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Chitosanase plays a pivotal role in the production of chitooligosaccharide. Nevertheless, there is untapped potential for enhancing both its catalytic efficiency and thermostability, which could significantly bolster its therapeutic and biotechnological applications. In this study, two computer-aided protein design methods, namely Fireprot and PROSS, were utilized to pinpoint 6 single Bacillus subtilis chitosanase BsCsn46A mutants (S126A, D191A, K70A, L159I, K104P, and A129L) as well as 4 multiple mutants (K70A/S126A, K70A/S126A/K104P, K70A/S126A/L159I, and K70A/S126A/K104P/L159). The wild-type (WT) and all 10 BsCsn46A mutants displayed robust adaptability across a broad pH range, exhibiting peak activity within the pH spectrum of 5.5 to 9.5. The results demonstrated that, compared to the WT, 9 out of 10 mutants exhibited significantly heightened chitosanase activity, with the sole exception being the D191A mutant, which displayed activity levels nearly identical to the WT. Notably, the A129L displayed an impressive 20% increase in the enzyme activity at elevated temperatures, specifically in the range of 55–80°C. Assessing protein stability, results indicated that all samples maintained stability when incubated at 30°C for 1 h. However, when subjected to a higher temperature of 40°C for 1 h, only the A129L mutant retained stability, which persisted even after an extended incubation period of 3 h at 40°C. Furthermore, a thermal stability analysis revealed noteworthy differences between the WT and the mutants. The WT chitosanase activity diminished by 50% after brief 30-min incubation at 50°C, whereas the K70A/S126A, K70A/S126A/K104P, and A129L mutants maintained 50% of their activity for approximately 2 h under the same conditions. In summary, the study provides valuable insights into the thermostability and catalytic activity of chitosanase, highlighting promising candidates for industrial chitosanase applications. [ABSTRACT FROM AUTHOR]

As high-value chitosan derivatives, chitooligosaccharides (COSs) with biodegradable, biocompatible, nontoxic, antimicrobial, and antioxidant activities have been widely applied in food-related fields. Chitosanases can hydrolyze chitosan to produce COSs. Herein, a chitosanase (LeCho1) from Lentinula edodes was successfully expressed in Escherichia coli and was then purified and characterized. LeCho1 had a low sequence identity with other chitosanases reported from the GH75 family. The recombinant protein showed a molecular mass of 27 kDa on SDS-PAGE. LeCho1 preferentially hydrolyzed chitosan with a high degree of deacetylation (DDA) and exhibited maximal activity (71.88 U/mg) towards 95% DDA chitosan at pH 3.0 and 50 °C. It possessed good stability at pH 2.0–6.0 and temperatures below 45 °C. Its hydrolytic activity was remarkably enhanced by the metal ion Mn2+ at 1 mM, while it was totally inhibited by 1 mM Fe3+ or 10 mM EDTA. Its Km and Vmax values were 0.04 μM and 76.81 μmol·min−1·mg−1, respectively, indicating good substrate affinity. LeCho1 degraded chitosan into COSs with degrees of polymerization (DPs) of 2–5, while it had no action on COSs with DPs of less than 5, revealing its endo-chitosanase activity. This study proved that chitosanase LeCho1 is a promising candidate in the industrial preparation of COSs due to its excellent properties. [ABSTRACT FROM AUTHOR]

Background: Chitosan oligosaccharides (COS) have great potential for applications in several fields, including agriculture, food industry or medicine. Nevertheless, the large-scale use of COS requires the development of cost-effective technologies for their production. The main objective of our investigation was to develop an effective method of enzymatic degradation of chitosan in a column reactor using Mucor circinelloides IBT-83 cells, immobilized in a polyurethane foam (PUF). These cells serve as a source of chitosanolytic enzymes. Results: The study revealed that the process of freeze-drying of immobilized mycelium increases the stability of the associated enzymes during chitosan hydrolysis. The use of stabilized preparations as an active reactor bed enables the production of COS at a constant level for 16 reactor cycles (384 h in total), i.e. 216 h longer compared to non-stabilized mycelium. In the hydrolysate, oligomers ranging in structure from dimer to hexamer as well as D-glucosamine were detected. The potential application of the obtained product in agriculture has been verified. The results of phytotests have demonstrated that the introduction of COS into the soil at a concentration of 0.01 or 0.05% w/w resulted in an increase in the growth of Lepidium sativum stem and root, respectively (extensions by 38 and 44% compared to the control sample). Conclusions: The research has verified that the PUF-immobilized M. circinelloides IBT-83 mycelium, which has been stabilized through freeze-drying, is a promising biocatalyst for the environmentally friendly and efficient generation of COS. This biocatalyst has the potential to be used in fertilizers. [ABSTRACT FROM AUTHOR]

To screen and identify high chitosanase producing strains and to optimize the fermentation condition and analyze the enzymatic properties of the target strain. In this study, high chitosanase-producing strain was screened from the sediment of shrimp ponds in Beihai, Guangxi through primary and secondary screening. The strain underwent identification, the enzyme production process of the strain was optimized through single-factor, Plackett-Burman and orthogonal optimization methods. The enzymatic properties and products of chitosanase produced by the strain were studied. The results showed that the strain of Gxun-5.3.3 with high chitosanase production was obtained by screening and was identified as Bacillus cereus. The optimal components for the enzyme-producing medium of B. cereus Gxun-5.3.3 was maltose 5 g/L, yeast powder 3 g/L, chitosan 15 g/L, and the initial pH and fermentation time were 6.0 and 84 h, respectively. Under the optimized conditions, the chitosanase activity reached 62.03±1.18 U/mL, which was 55.58% higher before optimization (39.87±1.24 U/mL). The protease produced by strain Gxun-5.3.3 had maximum activity at 60 ℃ and optimum pH at 5.0, respectively. Metal ions Fe2+ and Mn2+ had a significant (P

Abstract Background Chitosan oligosaccharides (COS) have great potential for applications in several fields, including agriculture, food industry or medicine. Nevertheless, the large-scale use of COS requires the development of cost-effective technologies for their production. The main objective of our investigation was to develop an effective method of enzymatic degradation of chitosan in a column reactor using Mucor circinelloides IBT-83 cells, immobilized in a polyurethane foam (PUF). These cells serve as a source of chitosanolytic enzymes. Results The study revealed that the process of freeze-drying of immobilized mycelium increases the stability of the associated enzymes during chitosan hydrolysis. The use of stabilized preparations as an active reactor bed enables the production of COS at a constant level for 16 reactor cycles (384 h in total), i.e. 216 h longer compared to non-stabilized mycelium. In the hydrolysate, oligomers ranging in structure from dimer to hexamer as well as D-glucosamine were detected. The potential application of the obtained product in agriculture has been verified. The results of phytotests have demonstrated that the introduction of COS into the soil at a concentration of 0.01 or 0.05% w/w resulted in an increase in the growth of Lepidium sativum stem and root, respectively (extensions by 38 and 44% compared to the control sample). Conclusions The research has verified that the PUF-immobilized M. circinelloides IBT-83 mycelium, which has been stabilized through freeze-drying, is a promising biocatalyst for the environmentally friendly and efficient generation of COS. This biocatalyst has the potential to be used in fertilizers.

