Detection of major xylanase-containing cellulose-binding domain from Penicillium verruculosum by combination of chromatofocusing and limited proteolysis |
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Authors: | Alejandro G Berlin Alexander V Gusakov Olga A Sinitsyna Arkady P Sinitsyn |
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Institution: | (1) Department of Chemistry, M. V. Lomonosov Moscow State University, Vorobyevy Gory, 119899 Moscow, Russia |
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Abstract: | Adsorption on microcrystalline cell ulose of enzyme components of cellulase complex from Penicillium verruculosum was studied by chromatofocusing on a Mono P column. The most strongly adsorbed and major component was identified as xylanase
(XYN) with MW 65 k Da and pl 4.5. The high adsorption degree of XYN on cellulose indicated the possible presence of a cellulose-binding domain in the
molecular sturcture. Limited proteolysis of XYN with papain was carried out. Kinetics of proteolysis was monitored by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and measuring activities toward insoluble xylan and
4-methylumbelliferyl-β-d-lactoside (MUF-LAC). During the proteolysis, formation of two polypeptides with MW 51 and 14k Da was observed. No loss of
activity toward thesolu blesubstrate was observed, wherease the activity toward xylan decreased rapidly. Adsorption distribution
coefficient (K
d) of the core protein separated by gel-filtration was found to be 15 times lower than the K
d for the initial nondigested XYN (0.02 and 0.29 L/g, respectively). The activity of core protein toward insoluble xylan was
close to zero, whereas the activity toward MUF-LAC was close to that exhibited by the original enzyme. The results presented
indicate a bifunctional organization of XYN, where one domain acts as a binding anchor for insoluble substrates and the other,
localized in the core protein, contains the active site. |
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Keywords: | Penicillium verruculosum xylanase cellulose-binding domain limited proteolysis papain enzyme adsorption |
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