Search Results (63)

The demand for sustainable, low-cost bioprocesses has encouraged the development of alternative enzyme production strategies. This study investigated the purification and characterization of a crude enzymatic extract (CEE) rich in α-amylase from Coprinus comatus, using wheat milling by-products as substrate. The CEE was obtained by submerged culture, followed by biomass removal, centrifugation, and ultrafiltration to yield a partially purified enzyme (PE). CEE and PE were evaluated for chemical composition, antioxidant and antibacterial activity, toxicity (Artemia salina assay), and enzymatic performance. Toxicity assays confirmed that CEE was non-toxic. Antioxidant activity reached 213.34 µmol TE·gDE⁻¹ (DPPH assay), with a total phenolic content of 8.01 mg GAE·gDE⁻¹. No antibacterial activity was detected. CEE hydrolyzed 96.31% of starch in 180 min, releasing 10.85 g·L⁻¹ glucose, while PE achieved 98% hydrolysis and released 14.5 g·L⁻¹. Optimal α-amylase activity occurred at 50 °C and pH 5.5 (CEE) or 5.0 (PE). Calcium ions improved the enzymatic activity and thermal stability. CEE retained over 60% activity after 721 days under refrigeration (4 °C) or freezing (0 °C). Although lyophilization enhanced enzyme concentration, it increased production costs. SDS-PAGE revealed bands of ~67 kDa (α-amylase) and ~35 kDa (glucoamylase). These findings support the use of CEE as a sustainable, low-cost biocatalyst for industrial use. Full article

Kluyveromyces marxianus is an emerging yeast cell host for diverse products, but multiple-gene expression in K. marxianus faces challenges due to limited current knowledge of cis-regulatory elements and insertion loci. Our previous study transferred an alien Saccharomyces cerevisiae chromosome I (R1) into K. marxianus, resulting in the creation of the monochromosomal hybrid yeast KS-R1. All R1 genes were actively transcribed, providing a series of loci with varying transcriptional activities. Here, we explore the use of R1 as a novel platform for stable, multi-gene integration and expression. By deleting three essential K. marxianus genes while complementing their functions with orthologs on R1, we achieved stable propagation of R1 in the absence of selective pressure. We characterized several loci on R1 that exhibit stable transcriptional activities under various conditions. GFP inserted in place of genes at six such loci demonstrated varying expression levels. Strains with GFP at two loci exhibited significantly higher expression than those with GFP at a single locus. Furthermore, we replaced five R1 genes with disulfide bond formation genes from Pichia pastoris at distinct loci, resulting in the active expression of all five genes and significantly enhanced production of heterologous glucoamylases BadGLA and TeGlaA. Our findings demonstrate that alien chromosomes offer a stable and versatile platform for the coordinated expression of multiple heterologous genes, serving as valuable tools for metabolic engineering and synthetic biology. Full article

The bioethanol produced from biomass fermentation is characterised by its renewable nature, as expressed in its life cycle. The Amazonian taro, with 72% starch content, can be an alternative to traditional raw materials such as corn and sugarcane. The objective of this study was to evaluate taro (Colocasia esculenta) as a raw material for bioethanol production through ultrasound-assisted enzymatic hydrolysis. For the enzymatic hydrolysis process, α-amylase and glucoamylase enzymes were used. Active dry yeast Saccharomyces cerevisiae was employed for fermentation. The chemical characterisation analysis of the flour with peel (WP) revealed high levels of protein (8.71%), crude fibre (4.21%) and total ash (5.56%). However, taro flour without peel (NP) revealed higher starch results (27.5%). The evaluation of reducing sugars during enzymatic hydrolysis showed better results in the samples with ultrasonic pretreatment, specifically in the sample with peel (WP-US) (1017.8 mg/L). The ethanol yield was significantly higher in the ultrasound-treated samples, achieving an approximately 35% increase in ethanol yield for both peeled and unpeeled samples compared to untreated samples. These findings open up new perspectives for using this tuber in the biofuel industry. Furthermore, they emphasise the need for continued research on biomass conversion processes. Full article