Chitosan oligosaccharide (COS) is a high value-added derivative with intriguing uses in the food, pharmaceutical, and cosmetic industries. Herein, a new chitosanase (CscA) from Kitasatospora setae KM-6054 was cloned and heterologously expressed in Escherichia coli BL21(DE3). CscA showed the highest sequence identity (87.7%) with the chitosanase from Amycolatopsis sp. CsO-2 and was recognized as a new glycoside hydrolase (GH) family 46 chitosanase. The recombinant chitosanase CscA was purified by Ni-NTA Sepharose column and showed a relative molecular weight of 31.07 kDa. CscA had the highest activity (241.39 U/mg) at pH 5.0 and 60 °C. CscA had a wide pH tolerance range, maintaining more than 70% activity in the pH range of 3–10. Furthermore, the enzyme decomposed chitosan to generate chitobiose ((GlcN)2), chitotriose ((GlcN)3), chitotetraose ((GlcN)4), and chitopentaose ((GlcN)5) products. CscA was shown to be a potential chitosanase for the efficient manufacture of COSs from chitosan, which has several uses in food and industry. [ABSTRACT FROM AUTHOR]

Marine polysaccharide degrading enzymes are pivotal for catalyzing glycosidic linkages' breakdown in polysaccharides, yielding low-degree oligosaccharides and monosaccharides. These enzymes show substantial potential in diverse biotechnological applications. Marine polysaccharides and their derivatives exhibit anti-inflammatory, antiviral, anticoagulant, and anticancer properties, and are used in food additives with potential health benefits such as prebiotic oligosaccharides. Research explores pharmaceutical applications of marine polysaccharide degrading enzymes, including novel drug delivery and therapeutic bioactive oligosaccharide production. These enzymes are crucial for depolymerizing marine polysaccharides, providing energy and nutrients to microorganisms, and contributing to nutrient cycling and ecological balance. The breakdown of marine polysaccharides is vital for the global carbon cycle and helps regulate atmospheric carbon dioxide levels, impacting environmental sustainability. This comprehensive review delves into marine polysaccharide degrading enzymes, emphasizing carrageenase, agarase, chitinase, chitosanase and alginate lyase. Challenges such as low yields, poor stability, and high costs prompt solutions through metabolic engineering, enzyme immobilization, and engineering, enhancing resistance and efficiency. These advancements amplify the value of marine polysaccharidedegrading enzymes in biotechnological and environmental contexts. [ABSTRACT FROM AUTHOR]

Background: High chitosanase-producing microorganisms from natural sources have extensive applications in food and agriculture. This study aimed to optimal conditions for high-activity chitosanase production. A named CGMCC21422 chitosanase-producing strain -2T-2 was isolated from soil and identified named as Aspergillus fumigatus chitosanase (A. fumigatus chitosanase). This enzymatic activity was validated in various culture conditions. It is stored in the China General Microbiological Culture Collection Center. The efficacy of A. fumigatus chitosanase in the degradation of chitosan was validated. Results: In this study, we determined that the optimal fermentation conditions of stain-2T-2 were 1.0% powered chitosan, 0.8% ammonium nitrate, 37°C culture temperature, initial pH 5.0, culture time 6 d, bottle volume 50 mL, and 2% inoculation dosage. Under these culture conditions, the highest enzyme activity of fermentation broth in the shaker flask reached 827.53 U/mL. The optimal reactive conditions of A. fumigatus-produced chitosanase are 55–60°C and pH 4.5. When the reactive temperature was over 60°C, the A. fumigatus chitosanase was easily inactivated. The chitosanase catalyzed substrate chitosan to produced ≈20% chito-oligosaccharide and ≥80% glucosamine salt samples in a variety of acidic solutions. These reactive products are not cytotoxic or mild to MH7A cells. Conclusions: A. fumigatus chitosanase strain -2T-2 is a strain with high chitosanase and can catalyze chitosan into chito-oligosaccharide in acidic solutions.How to cite: Yang, H., Wang, L., Xu, C, et al. Screening for Aspergillus fumigatus strain-2T-2 with high chitosanase production activity and its application in chitosan degradation. Electron J Biotechnol 2024; 68. https://doi.org/10.1016/j.ejbt.2024.01.003.

Abstract Chitooligosaccharides (COS) find numerous applications due to their exceptional properties. Enzymatic hydrolysis of chitosan by chitosanase is considered an advantageous route for COS production. Heterologous expression of chitosanase holds significant promise, yet studies using commonly employed Escherichia coli and Pichia pastoris strains encounter challenges in subsequent handling and industrial scalability. In this investigation, we opted for using the safe yeast strain Saccharomyces cerevisiae (GRAS), obviating the need for methanol induction, resulting in successful expression. Ultimately, utilizing the GTR-CRISPR editing system, shake flask enzyme activity reached 2 U/ml. The optimal chitosanase activity was achieved at 55℃ and pH 5, with favorable stability between 30 and 50 °C. Following a 2-h catalytic reaction, the product primarily consisted of chitobiose to chitotetraose, predominantly at the chitotriose position, with a slight increase in chitobiose content observed during the later stages of enzymatic hydrolysis. The results affirm the feasibility of heterologous chitosanase expression through Saccharomyces cerevisiae, underscoring its significant industrial potential. Graphical Abstract

As high-value chitosan derivatives, chitooligosaccharides (COSs) with biodegradable, biocompatible, nontoxic, antimicrobial, and antioxidant activities have been widely applied in food-related fields. Chitosanases can hydrolyze chitosan to produce COSs. Herein, a chitosanase (LeCho1) from Lentinula edodes was successfully expressed in Escherichia coli and was then purified and characterized. LeCho1 had a low sequence identity with other chitosanases reported from the GH75 family. The recombinant protein showed a molecular mass of 27 kDa on SDS-PAGE. LeCho1 preferentially hydrolyzed chitosan with a high degree of deacetylation (DDA) and exhibited maximal activity (71.88 U/mg) towards 95% DDA chitosan at pH 3.0 and 50 °C. It possessed good stability at pH 2.0–6.0 and temperatures below 45 °C. Its hydrolytic activity was remarkably enhanced by the metal ion Mn2+ at 1 mM, while it was totally inhibited by 1 mM Fe3+ or 10 mM EDTA. Its Km and Vmax values were 0.04 μM and 76.81 μmol·min−1·mg−1, respectively, indicating good substrate affinity. LeCho1 degraded chitosan into COSs with degrees of polymerization (DPs) of 2–5, while it had no action on COSs with DPs of less than 5, revealing its endo-chitosanase activity. This study proved that chitosanase LeCho1 is a promising candidate in the industrial preparation of COSs due to its excellent properties.

