Widespread utilization of polyethylene terephthalate (PET) has caused critical environmental pollution. The enzymatic degradation of PET is a promising solution to this problem. In this study, PETase, which exhibits much higher PET-hydrolytic activity than other enzymes, was successfully secreted into extracellular milieu from Bacillus subtilis 168 under the direction of its native signal peptide (named SPPETase). SPPETase is predicted to be a twin-arginine signal peptide. Intriguingly, inactivation of twin-arginine translocation (Tat) complexes improved the secretion amount by 3.8-fold, indicating that PETase was exported via Tat-independent pathway. To the best of our knowledge, this is the first report on the improvement of Tat-independent secretion by inactivating Tat components of B. subtilis 168 in LB medium. Furthermore, PET film degradation assay showed that the secreted PETase was fully active. This study paves the first step to construct an efficient engineered strain for PET degradation.

Protein stability and evolvability influence each other.A lthough protein dynamics play essential roles in various catalytically important properties,their high flexibility and diversity makes it difficult to incorporate such properties into rational engineering.T herefore,h ow to unlock the potential evolvability in auser-friendly rational design process remains achallenge.Inthis endeavor,wedescribe amethod for engineering an enantioselective alcohol dehydrogenase.I t enables synthetically important substrate acceptance for 4chlorophenyl pyridine-2-yl ketone,and perfect stereocontrol of both (S)-and (R)-configured products.T hermodynamic analysis unveiled the subtle interaction between enzyme stability and evolvability,while computational studies provided insights into the origin of selectivity and substrate recognition. Preparative-scale synthesis of the (S)-product (73 %y ield; > 99 %e e) was performed on ag ram-scale.T his proof-ofprinciple study demonstrates that interfaced proline residues can be rationally engineered to unlock evolvability and thus providea ccess to new biocatalysts with highly improved catalytic performance.

There are ongoing interests in improving the galactooligosaccharide (GOS) synthesis efficiency of β-galactosidase by protein engineering. In this study, an intelligent double-hydrophobic amino acid scanning strategy was proposed and employed to target nine residues forming the glycon-binding site (−1 subsite) of β-galactosidase Bgal1-3. Two mutants C510V and H512I with significantly improved GOS synthesis efficiency were obtained. When 40% (w/v) lactose was used as a substrate, Bgal1-3 reached a maximum GOS yield of 45.3% at 16 h, while the mutants reached higher yields in a much shorter time (59.1% at 10 h for C510V, 51.5% at 2 h for H512I). When skim milk was treated with these enzymes, more GOS was produced (19.9 g/L for C510V, 12.7 g/L for H512I) than that for Bgal1-3 (10.3 g/L) at a lactose conversion of 90%. These results validated hydrophobicity scanning as an efficient method to engineer β-galactosidases into promising catalysts for the preparation of GOS and GOS-enriched milk.