Search

Although chitooligosaccharide (COS) has attracted the attention of some researchers due to its good solubility and broad-spectrum antibacterial activity, our study found that Staphylococcus aureus treated with low concentration of COS actively entered the viable-but-nonculturable (VBNC) state to resist this environmental stress. In this study, the transcriptome of VBNC-state S. aureus after COS treatment was analyzed by RNA-sequencing. Compared with the control group, pathway enrichment analysis showed that COS-treated S. aureus adopted a series of adaptive adjustment strategies for survival, including significant up-regulation of the differential genes' expression of such as ABC transporters (metI, tagG), Sec dependent transport pathway (secDF), peptidoglycan synthesis pathway (murG) and alteration of their physiological metabolic patterns, where ATP depletion played a key role in the formation of the VBNC-state S. aureus. Further, by using oxidative phosphorylation uncoupling agent to adjust the initial level of ATP in S. aureus, it was found that the reduction of intracellular ATP level could accelerate the formation of VBNC state. Overall, our results preliminarily elucidated the molecular mechanism of COS inducing the VBNC-state S. aureus. It provided an important theoretical reference for further achieving effective bacterial inactivation by COS., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

Dialdehyde-based cross-linking agents are widely used in the cross-linking of amino group-containing macromolecules. However, the most commonly used cross-linking agents, glutaraldehyde (GA) and genipin (GP), have safety issues. In this study, a series of dialdehyde derivatives of polysaccharides (DADPs) were prepared by oxidation of polysaccharides, and their biocompatibility and cross-linking properties were tested using chitosan as a model macromolecule. The DADPs showed outstanding cross-linking and gelation properties comparable to GA and GP. The DADPs and hydrogels cross-linked with the DADPs exhibited excellent cytocompatibility and hemocompatibility with different concentrations while significant cytotoxicity was observed in GA and GP. The experimental results showed that the cross-linking effect of DADPs increased along with their oxidation degree. The outstanding cross-linking effect of the DADPs show a potential for use in the cross-linking of biomacromolecules with amino groups and could be an adequate alternative to existing cross-linkers., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Csn-PD, a glycoside family 46 chitosanase from Paenibacillus dendritiformis , exhibits endotype hydrolysis of chitosan and produces (GlcN) 2 as the major product. Here, we report the crystal structure of Csn-PD at 1.68 Å resolution. The structure contains 14 α-helices and two β-strands that fold into two globular domains with the substrate bound between them. To evaluate the function of a loop in the substrate-binding region (residues 112-116, NDKHP), a mutant Csn-PDL1, in which this loop was deleted, was generated. Hydrolysis of chitosan by the mutant yielded chitooligosaccharides (COSs) with higher degrees of polymerization (DP) than the wild-type enzyme. Excitingly, (GlcN) 6 was produced from smaller COSs via transglycosylation activity of the mutant. Hence, the catalytic performance of a chitosanase was altered by modification of a loop in the substrate-binding regions. Our novel data on a chitosanase with transglycosylation activity offer a promising way to produce COSs with high DP.

Endo-chitosanases (EC 3.2.1.132) are generally considered to selectively release functional chito-oligosaccharides (COSs) with degrees of polymerization (DPs) ≥ 2. Although numerous endo-chitosanases have been characterized, the digestion specificity of endo-chitosanases needs to be further explored. In this study, a GH46 endo-chitosanase OUC-CsnPa was cloned, expressed, and characterized from Paenibacillus sp. 1-18. The digestion pattern analysis indicated that OUC-CsnPa could produce monosaccharides from chitotetraose [(GlcN) 4 ], the smallest recognized substrate, in a random endo-acting manner. Especially, the enzyme specificities during chitosan digestion including the regulation of product abundance through a transglycosylation reaction were also evaluated. It was hypothesized that an insertion region in OUC-CsnPa may form a strong force to be involved in stabilizing (GlcN) 4 at its negative subsite for efficient hydrolysis. This is the first comprehensive report to reveal the digestion specificity and subsite specificity of monosaccharide production by endo-chitosanases. Overall, OUC-CsnPa described here highlights the previously unknown digestion properties of the endo-acting chitosanases and provides a unique example of possible structure-function relationships.

