Sulfated Laminarin Polysaccharides Reduce the Adhesion of Nano-COM Crystals to Renal Epithelial Cells by Inhibiting Oxidative and Endoplasmic Reticulum Stress.

Purpose: Adhesion between calcium oxalate crystals and renal tubular epithelial cells is a vital cause of renal stone formation; however, the drugs that inhibit crystal adhesion and the mechanism of inhibition have yet to be explored. Methods: The cell injury model was constructed using nano-COM crystals, and changes in oxidative stress levels, endoplasmic reticulum (ER) stress levels, downstream p38 MAPK protein expression, apoptosis, adhesion protein osteopontin expression, and cell-crystal adhesion were examined in the presence of Laminarin polysaccharide (DLP) and sulfated DLP (SDLP) under protected and unprotected conditions. Results: Both DLP and SDLP inhibited nano-COM damage to human kidney proximal tubular epithelial cell (HK-2), increased cell viability, decreased ROS levels, reduced the opening of mitochondrial membrane permeability transition pore, markedly reduced ER Ca2+ ion concentration and adhesion molecule OPN expression, down-regulated the expression of ER stress signature proteins including CHOP, Caspase 12, and p38 MAPK, and decreased the apoptosis rate of cells. SDLP has a better protective effect on cells than DLP. Conclusions: SDLP protects HK-2 cells from nano-COM crystal-induced apoptosis by reducing oxidative and ER stress levels and their downstream factors, thereby reducing crystal-cell adhesion interactions and the risks of kidney stone formation.

Corn Silk Polysaccharide Reduces the Risk of Kidney Stone Formation by Reducing Oxidative Stress and Inhibiting COM Crystal Adhesion and Aggregation.

The aim of this study is to explore the inhibition of nanocalcium oxalate monohydrate (nano-COM) crystal adhesion and aggregation on the HK-2 cell surface after the protection of corn silk polysaccharides (CSPs) and the effect of carboxyl group (-COOH) content and polysaccharide concentration. METHOD: HK-2 cells were damaged by 100 nm COM crystals to build an injury model. The cells were protected by CSPs with -COOH contents of 3.92% (CSP0) and 16.38% (CCSP3), respectively. The changes in the biochemical indexes of HK-2 cells and the difference in adhesion amount and aggregation degree of nano-COM on the cell surface before and after CSP protection were detected. RESULTS: CSP0 and CCSP3 protection can obviously inhibit HK-2 cell damage caused by nano-COM crystals, restore cytoskeleton morphology, reduce intracellular ROS level, inhibit phosphoserine eversion, restore the polarity of the mitochondrial membrane potential, normalize the cell cycle process, and reduce the expression of adhesion molecules, OPN, Annexin A1, HSP90, HAS3, and CD44 on the cell surface. Finally, the adhesion and aggregation of nano-COM crystals on the cell surface were effectively inhibited. The carboxymethylated CSP3 exhibited a higher protective effect on cells than the original CSP0, and cell viability was further improved with the increase in polysaccharide concentration. CONCLUSIONS: CSPs can protect HK-2 cells from calcium oxalate crystal damage and effectively reduce the adhesion and aggregation of nano-COM crystals on the cell surface, which is conducive to inhibiting the formation of calcium oxalate kidney stones.

Carboxymethylated Desmodium styracifolium polysaccharide reduces the risk of calcium oxalate kidney stone formation by inhibiting crystal adhesion and promoting crystal endocytosis.

The inhibition of cell surface crystal adhesion and an appropriate increase in crystal endocytosis contribute to the inhibition of kidney stone formation. In this study, we investigated the effects of different degrees of carboxymethylation on these processes. An injury model was established by treating human renal proximal tubular epithelial (HK-2) cells with 98.3 ± 8.1 nm calcium oxalate dihydrate (nanoCOD) crystals. The HK-2 cells were protected with carboxy (-COOH) Desmodium styracifolium polysaccharides at 1.17% (DSP0), 7.45% (CDSP1), 12.2% (CDSP2), and 17.7% (CDSP3). Changes in biochemical indexes and effects on nanoCOD adhesion and endocytosis were detected. The protection of HK-2 cells from nanoCOD-induced oxidative damage by carboxymethylated Desmodium styracifolium polysaccharides (CDSPs) is closely related to the protection of subcellular organelles, such as mitochondria. CDSPs can reduce crystal adhesion on the cell surface and maintain appropriate crystal endocytosis, thereby reducing the risk of kidney stone formation. CDSP2 with moderate -COOH content showed the strongest protective activity among the CDSPs.

