Increased thermal resistance and modification of the catalytic properties of a beta-glucosidase by random mutagenesis and in vitro recombination

The bglB gene from Paenibacillus polymyxa was subjected to random mutagenes is mediated by error prone polymerase chain reaction amplification and DNA shuffling. After this treatment, mutant variants of the encoded p-glucosida se with enhanced thermal resistance were selected. We identified five amino acid substitutions at four different positions of the sequence that increa sed the resistance of the enzyme to heat denaturation, Four of the mutation s, H62R, M319V, M319I, and M361I, did not change the kinetic parameters of the enzyme. However, mutant N223Y, which caused only a marginal increase in thermoresistance, showed an 8-fold decrease in K-m. Copies of the bglB gen e carrying each one of the individual mutations were recombined in vitro by DNA shuffling. As a result, we obtained an enzyme that simultaneously exhi bited a 20-fold increase in heat resistance and an 8-fold increase in the c atalytic efficiency. The structural basis of the properties conferred by th e mutations was analyzed using homology-based structural models. The four m utations causing a more pronounced effect on thermoresistance were located in loops, on the periphery of the (alpha/beta)(8) barrel that conforms the structure of the protein. Mutation N223Y, which modifies the catalytic prop erties of the enzyme, was on one of the barrel beta-strands that shape the active center.