The role of the Flb protein family in the life cycle of Aspergillus niger.

Genes flbA-E are involved in sporulation and vegetative growth in Aspergillus nidulans. Inactivation of either of these genes results in a fluffy phenotype with delayed or even abolished sporulation. Previously, a non-sporulating phenotype was obtained by inactivating flbA in Aspergillus niger, which was accompanied by lysis, thinner cell walls, and an increased secretome complexity. Here, we further studied the role of the flb genes of A. niger. Strains ΔflbA, ΔflbB and ΔflbE showed increased biomass formation, while inactivation of flbA-D reduced, or even abolished, formation of conidia. Strain ΔflbA was more sensitive to H2O2, DTT, and the cell wall integrity stress compounds SDS and Congo Red (CR). Also, ΔflbC was more sensitive to SDS, while ΔflbB, ΔflbD, and ΔflbE were more sensitive to CR. On the other hand, inactivation of flbE increased resistance to H2O2. Enzyme secretion was impacted when the Δflb strains were grown on xylose. Strain ΔflbE showed reduced xylanase, cellulase and amylase secretion. On the other hand, amylase secretion at the periphery of the ΔflbA colony was reduced but not in its center, while secretion of this enzyme was increased in the center of the ΔflbB colony but not at its periphery. Inactivation of flbC and flbD also impacted zonal cellulase and amylase activity. Together, the Flb protein family of A. niger function in biomass formation, sporulation, stress response, and protein secretion.

Linkage-Editing Pseudo-Glycans: A Reductive α-Fluorovinyl-C-Glycosylation Strategy to Create Glycan Analogs with Altered Biological Activities.

The acetal (O-glycoside) bonds of glycans and glycoconjugates are chemically and biologically vulnerable, and therefore C-glycosides are of interest as more stable analogs. We hypothesized that, if the O-glycoside linkage plays a vital role in glycan function, the biological activities of C-glycoside analogs would vary depending on their substituents. Based on this idea, we adopted a "linkage-editing strategy" for the creation of glycan analogs (pseudo-glycans). We designed three types of pseudo-glycans with CH2 and CHF linkages, which resemble the O-glycoside linkage in terms of bond lengths, angles, and bulkiness, and synthesized them efficiently by means of fluorovinyl C-glycosylation and selective hydrogenation reactions. Application of this strategy to isomaltose (IM), an inducer of amylase expression, and α-GalCer, which activates iNKT cells, resulted in the discovery of CH2-IM, which shows increased amylase production ability, and CHF-α-GalCer, which shows activity opposite that of native α-GalCer, serving as an antagonist of iNKT cells.

High level food-grade expression of maltogenic amylase in Bacillus subtilis through dal gene auxotrophic selection marker.

As a food-safe microorganism, Bacillus subtilis has been widely utilized in the production of food enzyme, where a food-grade expression system without antibiotic is required. However, there is no mature system for such expression, since the recombinant plasmid in existing food-grade expression system is unstable especially in high-density fermentation. In this study, we constructed a food-grade expression system based on the dal gene auxotrophic selection marker. Specifically, maltogenic amylase (AmyM) was expressed in dal deletion strain without antibiotic, yielding an activity of 519 U/mL. To increase the expression of AmyM, the promoter of amyM (gene encoding AmyM) was optimized. Furthermore, we found that excessive expression of dal gene was detrimental to the stability of plasmid, and the ribosome binding site (RBS) of dal was mutated with the reduced synthesis of D-alanine. After that, AmyM activity increased to 1364 U/mL with the 100 % stability of plasmid. The 3-L fermentor cultivation was performed with the highest value ever reported in food-grade microorganisms, an activity of 2388 U/mL, showing the scale-up production capability of this system. Besides, it is also able to apply the system for other food enzymes, which indicating the great generalizability of this system for different application.

From static to semi-dynamic in vitro digestion conditions relevant for the older population: starch and protein digestion of cooked lentils.

