreatment, starch pretreatment and mixed reaction system treatment to enhance starch hydrolysis efficiency. These different processes were studied to explore the mechanism of ultrasound in promoting enzymatic reactions. The hydrolysis degree of starch was determined via measuring the reducing sugar yield. Ultrasound caused enzyme inactivation under high temperatures, high ultrasonic power and long-time treatment, especially at high temperatures exceeding 65 °C. Ultrasound pretreatment of starch before enzymolysis led to the furtherance of starch hydrolysis degree. Meanwhile, sonicating the mixed enzymatic reaction system below 65 °C promoted starch hydrolysis significantly, inducing more than five- fold growth in the degree of starch hydrolysis as much as the ultrasound pretreatment caused. Molecular weights analysis conducted by the MALLS system reflected the enormous damage of starch molecules caused by ultrasound. The amylose contents and chain length distributions of samples were separately analyzed by iodine binding method and size exclusion chromatography. The results of the two experiments illustrate that ultrasound could promote the enzymatic hydrolysis of amylopectin, which is harder for glucoamylase to hydrolyze compared to amylose.

Glycogen storage disease type III (GSD III) is a rare autosomal recessive inborn error of glycogen degradation pathway due to deficiency or reduced activity of glycogen debranching enzyme (GDE) that results in accumulation of abnormal glycogen in the liver, muscle, and heart. The cardinal hallmarks are hepatomegaly, fasting hypoglycemia, seizures, growth retardation, progressive skeletal myopathy, and cardiomyopathy in few. To date, 258 mutations in amyloglucosidase (AGL) gene have been identified worldwide. However, the mutation spectrum in the Asian Indian region is yet to be well characterized. We investigated 24 patients of Asian origin from 21 unrelated families with a provisional diagnosis of GSD III based on clinical and biochemical criteria. Molecular diagnosis was assessed by bidirectional sequencing and the impact of novel missense variants on the tertiary (three-dimensional) structure of GDE was evaluated by molecular modeling approach. Eighteen different pathogenic variants were identified, out of which 78% were novel. Novel variants included five nonsense, three small duplications and two small deletions, a splice site variant, and three missense variants. Variations in Exons 4, 14, 19, 24, 27, and 33 accounted for 61% of the total pathogenic variants identified and Allele p.Gly798Alafs*3 showed a high allele frequency of 11%. Molecular modeling study of novel pathogenic missense variants indicated the probable underlying molecular mechanism of adverse impact of variations on the structure and catalytic function of human GDE. Our study is the first large study on GSD III from the Asian subcontinent, which further expands the mutation spectrum of AGL.

Introducing an efficient method for the rapid conversion of starch into gluconic acid is desirable to solve the current problems existing in traditional gluconic acid preparation processes. In this study, a robust and easy-to-use multienzymatic cascade reaction system of coimmobilized GA@GOx hybrid nanoflowers with a specific spatial distribution of enzymes by compartmentalization was constructed and applied to catalyze starch to gluconic acid in one pot. In the preparation processes, the glucose oxidase (GOx) hybrid nanoflowers were first synthesized via a self-assembly mechanism, and then, glucoamylase (GA) was adsorbed on the surface of GOx hybrid nanoflowers through the interaction of Cu2+ and amino acids of GA. The optimum preparation conditions and reaction parameters of the GA@GOx hybrid nanoflowers had been investigated. In addition, the morphology, composition, and crystallization of the GA@GOx hybrid nanoflowers had been fully studied. Based on the lower Km, the GA@GOx hybrid nanoflowers with compartmentalization had a better effect of the substrate channeling on the catalytic efficiency. The final results indicated that the overall enzyme activity of the GA@GOx hybrid nanoflowers increased by 1.5 times, and the conversion efficiency was 92.12% within 80 min significantly superior to the free multienzyme system, which showed the outstanding conversion of starch into gluconic acid in one pot.

The effects of multicycle ultrasound-assisted ice recrystallization (US+IR) combined with amyloglucosidase (AMG) or maltogenic α-amylase (MA) catalyzed hydrolysis on structure were investigated. Scanning electron microscopy (SEM) showed that the US+IR produced shallow indentations and grooves on the exterior of granules while the combination US+IR and enzyme hydrolysis created additional pores on starch granules. MA displayed a higher number of pores than AMG. The highest values of specific surface area (SBET) and the total pore volume were obtained for US+IR→MA (1.96 m2 g−1 and 7.26 × 10−3 cm3 g−1, respectively). The US+IR treatment significantly decreased the relative crystallinity, amylose content and swelling capacity. Those parameters were further efficiently decreased following enzymatic hydrolysis. The combined treatments generated products with higher initial gelatinization temperature (Ti) compared to the corresponding controls. The US+IR increased the digestion rate constant (k-value) compared to native starch. However, the combined treatment, US+IR→AMG, significantly decreased the k-value from 2.97 × 10−3 to 2.50 × 10−3 min−1 compared to its control. Our study demonstrates that US+IR treatment in combination with enzyme hydrolysis is a useful method to produce specifically functionalized porous rice starch that can be used as e.g. absorbents and for further chemical modifications.

