Date of Award
Doctor of Philosophy (PhD)
Roger A. Laine
Two chitin-binding proteins from Vibrio parahaemolyticus are secreted into the culture medium after the addition of chitin or oligomers of N-acetylglucosamine, but only to lower levels of expression with a few other saccharides. The first protein exhibited a very salt-resistant chitinase activity with activity maxima at pH 6 and 9. The enzyme is stable for 30 minutes at 40$\sp\circ$C, and maximal activity is reached at 50$\sp\circ$C. The chitinase hydrolyzes optimally chitin oligomer substrates with degree of polymerization of 4 and higher. The hydrolytic activity upon the chitotrimer is low; the dimer cannot be hydrolyzed by the enzyme. When incubated with chitotrimer, the protein exhibits transglycosylase activity. The chitinase from V. parahaemolyticus shows a strong sequence homology with chitinases from Serratia marcescens and Bacillus circulans. The other protein purified from Vibrio parahaemolyticus culture supernatant fractions is a chitin-binding protein with an apparent molecular weight of 134,000. The protein was either purified by conventional ammonium sulfate precipitation followed by fractionation on a gel filtration column, or by affinity chromatography on regenerated chitin, followed by differential elution with increasing concentrations of guanidine hydrochloride. Samples of this protein eluted with guanidine hydrochloride retained their binding capacity after dialysis. The protein could also be specifically eluted from chitin with a concentrated solution of chitin oligomers. The N-terminal amino acid sequence of the protein showed no strong homology to any known protein sequences in the GeneBank data bank, suggesting that it may be a novel carbohydrate-binding protein tentatively named chitovibrin. Chitovibrin exhibits similar chromatographic characteristics to chitinase, but polyclonal antibodies raised against chitinase do not cross-react with chitovibrin. The function of chitovibrin has yet to be determined.
Gildemeister, Otto Silva, "Study on Two Chitin-Binding Proteins From Vibrio Parahaemolyticus." (1991). LSU Historical Dissertations and Theses. 5235.