Purification Of Thermostable Chitinase From Thermomyces Lanuginosus And Cloning And Expression Of The Encoding Gene

Chitinases have been attracted increasing attention due to their application potential in such fields as plant pathogenic fungi and pest control, protoplasts generation from yeast and filamentous fungi and bioconversions of chitin waster in recent years. Chitin oligomers, which are enzymelytic products of chitin, have been universally applied in agriculture, medicine, chemical industry and environmental fields because of their biological activities. However, there are several limits, for example, degradation, high cost, and environmental pollution, on hydrolysis chitin by chemical method. In contrast, biodegradation process is superior to that not only increase utilization ratio of chitin but also gain greater benefit. Many organisms capable of producing chitinase were screened and isolated. But commercial chitinases from organisms growing at room temperature are limited on agricultural and industrial application due to high cost, low thermostability, shorter shelf-life and lower activity. It is implied that research on thermostable chitinase is of importance in industrial exploitation. Although 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 . Thermomyces lanuginosus is a thermophilic fungus with higher growth temperature, and is shown to produce thermostable protease, glucomylase, lipoxygenase and lipase. However, chitinase from the genus Thermomyces lanuginosus have not yet been studied. In the present study, an extracellular chitinase (CHIT) from Thermomyces lanuginosus was produced when Thermomyces lanuginosus was grown in minimal liquid medium containing colloidal chitin as the only carbon source. The enzyme was purified from the culture filtrate of the strain by fractional ammonium sulphate precipitation, DEAE-Sepharose chromatography and Pheny1-Sepharose chromatography. The purified enzyme was shown to be homogeneous by SDS-PAGE, and the molecular weight was estimated to be 48kDa. The chitinase exhibited optimum catalytic activity at pH 4.5 and 55℃. The half life time of the enzyme at 65℃was 25min. Different metal ions showed different effects on the chitinase activity. Ca2+ ,Ba2+,Na+,K+ enhanced the enzyme activity , whereas Fe2+,Ag+,Hg2+ cause obvious inhibition. The Vmax and Km values of chitinase on colloidal chitin (2.5%) were 22.12 μmol ml-1min-1 and 9.56 mg ml-1 respectively. The chitinase revealed antifungal activity against tested fungi to different extent. The first 15 amino acids from the N-terminal of the CHIT were AQGYLSVQYFVNWAI. The sequence showed strong similarity with some other fungal chitinase such as those from Aspergillus fumigatus, Coniothyrium minitans. Degenerate primers based on the N-terminal sequences of purified CHIT and a cDNA fragment encoding the CHIT gene was obtained through RT-PCR. The RACE was used to generate full-length cDNA clones. The cDNA of CHIT contained an ORF of 1326bp encoding 442 amino acids. The gene chit has been registered in GenBank with accession number DQ092332. The first 43 amino acids are presumed to be a signal peptide. The amino-acids sequence from 44 to 58 corresponded to the N-terminal sequence of the purified protein. After rocessing of the signal peptide sequence, the calculated size of CHIT is 46 kDa. The alignment results of putative amino acids sequence showed the catalytic domain of chit 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 Thermomyces lanuginosus chitinase could belong to chitinase 18 family. The chit gene and expression vector pET-30a(+) were digested with EcoRⅠand NotⅠ, and ligation was carried out in vitro. The recombinant plasmid pET/chit was generated, and transformed into E. coli BL21 (DE3)。The recombinant protein was produced in large amount after IPTG induction, approximately 46kDa protein was determined by SDS-PAGE, and this size was agreed with the protein molecular weight from the putative amino acid sequence; no interest protein was produced by inducing with IPTG after the pET-30a(+) was transformed into BL21. The solubility analysis showed the recombinant protein was presented in a fusion form due to the high induce temperature results in E. coli BL21 growth rapid and overexpression of recombinant protein in host cell.