Chitin deacetylase can be used in the green and efficient preparation of chitosan from chitin. Herein, a novel chitin deacetylase Sb CDA from Streptomyces bacillaris was heterologously expressed and comprehensively characterized. Sb DNA exhibits its highest deacetylation activity at 35 °C and pH 8.0. The enzyme activity is enhanced by Mn 2+ and prominently inhibited by Zn 2+ , SDS, and EDTA. Sb CDA showed better deacetylation activity on colloidal chitin, (GlcNAc) 5 , and (GlcNAc) 6 than other forms of the substrate. Molecular modification of Sb CDA was conducted based on sequence alignment and homology modeling. A mutant Sb CDA63G with higher activity and better temperature stability was obtained. The deacetylation activity of Sb CDA63G was increased by 133% compared with the original enzyme, and the optimal reaction temperature increased from 35 to 40 °C. The half-life of Sb CDA63G at 40 °C is 15 h, which was 5 h longer than that of the original enzyme. The improved characteristics of the chitin deacetylase Sb CDA63G make it a potential candidate to industrially produce chitosan from chitin.

Chitosanase, a glycoside hydrolase (GH), catalyzes the cleavage of β-1,4-glycosidic bonds in polysaccharides and is widely distributed in nature. Many organisms produce chitosanases, and numerous chitosanases in the GH families have been intensely studied. The reported chitosanases mainly cleaved the inter-glucosamine glycosidic bonds, while substrate specificity is not strictly unique due to the existence of bifunctional or multifunctional activity profiles. The promiscuity of chitosanases is essential for the different pathways of biomass polysaccharide conversion and understanding of the chitosanase evolutionary process. However, the reviews for this aspect are completely unknown. This review provides an overview of the promiscuous activities, also considering the substrate and product specificity of chitosanases observed to date. These contribute to important implications for the future discovery and research of promiscuous chitosanases and applications related to biomass conversion. KEY POINTS: • The promiscuity of chitosanases is reviewed for the first time. • The current review provides insights into the substrate specificity of chitosanases. • The mode-product relationship and prospect of promiscuous chitosanases are highlighted., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Intermediate-state bacteria produced in the process of bacteriostasis have always been a potential threat to public health, but they are very easy to be overlooked. As a natural and non-toxic biological antibacterial agent, chitooligosaccharide (COS) has attracted the public's attention. However, little is known about the microbial stress response during the antibacterial process of COS. In this study, the antibacterial mechanisms of COS were expounded, and the formation of sublethal and viable but nonculturable (VBNC) state were further investigated. The COS was shown to bind to the cell envelopes, and the permeability and integrity of bacterial cell membrane were damaged severely, accompanied by the increase of intracellular reactive oxygen species and decrease of adenosine triphosphate content. Flow cytometry analysis indicated that COS finally inactivated Escherichia coli through the sublethal injury process. While for Staphylococcus aureus, some cells were induced into VBNC state by COS, causing incomplete inactivation., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

In the present study, we carried out a comprehensive investigation of glycoside hydrolase (GH) 46 model-chitosanases based on cleavage specificity classification to understand their unknown bifunctional activity. We for the first time show that GH46 chitosanase CsnMHK1 from Bacillus circulans MH-K1, which was previously thought to be strictly exclusive to chitosan, can hydrolyze both chito- and cello-substrates. We determined the digestion direction of bifunctional chitosanase CsnMHK1 from class III and compared it with class II chitosanase belonging to GH8, providing insight into unique substrate specificities and a new perspective on its reclassification. The results lead us to challenge the current understanding of chitosanase substrate specificity based on GH taxonomy classification and suggest that the prevalence from the common bifunctional activity may have occurred. Altogether, these data contribute to the understanding of chitosanase recognition and hydrolysis toward chito- and cello-substrates, which is valuable for future studies on chitosanases.