To explore a valuable new enzyme method that effectively changes the molecular structure and absorption property of potato and sweet potato starch granules, the common combination of α-amylase (AA) and glucoamylase (GA), namely AA → GA (common method as contrast), the combination of glycosyltransferase (GT) and GA, namely GT → GA, or the combination of sequential AA, GT and GA (AA → GT → GA) treatment was utilized. The results indicated that three types of enzyme treatments increased the pore size, pore volume, specific surface area and relative crystallinity of potato and sweet potato starches, resulting in a significant increment in adsorption capacities. Compared to GT → GA or AA → GA treatment, AA → GT → GA treatment had the most significant effect on the hydrolysis of starches since a lower amylose content, higher branched degree and more amount of short side chains of starches obtained using AA → GT treatment might increase the amount of GA active sites. So, GT → AA → GA treatment had the most significant absorption impact on oil, dye and heavy metal ions of starches. Compared to native starch, the adsorption capacity of GT/AA/GA-treated starches increased by about 7–21 folds. Thus, a novel GT → AA → GA treatment may be a most effective method to prepare porous potato and sweet potato starches as desirable green adsorbents.

A tannase-encoding gene, AotanB, from Aspergillus oryzae RIB40 was overexpressed in A. oryzae AOK11 niaD-deficient mutant derived from an industrial strain under the control of an improved glucoamylase gene promoter PglaA142. The recombinant tannase, designated as rAoTanBO, was produced efficiently as an active extracellular enzyme. Purified rAoTanBO showed a smeared band with a molecular mass of approximately 80–100 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The rAoTanBO had a molecular mass of 65 kDa, after treatment with endo-β-N-acetylglucosaminidase H. Purified rAoTanBO exhibited maximum activity at 30–35°C and pH 6.0. The tannase activity of purified rAoTanBO towards natural and artificial substrates was 2–8 folds higher than that of the recombinant enzyme produced by Pichia pastoris, designated as rAoTanBP. N-terminus of the mature rAoTanBP had six more amino acids than the N-terminus of the mature rAoTanBO. Kinetic analyses showed that rAoTanBO had higher catalytic efficiency (kcat/Km) than rAoTanBP. rAoTanBO was stable up to 60°C and higher thermostability than rAoTanBP. N-linked oligosaccharides had no effect on the activity and stability of rAoTanBO and rAoTanBP.

Aspergillus section Nigri is a fungus used industrially because of its ability to produce enzymes such as cellulolytic, amylolytic and proteolytic enzymes. In this study, we obtained twenty-eight strains of Aspergillus section Nigri from the traditional Korean fermentation starter, nuruk, which is known as a mixed culture of enzymatic filamentous fungi and yeasts. All strains were identified as Aspergillus section Nigri through combined phylogenetic analysis using partial β-tubulin and calmodulin gene sequences. The cellulase, amylase and protease activities of Korean strains were measured and compared with ten reference strains of Aspergillus niger. Most Korean strains showed higher cellulolytic activity than reference strains, and Aspergillus neoniger KCN5 showed the highest β-glucosidase activity. Two-thirds of the Korean strains showed similar levels of α- and glucoamylase activity as the reference strains. The protease activity of Aspergillus section Nigri strains was the highest at pH 3.0, and A. niger KSJ2 showed the highest acidic protease activity. By comparing ten reference strains and twenty-eight Korean strains, our results suggested useful Aspergillus section Nigri strains from nuruk with high enzyme activity, such as KCN5 and KSJ2, and their potential for industrial applications as enzyme producers.