Pruning is a common forest-tending method; its purpose is to promote growth and improve the overall stand quality. Poplar is a fast-growing, broad-leaved tree species with high ecological and economic value. It is a common management method to promote its growth by pruning and adjusting the spatial structure of the stand, but its potential regulatory mechanism remains unclear. In this study, transcriptome and metabolome data of different parts at all pruning intensities were determined and analyzed. The results showed that 7316 differentially expressed genes were identified in this study. In the plant hormone signal transduction pathway, candidate genes were found in eight kinds of plant hormones, among which the main expression was gibberellin, auxin, and brassinosteroid. Some candidate gene structures (beta-glucosidase, endoglucanase, hexokinase, glucan endo-1, 3-beta-D-glucosidase, beta-fructofuranosidase, fructokinase, maltase-glucoamylase, phosphoglucomutase, and sucrose) were specifically associated with starch and sucrose biosynthesis. In the starch and sucrose biosynthesis pathway, D-fructose 6-phosphate, D-glucose 1,6-bisphosphate, and glucose-1-phosphate were the highest in stems and higher in the first round of pruning than in no pruning. The bHLH plays a key role in the starch and sucrose synthetic pathway, and AP2/ERF-ERF is important in the plant hormone signal transduction pathway. These results laid a foundation for understanding the molecular mechanism of starch and sucrose biosynthesis and provided a theoretical basis for promoting tree growth through pruning. Full article

The formation of atroposelective biaryl compounds in plants and fungi is well understood; however, polyketide aglycone synthesis and dimerization in bacteria remain unclear. Thus, the biosynthetic gene cluster (BGC) responsible for antibacterial setomimycin production from Streptomyces nojiriensis JCM3382 was examined in comparison with the BGCs of spectomycin, julichromes, lincolnenins, and huanglongmycin. The setomimycin BGC includes post-polyketide synthase (PKS) assembly/cycling enzymes StmD (C-9 ketoreductase), StmE (aromatase), and StmF (thioesterase) as key components. The heterodimeric TcmI-like cyclases StmH and StmK are proposed to aid in forming the setomimycin monomer. In addition, StmI (P-450) is predicted to catalyze the biaryl coupling of two monomeric setomimycin units, with StmM (ferredoxin) specific to the setomimycin BGC. The roles of StmL and StmN, part of the nuclear transport factor 2 (NTF-2)-like protein family and unique to setomimycin BGCs, could particularly interest biochemists and combinatorial biologists. α-Glucosidase, a key enzyme in type 2 diabetes, hydrolyzes carbohydrates into glucose, thereby elevating blood glucose levels. This study aimed to assess the α-glucosidase inhibitory activity of EtOAc extracts of JCM 3382 and setomimycin. The JCM 3382 EtOAc extract and setomimycin exhibited greater potency than the standard inhibitor, acarbose, with IC₅₀ values of 285.14 ± 2.04 μg/mL and 231.26 ± 0.41 μM, respectively. Molecular docking demonstrated two hydrogen bonds with maltase-glucoamylase chain A residues Thr205 and Lys480 (binding energy = −6.8 kcal·mol⁻¹), two π–π interactions with Trp406 and Phe450, and one π–cation interaction with Asp542. Residue-energy analysis highlighted Trp406 and Phe450 as key in setomimycin’s binding to maltase-glucoamylase. These findings suggest that setomimycin is a promising candidate for further enzymological research and potential antidiabetic therapy. Full article

