Gene_locus Report for: crysp-Q874E9

Cutinases are esterases that release fatty acids from the apoplastic layer in plants. As they accept bulky and hydrophobic substrates, cutinases could be used in many applications, ranging from valorization of bark-rich side streams to plastic recycling. Advancement of these applications with cutinases as biocatalysts, however, requires deeper knowledge of the enzymes' biodiversity and structure-function relationships. Here, we mined over 3000 members from Carbohydrate Esterase family 5 (CE5) for putative cutinases and condensed it to 151 genes from known or putative lignocellulose-targeting organisms. The 151 genes were subjected to a phylogenetic analysis. While cutinases with available crystal structures were phylogenetically closely related, we selected nine phylogenic diverse cutinases for characterization. The nine selected cutinases were recombinantly produced and their kinetic activity was characterized against para-nitrophenol substrates esterified with consecutively longer alkyl chains (pNP-C(2) to C(16)). The investigated cutinases each had a unique activity fingerprint against tested pNP-substrates. The five enzymes with the highest activity on pNP-C(12) and C(16), indicative of activity on bulky hydrophobic compounds, were selected for in-depth kinetic and structure-function analysis. All five enzymes showed a decrease in k(cat) values with increasing substrate chain length, while K(M) values and binding energies (calculated from in silico docking analysis) improved. Two cutinases from Fusarium solani and Cryptococcus sp. exhibited outstandingly low K(M) values, resulting in high catalytic efficiencies towards pNP-C(16). Docking analysis suggested that different clades of the phylogenetic tree may harbor enzymes with different modes of substrate interaction, involving a solvent-exposed catalytic triad, a lipase-like lid, or a clamshell-like active site possibly formed by flexible loops.

The structural and enzymatic characteristics of a cutinase-like enzyme (CLE) from Cryptococcus sp. strain S-2, which exhibits remote homology to a lipolytic enzyme and a cutinase from the fungus Fusarium solani (FS cutinase), were compared to investigate the unique substrate specificity of CLE. The crystal structure of CLE was solved to a 1.05 A resolution. Moreover, hydrolysis assays demonstrated the broad specificity of CLE for short and long-chain substrates, as well as the preferred specificity of FS cutinase for short-chain substrates. In addition, site-directed mutagenesis was performed to increase the hydrolysis activity on long-chain substrates, indicating that the hydrophobic aromatic residues are important for the specificity to the long-chain substrate. These results indicate that hydrophobic residues, especially the aromatic ones exposed to solvent, are important for retaining lipase activity.

A purified lipase from the yeast Cryptococcus sp. strain S-2 exhibited remote homology to proteins belonging to the cutinase family rather than to lipases. This enzyme could effectively degrade the high-molecular-weight compound polylactic acid, as well as other biodegradable plastics, including polybutylene succinate, poly (epsilon-caprolactone), and poly(3-hydroxybutyrate).

Polylactic acid (PLA) is a major contributor to the global bioplastic production capacity. However, post-consumer PLA waste is not fully degraded during traditional organic waste treatment processes and can persist in nature for many years. Efficient enzymatic hydrolysis of PLA would contribute to cleaner, more energy-efficient, environmentally friendly waste management processes. However, high costs and a lack of effective enzyme producers curtail the large-scale application of such enzymatic systems. This study reports the recombinant expression of a fungal cutinase-like enzyme (CLE1) in the yeast Saccharomyces cerevisiae, which produced a crude supernatant that efficiently hydrolyses different types of PLA materials. The codon-optimised Y294[CLEns] strain delivered the best enzyme production and hydrolysis capabilities, releasing up to 9.44 g/L lactic acid from 10 g/L PLA films with more than 40% loss in film weight. This work highlights the potential of fungal hosts producing PLA hydrolases for future commercial applications in PLA recycling.

Cutinases are esterases that release fatty acids from the apoplastic layer in plants. As they accept bulky and hydrophobic substrates, cutinases could be used in many applications, ranging from valorization of bark-rich side streams to plastic recycling. Advancement of these applications with cutinases as biocatalysts, however, requires deeper knowledge of the enzymes' biodiversity and structure-function relationships. Here, we mined over 3000 members from Carbohydrate Esterase family 5 (CE5) for putative cutinases and condensed it to 151 genes from known or putative lignocellulose-targeting organisms. The 151 genes were subjected to a phylogenetic analysis. While cutinases with available crystal structures were phylogenetically closely related, we selected nine phylogenic diverse cutinases for characterization. The nine selected cutinases were recombinantly produced and their kinetic activity was characterized against para-nitrophenol substrates esterified with consecutively longer alkyl chains (pNP-C(2) to C(16)). The investigated cutinases each had a unique activity fingerprint against tested pNP-substrates. The five enzymes with the highest activity on pNP-C(12) and C(16), indicative of activity on bulky hydrophobic compounds, were selected for in-depth kinetic and structure-function analysis. All five enzymes showed a decrease in k(cat) values with increasing substrate chain length, while K(M) values and binding energies (calculated from in silico docking analysis) improved. Two cutinases from Fusarium solani and Cryptococcus sp. exhibited outstandingly low K(M) values, resulting in high catalytic efficiencies towards pNP-C(16). Docking analysis suggested that different clades of the phylogenetic tree may harbor enzymes with different modes of substrate interaction, involving a solvent-exposed catalytic triad, a lipase-like lid, or a clamshell-like active site possibly formed by flexible loops.

Poly(lactic acid) (PLA) depolymerases are categorized into protease-type and lipase-type. Protease-types can hydrolyze poly(L-lactic acid) (PLLA) but not poly(D-lactic acid) (PDLA). Lipase-types, including cutinase-like enzyme (CLE) from Cryptococcus sp. strain S-2 preferentially hydrolyze PDLA. Both enzymes degraded not only PLA emulsion but also PLA film, in which amorphous region is preferentially attacked, but crystalline region can be also attacked. Stereocomplex PLA (sc-PLA) formed by 50:50 blending of PLLA and PDLA included no homo crystals, but a tiny homo crystallization peak appeared and crystallinity increased by 5% when attacked by CLE, although no significant change of molecular weight and crystalline size was found. Enantioselective degradation must occur in amorphous region of PLLA/PDLA film and preferentially hydrolyzed PDLA, resulting in a slightly excess amount of PLLA remained, which must be crystallized

The structural and enzymatic characteristics of a cutinase-like enzyme (CLE) from Cryptococcus sp. strain S-2, which exhibits remote homology to a lipolytic enzyme and a cutinase from the fungus Fusarium solani (FS cutinase), were compared to investigate the unique substrate specificity of CLE. The crystal structure of CLE was solved to a 1.05 A resolution. Moreover, hydrolysis assays demonstrated the broad specificity of CLE for short and long-chain substrates, as well as the preferred specificity of FS cutinase for short-chain substrates. In addition, site-directed mutagenesis was performed to increase the hydrolysis activity on long-chain substrates, indicating that the hydrophobic aromatic residues are important for the specificity to the long-chain substrate. These results indicate that hydrophobic residues, especially the aromatic ones exposed to solvent, are important for retaining lipase activity.

A purified lipase from the yeast Cryptococcus sp. strain S-2 exhibited remote homology to proteins belonging to the cutinase family rather than to lipases. This enzyme could effectively degrade the high-molecular-weight compound polylactic acid, as well as other biodegradable plastics, including polybutylene succinate, poly (epsilon-caprolactone), and poly(3-hydroxybutyrate).