Background Glucoamylase is an important industrial enzyme for the saccharification of starch during sugar production, but the production cost of glucoamylase is a major limiting factor for the growth of the starch-based sugar market. Therefore, seeking strategies for high-level expression of glucoamylase in heterologous hosts are considered as the main way to reduce the enzyme cost. Results ReGa15A from Rasamsonia emersonii and TlGa15B-GA2 from Talaromyces leycettanus have similar properties. However, the secretion level of ReGa15A was significantly higher than TlGa15B-GA2 in Pichia pastoris. To explore the underlying mechanisms affecting the differential expression levels of glucoamylase in P. pastoris, the amino acid sequences and three-dimensional structures of them were compared and analyzed. First, the CBM region was identified by fragment replacement as the key region affecting the expression levels of ReGa15A and TlGa15B-GA2. Then, through the substitution and site-directed mutation of the motifs in the CBM region, three mutants with significantly increased expression levels were obtained. The eight-point mutant TlGA-M4 (S589D/Q599A/G600Y/V603Q/T607I/V608L/N609D/R613Q), the three-point mutant TlGA-M6 (Q599A/G600Y/V603Q) and the five-point mutant TlGA-M7 (S589D/T607I/V608L/N609D/R613Q) have the same specific activity with the wild-type, and the enzyme activity and secretion level have increased by 4–5 times, respectively. At the same time, the expression levels were 5.8-, 2.0- and 2.4-fold higher than that of wild type, respectively. Meanwhile, the expression of genes related to the unfolded protein responses (UPR) in the endoplasmic reticulum (ER) did not differ significantly between the mutants and wild type. In addition, the most highly expressed mutant, TlGA-M7 exhibits rapidly and effectively hydrolyze raw corn starch. Conclusions Our results constitute the first demonstration of improved expression and secretion of a glucoamylase in P. pastoris by introducing mutations within the non-catalytic CBM. This provides a novel and effective strategy for improving the expression of recombinant proteins in heterologous host expression systems.

Enzymatic biofuel cells (EBFCs) provide a new strategy to enable direct biomass-to-electricity conversion, posing considerable demand on sequential enzymes. However, artificial blend of multi-enzyme systems often suffer biocatalytic inefficiency due to the rambling mixture of catalytic units. In an attempt to construct a high-performance starch/O

Glucoamylase is one of the most widely used enzymes in industry, but the development background and existing circumstances of industrial glucoamylase were not described by published articles. CiteSpace, a powerful tool for bibliometric, was used to analyze the past, existing circumstances, and trends of a professional field. In this study, 1820 Web-of-Science-indexed articles from 1991 to 2021 were collected and analyzed by CiteSpace. The research hotspots of industrial glucoamylase, like glucoamylase strain directional improvement, Aspergillus niger glucoamylase, glucoamylase immobilization, application of glucoamylase in ethanol production, and "customized production" of porous starch, were found by analyzing countries, institutions, authors, keywords, and references of articles. PRACTICAL APPLICATIONS: The research progress of glucoamylase with industrial potential was analyzed by CiteSpace, and a significant research direction of glucoamylase with industrial potential was found. This is helpful for academic and corporate audiences to understand the current situation of glucoamylase with industrial potential and carry out follow-up works.

The reactions of intestinal functional parameters to type 2 diabetes at a young age remain unclear. The study aimed to assess changes in the activity of intestinal enzymes, glucose absorption, transporter content (SGLT1, GLUT2) and intestinal structure in young Wistar rats with type 2 diabetes (T2D) and impaired glucose tolerance (IGT). To induce these conditions in the T2D (n = 4) and IGT (n = 6) rats, we used a high-fat diet and a low dose of streptozotocin. Rats fed a high-fat diet (HFD) (n = 6) or a standard diet (SCD) (n = 6) were used as controls. The results showed that in T2D rats, the ability of the small intestine to absorb glucose was higher in comparison to HFD rats (p < 0.05). This was accompanied by a tendency towards an increase in the number of enterocytes on the villi of the small intestine in the absence of changes in the content of SGLT1 and GLUT2 in the brush border membrane of the enterocytes. T2D rats also showed lower maltase and alkaline phosphatase (AP) activity in the jejunal mucosa compared to the IGT rats (p < 0.05) and lower AP activity in the colon contents compared to the HFD (p < 0.05) and IGT (p < 0.05) rats. Thus, this study provides insights into the adaptation of the functional and structural parameters of the small intestine in the development of type 2 diabetes and impaired glucose tolerance in young representatives.

Aureobasidium melanogenum TN3-1 isolated from a natural honey was a highly genome-duplicated yeast-like fungal strain and a very high pullulan producer. In this study, simultaneous removal of both duplicated AMY1 genes encoding α-amylase and duplicated PKS1 genes responsible for melanin biosynthesis in A. melanogenum TN3-1 rendered a mutant AMY-PKS-11 to transform 140.0 g/L of glucose to produce 103.50 g/L of pigment-free pullulan with molecular weight (Mw) of 3.2 × 105 g/mol. α-Amylase activity produced by the mutant AMY-PKS-11 and expression of the AMY1 genes and PKS genes in it was reduced, but expression of various genes responsible for pullulan biosynthesis in the mutant AMY-PKS-11 was up-regulated. The produced pullulan was used to make the capsule shells successfully and the prepared pullulan capsule shells had various advantages such as high strength, good oxygen barrier properties, raw materials availability, tightness, lightness and high water resistance and may be suitable for all the consumers. Therefore, the prepared capsule shells had highly potential applications in food and pharmaceutical industries.