An amylolytic industrial yeast strain named 1974-GA-temA, developed previously by our research team by coexpressing the α-amylase and glucoamylase genes, combines enzyme production, sweet potato residue (SPR) hydrolysis, and glucose fermentation into ethanol in a one-step process. This consolidated bioprocessing (CBP) method has great application potential in the commercial production of bioethanol from SPR, but important fermentation parameters should be optimized to further increase the ethanol concentration and yield. In this study, the effects of the initial fermentation pH, solid-to-liquid ratio, inoculation volume, addition of exogenous enzyme, and supplementation with metal ions were systemically investigated. Single-factor experiments revealed that the optimal pH was 4.0. In the solid-to-liquid ratio test, an increase in the solid-to-liquid ratio corresponded with a gradual increase in the ethanol concentration, peaking at 1:5. However, the ethanol yield gradually decreased, with the optimal solid-to-liquid ratio identified as 1:5. The ethanol concentration and yield reached 9.73 g/L and 5.84%, respectively. Additionally, an increase in the inoculum size resulted in increased ethanol concentration and yield, with the optimal inoculum level determined to be 10%. An ethanol concentration of 7.87 g/L was attained under these specified conditions, equating to an ethanol yield of 4.72%. Further analysis was conducted to assess the effects of exogenous cellulase, hemicellulase, and pectinase, both individually and in combination, on ethanol concentration and yield. The results indicated that pectinase had a particularly significant effect. The highest ethanol concentration was observed when all three enzymes were administered concurrently, yielding 27.27 g/L ethanol. Then, the role of metal ions in SPR fermentation was evaluated. The metal ions did not significantly affect the process, with the exception of copper ions. The addition of copper ions at a specific concentration of 0.2 g/100 g SPR increased the ethanol concentration. However, concentrations exceeding 0.2 g/100 g SPR inhibited yeast cell growth. Finally, orthogonal optimization was employed to determine the optimal combination of factors: pH, 4.0; solid-to-liquid ratio, 1:6; inoculation volume, 10%; cellulase and pectinase addition; and the absence of Cu²⁺ addition. Under these conditions, strain 1974-GA-temA produced 34.83 ± 0.62 g/L ethanol after 8 days of fermentation, corresponding to a 20.90% ± 0.37% ethanol yield. This value markedly exceeds the outcomes of all the conducted orthogonal experiments. The fermentation optimization experiments in this study are expected to increase ethanol production during the CBP fermentation of SPR. Full article

Biomass from various types of organic waste was tested for possible use in hydrogen production. The composition consisted of lignified samples, green waste, and kitchen scraps such as fruit and vegetable peels and leftover food. For this purpose, the enzymatic pretreatment of organic waste with a combination of five different hydrolytic enzymes (cellulase, amylase, glucoamylase, pectinase and xylase) was investigated to determine its ability to produce hydrogen (H₂) with the hydrolyzate produced here. In course, the anaerobic rod-shaped bacterium T. neapolitana was used for H₂ production. First, the enzymes were investigated using different substrates in preliminary experiments. Subsequently, hydrolyses were carried out using different types of organic waste. In the hydrolysis carried out here for 48 h, an increase in glucose concentration of 481% was measured for waste loads containing starch, corresponding to a glucose concentration at the end of hydrolysis of 7.5 g·L⁻¹. In the subsequent set fermentation in serum bottles, a H₂ yield of 1.26 mmol H₂ was obtained in the overhead space when Terrific Broth Medium with glucose and yeast extract (TBGY medium) was used. When hydrolyzed organic waste was used, even a H₂ yield of 1.37 mmol could be achieved in the overhead space. In addition, a dedicated reactor system for the anaerobic fermentation of T. neapolitana to produce H₂ was developed. The bioreactor developed here can ferment anaerobically with a very low loss of produced gas. Here, after 24 h, a hydrogen concentration of 83% could be measured in the overhead space. Full article

