Efficient Hydrolysis Research Articles

Selective Protonation of Catalytic Dyad for γ-Secretase-Mediated Hydrolysis Revealed by Multiscale Simulations.

γ-Secretase plays a crucial role in producing disease-related amyloid-β proteins by cleaving the amyloid precursor protein (APP). The enzyme employs its catalytic dyad containing two aspartates (Asp257 and Asp385) to hydrolyze the substrate by a general acid-base catalytic mechanism, necessitating monoprotonation of the two aspartates for efficient hydrolysis. However, the precise protonation states of the aspartates remain uncertain. In this study, we employed a multiscale computational approach to investigate the dependence of the catalytic efficiency of γ-secretase on the protonation states of its catalytic dyad. Over 200 ms unbiased atomistic simulations of the substrate-enzyme complex reveal diverse orientations of the scissile bond of the bound substrate and accessible structural ensembles of the catalytic dyad with Asp257-Asp385 distances fluctuating between 4 and 10 Å. With a quantum mechanics/molecular mechanics (QM/MM) approach accelerated by enhanced sampling techniques, we find that the first step of the hydrolysis reaction, i.e., the formation of a gem-diol intermediate, experiences a higher reaction barrier by ∼2 kcal/mol when Asp385 is protonated. Furthermore, we find that Arg269 of the enzyme is most likely responsible for this preference of the protonation state: its basic side chain is spatially close to that of Asp257 and specifically stabilizes the transition state electrostatically when Asp257 is protonated. Collectively, our study suggests that Asp257 is likely the favored protonation site for APP cleavage by γ-secretase.

Thermophilic aerobic digestion using aquaculture sludge from rainbow trout aquaculture facilities: effect of salinity

The objectives of this study were to evaluate the potential of using thermophilic aerobic digestion (TAD) to hydrolyze aquaculture sludge, and to investigate the hydrolysis efficiency and changes in microbial community structure during TAD at 0, 15, and 30 practical salinity units (psu). As digestion progressed, soluble organic matter concentrations in all reactors increased to their maximum values at 6 h. The hydrolysis efficiency at 6 h decreased as salinity increased: 2.42% at 0 psu, 1.78% at 15 psu, and 1.04% at 30 psu. The microbial community compositions at the genus level prominently differed in the relative abundances of dominant bacteria between 0 psu and 30 psu. The relative abundance of genera Iodidimonas and Tepidiphilus increased significantly as salinity increased. Increase in the salinity at which thermophilic aerobic digestion of aquaculture sludge was conducted altered the microbial community structure, which in turn decreased the efficiency of organic matter hydrolysis.

Simple enzymatic depolymerization process based on rapid ball milling pretreatment for high-crystalline polyethylene terephthalate fibers

The high crystallinity (30 %–50 %) of discarded polyester textile waste limits the industrialization of its clean enzymatic depolymerization. In this study, a simple process based on ball milling pretreatment was developed to achieve effective enzymatic hydrolysis of high-crystalline polyester fiber. Ball milling was selected for its short, mild, and chemical-free process, which achieved a remarkable 23.8-fold (60.9 %) increase in terephthalic acid (TPA) yield from waste polyethylene terephthalate (PET) degradation, along with high TPA purity in the released soluble compounds. Just 30 min of ball milling at room temperature induced polyester amorphization, resulting in polyester with 12 % lower crystallinity compared with untreated polyester (51 %), while simultaneously increasing the surface roughness of polyester, thereby enhancing the efficiency of enzymatic hydrolysis. The simple process for effective enzymatic-depolymerization of waste polyester fiber developed in this study has potential industrial applications

Improved volatile fatty acid production in anaerobic digestion via simultaneous temperature regulation and persulfate activation by biochar: Chemical and biological response mechanisms

