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

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.

Application of Spectroscopic Methods for Structural Analysis of Chitin and Chitosan

“…The ESI ion source is usually coupled to an ion trap analyser [235], but a quadrupole type of analyser can also be used [237]. For sample measurements several parameters should be optimized, for example, the source voltage (3.5–4 kV), nitrogen sheath gas rate and capillary temperature.…”

“…From MS studies it was found that immobilization of enzymes (papain [259], neutral protease [243]) enhanced depolymerization in the chitosan chain comparing with free enzymes [243,259]. The activities of enzymes isolated from different sources (commercial enzymes [261], isolated from living organisms: Vibrio harveyi [244], Serratia marcensces [251], Bacillus circulans [237], Amycolatopsis orientalis , Streptomyces sp. [241]) were tested by qualitative MS studies of obtained chitooligosaccharides.…”

Structural Determinants Responsible for Substrate Recognition and Mode of Action in Family 11 Polysaccharide Lyases

“…This conversion is based on protein engineering reducing the steric restriction at the subsite Ϫ3 specific for the exotype enzyme. More recently, endo/exo conversion in GH families 26, 46, and 74 has been demonstrated (42)(43)(44). Although our molecular conversion of exotype YesX to endotype YesW-like enzyme is similar to that of GH families 6 and 74 exohydrolase to endoenzyme by removing a specific loop for exotype enzyme enclosing the active cleft, this is the first example of endo/exo conversion in PLs.…”

Design and Evolution of Biocatalysts

“…The catalytic efficiency of 25B12 for the new substrate (0.04 s -1 mM -1 ) was lower than the wild-type enzyme for the original substrate (40.5 s -1 mM -1 ) and even lower than the 25B12 mutant for the original substrate (0.49 s -1 mM -1 ). In most cases of designing substrate specificity, the catalytic activity of the resulting enzyme was very low for the targeted substrate [2,17], and increased selectivity mainly resulted from decreased activity towards the original substrate [22,27]. Unknown remote mutations can cause changes in catalytic efficiency between the wild-type and designed enzymes.…”