Two novel thermostable chitinase genes from thermophilic fungi: cloning, expression and characterization.
Two chitinase genes, Tachit1 from Thermoascus aurantiacus var. levisporus and Ctchit1 from Chaetomium thermophilum were isolated. Tachit1 and Ctchit1 encode putative single-domain proteins (TaCHIT1 and CtCHIT1) of 399 and 402 amino acid residues, respectively. The catalytic domains of TaCHIT1 and CtCHIT1 are similar to those of other fungal chitinases in family 18 of glycosyl hydrolases. TaCHIT1 and CtCHIT1 have a molecular weight of about 48.4 and 47.3kDa, respectively when produced in recombinant Pichia pastoris. The enzymes exhibited optimum catalytic activity at pH 8.0 and 50 degrees C for TaCHIT1 and at pH 5.5 and 60 degrees C for CtCHIT1. TaCHIT1 retained 95.3% of its activity after 60 min at 50 degrees C. CtCHIT1 was stable at 50 degrees C and retained 96.7% of its activity after 60 min incubation at 60 degrees C. The TaCHIT1 and CtCHIT1 produced Glc-NAc2 as the major product, when colloidal chitin was used as the substrate. The enzyme could not hydrolyze pNp-(GlcNAc), but hydrolyzed colloidal chitin, powdery chitin and chitosan. These features make these proteins potentially useful for applications requiring chitin hydrolysis at elevated temperatures.
Li
An-Na
AN
Department of Environmental Biology, Shandong Agricultural University, Taian, Shandong 271018, China.
Yu
Kai
K
Liu
Hai-Quan
HQ
Zhang
Jie
J
Li
Hua
H
Li
Duo-Chuan
DC
D002384:Catalysis; D020134:Catalytic Domain; D002686:Chitin / Q000737:chemistry; D002688:Chitinases / Q000235:genetics*; D003001:Cloning, Molecular; D003102:Colloids / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D015966:Gene Expression Regulation, Fungal; D006867:Hydrolases / Q000235:genetics; D010843:Pichia / Q000201:enzymology* / Q000235:genetics*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors
D003102:Colloids; D011994:Recombinant Proteins; D002686:Chitin; D006867:Hydrolases; D002688:Chitinases