Carboxymethylated Rhizoma alismatis polysaccharides reduces the risk of calcium oxalate stone formation by reducing cellular inflammation and oxidative stress.

This study aims to elucidate the mechanism and potential of Rhizoma alismatis polysaccharides (RAPs) in preventing oxidative damage to human renal proximal tubule epithelial cells. The experimental approach involved incubating HK-2 cells with 100 nm calcium oxalate monohydrate for 24 h to establish a cellular injury model. Protection was provided by RAPs with varying carboxyl group contents: 3.57%, 7.79%, 10.84%, and 15.33%. The safeguarding effect of RAPs was evaluated by analyzing relevant cellular biochemical indicators. Findings demonstrate that RAPs exhibit notable antioxidative properties. They effectively diminish the release of reactive oxygen species, lactate dehydrogenase, and malondialdehyde, a lipid oxidation byproduct. Moreover, RAPs enhance superoxide dismutase activity and mitochondrial membrane potential while attenuating the permeability of the mitochondrial permeability transition pore. Additionally, RAPs significantly reduce levels of inflammatory factors, including NLRP3, TNF-α, IL-6, and NO. This reduction corresponds to the inhibition of overproduced pro-inflammatory mediator nitric oxide and the caspase 3 enzyme, leading to a reduction in cellular apoptosis. RAPs also display the ability to suppress the expression of the HK-2 cell surface adhesion molecule CD44. The observed results collectively underscore the substantial anti-inflammatory and anti-apoptotic potential of all four RAPs. Moreover, their capacity to modulate the expression of cell surface adhesion molecules highlights their potential in inhibiting the formation of kidney stones. Notably, RAP3, boasting the highest carboxyl group content, emerges as the most potent agent in this regard.

De Novo Synthesis of Dihydro-ß-ionone through Metabolic Engineering and Bacterium-Yeast Coculture.

Dihydro-ß-ionone is a common type of ionone used in the flavor and fragrance industries because of its characteristic scent. The production of flavors in microbial cell factories offers a sustainable and environmentally friendly approach to accessing them, independent of extraction from natural sources. However, the native pathway of dihydro-ß-ionone remains unclear, hindering heterologous biosynthesis in microbial hosts. Herein, we devised a microbial platform for de novo syntheses of dihydro-ß-ionone from a simple carbon source with glycerol. The complete dihydro-ß-ionone pathway was reconstructed in Escherichia coli with multiple metabolic engineering strategies to generate a strain capable of producing 8 mg/L of dihydro-ß-ionone, although this was accompanied by a surplus precursor ß-ionone in culture. To overcome this issue, Saccharomyces cerevisiae was identified as having a conversion rate for transforming ß-ionone to dihydro-ß-ionone that was higher than that of E. coli via whole-cell catalysis. Consequently, the titer of dihydro-ß-ionone was increased using the E. coli-S. cerevisiae coculture to 27 mg/L. Our study offers an efficient platform for biobased dihydro-ß-ionone production and extends coculture engineering to overproducing target molecules in extended metabolic pathways.

Multi-omic analysis revealed the therapeutic mechanisms of Alpinia oxyphylla fructus water extract against bladder overactivity in spontaneously hypertensive rats.

