Expression and Characterization of a Novel Cold-Adapted Chitosanase from Marine Renibacterium sp. Suitable for Chitooligosaccharides Preparation

(1) Background: Chitooligosaccharides (COS) have numerous applications due to their excellent properties. Chitosan hydrolysis using chitosanases has been proposed as an advisable method for COS preparation. Although many chitosanases from various sources have been identified, the cold-adapted ones with high stability are still rather rare but required. (2) Methods: A novel chitosanase named CsnY from marine bacterium Renibacterium sp. Y82 was expressed in Escherichia coli, following sequence analysis. Then, the characterizations of recombinant CsnY purified through Ni–NTA affinity chromatography were conducted, including effects of pH and temperature, effects of metal ions and chemicals, and final product analysis. (3) Results: The GH46 family chitosanase CsnY possessed promising thermostability at broad temperature range (0–50 °C), and with optimal activity at 40 °C and pH 6.0, especially showing relatively high activity (over 80% of its maximum activity) at low temperatures (20–30 °C), which demonstrated the cold-adapted property. Common metal ions or chemicals had no obvious effect on CsnY except Mn2+ and Co2+. Finally, CsnY was determined to be an endo-type chitosanase generating chitodisaccharides and -trisaccharides as main products, whose total concentration reached 56.74 mM within 2 h against 2% (w/v) initial chitosan substrate. (4) Conclusions: The results suggest the cold-adapted CsnY with favorable stability has desirable potential for the industrial production of COS.

Efficient Preparation of Chitooligosaccharide With a Potential Chitosanase Csn-SH and Its Application for Fungi Disease Protection

Chitosanase plays a vital role in bioactive chitooligosaccharide preparation. Here, we characterized and prepared a potential GH46 family chitosanase from Bacillus atrophaeus BSS. The purified recombinant enzyme Csn-SH showed a molecular weight of 27.0 kDa. Csn-SH displayed maximal activity toward chitosan at pH 5.0 and 45°C. Thin-layer chromatography and electrospray ionization–mass spectrometry indicated that Csn-SH mainly hydrolyzed chitosan into (GlcN)2, (GlcN)3, and (GlcN)4 with an endo-type cleavage pattern. Molecular docking analysis demonstrated that Csn-SH cleaved the glycoside bonds between subsites −2 and + 1 of (GlcN)6. Importantly, the chitosan hydrolysis rate of Csn-SH reached 80.57% within 40 min, which could reduce time and water consumption. The hydrolysates prepared with Csn-SH exhibited a good antifungal activity against Magnaporthe oryzae and Colletotrichum higginsianum. The above results suggested that Csn-SH could be used to produce active chitooligosaccharides efficiently that are biocontrol agents applicable for safe and sustainable agricultural production.

Increasing chitosanase production in Bacilluse cereus by a novel mutagenesis and screeen method

Background Chitosan hydrolysis by chitosanase is one of the most effective methods to produce chitosan oligosaccharides, however, the low yield of chitosanase cannot meet the current requirement. In this paper, a strain producing chitosanase was screened, and a novel mutagenesis system (1)) was selected to increase the yield of chitosanase. The hydrolyzed products from chitosan by chitosanase produced by mutan train were also analyzed by LC-MS. Results A strain of Bacillus cereus capable of producing chitosanase was screened and identified from soil samples. A mutant strain of Bacillus cereus was obtained by ARTP mutagenesis and bioscreening method, and chitosanase activity was 2.49 folds that of the original bacteria. After optimized fermentation conditions, the enzyme activity of the mutant strain was 3.3 folds that of the original bacteria. The relative molecular weight of the purified chitosanase was 43 kDa. Ten chitosan oligosaccharides(2–4 oligosaccharides) were obtained by hydrolyzing chitosan with it. Conclutions: The results showed that the ARTP 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 mutagenesis of other enzyme-producing microorganisms.

Membrane Technology Application for Fractionation Process to Obtain High Quality Glucosamine

Glucosamine, monosaccharide from chitosan obtained from the chitin deacetylation process, has been used widely in various fields such as nutrition, pharmacy, and cosmetics. Glucosamine can be obtained from the hydrolysis of chitosan. Enzymatic hydrolysis provides the advantage of mild reaction conditions, environmentally friendly, and high yield. But until now, the separation of glucosamine from the chitosan hydrolysis fraction has been an obstacle. Ultrafiltration membranes offer an efficient filtration process because they do not require additional chemicals. The performance of ultrafiltration membranes was analyzed from the fractionation process of chitosan hydrolysis. The PES membranes in 10, 25, and 50 kDa were used to filter hydrolyzed Low Molecular Weight Chitosan (LMWC) in varied concentrations. The experiment was carried out in crossflow membrane module for flat sheet at room temperature in 1 bar. The permeate flux during filtration decreased rapidly at the initial and gradually over time because of fouling and concentration polarization. The more concentrated hydrolyzed LMWC solution resulted higher percentage of rejection up to almost 20% at the same membrane MWCO while higher MWCO resulted lower rejection percentage for the same hydrolyzed LMWC concentration. The FTIR spectrum of the used membranes of all types had absorption bands of glucosamine which proved that the fractionation process occurred. The time retention in HPLC chromatograms of glucosamine produced were similar with standard glucosamine. Thus, ultrafiltration could be applied for hydrolyzed LMWC fractionation process.Keywords: fractionation; glucosamine; LMWC; MWCO; ultrafiltration

