Study On Liquid Fermentation, Purification And Gene Cloning Of Protease Of Paecilomyces Farinosus

In the present thesis, the biological characters of 15 strains Paecilomyces farinosuswere studied, and 24 isolates Paecilomyces farinosus were screened basing on the activityof sublitisin-like protease. The strain Pf96 was selected as a research object basing on theabove study. The optimal cultivation method of strain Pf96 was confirmed, and the Pr1protease from this isolates also was partly purified. The complete cDNA gene ofsublitisin-like protease was cloned from strain Pf96. The enzyme activities from 24 isolates of Paecilomyces farinosus with differentgeographic origins and hosts were detected. The results showed that the enzyme activity ofPf102 was the highest in these studied isolates, and Pf96 had higher enzyme activity too. Fifteen isolates of Paecilomyces farinosus differed from one another in conidiation,growth rate, drought resistance, UV-protective ability and germination rate. The resultsshowed that the optimal temperature was 20℃ for conidiation and growth, and thegermination rate of spores was in the range of 80%~100% in 24 hours. In addition thedrought resistance of Pf01 was the strongest among 15 isolates, while Pf127 was theweakest. The range of germination rates from 15 isolates was 44%~81% by UV irradiationfor 5 minutes. The strain Pf96 has the most excellent characters in conidiation, growth,germination rate and UV irradiation resistance among these studied isolates. Ultimately, the strain Pf96 was selected as a research object for studying the optimalcultivation method, purifying sublitisin-like protease, and cloning the gene ofsublitisin-like protease basing on comparing the biological characters and producing Pr1protease of different strains. In the same cultivation conditions, the relative activities of enzymes were used fortargets. Ultimately, the optimal cultivation method of Pf96 was: chitin 1%, temperature25℃, pH8.0, age 6d. The cuticle-degrading proteases from entomopathogenic fungus, Paecilomycesfarinosus, were induced by adding the cuticle of cicada into liquid media. After ammoniumsulfate precipitation with 80% saturation, dialysis, an ion-exchange chromatography withDEAE-cellulose, and gel filtration with Sephadex G-100, a protease was purified partly.Thepartly purified enzyme had a specific sublitisin-like protease activity which was 18.32times that of the culture supernatant with 4.42% primary yield, and showed two bands onPAGE. In order to clone a complete cDNA of sublitisin-like protease from Paecilomycesfarinosus, a pair of primers was designed according to the high conserved nucleotidesequences in GenBank, and RT-PCR, 3'/5'-RACE PCR were used. The cloned completecDNA with 2060bp length was obtained, and the GenBank accession number is DQ022189.Alignments with the deduced amino acid of mature proteins in four species of fungi show69%,71%,68% and 68%,identity with Metarhizium anisopliae var. anisopliae(CAB63913),Gibberella zeae(EAA68914), M.anisopliae var.acridum(CAC95047)and Podosporaanserina(AAC03564), respectively. Meanwhile, the active site residues for serine proteasescan be readily identified in the mature protease.