Chitooligosaccharides (COS) find numerous applications due to their exceptional properties. Enzymatic hydrolysis of chitosan by chitosanase is considered an advantageous route for COS production. Heterologous expression of chitosanase holds significant promise, yet studies using commonly employed Escherichia coli and Pichia pastoris strains encounter challenges in subsequent handling and industrial scalability. In this investigation, we opted for using the safe yeast strain Saccharomyces cerevisiae (GRAS), obviating the need for methanol induction, resulting in successful expression. Ultimately, utilizing the GTR-CRISPR editing system, shake flask enzyme activity reached 2 U/ml. The optimal chitosanase activity was achieved at 55℃ and pH 5, with favorable stability between 30 and 50 °C. Following a 2-h catalytic reaction, the product primarily consisted of chitobiose to chitotetraose, predominantly at the chitotriose position, with a slight increase in chitobiose content observed during the later stages of enzymatic hydrolysis. The results affirm the feasibility of heterologous chitosanase expression through Saccharomyces cerevisiae, underscoring its significant industrial potential. [ABSTRACT FROM AUTHOR]

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Abstract Purpose The present research work focuses on the extraction of chitosanase enzyme from soil fungi. Chitosan hydrolysis by chitosanase is one of the most effective methods to produce chitosan oligosaccharides which are new biomaterials that have many biological activities such as antitumour, antioxidant, antidiabetic and antimicrobial. Method A strain producing chitosanase was screened and identified as Fusarium oxysporum D18 with an accession number OL343607. Various physiological parameters (incubation type, carbon source, additive nitrogen source, statistical evaluation, solid state fermentation) were assessed to increase chitosanase production. Results Fusarium oxysporum D18 produced a considerable value of chitosanase (1.220 U/ml). After 7 days of incubation, the best carbon source was lactose, and the best nitrogen source was ammonium chloride. Statistical evaluation was carried out by using Plackett–Burman and Box-Behnken designs. The highest chitosanase production (1.994 U/ml) was induced by the medium composition g/l: KH2PO4 (1.5), MgSO4 (0.269), lactose (18), NH4Cl (1.26), pH (6.68), using a 5-day-old inoculum and chitosanase activity was 1.63 folds that of the original medium. The production of chitosanase by Fusarium oxysporum D18 in solid state cultures using different solid substrates was studied and the best solid substrate for higher chitosanase activity (2.246 U/ml) was raw shrimp heads and shells and chitosanase activity was 1.13 folds that of the optimized liquid cultures. An extracellular chitosanase was isolated and partially purified by using 75% saturation of ammonium sulphate. The highest chitosanase activity (3.667 U/ml) with a specific activity of 0.390 U/mg protein was obtained at enzyme protein concentration of 9.391 mg/ml, substrate concentration of 1.2 % (w/v), V max of the enzyme of approximately 0.430 U/mg protein, and K M of 0.26 % (w/v), at pH 5.6 and reaction temperature of 50 °C. The activity of the purified and characterized chitosanase increased by 3 times than that the original isolate activity. The enzyme was thermostable and retained about 55% of its original activity after heating at 70 °C for 15 min. The enzyme preparations were activated by Ca2+ ions and inactivated by Zn+2, Cu+2 ions, and EDTA. Conclusion An antitumour activity of chitooligosaccharides produced by the chitosanase was applied to the MCF-7 (breast carcinoma cells) and they had a cytotoxicity inhibitory effect against them about IC50 = 448 μg/ml.

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]

Chitooligosaccharides (COSs) are b-1,4-linked homo-oligosaccharides of N-acetylglucosamine (GlcNAc) or glucosamine (GlcN), and also include hetero-oligosaccharides composed of GlcNAc and GlcN. These sugars are of practical importance because of their various biological activities, such as antimicrobial, anti-inflammatory, antioxidant and antitumor activities, as well as triggering the innate immunity in plants. The reported data on bioactivities of COSs used to contain some uncertainties or contradictions, because the experiments were conducted with poorly characterized COS mixtures. Recently, COSs have been satisfactorily characterized with respect to their structures, especially the degree of polymerization (DP) and degree of N-acetylation (DA); thus, the structure–bioactivity relationship of COSs has become more unambiguous. To date, various green-chemical strategies involving enzymatic synthesis of COSs with designed sequences and desired biological activities have been developed. The enzymatic strategies could involve transglycosylation or glycosynthase reactions using reducing end-activated sugars as the donor substrates and chitinase/chitosanase and their mutants as the biocatalysts. Site-specific chitin deacetylases were also proposed to be applicable for this purpose. Furthermore, to improve the yields of the COS products, metabolic engineering techniques could be applied. The above-mentioned approaches will provide the opportunity to produce tailor-made COSs, leading to the enhanced utilization of chitin biomass. [ABSTRACT FROM AUTHOR]

The aims of this study were to obtain chitooligosaccharides (COS) from chitosan (CH) with improved functional properties and comparison of the use of two different enzymes: commercial cellulase (CL) and the dedicated enzyme chitosanase (CS). After enzymatic reaction, chitosan oligomers (NFs) were isolated by methanol into two fractions: precipitate (HMF) and supernatant (LMF). The occurrence of a hydrolysis reaction was confirmed by an increased reducing sugar content and viscosity reduction of chitosan oligomers. CPMAS 13C NMR analysis confirmed the dissimilar cleavage mechanism of the enzymes used. LMF and NF fractions were characterised by improved solubility in water (94.56%) compared to the HMF and CH samples (70.64%). Thermogravimetric analysis (TGA) showed that the HMF decomposed in two-stage process while CH, NF, and LMF decomposed in a three-stage process. The greatest mass loss of LMF samples (58.35%) suggests their sensitivity to high-temperature treatments. COS were a mixture of DP (degrees of polymerisation) from 3 to 18 hetero-chitooligomers, with an average Mw of <3 kDa. CL consisted of more low-DP products (DP 3–7) than COS made with CS. LMF characterised by DP~2 showed lower DPPH radical scavenging activity than HMF and NF with DP 3–7. The ability to reduce Escherichia coli increased in the given order: LMF > NF > HMF > CH. [ABSTRACT FROM AUTHOR]

