Thermostable, salt tolerant, wide pH range novel chitobiase from Vibrio parahemolyticus: isolation, characterization, molecular cloning, and expression
| Autor: | B C, Zhu, J Y, Lo, Y T, Li, S C, Li, J M, Jaynes, O S, Gildemeister, R A, Laine, C Y, Ou |
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| Rok vydání: | 1992 |
| Předmět: |
Hot Temperature
Molecular Sequence Data Osmolar Concentration Gene Expression Regulation Bacterial Hydrogen-Ion Concentration Sodium Chloride Cell Fractionation Recombinant Proteins Substrate Specificity Enzyme Activation Kinetics Transformation Genetic Bacterial Proteins Carbohydrate Sequence Genes Bacterial Acetylglucosaminidase Enzyme Stability Escherichia coli Amino Acid Sequence Isoelectric Point Vibrio parahaemolyticus Cloning Molecular |
| Zdroj: | Journal of biochemistry. 112(1) |
| ISSN: | 0021-924X |
| Popis: | A chitobiase gene from Vibrio parahemolyticus was cloned into plasmid pUC18 in Escherichia coli strain DH5 alpha. The plasmid construct, pC120, contained a 6.4 kb Vibrio DNA insert. The recombinant gene expressed chitobiase [EC 3.2.1.30] activity similar to that found in the native Vibrio. The enzyme was purified by ion exchange, hydroxylapatite and gel permeation chromatographies, and exhibited an apparent molecular weight of 80 kDa on SDS-polyacrylamide gel electrophoresis. Chitobiose and 6 more substrates, including beta-N-acetyl galactosamine glycosides, were hydrolyzed by the recombinant chitobiase, indicating its putative classification as an hexosaminidase [EC 3.2.1.52]. The enzyme was resistant to denaturation by 2 M NaCl, thermostable at 45 degrees C and active over a very unusual (for glycosyl hydrolases) pH range, from 4 to 10. The purified cloned chitobiase gave 4 closely focussed bands on an isoelectric focusing gel, at pH 4 to 6.5. The N-terminal 43 amino acid sequence shows no homology with other proteins in commercial databanks or in the literature, and from its N-terminal sequence, appears to be a novel protein, unrelated in sequence to chitobiases from other Vibrios reported and unrelated to hexosaminidases from other organisms. |
| Databáze: | OpenAIRE |
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