Structural simulation and protein engineering to convert an endo-chitosanase to an exo-chitosanase

Yueh Yun Yao, Keshab Lal Shrestha, Yue Jin Wu, Huei Ju Tasi, Chun Chen Chen, Jinn-Moon Yang, Akikazu Ando, Chih Yu Cheng, Yaw-Kuen Li*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

13 Scopus citations

Abstract

To obtain an enzyme for the production of chito-disaccharides (GlcN 2 ) 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 D 115 and T 222 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 GlcN 2 , GlcN 3 and GlcN 4 was obtained with WT-Csn, whereas Chim-Csn formed, with a smaller catalytic rate (3% of WT-Csn activity), GlcN 2 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 GlcN 2 from chitosan as the dominant product.

Original languageEnglish
Pages (from-to)561-566
Number of pages6
JournalProtein Engineering, Design and Selection
Volume21
Issue number9
DOIs
StatePublished - 1 Sep 2008

Keywords

  • Chitosan
  • Exo-chitosanase
  • Mass spectrometry
  • Structural modeling

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