Cu2(OH)3NO3 nanozyme sensor array for the discrimination of multiple sulfides in food.

In the food industry, sulfides are commonly used as preservatives and flavor regulators. However, long-term excessive intake of sulfides can lead to serious health problems. Therefore, developing efficient sulfide detection methods is particularly important. Here, we have effectively synthesized a novel bifunctional copper hydroxide nitrate (Cu2(OH)3NO3) nanozyme with outstanding peroxidase-like and laccase-like behaviors in basic deep eutectic solvents (DES). Because the various types of sulfides have diverse regulatory effects on the two catalytic behaviors of Cu2(OH)3NO3, a two channel nanozyme sensor array based on the peroxidase-like and laccase-like behaviors of Cu2(OH)3NO3 was constructed and successfully used for the identification of six kinds of sulfides (Na2S, Na2S2O3, Na2SO3, Na2SO4, NaHSO3, and Na2S2O8). Remarkably, the sensor array has achieved successful discrimination among six sulfides present in wine, egg, and milk samples. Finally, the sensor array has successfully distinguished and differentiated three actual samples (wine, egg, and milk). This study is of great significance in promoting the efficient construction of array units and improving the effective identification of sulfides in complex food samples.

IPAD-DB: a manually curated database for experimentally verified inhibitors of proteins associated with Alzheimer's disease.

Alzheimer's disease (AD) is a universal neurodegenerative disease with the feature of progressive dementia. Currently, there are only seven Food and Drug Administration-approved drugs for the treatment of AD, which merely offer temporary relief from symptom deterioration without reversing the underlying disease process. The identification of inhibitors capable of interacting with proteins associated with AD plays a pivotal role in the development of effective therapeutic interventions. However, a vast number of such inhibitors are dispersed throughout numerous published articles, rendering it inconvenient for researchers to explore potential drug candidates for AD. In light of this, we have manually compiled inhibitors targeting proteins associated with AD and constructed a comprehensive database known as IPAD-DB (Inhibitors of Proteins associated with Alzheimer's Disease Database). The curated inhibitors within this database encompass a diverse range of compounds, including natural compounds, synthetic compounds, drugs, natural extracts and nano-inhibitors. To date, the database has compiled >4800 entries, each representing a correspondent relationship between an inhibitor and its target protein. IPAD-DB offers a user-friendly interface that facilitates browsing, searching and downloading of its records. We firmly believe that IPAD-DB represents a valuable resource for screening potential AD drug candidates and investigating the underlying mechanisms of this debilitating disease. Access to IPAD-DB is freely available at http://www.lamee.cn/ipad-db/ and is compatible with all major web browsers. Database URL: http://www.lamee.cn/ipad-db/.

Rational Design of a Yeast-derived 3',5'-bisphosphate Nucleotidase with Improved Substrate Specificity.

In recent years, a convenient phosphatase-coupled sulfotransferase assay method has been proven to be applicable to most sulfotransferases. The central principle of the method is that phosphatase specifically degrades 3'-phosphoadenosine-5'-phosphate (PAP) and leaves 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Our group previously acquired a yeast 3',5'-bisphosphate nucleotidase (YND), which showed a higher catalytic activity for PAP than PAPS and could be a potential phosphatase for the sulfotransferase assay. Here, we obtained a beneficial mutant of YND with markedly improved substrate specificity towards PAP via rational design. Of 9 chosen mutation sites in the active site pocket, the mutation G236D showed the best specificity for PAP. After optimization of the reaction conditions, the mutant YNDG236D displayed a 4.8-fold increase in the catalytic ratio PAP/PAPS compared to the wild-type. We subsequently applied YNDG236D to the assay of human SULT1A1 and SULT1A3 with their known substrate 1-naphthol, indicating that the mutant could be used to evaluate sulfotransferase activity by colorimetry. Analysis of the MD simulation results revealed that the improved substrate specificity of the mutant towards PAP may stem from a more stable protein conformation and the changed flexibility of key residues in the entrance of the substrate tunnel. This research will provide a valuable reference for the development of efficient sulfotransferase activity assays.

Quantum chemical modeling of enantioselective sulfoxidation and epoxidation reactions by indole monooxygenase VpIndA1.