In the context of adequately feeding the rising older population, lentils have an important potential as sources of (plant-based) protein as well as slowly digestible bio-encapsulated starch and fibre. This study evaluated in vitro digestion of protein and starch in lentils under conditions representing the gastrointestinal tract of older adults. Both static and semi-dynamic simulations were applied to analyze the effect of specific gastrointestinal conditions (healthy versus older adult) on macronutrient digestion patterns. Gastric proteolysis was strongly dependent on applied gastric pH (gradient), leading to a lower extent of protein hydrolysis for simulations relevant for older adults. Fewer and smaller (lower degree of polymerization, DP) bioaccessible peptides were formed during gastric proteolysis under older adult compared to healthy adult conditions. These differences, developed during the in vitro gastric phase, were compensated during small intestinal digestion, yielding similar final proteolysis levels regardless of the applied simulation conditions. In contrast, in the presence of saliva, amylolysis was generally accelerated under older adult conditions. Moreover, the current work highlighted the importance of considering saliva (or salivary amylase) incorporation in simulations where the applied gastric pH (gradient) allows salivary amylase activity. Under both healthy and older adult conditions, in vitro starch hydrolysis bio-encapsulated in cotyledon cells of cooked lentils was attenuated, compared to a white bread reference.

Rab7 localized on zymogen granules is involved in maturation but not in autophagy or regulated exocytosis in pancreatic acinar cells.

Rab7 is known to function in the autophagy and endocytosis pathways in eukaryocytes and is related to various diseases. We recently reported that Rab7 plays a protective role against acute pancreatitis. However, its physiological function in exocytic cells remains unclear. Therefore, we investigated the role of Rab7 in pancreas-specific Rab7 knockout mice (Rab7Δpan). Immunofluorescence microscopy revealed that Rab7 colocalized with amylase in pancreatic acinar cells of wild-type mice, but not in Rab7Δpan mice. Western blotting confirmed Rab7 localization in the zymogen granule (ZG) membranes of wild-type mice. Cholecystokinin (CCK)-stimulated amylase secretion examined using isolated pancreatic acini was similar in Rab7Δpan and wild-type mice. In contrast, electron microscopy revealed that the diameters of ZGs were shorter and the number of ZGs was larger in the pancreatic acinar cells of Rab7Δpan mice than in those of wild-type mice. However, the number of ZGs decreased in both Rab7Δpan and wild-type mice after 24 h of starvation. In addition, the amount of amylase in the pancreas was decreased in both Rab7Δpan and wild-type mice. These data indicate that Rab7 localized on ZGs plays a crucial role in the maturation of ZGs but not in their autophagy or regulated exocytosis in pancreatic acinar cells.

Challenges and prospects of microbial α-amylases for industrial application: a review.

α-Amylases are essential biocatalysts representing a billion-dollar market with significant long-term global demand. They have varied applications ranging from detergent, textile, and food sectors such as bakery to, more recently, biofuel industries. Microbial α-amylases have distinct advantages over their plant and animal counterparts owing to generally good activities and better stability at temperature and pH extremes. With the scope of applications expanding, the need for new and improved α-amylases is ever-growing. However, scaling up microbial α-amylase technology from the laboratory to industry for practical applications is impeded by several issues, ranging from mass transfer limitations, low enzyme yields, and energy-intensive product recovery that adds to high production costs. This review highlights the major challenges and prospects for the production of microbial α-amylases, considering the various avenues of industrial bioprocessing such as culture-independent approaches, nutrient optimization, bioreactor operations with design improvements, and product down-streaming approaches towards developing efficient α-amylases with high activity and recyclability. Since the sequence and structure of the enzyme play a crucial role in modulating its functional properties, we have also tried to analyze the structural composition of microbial α-amylase as a guide to its thermodynamic properties to identify the areas that can be targeted for enhancing the catalytic activity and thermostability of the enzyme through varied immobilization or selective enzyme engineering approaches. Also, the utilization of inexpensive and renewable substrates for enzyme production to isolate α-amylases with non-conventional applications has been briefly discussed.

The cholinesterase and C-reactive protein score is a potential predictor of pseudoaneurysm formation after pancreaticoduodenectomy in patients with soft pancreas.