Effect of Piper betle Leaf Extract-Loaded Chitosan Nano Particle as Fruit-Coating Assay to Control Anthracnose on Mango

Green betel leaf (Piper betle L.) is one of the plants being used for traditional herbal medicine. This study aimed to determine betel leaf extract-chitosan nanoparticles to control anthracnose disease on mango. Chitosan nanoparticles were prepared by ionic gelation method using sodium tripolyphosphate as cross-linking agent. Characterization of the betel leaf extract was done by pyrolysis GC-MS; while chitosan nanoparticles were characterized using FTIR spectroscopy, SEM, and PSA analysis. The results showed that the green betel leaf extract contains 71.18 + 0.3% of antifungal phenolic compounds. The most phenolic compounds and their derivatives in the betel leaf extract was 1-hydroxy-4-methylbenzotriazole. Chitosan hydrolysis reduced the chitosan MW from 754.89 kDa to 245.85 kDa. Based on FTIR analysis, hydrolysis treatment and the addition of extract affected the existence of chitosan functional group, and wave numbers. The absorption of aromatic groups was observed at 1000 - 650 cm-1 wave numbers. The sizes of particle were ranged from 101 + 6.25 nm to 431.1 + 4.32 nm. The size of chitosan without hydrolysis was bigger than that of chitosan with hydrolysis. The SEM morphology of the chitosan nanoparticle-betel leaf extract was spherical shape. Chitosan hydrolysis treatment had a higher antifungal effect than that of chitosan without hydrolysis. The mass ratio of chitosan nanoparticles and betel leaf extracts (3:1; v/v) of both without hydrolysis and with hydrolysis was found as a good formula in suppressing anthracnose on mangoes with the degree of disease inhibition of 85.88% and 98.82%, respectively. The betel leaf extract-loaded chitosan nanoparticle treatment may offer the fruit shelf life up to 6 days.

Microwave Irradiation-Assisted Chitosan Hydrolysis Using Cellulase Enzyme

The influence of microwave irradiation on the chitosan hydrolysis catalyzed by cellulase enzyme was studied. The hydrolyzed chitosan was characterized by measuring its viscosity and reducing sugar. Further, it was also characterized by Fourier-Transform Infrared Spectroscopy (FT-IR), X-ray Diffraction (XRD), and Scanning Electron Microscope (SEM). The classical Michaelis-Menten kinetic parameters were measured by analyzing the amount of reducing sugars. The results were compared with the hydrolysis by using conventional shaker incubator. The hydrolysis reaction time needed to obtain similar reducing sugar yield was significantly lower for microwave irradiation than shaker incubator. On the other hand, the reduction rate of the relative viscosity was significantly higher for the hydrolysis of chitosan using shaker incubator. A significant difference in chemical structure was observed between hydrolysis using microwave irradiation and shaker incubator. Overall, the result showed that the hydrolysis behavior of chitosan using microwave irradiation is significantly different with using shaker incubator. Copyright © 2018 BCREC Group. All rights reservedReceived: 19th March 2018; Revised: 19th June 2018; Accepted: 25th June 2018How to Cite: Rokhati, N., Pramudono, B., Istirokhatun, T., Susanto, H. (2018). Microwave Irradiation-Assisted Chitosan Hydrolysis Using Cellulase Enzyme. Bulletin of Chemical Reaction Engineering & Catalysis, 13 (3): 466-474 (doi:10.9767/bcrec.13.3.2378.466-474)Permalink/DOI: https://doi.org/10.9767/bcrec.13.3.2378.466-474

Performance Comparison of Commercial Enzymes for The Synthesis of Glucosamine by Chitosan Hydrolysis in The Presence of Surfactant

Glucosamine has attracted much attention due to its potential biological, pharmaceutical and nutritional effects. In this research, glucosamine was prepared by enzymatic hydrolysis of low molecule weight chitosan with nonionic surfactant (Tween 80) addition. The change in reducing sugar content was used as an indicator for the hydrolysis reaction rate. The results showed that addition of Tween 80 (1%, w/w) increases the formation of reducing sugars approximately two times higher than without Tween 80 addition. Hydrolysis using a combination of cellulase and β-glucosidase showed higher reaction rate than using a combination of cellobiohydrolase and β-glucosidase or using β-glucosidase only. The combination of cellulase and β-glucosidase showed optimum hydrolysis at reaction time of 24 hours. High-performance liquid chromatography (HPLC) analysis of the glucosamine product showed typical peak distributions the same as those of commercial standard glucosamine hydrochloride.

Optimization of Chitosan hydrolysis by Cellulase Enzyme to produce Chitooligosaccharide

In order to increase the water solubility of chitosan and potential application to products that is good for human health. Chitosan was caried out the optimization of hydrolysis by cellulase to produce chitooligosaccharide. Chitosan with degree of deacetyl more than 80% and cellulase used in this study. Surface Response Method (RSM) - Central Composite Design (CCD) option is used to optimize the hydrolysis. Results of the study showed optimal values for hydrolysis such as 49 oC temperature, 5.9 pH, 0.76% substrate concentration, and 8.97 U/g enzyme concentration, 180 minutes hydrolysis time. More than 90% of the oligosaccharides produced were in the range less than 10 kDa. The research results are the premise for production of COS powder to water-soluble.