Increasing volatile fatty acid (VFA) production via persulfate activation (i.e., chemical effect) in anaerobic digestion (AD) is an emerging resource utilization method. However, the reaction mechanisms responsible for improving VFA production in AD via simultaneous temperature regulation and persulfate activation by biochar remain unclear. In this study, three PB15 treatment systems of low temperature (15 °C), medium temperature (35 °C) and high temperature (55 °C) were set to explore the relationship between VFAs production and treatment temperature and the influence of temperature on the reaction mechanism. The results show that the improvement of hydrolysis and acidification efficiency of the system in the medium temperature system is the highest. The VFA yield and acid production rate in the treatment group at 35°C were 2.49 and 5.22 times higher than those in the control group, respectively. The chemical effect effectively initiated the anaerobic acid production process and maintained the dominant role of the biological effect. The activity of persulfate is too low at low temperature, and its decomposition is too fast at high temperature. Plenty of free radicals lead to enhanced oxidation of the system, which may kill the fermentation bacteria. The NCM model indicates that microbial stability is reduced in high temperature systems. The SEM model showed that temperature change mainly affected substrate degradation by hydrolytic bacteria and indirectly affected acid production by acid-producing bacteria. This study provides a new strategy for realizing pollutant recycling and increasing VFAs production in cold area.

In-depth recognition of mixed surfactants maintaining the enzymatic activity of cellulases through stabilization of their spatial structures

Mixed surfactants improve the enzymatic hydrolysis of lignocellulosic substrates by enhancing cellulase stability against heat, pH, shear, and air–liquid interface stress. Under conditions of multiple factorial stresses (50 °C, pH 4.8, 180 rpm, and 15.5 cm2 air–liquid interface), cellulase with ternary surfactants (Tween 60/Triton X-114/CTAB, the molar ratio 14:5.5:1) retained 84 % of its activity after 48 h of incubation, representing 1.15 and 1.29 folds that of the cellulase activity with the single Tween 60 and with no surfactants, respectively. This is attributed to the fact that ternary surfactants possess better rheology modulation and air–liquid interface competitiveness. In addition, the computational approach demonstrated that the ternary surfactants were capable of forming stronger hydrophobic and hydrogen-bond interactions with cellulase enzymes, thus maintaining its secondary structure and preventing the detrimental α-helix to β-sheet transformation known to compromise cellulase activity. This synergy offers valuable insights into surfactant-cellulase interactions and supports efficient enzymatic hydrolysis in biorefineries.

A novel mechanocatalytic process by vibratory disk mill for efficient hydrolysis of cellulose, chitin and xylan

Polysaccharides are primary sources to manufacture chemical products with low carbon footprints, owing to their carbons from atmospheric carbon dioxide fixed through photosynthesis. Breaking down such polysaccharides into water-soluble saccharides through the hydrolysis of glycosidic bonds is one of key steps to produce valuable chemicals; however, previous chemical and enzymatic processes have been suffered from their fundamental drawbacks (e.g. not environmentally benign, high production cost) and are not regarded as feasible approaches. Recently, mechanocatalytic process, which proceeds hydrolysis of polysaccharides by simply milling with solid acids, attracts attentions owing to its cost-effectiveness and environmental beingness. Herein, we propose the novel mechanocatalytic system to efficiently proceed the hydrolysis of glycosidic bonds in polysaccharides by using “vibratory disk mill”, which can produce strong shear and impact force. By milling polysaccharides (cellulose, chitin and xylan) with solid acid kaolin, the polysaccharides can be efficiently hydrolysed to water-soluble saccharides with a rate twice higher than the previous milling devices, achieving quantitative conversion of polysaccharide to water soluble products (solubility: >99 %). We elucidated the reaction mechanism of polysaccharides with kaolin by X-ray diffractograms: forces produced by the vibratory disk mill can efficiently delaminate layered aluminosilicate nanosheets in kaolin, leading to better accessibility of polysaccharides on the acidic surface of the nanosheets. This novel mechanocatalytic system can also be applicable to produce oligosaccharides directly from crude biomass samples (birchwood chips and shrimp shells) without any chemical purification steps, demonstrating that this system can be used in industrial biomass processing.