OBJECTIVE: Alpinia oxyphylla fructus without impurities and shells is called "Yi-Zhi-Ren" (YZR) in Chinese, and traditionally used to alleviate enuresis. The aim of this study was to investigate the effects and underlying mechanisms of YZR in the treatment of overactive bladder (OAB) in spontaneously hypertensive rats (SHR), a vascular disorder-related OAB model. METHODS: A 3-week administration of YZR water extract (p.o.) was done, followed by urodynamics to measure bladder parameters. Changes in bladder structure were observed through H&E staining and Masson's staining. An integrated approach involving network pharmacology, transcriptomics and metabolomics was employed to elucidate the potential mechanisms of YZR, and the key proteins involved in the mechanisms were validated by Western blotting. Additionally, network pharmacology was used to predict the relationship between YZR's active components and validated proteins. RESULTS: YZR treatment significantly improved the bladder storage parameters, tightened the detrusor layer, reduced inflammatory infiltration, and decreased collagen proportion in the SHR bladder. These results indicated that YZR water extract can alleviate OAB symptoms and improve bladder structure. Integrated analysis suggested that YZR may affect extracellular matrix-receptor interaction and calcium signaling pathway. Western blotting results further confirmed that the reduction in key proteins, such as TGFß1, p-SMAD3, collagen III, Gq and PLCß1, involved in collagen synthesis and calcium signaling pathways after YZR treatment. Network pharmacology predicted that sitosterol, chrysin, and nootkatone were potential components responsible for YZR's therapeutic effect on OAB. CONCLUSION: YZR's mechanisms of action in treating OAB involved the TGFß1-SMAD3 signaling pathway-related collagen synthesis and Gq-PLCß1 calcium signaling pathway, which are associated with detrusor contraction frequency and strength, respectively.

Multi-omic approaches provide insights into the molecular mechanisms of Sojae semen germinatum water extract against overactive bladder.

Sojae semen germinatum (SSG) is derived from mature soybean seeds that have been germinated and dried, typically with sprouts measuring approximately 0.5 cm in length. SSG is traditionally known for its properties in clearing heat and moisture. Nevertheless, limited information was reported on the effects and mechanisms of SSG in alleviating urinary symptoms. This study employed urodynamic parameters to investigate the therapeutic effect of SSG water extract on overactive bladder (OAB) in the rat model with benign prostatic hyperplasia. Through a combination of transcriptomic and metabolomic analyses, the pathways and key proteins of the SSG treatment for OAB were identified and validated by ELISA and Western blotting. Furthermore, network pharmacology elucidated the roles of SSG's isoflavones acting on the target which was identified by above-mentioned multi-omics analysis. Our results indicate that SSG water extract significantly mitigated OAB by down-regulating the PGE2/EP1/PLCß2/p-MLC signaling pathway. It was speculated that the active ingredient in the SSG on EP1 was genistein. This study provided valuable insights into the molecular mechanisms of SSG water extract, emphasizing the multi-target characteristics and critical pathways in improving OAB. Furthermore, this study contributes to the potential utilization of SSG as a functional food.

Carotenoid Cleavage Dioxygenase 1 and Its Application for the Production of C13-Apocarotenoids in Microbial Cell Factories: A Review.

C13-apocarotenoids are naturally derived from the C9-C10 (C9'-C10') double-bond cleavage of carotenoids by carotenoid cleavage dioxygenases (CCDs). As high-value flavors and fragrances in the food and cosmetic industries, the sustainable production of C13-apocarotenoids is emerging in microbial cell factories by the carotenoid cleavage dioxygenase 1 (CCD1) subfamily. However, the commercialization of microbial-based C13-apocarotenoids is still limited by the poor performance of CCD1, which severely constrains its conversion efficiency from precursor carotenoids. This review focuses on the classification of CCDs and their cleavage modes for carotenoids to generate corresponding apocarotenoids. We then emphatically discuss the advances for C13-apocarotenoid biosynthesis in microbial cell factories with various strategies, including optimization of CCD1 expression, improvement of CCD1's catalytic activity and substrate specificity, strengthening of substrate channeling, and development of oleaginous microbial hosts, which have been verified to increase the conversion rate from carotenoids. Lastly, the current challenges and future directions will be discussed to enhance CCDs' application for C13-apocarotenoids biomanufacturing.

Size-Dependent Cytotoxicity, Adhesion, and Endocytosis of Micro-/Nano-hydroxyapatite Crystals in HK-2 Cells.

