Abstract:[Background] Agarases belong to glycoside hydrolases with broad application potentials in scientific research, health-food, pharmaceutical and cosmetic industries. Although a β-agarase YM01-3 from Catenovulum agarivorans YM01T was obtained in previous studies with an activity up to 1.14×104 U/mg, its catalytic mechanism is still unclear. [Objective] In order to clarify the catalytic mechanism of YM01-3, the key sites affecting the activity should be identified. [Methods] A random mutation library in Bacillus brevis was constructed by error-prone PCR to select mutants that completely lost enzyme activity. In total 227 mutants showing lower activity were selected from about 10 000 clones and 80 mutants were sequenced and analyzed. The putative key sites were confirmed by mutation construction. [Results] Compared to the original enzyme YM01-3, two mutants at 137 and 237 sites lost more than 90% catalytic activity. Y137 and D237, located in the catalytic cavity, were proved to be the novel key sites closely related to YM01-3 hydrolytic activity. [Conclusion] Our findings provide reference for further studying catalytic mechanism and molecular modification of β-agarase.
