Tiruchirappalli, Tamil Nadu | India
Primary Affiliation: Bharathidasan University - Tiruchirappalli, Tamil Nadu , India
PubMed Central Citations
2PubMed Central Citations
Biotechnol Appl Biochem 2015 Jul-Aug;62(4):523-9. Epub 2015 Jul 26.
Department of Molecular Microbiology, School of Biotechnology, Madurai Kamaraj University, Madurai, India.
2015 Jul-Aug;62(4):523-9. Epub 2015 Jul 26
Biotechnol. Appl. Biochem.
A recombinant, thermostable fungal chitinase from the thermophilic fungus, Thermomyces lanuginosus, was immobilized on glutaraldehyde cross-linked chitosan beads, and the properties of the immobilized chitinase were studied. The enzyme was found to be almost completely immobilized in 6 H under shaking condition at 30 °C. The immobilized enzyme exhibited much wider pH optimum and was more stable at alkaline pH values as compared with the soluble enzyme. Both the forms of the enzyme were optimally active at 60 °C and stable at 50 °C for 3 H, and after 3 H, the activity of the soluble enzyme declined sharply, whereas the immobilized chitinase was stable up to 6 H without any significant loss in the activity. KM and Vmax values of the immobilized enzyme were 1.18 mM and 445.7 μmol/Min/mg of protein, respectively. The immobilized enzyme was stable at least for 1 month at 4 °C without any significant loss in the activity.
Ind J Appl Res. 4(11): 55-63
Indian Journal of Applied Research
A chitinase gene of the thermophilic fungus, T. lanuginosus, was cloned and overexpressed in Saccharomyces cerevisiae. The overexpressed recombinant chitinase was purified by ammonium sulphate precipitation, ion exchange and hydrophobic interaction column chromatographic techniques. The enzyme was purified 20-fold with a specific activity of 69817. The recombinant chitinase was homogeneous as judged by SDS-PAGE and the molecular mass of the protein was ~ 39 kDa. The purified chitinase was optimally active at pH 6.0 and at 60 ˚C. KM and Vmax of the purified recombinant were found to be 0.66 mM and 81.6 moles/min/mg protein, respectively. The enzyme was found to be highly thermostable and retained about 80% of the activity even after 3 h at 50 ˚C. The enzyme was found to be glycosylated. MALDI-MS analysis confirmed that the purified enzyme belongs to an extracellular, family 18 chitinase type. Atomic force microscopic data clearly indicate the degradation of the native chitin polymer by the recombinant purified chitinase to much smaller units. Efficient hydrolysis of native chitins by the recombinant chitinase suggests potential industrial applications for this enzyme requiring chitin processing at elevated temperatures. CD spectra of the native and denatured recombinant chitinase suggest that the beta structures play an important role on the stability of the enzyme at high temperatures.
International Journal of ChemTech Research CODEN (USA): IJCRGG ISSN : 0974-4290 Vol.6, No.8, pp 4042
International Journal of ChemTech Research
A thermophilic fungal chitinase was cloned, sequenced and overexpressed in Saccharomyces cerevisiae. Multiple sequence alignment of the recombinant chitinase revealed that the catalytic domain is 100% conserved with other family GH18 chitinases. Phylogenetic analysis revealed that the recombinant chitinase exhibited a very close proximity to the chitinases of other thermophilic fungi and higher fungi. Glycosylation of the recombinant chitinase was analyzed by in silico methods and confirmed by experimental data. Homology modeling of the chitinase showed the typical (α/β)8 TIM barrel structure. Molecular docking of the enzyme revealed the involvement of a glutamic acid of the catalytic domain in the catalytic process.
J Microb Biochem Technol 4: 086-091. doi: 10.4172/1948-5948.1000076
Journal of Microbial & Biochemical Technology
A chitinase gene from the thermophilic fungus, Thermomyces lanuginosus ATCC 44008 has been cloned and overexpressed in Saccharomyces cerevisiae SEY 2101. The recombinant chitinase was produced as soluble secreted protein. The enzyme activity was found to be maximum on fourth day at 30°C in the induction medium. The overexpressed chitinase displayed optimum activity at pH 6.5 and at 60°C. The recombinant chitinase attributed remarkable thermostability by holding more than 60% of the enzyme activity after 6 h at 50°C. The molecular mass of the overexpressed chitinase was 42 kDa as measured by SDS-PAGE. The kinetic parameters such as KM and Vmax of the enzyme were found to be 0.403 mM and 8.74 mmoles/min/mg protein, respectively. Synthesis of the recombinant chitinase was strongly repressed by glucose. The eukaryotic translational inhibitor, cycloheximide in the induction medium showed 10% of higher activity whereas 30% of the activity was inhibited by transcriptional inhibitors, viz. 8-azaguanine and 8-hydroxyquinoline. The overexpressed recombinant chitinase may find potential application in pharmaceutical industry to prepare chitooligosaccharides.