Indole monooxygenases (IMOs) are enzymes from the family of Group E monooxygenases, requiring flavin adenine dinucleotide (FAD) for their activities. IMOs play important roles in both sulfoxidation and epoxidation reactions. The broad substrate range and high selectivity of IMOs make them promising biocatalytic tools for synthesizing chiral compounds. In the present study, quantum chemical calculations using the cluster approach were performed to investigate the reaction mechanism and the enantioselectivity of the IMO from Variovorax paradoxus EPS (VpIndA1). The sulfoxidation of methyl phenyl sulfide (MPS) and the epoxidation of indene were chosen as the representative reactions. The calculations confirmed that the FADOOH intermediate is the catalytic species in the VpIndA1 reactions. The oxidation of MPS adopts a one-step mechanism involving the direct oxygen-transfer from FADOOH to the substrate and the proton transfer from the -OH group back to FAD, while the oxidation of indene follows a stepwise mechanism involving a carbocation intermediate. It was computationally predicted that VpIndA1 prefers the formation of (S)-product for the MPS sulfoxidation and (1S,2R)-product for the indene epoxidation, consistent with the experimental observations. Importantly, the factors controlling the stereo-preference of the two reactions are identified. The findings in the present study provide valuable insights into the VpIndA1-catalyzed reactions, which are essential for the rational design of this enzyme and other IMOs for industrial applications. It is also worth emphasizing that the quantum chemical cluster approach is again demonstrated to be powerful in studying the enantioselectivity of enzymatic reactions.

A Critical Review on Immobilized Sucrose Isomerase and Cells for Producing Isomaltulose.

Isomaltulose is a novel sweetener and is considered healthier than the common sugars, such as sucrose or glucose. It has been internationally recognized as a safe food product and holds vast potential in pharmaceutical and food industries. Sucrose isomerase is commonly used to produce isomaltulose from the substrate sucrose in vitro and in vivo. However, free cells/enzymes were often mixed with the product, making recycling difficult and leading to a significant increase in production costs. Immobilized cells/enzymes have the following advantages including easy separation from products, high stability, and reusability, which can significantly reduce production costs. They are more suitable than free ones for industrial production. Recently, immobilized cells/enzymes have been encapsulated using composite materials to enhance their mechanical strength and reusability and reduce leakage. This review summarizes the advancements made in immobilized cells/enzymes for isomaltulose production in terms of refining traditional approaches and innovating in materials and methods. Moreover, innovations in immobilized enzyme methods include cross-linked enzyme aggregates, nanoflowers, inclusion bodies, and directed affinity immobilization. Material innovations involve nanomaterials, graphene oxide, and so on. These innovations circumvent challenges like the utilization of toxic cross-linking agents and enzyme leakage encountered in traditional methods, thus contributing to enhanced enzyme stability.

Nanozymes sensor array for discrimination and intelligent sensing of phenolic acids in food.

Although nanozymes sensor arrays have the potential to recognize multiple target substances simultaneously, they currently rarely identify phenolic acids in food due to limited catalytic performance and complex preparation conditions of nanozymes. Here, inspired by the structure of polyphenol oxidase, we have successfully prepared a novel gallic acid-Cu (GA-Cu) nanozyme with laccase-like activity. Due to the different catalytic efficiency of GA-Cu nanozymes towards six common phenolic acids, a three-channel colorimetric sensor array was constructed using reaction kinetics as the sensing unit to achieve high-throughput detection and identification of six phenolic acids within a concentration range from 1 to 100 µM. This method avoids the creation of numerous sensing units. Notably, the successful discrimination of six phenolic acids in samples of juice, beer, and wine has been achieved by the sensor array. Finally, aided by smartphones, a portable technique has been devised for the detection of phenolic acids.

Brazilin-7-2-butenoate inhibits amyloid ß-protein aggregation, alleviates cytotoxicity, and protects Caenorhabditis elegans.

The fibrillogenesis of amyloid ß-protein (Aß) gradually accumulates to form neurotoxic Aß aggregates in the human brain, which is the direct cause of Alzheimer's disease (AD) related symptoms. There are currently no effective therapies for AD. Brazilin, a natural polyphenol, inhibits Aß fibrillogenesis, disrupts the mature fibrils and alleviates the corresponding cytotoxicity, but it also has the high toxic. Therefore, brazilin-7-2-butenoate (B-7-2-B), a brazilin derivative, was designed and synthesized. B-7-2-B exhibited lower toxicity and stronger inhibitory effect on Aß aggregation than brazilin. B-7-2-B could prevent the formation of Aß fibrils and oligomers, and depolymerize pre-formed aggregates in a dose-dependent manner. Furthermore, B-7-2-B prominently alleviated the cytotoxicity and the oxidative stress induced by Aß aggregates in PC12 cells. The protective impacts of B-7-2-B were further demonstrated by using the Caenorhabditis elegans model, including decreasing the extent of Aß aggregation, improving the motility and sensation disorders. Eventually, B-7-2-B was proven to be no apparent damage to worms. In summarize, it can be concluded that B-7-2-B has the potential as a drug for treating AD.

