Thermus sp. strain Rt41A produces an extracellular thermostable alkaline proteinase. The enzyme has a high isoelectric point (10.25 -10.5) which can be exploited in purification by using cationexchange chromatography. The proteinase was purified to homogeneity and has a molecular mass of 32.5 kDa by SDS/PAGE. It is a glycoprotein, containing 0.7% carbohydrate as glucose equivalents, and has four half-cystine residues present as two disulphide bonds. Maximum proteolytic activity was observed at pH 8.0 against azocasein and greater than 75% of this activity was retained in the pH range 7.0-10.0. Substrate inhibition was observed with casein and azocasein. The enzyme was stable in the pH range 5.0-10.0 and maximum activity, in a 10-min assay, was observed at 90°C with 5 mM CaCI, present. No loss of activity was observed after 24 h at 70°C and the half-lives at 80°C and 90°C were 13.5 h and 20 min, respectively. Removal of Ca2+ reduced the temperature for maximum proteolytic activity against azocasein to 60 "C and the half-life at 70 "C was 2.85 min. The enzyme was stable at low and high ionic strength and in the presence of denaturing reagents and organic solvents.Rt41A proteinase cleaved a number of synthetic amino acid p-nitrophenol esters, the kinetic data indicating that small aliphatic or aromatic amino acids were the preferred residue at the P1 position. The kinetic data for the hydrolysis of a number of peptide p-nitroanilide substrates are also reported. Primary cleavage of the oxidized insulin B chain occurred at sites where the P1' amino acid was aromatic. Minor cleavage sites (24 h incubation) were for amino acids with aliphatic side chains at the P1' position. The esterase and insulin cleavage data indicate the specificity is similar for both the P1 and P1' sites.Generally there is no firm evidence to suggest that an enzyme isolated from an extremely thermophilic organism will have a higher specific activity than its mesophilic counterpart (when assayed at a temperature appropriate to the growth temperature of the producing organism). However, mesophilic proteins tend to denature and become more susceptible to proteolysis at the temperatures at which thermostable enzymes operate optimally. Thus, proteinases from extreme thermophiles have higher specific activities against mesophilic proteins than do most microbial proteinases [l]. The inherent stability of thermophilic proteinases to high temperatures as well as to detergents, organic solvents and chaotropic agents, makes these enzymes potentially useful in a range of biotechnological applications [2, 31.

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. NH 2 -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. The K m and k cat for 4-nitrophenyl-␤-D-N,N-diacetylchitobiose were 4.28 mM and 1.7 s ؊1 respectively, and for 4-nitrophenyl-␤-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.Chitin is the second most abundant polysaccharide found in nature and consists of variable-length linear chains of ␤-1,4-linked polymers of N-acetylglucosamine hydrogen bonded into an ordered insoluble crystalline structure. The enormous amounts of chitin produced annually in the biosphere are degraded by chitinases. Chitinases are ubiquitous in nature, being found in eucaryotes, procaryotes, archaea, and viruses. They consist of a group of hydrolytic enzymes that are able to break down polymeric chitin to chitin oligosaccharides, diacetylchitobiose, and N-acetylglucosamine. Endochitinases catalyze the hydrolysis of chitin at random sites along the polymer, whereas exochitinases (␤-1,4-N-acetylglucosaminidases) remove single N-acetylglucosamine residues from the nonreducing ends of chitin chains. Chitobiosidases that remove diacetylchitobiose from the nonreducing ends of chitin oligosaccharides are often considered exo-or endochitinases. They should be described as exochitinases only if it can be demonstrated that the specificity is restricted to the removal of diacetylchitobiose from the nonreducing ends of chitin and chitin oligosaccharides. Efficient breakdown of chitin to metabolizable monomers requires the action of both endochitinases and exochitinases to release monomeric N-acetylglucosamine, which can then be metabolized to generate energy, CO 2 , H 2 O, and NH 3 .

This study examines the safety and immunogenicity of an oral, whole-cell Pseudomonas aeruginosa vaccine administered to healthy volunteers. Thirty subjects received an oral dose of Pseudostat in two timed, measured doses with serological follow-up to 56 days postvaccination. Following vaccination, several individuals were identified as antibody responders for all three immunoglobulin (Ig) isotypes tested, specifically against whole-cell P. aeruginosa extract and outer membrane proteins F and I. The mean pooled lipopolysaccharide antigen-specific IgA showed the most significant and constant increases in titer postdose, with a similar increase in titer for whole-cell P. aeruginosa extract-specific IgA. The results demonstrated an increased phagocytic ability of the selected macrophage cell line in post vaccination sera. Furthermore a significant increase in intracellular macrophage killing of opsonized P. aeruginosa was also demonstrated (82% on day 14 postdose) in the presence of the postvaccination sera. The safety component of the study did not show any vaccine-attributable adverse effects in any of the subjects, as documented by clinical evidence, hematology, and biochemistry profiles. We conclude that Pseudostat is safe and immunogenic in humans at this dose and that further studies to determine the appropriate dosage and efficacy are needed. In our study, we have shown that the most significant and sustained responses to oral vaccination in human adult volunteers were serum IgA levels and that pooled sera collected postimmunization have an increased capacity to promote opsonophagocytotic killing of P. aeruginosa.

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The Thermus isolate Rt4A2 was found to produce an extracellular chelator-resistant proteinase. The proteinase was purified to homogeneity by (NH4)2SO4 precipitation, cation-exchange chromatography, gel-filtration chromatography, and weak anion-exchange chromatography. The Rt4A2 proteinase was found to have properties typical of an alkaline serine proteinase. It had a pH optimum of 9.0 and was specifically inhibited by phenylmethanesulphonyl fluoride. Its isoelectric point was greater than 10.25. Its molecular-mass was 31.6 kDa as determined by SDS/PAGE. N-terminal sequencing has shown it to have high sequence similarity with other serine proteinases from Thermus species. The proteinase hydrolysed a number of substrates including fibrin, casein, haemoglobin, collagen, albumin and the synthetic chromogenic peptide substrate Suc-Ala-Ala-Pro-Phe-NH-Np. The specific activity of the purified proteinase using azocasein as substrate was 313 units/mg. Substrate inhibition was observed above an azocasein concentration of 0.05% (w/v). Esterase activity was directed mainly towards those substrates containing the aliphatic or aromatic residues of alanine, glycine, tryptophan, tyrosine and phenylalanine. Thermostability half-lives of greater than 7 days at 70 degrees C, 43 h at 80 degrees C and 90 min at 90 degrees C were found in the presence of 5 mM CaCl2. At 90 degrees C increasing the CaCl2 concentration 100-fold (0.5 mM to 50 mM) caused a 4.3-fold increase in the half-life of the enzyme from 30 to 130 min. Half-lives of 19.4 min at 100 degrees C and 4.4 min at 105 degrees C were found in the presence of 50 mM CaCl2. The metal chelators EGTA and EDTA reduced the stability at higher temperatures but had no effect on the activity of the proteinase. Activity was not stimulated by common metal activators such as Ca2+, Mg2+ and Zn2+.