Cloning and expression of a novel chitinase chi58 from Chaetomium cupreum in Pichia pastoris.
A gene encoding a novel chitinase chi58 was cloned from the fungus Chaetomium cupreum by using inverse PCR. The DNA sequence of chi58 contains a 1,602 bp open reading frame and two introns that are 52 and 201 bp in length. Regarding our in silico analysis, chi58 is a modular enzyme composed of a family-18 catalytic domain, which is responsible for chitinase activity, and a chitin-binding domain containing several cysteines. Apparently, the function of these domains is to anchor the enzyme tightly onto the large insoluble polymeric substrate. Chi58 has a pI of 4.47 and a deduced molecular mass of 58 kDa. The optimal pH and temperature conditions were determined to be 5.8 and 45 degrees C, respectively, when colloidal chitin was used as the substrate. SDS-PAGE and zymogram analyses indicated the presence of a single active chitinase. Cells with pPIC9K-chi58 produced an extracellular chitinase that had an activity of 39 U/ml protein. Metal ions such as Ba(2+), Mg(2+), K(+), Cu(2+), Fe(3+), Zn(2+), and Co(2+) also influenced the activity of the recombinant enzyme.
Wang
Yan-Jun
YJ
Department of Life Science and Engineering, Harbin Institute of Technology, Harbin, China. hrbwyj@yahoo.com.cn
Yang
Qian
Q
D000595:Amino Acid Sequence; D002597:Chaetomium / Q000201:enzymology* / Q000235:genetics; D002688:Chitinases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D007221:Industry; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D010572:Pest Control, Biological; D010843:Pichia / Q000235:genetics*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D008670:Metals; D011994:Recombinant Proteins; D002688:Chitinases