Cutinases (EC 3.1.1.74) are extracellular enzymes that belong to alpha/beta hydrolases. They are serine esterases with the classical Ser-His-Asp triad similar to several lipases and serine proteases. In nature, cutinases catalyse the hydrolysis of the polyesters of the cuticle and the suberin layers, which protect plant surfaces. Cutinase production is typical for plant pathogenic fungi, but also, bacterial cutinases and cutinases from plant pollen have been discovered. Cutinases are promiscuous esterases catalysing reactions with a wide range of different substrates, such as short-chain soluble esters, water-insoluble medium and long-chain triacylglycerols, polyesters and waxes. In the current work, an overview is given on suggested applications of cutinases in the textile industry, in laundry detergents, in processing of biomass and food, in biocatalysis and in detoxification of environmental pollutants. The applications are discussed from the point of view of cutinase properties-which properties of cutinases are already advantageous and which would be desired. In addition, improvements that have been made on cutinase performance by protein and reaction engineering are reviewed.
A cutinase gene (ScCut1) was amplified by PCR from the genomic DNA of the ascomycetous plant pathogen Sirococcous conigenus VTT D-04989 using degenerate primers designed on the basis of conserved segments of known cutinases and cutinase-like enzymes. No introns or N- or O-glycosylation sites could be detected by analysis of the ScCut1 gene sequence. The alignment of ScCut1 with other fungal cutinases indicated that ScCut1 contained the conserved motif G-Y-S-Q-G surrounding the active site serine as well as the aspartic acid and histidine residues of the cutinase active site. The gene was expressed in Pichia pastoris, and the recombinantly produced ScCut1 enzyme was purified to homogeneity by immobilized metal affinity chromatography exploiting a C-terminal His-tag translationally fused to the protein. The purified ScCut1 exhibited activity at acidic pH. The K(m) and V(max) values determined for pNP-butyrate esterase activity at pH 4.5 were 1.7 mM and 740 nkat mg(-)(1), respectively. Maximal activities were determined at between pH 4.7 and 5.2 and at between pH 4.1 and 4.6 with pNP-butyrate and tritiated cutin as the substrates, respectively. With both substrates, the enzyme was active over a broad pH range (between pH 3.0 and 7.5). Activity could still be detected at pH 3.0 both with tritiated cutin and with p-nitrophenyl butyrate (relative activity of 25 %) as the substrates. ScCut1 showed activity towards shorter (C2 to C3) fatty acid esters of p-nitrophenol than towards longer ones. Circular dichroism analysis suggested that the denaturation of ScCut1 by heating the protein sample to 80 degrees C was to a great extent reversible.
Cutinases belong to the alpha/beta-hydrolase fold family of enzymes and degrade cutin and various esters, including triglycerides, phospholipids and galactolipids. Cutinases are able to degrade aggregated and soluble substrates because, in contrast with true lipases, they do not have a lid covering their catalytic machinery. We report here the structure of a cutinase from the fungus Trichoderma reesei (Tr) in native and inhibitor-bound conformations, along with its enzymatic characterization. A rare characteristic of Tr cutinase is its optimal activity at acidic pH. Furthermore, Tr cutinase, in contrast with classical cutinases, possesses a lid covering its active site and requires the presence of detergents for activity. In addition to the presence of the lid, the core of the Tr enzyme is very similar to other cutinase cores, with a central five-stranded beta-sheet covered by helices on either side. The catalytic residues form a catalytic triad involving Ser164, His229 and Asp216 that is covered by the two N-terminal helices, which form the lid. This lid opens in the presence of surfactants, such as beta-octylglucoside, and uncovers the catalytic crevice, allowing a C11Y4 phosphonate inhibitor to bind to the catalytic serine. Taken together, these results reveal Tr cutinase to be a member of a new group of lipolytic enzymes resembling cutinases but with kinetic and structural features of true lipases and a heightened specificity for long-chain triglycerides.
Isolates from gardening waste compost and 38 culture collection microbes were grown on agar plates at pH 4.0 with the cutinase model substrate polycaprolactone as a carbon source. The strains showing polycaprolactone hydrolysis were cultivated in liquid at acidic pH and the cultivations were monitored by assaying the p-nitrophenyl butyrate esterase activities. Culture supernatants of four strains were analyzed for the hydrolysis of tritiated apple cutin at different pHs. Highest amounts of radioactive hydrolysis products were detected at pHs below 5. The hydrolysis of apple cutin by the culture supernatants at acidic pH was further confirmed by GC-MS analysis of the hydrolysis products. On the basis of screening, the acidic cutinase from Aspergillus niger CBS 513.88 was chosen for heterogeneous production in Pichia pastoris and for analysis of the effects of pH on activity and stability. The recombinant enzyme showed activity over a broad range of pHs with maximal activity between pH 5.0 and 6.5. Activity could be detected still at pH 3.5.
