Chitosanase Production Research Articles

Screening for Aspergillus fumigatus strain-2T-2 with high chitosanase production activity and its application in chitosan degradation

BackgroundHigh 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. ResultsIn 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. ConclusionsA. 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.

Production, purification, and characterization of acid-active chitosanase from Lentzea sp. OUR-I1 using squid pen waste as low-cost nutrient source with simultaneous recovery of bioactive compounds from microbial fermentation

Squid pen waste is a promising source of chitin and protein that can be converted to a vast array of industrially important products. This study used squid pen powder (SPP) as an inexpensive nutrient source and inducer for Lentzea sp. OUR-I1 to produce chitosanase (chitosanase OUR-I1). The highest chitosanase level was obtained from a medium containing 1.5% SPP, and 0.1% (NH4)2SO4 with initial pH 5.0 and incubation at 30 °C for 5 days. The enzyme possessed properties desirable in industrial application such as being acid active and acid tolerant (pH 3.0–5.5) with moderate thermal stability up to 60 °C. It was found that chitosanase OUR-I1 expressed an exceptional hydrolytic ability against native chitosan extracted from SPP and generated glucosamine (COS-DP1) and its dimer (COS-DP2) as main hydrolysis products which displayed great scavenging activities against DPPH and ABTS with EC50 values of 3.74 mg/mL and 9.82 mg/mL, respectively. Besides, protein hydrolysate of 5.7 mg/mL and reducing sugar of 0.18 mg/mL were generated from SPP during chitosanase production. Notably, the recovered fraction during purification step exhibited excellent antioxidant activities against DPPH (E50 = 0.99 mg/mL) and ABTS (E50 = 0.02 mg/mL). The findings showed the potential use of Lentzea sp. OUR-I1 as a candidate microbial tool for bioconversion of squid pen waste into chitosanase and hydrolysis products with high antioxidant activities. The present study has shown the cost-effective approach for chitosanase production with simultaneous recovery of industrially useful co-products.

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.

Constitutive chitosanase from Bacillus thuringiensis B-387 and its potential for preparation of antimicrobial chitooligomers.

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 40kDa. 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 ~ 43µM/mL × min, KM ~ 0.22mg/mL, kcat ~ 4.79 × 104s-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 14-46kDa 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.

Heterologous Expression and Characterization of a High-Efficiency Chitosanase From Bacillus mojavensis SY1 Suitable for Production of Chitosan Oligosaccharides.

Chitosanase plays an important role in enzymatic production of chitosan oligosaccharides (COSs). The present study describes the gene cloning and high-level expression of a high-efficiency chitosanase from Bacillus mojavensis SY1 (CsnBm). The gene encoding CsnBm was obtained by homologous cloning, ligated to pPICZαA, and transformed into Pichia pastoris X33. A recombinant strain designated X33-C3 with the highest activity was isolated from 120 recombinant colonies. The maximum activity and total protein concentration of recombinant strain X33-C3 were 6,052 U/ml and 3.75 g/l, respectively, which were obtained in fed-batch cultivation in a 50-l bioreactor. The optimal temperature and pH of purified CsnBm were 55°C and 5.5, respectively. Meanwhile, CsnBm was stable from pH 4.0 to 9.0 and 40 to 55°C. The purified CsnBm exhibited high activity toward colloidal chitosan with degrees of deacetylation from 85 to 95%. Furthermore, CsnBm exhibited high efficiency to hydrolyze different concentration of colloidal chitosan to produce COSs. The result of this study not only identifies a high-efficiency chitosanase for preparation of COSs, but also casts some insight into the high-level production of chitosanase in heterologous systems.

Solid-State Fermentation Production of Chitosanase by <i>Streptomyces</i> with Waste Mycelia of <i>Aspergillus niger</i>

Solid-state fermentation was carried out using mycelium powder of Aspergillus niger as substrate for the production of chitosanase of Streptomyces. Results of the experiments indicated that the optimal medium consisted of wheat bran and mycelium powder of Aspergillus niger with initial moisture content of 60% - 70%. The enzyme activity reached 41.33 U per gram dry medium after cultured for 5 days at 28&#176C - 30&#176C and an initial pH 6.5. Chitosanase was detected on the second day of incubation and had maximal activity at 5 days and decreased gradually within a 1 month period. Solid-state fermentation is maybe an economic alternative in the production.