Differential diagnosis of rare adrenal cellular schwannomas: A case report.

BACKGROUND: Adrenal cellular schwannomas are exceptionally rare stromal tumors that are often misdiagnosed due to the lack of specific radiological, serological, or clinical features. In this report, we describe the differential diagnosis of a rare adrenal cellular schwannoma. METHODS: A 69-year-old man with a history of persistent hypertension, chronic kidney disease, hypertensive heart disease, and cardiac insufficiency was hospitalized due to bilateral lower extremity edema lasting for 3 months. Plain computed tomography at that time revealed a space-occupying lesion in the right adrenal gland. As serum levels of catecholamines, cortisol, and adrenocorticotropic hormone were within normal ranges, the edema was attributed to the chronic kidney disease and cardiac insufficiency, and the patient was referred to our hospital for surgical treatment. Contrast-enhanced computed tomography revealed heterogeneous enhancement in the adrenal mass indicating pheochromocytoma. An irregularly shaped 5 cm mass with a complete capsule in the right adrenal gland was laparoscopically resected. The postoperative histopathological diagnosis was adrenal cellular schwannoma. RESULTS: The postoperative course was unremarkable and the tumor did not recur during 5 years of follow-up. CONCLUSION: Adrenal cellular schwannoma is a very rare tumor that is extremely difficult to preoperatively diagnose. Histological and immunohistochemical analyses are required for differential diagnosis and confirmation. Cellular schwannomas can transform into malignant peripheral nerve sheath tumors, but not often. Consequently, regular postoperative follow-up is required for such patients, especially imaging.

Natural flavonoid hesperetin blocks amyloid ß-protein fibrillogenesis, depolymerizes preformed fibrils and alleviates cytotoxicity caused by amyloids.

The aggregation of ß-amyloid (Aß) peptides to form amyloid plaques is one of the primary hallmarks for Alzheimer's disease (AD). Dietary flavonoid supplements containing hesperetin have an ability to decline the risk of developing AD, but the molecular mechanism is still unclear. In this work, hesperetin, a flavanone abundant in citrus fruits, has been proven to prevent the formation of Aß aggregates and depolymerized preformed fibrils in a concentration-dependent fashion. Hesperetin inhibited the conformational conversion from the natural structure to a ß-sheet-rich conformation. It was found that hesperetin significantly reduced the cytotoxicity and relieved oxidative stress eventuated by Aß aggregates in a concentration-dependent manner. Additionally, the beneficial effects of hesperetin were confirmed in Caenorhabditis elegans, including the inhibition of the formation and deposition of Aß aggregates and extension of their lifespan. Finally, the results of molecular dynamics simulations showed that hesperetin directly interacted with an Aß42 pentamer mainly through strong non-polar and electrostatic interactions, which destroyed the structural stability of the preformed pentamer. To summarize, hesperetin exhibits great potential as a prospective dietary supplement for preventing and improving AD.

Ultrasound-Assisted Multi-Enzyme Extraction for Highly Efficient Extraction of Polysaccharides from Ulva lactuca.

Ulva polysaccharides present several physiological activities including antiviral, antitumor and anti-plasmodial effects. However, current processing usually results in low yields and high prices, thus lacking commercialization potential. The aim of this study was to develop an efficient method for the extraction of Ulva polysaccharides with high biological activity. The effect of cell wall-degrading enzymes including cellulase, hemicellulase, pectinase and protease on Ulva polysaccharide extraction was studied by statistical mixing design. Using the most effective enzyme preparations as the basic components, the optimal proportions of the enzyme mixture were determined as follows: cellulase 35.3%, pectinase 34.5%, alkaline protease 30.2%, which increased the polysaccharide yield from 6.43% in the absence of enzymes to 26.68%. Subsequently, through response surface analysis, the optimal conditions were determined: enzyme concentration of 1.5%, enzymatic time of 1.1 h, ultrasonic time of 90 min and enzymatic temperature of 60 °C. Under the optimal extraction conditions, the extraction yield of Ulva polysaccharides could be increased to 30.14%. Moreover, extracted polysaccharides exhibit strong antioxidant properties in DPPH, ABTS, hydroxyl radical, superoxide radical and H2O2-induced cellular damage models. This study laid a solid foundation for the use and development of Ulva polysaccharides.

