Identification and Characterization of Acidic Hydrolases with Chitinase and Chitosanase Activities from Sweet Orange Callus Tissue1

Oßwald, Wolfgang; Shapiro, Jeffrey P.; Doostdar, Hamed; McDonald, Roy E.; Niedz, Randall P.; Nairn, Campbell J.; Hearn, C. J.; Mayer, Richard T.

doi:10.1093/oxfordjournals.pcp.a078662

Cited by 54 publications

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“…Chitosanases have been found and cloned from several organisms, such as viruses (9), bacteria (10), fungi (11)(12)(13)(14), and plants (15). According to amino acid sequence homologies (16), chitosanases have been classified into five glycoside hydrolase families: GH-5, 2 GH-8, GH-46, GH-75, and GH-80.…”

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confidence: 99%

Exploration of Glycosyl Hydrolase Family 75, a Chitosanase from Aspergillus fumigatus

Cheng

Chang

et al. 2006

Journal of Biological Chemistry

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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.

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confidence: 99%

Exploration of Glycosyl Hydrolase Family 75, a Chitosanase from Aspergillus fumigatus

Cheng

Chang

et al. 2006

Journal of Biological Chemistry

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“…Most chitosanases are found in microorganisms (8 -11), and a few are found in plants (12)(13)(14)(15). The complete amino acid sequences have been reported for procaryotic chitosanases from Bacillus circulans MH-K1 (MH-K1 chitosanase) 1 (16), Streptomyces sp.…”

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Crystal Structure of Chitosanase from Bacillus circulans MH-K1 at 1.6-Å Resolution and Its Substrate Recognition Mechanism

Saito¹,

Kita²,

Higuchi³

et al. 1999

Journal of Biological Chemistry

105

113

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Chitosanase from Bacillus circulans MH-K1 is a 29-kDa extracellular protein composed of 259 amino acids. The crystal structure of chitosanase from B. circulans MH-K1 has been determined by multiwavelength anomalous diffraction method and refined to crystallographic R ‫؍‬ 19.2% (R free ‫؍‬ 23.5%) for the diffraction data at 1.6-Å resolution collected by synchrotron radiation. The enzyme has two globular upper and lower domains, which generate the active site cleft for the substrate binding. The overall molecular folding is similar to chitosanase from Streptomyces sp. N174, although there is only 20% identity at the amino acid sequence level between both chitosanases. However, there are three regions in which the topology is remarkably different. In addition, the disulfide bridge between Cys 50 and Cys 124 joins the ␤1 strand and the ␣7 helix, which is not conserved among other chitosanases. The orientation of two backbone helices, which connect the two domains, is also different and is responsible for the differences in size and shape of the active site cleft in these two chitosanases. This structural difference in the active site cleft is the reason why the enzymes specifically recognize different substrates and catalyze different types of chitosan degradation.

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“…The BCLVC sequence shows a high degree of homology to published sequences of basic class III chitinase/lysozymes (Bernasconi et al 1987;M6traux et al 1989;Samac et al 1990;Jekel et al 1991), including two acidic class III chitinases isolated from the same citrus cell line (Osswald et al 1994). The acidic citrus enzymes, however, had no lysozyme activity, but did exhibit chitinase and chitosanase activities.…”

Section: Resultsmentioning

confidence: 90%

Characterization of seven basic endochitinases isolated from cell cultures of Citrus sinensis (L.)

Mayer

McCollum

Niedz

et al. 1996

Planta

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Seven endochitinases (EC 3.2.1.14) (relative molecular masses 23,000-28,000 and isoelectric points 10.3-10.4) were purified from nonembryogenic Citrus sinensis L. Osbeck cv. Valencia callus tissue. The basic chitinase/lysozyme from this tissue (BCLVC) exhibited lysozyme, chitinase and chitosanase activities and was determined to be a class III chitinase. While BCLVC acted as a lysozyme at pH 4.5 and low ionic strength (0.03) it acted as a chitinase/chitosanase at high ionic strengths (0.2) with a pH optimum of ca. 5. The lysozyme activity of BCLVC was inhibited by histamine, imidazole, histidine and the N-acetyl-D-glucosamine oligosaccharide (GlcNAc)3. The basic chitinase from cv. Valencia callus, BCVC-2, had an N-terminal amino acid sequence similar to tomato and tobacco AP24 proteins. The sequences of the other five chitinases were N-terminal blocked. Whereas BCLVC was capable of hydrolyzing 13.8-100% acetylated chitosans and (GlcNAc)4-6 oligosaccharides, BCVC-2 hydrolyzed only 100% acetylated chitosan, and the remaining enzymes expressed varying degrees of hydrolytic capabilities. Experiments with (GlcNAc)2-6 suggest that BCLVC hydrolysis occurs in largely tetrasaccharide units whereas hydrolysis by the other chitinases occurs in disaccharide units. Cross-reactivities of the purified proteins with antibodies for a potato leaf chitinase (AbPLC), BCLVC, BCVC-3, and tomato AP24 indicate that these are separate and distinct proteins.

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Identification and Characterization of Acidic Hydrolases with Chitinase and Chitosanase Activities from Sweet Orange Callus Tissue1

Cited by 54 publications

References 0 publications

Exploration of Glycosyl Hydrolase Family 75, a Chitosanase from Aspergillus fumigatus

Exploration of Glycosyl Hydrolase Family 75, a Chitosanase from Aspergillus fumigatus

Crystal Structure of Chitosanase from Bacillus circulans MH-K1 at 1.6-Å Resolution and Its Substrate Recognition Mechanism

Characterization of seven basic endochitinases isolated from cell cultures of Citrus sinensis (L.)

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