In this work four commercial cellulase-hemicellulase mixtures with different activity profiles were used for solubilization of carbohydrates from brewers' spent grain (BSG). After the enzyme treatment, both the solubilised fraction and the unhydrolysed residue were characterized. Treatment with 5,000 nkat/g xylanase for 5 h at 50°C resulted in the solubilisation of 13-14% of the BSG dry weight as monosaccharides. This corresponded to the solubilisation of 26-28% of the original carbohydrates and 30-34% of original arabinoxylans, depending on the enzyme cocktail used. The relatively low hydrolysis level indicates that the majority of the BSG biomass is rather recalcitrant towards the cellulose-hemicellulase enzyme mixtures applied in this study. The enzyme activity profile had a crucial impact on the chemistry of the oligosaccharides produced through the solubilisation of BSG. The presence of feruloyl esterase (FAE) activity in the enzyme cocktail resulted in the production of free ferulic acid, arabinoxylo-oligosaccharides and their corresponding monomers. However, when the enzyme mixture was devoid of FAE activity, ferulic acid was still bound to the oligosaccharides. The unhydrolysed fraction was still found to contain over 40% of carbohydrates after enzymatic treatment despite the extensive enzyme dosages used. The protein fraction remained largely unaffected (i.e. insoluble) by the carbohydrate-disrupting enzyme treatments. In addition to the recalcitrant carbohydrates, the residue was enriched with lignin and lipid type structures.

The fate of black currant ( Ribes nigrum L.) and bilberry ( Vaccinium myrtillus L.) flavonols in enzyme-aided processing was studied. The flavonols were quantified and characterized by high-performance liquid chromatography equipped with a diode array detector and an electrospray ionization mass spectrometer. A tentative identification for 14 black currant and 19 bilberry flavonols is presented representing 11 previously unpublished conjugates. For the first time in any berry, the presence of laricitrin conjugates is reported. The enzyme-aided processing affected the flavonol extractability, elevating the yield in juices and decreasing that in press residues. Importantly, no significant loss of the berry flavonols was observed during the experiments, although some hydrolysis of flavonol conjugates was recorded. To maximize the effect on flavonol extractability, higher enzyme dosages were needed for black currants than for bilberries. The data show that the flavonol extractability and hydrolysis are dependent on the texture of raw material, the glycosylation pattern of the conjugates, and the activity profile of the enzyme preparation.

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...

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