The Cloning, Expression And Characterization Of Thermostable Chitinase From Chaetomium Thermophilum

Chitin is the second most abundant carbohydrate available after cellulose on earth, and can be hydrolyzed into N-Acetyl-β-D-Glucosamine by chitinase in fungi.Because of limiting in hydrolysis chitin by chemical method, for example, degradation, high cost, and environmental pollution. In contrast, biodegradation process is superior to that not only increase utilization ratio of chitin but also gain greater benefit. So chtinases have been universally applied in agriculture, medicine, chemical industry and environmental fieldsAlthough a large number of chitin-hydrolyzing enzymes have been extensively isolated from eucaryotes and bacteria, and their corresponding genes have been cloned and characterized, few studies on thermostable chitinase from thermophilies are reported, except for the enzymes from the hyperthermophilic archaeon and bacteria, such as Thermococcus chitonophagus, Thermococcus kodakaraensis KOD1, Streptomyces and Bacillus . and only one thermostable chitinse form fungi is reported by our lab by Guo Runfang.Chaetomium thermophilum is a thermophilic fungus with higher growth temperature, and is shown to produce thermostable protease, glucomylase, SOD and lipase. However, chitinase from the genus C. thermophilum have not yet been studied. In the present study, an endochitinase (CHIT) from C. thermophilum was produced when C. thermophilum was grown in minimal liquid medium containing colloidal chitin as the only carbon source.Degenerate primers based on existed cellobiohydrolases present in the databases. A 1545-length cDNA fragment encoding the chit gene was obtained through RT-PCR. And RACE-PCR. The gene has been registered in GenBank with accession number EU697741. Then partial genomic DNA of chit gene was cloned and the length was 1263bp with one introns. The gene has been registered in GenBank with accession number EU697742.The putative amino acids sequence had the catalytic domain of chit ,not a signal peptide CHBD and Ser/Thr domain. The alignment results of amino acids showed that it was high homology with the catalytic domains of the other chitinases in family-18, contained 2 conserved motifs related with catalytic activity of chitinase. Among these conserve sequences, the invariant carboxylic amino acid residue Asp and Glu have been proved essential in the acid-base catalytisis of chitinase and therefore C. thermophilum chitinase could belong to chitinase 18 family.The chit gene and expression vector pPIC9K were digested with EcoRⅠand NotⅠ, and ligation was carried out in vitro. The recombinant expression plasmid pPIC9K/chit was constructed and sequence to confirm the correct reading frame. The construct plasmid pPIC9K/chit was linearized with a restriction enzyme Sac I (insertion at 5'AOX1), and transforming Pichia GS115 competent cell by tip-and-run method, and Screening for His+Mut+ transformants on MD and MM plates. The parent vector was linearized with the same restriction enzyme and transformed GS115 as a control. PCR analysis of Pichia integrants and G418 screening determined the multicopy integrants to induce by methanol. These integrants were used to analyze expression levels every 24 hours of interest protein and determine the engineering strains with high expression level, such as GS-CH-4. The genetic and chitinase expression stability of recombinant Pichia pastoris GS-CH-4 were tested and characterized.Streak for single colonies of His+ transformant GS-CH-4 on YPD plate for 8 generations, PCR analysis showed the interest gene was integrated in Pichia genome, and the expression level was also kept stable. The expression chitinase exhibited optimum catalytic activity at pH 5.5 and 60℃. The enzyme was stable at 50℃and the half life time at 65℃was 40 min. Different metal ions showed different effects on the chitinase activity. Ba2+,Fe2+enhanced the enzyme activity , whereas Ag+,Hg2+ cause obvious inhibition.The C. thermophilum chitinase was secreted into the culture medium by the yeast Pichia pastoris in a functionally activity form, and the specific properties of expression chitinase was the same as those of the native strain C. thermophilum. It implied the expression chtinase could be applied widely for the chitin industry and biological fields. Therefore, we hope to reconstruct the chit gene or optimize the expression condition to obtain the yeast engineering strains suitable for industrial application.