Heterologous expression and fibrillary characterization of the microtubule-binding domain of tau associated with tauopathies.

BACKGROUND: Neurofibrillary tangles (NFTs) are one of the most common pathological characteristics of Alzheimer's disease. The NFTs are mainly composed of hyperphosphorylated microtubule-associated tau. Thus, recombinant tau is urgently required for the study of its fibrillogenesis and its associated cytotoxicity. METHODS AND RESULTS: Heterologous expression, purification, and fibrillation of the microtubule-binding domain (MBD) of tau (tauMBD) were performed. The tauMBD was heterologously expressed in E. coli. Ni-chelating affinity chromatography was then performed to purify the target protein. Thereafter, tauMBD was systematically identified using the SDS-PAGE, western blot and MALDI-TOF MS methods. The aggregation propensity of the tauMBD was explored by both the thioflavin T fluorescence and atomic force microscopy experiments. CONCLUSIONS: The final yield of the recombinant tauMBD was ~ 20 mg L-1. It is shown that TauMBD, in the absence of an inducer, self-assembled into the typical fibrils at a faster rate than wild-type tau. Finally, the in vitro cytotoxicity of tauMBD aggregates was validated using PC12 cells. The heterologously expressed tau in this study can be further used in the investigation of the biophysical and cellular cytotoxic properties of tau.

Smartphone-assisted nanozyme sensor array constructed based on reaction kinetics for the discrimination and identification of phenolic compounds.

Although the research on nanozymes has attracted widespread attention in recent years, the development of highly active and multifunctional nanozymes remains a challenge. Here, a bifunctional AMP-Cu nanozyme with laccase- and catecholase-like activities was successfully prepared at room temperature with Cu2+ as the metal ion and adenosine-5'-monophosphate (AMP) as the ligand molecule. Based on the excellent catalytic performance of AMP-Cu, a three-channel colorimetric sensor array was constructed using reaction kinetics as the sensing unit to achieve high-throughput detection and identification of six common phenolic compounds at low concentrations. This strategy simplifies the construction of sensor array and demonstrates the capacity to obtain multidimensional data from a single material. Finally, with the assistance of smartphones and homemade dark boxes, a portable on-site detection method for phenolic compounds was developed. This work would contribute to the development of portable sensors and the highly efficient identification of phenolic compounds in complex samples.

Improving the thermal stability and catalytic activity of ulvan lyase by the combination of FoldX and KnowVolution campaign.

Thermal stability is one of the most important properties of ulvan lyases for their application in algae biomass degradation. The Knowledge gaining directed eVolution (KnowVolution) protein engineering strategy could be employed to improve thermostability of ulvan lyase with less screening effort. Herein, the unfolding free energies (ΔΔG) of the loop region were calculated using FoldX and four sites (D103, G104, T113, Q229) were selected for saturation mutagenesis, resulting in the identification of a favorable single-site mutant Q229M. Subsequently, iteration mutation was carried out with the mutant N57P (previously obtained by our group) to further enhance the performance of ulvan lyase. The results showed that the most beneficial variant N57P/Q229M exhibited a 1.67-fold and 2-fold increase in residual activity compared to the wild type after incubation at 40 °C and 50 °C for 1 h, respectively. In addition, the variant produced 1.06 mg/mL of reducing sugar in 2 h, which was almost four times as much as the wild type. Molecular dynamics simulations revealed that N57P/Q229M mutant enhanced the structural rigidity by augmenting intramolecular hydrogen bonds. Meanwhile, the shorter proton transmission distance between the general base of the enzyme and the substrate contributed to the glycosidic bond breakage. Our research showed that in silico saturation mutagenesis using position scan module in FoldX allowed for faster screening of mutants with improved thermal stability, and combining it with KnowVolution enabled a balanced effect of thermal stability and enzyme activity in protein engineering.

Brazilin-7-acetate, a novel potential drug of Parkinson's disease, hinders the formation of α-synuclein fibril, mitigates cytotoxicity, and decreases oxidative stress.

