Hydrolysis Of Chitosan Research Articles

Enzymatic Production of Chitooligosaccharide Using a GH Family 46 Chitosanase from Paenibacillus elgii and Its Antioxidant Activity

Chitooligosaccharide (COS), a natural antioxidant, is a hydrolysis product of chitosan created using enzymatic or chemical methods. COS has received considerable attention recently, making its efficient bioproduction of great value. This study investigated the optimal conditions for the enzymatic method using a GH family 46 chitosanase from Paenibacillus elgii TKU051 to prepare COS based on the response surface methodology (RSM). The results showed optimal values for chitosan hydrolysis, such as a pH of 5.5, an incubation temperature of 58.3 °C, an [E]/[S] ratio of 118.494 (U/g), and an incubation time of 6.821 h. Under the optimal conditions, the highest reducing sugar level (per substrate, w/w) of the chitosan hydrolysis process that could be reached was 690.587 mg/g. The composition of the obtained COS was analyzed using the thin-layer chromatography (TLC) method, yielding (GlcN)2 and (GlcN)3 as the products. The ascorbic acid equivalent antioxidant capacity (AEAC) of the obtained COS was found to be 1246 mg/100 g (via a DPPH (2,2-diphenyl-1-picrylhydrazyl) radical-scavenging assay) and 3673 mg/100 g (via an ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) radical-scavenging assay). This green and efficient bioproduction method may possess excellent potential for application in bioactive COS preparation.

Continuous production of chitooligosaccharides in a column reactor by the PUF-immobilized whole cell enzymes of Mucor circinelloides IBT-83

BackgroundChitosan 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.ResultsThe 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).ConclusionsThe 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.

Effects of chitosan hydrolysate on control of postharvest infection caused by Botrytis cinerea and physiological responses of wounded tomato fruit

Chitosan is considered an eco-friendly plant protection agent. Chitosan hydrolysate (CH) with an average molecular weight (MW) of the main fraction of 135 × 103 Da, with a deacetylation degree (DD) of 93 % is an unfractionated product of nitric acid hydrolysis of high molecular weight chitosan. The effect of the CH on gray mold caused by Botrytis cinerea in tomato fruit stored at 25 °C was investigated. Chitosan provided effective control of B. cinerea on tomato fruit. The CH treatment of wounded tomato fruit had a protective effect, reducing the percentage of infected fruit on the 7 d after treatment to 50 %, compared with untreated wounded fruit (69 %) and water-treated fruit (88 %). The protective effect of the CH in tomato fruit may be due to a direct fungitoxic action against the pathogen. It was found that the CH induced an immune response in tomato fruit via the accumulation of phenolic compounds. The level of phenolic compounds in the CH group was higher than in the untreated wound group from day 1 to 5, with a maximum reached on day 3 (113 mg-eq GA kg-1). No significant differences were found in the activities of peroxidase, phenylalanine ammonia lyase, polyphenol oxidase, β-1,3-glucanase and chitinase. Thus, the CH affects the processes occurring during fruit damage and contributes to the preservation of the consumer qualities of fruit.

Expression of chitosanase from Aspergillus fumigatus chitosanase in Saccharomyces cerevisiae by CRISPR-Cas9 tools.

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.

Chitinous material bioconversion by three new chitinases from the yeast Mestchnikowia pulcherrima

BackgroundChitinases are widely distributed enzymes that perform the biotransformation of chitin, one of the most abundant polysaccharides on the biosphere, into useful value-added chitooligosaccharides (COS) with a wide variety of biotechnological applications in food, health, and agricultural fields. One of the most important group of enzymes involved in the degradation of chitin comprises the glycoside hydrolase family 18 (GH18), which harbours endo- and exo-enzymes that act synergistically to depolymerize chitin. The secretion of a chitinase activity from the ubiquitous yeast Mestchnikowia pulcherrima and their involvement in the post-harvest biological control of fungal pathogens was previously reported.ResultsThree new chitinases from M. pulcherrima, MpChit35, MpChit38 and MpChit41, were molecularly characterized and extracellularly expressed in Pichia pastoris to about 91, 90 and 71 mU ml− 1, respectively. The three enzymes hydrolysed colloidal chitin with optimal activity at 45 ºC and pH 4.0-4.5, increased 2-times their activities using 1 mM of Mn2+ and hydrolysed different types of commercial chitosan. The partial separation and characterization of the complex COS mixtures produced from the hydrolysis of chitin and chitosan were achieved by a new anionic chromatography HPAEC-PAD method and mass spectrometry assays. An overview of the predicted structures of these proteins and their catalytic modes of action were also presented. Depicted their high sequence and structural homology, MpChit35 acted as an exo-chitinase producing di-acetyl-chitobiose from chitin while MpChit38 and MpChit41 both acted as endo-chitinases producing tri-acetyl-chitotriose as main final product.ConclusionsThree new chitinases from the yeast M. pulcherrima were molecularly characterized and their enzymatic and structural characteristics analysed. These enzymes transformed chitinous materials to fully and partially acetylated COS through different modes of splitting, which make them interesting biocatalysts for deeper structural-function studies on the challenging enzymatic conversion of chitin.

