Overexpression and kinetic analysis of Ideonella sakaiensis PETase for polyethylene terephthalate (PET) degradation

Abstract

Ideonella sakaiensis PET hydrolase (IsPETase) is a well-characterized enzyme for effective PET biodegradation. However, the low soluble expression level of the enzyme hampers its practical implementation in the biodegradation of PET. Herein, the expression of IsPETase^Mut, one of the most active mutants of IsPETase obtained so far, was systematically explored in E. coli by adopting a series of strategies. A notable improvement of soluble IsPETase^Mut was observed by using chaperon co-expression and fusion expression systems. Under the optimized conditions, GroEL/ES co-expression system yielded 75 ± 3.4 mg·L^-1 purified soluble IsPETase^Mut (GroEL/ES), and NusA fusion expression system yielded 80 ± 3.7 mg·L^-1 purified soluble NusA-IsPETase^Mut, which are 12.5- and 4.6-fold, respectively, higher than its commonly expression in E. coli. The two purified enzymes were further characterized. The results showed that IsPETase^Mut (GroEL/ES) displayed the same catalytic behavior as IsPETase^Mut, while the fusion of NusA conferred new enzymatic properties to IsPETase^Mut. Although NusA-IsPETase^Mut displayed a lower initial hydrolysis capacity than IsPETase^Mut, it showed a 1.4-fold higher adsorption constant toward PET. Moreover, the product inhibition effect of terephthalic acid (TPA) on IsPETase was reduced with NusA-IsPETase^Mut. Taken together, the latter two catalytic properties of NusA-IsPETase^Mut are more likely to contribute to the enhanced product release by NusA-IsPETase^Mut PET degradation for two weeks.

Keywords: Biodegradation of PET; Expression optimization; GroEL/ES co-Expression; IsPETase; NusA fusion system.