Parkinson's disease (PD) is a prevalent neurodegenerative disorder characterized by the accumulation of α-synuclein (α-Syn) aggregates. However, there are currently no effective therapies for PD. Brazilin, an inhibitor of α-Syn aggregation, is unstable and toxic. Therefore, we have developed and synthesized derivatives of brazilin. One of these derivatives, called brazilin-7-acetate (B-7-A), has shown reduced toxicity and a stronger effect on inhibiting α-Syn aggregation. It showed that B-7-A prevented the formation of α-Syn fibers and disrupted existing fibers in a dosage-dependent manner. Additionally, B-7-A significantly reduced the cytotoxicity of α-Syn aggregates and alleviated oxidative stress in PC12 cells. The beneficial effects of B-7-A were also confirmed using the Caenorhabditis elegans model. These effects included preventing the accumulation of α-Syn clumps, improving behavior disorder, increasing lifespan, reducing oxidative stress, and protecting against lipid oxidation and loss. Finally, B-7-A showed good ADMET properties in silico. Based on these findings, B-7-A exhibits potential as a prospective treatment for PD.

Exploring the molecular mechanism of cold-adaption of an alkaline protease mutant by molecular dynamics simulations and residue interaction network.

Psychrophilic proteases have attracted enormous attention in past decades, due to their high catalytic activity at low temperatures in a wide range of industrial processes, especially in the detergent and leather industries. Among them, H5 is an alkaline protease mutant, which featuring psychrophilic-like behavior, but the reasons that H5 with higher activity at low temperatures are still poorly understood. Herein, the molecular dynamics (MD) simulations combined with residue interaction network (RIN) were utilized to investigate the mechanisms of the cold-adaption of mutant H5. The results demonstrated that two loops involved in the substrate binding G100-S104 and S125-S129 in H5 had higher mobility, and the distance enlargement between the two loops modulated the substrate's accessibility compared with wild type counterpart. Besides, H5 enhanced conformational flexibility by weakening salt bridges and increasing interaction with the solvent. In particular, the absence of Lys251-Asp197-Arg247 salt bridge network may contribute to the structural mobility. Based on the free energy landscape and molecular mechanics Poisson-Boltzmann surface area of the wild type and H5, it was elucidated that H5 possessed a large population of interconvertible conformations, resulting in the weaker substrate binding free energy. The calculated RIN topology parameters such as the average degree, average cluster coefficient, and average path length further verified that the mutant H5 attenuated residue-to-residue interactions. The investigation of the mechanisms by which how the residue mutation affects the stability and activity of enzymes provides a theoretical basis for the development of cold-adapted protease.

Heterologous expression and characterization of an M4 family extracellular metalloprotease for detergent application.

Proteolytic enzymes stand out as the most widely employed category utilized in manufacturing industry. A new protease was separated from Planococcus sp.11815 strain and named as nprS-15615 in this research. The gene of this protease has not been reported, and its enzymatic properties have been studied for the first time. To enhance enzyme production, the Planococcus sp. protease gene was expressed in Bacillus licheniformis 2709. The expression level of nprS-15615 was observed under the control of regulatory elements PaprE. nprS-15615 protease activity reached 1186.24±32.87 U/mL after 48 hours of cultivation in shake flasks which was nearly four times the output of the original bacteria (291.38±25.73U/mL). The optimum temperature and pH of the recombinant protease were 30 ℃ and 8.0, respectively.The enzyme exhibited the highest capacity for hydrolyzing casein and demonstrated resilience towards a NaCl concentration of 10.0% (wt/v). Furthermore, in the presence of 0.5% surfactants, the recombinant protease activity can maintain above 75%, and with the existence of 0.5% liquid detergents, there was basically no loss of enzyme activity which indicated that nprS-15615 had good compatibility with surfactants and liquid detergents. In addition, npS-15615 performed well in the washing experiment, and the washing effect at 20 ℃ can be significantly improved by adding crude enzyme solution in the washing process.

Citrus Flavonoid Hesperetin Inhibits α-Synuclein Fibrillogenesis, Disrupts Mature Fibrils, and Reduces Their Cytotoxicity: In Vitro and In Vivo Studies.

Misfolding and subsequent fibrillogenesis of α-synuclein (αSN) significantly influence the development of Parkinson's disease (PD). This study reports the inhibitory effect of citrus flavonoid hesperetin (Hst) on αSN fibrillation. Based on thioflavin T fluorometry and atomic force microscopy studies, Hst inhibited αSN fibrillation by interfering with initial nucleation and slowing the elongation rate. Furthermore, the inhibitory effect was concentration-dependent with a half-maximal inhibitory concentration of 24.4 µM. Cytotoxicity experiments showed that 100 µM Hst significantly reduced the cytotoxicity of αSN aggregates and maintained 98.4% cell activity. In addition, Hst disassembled the preprepared αSN fibrils into smaller and less-toxic aggregates. Excitingly, supplementation with 100 µM Hst inhibited the accumulation of 36.3% αSN in NL5901 and restored the amyloid-induced reduction in NL5901 lipid abundance, extending the mean lifespan of NL5901 to 23 d. These findings could support the use of Hst as a dietary supplement to regulate αSN fibrillation and prevent the development of PD.