Nano-hydroxyapatite (nano-HAP) is often used as a crystal nest to induce calcium oxalate (CaOx) kidney stone formation, but the mechanism of interaction between HAP crystals of different properties and renal tubular epithelial cells remains unclear. In this study, the adhesion and endocytosis of HAP crystals with sizes of 40 nm, 70 nm, 1 µm, and 2 µm (HAP-40 nm, HAP-70 nm, HAP-1 µm, and HAP-2 µm, respectively) to human renal proximal tubular epithelial cells (HK-2) were comparatively studied. The results showed that HAP crystals of all sizes promoted the expression of osteopontin and hyaluronic acid on the cell surface, destroyed the integrity of the lysosomes, and induced the apoptosis and necrosis of cells. Nano-HAP crystals had a higher specific surface area, a smaller contact angle, a higher surface energy, and a lower Zeta potential than those of micro-HAP. Therefore, the abilities of HK-2 cells to adhere to and endocytose nano-HAP crystals were greater than their abilities to do the same for micro-HAP crystals. The order of the endocytosed crystals was as follows: HAP-40 nm > HAP-70 nm > HAP-1 µm > HAP-2 µm. The endocytosed HAP crystals entered the lysosomes. The more crystal endocytosis and adhesion there is, the more toxic it is to HK-2 cells. The results of this study showed that nanosized HAP crystals greatly promoted the formation of kidney stones than micrometer-sized HAP crystals.

On the Optimal Error Exponent of Type-Based Distributed Hypothesis Testing.

Distributed hypothesis testing (DHT) has emerged as a significant research area, but the information-theoretic optimality of coding strategies is often typically hard to address. This paper studies the DHT problems under the type-based setting, which is requested from the popular federated learning methods. Specifically, two communication models are considered: (i) DHT problem over noiseless channels, where each node observes i.i.d. samples and sends a one-dimensional statistic of observed samples to the decision center for decision making; and (ii) DHT problem over AWGN channels, where the distributed nodes are restricted to transmit functions of the empirical distributions of the observed data sequences due to practical computational constraints. For both of these problems, we present the optimal error exponent by providing both the achievability and converse results. In addition, we offer corresponding coding strategies and decision rules. Our results not only offer coding guidance for distributed systems, but also have the potential to be applied to more complex problems, enhancing the understanding and application of DHT in various domains.

Enabling country-scale land cover mapping with meter-resolution satellite imagery.

High-resolution satellite images can provide abundant, detailed spatial information for land cover classification, which is particularly important for studying the complicated built environment. However, due to the complex land cover patterns, the costly training sample collections, and the severe distribution shifts of satellite imageries caused by, e.g., geographical differences or acquisition conditions, few studies have applied high-resolution images to land cover mapping in detailed categories at large scale. To fill this gap, we present a large-scale land cover dataset, Five-Billion-Pixels. It contains more than 5 billion labeled pixels of 150 high-resolution Gaofen-2 (4 m) satellite images, annotated in a 24-category system covering artificial-constructed, agricultural, and natural classes. In addition, we propose a deep-learning-based unsupervised domain adaptation approach that can transfer classification models trained on labeled dataset (referred to as the source domain) to unlabeled data (referred to as the target domain) for large-scale land cover mapping. Specifically, we introduce an end-to-end Siamese network employing dynamic pseudo-label assignment and class balancing strategy to perform adaptive domain joint learning. To validate the generalizability of our dataset and the proposed approach across different sensors and different geographical regions, we carry out land cover mapping on five megacities in China and six cities in other five Asian countries severally using: PlanetScope (3 m), Gaofen-1 (8 m), and Sentinel-2 (10 m) satellite images. Over a total study area of 60,000 km2, the experiments show promising results even though the input images are entirely unlabeled. The proposed approach, trained with the Five-Billion-Pixels dataset, enables high-quality and detailed land cover mapping across the whole country of China and some other Asian countries at meter-resolution.

A fluorescent probe based on a phenylalanine derivative is capable of sequential detection of Zn2+ and Cys/His.