Chitosanases play a significant part in the hydrolysis of chitosan to form chitooligosaccharides (COS) that possess diverse biological activities. This study aimed to enhance the productivity of Paenibacillus elgii TKU051 chitosanase by fermentation from chitinous fishery wastes. The ideal parameters for achieving maximum chitosanase activity were determined: a squid pens powder amount of 5.278% (w/v), an initial pH value of 8.93, an incubation temperature of 38 °C, and an incubation duration of 5.73 days. The resulting chitosanase activity of the culture medium was 2.023 U/mL. A chitosanase with a molecular weight of 25 kDa was isolated from the culture medium of P. elgii TKU051 and was biochemically characterized. Liquid chromatography with tandem mass spectrometry analysis revealed that P. elgii TKU051 chitosanase exhibited a maximum amino acid identity of 43% with a chitosanase of Bacillus circulans belonging to the glycoside hydrolase (GH) family 46. P. elgii TKU051 chitosanase demonstrated optimal activity at pH 5.5 while displaying remarkable stability within the pH range of 5.0 to 9.0. The enzyme displayed maximum efficiency at 60 °C and demonstrated considerable stability at temperatures ≤40 °C. The presence of Mn2+ positively affected the activity of the enzyme, while the presence of Cu2+ had a negative effect. Thin-layer chromatography analysis demonstrated that P. elgii TKU051 chitosanase exhibited an endo-type cleavage pattern and hydrolyzed chitosan with 98% degree of deacetylation to yield (GlcN)2 and (GlcN)3. The enzymatic properties of P. elgii TKU051 chitosanase render it a promising candidate for application in the production of COS. [ABSTRACT FROM AUTHOR]

Chitooligosaccharides (COS), a high-value chitosan derivative, have many applications in food, pharmaceuticals, cosmetics and agriculture owing to their unique biological activities. Chitosanase, which catalyzes the hydrolysis of chitosan, can cleave β-1,4 linkages to produce COS. In this study, a chitosanase-producing Bacillus paramycoides BP-N07 was isolated from marine mud samples. The chitosanase enzyme (BpCSN) activity was 2648.66 ± 20.45 U/mL at 52 h and was able to effectively degrade chitosan. The molecular weight of purified BpCSN was approximately 37 kDa. The yield and enzyme activity of BpCSN were 0.41 mg/mL and 8133.17 ± 47.83 U/mg, respectively. The optimum temperature and pH of BpCSN were 50 °C and 6.0, respectively. The results of the high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC) of chitosan treated with BpCSN for 3 h showed that it is an endo-chitosanase, and the main degradation products were chitobiose, chitotriose and chitotetraose. BpCSN was used for the preparation of oligosaccharides: 1.0 mg enzyme converted 10.0 g chitosan with 2% acetic acid into oligosaccharides in 3 h at 50 °C. In summary, this paper reports that BpCSN has wide adaptability to temperature and pH and high activity for hydrolyzing chitosan substrates. Thus, BpCSN is a chitosan decomposer that can be used for producing chitooligosaccharides industrially. [ABSTRACT FROM AUTHOR]

Purpose: The present research work focuses on the extraction of chitosanase enzyme from soil fungi. Chitosan hydrolysis by chitosanase is one of the most effective methods to produce chitosan oligosaccharides which are new biomaterials that have many biological activities such as antitumour, antioxidant, antidiabetic and antimicrobial. Method: A strain producing chitosanase was screened and identified as Fusarium oxysporum D18 with an accession number OL343607. Various physiological parameters (incubation type, carbon source, additive nitrogen source, statistical evaluation, solid state fermentation) were assessed to increase chitosanase production. Results: Fusarium oxysporum D18 produced a considerable value of chitosanase (1.220 U/ml). After 7 days of incubation, the best carbon source was lactose, and the best nitrogen source was ammonium chloride. Statistical evaluation was carried out by using Plackett–Burman and Box-Behnken designs. The highest chitosanase production (1.994 U/ml) was induced by the medium composition g/l: KH2PO4 (1.5), MgSO4 (0.269), lactose (18), NH4Cl (1.26), pH (6.68), using a 5-day-old inoculum and chitosanase activity was 1.63 folds that of the original medium. The production of chitosanase by Fusarium oxysporum D18 in solid state cultures using different solid substrates was studied and the best solid substrate for higher chitosanase activity (2.246 U/ml) was raw shrimp heads and shells and chitosanase activity was 1.13 folds that of the optimized liquid cultures. An extracellular chitosanase was isolated and partially purified by using 75% saturation of ammonium sulphate. The highest chitosanase activity (3.667 U/ml) with a specific activity of 0.390 U/mg protein was obtained at enzyme protein concentration of 9.391 mg/ml, substrate concentration of 1.2 % (w/v), Vmax of the enzyme of approximately 0.430 U/mg protein, and KM of 0.26 % (w/v), at pH 5.6 and reaction temperature of 50 °C. The activity of the purified and characterized chitosanase increased by 3 times than that the original isolate activity. The enzyme was thermostable and retained about 55% of its original activity after heating at 70 °C for 15 min. The enzyme preparations were activated by Ca2+ ions and inactivated by Zn+2, Cu+2 ions, and EDTA. Conclusion: An antitumour activity of chitooligosaccharides produced by the chitosanase was applied to the MCF-7 (breast carcinoma cells) and they had a cytotoxicity inhibitory effect against them about IC50 = 448 μg/ml. Highlights: (1) We screened and identified a chitosanase producing strain from the soil as Fusarium oxysporum D18 with an accession number OL343607. (2) The enzyme activity of chitosanase was greatly improved by optimizing the fermentation medium of the fungus. (3) The activity of chitosanase was optimized by statistical evaluation that was carried out by using Plackett-Burman and Box-Behnken designs. (4) The production of chitosanase by Fusarium oxysporum D18 in solid state cultures using different solid substrates was studied for increasing activity. (5) Partial purification of the enzyme was performed by using 75 % saturation of ammonium sulphate. (6) Physical and chemical properties of the enzyme were performed leading to a highly thermostable chitosanase. (7) An antitumour chitosanase activity was applied to the MCF-7. [ABSTRACT FROM AUTHOR]

