Site-directed mutagenesis of a fungal beta-1,4-endoglucanase increases the minimum size required for the substrate.

Conserved regions within a family-5 beta-1,4,-endoglucanase (Eg11), produced by the fungus Macrophomina phaseolina, have been modified through site-directed mutagenesis, resulting in production of an enzyme with novel substrate requirements. The engineered form was generated through mutagenesis of D232A, which lies within the substrate-binding cleft of Eg11. Wild-type Eg11 requires a minimum substrate size of five glucosyl units, while the engineered form requires a minimum of six glucosyl units. Screening was facilitated by the unique ability to obtain functional expression of the fungal endoglucanase in Escherichia coli. Wild-type and mutated Eg11 have equivalent activity on cellohexaose, and both release cellobiose from the reducing end of the cellodextrin. This is the first example of protein engineering of an endoglucanase that results in a novel minimum substrate requirement for cellohexaose. This substrate specificity has not been reported for any native endoglucanases, thus the modified Eg11 may prove useful in applications requiring specific hydrolysis of complex carbohydrates such as beta-glucans.