The aim of this study was to investigate the potential of Amaranthus cruentus flavonoids (quercetin, kaempferol, catechin, hesperetin, naringenin, hesperidin, and naringin), cinnamic acid derivatives (p-coumaric acid, ferulic acid, and caffeic acid), and benzoic acids (vanillic acid and 4-hydroxybenzoic acid) as antioxidants, antidiabetic, and antihypertensive agents. An analytical method for simultaneous quantification of flavonoids, cinnamic acid derivatives, and benzoic acids for metabolomic analysis of leaves and inflorescences from A. cruentus was developed with HPLC-UV-DAD. Evaluation of linearity, limit of detection, limit of quantitation, precision, and recovery was used to validate the analytical method developed. Maximum total flavonoids contents (5.2 mg/g of lyophilized material) and cinnamic acid derivatives contents (0.6 mg/g of lyophilized material) were found in leaves. Using UV-Vis spectrophotometry, the maximum total betacyanin contents (74.4 mg/g of lyophilized material) and betaxanthin contents (31 mg/g of lyophilized material) were found in inflorescences. The leaf extract showed the highest activity in removing DPPH radicals. In vitro antidiabetic activity of extracts was performed with pancreatic α-glucosidase and intestinal α-amylase, and compared to acarbose. Both extracts exhibited a reduction in enzyme activity from 57 to 74%. Furthermore, the in vivo tests on normoglycemic murine models showed improved glucose homeostasis after sucrose load, which was significantly different from the control. In vitro antihypertensive activity of extracts was performed with angiotensin-converting enzyme and contrasted to captopril; both extracts exhibited a reduction of enzyme activity from 53 to 58%. The leaf extract induced a 45% relaxation in an ex vivo aorta model. In the molecular docking analysis, isoamaranthin and isogomphrenin-I showed predictive binding affinity for α-glucosidases (human maltase-glucoamylase and human sucrase-isomaltase), while catechin displayed binding affinity for human angiotensin-converting enzyme. The data from this study highlights the potential of A. cruentus as a functional food. Full article

Background: Oat-based Milk Alternatives (OMAs) provide multiple health benefits arising from oat’s unique compounds: avenanthramides, avenacosides, and dietary fibre β-glucan. Avenanthramides–polyphenols, unique to oats, provide anti-inflammatory and antioxidant effects, whilst avenacosides are saponins with anti-bacterial and anti-fungal properties. β-Glucans assist in lowering blood cholesterol and lead to the prevention of diabetes and cardiovascular diseases. However, oats undergo many stages of processing to ensure a sensory appealing and safe OMA product, including enzymatic treatment, heating, high shear, decanting of larger solids, and homogenisation. It is possible that throughout these stages, compounds may be affected by degradation or lost entirely. Objective: The concentration of avenanthramides, avenacosides, and β-glucans in the OMA samples was measured at each of the 12 stages of an OMA production, with a comparison of short ultra-heat treatment (UHT) and prolonged high heat treatment, to assess how they may be affected. Design: OMA samples were produced from basic ingredients within the pilot plant. Liquid chromatography–mass spectrometry was used to measure the concentration of avenanthramides and avenacosides. β-Glucan was determined spectrophotometrically using the Megazymes assay. Results: Avenanthramides and avenacosides were found to significantly increase in concentration after initial enzymatic treatment with alpha-amylase, whilst avenanthramides also increased post 90 °C treatment, and decanting – suggesting that these compounds are not being lost in the removed solids. However, avenanthramides decreased after UHT and prolonged heat treatment, suggesting they may be susceptible to degradation from prolonged heat and temperatures above 120 °C. β-Glucans concentrations decreased post glucoamylase treatment, and decanting – suggesting that β-glucans are lost within the decanted slurry, and increased after treatment with alpha-amylase, 90 °C and high shear mixing. Conclusion: With this information, future products may be optimised to preserve these components to improve the health benefits of oat-based milk alternatives. Full article

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. Full article

The present study employed Hunan local Tuqu for fermentation and investigated the physicochemical properties, microbial community composition, and volatile flavor compounds of the fermented grains, as well as the correlation between the physicochemical indicators and the microbial community. The findings reveal that the activities of α-amylase and glucoamylase were highest during the initial stages of the fermentation process. The acid protease activity increased to 30.6 U/g on the second day and then decreased. Cellulose and lipase activities both showed an increasing trend. The moisture content increased sharply to 73.41% and then remained relatively stable. The acidity was highest on the eighth day. Fifty genera of bacteria and twenty-two genera of fungi were detected. Lactobacillus was dominant among bacteria, and Saccharomyces was dominant among fungi. A correlation analysis showed that there were positive correlations between moisture, acidity, cellulose, lipase activities and Lactobacillus, and there were positive correlations between moisture content, acidity, cellulase activity, acidic protease activity and Saccharomyces. A total of 46 volatile flavor compounds were detected, of which 6 alcohols and 14 esters constituted the major portion, and 9 key flavor compounds with an ROAV > 1 were identified throughout the fermentation process. Isoamyl acetate had the highest ROAV and made the greatest contribution to the flavor. Full article