BACKGROUND: Pseudoaneurysm (PA) rupture after pancreaticoduodenectomy (PD) is a life-threatening complication. Most PA cases originate from postoperative pancreatic fistulas (POPFs). Although several risk factors for POPF have been identified, specific risk factors for PA formation remain unclear. Therefore, we retrospectively analyzed PD cases with soft pancreas and proposed a novel strategy for early detection of PA formation. METHODS: Overall, 120 patients underwent PD between 2010 and 2020 at our institution; of these, 65 patients with soft pancreas were enrolled. We evaluated the clinicopathological factors influencing PA formation and developed a risk score to predict PA formation. RESULTS: In total, 11 of the 65 patients developed PAs (PA formation group: PAG), and 8 of these 11 PAs ruptured. The median time to PA formation was 15 days, with a minimum of 5 days. The PAG was significantly older than the non-PA formation group, were predominantly men, and had comorbid diabetes mellitus. Pre- and intra-operative findings were similar between the two groups. Importantly, no significant differences were found in postoperative drain amylase levels and total drain amylase content. Cholinesterase and C-reactive protein (CRP) levels on postoperative day (POD) 3 were significantly different between the two groups. Multivariate analysis showed that cholinesterase ≤ 112 U/L and CRP ≥ 16.0 mg/dl on POD 3 were independent predictors of PA formation. CONCLUSIONS: Decreased cholinesterase and elevated CRP on POD 3 (Cho-C score) are useful predictors of PA formation in cases with soft pancreas. In such cases, periodic computed tomography evaluations and strict drain management are necessary to prevent life-threatening hemorrhage.

Comparative Evaluation of Salivary Parameters in Tobacco Substance Abusers.

BACKGROUND: Tobacco use by youth is ever-demanding, and it is increasingly distributed not only in India but also globally. Saliva is a complex oral bio-fluid, freely available, performing absolute tasks for maintaining oral health and homeostasis. It contains a plethora of significant constituents such as proline-rich proteins (PRPs), immunoglobulins, IgA, enzymes lysozyme, lactoferrin, peroxidases, amylase, etc. The basic ecological balance of the oral cavity is stabilized via salivary clearance by reduced aggregation and adherence of microorganisms by direct microbial activity. This balance of oral activity is also done by indirect mechanisms by immunological as well as non-immunological means and also by effectively regulating salivary pH flow rate. This institutional observational study was planned to assess and compare salivary parameters (pH, salivary flow rate), total proteins, α-amylase, calcium, phosphate, and IgA, of unstimulated whole saliva of both tobacco abusers and tobacco non-users. METHODS: The Study consisted of 270 participants (Tobacco habit) group, n = 135 and Control (Healthy) group, n = 135 and were in the age range of 20-50 years. They were assessed for oral health status, followed by the analysis of salivary pH, flow rate, total proteins, amylase, calcium, phosphates, and IgA of unstimulated whole saliva. RESULTS: Comparative evaluation of salivary parameters among groups found that varying tobacco abusers had increased salivary amylase, protein levels, and phosphate whereas decreased salivary pH, flow rate, IgA, and in the whole unstimulated saliva samples than those of non-tobacco users. This difference among groups was statistically significant. (p < 0.05), and calcium levels were not altered significantly. CONCLUSIONS: This study concludes that salivary parameters are altered in tobacco abusers when compared to those of non-abusers, and it was more significant in smokeless tobacco abusers than in any other form of tobacco abuse.

Purification and characterization of amylases from three freshwater fish species providing new insight application as enzyme molecular markers for zymography.

Purification of amylases from digestive tracts of three freshwater fish species with Q-Sepharose Fast Flow and Sephacryl S-200 columns displayed two isoforms of amylases from Osteochilus hasselti (O1, O2) and three isoforms of those from both Hampala dispar (UB, H1, H2) and Puntioplites proctozystron (P1, P2, P3). The optimum pH values displayed at 7.0 and 8.0, while the optimum temperatures revealed at 40 and 50 °C. Almost isoenzyme activities were activated by NaCl and CaCl2, whereas EDTA and SDS strongly inhibited all enzymatic activities. Verification with an atomic absorption spectrophotometry exhibited the presence of Ca2+ ions in the range of 0.02-13.53 ppm per mg protein indicating that amylases are Ca2+ dependent. Molecular weight analysis revealed 12 to 147 kDa. The UB, O1, and H2 amylases with appropriate molecular masses of 64, 49, and 25 kDa validated with LC-MS/MS were selected. Three certain enzymes revealed high stability in a sample buffer after five cycles of freeze-thawing process upon storage at - 20 °C for 12 weeks. No protein degradation was observed on polyacrylamide gel, and the enzymes still displayed sharp and clear bands on zymograms. The result suggested that the purified fish amylases, which expressed high activities and stabilities, were potentially used as enzyme molecular weight markers for zymography.

Promoter-proximal introns impact recombinant amylase expression in Saccharomyces cerevisiae.

