PEDS Advance Access published online on June 6, 2008
Protein Engineering Design and Selection, doi:10.1093/protein/gzn033
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Structural simulation and protein engineering to convert an endo-chitosanase to an exo-chitosanase
1Department of Applied Chemistry 2Department of Biological Science and Technology, Institute of Bioinformatics, National Chiao Tung University, 1001 Ta-Hseh Road, Hsinchu, Taiwan 3Department of Agricultural Chemistry, Faculty of Horticulture, Chiba University, Matsudo 271, Japan 4Department of Marine Biotechnology, National Kaohsiung Marine University, 142 Hai-Chuan Road, Nan-Tzu Dist, Kaohsiung, Taiwan
5 To whom correspondence should be addressed. E-mail: ykl{at}cc.nctu.edu.tw (Y.-K. Li)/cycheng{at}mail.nkmu.edu.tw (C.-Y. C)
To obtain an enzyme for the production of chito-disaccharides (GlcN2) by converting endo-chitosanase to exo-chitosanase, we chose an endo-chitosanase from Bacillus circulans MH-K1 (Csn) as the candidate for protein engineering. Using molecular modeling, two peptides with five amino acids (PCLGG) and six amino acids (SRTCKP) were designed and inserted after the positions of D115 and T222 of Csn, respectively. The inserted fragments are expected to form loops that might protrude from opposite walls of the substrate-binding cleft, thus forming a ‘roof’ over the catalytic site that might alter the product specificity. The chimeric chitosanase (Chim-Csn) and wild-type chitosanase (WT-Csn) were both over-expressed in Escherichia coli and purified nearly to homogeneity. The products formed from chitosan were analyzed by ESI-MS (electrospray ionization-mass spectrometry). A mixture of GlcN2, GlcN3 and GlcN4 was obtained with WT-Csn, whereas Chim-Csn formed, with a smaller catalytic rate (3% of WT-Csn activity), GlcN2 as the dominant product. Measurements of viscosity showed that, with similar amounts of enzyme activity, Chim-Csn catalyzed the hydrolysis of chitosan with a smaller rate of viscosity decrease than WT-Csn. The results indicate that, on inserting two surface loops, the endo-type chitosanase was converted into an exo-type chitosanase, which to our knowledge is the first chitosanase that releases GlcN2 from chitosan as the dominant product.
Keywords: chitosan/exo-chitosanase/mass spectrometry/structural modeling
Received September 24, 2007; revised April 23, 2008; accepted May 7, 2008.
Abbreviations: GlcN, 2-amino-2-deoxy-D-glucopyranose; GlcNn, β-1,4-linked oligomer of GlcN with a polymerization degree n.