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. Full article

Peony seed meal (PSM) is the by-product obtained from peony seeds after oil extraction. In this study, PSM was incorporated into traditional koji-making, and its impacts on koji enzyme activities and flavor compounds in final products were investigated. In the process of koji fermentation, the optimal addition ratio of PSM to soybean was determined as 7:3. Under this ratio, the maximum enzyme activities of neutral protease, amylase, and glucoamylase were 1177.85, 686.58, and 1564.36 U/g, respectively, and the koji obtained was subjected to maturation. During post-fermentation, changes in the fermentation characteristics of the paste samples were monitored, and it was found that compared to the soybean paste without PSM, the enzyme activities maintained at a relatively good level. The PSM soybean paste contained a total of 80 flavor compounds and 11 key flavor compounds (OAV ≥ 1), including ethyl isovalerate, isovaleric acid, hexanal, phenylacetaldehyde, 3-Methyl-1-butanol 4-heptanone, 2-pentylfuran, methanethiol ester caproate, isoamyl acetate, 3-methyl-4-heptanone, and isovaleraldehyde. These findings could be used to improve the quality of traditional fermented paste, enrich its flavor, and simultaneously promote PSM as a valuable resource for fermented foods. Full article

Daqu is a traditional starter for Baijiu fermentation and is produced by spontaneous fermentation of ground and moistened barley or wheat. The quality of Daqu is traditionally evaluated based on physicochemical and subjective sensory parameters without microbiological analysis. Here, we compared the physicochemical characteristics of qualified (QD) and inferior (ID) Daqu, their microbial communities based on plate counting and PacBio SMRT sequencing of rRNA gene libraries, and their impacts on Baijiu fermentation. The results showed that the glucoamylase and α-amylase activities of QD were significantly higher than those of ID. The counts of yeasts and relative abundances of functional microbes, especially the amylolytic bacterium Bacillus licheniformis and fungi Saccharomycopsis fibuligera and Lichtheimia ramosa, were significantly higher in QD than in ID. The laboratory-scale Baijiu fermentation tests showed that the relative abundances of the amylolytic microbes were higher in the QD than the ID fermentation set, resulting in more efficient fermentation, as indicated by more weight loss and higher moisture content in the former. Consequently, more glycerol, acetic acid, ethanol, and other volatile compounds were produced in the QD than in the ID fermentation set. The results suggest that Daqu quality is determined by, and can be evaluated based on, its microbial community. Full article

Nanobodies, also referred to as VHH antibodies, are the smallest fragments of naturally produced camelid antibodies and are ideal affinity reagents due to their remarkable properties. They are considered an alternative to monoclonal antibodies (mAbs) with potential utility in imaging, diagnostic, and other biotechnological applications given the difficulties associated with mAb expression. Aspergillus oryzae (A. oryzae) is a potential system for the large-scale expression and production of functional VHH antibodies that can be used to meet the demand for affinity reagents. In this study, anti-RNase A VHH was expressed under the control of the glucoamylase promoter in pyrG auxotrophic A. oryzae grown in a fermenter. The feature of pyrG auxotrophy, selected for the construction of a stable and efficient platform, was established using homologous recombination. Pull-down assay, size exclusion chromatography, and surface plasmon resonance were used to confirm the binding specificity of anti-RNase A VHH to RNase A. The affinity of anti-RNase A VHH was nearly 18.3-fold higher (1.9 nM) when expressed in pyrG auxotrophic A. oryzae rather than in Escherichia coli. This demonstrates that pyrG auxotrophic A. oryzae is a practical, industrially scalable, and promising biotechnological platform for the large-scale production of functional VHH antibodies with high binding activity. Full article