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Abstract Background An important conceptual advance in health and the environment has been recognized that enzymes play a key role in the green processing industries. Of particular interest, chitosanase is beneficial for recycling the chitosan resource and producing chitosan oligosaccharides. Also, chitosan gene expression and molecular characterization will promote understanding of the biological function of bacterial chitosanase as well as explore chitosanase for utilizing chitosan resources. Results A chitosanase-producing bacterium TY24 was isolated and identified as Bacillus cereus. Moreover, the chitosanase gene was cloned and expressed in Escherichia coli. Sequence analysis reveals that the recombinant chitosanase (CHOE) belongs to the glycoside hydrolases 8 family. The purified CHOE has a molecular weight of about 48 kDa and the specific activity of 1150 U/mg. The optimal pH and temperature of CHOE were 5.5 and 65 °C, respectively. The enzyme was observed stable at the pH range of 4.5–7.5 and the temperature range of 30–65 °C. Especially, the half-life of CHOE at 65 °C was 161 min. Additionally, the activity of CHOE was remarkably enhanced in the presence of Mn2+, Cu2+, Mg2+ and K+, beside Ca2+ at 5 mM. Especially, the activity of CHOE was enhanced to more than 120% in the presence of 1% of various surfactants. CHOE exhibited the highest substrate specificity toward colloid chitosan. Conclusion A bacterial chitosanase was cloned from B. cereus and successfully expressed in E. coli (BL21) DE3. The recombinant enzyme displayed good stability under acid pH and high-temperature conditions.

Chitosanase could degrade chitosan efficiently under mild conditions to prepare chitosan oligosaccharides (COSs). COS possesses versatile physiological activities and has wide application prospects in food, pharmaceutical and cosmetic fields. Herein, a new glycoside hydrolase (GH) family 46 chitosanase (CscB) was cloned from Kitasatospora setae KM-6054 and heterologously expressed in Escherichia coli. The recombinant chitosanase CscB was purified by Ni-charged magnetic beads and showed a relative molecular weight of 29.19 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). CscB showed the maximal activity (1094.21 U/mg) at pH 6.0 and 30 °C. It was revealed that CscB is a cold-adapted enzyme. CscB was determined to be an endo-type chitosanase with a polymerization degree of the final product mainly in the range of 2–4. This new cold-adapted chitosanase provides an efficient enzyme tool for clean production of COSs. [ABSTRACT FROM AUTHOR]

Environmentally friendly and efficient biodegradation with chitosanase for degrading chitosan to oligosaccharide has been gaining more importance. Here, we studied a chitosanase from Aspergillus fumigatus with potential for production, but does not have the ideal thermal stability. The structure predicted by the Alphafold2 model, especially the binding site and two catalytic residues, has been found to have a high similarity with the experimental structure of the chitosanase V-CSN from the same family. The effects of temperature on structure and function were studied by dynamic simulation and the results showed that the binding site had high flexibility. After heating up from 300 K to 350 K, the RMSD and RMSF of the binding site increased significantly, in particular, the downward shift of loop6 closed the binding site, resulting in the spatial hindrance of binding. The time proportions of important hydrogen bonds at the binding site decreased sharply, indicating that serious disruption of hydrogen bonds should be the main interaction factor for conformational changes. The residues contributing energetically to binding were also revealed to be in the highly flexible region, which inevitably leads to the decrease in the activity stability at high temperature. These findings provide directions for the modification of thermal stability and perspectives on the research of proteins without experimental structures. [ABSTRACT FROM AUTHOR]

Chitosanases are potential candidates for chitooligosaccharides (COS) production‐based industries, therefore, the discovery of chitosanases having commercial potential will remain a priority worldwide. This study aims to characterize different chitosanases of Bacillus strains for COS production. Six different indigenous Bacillus strains (B. cereus EGE‐B‐6.1m, B. cereus EGE‐B‐2.5m, B. cereus EGE‐B‐5.5m, B. cereus EGE‐B‐10.4i, B. thuringiensis EGE‐B‐3.5m, and B. mojavensis EGE‐B‐5.2i) were used to purify and characterize chitosanases. All purified chitosanases have a similar molecular weight (37 kDa) as determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. However, other characteristics such as optimum temperature and pH, kinetic parameters (Km and Vmax), temperature, and pH stabilities were dissimilar among the strains of different Bacillus species and within the same species. Furthermore, chitosanases of all strains were able to successfully hydrolyze chitosan to COS and oligomers of the degree of polymerization 2–6 were detected with chitobiose and chitotriose as major hydrolysis products. The relative yields of COS were in a range of 19%–31% and chitosanase of B. thuringiensis EGE‐B‐3.5m turned out to be the best enzyme in terms of its characteristics and COS production potential with maximum relative yield (31%). Results revealed that Bacillus chitosanases could be used directly for efficient bioconversion of chitosan into COS and will be valuable for large‐scale production of biologically active COS. [ABSTRACT FROM AUTHOR]

Copyright of Modern Food Science & Technology is the property of Editorial Office of Modern Food Science & Technology and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Chinese Journal of Bioprocess Engineering is the property of Chinese Journal of Bioprocess Engineering Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

At present, chitosanase used to prepare chito-oligosaccharides has the disadvantage of low enzyme activity and poor acid stability. In this study, chitosanase-producing strain was screened from mud samples from Lianyungang Haizhou Bay, the highest chitosanase-producing strain was identified, and the acid stability and enzyme properties of the strain were studied. The screening was carried out by using the transparent zone method, and shaking flask fermentation. The chitosanase producing strain CLT08 was obtained. Then, the strain CLT08 was identified as Paenibacillus chitinolyticus with morphological characteristics, physiological and biochemical determination and 16S rDNA sequence amplification and analysis. The optimum temperature and pH for chitosanase production of strain CLT08 were 45 ℃ and pH4.0; Zn2+ had a significant activation effect on the enzyme, and Ba2+, Co2+, Fe2+ had significant effects inhibition. The degree of polymerization of chitosan was≥6 after being hydrolyzed by CLT08 chitosanase. The results of this study laid the foundation for further research on the enzyme.