Here, a multifunctional film (MFF) as an alternative tissue adhesive in the form of an interpenetrating network consisting of self-crosslinked aldehyde-functionalized chitosan (AC) and crosslinked poly(acrylic acid) (PAA) further coordinated with Ag + is reported. The MFF combines enhanced toughness and stretchability, which is attributed to the synergistic effects of the double-network design. Covalent crosslinking maintains the overall integrity of the MFF matrix, while noncovalent crosslinking dissipates energy under deformation. Upon contact, the MFF quickly dries the tissue surface followed by instant physical crosslinking to the tissue. Subsequent covalent crosslinking between the aldehyde groups of the MFF and the primary amine groups on the surface of the tissue further stabilizes the adhesion. Meanwhile, Ag + provides strong antibacterial properties to the MFF. Notably, in vivo studies demonstrate that the MFF allows facile and tough attachment to the wet and dynamic surface of rabbit liver and presents superior hemostasis and sealing properties. Furthermore, the MFF can be safely degraded without causing abnormal defects in vivo. The outstanding physicochemical properties of the MFF can potentially be a good alternative to existing sutures or staples and has potential for use in clinical practice., (© 2022 Wiley-VCH GmbH.)

Chitosanases are critical tools for the preparation of active oligosaccharides, whose composition is related to the cleavage pattern of the enzyme. Although numerous chitosanases have been characterized, the glycoside hydrolase (GH) family 5 chitosanases with other activities have rarely been investigated. Herein, a novel and second GH5 chitosanase OUC-Csngly from Streptomyces bacillaris was cloned and further characterized by expression in Escherichia coli BL21 (DE3). Interestingly, OUC-Csngly possessed dual chitosanase and cellulase activities. Molecular docking analysis showed that the C-2 group of sugar units affected the binding of the enzyme to oligosaccharides, which could result in different cleavage patterns toward chito-oligosaccharides (COSs) and cello-oligosaccharides. Further, we characterized OUC-Csngly's distinctive cleavage patterns toward two different types of oligosaccharides. Meanwhile, endo-type chitosanase OUC-Csngly generated (GlcN) - (GlcN) 4 from chitosan, was significantly different from other chitosanases. To our knowledge, this is the first report to investigate the different cleavage patterns of chitosanase for COSs and cello-oligosaccharides.Key points• The molecular docking showed C-2 group of sugar units in substrate affecting the cleavage pattern.• The first chitosanase exhibited different cleavage patterns towards chito- and cello-oligosaccharides.• The groups at C-2 influence the subsite composition of the enzyme's active cleft., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Herein, the split aptamers, chitosan oligosaccharide, and AuNPs were combined as nanocomposites that present different formations to develop a label-free colorimetric aptasensor for rapid detection of small molecules. Kanamycin was chosen as a model target. Computational studies were performed to assist in the design of orientated immobilization of the split aptamers onto the AuNPs surface. Chitosan oligosaccharide was initially applied as an aggregation inducer of AuNPs, and chitopentaose was screened as the optimal. Under optimized conditions, the proposed aptasensor showed high sensitivity and selectivity, with a limit of detection of 20.58 nM, a linear range of 25-800 nM, and good recoveries of 98.49-104.9% and 85.69-107.0% when employed to detect kanamycin in tap water and milk samples, respectively. Only 55 min was needed for the whole assay. More importantly, this study can serve as a novel and robust reference for the aptasensing detection of other small molecules., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Many by-products that are harmful to the environment and human health are generated during food processing. However, these wastes are often potential resources with high-added value. For example, crustacean waste contains large amounts of chitin. Chitin is one of the most abundant polysaccharides in natural macromolecules, and is a typical component of crustaceans, mollusks, insect exoskeleton and fungal cell walls. Chitosan is prepared by deacetylation of chitin and a copolymer of D-glucosamine and N-acetyl-D-glucosamine through β-(1 → 4)-glycosidic bonds. Chitosan has better solubility, biocompatibility and degradability compared with chitin. This review introduces the preparation, physicochemical properties, chemical and physical modification methods of chitosan, which could help us understand its biological activities and applications. According to the latest reports, the antibacterial activity, antioxidant, immune and antitumor activities of chitosan and its derivatives are summarized. Simultaneously, the various applications of chitosan and its derivatives are reviewed, including food, chemical, textile, medical and health, and functional materials. Finally, some insights into its future potential are provided, including novel modification methods, directional modification according to structure-activity relationship, activity and application development direction, etc., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Chitosan derivates with varying degrees of polymerization (DP) have attracted great concern due to their excellent biological activities. Increasing the abundance of chitosanases with different degradation modes contributes to revealing their catalytic mechanisms and facilitating the production of chitosan derivates. However, the identification of endo-chitosanases capable of producing chitobiose and D-glucosamine (GlcN) from chitosan substrates has remained elusive. Herein, an endo-chitosanase (CsnCA) belonging to the GH46 family was identified based on structural analysis in phylogenetic evolution. Moreover, we demonstrate that CsnCA acts in a random endo-acting manner, producing chitosan derivatives with DP ≤ 2. The in-depth analysis of CsnCA revealed that (GlcN)3 serves as the minimal substrate, undergoing cleavage in the mode that occupies the subsites − 2 to + 1, resulting in the release of GlcN. This study succeeded in discovering a chitosanase with distinctive degradation modes, which could facilitate the mechanistic understanding of chitosanases, further empowering the production of chitosan derivates with specific DP. Key points: • Structural docking and evolutionary analysis guide to mining the chitosanase. • The endo-chitosanase exhibits a unique GlcN-producing cleavage pattern. • The cleavage direction of chitosanase to produce GlcN was identified. [ABSTRACT FROM AUTHOR]

