Keith Peek

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

ABSTRACT A chitinase antigen has been identified in Pseudomonas aeruginosa strain 385 using sera from animals immunized with a whole-cell vaccine. The majority of the activity was shown to be in the cytoplasm, with some activity in the membrane fraction. The chitinase was not secreted into the culture medium. Purification of the enzyme was achieved by exploiting its binding to crab shell chitin. The purified enzyme had a molecular mass of 58 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a pI of 5.2. NH2-terminal amino acid sequencing revealed two sequences of M(I/L)RID and (Q/M/V)AREDAAAAM that gave an exact match to sequences in a translated putative open reading frame from the P. aeruginosa genome. The chitinase was active against chitin azure, ethylene glycol chitin, and colloidal chitin. It did not display any lysozyme activity. Using synthetic 4-methylumbelliferyl chitin substrates, it was shown to be an endochitinase. TheKm and kcat for 4-nitrophenyl-β-d-N,N′-diacetylchitobiose were 4.28 mM and 1.7 s−1 respectively, and for 4-nitrophenyl-β-d-N,N′,N′′-triacetylchitotriose, they were 0.48 mM and 0.16 s−1 respectively. The pH optimum was determined to be pH 6.75, and 90% activity was maintained over the pH range 6.5 to 7.1. The enzyme was stable over the pH range 5 to 10 for 3 h and to temperatures up to 50°C for 30 min. The chitinase bound strongly to chitin, chitin azure, colloidal chitin, lichenan, and cellulose but poorly to chitosan, xylan, and heparin. It is suggested that the chitinase functions primarily as a chitobiosidase, removing chitobiose from the nonreducing ends of chitin and chitin oligosaccharides.

Laboratory-scale investigations into the use of extremely thermophilic proteinases for cleaning ultrafiltration membranes fouled during whey processing

Abstract The potential of proteolytic enzymes from extremely thermophilic eubacteria to clear ultrafiltration membranes fouled during the processing of whey was investigated. The enzymes were initially tested for their ability to hydrolyse freeze-dried preparations of material removed from fouled ultrafiltration membranes. Under the conditions used, the serine proteinases from Thermus strains were more efficient than the metalloproteinase from Bacillus strain EA.1, and were subsequently investigated for their ability to clear fouled membrane discs using purpose-built laboratory scale equipment. The proteinase from Thermus strain Rt4.1A proved most effective and gave a linear increase of water flux with time when used to treat fouled membranes at 70°C. The rate of clearing and final flux levels obtained using enzyme alone were lower than those obtained using alkaline-EDTA. The performance of the enzyme was, however, synergistically enhanced using the anionic detergent sodium dodecyl sulphate in combination with the enzyme.

The use of phenyl-Sepharose for the affinity purification of proteinases

Phenyl-Sepharose is most often used as an adsorbent for hydrophobic interaction chromatography (HIC). We report on its effective use for the affinity purification of some extracellular thermostable proteinases from bacterial sources. Proteinases belonging to the serine, aspartate and metallo mechanistic classes were effectively retained by the media. Purification factors in the range of 2.9-60 and enzyme activity yields in excess of 88% were obtained. In some cases homogeneous enzyme was obtained from culture supernatants in a single step. A number of other proteinases from mammalian sources were also retained. The specificity of the enzyme/support interaction was studied. Proteinases complexed with peptide inhibitors (pepstatin and chymostatin) showed reduced binding to phenyl Sepharose indicating interaction with the active site cleft whereas modification with low molecular weight active site directed inactivators such as PMSF and DAN did not, indicating that binding may not be dependent on the catalytic site. Pepsinogen and the pro-enzyme form of the serine proteinase from the thermophilic Bacillus sp. strain Ak.1 were not retained by the media and could be resolved in an efficient manner from their active counterparts.