Consolidated bioprocessing (CBP) of starch requires recombinant Saccharomyces cerevisiae strains that produce raw starch-degrading enzymes and ferment the resultant sugars to ethanol in a single step. In this study, the native S. cerevisiae COX4 and RPS25A promoter-proximal introns were evaluated for enhanced expression of amylase genes (ateA, temA or temG_Opt) under the control of an S. cerevisiae promoter (ENO1P, TEF1P, TDH3P, or HXT7P). The results showed that different promoters and promoter-intron combinations differentially affected recombinant amylase production: ENO1P-COX4i and TDH3P-RPS25Ai were the best promoters for AteA, followed closely by HXT7P. The latter was also the best promoter for TemA and TemG production, followed closely by TDH3P-RPS25Ai for both these enzymes. Introducing promoter-proximal introns increased amylase activity up to 62% in Y294[ENO-COX-AteA] and Y294[TDH3-RPS-TemA], a significant improvement relative to the intron-less promoters. Strains co-expressing both an α-amylase and glucoamylase genes yielded up to 56 g/L ethanol from 20% w/v raw starch, with a higher carbon conversion observed with strains co-expressing TDH3P-RPS25Ai-temG_Opt than HXT7P-temG_Opt. The study showed that promoter-proximal introns can enhance amylase activity in S. cerevisiae and suggest that these alternative cassettes may also be considered for expression in more efficient ethanol-producing industrial yeast strains for raw starch CBP.

Protein secretion zones during overexpression of amylase within the Gram-positive cell wall.

BACKGROUND: Whereas the translocation of proteins across the cell membrane has been thoroughly investigated, it is still unclear how proteins cross the cell wall in Gram-positive bacteria, which are widely used for industrial applications. We have studied the secretion of α-amylase AmyE within two different Bacillus strains, B. subtilis and B. licheniformis. RESULTS: We show that a C-terminal fusion of AmyE with the fluorescent reporter mCherry is secreted via discrete patches showing very low dynamics. These are visible at many places within the cell wall for many minutes. Expression from a high copy number plasmid was required to be able to see these structures we term "secretion zones". Zones corresponded to visualized AmyE activity on the surface of cells, showing that they release active enzymes. They overlapped with SecA signals but did not frequently co-localize with the secretion ATPase. Single particle tracking showed higher dynamics of SecA and of SecDF, involved in AmyE secretion, at the cell membrane than AmyE. These experiments suggest that SecA initially translocates AmyE molecules through the cell membrane, and then diffuses to a different translocon. Single molecule tracking of SecA suggests the existence of three distinct diffusive states of SecA, which change during AmyE overexpression, but increased AmyE secretion does not appear to overwhelm the system. CONCLUSIONS: Because secretion zones were only found during the transition to and within the stationary phase, diffusion rather than passive transport based on cell wall growth from inside to outside may release AmyE and, thus, probably secreted proteins in general. Our findings suggest active transport through the cell membrane and slow, passive transition through the cell wall, at least for overexpressed proteins, in bacteria of the genus Bacillus.

Changes in digestive enzyme activities during the early ontogeny of milkfish, Chanos chanos larvae.

Knowledge of the developmental ontogeny of the digestive system and nutritional requirements of marine fish larvae is a primary requisite for their successful rearing under an optimal feeding regime. In this context, we assessed the activity profile of key digestive enzymes viz., trypsin, chymotrypsin, leucine aminopeptidase, lipase, amylase, and alkaline phosphatase during the early ontogeny of milkfish, Chanos chanos (0 day, 3 days, 6 days, 9 days, 12 days, 15 days, 18 days, 21 days, 25 days, and 30 days post-hatch). Larvae for this study were obtained from the successful breeding of milkfish at ICAR-Central Institute of Brackishwater Aquaculture, India. Growth curves (length and weight) of the larvae indicated a positive morphological development under a standardized feeding regime that comprised Chlorella salina, Brachionus plicatilis, Artemia salina nauplii, and commercial weaning feed for different larval stages. With respect to protein digestion, the specific activity of pancreatic enzymes trypsin and chymotrypsin and intestinal brush border leucine aminopeptidase showed two peaks at 3 dph and 15 dph, following the introduction of rotifer and Artemia nauplii. Similar bimodal peaks were observed for alkaline phosphatase and amylase activities, with the first peak at 3 dph and the second peak at 18 dph and 21 dph, respectively. Whereas in the case of lipase, high activity levels were observed at 0 dph, 3 dph, and 18 dph, with subsequent decreases and fluctuations. Overall, as most of the enzymes were found to have peak activities at 15 to 21 dph, this period can be potentially considered as the developmental window for weaning larvae from live to formulated feeds in milkfish hatcheries.

