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Isolation and Identification of a Chitinase-producing Strain and Characteration of Its Chitinase

Author: XiongJing
Tutor: XiaJinLan
School: Central South University
Course: Microbiology
Keywords: Aspergillus fumigatus Chitinase Chitin Purification
CLC: Q93
Type: Master's thesis
Year: 2009
Downloads: 250
Quote: 4
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Abstract

Chitin nature, rich in natural resources, renewable, biocompatible, biodegradable, bio-functional characteristics, is irreplaceable in many high-tech fields of biomedical and environment-friendly material, has a wide range of purposes. Chitinase is the chitinase-specific degradation enzyme required. To carry out the the chitinase study, the conversion of chitin resources, has become a the global popular research topic. Screened from the Hunan shrimp farms, rivers, and other places to a production of fungal chitinase CS-01, and its classification and identification; separation and purification of the strains produced chitinase, studied the temperature and the pH of the chitinase degradation of colloidal chitin activity and glucose and colloidal chitin substrate of the strain chitinase expression. (1) 12 samples by enrichment culture using flat transparent circle method for screening selective solid plate chitin as sole carbon source 7 strains form obviously transparent circle that 7 strains rescreening, a production of enzyme activity is relatively high fungal CS-01. (2) the use of means of morphological and molecular biology of this strain were identified by their colony characteristics, the individual morphological observation and ITS rDNA sequence analysis, and ultimately determine the strain Aspergillus fumigatus. (3) using the salting-out, gel filtration chromatography, dialysis, polyethylene glycol, concentrated, ion exchange chromatography and freeze-dried, isolated and purified chitinase. Native-PAGE and SDS-PAGE analysis identified the chitinase 45 kDa monomeric enzyme. (4) to concentrated pure enzyme were used to study the influence of temperature and pH on the the chitinase apparent vitality. The results showed that: when the enzyme reaction temperature reached 55 ° C in a buffer solution of pH 5, the largest apparent activity; in the 45-65 ℃ relatively apparent activity in more than 80%, indicating that the initial enzyme solution has a wide high-temperature adaptation range, which is the direct use of this strain for fermentation degradation of colloidal chitin significance. (5) The Aspergillus fumigatus strain CS-01 cultured in the medium of the different carbon sources and found that When the culture with 1% glucose as carbon source, the entire culture process never detected chitinase enzyme activity; while 1% colloidal chitin as a carbon source for culture, cultured to 12h have chitinase vitality, and the maximum (0.128 U / mL) at 36h; colloidal chitin and glucose mixed carbon source the culture medium the generation train early chitinase enzyme activity is not detected. Determination of reducing sugar in the mixed carbon source was found only when the glucose in the medium is exhausted, in order to detect the vitality of the chitinase generated. The above results show that, CS-01 production are inducible expression of chitinase.

Isolation and Identification of a Chitinase-producing Strain and Characteration of Its Chitinase

Abstract

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