A facile and dual fluorescent chemosensor (named 7-IDF) based on a phenylalanine derivative with an indole group was designed and synthesized. 7-IDF can selectively and sensitively detect Zn2+ via obvious fluorescence enhancement in an aqueous solution. Remarkably, the 7-IDF-Zn complex with blue luminescence has higher selectivity toward cysteine (Cys) and histidine (His) than for other amino acids. Intriguingly, 7-IDF can also be used as an excellent probe to detect Zn2+ in real water samples. Moreover, 7-IDF and 7-IDF-Zn possess excellent biocompatibility and cell permeability, and 7-IDF can consecutively detect Zn2+ and Cys/His in Hela cells through fluorescence imaging experiments. This study suggests that the phenylalanine-based chemosensor possesses great potential applications for the sequential detection of Zn2+ and Cys/His in biosystems.

Myoglobin mutant with enhanced nitrite reductase activity regulates intracellular oxidative stress in human breast cancer cells.

Heme proteins play vital roles in regulating the reactive oxygen/nitrogen species (ROS/RNS) levels in cells. In this study, we overexpressed human wild-type (WT) myoglobin (Mb) and its double mutant, F43H/H64A Mb with enhanced nitrite reductase (NIR) activity, in the typical representative triple-negative breast cancer cell, MDA-MB-231 cells. The results showed that the overexpression of F43H/H64A Mb increased the level of nitric oxide (NO) and the degree of oxidative stress, and then activated Akt/MAPK mediated apoptotic cascade, whereas WT Mb showed the opposite effect. This study indicates that Mb plays an important role in maintaining the balance of the cellular redox system and could thus be a valuable target for cancer therapy.

Regulating Effect of Cytochrome b5 Overexpression on Human Breast Cancer Cells.

Imbalance in the cellular redox system is thought to be associated with the induction and progression of breast cancers, and heme proteins may regulate the redox balance. Cytochrome b5 (Cyt b5) is a small mitochondrial heme protein. Its function and regulating mechanism in breast cancer remain unknown. In this study, we elucidated the level of endogenous oxidative stress in breast cancer cells, MCF-7 cells (hormone receptor-positive cells) and MDA-MB-231 cells (triple-negative cells), and investigated the difference in Cyt b5 content. Based on the low content of Cyt b5 in MDA-MB-231 cells, the overexpression of Cyt b5 was found to regulate the oxidative stress and apoptosis cascades, including ERK1/2 and Akt signaling pathways. The overexpressed Cyt b5 MDA-MB-231 cells were shown to exhibit decreased oxidative stress, less phosphorylation of ERK1/2 and Akt, and less cleavage of caspases 3 and 9 upon treatment with H2O2, as compared to those of normal MDA-MB-231 cells. Moreover, the overexpressed Cyt b5 most likely functioned by interacting with its protein partner, Cyt c, as suggested by co-immunoprecipitation studies. These results indicated that Cyt b5 has different effects on breast cancer cells of different phenotypes, which provides useful information for understanding the multiple roles of Cyt b5 and provides clues for clinical treatment.

One-pot synthesis of dihydro-ß-ionone from carotenoids using carotenoid cleavage dioxygenase and enoate reductase.

Dihydro-ß-ionone is a characteristic aroma compound of Osmanthus fragrans and is widely applied in the flavor & fragrance industry. However, the main focus is on chemical synthesis due to the metabolic pathways of dihydro-ß-ionone is still unclear. Here, we explored the one-pot synthesis system for dihydro-ß-ionone production using carotenoid cleavage dioxygenase (CCD) and enoate reductase. After screening the CCD enzyme, PhCCD1 from the Petunia hybrid was identified as the suitable enzyme for the first step of dihydro-ß-ionone synthesis due to the high enzyme activity for carotenoid. The PhCCD1 was expressed in Escherichia coli and further characterized. The optimal activity of PhCCD1 was observed at pH 6.8 and 45 °C. The enzyme was stable over the pH range of 6.0-8.0 and had good thermal stability below 40 °C. Then, we optimized the coupled reaction conditions for dihydro-ß-ionone production by PhCCD1 and enoate reductase AaDBR1 from Artemisia annua. Furthermore, we introduced the NADPH regeneration system with a 1.5-fold enhancement for dihydro-ß-ionone production. Collectively, approximately 13.34 mg/L dihydro-ß-ionone was obtained by the one-pot biosystem with a corresponding molar conversion of 85.8%. For the first time, we successfully designed and constructed a new synthesis pathway for dihydro-ß-ionone production in vitro. The coupled catalysis reported herein illustrates the feasibility of producing dihydro-ß-ionone from carotenoids and guides further engineering in the food industry.