Molecular Cloning, Characterization, and Application of a Novel Multifunctional Isoamylase (MIsA) from Myxococcus sp. Strain V11

A novel multifunctional isoamylase, MIsA from Myxococcus sp. strain V11, was expressed in Escherichia coli BL21(DE3). Sequence alignment revealed that MIsA is a typical isoamylase that belongs to glycoside hydrolase family 13 (GH 13). MIsA can hydrolyze the α-1,6-branches of amylopectin and pullulan, as well as the α-1,4-glucosidic bond in amylose. Additionally, MIsA demonstrates 4-α-D-glucan transferase activity, enabling the transfer of α-1,4-glucan oligosaccharides between molecules, particularly with linear maltooligosaccharides. The Km, Kcat, and Vmax values of the MIsA for amylopectin were 1.22 mM, 40.42 µmol·min–1·mg–1, and 4046.31 mM·min–1. The yields of amylopectin and amylose hydrolyzed into oligosaccharides were 10.16% and 11.70%, respectively. The hydrolysis efficiencies were 55%, 35%, and 30% for amylopectin, soluble starch, and amylose, respectively. In the composite enzyme hydrolysis of amylose, the yield of maltotetraose increased by 1.81-fold and 2.73-fold compared with that of MIsA and MTHase (MCK8499120) alone, respectively.

Enhancement of hydrogen production in dark fermentation of corn stover hydrolysates through composite electric field pretreatment: Improving enzymatic efficiency and regulating microbial metabolic balance

This study investigates the effects of composite electric field pretreatment (CEP) on hydrogen production from corn stover enzymatic hydrolysates in dark fermentation. The findings reveal that under optimal conditions, the CEP group achieved a cumulative hydrogen yield of 77.3 ± 2.6 mL/g TS, marking a 55.3 % increase compared to the control group without the electric field. CEP significantly enhances the fermentation capacity of enzymatic hydrolysates during the acid-stage of dark fermentation by disrupting the lignin structure and optimizing cellulase hydrolysis efficiency. Additionally, pH self-regulation is facilitated through the interaction between volatile fatty acids (VFAs) and ammonia nitrogen. Microbial community analysis revealed that CEP shifts the metabolic balance between Clostridium_sensu_stricto_1 and Lactococcus, leading to increased hydrogen yield and concentration during acid fermentation. Notably, Terrisporobacter exhibited a superior acid-producing capability compared to Bacteroides during the dark fermentation of enzymatic hydrolysates. This study provides a new perspective for the practical application of corn stover.

Elucidating the synergic mechanism of sodium pyrophosphate assisted enzymolysis on the waste activated sludge fermentation enhancement: Insights from organic conversion and metagenomic analysis

Lysozyme has been identified as a promising approach for enhancing waste activated sludge (WAS) anaerobic fermentation. However, the extracellular polymeric substance (EPS), functioning as a ‘protective shell’ for microbial cells, hindered the lysozyme transfer and subsequent interaction with cell wall, resulting in excessive dosage and limited efficiency in practical engineering applications. This study innovatively proposed a sodium pyrophosphate (SP) assisted lysozyme treatment, aiming to overcome such limitations and enhance the solubilization and hydrolysis efficiency. As a result, 3583.5 mg COD/L short-chain fatty acids (SCFAs) and 23.1 % acidification rate were achieved after 2-day anaerobic fermentation within SP-lysozyme treatment. Mechanism investigation revealed that SP facilitated EPS disruption by inducing a loosening of the protein structure, leading to the enhanced solubilization of organic matter within the EPS and microbial cells. Consequently, the release of substantial substrates contributed to the enrichment of functional microbes associated with hydrolysis and acidogenesis, accompanied by the augmentation of corresponding metabolic functions. Besides, the key functional genes analysis demonstrated the amino acid metabolism, glycolysis, pyruvate metabolism and SCFAs transport were facilitated and the methanogenesis was inhibited on the contrary with SP-lysozyme treatment, which was undoubtedly responsible for the superior SCFAs accumulation in anaerobic fermentation process. Considering the carbon resource recovery in SCFAs form, such novel SP-lysozyme treatment exhibited 325.1 CNY /ton SS (45.5 USD / ton SS) economic benefit and avoided 0.12 CO2/ton SS carbon emission, suggesting its promising application potential in future WAS management.