Natural macromolecules such as polysaccharides are bioactive substances obtained from the agricultural feed stocks and\ or crustacean shell wastes, thus chitin biomass as a rich renewable resource; is an additional abundant natural polysaccharide in the environment. Despite the recent interests in the biomedical applications of chitin, complexity of chitosan and their derivatives in terms of controlling their chemical synthesis due to the formation of secondary compounds is a major problem, thus this research aimed to focus on the microbial biosynthesis of chitinous products and their derivatives from the seafood wastes. In the current research work; chitin, chitosan and chitooligosaccharides (COS) were biosynthesized from Archachatina marginata exoskeletons; through chemical deacetylation and microbial transformation techniques. During this study, Archachatina marginata exoskeleton was analyzed according to the method adopted by the Association of Official Analytical Chemists. In addition, a synthetic route for chitooligosaccharides production was investigated using a previously characterized Saccharomyces cerevisiae KR13 strain. The crude extract obtained after the biotransformation period was subjected to derivatization, and then the chitooligosaccharides yield, enzymes activities and their molecular weights were determined using Gas chromatography (GC). Moreover, the size exclusion chromatography revealed the presence of several enzymes, including chitin deacetylase; chitooligosaccharide deacetylase, chitinase and chitosanase; in addition to chitooligosaccharides derivatives such as; chitohexose, chitopentose, chitotetrose, chitotriose, chitobiose and chitosan with varying concentrations. Based on the results obtained in this study, manipulation of S. cerevisiae KR13 strain clarified the poor solubility of chitin and chitosan, which makes a challenge in using them in the biomedical applications, despite their important functional activities.

Abstract Chitooligosaccharides (COSs) and d‐glucosamine (GlcN), the most valuable biomolecules due to their various physiological functions, can be available inexpensively from chitosan by bio‐enzymatic degradation, in which chitosanases play a key role. On the other hand, pretreatment of chitosan is the necessary procedure in the bioproduction by enzymatic hydrolysis. Therefore, the green and efficient pretreatment strategy as well as superior enzyme activity are the most critical aspects in scaled up production. This study investigated environmentally friendly and efficient enzymatic hydrolysis of powdery chitosan (PC) pretreated by ball‐milling. First, heterologous expression yielded OUC‐CsnPT, a glycoside hydrolase family 46 chitosanase with a predominant specific activity of 5346.56 U/mg at pH 6.0°C and 45°C. The endo‐type chitosanase OUC‐CsnPT hydrolyzed chitosan to produce GlcN and chitobiose [(GlcN)2]. Then, ball‐milling was employed to pretreat PC to promote chitosanase OUC‐CsnPT hydrolysis, and the effectiveness was characterized by scanning electron microscope, X‐ray diffraction, and FTIR‐attenuated total reflection. Through the development of the novel route, the yield of COSs and GlcN for ball‐milling powdery chitosan increased by 10.8‐folds with a concentration of 14.5 μmol/ml. Hence, this technique presents a promising strategy suited to be a straightforward and environmentally friendly option for oligosaccharides production, broadening the theoretical basis for PC biodegradation.

Chitooligosaccharides (COS), a high-value chitosan derivative, have many applications in food, pharmaceuticals, cosmetics and agriculture owing to their unique biological activities. Chitosanase, which catalyzes the hydrolysis of chitosan, can cleave β-1,4 linkages to produce COS. In this study, a chitosanase-producing Bacillus paramycoides BP-N07 was isolated from marine mud samples. The chitosanase enzyme (BpCSN) activity was 2648.66 ± 20.45 U/mL at 52 h and was able to effectively degrade chitosan. The molecular weight of purified BpCSN was approximately 37 kDa. The yield and enzyme activity of BpCSN were 0.41 mg/mL and 8133.17 ± 47.83 U/mg, respectively. The optimum temperature and pH of BpCSN were 50 °C and 6.0, respectively. The results of the high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC) of chitosan treated with BpCSN for 3 h showed that it is an endo-chitosanase, and the main degradation products were chitobiose, chitotriose and chitotetraose. BpCSN was used for the preparation of oligosaccharides: 1.0 mg enzyme converted 10.0 g chitosan with 2% acetic acid into oligosaccharides in 3 h at 50 °C. In summary, this paper reports that BpCSN has wide adaptability to temperature and pH and high activity for hydrolyzing chitosan substrates. Thus, BpCSN is a chitosan decomposer that can be used for producing chitooligosaccharides industrially.

Chitooligosaccharides (COSs) are b-1,4-linked homo-oligosaccharides of N-acetylglucosamine (GlcNAc) or glucosamine (GlcN), and also include hetero-oligosaccharides composed of GlcNAc and GlcN. These sugars are of practical importance because of their various biological activities, such as antimicrobial, anti-inflammatory, antioxidant and antitumor activities, as well as triggering the innate immunity in plants. The reported data on bioactivities of COSs used to contain some uncertainties or contradictions, because the experiments were conducted with poorly characterized COS mixtures. Recently, COSs have been satisfactorily characterized with respect to their structures, especially the degree of polymerization (DP) and degree of N-acetylation (DA); thus, the structure–bioactivity relationship of COSs has become more unambiguous. To date, various green-chemical strategies involving enzymatic synthesis of COSs with designed sequences and desired biological activities have been developed. The enzymatic strategies could involve transglycosylation or glycosynthase reactions using reducing end-activated sugars as the donor substrates and chitinase/chitosanase and their mutants as the biocatalysts. Site-specific chitin deacetylases were also proposed to be applicable for this purpose. Furthermore, to improve the yields of the COS products, metabolic engineering techniques could be applied. The above-mentioned approaches will provide the opportunity to produce tailor-made COSs, leading to the enhanced utilization of chitin biomass.

Atmospheric and room temperature plasma(ARTP) Bacillus cereus was used to screen the strains with increased enzyme activity. The results showed that culturing Bacillus cereus in the seed solution for 6 hours could reach the middle and late logarithmic phase. When the mutagenesis time of normal temperature and atmospheric pressure plasma was 60 s, the lethality of Bacillus cereus reached 85%. A mutant strain with a 13.19% increase in chitosanase activity was screened. Then the stability of the enzyme activity was verified. The average enzyme activity after six generations of culture was 9.643367 U/mL, and the fluctuation was within 5%, indicating that its enzyme production was high and the stability was strong, and it could be used for subsequent starting strains. After the mutagenesis, the strain was observed by electron microscope, and it was found that the morphology of the colony did not change significantly, but the individual morphology of the strain had changed, and it was slenderer than the original strain, which proved that the increase in enzyme activity was the effect of ARTP mutagenesis.