Cloning and Characterization of a Novel Carotenoid Cleavage Dioxygenase 1 from Helianthus annuus.

Natural ß-ionone, a high-value flavoring agent, has been widely applied in the food, cosmetics, and perfume industry. However, attempts to overproduce ß-ionone in microorganisms have been limited by the efficiency of carotenoid cleavage dioxygenases (CCDs), which catalyzes ß-carotene in the biosynthesis pathway. In order to obtain CCD genes responsible for the specific cleavage of carotenoids generating ß-ionone, a novel carotenoid cleavage dioxygenase 1 from Helianthus annuus was cloned and overexpressed in Escherichia coli BL21(DE3). The recombinant CCD was able to cleave a variety of carotenoids at the 9, 10 (9', 10') sites to produce C13 products in vitro, including ß-ionone, pseudoionone, 3-hydroxy-4-oxo-ß-ionone, 3-hydroxy-ß-ionone, and 3-hydroxy-α-ionone, which vary depending on the carotenoid substrates. In comparison with lycopene and zeaxanthin, HaCCD1 also showed the high specificity for ß-carotene to cleave the 9, 10 (9', 10') double bond to produce ß-ionone in E. coli accumulating carotenoids. Finally, the expression of HaCCD1 in E. coli was optimized, and biochemical characterizations were further clarified. The optimal activity of HaCCD1 was at pH 8.8 and 50 °C. The Vmax for ß-apo-8'-carotenal was 10.14 U/mg, while the Km was 0.32 mM. Collectively, our study provides a valuable enzyme for the synthesis of natural ß-ionone by biotransformation and synthetic biology platform.

Improvements in xylose stability and thermalstability of GH39 ß-xylosidase from Dictyoglomus thermophilum by site-directed mutagenesis and insights into its xylose tolerance mechanism.

ß-Xylosidases are often inhibited by its reaction product xylose or inactivated by high temperature environment, which limited its application in hemicellulosic biomass conversion to fuel and food processing. Remarkably, some ß-xylosidases from GH39 family are tolerant to xylose. Therefore, it is of great significance to elucidate the effect mechanism of xylose on GH39 ß-xylosidases to improve their application. In this paper, based on the homologous model and prediction of protein active pocket constructed by I-TASSA and PyMOL, two putative xylose tolerance relevant sites (283 and 284) were mutated at the bottom of the protein active pocket, where xylose sensitivity and thermostability of Dictyoglomus thermophilum ß-xylosidase Xln-DT were improved by site-directed mutagenesis. The Xln-DT mutant Xln-DT-284ASP and Xln-DT-284ALA showed high xylose tolerance, with the Ki values of 4602 mM and 3708 mM, respectively, which increased by 9-35% compared with the wildtype Xln-DT. The thermostability of mutant Xln-DT-284ASP was significantly improved at 75 and 85 °C, while the activity of the wild enzyme Xln-DT decreased to 40-20%, the activity of the mutant enzyme still remained 100%. The mutant Xln-DT-284ALA showed excellent stability at pH 4.0-7.0, but Xln-DT-284ASP showed slightly decreased activity. Furthermore, in order to explore the key sites and mechanism of xylose's effect on ß-xylosidase activity, the interaction between xylose and enzyme was simulated by molecular docking. Besides binding to the active sites at the bottom of the substrate channel, xylose can also bind to sites in the middle or entrance of the channel with different affinities, which may determine the xylose inhibition of ß-xylosidase. In conclusion, the improved xylose tolerance of mutant enzyme could be more advantageous in the degradation of hemicellulose and the biotransformation of other natural active substances containing xylose. This study supplies new insights into general mechanism of xylose effect on the activity of GH 39 ß-xylosidases as well as related enzymes that modulate their activity via feedback control mechanism.