Purification and characterization of α-galactosidases from Penicillium griseoroseum for efficient soymilk hydrolysis

Soybean utilization is limited by the presence of raffinose oligosaccharides (RFO), which are not digested by humans and cause gastrointestinal discomfort. This study explores the potential of α-galactosidases from Penicillium griseoroseum for RFO hydrolysis in soymilk. Two distinct α-galactosidase enzymes, designated α-Gal1 and α-Gal2, were purified using a combination of ion-exchange chromatography and native polyacrylamide gel electrophoresis. Both enzymes exhibited characteristics of multimeric proteins and displayed similar biochemical properties. Optimal activity was observed at a pH range of 4.5–5.0 and a temperature range of 40–45 °C. Notably, α-Gal1 demonstrated high thermostability with a half-life of 16 h at 40 °C. The α-galactosidases displayed different substrate affinitiesfor the substrates ρ-NP-αGal, o-NP-αGal, rD-raffinose, d-stachyose, and mD-melibiose. The Michaelis-Menten constant (Km) values for α-Gal1 were 1.06, 1.31, 28.74, 19.88, and 4.77 mmol/L, respectively, while those for α-Gal2 were 0.8, 1.26, 30.46, 21.74 and 5.01 mmol/L, respectively. Both α-Gal1 and α-Gal2 were strongly inhibited by metal ions (Ag⁺, Cu2⁺, Fe2⁺, and Hg2⁺) and moderately inhibited by d-melibiose. Importantly, both enzymes efficiently hydrolyzed RFOs, achieving complete d-stachyose elimination from soymilk after a 6-h incubation. These findings propose the promising application of these α-galactosidases in industrial soymilk production, potentially enhancing its nutritional value and alleviating gastrointestinal issues in consumers.

Computational Analysis of Sarin, Soman, and Their Water Mixtures in NU-1000: Interaction Mechanisms, Distribution Patterns, and Pairing Effects.

Due to their extraordinary structural stability under humid conditions, zirconium-based metal-organic frameworks (Zr-MOFs) have been widely investigated for the hydrolytic degradation of nerve agents. That said, mechanisms of hydrolysis in the solid state and the participation of environmental water are not well understood. This work utilizes computational techniques to evaluate the behavior of water and two organophosphorus nerve agents (sarin and soman) in NU-1000, a Zr-MOF with the characteristic attributes for hydrolytic efficiency under humid conditions. Density functional theory (DFT) calculations reveal that soman binds more favorably to NU-1000 active sites than sarin, resulting in different preferential locations of each nerve agent within the framework. The strength of nerve agent binding is also found to vary depending on the site environment, with more favorable binding of both nerve agents occurring in the c-pores of NU-1000 than in the mesopores. Molecular dynamics (MD) simulation results further illustrate that free water molecules in NU-1000 prioritize interactions with nerve agents. Given the variation in their affinity for active site interactions, the introduction of different nerve agents to the framework results in substantial differences in water distribution and behavior. The results give insight into potential variances in the functionality of NU-1000 toward the hydrolysis of each nerve agent. More importantly, they emphasize the significance of considering the role of environmental water in hydrolysis and the possibility of diverse reaction variables based on the type of nerve agent and the properties of the MOF.

More is not always better. Multiple carbohydrate-binding domains for efficient starch hydrolysis

Lactobacillus amylovorus α-amylase is an endoenzyme with a peculiar starch-binding domain containing five identical family 26 carbohydrate-binding modules (CBM26). To investigate the impact of CBMs on catalytic activity, C-terminally truncated derivatives were constructed. The catalytic domain alone shows low affinity for the substrate and a very slow reaction rate, highlighting the importance of CBMs in maintaining the enzyme's optimal conformation and dynamics for efficient catalysis. CBMs enhance enzyme performance, as indicated by improved catalytic efficiency (kcat/Km). Interestingly, the enzyme variant LaCD3CBM, with three CBMs, exhibits the best catalytic efficiency on soluble starch, outperforming the wild-type amylase, with five CBMs. In the case of insoluble starch, the catalytic domain alone could not hydrolyze it and even adding a CBM, the release of reducing sugars was very inefficient. However, this efficiency was significantly improved by two orders of magnitude for the three-CBM variant and the wild-type amylase. CBMs also played a crucial role in protein thermostability, contributing to a higher melting temperature of the catalytic domain with just a single CBM. Notably, thermostability did not increase with the number of CBMs. In conclusion, spatial arrangement and interactions between the catalytic domain and CBMs significantly influenced enzymatic efficiency with both soluble and insoluble substrates. These interactions optimize enzymatic activity and improve thermostability.