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. [ABSTRACT FROM AUTHOR]

Background: An important conceptual advance in health and the environment has been recognized that enzymes play a key role in the green processing industries. Of particular interest, chitosanase is beneficial for recycling the chitosan resource and producing chitosan oligosaccharides. Also, chitosan gene expression and molecular characterization will promote understanding of the biological function of bacterial chitosanase as well as explore chitosanase for utilizing chitosan resources. Results: A chitosanase-producing bacterium TY24 was isolated and identified as Bacillus cereus. Moreover, the chitosanase gene was cloned and expressed in Escherichia coli. Sequence analysis reveals that the recombinant chitosanase (CHOE) belongs to the glycoside hydrolases 8 family. The purified CHOE has a molecular weight of about 48 kDa and the specific activity of 1150 U/mg. The optimal pH and temperature of CHOE were 5.5 and 65 °C, respectively. The enzyme was observed stable at the pH range of 4.5–7.5 and the temperature range of 30–65 °C. Especially, the half-life of CHOE at 65 °C was 161 min. Additionally, the activity of CHOE was remarkably enhanced in the presence of Mn2+, Cu2+, Mg2+ and K+, beside Ca2+ at 5 mM. Especially, the activity of CHOE was enhanced to more than 120% in the presence of 1% of various surfactants. CHOE exhibited the highest substrate specificity toward colloid chitosan. Conclusion: A bacterial chitosanase was cloned from B. cereus and successfully expressed in E. coli (BL21) DE3. The recombinant enzyme displayed good stability under acid pH and high-temperature conditions. [ABSTRACT FROM AUTHOR]

The article proves the ability of the entomopathogenic strain B. thuringiensis var. dendrolimus B-387 to high the constitutive production (3–12.5 U/mL) of extracellular chitosanase, that was found for the first time. The enzyme was purified in 94-fold by ultrafiltration, affinity sorption and cation-exchange chromatography and characterized biochemically. The molecular mass of the chitosanase determined using SDS-PAGE is 40 kDa. Temperature and pH-optima of the enzyme are 55 °C and pH 6.5, respectively; the chitosanase was stable under 50–60 °C and pH 4–10.5. Purified chitosanase most rapidly (Vmax ~ 43 µM/mL × min, KM ~ 0.22 mg/mL, kcat ~ 4.79 × 104 s−1) hydrolyzed soluble chitosan of the deacetylation degree (DD) 85% by endo-mode, and did not degrade colloidal chitin, CM-cellulose and some other glucans. The main reaction products of the chitosan enzymolysis included chitobiose, chitotriose and chitotetraose. In addition to small chitooligosaccharides (CHOs), the studied chitosanase also generated low-molecular weight chitosan (LMWC) with average Mw in range 14–46 kDa and recovery 14–35%, depending on the enzyme/substrate ratio and incubation temperature. In some cases, the chitosan (DD 85 and 50%) oligomers prepared using crude chitosanase from B. thuringiensis B-387 indicated higher antifungal and antibacterial activities in vitro in comparison with the initial polysaccharides. The data obtained indicate the good prospect of chitosanase B-387 for the production of bioactive CHOs. [ABSTRACT FROM AUTHOR]

Chitosanase could cleave β-1,4-linkage of chitosan to produce chitooligosaccharides (COS) with diverse biological activities. However, there are many limitations on the use of free chitosanase in industrial production. Enzyme immobilization is generally considered a valuable strategy in industrial-scale applications. In this study, the chitosanase Csn-BAC from Bacillus sp. MD-5 was immobilized on Fe3O4-SiO2 magnetic nanoparticles (MNPs) to enhance its properties, which could be recovered easily from reaction media using magnetic separation. The activities of Csn-BAC immobilized with MNPs (MNPs@Csn-BAC) were determined with temperature and pH, and the thermal- and pH-stabilities, respectively. The reusability of the MNPs@Csn-BAC was determined in repeated reaction cycles. Immobilization enhanced the thermal and pH stability of Csn-BAC compared with the free enzyme. After eight reaction cycles using MNPs@Csn-BAC, the residual enzyme activity was 72.15%. Finally, the amount of COS released by MNPs@Csn-BAC was 1.86 times higher than that of the free Csn-BAC in the catalytic performance experiment. The immobilized Csn-BAC exhibits broad application prospects in the production of COS. [ABSTRACT FROM AUTHOR]

Chitosan oligosaccharides (COSs) are widely applied in many areas due to its various biological activities. Controllable preparation of COSs with desired degree of polymerization (DP) via suitable chitosanase is of great value. Herein, a novel glycoside hydrolase (GH) family 46 chitosanase (SlCsn46) from marine Streptomyces lydicus S1 was prepared, characterized and used to controllably produce COSs with different DP. The specific activity of purified recombinant SlCsn46 was 1,008.5 U/mg. The optimal temperature and pH of purified SlCsn46 were 50◦C and 6.0, respectively. Metal ions Mn2+ could improve the stability of SlCsn46. Additionally, SlCsn46 can efficiently hydrolyze 2% and 4% colloidal chitosan to prepare COSs with DP 2–4, 2–5, and 2–6 by adjusting the amount of SlCsn46 added. Moreover, COSs with DP 2–4, 2–5, and 2–6 exhibited potential application value for prolonging the shelf-life of pre-packaged Tofu. The water-holding capacity (WHC), sensorial properties, total viable count (TVC), pH and total volatile base nitrogen (TVB-N) of prepacked tofu incorporated with 4 mg/mL COSs with DP 2–4, 2–5, and 2–6 were better than those of the control during 15 days of storage at 10◦C. Thus, the controllable hydrolysis strategy provides an effective method to prepare COSs with desired DP and its potential application on preservation of pre-packed tofu. [ABSTRACT FROM AUTHOR]

In order to improve the thermostability of Streptomyces avermitilis chitosanase SaCsn46A,the protein structure of SaCsn46A was simulated by using SWISS-MODEL online server using Streptomyces sp .SirexAA-E chitosanase（PDB accession number:4ilily .1A）as template .The predicted protein structure of SaCsn46A was submitted to online prediction software PoPMuSiC-2 .1to search for amino acid residues that had significant influence on the stability of SaCsn46A,and mutants were constructed by site-directed mutation .The wild-type chitosanase and its mutant were heterologous expressed in Escherichia coli Rosetta,respectively .The recombinant protein were purified by Ni-NTA affinity chromatography .Enzymatic properties of wild-type chitosanase and the mutant G237A were determined and the optimum temperature of G237A was increased by 5℃．And temperature stability and pH stability significantly improved .In order to further improve the expression efficiency of the mutant,the inducement conditions of the mutant expressing strain were optimized .The optimal conditions were determined as follows:IPTG concentration 0 .7mmol/L,induction temperature 20 ℃,induction time 8h . [ABSTRACT FROM AUTHOR]

