Simultaneous enhancement of barley ß-amylase thermostability and catalytic activity by R115 and T387 residue sites mutation.

ABSTRACT

OBJECTIVE: To simultaneously increase the thermostability and catalytic activity of barley ß-amylase. METHODS: The amino acid sequences of various barley ß-amylases with different enzyme properties were aligned, two amino acid residues R115 and T387 were identified to be important for barley ß-amylase properties. R115C and T387V were then generated using site-directed and saturation mutagenesis. RESULTS: R115C and T387V mutants increased the enzyme catalytic activity and thermostability, respectively. After combinational mutagenesis, the T50 value and t(1/2,60oC) value of R115C/T387V mutant reached 59.4 °C and 48.8 min, which were 3.6 °C higher and 29.5 min longer than those of wild-type. The kcat/Km value of mutant R115C/T387V were 59.82/s·mM, which were 54.7% higher than that of wild-type. The increased surface hydrophobicity and newly formed strong hydrogen bonds and salt bridges might be responsible for the enzyme thermostability improvement while the two additional hydrogen bonds formed in the active center may lead to the catalytic property enhancement. CONCLUSIONS: The mutant R115C/T387V showed high catalytic activity and thermostability indicating great potential for application in industry.