Chitosanase Research Articles

Modulation of the Host Defence System by Nematophagous Fungi and Chitosan

Nematophagous fungi (NFs), which are responsible for soil suppression of plant-parasitic nematodes, are multitrophic biocontrol agents. This raises the question of the transition between lifestyles (e.g., endophytism vs. egg parasitism). The NF Pochonia chlamydosporia colonises food crops and promotes their growth and yield. When colonising the plant, P. chlamydosporia induces the plant immunity (PI). However, it also evades the PI. To do this, both endophytic NF and pathogenic fungi (PF) secrete LysM effectors (LysM-effs). LysM effectors have been shown to have diverse functions in different organisms, including the protection of fungal chitin from plant chitinases. P. chlamydosporia is resistant to chitosan, which modulates gene expression in fungi and plants and has antimicrobial properties. P. chlamydosporia chitin deacetylases (CDA) and chitosanases (CSN) also help P. chlamydosporia evade plant immunity, resist exogenous chitosan, and are induced during fungal infection of nematode eggs. NF-chitosan formulations are new biomanagement tools against plant parasitic nematodes, fungal wilt pathogens and insect pests that currently threaten food security crops. Furthermore, omics techniques are useful tools to elucidate the role of CDAs, CSNs, LysM-effs, adhesion proteins and carbohydrate-active enzymes in pathogen–BCA–plant interactions, adhesion and infection to nematode eggs and their modulation by chitosan.

Functional Properties of Chitosan Oligomers Obtained by Enzymatic Hydrolysis.

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.

Gene cloning and molecular characterization of a thermostable chitosanase from Bacillus cereus TY24

BackgroundAn 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.ResultsA 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.ConclusionA 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.

Preparation and Composition Analysis of Chitooligosaccharides with Low Degree of Deacetylation by Hydrolysis of Bacillus subtilis Chitosanase

Preparation and Composition Analysis of Chitooligosaccharides with Low Degree of Deacetylation by Hydrolysis of Bacillus subtilis Chitosanase

Investigating chitin deacetylation and chitosan hydrolysis during vegetative growth in Magnaporthe oryzae.

SummaryChitin deacetylation results in the formation of chitosan, a polymer of β1,4‐linked glucosamine. Chitosan is known to have important functions in the cell walls of a number of fungal species, but its role during hyphal growth has not yet been investigated. In this study, we have characterized the role of chitin deacetylation during vegetative hyphal growth in the filamentous phytopathogen Magnaporthe oryzae. We found that chitosan localizes to the septa and lateral cell walls of vegetative hyphae and identified 2 chitin deacetylases expressed during vegetative growth—CDA1 and CDA4. Deletion strains and fluorescent protein fusions demonstrated that CDA1 is necessary for chitin deacetylation in the septa and lateral cell walls of mature hyphae in colony interiors, whereas CDA4 deacetylates chitin in the hyphae at colony margins. However, although the Δcda1 strain was more resistant to cell wall hydrolysis, growth and pathogenic development were otherwise unaffected in the deletion strains. The role of chitosan hydrolysis was also investigated. A single gene encoding a putative chitosanase (CSN) was discovered in M. oryzae and found to be expressed during vegetative growth. However, chitosan localization, vegetative growth, and pathogenic development were unaffected in a CSN deletion strain, rendering the role of this enzyme unclear.

Improved extracellular expression and high-cell-density fed-batch fermentation of chitosanase from Aspergillus Fumigatus in Escherichia coli.

Chitosanase (CSN) from Aspergillus fumigatus has good thermal stability, wide pH range duration, and effective hydrolysis for chitosan. Inhere, CSN was successfully expressed in Escherichia coli followed by extracellular secretion under the guidance of an N-terminal signal peptide PelB, which effectively prompted its secretion out of E. coli cells. To facilitate its later purification, N-terminal or C-terminal 6xHis epitope tag was added to the PelB-CSN protein complex. Our results indicated that PelB-CSN without 6xHis-tag (PelB-CSN) or with N-terminal 6xHis-tag (PelB-CSN-N) can both be effectively secreted into the medium, while CSN with 6xHis-tag anchored at C-terminus was expressed as inclusion bodies. Process optimization strategies were further developed to improve the secretion efficiency of recombinant PelB-CSN-N in E. coli. Under the induction of 10g/L lactose in shake-flask culture, the extracellular activity of CSN reached 6015U/mL at 25°C in TB medium containing 1% glycine. Moreover, a fed-batch fermentation strategy for high-cell-density cultivation was applied in a 5-L fermenter, increasing the extracellular CSN activity to 14,000U/mL in 2-day fermentation with the optimal addition of lactose and glycine.

Conversion of squid pen by using Serratia sp. TKU020 fermentation for the production of enzymes, antioxidants, and N-acetyl chitooligosaccharides

A chitinase (CHT), a chitosanase (CHS) and a protease (PRO) were purified from the culture supernatant of Serratia sp. TKU020 with squid pen as the sole carbon/nitrogen source. The molecular masses of CHT, CHS and PRO determined by SDS-PAGE were approximately 65 kDa, 55 kDa and 55 kDa, respectively. CHT and CHS were inhibited by Mn 2+, EDTA and PRO was inhibited by Mg 2+, EDTA. The antioxidant activity of TKU020 culture supernatant was 78% (DPPH scavenging ability). N-Acetylglucosamine (GlcNAc) and N-acetyl chitobiose (GlcNAc) 2 were also produced from the culture supernatant by using TKU020 strain fermentation. The maximum production of GlcNAc and (GlcNAc) 2 was 1.3 mg/mL and 2.7 mg/mL, respectively, after 4 days of fermentation. With this method, we have shown that squid pen wastes can be utilized and it is effective in the production of enzymes, antioxidants, and N-acetyl chitooligosaccharides, facilitating its potential use in industrial applications and functional foods.

Chitosanase from &lt;i&gt;Streptomyces&lt;/i&gt; sp. strain N174: a comparative review of its structure and function

Chitosanase from &lt;i&gt;Streptomyces&lt;/i&gt; sp. strain N174: a comparative review of its structure and function

Primary sequence of the chitosanase from Streptomyces sp. strain N174 and comparison with other endoglycosidases

A 1.6-kb DNA fragment from the soil actinomycete, Streptomyces sp. strain N174, containing the gene ( csn) encoding an extracellular chitosanase (CSN), has been isolated and its complete nucleotide sequence determined. The gene was expressed in Escherichia coli and Streptomyces lividans using appropriate vectors. The sequence was found to contain one large ORF which encodes a protein of 238 amino acids (aa). The deduced aa sequence begins with a signal peptide which has an unusual C-terminal segment, well recognized by the Streptomyces secretion system, but poorly in Escherichia coli. The strain N174 CSN aa sequence was compared with those of other proteins and significant homology was found only with the Bacillus circulans MH-K1 CSN but not with known chitinases or lysozymes. This suggests that CSN form a new enzyme family distinct from other endoglycosidases.