NovelCoprinopsis cinereaPolyesterase That Hydrolyzes Cutin and Suberin

Abstract

Three cutinase gene-like genes from the basidiomycete Coprinopsis cinerea (Coprinus cinereus) found with a similarity search were cloned and expressed in Trichoderma reesei under the control of an inducible cbh1 promoter. The selected transformants of all three polyesterase constructs showed activity with p-nitrophenylbutyrate, used as a model substrate. The most promising transformant of the cutinase CC1G_09668.1 gene construct was cultivated in a laboratory fermentor, with a production yield of 1.4 g liter ؊l purified protein. The expressed cutinase (CcCUT1) was purified to homogeneity by immobilized metal affinity chromatography exploiting a C-terminal His tag. The N terminus of the enzyme was found to be blocked. The molecular mass of the purified enzyme was determined to be around 18.8 kDa by mass spectrometry. CcCUT1 had higher activity on shorter (C 2 to C 10 ) fatty acid esters of p-nitrophenol than on longer ones, and it also exhibited lipase activity. CcCUT1 had optimal activity between pH 7 and 8 but retained activity over a wide pH range. The enzyme retained 80% of its activity after 20 h of incubation at 50°C, but residual activity decreased sharply at 60°C. Microscopic analyses and determination of released hydrolysis products showed that the enzyme was able to depolymerize apple cutin and birch outer bark suberin.Cutin and suberin are lipid-derived insoluble polyesters that serve as structural components of the outer barriers of plants. Significant amounts of cutin are present in different agricultural and food raw materials, such as cereals, berries, fruits, and vegetables, and their processing by-products, whereas the bark of cork oak and birch is rich in suberin. These polymers are estimated to be the third most abundant natural polymers after cellulose and lignin (22). The weight of an isolated cuticle ranges from 0.45 to 0.8 mg cm Ϫ2 (leaf cuticles) to 2 mg cm Ϫ2(fruit cuticles), of which 40 to 80% is formed by cutin (23).Cutin is primarily composed of ester-linked fatty acids, of which mono-, di-, and trihydroxy and epoxy fatty acids of the C 16 and C 18 families are the most abundant (25). Suberin consists of both a polyaliphatic fatty acid domain similar to that of cutin, with epoxy, hydroxy, and carboxylic acid functionalities, and a polyphenolic domain (4). The main suberin monomers are aliphatic long-chain ␣,-diacids and -hydroxyacids, together with glycerol (20). Cutin can be depolymerized by chemical cleavage of the ester bonds. However, epoxy, hydroxy, and carboxylic acid functionalities are not always retained when harsh chemical treatments are applied. Thus, enzymatic depolymerization of cutin is an attractive option and could result in formation of both monomers and oligomers with unmodified functionalities (26). Partial depolymerization of cork suberin by methanolysis has been found to form linear esters, glyceryl esters, and feruloyl esters, in which ferulic acid is linked to hydroxy acids (20).Cutinases and suberinases are polyesterases which are able to degrade or partially de...

Two Novel Class II Hydrophobins from Trichoderma spp. Stimulate Enzymatic Hydrolysis of Poly(Ethylene Terephthalate) when Expressed as Fusion Proteins

“…However, considering that the molecular mass of GST is 26 kDa, whereas that of HFB4 is 9 kDa, the actual active concentration of HFB4 is only a fourth of that, theoretically reducing the A 50 concentrations to 8 to 30 mg/ liter. This concentration is comparable to that reported by previous studies to stimulate cutinase hydrolysis of cutin and suberin by HFB2 from T. reesei (24). However, the extent of stimulation of PET hydrolysis by HFB4 was higher (2.5-fold versus 1.4-to 1.7-fold).…”

“…It is tempting to speculate that the expanded arsenal of hydrophobins may be involved in this property. In fact, hydrolysis of the natural polyesters cutin and suberin by a Coprinopsis cinerea polyesterase was enhanced in the presence of T. reesei HFB2 (24).…”

Three New Cutinases from the Yeast Arxula adeninivorans That Are Suitable for Biotechnological Applications

“…In contrast, Acut1-6hp and Acut2-6hp had higher activity with the C 6 ester pNP-caproate; however, in the case of triacylglycerides, the result for Acut1-6hp and Acut2-6hp was similar to that for Acut3-6hp and FsCut-6hp. Several known cutinases, for example, TtCutA from Thielavia terrestris (5) and PaE from Pseudozyma antarctica (6), have the same substrate profile, but there are also other cutinases or cutinase-like enzymes which have slightly different affinities, with higher levels of activity toward pNP-acetate being detected (25,45,46). Martinez et al (47) suggested that the low levels of activity of cutinases toward pNP esters of long-chain fatty acids are related to the lack of a large hydrophobic area around the active site, which is present in lipases.…”

Which properties of cutinases are important for applications?

“…In addition, large volumes of suberin containing materials are available as the by-products of cork, saw-mill, paper and pulp industries (Gandini et al 2006;Kolattukudy 2001). It has therefore been suggested that these materials could be fractionated, and the isolated cutin and suberin could be used for the production of commercially valuable materials and compounds (Kolattukudy 2001;Kontkanen et al 2009). …”