Increasing chitosanase production in Bacillus cereus by a novel mutagenesis and screen method

ABSTRACT Chitosan hydrolysis by chitosanase is one of the most effective methods to produce chitosan oligosaccharides. One of the prerequisites of enzyme fermentation production is to select and breed enzyme-producing cells with good performance. So in the process of fermentation production, the low yield of chitosanase cannot meet the current requirement. In this paper, a strain producing chitosanase was screened and identified, and a novel mutagenesis system (Atmospheric and Room Temperature Plasma (ARTP)) was selected to increase the yield of chitosanase. Then, the fermentation medium was optimized to further improve the enzyme activity of the strain. A strain of Bacillus cereus capable of producing chitosanase was screened and identified from soil samples. A mutant strain of B.cereus was obtained by Atmospheric and Room Temperature Plasma mutagenesis and bioscreening method, and chitosanase activity was 2.49 folds that of the original bacterium. After an optimized fermentation medium, the enzyme activity of the mutant strain was 1.47 folds that of the original bacterium. Combined with all the above optimization experiments, the enzyme activity of mutant strain increased by 3.66 times. The results showed that the Atmospheric and Room Temperature Plasma mutagenesis and bioscreening method could significantly increase the yield of chitosanase in B.cereus, and had little effect on the properties of the enzyme. These findings have potential applications in the mutagenesis of other enzyme-producing microorganisms.

Statistical optimization of halophilic chitosanase and protease production by Bacillus cereus HMRSC30 isolated from Terasi simultaneous with chitin extraction from shrimp shell waste

Chitosanase is an industrially important enzyme involved in the production of bioactive chitooligosaccharides. In the present study, Bacillus cereus HMRSC30 isolated from the Indonesian shrimp paste was found to have high ability to assimilate shrimp shell powder (SSP), and selected as the microbial tool to produce low-cost chitosanase under statistical optimization. The highest chitosanase production (4.85 U/mL) was achieved under the optimal conditions: 20 g/L SSP, 3 g/L (NH4)2SO4, 0.3 g/L Mg2SO4, 1.5 g/L KH2PO4, 1.5 g/L K2HPO4, pH 5.5 with a 15% (v/v) inoculum size at 35 °C for 3 days incubation. Besides chitosanase, a high amount of protease (33.47–72.19 U/mL) was also synthesized during 7 days fermentation. The optimal fermentation conditions also simultaneously deproteinized (76.36–97.86%) and demineralized (24.07–67.22%) the SSP. These observations indicated the potential of the strain HMRSC30 in a biological process for the enzyme production simultaneous with chitin extraction. The purified chitosanase with an apparent MW of 41 kDa exhibited the highest specificity to 75–85% deacetylated chitosan in soluble form (100%), followed by colloidal form (73.6%) and soluble chitin (73.4%) in an acidic pH of 5.5 at 55 °C. Additionally, it was discovered that the chitosanase obtained was a halophilic enzyme which was fully activated by 2.5% (w/v) NaCl and displayed activity of 81.73% even in the presence of 7.5% (w/v) NaCl. The enzyme yielded a putative dimer, and trimer of glucosamine as the predominant hydrolysis products. This study revealed that the properties of chitosanase from the strain HMRSC30 would be useful in industrial applications.

Production of Chitosanase by Lentzea sp. OUR-I1 Using Acid-Pretreated Shrimp Shell in an Air-Lift Bioreactor and the Feasibility of Utilizing the Residual Biomass

Chitosanase is an attractive enzymatic tool for the production of bioactive chitooligosaccharides. Nevertheless, its industrial use is restricted by its high cost and insufficient availability because chitosanase production needs an expensive inducer, chitosan. Therefore, this study developed a process to produce chitosanase from abundant and inexpensive shrimp shell waste entailing the pretreatment of shrimp shell powder (SSP) with 0.4 M acetic acid (AcSSP), which increased the substrate bioavailability and enhanced the chitosanase production by Lentzea sp. OUR-I1 four-fold. The initial flask-based process was scaled up into an air-lift bioreactor (ALB) and the maximum chitosanase activity (0.974 U/mL) was observed at an aeration rate of 2.0 vvm. This is the first report indicating the suitability of ALB for chitosanase production by Lentzea sp. OUR-I1. Interestingly, Lentzea sp. OUR-I1 also produces a yellow pigment with prospective structure as carotenoids, indicating that the mycelium could be reused in the production of pigments. In addition, the fermented AcSPP displayed the FTIR spectrum of chitin and has high potential as an absorbent for methyl orange, methylene blue and coomassie brilliant blue, with a maximum decolorization efficiency of 73.21, 64.59 and 87.93%, respectively. The process developed represents a cost effective and environmentally friendly method of valorizing a waste product from the food industry into valuable bioproducts.