Nanobody Conjugates for Targeted Cancer Therapy and Imaging.

Conventional antibody-based targeted cancer therapy is one of the most promising avenues of successful cancer treatment, with the potential to reduce toxic side effects to healthy cells surrounding tumor cells. However, the full potential of antibodies is severely limited due to their large size, low stability, slow clearance, and high immunogenicity. Alternatively, recently discovered nanobodies, which are the smallest naturally occurring antigen-binding format, have shown great potential for addressing these limitations. Bioconjugation of nanobodies to functional groups such as toxins, enzymes, radionucleotides, and fluorophores can improve the efficacy and potency of nanobodies, enhance their in vivo pharmacokinetics, and expand the range of potential applications. Herein, we review the superior characteristics of nanobodies in comparison to conventional antibodies and provide insight into recent developments in nanobody conjugates for targeted cancer therapy and imaging.

High-level expression of a novel multifunctional GH3 family ß-xylosidase/α-arabinosidase/ß-glucosidase from Dictyoglomus turgidum in Escherichia coli.

A novel ß-xylosidase Dt-2286 from Dictyoglomus turgidum was cloned and overexpressed in Escherichia coli BL21 (DE3). Dt-2286 belonging to glycoside hydrolase (GH) family 3 encodes a polypeptide with 762 amino acid residues with a molecular weight of 85.1 kDa. By optimization of the growth and induction conditions, the activity of ß-xylosidase reached 273 U/mL, which is the highest yield reported to date from E. coli in a shake-flask. The optimal activities of the purified Dt-2286 were found at pH 5.0 and 98 °C. It also shows excellent thermostable/haloduric/organic solvent-tolerance. Dt-2286 was revealed to be a multifunctional enzyme with ß-xylosidase, α-arabinofuranoside, α-arabinopyranoside and ß-glucosidase activities, and Kcat/Km was 5245.316 mM-1 s-1, 2077.353 mM-1 s-1, 1626.454 mM-1 s-1, and 470.432 mM-1 s-1 respectively. Dt-2286 showed significant synergistic effects on the degradation of xylans, releasing more reduced sugars (up to 15.08 fold) by simultaneous addition with endoxylanase. Moreover, this enzyme has good activity in the hydrolysis of epimedium B, demonstrating its versatility in practical applications.

Co-production of Xylooligosaccharides and Xylose From Poplar Sawdust by Recombinant Endo-1,4-ß-Xylanase and ß-Xylosidase Mixture Hydrolysis.

As is well-known, endo-1,4-ß-xylanase and ß-xylosidase are the rate-limiting enzymes in the degradation of xylan (the major hemicellulosic component), main functions of which are cleavaging xylan to release xylooligosaccharides (XOS) and xylose that these two compounds have important application value in fuel, food, and other industries. This study focuses on enzymatic hydrolysis of poplar sawdust xylan for production of XOS and xylose by a GH11 endo-1,4-ß-xylanase MxynB-8 and a GH39 ß-xylosidase Xln-DT. MxynB-8 showed excellent ability to hydrolyze hemicellulose of broadleaf plants, such as poplar. Under optimized conditions (50°C, pH 6.0, dosage of 500 U/g, substrate concentration of 2 mg/mL), the final XOS yield was 85.5%, and the content of XOS2-3 reached 93.9% after 18 h. The enzymatic efficiency by MxynB-8 based on the poplar sawdust xylan in the raw material was 30.5%. Xln-DT showed excellent xylose/glucose/arabinose tolerance, which is applied as a candidate to apply in degradation of hemicellulose. In addition, the process and enzymatic mode of poplar sawdust xylan with MxynB-8 and Xln-DT were investigated. The results showed that the enzymatic hydrolysis yield of poplar sawdust xylan was improved by adding Xln-DT, and a xylose-rich hydrolysate could be obtained at high purity, with the xylose yield of 89.9%. The enzymatic hydrolysis yield was higher (32.2%) by using MxynB-8 and Xln-DT together. This study provides a deep understanding of double-enzyme synergetic enzymolysis of wood polysaccharides to valuable products.