Improving the technology for obtaining of low molecular weight chitosan under conditions of controlled enzymatic cleavage

Relevance. Annotation. Experimental data on improving the production technology of regulated enzymatic cleavage of chitosan to obtain its low-molecular derivatives are presented. Experimental studies were carried out in the conditions of the Department of Disease Diagnostics, Therapy, Obstetrics and Animal Reproduction of the Moscow State Academy of Veterinary Medicine and Biotechnology — MVA named after K.I. Skryabin and on the basis of the All-Russian Research and Technological Institute of the Biological Industry.Methods. Acid-soluble chitosan from king crab shell (MM 700 kDa, SDA 85%), chitosan succinate (MM 330 kDa, SZ 75.2%), low-molecular food chitosan (hydrochloride) (MM 50 kDa) were used as raw materials for obtaining new modifications of chitosan, gel chitosan (2% solution in 2% acetic acid) manufactured by Bioprogress LLC.Results. As a result of the experiment, the technological parameters of obtaining low-molecular chitosan by enzymatic hydrolysis were selected. The greatest decrease in the dynamic viscosity of chitosan was reliably observed in the pilot-industrial series II (by 29.3 times), and the pilot-industrial series I and III — by 6.9 and 10.6 times, respectively. As a result of enzymatic hydrolysis of chitosan in the pilot series II, it was possible to reduce the molecular weight from 700 to 24 kDa, and in series I and III — to 102 and 66 kDa. Based on the research results, a technological scheme for obtaining low-molecular-weight chitosan derivatives by enzymatic hydrolysis has been developed, tested and proposed for industrial use, technical conditions have been developed.

Valorization of marine by-products using green technology

In this work, two cases of marine by-product valorization using green technologies were highlighted, i.e., utilization of high-pressure carbon dioxide (HPCD) for demineralization of goldstripe sardinella (Sardinella gibbosa) scales and the use of non-specific enzymes for chitosan hydrolysis. After demineralization, type I collagen can be recovered from sardine scales. This work used HPCD process (of up to 10 bar or 1 MPa) to remove minerals from scales instead of the traditional strong acid method. Results from initial phase of the work showed that the process efficiency was significantly affected by pressure applied and treatment time (p < 0.05). And the demineralization efficiency reached was significantly lower than that achieved by treatment with hydrochloric acid (p < 0.05). In the second study, the chitosanolytic activity of four non-specific enzymes were assessed for chitosan hydrolysis. The results indicated that acid protease and cellulase could be a potential choice to produce chitooligosaccharides (COS) from shrimp shell.

Chitosanase Production from the Liquid Fermentation of Squid Pens Waste by Paenibacillus elgii.

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.

Production, Characterization and Application of a Novel Chitosanase from Marine Bacterium Bacillus paramycoides BP-N07.

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.

Production, statistical evaluation and characterization of chitosanase from Fusarium oxysporum D18

PurposeThe 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.MethodA 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.ResultsFusarium 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.ConclusionAn 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.

Crude Enzyme Concentrate of Filamentous Fungus Hydrolyzed Chitosan to Obtain Oligomers of Different Sizes.

Chitosan is a non-cytotoxic polysaccharide that, upon hydrolysis, releases oligomers of different sizes that may have antioxidant, antimicrobial activity and the inhibition of cancer cell growth, among other applications. It is, therefore, a hydrolysis process with great biotechnological relevance. Thus, this study aims to use a crude enzyme concentrate (CEC) produced by a filamentous fungus to obtain oligomers with different molecular weights. The microorganism was cultivated in a liquid medium (modified Czapeck-with carboxymethylcellulose as enzyme inducer). The enzymes present in the CEC were identified by LC-MS/MS, with an emphasis on cellobiohydrolase (E.C 3.2.1.91). The fungus of the Aspergillus genus was identified by amplifying the ITS1-5.8S-ITS2 rDNA region and metaproteomic analysis, where the excreted enzymes were identified with sequence coverage greater than 84% to A. nidulans. Chitosan hydrolysis assays compared the CEC with the commercial enzyme (Celluclast 1.5 L®). The ability to reduce the initial molecular mass of chitosan by 47.80, 75.24, and 93.26% after 2.0, 5.0, and 24 h of reaction, respectively, was observed. FTIR analyses revealed lower absorbance of chitosan oligomers' spectral signals, and their crystallinity was reduced after 3 h of hydrolysis. Based on these results, we can conclude that the crude enzyme concentrate showed a significant technological potential for obtaining chitosan oligomers of different sizes.