Early postoperative risk stratification in patients with pancreatic fistula after pancreaticoduodenectomy.

BACKGROUND: Early stratification of postoperative pancreatic fistula according to severity and/or need for invasive intervention may improve outcomes after pancreaticoduodenectomy. This study aimed to identify the early postoperative variables that may predict postoperative pancreatic fistula severity. METHODS: All patients diagnosed with biochemical leak and clinically relevant-postoperative pancreatic fistula based on drain fluid amylase >300 U/L on the fifth postoperative day after pancreaticoduodenectomy were identified from a consecutive cohort from Birmingham, UK. Demographics, intraoperative parameters, and postoperative laboratory results on postoperative days 1 through 7 were retrospectively extracted. Independent predictors of clinically relevant-postoperative pancreatic fistula were identified using multivariable binary logistic regression and converted into a risk score, which was applied to an external cohort from Verona, Italy. RESULTS: The Birmingham cohort had 187 patients diagnosed with postoperative pancreatic fistula (biochemical leak: 99, clinically relevant: 88). In clinically relevant-postoperative pancreatic fistula patients, the leak became clinically relevant at a median of 9 days (interquartile range: 6-13) after pancreaticoduodenectomy. Male sex (P = .002), drain fluid amylase-postoperative day 3 (P < .001), c-reactive protein postoperative day 3 (P < .001), and albumin-postoperative day 3 (P = .028) were found to be significant predictors of clinically relevant-postoperative pancreatic fistula on multivariable analysis. The multivariable model was converted into a risk score with an area under the receiver operating characteristic curve of 0.78 (standard error: 0.038). This score significantly predicted the need for invasive intervention (postoperative pancreatic fistula grades B3 and C) in the Verona cohort (n = 121; area under the receiver operating characteristic curve: 0.68; standard error = 0.06; P = .006) but did not predict clinically relevant-postoperative pancreatic fistula when grades B1 and B2 were included (area under the receiver operating characteristic curve 0.52; standard error = 0.07; P = .802). CONCLUSION: We developed a novel risk score based on early postoperative laboratory values that can accurately predict higher grades of clinically relevant-postoperative pancreatic fistula requiring invasive intervention. Early identification of severe postoperative pancreatic fistula may allow earlier intervention.

The effects of eugenol on histological, enzymatic, and oxidative parameters in the major salivary glands and pancreas of healthy male Wistar rats.

OBJECTIVE: We evaluated the effects of eugenol on histological, enzymatic, and oxidative parameters in the pancreas, parotid, submandibular, and sublingual glands of healthy male rats. DESIGN: Twenty-four adult Wistar rats were assigned into four groups (n = 6/group). Control rats received 2% Tween-20 (eugenol vehicle), whereas the other animals received 10, 20, and 40 mg kg-1 eugenol through gavage daily for 60 d. Major salivary and pancreatic glands were weighed and preserved fixed for microscopic analysis and frozen for in vitro assays. RESULTS: Eugenol did not alter glands' weight and serum amylase activity regardless of the concentration. The highest dose of eugenol caused an increase in pancreatic amylase activity and a reduction of lipase activity from serum and pancreas. Eugenol at 40 mg kg-1 diminished the activity of SOD and FRAP in the submandibular gland and CAT and FRAP in the sublingual gland. However, it did not exert any effect on GST regardless of the gland. Additionally, 40 mg kg-1 eugenol increased MDA levels in pancreatic, parotid, and submandibular glands and NO levels in the sublingual. The concentrations of eugenol induced distinct responses in the glands regarding the activity of Na+/K+, Mg2+, and total ATPase activity. They also affected histomorphometrical and histochemistrical parameters in the submandibular gland only. CONCLUSIONS: Results indicated that 40 mg kg-1 eugenol altered most of the biochemical and oxidatived parameters of digestive glands. Only submandibular glands presented histological changes after eugenol exposure suggesting potential implications for its function.

Incidence and risk factors of postoperative hyperamylasemia and pancreatitis following total knee arthroplasty: a retrospective study.

