Studies On Purification, Characterization, And Gene Cloning Of Chitinase From Isaria Farinosa

In the present thesis, 28 strains of Isaria farinosa were studied for their chitinases activities, and strain RCEF0622 of Isaria farinosa with the highest chitinase activity was chosen to study the optimum of fermentation conditions for chitinase, chitinase purification and enzymological character. The cDNA gene of chitinase from isolate RCEF0685 was also clonied.Chitinases were induced when Isaria farinosa grew on a complete medium covered by cellophane to pre-logarithmic phase at first, and then was transferred to the medium containing colloidal chitin as a sole carbon and nitrogen source.The effects of pH, liquid medium capacity, inocula amount and temperature were studied for its optimal fermentation conditions by single factor level. Basing on the above study, its optimal fermentation conditions were further studied by orthogonal experiments.The results showed that the optimal culture conditions were the following: the colloid chitin 2%, initial pH of culture 6.0, inoculating amount 6%, 100 ml flask containing 15 ml cultural liquid, temperature 19℃, and cultural time 36h. The optimal fermentation process of strain RCEF0622 showed the enzyme activity of chitinase increased to a great extent by 55.61%, and the productive of protein also increased 55.34%. In fact, its specific activity did not increase, and was kept in a same level. The chitinase was determined as a kind of endochitinase with special test method.With the process of enzyme purification, the optimal concentration was 80% saturation determined by two ammonium sulfate precipitation experiments, the lowest specific activity of cultural supernatant, the most specific activity of precipitation and the most yields. The pH 8.0 was determined by the optimal adsorption for DEAE-Sepharose Fast Flow.An extracellular chitinase was purified by ammonium sulfate fraction, DEAE-Sepharose Fast Flow chromatography, and Sephadex G-100 chromatography. The purified enzyme had a specific activity of chitinase which was 12.65 times that of the culture supernatant with 5.9% primary yield, and showed one band on PAGE. Its molecular weight was estimated to be about 38.5kD by SDS-PAGE. When colloidal chitin was substrate, the michaelis constant (Km) of chitinase from strain RCEF0622 was 0.73mg/ml. The optimal reaction temperature was 40℃, and the optimal pH for reaction was 4.6. The enzyme activity was stable at a pH between 4 and 8 and under temperature 30℃. Boracic acid can also promote for enzyme activity more or less. The enzyme activity was enhanced by Mg2+, Mn2+, Al3+, K+, Ca2+and Ba2+, and Mn2+was obviously for the enzyme activity elevation, especially. However, enzyme activity was strongly inhibited by Zn2+ and Cu2+.This thesis also reports the complete full-length cDNA coding the chitinases produced by the biocontrol agent Isaria farinosa using SMART RACE RT-PCR. Analysis of the cloned complete cDNA, with a whole sequence of 1549bp, showed that it encompassed an open reading frame (ORF) with 1272bp encoding 423 amino acids with a stretch of 22 amino acid residues displaying characteristics of signal peptide. Tool revealed that the mature chitinase (without signal sequence) has a molecular mass of 43.9 kDa with a calculated pI of 5.67. This sequence contained two highly conserved regions of the active domain of the family 18 glycosyl hydrolases including a presumed enzymatic active site and a potential chitin-binding domain. The cloned Isaria chinitase belongs to the class V in 18 family of glycosyl hydrolase. Alignments with the deduced amino acid of mature proteins in 5 species of fungi showed 91%, 89%, 80%, 76% and 75%, respectively, identical with those of Torrubiella confragosa(AAV98691),Aphanocladium album(CAA45468);Verticillium fungicola(AAP45631),Nomuraea rileyi(AAP04616)and Beauveria bassiana(AAN41261), respectively.