Enhanced glucosamine production through synergistic action of Aspergillus terreus chitozymes

Chitin rich wastes generated from seafood processing industries have become critical concern for coastal area pollution however their transformation into glucosamine (GlcN) could be considered as a value-addition approach. Although, GlcN production through chemical means is in wide practice but, harsh reaction conditions and variable product quality led to urge of biological synthesis. Moreover, the enzymatic means of production need to be explored further to achieve increased production levels of chitozymes that could be employed for GlcN generation. The present study is aimed to develop an integrated process comprised of chemo-thermal pretreatment (to overcome chitin recalcitrance) followed by enzymatic hydrolysis through myco-chitozymes to augment chitin degradation for production of high GlcN titres. Aspergillus terreus has been observed to produce comparatively high titres of chitinase (229 Ul−1), chitosanase (35 Ul−1) and GlcN (213 mg l−1) from shrimp shell chitin as compared to Fusarium pallidoroseum. Utilization of sulphuric acid (2%, w/v) in combination of thermal pretreatment (121 °C, 15 psi, 60 min) was appeared as the most effective pretreatment strategy and resulted into enhanced chitinase (320 Ul−1) and chitosanase (58 Ul−1) production. Augmentation of GlcN production (772 mg l−1) was achieved by synergistic action of chitinase and chitosanase at 2:1 ratio onto pretreated chitin. Thin layer chromatography (TLC) revealed significant production of GlcN via enzymatic hydrolysis. Enhancement in GlcN production through developed chemo-enzymatic method not only provides an eco-friendly, sustainable solution to reduce coastal area pollution but also paves the way to ameliorate industrial production of GlcN in an economic way.

Enzymatic production of low-Mw chitosan-derivatives: Characterization and biological activities evaluation

This study reports the enzymatic depolymerization of chitosan by the chitosanolytic preparation of Bacillus licheniformis GA11. For this purpose, chitosanase production and biochemical characterization of the crude enzyme preparation from GA11 were firstly investigated. The highest chitosanase production was obtained in a culture medium containing 50.0 g/l of a mixture of soluble starch/shrimp shells/crab shells and 5.0 g/l tryptone, after incubation during 5 days, at 30 °C with a pHi of 6.0 and under continuous agitation at 200 rpm. Then, the chitosanolytic preparation, exhibiting maximum activity at 65 °C and pH 5.0, was used to hydrolyze chitosan, leading to various chitosan-depolymerization products (CDP) with different physicochemical characteristics. Finally, the antioxidant and antimicrobial potentials of CDP were evaluated, allowing to conclude that the molecular weight and the acetylation degree highly affect the biological activities of CDP.

In vitro evaluation of some isolated bacteria against the plant parasite nematode Meloidogyne incognita

BackgroundMicrobial cultures are extensively used as environment friendly ways for biological control of parasitic pests, including the root-knot nematodes, alternative to the use of chemical nematicides. The present study was conducted to isolate some lytic rhizobacteria and examine their nematicidal activity against Meloidogyne incognita J2 mortality and egg hatching in in vitro test.ResultsLytic Rhizobacteria were isolated from soil samples adhering to tomato plant roots from different localities at Giza governorate, Egypt. Six bacterial isolates, exhibited high efficacy against root-knot nematodes, were identified based on the analysis of the 16S rRNA gene sequence as Pseudomonas aeruginosa, Paenibacillus polymyxa, Lysinibacillus sphaericus, Bacillus cereus, Bacillus subtilis, and Achromobacter xylosoxidans. These strains showed high production of chitinase, chitosanase, and protease using colloidal chitin and soluble chitosan as carbon sources. Two strains, Paenibacillus polymyxa and Bacillus subtilis, produced the highest levels of chitinase in the media. Except for Pseudomonas aeruginosa, all the bacterial strains produced high levels of chitosanase.ConclusionThe results revealed that high amounts of protease were, however, secreted by Pseudomonas aeruginosa as compared to the other strains. In in vitro tests, all the bacterial culture filtrates potentially displayed nematicidal effect in M. incognita egg hatching and an obvious increase in J2 mortality as compared to control. Paenibacillus polymyxa caused 100% juvenile mortality followed by Bacillus subtilis 97.25%, Bacillus cereus 94%, Achromobacter xylosoxidans 93%, Lysinibacillus sphaericus 92%, and Pseudomonas aeruginosa 84.29% after 48 h of exposure, as compared to control.