In this study, we aimed to clone and further investigate the novel chitosanase Po CSN75A from Penicillium oxalicum M2 that we have previously identified and characterized. Sequence alignment and bioinformatics analysis revealed the conserved regions, enzyme classification, and other properties of Po CSN75A. Furthermore, the gene encoding Po CSN75A was heterologously expressed in Pichia pastoris GS115 and successfully purified via Ni-NTA chromatography. The purified recombinant Po CSN75A had an optimum pH and temperature of 5.5 and 60 °C, respectively. The enzyme was stable within a pH range of 3.0–6.0 and thermostable at 45 °C and below. Additionally, treatments with cations (Ca2+, Mn2+), non-ionic surfactants (Tween 20/40/60/80, Triton X-100), and common reducing agents (dithiothreitol [DTT], β-mercaptoethanol [β-ME]) were observed to significantly enhance the catalytic ability of the recombinant protein, which exhibited an obvious substrate preference for chitosan. For the enzyme kinetic parameters, the V max and K m values against colloidal chitosan were determined to be 4.36 U/mL and 0.27 mg/mL, respectively. Molecular docking (MD) for the overall model of enzymes and substrates identified several potentially important amino acid residues (D148, D150, T149 and E159). The recombinant protein was also found to exhibit a typical endohydrolytic pattern, with a chitooligosaccharide dimer and trimer as the final hydrolytic products. [Display omitted] ● A novel chitosanase gene from Penicillium oxalicum M2 was cloned. ● Po CSN75A expression in P. pastoris was 4.62 folds that of the original strain ● MD expanded the understanding of the catalytic mechanism of Po CSN75A. [ABSTRACT FROM AUTHOR]

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This study discovered a novel chitosanase from Penicillium oxalicum M2 based on a new screening strategy. An extracellular chitosanase was isolated and purified from the fermentation broth of Penicillium oxalicum M2. A 19.34-fold purification was achieved on a cation exchange column. Using sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, chitosanase was determined at approximately 42 kDa without any subunits. The sequence of peptide in the protein was identified as SALNKNYITNFSTLR by MALTI-TOF/TOF MS. The maximum catalytic activity of the purified enzyme was 60.45 U/mg at the optimum pH and temperature of 5.5 and 60 °C. The enzyme activity held stability in the range of 35–50 °C and pH 3–4.5. Ca2+, Mn2+, non-ionic surfactants (Tween 20/40/60/80 and Trition X-100) and some common reducing agents (DTT and β-ME) could significantly activate chitosanase. The purified enzyme showed rigorous specificity to chitosan as a substrate. The hydrolysate in the final stage of hydrolysis consisted of chitooligosaccharides with a degree of polymerization ranging from 2 to 5 and without glucosamine or acetylglucosamine. The monomeric enzyme obtained by one-step purification reveal applications potential in sugar industry, and expanded our understanding of the GH75 family chitosanases simultaneously. [ABSTRACT FROM AUTHOR]

Chitosan is a kind of linear alkaline polysaccharide, which consists of N -acetyl- D -glucosamine units and D -glucosamine units. Both chitosan and its degradation products (chitosan oligosaccharides) possess versatile biological activities such as antitumor, antioxidant, antimicrobial, and anti-inflammatory activities. Therefore, chitosan and its chitosan oligosaccharides held great potential in food, medicine, agriculture and other fields. Chitosanases are a class of glycoside hydrolases that could specifically degrade deacetylated chitosan and they could break the β-1, 4-glycosidic bonds between glucosamines. Chitosanases have been widely used in various fields, such as preparation of chitosan oligosaccharides (COS) with unique physiological activities, as biocontrol agents for plant pest and disease resistance, to facilitate chitosan-mediated gene delivery and to prepare protoplasts. Therefore, researches on the fundamentals and applications of chitosanases have received extensive and increasing attention. It is very important to summarize and discuss the results of the previous studies to carry out the subsequent work. In this review, we discuss the research progress on the structures, catalytic mechanisms, molecular modifications and applications of the chitosanases. This comprehensive information should be helpful to expand the applications of chitosanases. No data was used for the research described in the article. Data will be made available on request. • The structural features of chitosanases have been systematically discussed. • The catalytic mechanism of different types of chitosanases has been elucidated. • The enzymatic preparation of chitosan oligosaccharides has been summarized. [ABSTRACT FROM AUTHOR]

Marine crustacean waste has not been fully utilized and is a rich source of chitin. Enzymatic degradation has attracted the wide attention of researchers due to its unique biocatalytic ability to protect the environment. Chitosan (CTS) and its derivative chitosan oligosaccharides (COSs) with various biological activities can be obtained by the enzymatic degradation of chitin. Many studies have shown that chitosan and its derivatives, chitosan oligosaccharides (COSs), have beneficial properties, including lipid-lowering, anti-inflammatory and antitumor activities, and have important application value in the medical treatment field, the food industry and agriculture. In this review, we describe the classification, biochemical characteristics and catalytic mechanisms of the major degrading enzymes: chitinases, chitin deacetylases (CDAs) and chitosanases. We also introduced the technology for enzymatic design and modification and proposed the current problems and development trends of enzymatic degradation of chitin polysaccharides. The discussion on the characteristics and catalytic mechanism of chitosan-degrading enzymes will help to develop new types of hydrolases by various biotechnology methods and promote their application in chitosan. [ABSTRACT FROM AUTHOR]

Copyright of South China Fisheries Science is the property of South China Fisheries Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Chitosanase was widely used in the production of bioactive chitooligosacchride (CHOS) due to their safety, controllability, environmental protection, and biodegradability. Studies showed that the bioactivity of CHOS is closely related to its degree of polymerization. Therefore, the production of ideal polymerized CHOS becomes our primary goal. In this study, the glycosyl hydrolase (GH) family 5 chitosanase was successfully expressed heterologously in Pichia pastoris. After 96 h of high-density fermentation, the chitosanase activity reached 90.62 U·mL−1, the protein content reached 9.76 mg·mL−1. When 2% chitosan was hydrolyzed by crude enzyme (20 U/mL), the hydrolysis rate reached 91.2% after 8 h, producing a mixture of CHOS with 2–4 desirable degrees of polymerization (DP). Then, the antioxidant activity of CHOS mixture was investigated, and the results showed that the antioxidant effect was concentration-dependent and had great application potential in the field of nutrition. [Display omitted] • The Csn5 was successfully expressed in Pichia pastoris GS115. • The final hydrolysate of chitosan (DDA > 95%) was chitobiose, chitotriose and chitotetraose hydrolyzed by Csn5. • The hydrolysis rate of Csn5 toward chitosan (DDA>95%) reached 82.5% and 91.2% after 2 h and 4 h, respectively. • The final hydrolysate of showed excellent antioxidant effect. [ABSTRACT FROM AUTHOR]