Evaluation of the probiotic ability of Cellulolytic Bacillus subtilis SBMP4 (FSP20) isolated from waste-dump site in FIIRO, Lagos Nigeria

BACKGROUND: Current invention in the field of biotechnology includes research into the probiotic potentials of enzyme-producing bacteria species. OBJECTIVE: This study evaluated the probiotic ability of cellulolytic Bacillus subtilis SBMP4 isolated from waste dump site. METHODS: Isolated Bacillus species was screened for cellulolytic ability on carboxyl methylcellulose agar (CMCA), and hydrolysis efficiency was calculated. Probiotic ability of cellulolytic Bacillus species was carried out as follows: pathogenic potentials, bacteriocin producing ability, antibiotic susceptibility testing, gastric acid, and bile tolerance. Molecular identification was done by means of 16S rRNA gene sequencing, blast search analysis and phylogenetic analysis. RESULTS: Zone of clearance of 30 ± 2.08 mm on CMCA with hydrolysis efficiency of 114 % depicted the isolate’s cellulolytic potential. Bacillus subtilis SBMP4 showed γ-hemolytic phenotype with negative lecithinase activity. Antibacterial activity of bacteriocin observed against Staphylococcus aureus ATCC 25923 reference culture, had maximum inhibition zone of 14±1.0 mm at 72 hour of incubation. Substantial susceptibility towards 9 out of 10 commonly used antibiotics further depicted the isolate’s probiotic ability. Bacillus subtilis SBMP4 exhibited good gastric acid tolerance (pH 2.5) of 5.857 Log cycle/ml and good tolerance to 5 % and 10 % bile salt concentrations with 6.134 and 6.301 Log cycle/ml of viable bacteria cells. Molecular identification of the selected bacteria species revealed it to be Bacillus subtilis SBMP4 with RefSeq accession number NR_118383, showing maximum sequence similarity of 98%. CONCLUSION: Cellulolytic Bacillus subtilis SBMP4 possesses good probiotic ability and has the potentials of being applied as additive in livestock feeding to enhance overall productivity. KEYWORDS: Cellulolytic, Probiotic, CelA gene, Bacteriocin, Bacillus subtilis

Lysozyme coupling protease pretreatment to relieve the humic acid inhibition on excess sludge anaerobic fermentation

The asynchronous dosed protease and lysozyme combination pretreatment was proved to be effective in enhancing the anaerobic fermentation of waste activated sludge (WAS). However, humic acid (HA) in the sludge could interact with hydrolase and restrain the hydrolysis efficiency, thus inhibiting short-chain fatty acids (SCFAs) production. This study investigated the effectiveness and mechanism of enzymatic pretreatment against HA. Results showed that the enzyme cocktail method increased the extracellular bioavailable contents by 34 %, which raised SCFAs production by 89.69 % (1269.65 mg COD /L). The balanced ratio of hydrolysis and fermentation communities suggested that the small molecular organics generated by the hydrolysis community could be sufficiently utilized by fermentation communities. The metabolism of amino acids and glucose was facilitated, and the activities of key enzymes were enhanced. These results clarified the effect of asynchronous enzyme cocktail pretreatment against HA inhibition and contributed to SCFAs production, which offered fresh perspectives on carbon recovery.

Comparative evaluation of the extracellular production of a polyethylene terephthalate degrading cutinase by Corynebacterium glutamicum and leaky Escherichia coli in batch and fed-batch processes

