ABSTRACT
An aprE mutant from B. subtilis 168 lacking the connecting loop Leu(75)-Leu(82) which is predicted to encode a Ca(2+) binding site was constructed. Expression of the mutant gene (aprEDeltaLeu(75)-Leu(82)) produced B. subtilis colonies lacking protease activity. Intrinsic fluorescence analysis revealed spectral differences between wild-type AprE and AprEDeltaL(75)-L(82). An AprEDeltaL(75)-L(82) variant with reestablished enzyme activity was selected by directed evolution. The novel mutations Thr(66)Met/Gly(102)Asp located in positions which are predicted to be important for catalytic activity were identified in this variant. Although these mutations restored hydrolysis, they had no effect with respect to thermal inactivation of AprEDeltaL(75)-L(82) T(66)M G(102)D. These results support the proposal that in addition to function as a calcium binding site, the loop that connects beta-sheet e3 with alpha-helix c plays a structural role on enzyme activity of AprE from B. subtilis 168.
Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Calcium/metabolism , Directed Molecular Evolution/methods , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Mutagenesis, Site-Directed/methods , Bacillus subtilis/enzymology , Bacillus subtilis/genetics , Bacterial Proteins/chemistry , Binding Sites , Enzyme Stability , Kinetics , Membrane Transport Proteins/chemistry , Models, Molecular , Protein Folding , Spectrometry, Fluorescence , Structure-Activity RelationshipABSTRACT
The soil microorganism Myxobacter Sp. AL-1 regulates in a differential manner the production of five extracellular cellulases during its life cycle. The nucleotide sequence of a cel9-cel48 cluster from the genome of this microorganism was recently obtained. Cel48 was expressed in Escherichia coli to generate a His(6)-Cel48 protein and the biochemical properties of the pure protein were determined. Cel48 was more efficient in degrading acid-swollen avicel (ASC) than carboxymethylcellulose (CMC). On the other hand, cel9 was expressed in Bacillus subtilis from an IPTG-inducible promoter. Zymogram analysis showed that after IPTG-induction, Cel9 existed in both the cell fraction and the culture medium of B. subtilis and the secreted protein was purified to homogeneity by FPLC-ionic exchange chromatography. The exocellobiohydrolase Cel48 showed a synergism of 1.68 times with the endocellulase Cel9 during ASC degradation using an 8.1-fold excess of Cel48 over Cel9. Western blot analysis revealed that both proteins were synthesized and secreted to the culture medium of Myxobacter Sp. AL-1. These results show that the cel9-cel48 cluster encodes functional endo- and exo-acting cellulases that allows Myobacter Sp. AL-1 to hydrolyse cellulose.