BACKGROUND: Postoperative hyperamylasemia and pancreatitis are recognized complications after abdominal and spinal surgeries. The aim of this study is to investigate the incidence and identify risk factors for postoperative hyperamylasemia and pancreatitis following total knee arthroplasty. METHODS: 170 patients undergoing total knee arthroplasty were retrospectively identified from our database from January 2017 to January 2021. Patients were divided into normal and hyperamylasemia groups based on the presence of serum amylase level within or greater than the normal range. The diagnosis of postoperative pancreatitis was based on the 2012 revised Atlanta Classification of Acute Pancreatitis. Patient demographics, perioperative parameters were investigated with student t test, chi square test and multivariate logistic regression analysis. RESULTS: 43 patients (25.3%) exhibited postoperative hyperamylasemia while eight patients (4.7%) exhibited serum amylase < 5 times the normal upper limit. One patient (0.6%) was designated as having postoperative pancreatitis. More patients with Hypertriglyceridemia (HTG) were noted in hyperamylasemia group (P = 0.009) compared with normal group. Hyperamylasemia group showed higher preoperative serum amylase (74.95 vs. 55.62 IU/L, P < 0.001), higher intra-operative blood loss (IBL) (117.67 vs. 77.01 mL, P = 0.040) and longer surgical duration (132.98 vs. 107.01 min, P = 0.041). Multivariate logistic analysis revealed that HTG (OR = 0.189, P = 0.006), preoperative serum amylase (OR = 1.042, P < 0.001) and IBL (OR = 1.004, P = 0.022) were independent risk factors for postoperative hyperamylasemia. CONCLUSIONS: A significant percentage of patients developed hyperamylasemia after total knee arthroplasty. Patients with HTG, higher preoperative serum amylase and higher IBL had an increased risk of developing postoperative hyperamylasemia and pancreatitis.

Large scale microfluidic CRISPR screening for increased amylase secretion in yeast.

Key to our ability to increase recombinant protein production through secretion is a better understanding of the pathways that interact to translate, process and export mature proteins to the surrounding environment, including the supporting cellular machinery that supplies necessary energy and building blocks. By combining droplet microfluidic screening with large-scale CRISPR libraries that perturb the expression of the majority of coding and non-coding genes in S. cerevisiae, we identified 345 genes for which an increase or decrease in gene expression resulted in increased secretion of α-amylase. Our results show that modulating the expression of genes involved in the trafficking of vesicles, endosome to Golgi transport, the phagophore assembly site, the cell cycle and energy supply improve α-amylase secretion. Besides protein-coding genes, we also find multiple long non-coding RNAs enriched in the vicinity of genes associated with endosomal, Golgi and vacuolar processes. We validated our results by overexpressing or deleting selected genes, which resulted in significant improvements in α-amylase secretion. The advantages, in terms of precision and speed, inherent to CRISPR based perturbations, enables iterative testing of new strains for increased protein secretion.

The Distinctive Permutated Domain Structure of Periplasmic α-Amylase (MalS) from Glycoside Hydrolase Family 13 Subfamily 19.

Periplasmic α-amylase MalS (EC. 3.2.1.1), which belongs to glycoside hydrolase (GH) family 13 subfamily 19, is an integral component of the maltose utilization pathway in Escherichia coli K12 and used among Ecnterobacteriaceae for the effective utilization of maltodextrin. We present the crystal structure of MalS from E. coli and reveal that it has unique structural features of circularly permutated domains and a possible CBM69. The conventional C-domain of amylase consists of amino acids 120-180 (N-terminal) and 646-676 (C-terminal) in MalS, and the whole domain architecture shows the complete circular permutation of C-A-B-A-C in domain order. Regarding substrate interaction, the enzyme has a 6-glucosyl unit pocket binding it to the non-reducing end of the cleavage site. Our study found that residues D385 and F367 play important roles in the preference of MalS for maltohexaose as an initial product. At the active site of MalS, ß-CD binds more weakly than the linear substrate, possibly due to the positioning of A402. MalS has two Ca2+ binding sites that contribute significantly to the thermostability of the enzyme. Intriguingly, the study found that MalS exhibits a high binding affinity for polysaccharides such as glycogen and amylopectin. The N domain, of which the electron density map was not observed, was predicted to be CBM69 by AlphaFold2 and might have a binding site for the polysaccharides. Structural analysis of MalS provides new insight into the structure-evolution relationship in GH13 subfamily 19 enzymes and a molecular basis for understanding the details of catalytic function and substrate binding of MalS.

Microscopic assessment of the degradation of millet starch granules by endogenous and exogenous enzymes during mashing.

