An Aspergillus chitosanase with potential for large‐scale preparation of chitosan oligosaccharides

Abstract

A chitosan-degrading fungus, designated Aspergillus sp. Y2K, was isolated from soil. The micro-organism was used for producing chitosanase (EC 3.2.1.132) in a minimal medium containing chitosan as the sole carbon source. The induced chitosanase was purified to homogeneity from the culture filtrate by concentration and cationic SP-Sepharose chromatography. The purified enzyme is a monomer with an estimated molecular mass of 25 kDa by SDS/PAGE and of 22 kDa by gel-filtration chromatography. pI, optimum pH and optimum temperature values were 8.4, 6.5 and 65-70 degrees C, respectively. The chitosanase is stable in the pH range from 4 to 7.5 at 55 degrees C. Higher deacetylated chitosan is a better substrate. Chitin, xylan, 6-O-sulphated chitosan and O-carboxymethyl chitin were indigestible by the purified enzyme. By endo-splitting activity, the chitosanase hydrolysed chitosan to form chitosan oligomers with chitotriose, chitotetraose and chitopentaose as the major products. The enzyme hydrolyses chitohexaose to form chitotriose, while the chitopentaose and shorter oligomers remain intact. The N-terminal amino acid sequence of the enzyme was determined as YNLPNNLKQIYDDHK, which provides useful information for further gene cloning of this enzyme. A 275 g-scale hydrolysis of chitosan was performed. The product distribution was virtually identical to that of the small-scale reaction. Owing to the simple purification process and high stability of the enzyme, it is potentially valuable for industrial applications.

Chitinases Are Essential for Sexual Development but Not Vegetative Growth in Cryptococcus neoformans

“…strain JG. Although these chitosanases have been shown to degrade chitosan, their in vitro physiological relevance has not been elucidated (8,60).…”

Production and Characterization of Partially Purified Chitosanase Enzyme of <i>Bacillus cereus</i> A4/B4 Isolated from Biowaste Soil Samples:Bioactive Chitooligosaccharide

“…The CDA broth was taken at various pH (4,5,6,7,8) and the selected strains was inoculated and were incubated at room temperature for 8 days. Every 24 hours, the enzyme activity was measured.…”

“…From the suspension 0.1ml was spread plated on to the CDA [6] plates having basal medium M9 along with (0.5% ,1% ,2%) substrate chitosan or colloidal chitosan of 0.5% and 1% [7][8][9], Minimal Salt Chitosan (MS) medium [10,11], Minimal Synthetic (MSM) Medium [12] and all the plates were incubated at 30° C for 3-5 days.…”

Classification of Chitosanases by Hydrolytic Specificity towardN1,N4-Diacetylchitohexaose

“…Chitosanases (EC 3.2.1.132) are glycosyl hydrolases that catalyze the endohydrolysis of -1,4 linkages between GlcN residues in a partially Nacetylated chitosan (Enzyme Nomenclature, http:// www.chem.qmul.ac.uk/iubmb/enzyme/). They are produced by various bacteria [1][2][3][4][5][6][7][8][9] and fungi, [10][11][12][13][14][15] and are useful in the preparation of biofunctional chitooligosaccharides. Fukamizo et al 16) have proposed that chitosanases can be classified into three subclasses according to their specificity for the hydrolysis of the -glycosidic linkages in partially N-acetylated chitosan molecules: subclass I chitosanases [16][17][18][19][20] split both GlcN-GlcN and GlcNAc-GlcN bonds, subclass II chitosanases 3) split only the GlcN-GlcN bond, and subclass III chitosanases 5,[21][22][23] split both GlcN-GlcN and GlcN-GlcNAc bonds.…”