Chitin deacetylase can be used in the green and efficient preparation of chitosan from chitin. Herein, a novel chitin deacetylase SbCDA from Streptomyces bacillaris was heterologously expressed and comprehensively characterized. SbDNA exhibits its highest deacetylation activity at 35 °C and pH 8.0. The enzyme activity is enhanced by Mn2+ and prominently inhibited by Zn2+, SDS, and EDTA. SbCDA showed better deacetylation activity on colloidal chitin, (GlcNAc)5, and (GlcNAc)6 than other forms of the substrate. Molecular modification of SbCDA was conducted based on sequence alignment and homology modeling. A mutant SbCDA63G with higher activity and better temperature stability was obtained. The deacetylation activity of SbCDA63G was increased by 133% compared with the original enzyme, and the optimal reaction temperature increased from 35 to 40 °C. The half-life of SbCDA63G at 40 °C is 15 h, which was 5 h longer than that of the original enzyme. The improved characteristics of the chitin deacetylase SbCDA63G make it a potential candidate to industrially produce chitosan from chitin. [ABSTRACT FROM AUTHOR]

Here, a novel double‐crosslinked hydrogel dressing with enhanced antibacterial and anti‐inflammatory activities is reported. Carboxymethyl chitosan is crosslinked with silane and Ca2+ to form a double‐crosslinked hydrogel, and silver nanoparticles are loaded to introduce antibacterial properties to the hydrogel. The covalent crosslinking preserves the integrity of the hydrogel matrix, while the ionic crosslinking dissipates the energy under deformation. It combines high stretchability, toughness, tissue adhesion, and antimicrobial function. This hydrogel exhibits excellent cytocompatibility and hemocompatibility. Moreover, in vitro and in vivo investigations demonstrate its efficacy in accelerating bacterial‐infected wound healing. Given its excellent cytocompatibility and mechanical properties, this hydrogel is anticipated to be widely used in bacteria‐associated wound management. [ABSTRACT FROM AUTHOR]

