Gene Cloning And Expression Of Chitinase From Thermoascus Aurantiacus Var. Levisporus

Chitin is widely distributed in nature. It is believed to be the second most abundant and renewable polymer on earth, next to cellulose. Chitin-degrading enzymes-chitinases attract increasing attention due to their application potential in such fields as plant pathogenic fungi and pest control, curing human diseases caused by fungi, protoplasts generation from yeast and filamentous fungi. Separating new chitinase genes and constructing high-effciency expression system which can high-efficient express chitinase is significant to the industrialization of chitinase.Thermoascus aurantiacus var. levisporus is a thermophilic fungus which is widely distributed and grows well at 45℃～50℃. In this study, a chitinase gene Tachit was isolated for the fist time from Thermoascus aurantiacus var. levisporus using RACE method. The nucleotide sequence of Tachit has been deposited in the GenBank database under accession number EF608144. The recombinant vector pET22b (+)/Tachit was constructed and transformed into E. coli BL21. Recombinant E. coli was induced by IPTG for chintinase expression. Analysed by SDS-PAGE, a 44kDa product was detected. The expression products were mostly composed of inclusion body. Recombinant vector pPIC3.5K/Tachit and pPIC9K/Tachit were constructed to transform Pichia pastoris GS115. Induced with methanol, the supernatant was concentrated by ammonium sulfate precipitation. The precipitated proteins were collected by centrifugation and purified using gel filtration. SDS-PAGE analyse showed a 45kDa single band of chintnase. The recombinant chitinase exhibited optimal activity at 50℃and pH8.0. It's stable under 50℃and between pH6～11.