Enzymatic activity and secondary metabolite profile of Trichoderma asperellum in presence of chitosan

The present research was aimed to study enzymatic activity (cellulase and chitosanase) and secondary metabolite production of Trichoderma asperellum in presence of chitosan. The qualitative assays for cellulase and chitosanase showed enhanced enzymatic activity of the fungus. The H/C ratio obtained was 1.76 and 1.49 at 500 ppm and 250 ppm of chitosan respectively, which was significantly higher, as compared to control (H/C ratio 1.2). For chitosanase activity, the largest purple zone (55.8 mm) was recorded in case of T. asperellum + chitosan 500 ppm which was significantly higher than other treatments. Besides enhancing enzymatic activity, chitosan also increased the production of two important secondary metabolites of T. asperellum viz. Tetradecanoic acid (myristic acid) and 1,2-benzenedicarboxylic acid. GC–MS results revealed that the maximum peak area of chromatogram was covered by tetradecanoic acid (28.23%) followed by 1,2-benzenedicarboxylic acid (18.16%) showing the high concentration of these metabolites as compared to other compounds. The enhanced production of cellulase, chitosanase and secondary metabolites by T. asperellum in presence of chitosan could be exploited for further research in improvement of bio-control activity of bio-agents for integrated plant disease management.

Microbial valorization of shrimp byproducts via the production of thermostable chitosanase and antioxidant chitooligosaccharides

Abstract Chitinous materials present in marine byproducts attract the research focus for their potential agricultural and biomedical applications. In the present study, shrimp processing byproduct was used for the production of chitosanase by solid state fermentation using Bacillus cereus strain SSW1. The isolated strain was identified by Gram stain, MALDI-TOF mass spectrometry and 16S DNA sequencing. The enzyme production was statistically optimized to reach 39.774U/g dry substrate by 5.3 fold of increase. The activity of the partially purified enzyme was optimized in respect to the effect of temperature, pH and substrate concentration in which the enzyme was indicated to be thermo-active and thermo-stable. Although several chitosanases have been previously studied, thermostable enzymes are still rather rare. The Km, Vmax and Kcat values were calculated to be 7.55 mg/mL, 30.3U/mL/min and 1.9min-1 and the half lives of the enzyme at 55, 60 and 65 °C were 693.15, 346.57 and 77.02min-1 respectively, confirming the thermo-stability of the produced enzyme. The antifungal activity of the produced enzyme was indicated against the phytopathogenic fungus Fusarium oxysporum. Additionally, chitooligosaccharides were obtained by the enzymatic hydrolysis of chitosan and by the bacterial hydrolysis of shrimp byproducts that was confirmed by thin layer chromatography analysis and Fourier Transform Infrared Spectroscopy. Finally, the purified chitooligosaccharides showed in vitro antioxidant activity assessed by 1,1-diphenyl-2-picrylhydrazyl free radical scavenging assay.

