Thermostable beta-cyclodextrin conjugates of two similar plant amine oxidases and their properties.
Syntheses of conjugates of garden pea (Pisum sativum) and grass pea (Lathyrus sativus) amine oxidases (PSAO and GPAO respectively) with BCD (beta-cyclodextrin), performed to improve the thermostability of the enzymes, are described in the present study. Periodate-oxidized BCD reacted with the enzyme proteins via free primary amino groups in a buffered solution containing cyanoborohydride as a reductant. Although the specific activities of PSAO and GPAO partially decreased after modification, Km values determined for the best diamine substrates remained almost unchanged. Both the BCD conjugates could be incubated at 65 degrees C for 30 min without considerable inactivation, and the residual activity remained detectable even after incubation at 75 degrees C. The conjugates contained approx. 30% of neutral sugars. Molecular masses of BCD-PSAO and BCD-GPAO (180 kDa), as estimated by gel-permeation chromatography, were higher compared with the value of 145 kDa for the native enzymes. This was in good correlation with the number of modified lysine residues determined by a spectrophotometric method. Peptide mass fingerprints of tryptic digests of BCD-PSAO and BCD-GPAO were less specific than those of the native enzymes when compared with the database sequence of PSAO. As a consequence of the modification, many unidentified peaks were observed in the digests of the studied conjugates that were not seen in the digests of native PSAO and GPAO. Only some of these peaks overlapped between BCD-PSAO and BCD-GPAO. The BCD conjugates described in the present study represent suitable candidates for biotechnological applications, e.g. in analyses using biosensors, which might benefit from increased storage stability and amine oxidation at high temperatures.
Sebela
Marek
M
Department of Biochemistry, Faculty of Science, Palacký University, Slechtitelů 11, CZ-783 71 Olomouc, Czech Republic. sebela@prfholnt.upol.cz
Kopecný
David
D
Lamplot
Zbynek
Z
Havlis
Jan
J
Thomas
Henrik
H
Shevchenko
Andrej
A
D004789:Enzyme Activation; D004795:Enzyme Stability; D007527:Isoenzymes / Q000737:chemistry; D027807:Lathyrus / Q000201:enzymology*; D018532:Pisum sativum / Q000201:enzymology*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013696:Temperature; D047392:beta-Cyclodextrins / Q000737:chemistry*
D007527:Isoenzymes; D047392:beta-Cyclodextrins; C031215:betadex