Engineering a Chimeric Lipase-cutinase (Lip-Cut) for Efficient Enzymatic Deinking of Waste Paper

Abstract

Lipase and cutinase belong to the esterase family and have biological applications in many fields. To develop more efficient biocatalysts that can be used for waste paper deinking, a chimeric lipase-cutinase (LipCut) was constructed and successfully overexpressed in Pichia pastoris. The chimeric Lip-Cut exhibited lipase and cutinase activities that were 127% and 210% higher than their parent enzymes, respectively. Cut was superior to Lip in ink removal and improvement of paper brightness than Lip. The Lip-Cut displayed a better ink removal efficiency and paper brightness than that of the Lip, Cut, and Lip/Cut mixture. When the chimeric Lip-Cut was used, the ink removal efficiencies were 25.8% and 16.2% higher than that of the control-treated laser-printed paper and newspaper, which had sheet brightness values of 88% ISO and 59% ISO, respectively. The results demonstrated that the proper construction of bi-functional Lip-Cut could enhance the catalytic properties through the synergistic action of the two moieties because of the complementary advantages in the substrate specificities and catalysis patterns of both enzymes. This may provide an effective way to engineer more efficient bi-functional lipases and cutinases for deinking waste paper.

A Middle-Aged Enzyme Still in Its Prime: Recent Advances in the Field of Cutinases

“…Reactions with MtCUT were conducted in pH 7.5 at 30 • C for 3 h, and they resulted in ink removal rates of close to 80% for both types of paper. Liu and coworkers studied the efficiency of T. terrestris cutinase and Thermomyces lanuginosus lipase for the deinking of laser-printed paper and newspaper pulps [136]. Researchers first noticed that even though lipase had very low activity on PVAc, there seemed to be a synergistic effect between the two enzymes.…”

Current Knowledge on Polyethylene Terephthalate Degradation by Genetically Modified Microorganisms

“…Despite the similarities, an important difference is the width of the active site cleft, in comparison with cutinase from TfH; this slot is three times larger at its widest point in PETase ( Austin et al, 2018 ). Furthermore, the residues surrounding the nucleophilic serine in the catalytic triad were found to be considerably different, which affects the substrate selectivity represented by these enzymes ( Liu et al, 2018b ).…”

“…Especially, the regions on the surface outside the active site of the enzymes and binding modules are essential, both in interaction with the polymer and during the hydrolysis ( Acero et al, 2013 ). For instance, Liu et al (2018a) assumed in their study that the wide substrate-binding pocket of PETase is critical for PET hydrolysis ( Liu et al, 2018b ). In contrast, Austin et al (2018) narrowed the binding cleft and observed improvement in PET degradation ( Austin et al, 2018 ).…”

“…The surface of the PET film was roughened and eroded, and a large number of holes were observed ( Ma et al, 2018 ). In the study of Liu et al (2018b) structure-guided site-directed mutagenesis was used to improve PETase catalytic efficiency. Several mutants were created with mutations in the active sites, substrate binding pockets or in the residues involved in stabilizing the rigidity of the active site.…”

“…An artificial chimeric enzyme was also constructed by Liu et al (2018a) . In their study lipase (Lip) from Thermomyces lanuginosus and cutinase (Cut) from Thielavia terrestris NRRL 8126 were used for the construction of bifunctional lipase-cutinase (Lip-Cut) by end-to-end fusion and overexpression in Pichia pastoris ( Liu et al, 2018b ). Lip-Cut exhibited a more efficient degradation ability towards poly(ε-caprolactone) (PCL).…”