The high gelatinization temperature (GT) of millet starch prevents the usage of infusion or step mashes as an effective means to generate fermentable sugars (FS) in brewing because the malt amylases lack thermostability at GT. Here, we investigate processing modifications to determine if millet starch can be efficiently degraded below GT. We determined that producing finer grists through milling did not introduce enough granule damage to markedly change gelatinization characteristics, though there was improved liberation of the endogenous enzymes. Alternatively, exogenous enzyme preparations were added to investigate their ability to degrade intact granules. At the recommended dosages (0.625 µL/g malt), significant FS concentrations were observed, although at lower concentrations and with a much-altered profile than possible with a typical wort. When exogenous enzymes were introduced at high (10×) addition rates, significant losses of granule birefringence and granule hollowing were observed well below GT, suggesting these exogenous enzymes can be utilized to digest millet malt starch below GT. The exogenous maltogenic α-amylase appears to drive the loss of birefringence, but more research is needed to understand the observed predominate glucose production.

The secretory ability of newly formed secretory granules is regulated by pro-cathepsin B and amylase in parotid glands.

Parotid glands are exocrine glands that release saliva into the oral cavity. Acinar cells of parotid glands produce many secretory granules (SGs) that contain the digestion enzyme amylase. After the generation of SGs in the Golgi apparatus, they mature by enlarging and membrane remodeling. VAMP2, which is involved in exocytosis, accumulates in the membrane of mature SGs. The remodeling of SG membranes is regarded as a preparation process for exocytosis but its detailed mechanism remains unknown. To address that subject, we investigated the secretory ability of newly formed SGs. Although amylase is a useful indicator of secretion, the cell leakage of amylase might affect the measurement of secretion. Thus, in this study, we focused on cathepsin B (CTSB), a lysosomal protease, as an indicator of secretion. It has been reported that some procathepsin B (pro-CTSB), which is a precursor of CTSB, is initially sorted to SGs after which it is transported to lysosomes by clathrin-coated vesicles. Because pro-CTSB is processed to mature CTSB after its arrival in lysosomes, we can distinguish between the secretion of SGs and cell leakage by measuring the secretion of pro-CTSB and mature CTSB, respectively. When acinar cells isolated from parotid glands were stimulated with isoproterenol (Iso), a ß-adrenergic agonist, the secretion of pro-CTSB was increased. In contrast, mature CTSB was not detected in the medium although it was abundant in the cell lysates. To prepare parotid glands rich in newly formed SGs, the depletion of per-existing SGs was induced by an intraperitoneal injection of Iso into rats. At 5 h after that injection, newly formed SGs were observed in parotid acinar cells and the secretion of pro-CTSB was also detected. We confirmed that the purified newly formed SGs contained pro-CTSB, but not mature CTSB. At 2 h after Iso injection, few SGs were observed in the parotid glands and the secretion of pro-CTSB was not detected, which proved that the Iso injection depleted pre-existing SGs and the SGs observed at 5 h were newly formed after the Iso injection. These results suggest that newly formed SGs have a secretory ability prior to membrane remodeling.

Exploring the interactive mechanism of acarbose with the amylase SusG in the starch utilization system of the human gut symbiont Bacteroides thetaiotaomicron through molecular modeling.

The α-amylase, SusG, is a principal component of the Bacteroides thetaiotaomicron (Bt) starch utilization system (Sus) used to metabolize complex starch molecules in the human gastrointestinal (GI) tract. We previously reported the non-microbicidal growth inhibition of Bt by the acarbose-mediated arrest of the Sus as a potential therapeutic strategy. Herein, we report a computational approach using density functional theory (DFT), molecular docking, and molecular dynamics (MD) simulation to explore the interactive mechanism between acarbose and SusG at the atomic level in an effort to understand how acarbose shuts down the Bt Sus. The docking analysis reveals that acarbose binds orthosterically to SusG with a binding affinity of -8.3 kcal/mol. The MD simulation provides evidence of conformational variability of acarbose at the active site of SusG and also suggests that acarbose interacts with the main catalytic residues via a general acid-base double-displacement catalytic mechanism. These results suggest that small molecule competitive inhibition against the SusG protein could impact the entire Bt Sus and eliminate or reduce the system's ability to metabolize starch. This computational strategy could serve as a potential avenue for structure-based drug design to discover other small molecules capable of inhibiting the Sus of Bt with high potency, thus providing a holistic approach for selective modulation of the GI microbiota.