BackgroundWith a growing global population, the generation of plastic waste and the depletion of fossil resources are major concerns that need to be addressed by developing sustainable and efficient plastic recycling methods. Biocatalytic recycling is emerging as a promising ecological alternative to conventional processes, particularly in the recycling of polyethylene terephthalate (PET). However, cost-effective production of the involved biocatalyst is essential for the transition of enzymatic PET recycling to a widely used industrial technology. Extracellular enzyme production using established organisms such as Escherichia coli or Corynebacterium glutamicum offers a promising way to reduce downstream processing costs.ResultsIn this study, we compared extracellular recombinant protein production by classical secretion in C. glutamicum and by membrane leakage in E. coli. A superior extracellular release of the cutinase ICCGDAQI was observed with E. coli in batch and fed-batch processes on a litre-scale. This phenomenon in E. coli, in the absence of a signal peptide, might be associated with membrane-destabilizing catalytic properties of the expressed cutinase. Optimisations regarding induction, expression temperature and duration as well as carbon source significantly enhanced extracellular cutinase activity. In particular, in fed-batch cultivation of E. coli at 30 °C with lactose as carbon source and inducer, a remarkable extracellular activity (137 U mL−1) and cutinase titre (660 mg L−1) were achieved after 48 h. Literature values obtained with other secretory organisms, such as Bacillus subtilis or Komagataella phaffii were clearly outperformed. The extracellular ICCGDAQI produced showed high efficacy in the hydrolysis of PET textile fibres, either chromatographically purified or unpurified as culture supernatant. In less than 18 h, 10 g L−1 substrate was hydrolysed using supernatant containing 3 mg cutinase ICCGDAQI at 70 °C, pH 9 with terephthalic acid yields of up to 97.8%.ConclusionExtracellular production can reduce the cost of recombinant proteins by simplifying downstream processing. In the case of the PET-hydrolysing cutinase ICCGDAQI, it was even possible to avoid chromatographic purification and still achieve efficient PET hydrolysis. With such production approaches and their further optimisation, enzymatic recycling of PET can contribute to a more efficient and environmentally friendly solution to the industrial recycling of plastics in the future.

Mechanisms of efficient polyacrylamide degradation: From multi-omics analysis to structural characterization of two amidohydrolases

Polyacrylamide (PAM) is a high molecular weight polymer with extensive applications. However, inefficient natural degradation of PAM results in its environmental accumulation. Here, using multi-omics analysis, we constructed the PAM biodegradation pathway in Klebsiella sp. PCX, an efficient PAM-degrading bacterium. Subsequently, two unclassified amidohydrolases (PCX00451 and PCX04581) were identified as key factors for rapid PAM biodegradation, both of which possessed much higher hydrolysis efficiency for PAM than for small molecule amide compounds. Besides, crystal structures of PCX00451 and PCX04581 were solved. Both two amidohydrolases were consisted with a twisted triosephosphateisomerase (TIM)-barrel and a smaller β-sandwich domain. And their binding pockets were in the conserved metal center of TIM-barrel domain. Moreover, Asp267 of PCX00451 and Asp282 of PCX04581 were examined as active sites for acid/base catalysis. Our research characterized the molecular mechanisms of two efficient amidohydrolases, providing theoretical basis and valuable tools for PAM bioremediation.

Fabrication of Thermo-Responsive Polymer-MOF@cellulase Composites with Improved Catalytic Performance for Hydrolysis of Cellulose.

Metal-organic frameworks (MOFs) are considered as an ideal enzyme support because of their porous structural superiority. However, MOFs@enzyme composites have usually compromised their hydrolysis efficiency due to the narrow space inducing unfavourable enzyme conformations. Herein, a thermo-responsive poly(N,N-dimethylacrylamide) (PD) was fixed onto the surface of UiO-66-NH2 (UiO) through a post-synthetic modification protocol. Using poly(2-vinyl-4,4 dimethylazlactone) (V) as a linker, PVD-UiO@cellulase composites were fabricated after cellulase was immobilized onto the UiO surface through covalent bonding. The composites conferred favorable cellulase conformations, boosting hydrolysis efficiency and stability, which relied on the soft PVD shell and confinement effect yielded by the curled PVD chains at high temperatures. Compared with free cellulase, the proposed composites exhibited a 33.1-fold enhancement of the Kcat values at 50 °C. The PVD-UiO@cellulase composites were applied to the hydrolysis of cellulose in the stalks and leaves of Epipremnum aureum. The results highlight the potential of smart PVD-UiO@cellulase composites in the hydrolysis of cellulose, affording a valuable platform for the preparation of unique MOFs@enzyme composites and their industrial applications.