Enhancing Enzyme Activity and Thermostability of Bacillus amyloliquefaciens Chitosanase BaCsn46A Through Saturation Mutagenesis at Ser196

Chitosanase plays an important role in chitooligosaccharides (COS) production. We found that the chitosanase (BaCsn46A) of Bacillus amyloliquefacien was a good candidate for chitosan hydrolysis of COS. In order to further improve the enzyme properties of BaCsn46A, the S196 located near the active center was found to be a critical site impacts on enzyme properties by sequence alignment analysis. Herein, saturation mutation was carried out to study role of 196 site on BaCsn46A catalytic function. Compared with WT, the specific enzyme activity of S196A increased by 118.79%, and the thermostability of S196A was much higher than WT. In addition, we found that the enzyme activity of S196P was 2.41% of that of WT, indicating that the type of amino acid in 196 site could significant affect the catalytic activity and thermostability of BaCsn46A. After molecular docking analysis we found that the increase in hydrogen bonds and decrease in unfavorable bonds interacting with the substrate were the main reason for the change of enzyme properties which is valuable for future studies on Bacillus species chitosanase.

Identification of a Key Loop for Tuning Transglycosylation Activity in the Substrate-Binding Region of a Chitosanase.

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.

Cytotoxicity on Cancer Cells according to Average Molecular Weight of Chitosan Hydrolysates

To confirm the molecular characteristics of chitooligosaccharides (COS) derived from the hydrolysis of high molecular weight chitosan (HMWC), measuring average molecular weight (MW) using gel permeation chromatography (GPC) and their cytotoxicity was confirmed by treatment with several cancer cells. To evaluate the cytotoxicity of Chi-S according to different MW on various cancer cells, two types of chitooligosaccharide (COS) named Chi-S and COS-S were subjected in a concentration-dependent manner. It was divided into chitinous active substance (Chi-S) containing N-acetylglucosamine and chitosan oligosaccharide substance (COS-S), respectively. The average MW of Chi-S obtained by enzymatic treatment of HMWC for 24 h was confirmed to be about 2.7 kDa. Chi-S showed no cytotoxicity against all treated cancer cells. However, rather than showing cytotoxicity in immune macrophages RAW264.7, significant cell proliferation was observed. On the other hand, COS-S (average MW of approximately 7.3-kDa) obtained by enzymatic treatment of HMWC for 12 h showed significant cytotoxicity against all cancer cells including immune macrophages RAW264.7 at a high concentration range of 100 mg/mL or more. Furthermore, no significant changes were observed in breast cancer cells and normal cells treated with the control group. Therefore, based on the results, we estimate that Chi-S may be involved in indirect anticancer activity by selective or adaptive immunological activity according to the molecular characteristics. Our data will be a good guide for subsequent studies that can focus on the particular activity of molecules according to the difference between chitosan MW and the deacetylation degree (DD) of HMWC.

Kinetic Modeling of an Enzyme Membrane Reactor for the Selective Production of Oligosaccharides

An enzyme membrane reactor is an attractive tool for producing oligosaccharides from biomass-based polysaccharides. However, kinetic modeling and reactor design based on the rate equations have rarely been reported for enzyme membrane reactors because of the difficulty in tracing the depolymerization process. In this study, a simplified reaction model based on Michaelis–Menten-type kinetics has been built to simulate the enzyme membrane reactor. Ramping various species into reactant, target, and byproduct worked well for discussing reactor performance. The use of a membrane with a molecular weight cut-off (MWCO) of 10 kDa with continuous feeding of the reactant was suggested for the efficient production of chitosan hexamer and pentamer by enzymatic hydrolysis of chitosan.

Chitosan capped silver nanoparticles: Adsorption and photochemical activities

Chitosan capped silver nanoparticles (Chi-Ag) were prepared using AgNO3 and sodium borohydride. Chitosan was detected by using ninhydrin test, thermal gravimetric analysis and measurement of relative viscosity. Chi-Ag was used for removal of cadmium (Cd2+) at room temperature. The maximum monolayer adsorption capacity, and sorption intensity were estimated to be 119.04 mg/g and 1.6, respectively, from Langmuir and Freundlich adsorption isotherm models. The kinetics of Cd2+ adsorption onto Chi-Ag was proceeds through the pseudo-second-order kinetic model. Boyd and Elovich models suggest the adsorption and/or coordination of Cd2+ with the NH2 and OH groups of chitosan along with AgNPs proceeds through the film diffusion and chemisorption process. The average viscosity molecular weight of chitosan and Chi-Ag decreased with increased potassium persulfate (K2S2O8) and hydrogen peroxide (H2O2) concentration. The presence of H2O2 and K2S2O8 promoted the hydrolysis of chitosan due to the cleavage of glycosidic bond by generated HO and SO4− radicals.

