Transglycosylation by chitinase D from Serratia proteamaculans improved through altered substrate interactions

Abstract

We describe improvement of the transglycosylation (TG) by chitinase D from Serratia proteamaculans (SpChiD). The SpChiD produced less proportion of TG products up to 90 min, with 2 mM chitotetraose as the substrate and hydrolysed the products subsequently. Of the five residues targeted at the catalytic center, E159D resulted in substantial loss of both hydrolytic and TG activities. Y160A resulted in a product profile similar to SpChiD and a rapid turnover of substrate with slightly increased TG activity. Rest of the three mutants, M226A, Y228A and R284A displayed improved TG and decreased hydrolytic ability. Four of the five amino acid substitutions, F64W, F125A, G119S and S116G, at the catalytic groove increased TG activity, while W120A completely lost the TG activity with a concomitant increase in hydrolysis. Mutation of W247 at the solvent accessible region significantly reduced the hydrolytic activity with increased TG activity. The mutants M226A, Y228A, F125A, S116G, F64W, G119S, R284, and W247A accumulated approximately double the concentration of TG products like chitopentaose and chitohexaose, compared to SpChiD, respectively. The double mutant E159D/F64W regained the activity with accumulation of 6.0% of chitopentaose at 6 h, similar to SpChiD at 30 min. Loss of chitobiase activity was unique to Y228A. Substitution of amino acids at catalytic center and/or groove substantially improved the TG activity of SpChiD, both in terms of quantity of TG products produced and the extended duration of TG activity.