Isolation And Identification Of A Chitinase-producing Strain And Characteration Of Its Chitinase

Chitin is one kind of widespread natural resource, with a series of excellent properties viz. biorenewability, biocompatibility, biodegradability, which offers application potentials in diversified areas, especially applied in preparation of biorelated and environment-friendly materials. Chitinase selectively splits chitin. The significance of realizing chitin conversion by chitinase is subject of significant concern to many people all over the world.In this dissertation, a chitinase-producing strain CS-01 from Hunan Province of China was isolated and identified. Chitinase secreted by the strain was purified and its partial properties were investigated. Then, effects of pH and temperature on the activity of the concentrated purified enzyme were studied. Additionally, effect of substrates viz. glucose and colloidal chitin on the CS-01 chitinase expression was also studied. The main results are listed as follows.(1) After the twelve samples cultured in the enrichment medium, the transparent flat circle was adopted to do the primary screening and 7 strains were got when chitin was used as the sole carbon source of selective solid medium to form clear transparent circle. After secondary screening of the 7 strains, a high-activity fungi strain named CS-01 was selected and maintained on colloidal chitin agar slant for further study.(2) Morphological and molecular biology methods were used to identify the colony characteristics, individual forms of observation and ITS rDNA sequence analysis of CS-01, and ultimately classified the strain as Aspergillus fumigatus.(3) Purification of the CS-01 chitinase was carried out by salting out, gel filtration chromatography, dialysis, PEG concentration, ion-exchange chromatography, freeze-drying, followed with electrophoresis analysis. Native-PAGE and SDS-PAGE indicated that the chitinase from A. fumigatus CS-01 was monomer with 45 kDa.(4) The effects of pH and temperature on the apparent activity of the concentrated purified enzyme were investigated. It was found that the maximum activity of the concentrated crude enzyme was appeared at pH 5 and 55℃. In the buffer pH 5, over 80% of the maximum activity was shown at 45-65℃. The advantage of its high adaptability to a wide range of high temperatures makes the strain useful in direct chitin hydrolysis by its fermentation broth.(5) A. fumigatus CS-01 is cultivated by different carbon sources. Chitinase activity present in the supernatant of 1% glucose cultivation could not detected all the time, while the culture supernatant from 1% colloidal chitin cultivation possessed enzymatic activity after incubated for 12 h and chitinase activity reached to its maximum (0.128 U/mL) at 36 h,. A. fumigatus CS-01 cultivated by mixed carbon source shows chitinase activity at late stage of growth. The above results indicate that the chitinase expression is chitin-inducible.