Effect of enzymatic-ultrasonic hydrolyzed chitooligosaccharide on rheology of gelatin incorporated yogurt and 3D printing

Bioactive chitooligosaccharide from enzymatic-ultrasonic hydrolysis of chitosan could work as modification agent for functionalizing the stirred yogurt and promoting the practical application. Microstructure, rheological property, texture, sensory quality , storage stability and 3D printability of the functionalized yogurt were investigated. Compared with chitosan, the obtained chitooligosaccharide has microscopic spherical shapes, more active groups and higher antioxidant activity . Introducing chitooligosaccharide-gelatin composite into yogurt in proper sequence could induce the great microscopic morphology, outstanding particle size distribution of yogurt and satisfactory gel structure. Rheology results further indicate that chitooligosaccharide could promote the structured matrix, great shearing-thinning performance, excellent deformation resistance, enhanced network, high thermo-reversible recuperative structure, great thixotropy and outstanding mechanical strength of yogurt. The fitted flow index and yield stress by Power-law and Herschel-bulkley illustrate the favorable shearing dependence, good extrudability, and satisfactory structure maintainability. Moreover, the prepared yogurt has satisfying texture and sensory quality, and could maintain probiotics at high level and thus great storage stability. The further 3D printed yogurt products on bread, and the further developed yogurts with bioactive ingredients suggest the promise in accelerating more types of nutritional dairy products with high quality and high application value. • Chitooligosaccharide is obtained from enzymatic-ultrasonic hydrolysis of chitosan. • Chitooligosaccharide enhances yogurt with good size distribution and gel structure. • Yogurt has great shear-thinning ability, elasticity, reversibility and thixotropy. • Yogurt possesses satisfying texture, sensory quality and favorable storage stability. • 3D printing of yogurts with high shape retention shows food customization potential.

꾸지뽕나무 열매 추출물의 Chlorogenic Acid, Rutin 함량 분석 및 생리활성

This study was carried out to very precisely understand the effect of solvent selection on the molecular properties in the investigation of the biological activity according to the molecular size of chitosan. Chitosan hydrolysates derived from the hydrolysis of high molecular weight chitosan (HMWC) were prepared in enzymatic hydrolysis under solvent selective conditions. Chitosan hydrolysates were characterized by HPLC and Matrix-Assisted Laser Desorption/ Ionization-Time of Flight (MALDI-TOF) mass spectrometry analyses, respectively. HPLC analysis did not show a significant difference in the size of hydrolysates according to the type of chitosan. On the other hand, it was confirmed that each molecular size was significantly different from the results presented by HPLC with MALDI-TOF mass spectrometry analysis. In addition, the effect on the antibacterial activity of the hydrolysates of chitosan, designated to “active molecular chitosan (AMC)”, by the solvent selected for the enzymatic reaction also showed a significant difference. Therefore, it is intended to suggest that the selection of solvents for the enzymatic reaction for chitosan hydrolysis and their antibacterial activity is very important.

Heterogenously‐expressed chitosanase combining a green ball milling method for enzymatic degradation

AbstractChitooligosaccharides (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.

Effect of Surfactant HLB Value on Enzymatic Hydrolysis of Chitosan

Nonionic surfactants are reported as being able to enhance enzyme stability and increase the conversion of enzymatic reactions. Surfactant-assisted enzymatic hydrolysis conversion is affected by surfactant HLB values. This work investigated the influence of nonionic surfactants with different HLB values on chitosan enzymatic hydrolysis using cellulase enzyme by measuring the reducing sugars formation, viscosity, and molecular weight of hydrolyzed chitosan. A characterization analysis of hydrolyzed products was also carried out. A higher HLB value exhibits a better enzymatic chitosan hydrolysis performance, shown by the decrease in a solution’s viscosity and the increase in reducing sugar formation. Increasing the surfactant concentration will also increase the hydrolysis rate. Nonionic surfactants can protect cellulase enzyme from the denaturation of temperature and stirring influence. The higher the HLB value, the lower the molecular weight of the hydrolyzed chitosan. The result of UV–Vis demonstrated aldehyde groups formation during hydrolysis. The SEM analysis showed that the chitosan, hydrolyzed using different HLB values of surfactants, had different surface morphologies. However, it did not change the chemical structure of the hydrolysis product seen by the FTIR analysis. The XRD patterns showed that the relative crystallinity of raw chitosan decreased when hydrolyzed with surfactants.