Elucidating Thermothielavioides terrestris secretome changes for improved saccharification of mild steam-pretreated spruce

BackgroundThe efficient use of softwood in biorefineries is hampered by its recalcitrance to enzymatic saccharification. In the present study, the fungus Thermothielavioides terrestris LPH172 was cultivated on three steam-pretreated spruce materials (STEX180°C/auto, STEX210°C/auto, and STEX210°C/H2SO4), characterized by different hemicellulose content and structure, as well as on untreated biomass. The aim of the study was to map substrate-induced changes in the secretome of T. terrestris grown on differently treated spruce materials and to evaluate the hydrolytic efficiency of the secretome as supplement for a commercial enzyme mixture.ResultsThe cultivation of T. terrestris was monitored by endo-cellulase, endo-xylanase, endo-mannanase, laccase, and peroxidase activity measurements. Proteomic analysis was performed on the secretomes induced by the spruce materials to map the differences in enzyme production. Growth of T. terrestris on STEX180°C/auto and STEX210°C/auto induced higher expression level of mannanases and mannosidases of the GH5_7 CAZy family compared to cultivation on the other materials. Cultivation on untreated biomass led to overexpression of GH47, GH76, and several hemicellulose debranching enzymes compared to the cultivation on the pretreated materials. T. terrestris grown on untreated, STEX180°C/auto and STEX210°C/auto induced three arabinofuranosidases of the GH43 and GH62 families; while growth on STEX210°C/H2SO4 induced a GH51 arabinofuranosidase and a GH115 glucuronidase. All secretomes contained five lytic polysaccharide monooxygenases of the AA9 family. Supplementation of Celluclast® + Novozym188 with the secretome obtained by growing the fungus grown on STEX180°C/auto achieved a twofold higher release of mannose from spruce steam-pretreated with acetic acid as catalyst, compared to the commercial enzyme cocktail alone.ConclusionsMinor changes in the structure and composition of spruce affect the composition of fungal secretomes, with differences in some classes explaining an increased hydrolytic efficiency. As demonstrated here, saccharification of spruce biomass with commercial enzyme cocktails can be further enhanced by supplementation with tailor-made secretomes.

Enhanced Hydrolysis of Carbonyl Sulfide in Coking Oven Gas Utilizing an Efficient Ca-Ba-γ-Al2O3 Catalyst

China possesses a substantial capacity for coke production, resulting in the annual generation of over 100 billion standard cubic meters of the by-product coke oven gas. The comprehensive utilization of this gas has emerged as a matter of significant concern within the coking industry. The removal of carbonyl sulfide (COS) from coke oven gas is crucial for enhancing gas quality, mitigating equipment corrosion, minimizing environmental pollution, elevating the quality of recovered products, and fostering the production of high-quality steel. A novel Ca-Ba-γ-Al2O3 catalyst has been devised, employing γ-Al2O3 as the catalyst matrix and integrating calcium hydroxide (Ca(OH)2) alongside barium hydroxide octahydrate (Ba(OH)2·8H2O) as the alkaline activating components. The impact of various factors, including reaction temperature, humidity, and the number of activating components loaded, on the hydrolysis efficiency of COS has been meticulously investigated. Furthermore, the catalytic reaction mechanism has been elucidated utilizing advanced characterization techniques such as X-ray diffraction (XRD) and Brunauer–Emmett–Teller (BET) analysis. The outcomes of this research reveal that, under optimal conditions of a reaction temperature of 55 °C and a humidity of 56%, the Ca-Ba-γ-Al2O3 catalyst achieves a remarkable COS hydrolysis efficiency of 95.22%.

Removal of raffinose and stachyose from soy-based beverages using soluble and immobilized MelA α-D-galactosidase from Lactiplantibacillus plantarum WCFS1

The hydrolysis of α-galactose-carbohydrates in soy-based beverages using the enzyme MelA α-D-galactosidase (Lp_3485) from Lactiplantibacillus plantarum WCFS1 is presented. MelA is a highly unstable trimeric enzyme whose activity and stability are strongly dependent on the dilution and polarity of the reaction medium. Considering this, the enzyme was immobilized and stabilized using different immobilization protocols. Post-immobilization techniques consisting of cross-linking with different polyfunctional polymers were used. The optimal derivative was used to hydrolyse different oligosaccharides of the raffinose family (raffinose and stachyose) present in soy drink preparations. Remarkably, MelA showed specific activity and high hydrolytic efficiency towards RFOs in soy-based beverages at a relatively low temperature (20 °C). Therefore, these results suggest that this enzyme could be preferentially used in the early stage of soybean processing to remove RFOs from soy-based beverages and soy-derived products, thereby improving their nutritional value.