Production of chitosanase from termophylic bacteria isolated from Bora Hotspring

The production of chitosanase enzyme was carried out by utilizing isolate of thermophilic bacteria from Bora hot spring in order to determine the optimum condition of its production process. B1211 bacterial isolates with the highest Chitinolytic Index values of 1.71 were used in crude chitosanase production. The crude chitosanase production was performed using a Completely Randomized Design (RAL) consisting of four independent variables, in the form of chitosan substrate concentration (0.25%, 0.50%, 0.75%, 1.00%), agitation speed (60, 90, 120, and 150 rpm), fermentation time (1, 3, 5, 7, and 9 days), and fermentation temperature (30, 40, 50, and 60°C). The activity of chitosanase as dependent variable was determined by Schales method, which referred to standard curve of D-glucosamine. The results showed that 1% chitosan substrate concentration yielded crude chitosanase with the highest activity of 0.408 U/mL and dissolved protein content of 9.887 mg/mL. Furthermore, the best agitation speed (90 rpm) resulted in 0.385 U/mL chitosanase activity and dissolved protein content of 23.74 mg/mL. In the fermentation time treatment, the 5-day fermentation time resulted in the highest chitosanase activity of 0.384 U/mL with dissolved protein content of 36.52 mg/mL. The fermentation temperature determination showed that the temperature of 60 °C as the preferred treatment with chitosanase activity of 0.569 U/mL and dissolved protein content of 5.752 mg/mL.

Production of a Thermostable Chitosanase from Shrimp Heads via Paenibacillus mucilaginosus TKU032 Conversion and its Application in the Preparation of Bioactive Chitosan Oligosaccharides.

Chitosanase has attracted great attention due to its potential applications in medicine, agriculture, and nutraceuticals. In this study, P. mucilaginosus TKU032, a bacterial strain isolated from Taiwanese soil, exhibited the highest chitosanase activity (0.53 U/mL) on medium containing shrimp heads as the sole carbon and nitrogen (C/N) source. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, a chitosanase isolated from P. mucilaginosus TKU032 cultured on shrimp head medium was determined at approximately 59 kDa. The characterized chitosanase showed interesting properties with optimal temperature and thermal stability up to 70 °C. Three chitosan oligosaccharide (COS) fractions were isolated from hydrolyzed colloidal chitosan that was catalyzed by TKU032 chitosanase. Of these, fraction I showed the highest α-glucosidase inhibitor (aGI) activity (65.86% at 20 mg/mL); its inhibitory mechanism followed the mixed noncompetitive inhibition model. Fractions II and III exhibited strong 2,2-diphenyl1-picrylhydrazyl (DPPH) radical scavenging activity (79.00% at 12 mg/mL and 73.29% at 16 mg/mL, respectively). In summary, the COS fractions obtained by hydrolyzing colloidal chitosan with TKU032 chitosanase may have potential use in medical or nutraceutical fields due to their aGI and antioxidant activities.

Production, Optimization and Characterisation of Chitosanase of Bacillus sp. and its Applications in Nanotechnology

Chitosanases is a class of enzymes which hydrolyse chitosan, a natural biopolymer consisting of d-glucosamine in various degrees. In this study, chitosanase producing Bacillus sp. was isolated from soil sample. Chitosanase production was optimized using response surface methodology and the produced chitosanase was characterized. The crude enzyme was found to possess antibacterial and antifungal activity. Chitosanase enzyme was used for trimming chitosan based polymeric nanoparticles produced using sodium trimetaphosphate chelator. Chitosanase enzyme was also utilized for synthesis of silver nanoparticles which were then characterized by UV–Vis, FTIR, SEM, TEM and AFM. The produced nanoparticles were checked for antibacterial and antifungal activity.

Examining the interplay between Streptococcus agalactiae, the biopolymer chitin and its derivative

Streptococcus agalactiae is a highly pathogenic bacterium of aquatic species and terrestrial animals worldwide, whereas chitin and its derivative chitosan are among the most abundant biopolymers found in nature, including the aquatic milieu. The present investigation focused on the capability of S. agalactiae to degrade and utilize these polymers. Growth of S. agalactiae in the presence of colloid chitin, chitosan, or N‐acetyl‐glucosamine (GlcNAc) was evaluated. Chitosanase production was measured daily over 7 days of growth period and degraded products were evaluated with thin later chorography. Chitin had no effect on the growth of S. agalactiae. Degraded chitin, however, stimulated the growth of S. agalactiae. S. agalactiae cells did not produce chitinase to degrade chitin; however, they readily utilize GlcNAc (product of degraded chitin) as sole source of carbon and nitrogen for growth. Chitosan at high concentrations had antibacterial activities against S. agalactiae, while in the presence of lower than the inhibitory level of chitosan in the medium, S. agalactiae secrets chitosanase to degrade chitosan, and utilizes it to a limited extent to benefit growth. The interaction of S. agalactiae with chitin hydrolytes and chitosan could play a role in the diverse habitat distribution and pathogenicity of S. agalactiae worldwide.