Analysis of the hyperthermophilic chitinase from Pyrococcus furiosus: activity toward crystalline chitin.
Chitinase [EC 3.2.1.14] is an enzyme that can hydrolyze the beta-1,4 linkage between N-acetyl-D-glucosamine in chitin. In the genome database of the hyperthermophilic archaeon Pyrococcus furiosus, we found two adjacent genes (PF1233 and PF1234) homologous to those of the chitinase of Thermococcus kodakaraensis. In the cultured medium of P. furiosus, however, no chitinase activity was detected. On analysis of the structural gene of P. furiosus, it appears that one nucleotide insertion in PF1234 caused a frame shift and separated a gene. By deletion of one nucleotide in PF1234, the best match was achieved between chitinases of T. kodakaraenesis and P. furiosus. We succeeded in constructing an artificial recombinant chitinase exhibiting hydrolytic activity toward not only colloidal but also crystalline chitins at high temperature. Furthermore, by analyzing the characteristics of the domains, a recombinant enzyme comprising two domains exhibiting high activity toward crystalline chitin was prepared.
Oku
Takashi
T
National Institute of Advanced Industrial Science and Technology, AIST, Ikeda, Osaka 563-8577, Japan.
Ishikawa
Kazuhiko
K
D000595:Amino Acid Sequence; D002686:Chitin / Q000737:chemistry*; D002688:Chitinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D003102:Colloids; D003460:Crystallization; D004795:Enzyme Stability; D016368:Frameshift Mutation; D006868:Hydrolysis; D008969:Molecular Sequence Data; D020027:Pyrococcus furiosus / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D019712:Thermococcus / Q000201:enzymology
D003102:Colloids; D011994:Recombinant Proteins; D002686:Chitin; D002688:Chitinases