Aer_2022_Environ.Res_212_113472

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 75s+/-s3.4smg.L(-1) purified soluble IsPETase(Mut) (GroEL/ES), and NusA fusion expression system yielded 80s+/-s3.7smg.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.