No 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.

[structure: see text] A new synthetic route was developed for the preparation of activity probe 1 for beta-glucosidase in this study. The key glycosidation step begins with benzyl p-hydroxyphenylacetate. Benzylic functionalization for the construction of the trapping device was achieved at later stages. Probe 1 was shown to be able to label the target enzyme. This cassette-like design offers great flexibility for future alterations. It would allow the synthetic scheme to expand to other glycosidase probes with different linker/reporter combinations.

Tandem mass spectrum analysis of the enzymatic hydrolysate revealed that the recombinant CSN can cleave linkages of GlcNAcGlcN and GlcN-GlcN in its substrate, suggesting that it is a subclass I chitosanase. In addition, an extensive site-directed mutagenesis study on 10 conserved carboxylic amino acids of glycosyl hydrolase family 75 was performed. This showed that among these various mutants, D160N and E169Q lost nearly all activity. Further investigation using circular dichroism measurements of D160N, E169Q, wild-type CSN, and other active mutants showed similar spectra, indicating that the loss of enzymatic activity in D160N and E169Q was not because of changes in protein structure but was caused by loss of the catalytic essential residue. We conclude that Asp 160 and Glu 169 are the essential residues for the action of A. fumigatus endo-chitosanase.Chitosanase (EC 3.2.1.132) is a hydrolytic enzyme acting on the ␤-1,4-glycosidic linkage of chitosan, a linear biopolymer of ␤-1,4-linked GlcN, to release chito-oligosaccharides. The oligomers GlcNAc and GlcN have interesting biological activities (1), including anti-tumor effects (2, 3), hypo-cholesterolemic effects (4), anti-microbial activities (5, 6), disease-resistance responses, and as phytoalexin elicitors in higher plants (7,8). Hence chitosanase, chito-oligosaccharides and their derivatives have attracted interest from the food and pharmaceutical industries because they can be used as edible additives, agricultural immunity controls and promise many other prospective applications as prophylactic agents for liver diseases (4), atherosclerosis, and hypertension.

No abstract

Fucosylated glycoconjugates have critical roles in biological processes, but a limited availability of alpha-l-fucosidase has hampered research on this human enzyme (h-Fuc) at a molecular level. After overexpressing h-Fuc in Escherichia coli as an active form, we investigated the catalytic function of this recombinant enzyme. Based on sequence alignment and structural analysis of close homologues of h-Fuc, nine residues of glutamate and aspartate in h-Fuc were selected for mutagenic tests to determine the essential residues. Among the mutants, D225N, E289Q, and E289G lost catalytic activity significantly; their k(cat) values are 1/5700, 1/430, and 1/340, respectively, of that of the wild-type enzyme. The Brønsted plot for k(cat)/K(m) for the E289G mutant is linear with beta(lg) = -0.93, but that for k(cat) is biphasic, with beta(lg) for poor substrates being -0.88 and for activated substrates being -0.11. The small magnitude of beta(lg) for the activated substrates may indicate that the rate-limiting step of the reaction is defucosylation, whereas the large magnitude of the latter beta(lg) value for the poor substrates indicates that the rate-limiting step of the reaction becomes fucosylation. The kinetic outcomes support an argument that Asp(225) functions as a nucleophile and Glu(289) as a general acid/base catalyst. As further evidence, azide significantly reactivated D225G and E289G, and (1)H NMR spectral analysis confirmed the formation of beta-fucosyl azide and alpha-fucosyl azide in the azide rescues of D225G and E289G catalyses, respectively. As direct evidence to prove the function of Glu(289), an accumulation of fucosyl-enzyme intermediate was detected directly through ESI/MS analysis.

A eukaryotic catechol 1,2-dioxygenase (1,2-CTD) was produced from a Candida albicans TL3 that possesses high tolerance for phenol and strong phenol degrading activity. The 1,2-CTD was purified via ammonium sulfate precipitation, Sephadex G-75 gel filtration, and HiTrap Q Sepharose column chromatography. The enzyme was purified to homogeneity and found to be a homodimer with a subunit molecular weight of 32,000. Each subunit contained one iron. The optimal temperature and pH were 25 degrees C and 8.0, respectively. Substrate analysis showed that the purified enzyme was a type I catechol 1,2-dioxygenase. This is the first time that a 1,2-CTD from a eukaryote (Candida albicans) has been characterized. Peptide sequencing on fragments of 1,2-CTD by Edman degradation and MALDI-TOF/TOF mass analyses provided information of amino acid sequences for BLAST analysis, the outcome of the BLAST revealed that this eukaryotic 1,2-CTD has high identity with a hypothetical protein, CaO19_12036, from Candida albicans SC5314. We conclude that the hypothetical protein is 1,2-CTD.

No abstract