Ultrahigh-throughput screening-based directed evolution of D-allulose 3-epimerase
D-allulose is a low-calorie sweetener with a variety of physiological functions,such as maintaining blood glucose balance and regulating lipid metabolism.Currently,the industrial production of D-allulose is mainly achieved by catalyzing the epimerization of D-fructose by D-allulose 3-epimerase(DAEase),but the application performance of DAEase still needs to be further improved to meet the growing market demand.Directed evolution is an effective molecular modification strategy,but the screening method is one of the key fac-tors limiting its efficiency.By constructing and optimizing the D-allulose-responsive biosensor,combing microtiter plate screening,the DAEase from Clostridium cellulolyticum H10(CcDAEase)library was ultrahigh-throughput screened,and a dominant mutant H209Q with a 14.6％increase in specific activity was finally obtained,its optimum temperature and optimum pH were 60 ℃ and 7.5,respectively,and the structure-activity relationship of this mutant was further analyzed.This study proves that the transcription factor-based biosensor can be employed as an effective directed evolution screening tool,which provides the possibility to further optimize the performance of DAEase,and also provides a theoretical basis and technical support for the industrial preparation of D-allulose.
万方数据
维普