Chitosanases play an important role in chitosan degradation, and the enzymatic degradation products of chitosan show various biological activities. Here, a novel glycoside hydrolase family 46 chitosanase (named Csn-BAC) from Bacillus sp. MD-5 was heterologously expressed in Escherichia coli BL21 (DE3). The recombinant enzyme was purified by Ni-NTA affinity chromatography, and its molecular weight was estimated to be 35 kDa by SDS-PAGE. Csn-BAC showed maximal activity toward colloidal chitosan at pH 7 and 40 °C. The enzymatic activity of Csn-BAC was enhanced by Mn2+, Cu2+ and Co2+ at 1 mM, and by Mn2+ at 5 mM. Thin-layer chromatography and electrospray ionization-mass spectrometry results demonstrated that Csn-BAC exhibited an endo-type cleavage pattern and hydrolyzed chitosan to yield, mainly, (GlcN) 2 and (GlcN) 3. The enzymatic properties of this chitosanase may make it a good candidate for use in oligosaccharide production-based industries. • A novel chitosanase Csn-BAC from Bacillus sp. MD-5 was cloned and purified. • Csn-BAC is a novel member of GH46 family. • Csn-BAC is quite stable over a 30–50 °C range. • Csn-BAC exhibits an endo-type cleavage pattern, which yields (GlcN) 2 and (GlcN) 3. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • A novel recombinant chitosanase (Csn21c) was identified and characterized. • The chitosanase owned good pH stability and thermal stability. • GH family 46 chitosanase (Csn21c) displayed an exo-type cleavage pattern. • This study provided a tool for production of glucosamine from chitosan. Abstract A gene encoding chitosanase from Streptomyces albolongus was cloned, sequenced and expressed in Escherichia coli. The novel recombinant enzyme (Csn21c) was purified by Ni-NTA Superflow Column and showed a molecular mass of 29.6 kDa by SDS-PAGE. The enzyme Csn21c showed the optimal activity in 50 mmol/L Tris-HCl buffer, pH 8.0, and 50 °C and it was strongly activated (2-fold) by Mn2+. It belonged to glycoside hydrolase 46 family according to NCBI database (http://www.ncbi.nlm.nih.gov/) and displayed an exo-type cleavage pattern, hydrolyzing chitosan mainly into d -glucosamine (GlcN) and chitobiose ((GlcN) 2) as confirmed by TLC and MS analysis. This study demonstrated that Csn21c can be an effective tool to produce abundant glucosamine and chitooligosaccharides (COS) from chitosan. [ABSTRACT FROM AUTHOR]

In this study, a post-selection strategy based on increasing key bases was proposed to improve aptamers' binding affinity. Phosphatidylserine (PS) was targeted as a model. Key bases of the original anti-PS aptamer were identified based on the molecular docking study, taken as repeating motif, and grafted onto the aptamer, with expectation to enhance the binding ability of the aptamer. An aptamer named PS-Apt-RKB2 was generated with significantly enhanced affinity. Biolayer interferometry assay results showed that the PS-Apt-RKB2 (Kd= 220.6 nM) exhibited a 29.59 times higher affinity over the original anti-PS aptamer (Kd= 6528 nM). Then, the PS-Apt-RKB2 was combined with chitosan oligosaccharide (COS) and AuNPs@Fe2+ to construct a label-free and COS-AuNPs@Fe2+ nanozyme-based colorimetric aptasensor. Under optimum conditions, the established aptasensor showed high sensitivity and demonstrated great application potentials in practice, with a limit of detection as low as 44.46 pM, and comparison rates of 91.21–105.4% when analyzing PS in real-world bioconversion samples with concentrations verified by high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD). This study can be further referred for developing well-performed aptamers and aptasensors for other targets. [Display omitted] • A post-selection strategy by increasing key base motifs was proposed. • A high affinity aptamer for phosphatidylserine (PS) was obtained with the strategy. • The aptamer showed a 29.59 times higher affinity than the original anti-PS aptamer. • A colorimetric aptasensor was developed with the aptamer and COS-AuNPs@Fe2+ nanozyme. • The aptasensor achieved highly sensitive detection of PS in bioconversion samples. [ABSTRACT FROM AUTHOR]