Thermal stability engineering of Glomerella cingulata cutinase

Abstract

Cutinase has been ascertained as a biocatalyst for biotechnological and industrial bioprocesses. The Glomerella cingulata cutinase was genetically modified to enhance its enzymatic performance to fulfill industrial requirements. Two sites were selected for mutagenesis with the aim of altering the surface electrostatics as well as removing a potentially deamidation-prone asparagine residue. The N177D cutinase variant was affirmed to be more resilient to temperature increase with a 2.7-fold increase in half-life at 50°C as compared with wild-type enzyme, while, the activity at 25°C is not compromised. Furthermore, the increase in thermal tolerance of this variant is accompanied by an increase in optimal temperature. Another variant, the L172K, however, exhibited higher enzymatic performance towards phenyl ester substrates of longer carbon chain length, yet its thermal stability is inversely affected. In order to restore the thermal stability of L172K, we constructed a L172K/N177D double variant and showed that these two mutations yield an improved variant with enhanced activity towards phenyl ester substrates and enhanced thermal stability. Taken together, our study may provide valuable information for enhancing catalytic performance and thermal stability in future engineering endeavors.

Expression and characterization of a cutinase (AnCUT2) from Aspergillus niger

“…AnCUT2 was shown to retain more than 60% of its relative activity after 1 h incubation at different pHs. Although it is a mesophilic enzyme, AnCUT2 exhibits remarkable stability after 1 h incubation at 40°C compared to G. cingulata cutinase [29] and F. solani [30]. Similar data have been previously observed in some fungal cutinases [30,31].…”

Microbial host selection and periplasmic folding in Escherichia coli affect the biochemical characteristics of a cutinase from Fusarium oxysporum

“…Pichia pastoris have proven to be a safe choice for the expression of fungal cutinases, such as the ones from Alternaria brassicicola [30] and Sirococcus conigenus [31]. Glomerella cingulata cutinase has been expressed both in E. coli Origami B cells and in P. pastoris [32,33], while the well-studied F. solani cutinase has been expressed in E. coli (WK-6) [34].…”

“…Chin et al (2013) [32] have also shown that different mutants of G. cingulata cutinase, with different thermostability profiles, exhibit differentiated substrate specificity. In particular, the most thermostable mutant showed higher activity for longer carbon…”

Structural and functional studies of a Fusarium oxysporum cutinase with polyethylene terephthalate modification potential

“…A crude cutinase preparation from a wild-type F. oxysporum strain CBMAI 1274, exhibited optimal activity at 28 ο C [60], while cut1 from F. solani expressed in P. pastoris showed an optimum temperature of 40 ο C [61]. Fungal cutinases that have been heterologously expressed in different expression systems exhibit optimal catalytic activity at temperatures varying from 25 ο C (a cutinase from G. cingulata expressed in E. coli Origami B [62]) to 50 ο C (a cutinase from H.…”