Lyophilization process optimization and molecular dynamics simulation of mRNA-LNPs for SARS-CoV-2 vaccine.

Some studies have shown that lyophilization significantly improves the stability of mRNA-LNPs and enables long-term storage at 2-8 °C. However, there is little research on the lyophilization process of mRNA-lipid nanoparticles (LNPs). Most previous studies have used empirical lyophilization with only a single lyoprotectant, resulting in low lyophilization efficiency, often requiring 40-100 h. In the present study, an efficient lyophilization method suitable for mRNA-LNPs was designed and optimized, shortening the total length of the lyophilization process to 8-18 h, which significantly reduced energy consumption and production costs. When the mixed lyoprotectant composed of sucrose, trehalose, and mannitol was added to mRNA-LNPs, the eutectic point and collapse temperature of the system were increased. The lyophilized product had a ginger root-shaped rigid structure with large porosity, which tolerated rapid temperature increases and efficiently removed water. In addition, the lyophilized mRNA-LNPs rapidly rehydrated and had good particle size distribution, encapsulation rate, and mRNA integrity. The lyophilized mRNA-LNPs were stable at 2-8 °C, and they did not reduce immunogenicity in vivo or in vitro. Molecular dynamics simulation was used to compare the phospholipid molecular layer with the lyoprotectant in aqueous and anhydrous environments to elucidate the mechanism of lyophilization to improve the stability of mRNA-LNPs. This efficient lyophilization platform significantly improves the accessibility of mRNA-LNPs.

Synthesis and its biological activity of carboxymethyl hemicellulose p-hydroxybenzoate (P-CMHC).

Hemicellulose extracted from ecalyptus APMP pulping waste liquor and undergoes etherification modification to produce carboxymethyl hemicellulose (CMHC). Subsequently, CMHC undergoes esterification reaction with p-hydroxybenzoic acid to synthesize a novel polysaccharide-based preservative known as carboxymethyl hemicellulose p-hydroxybenzoate (P-CMHC). The synthesis conditions of P-CMHC were optimized using the response surface methodology, resulting in an optimal esterification condition that achieved a degree of substitution of 0.232. P-CMHC exhibits excellent antioxidant activity, including 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl radical scavenging activities. Additionally, it demonstrates favorable hygroscopic and moisturizing properties. Thiazole blue (MTT) experiments evaluating cell proliferation rate indicate that P-CMHC possesses negligible cytotoxicity, making it a promising, safe, and healthy preservative. Consequently, it can be considered as a new material for applications in the fields of biomedicine, food, and cosmetics.

Enhancement of rice husks saccharification through hydrolase preparation assisted by lytic polysaccharide monooxygenase.

Rice husk is an abundant agricultural waste generated from rice production, but its application is limited. Considering its complex components, the rice husk was hydrolyzed by different enzymes to enhance its saccharification. In this study, saccharification of the rice husk by cellulase, xylosidase, and xylanase was first investigated. The synergistic effect of LPMO on the above hydrolases and different enzyme combinations in the saccharification process was then explored. Thereafter, the formulation of the enzyme cocktail and the degradation conditions were optimized to obtain the highest saccharification efficiency. The results showed that the optimum enzyme cocktail consists of Celluclast 1.5 L (83.3 mg/g substrate), the key enzymes in the saccharification process, worked with BpXyl (20 mg/g substrate), BpXyn11 (24 mg/g substrate), and R17L/N25G (4 mg/g substrate). The highest reducing sugar concentration (1.19 mg/mL) was obtained at pH 6.0 and 60 â„ƒ for 24 h. Fourier transform infrared spectroscopy and scanning electron microscopy were employed to characterize the structural changes in the rice husk after degradation. The results showed that the key chemical bonds in cellulose and hemicellulose were broken. This study illuminated the concept of saccharifying lignocellulose from rice husk using LPMO synergistically assisted combined-hydrolase including cellulase, xylosidase, and xylanase, and provided a theoretical basis for lignocellulose biodegradation.