Purification, characterization, and primary structure of a chitinase from Pseudomonas sp. YHS-A2

Abstract

A chitinase gene (chiA) from Pseudomonas sp. YHS-A2 was cloned into Escherichia coli using pUC19. The nucleotide sequence determination revealed a single open reading frame of chiA comprised of 1902 nucleotide base pairs and 633 deduced amino acids with a molecular weight of 67,452 Da. Amino acid sequence alignment showed that ChiA contains two putative chitin-binding domains and a single catalytic domain. Two proline-threonine repeat regions, which are linkers between catalytic and substrate-binding domains in some cellulases and xylanases, were also found. From E. coli, ChiA was purified 12.8-fold relative to the periplasmic fraction. The Michaelis constant and maximum initial velocity for p-nitrophenyl-N,N'-diacetylchitobiose were 1.06 mM and 44.4 micromol/h per mg protein, respectively. The purified ChiA binds not only to colloidal chitin but also to other substrates (avicel, chitosan, and xylan), but the binding affinity of avicel, chitosan, and xylan is around 10 times lower than that of colloidal chitin. The reaction of ChiA with colloidal chitin and chitooligosaccharides (trimer-hexamer) produced an end product of N,N'-diacetylchitobiose, indicating that ChiA is a chitobiosidase.

Identification and Characterization of a Chitinase Antigen from Pseudomonas aeruginosa Strain 385

“…Although there have been relatively few detailed investigations of the subcellular location of bacterial chitinases, most described thus far are secreted, including those for other P. aeruginosa strains (35,37) and Pseudomonas sp. (25). Notable exceptions can be found in the marine bacterium V. furnissii, which has two periplasmic chitinases (22), and ChiB from S. marcescens isolate BJL200 which was found mostly in the periplasm (less than 1% was found extracellularly) (3): however, this enzyme is secreted in other strains (36).…”

“…(34), Clostridium paraputrificum (27), and Pseudomonas sp. (25) were shown to have affinity for cellulose and avicel. In view of these data, it is perhaps not surprising that the chitinase from P. aeruginosa can also bind to cellulose.…”

Putative Exposed Aromatic and Hydroxyl Residues on the Surface of the N-Terminal Domains of Chi1 from Aeromonas caviae CB101 Are Essential for Chitin Binding and Hydrolysis

“…all of these enzymes belong to family 18 subfamily A (7,8,15). Excluding the catalytic domains, all three bacterial chitinases whose structures have been resolved have substrate binding domains either at their N (ChiA) or C (ChiB, ChiA1) termini, and this modular organizing feature is common for most of the known bacterial chitinases (2,11,12,14,15,16).…”

Biochemical characterisation of two forms of halo- and thermo-tolerant chitinase C ofSalinivibrio costicola expressed inEscherichia coli

“…Analysis of the kinetic data at lower substrate concentrations (5-30 µM) by the LineweaverBurk transformation indicated similar K m values for Chi-I and Chi-II of 30 and 31.8 µM while V max were 10 and 9.2 µmol/h/mg protein, respectively. K m and V max against this substrate for ChiA from Pseudomonas (Lee et al, 2000) were 1.06 mM and 44.4 µmol/h/mg protein. As a low K m indicate a high affinity of the enzyme for its substrate, Chi-I and Chi-II from S. costicola showed higher affinity for the substrate than that of chitinases from Pseudomonas.…”

“…Chi-I and Chi-II at high activity were able to hydrolyse the dimer or trimer to a monomer and a dimer with remaining trace amount of substrates (dimmer or trimer). ChiA from Pseudomonas (Lee et al, 2000) exhibited only chitobiosidase activity while chitinase from V. parahaemolyticus (Zhu et al, 1992) functioned as a b-N-acetylhexosaminidase. ChiA from S. marcescens exhibited both exo-and endo-chitinolytic acitivity.…”