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Three genes were cloned and expressed in Trichoderma reesei to identify novel cutinolytic polyesterases that could be exploited in treating agricultural, food, and forest raw materials as well as their processing by-products. The results from the microscopic analyses and determination of released hydrolysis products showed that the enzyme cutinolytic polyesterases was able to depolymerize apple cutin and birch outer bark suberin.

Abstract: Bovine milk fat has one of the most complex compositions of all natural fats. It is composed of more than 400 different fatty acids, present primarily as triacylglycerols, which gives milk fat highly diverse functional and nutritional properties. The complexity of milk fat provides both opportunities and challenges for modifying its composition for different applications. Due to the versatility of lipid compounds, milk fat can be considered as a good source of essential fatty acids and fat-soluble vitamins. In addition, milk fat has physically and chemically favourable properties, and it also has good sensory quality. However, the negative nutritional image of milk fat, especially certain saturated long-chain fatty acids, and poor spreadability of butter have driven development of technologies to produce milk fat fractions with different physico-chemical or nutritional properties. This article reviews the current literature on enzymatic and physical means of fractionating bovine milk fat into different fractions, and highlights their fields of usage. [ABSTRACT FROM AUTHOR]

Milk fat was treated with the sn- 2 specific PAN-2 lipase from Pseudozyma antarctica with the aim of achieving selective removal of saturated fatty acids with chain lengths between C12 and C16, which are abundant at the sn- 2 position. Hydrolysis was also carried out using the non-specific CRU-NS from Candida rugosa and the 1,3-specific TLA-1,3 from Thermomyces lanuginosus . PAN-2 treatment decreased the proportion of the target fatty acids considerably (content of 28%, w/w) and increased the proportion of unsaturated ones (content of 52%, w/w) at the degree of hydrolysis of 48% (mol/mol) in relation to the unhydrolysed substrate (47%, w/w, C12:0-C14:0 and 32%, w/w, unsaturated). The fatty acid distributions of the TLA-1,3 and CRU-NS treated samples were similar to untreated milk fat. The proportion of crystalline fat was smaller and the firmness lower in the PAN-2 treated milk fat samples than in the untreated milk fat at 5 °C. [ABSTRACT FROM AUTHOR]

Cutinases belong to the α/β-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 β-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 β-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. [ABSTRACT FROM AUTHOR]

Abstract: Over 250 microbial strains were screened for lipase activity specific towards saturated medium and long-chain fatty acids. Strains showing trimyristin (C14:0) hydrolysis on agar medium were further streaked on tricaprylin (C8:0) and on olive oil (mostly C18:1) agar to identify strains hydrolysing C12–C15 fatty acids with modest or no activity towards short-chain and unsaturated fatty acids. The twenty strains showing the desired specificity were grown as liquid cultures and analysed for p-nitrophenyl (p-NP) myristate hydrolysis in the presence of Triton X-100. Cells from the cultures showing the highest activity towards p-NP myristate were analysed for specificity towards p-NP esters of different fatty acid chain lengths. On the basis of the p-NP ester profiling, four lipase samples were selected for hydrolysis experiments on butter oil. Of these, the sample obtained from the cultivation of Didpodascus capitatus met the screening criteria: C12:0–C16:0 specificity and no preference towards C18:0 and C18:1 fatty acids. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Green labeled pectins were extracted by an environmentally friendly way using proteases and cellulases being able to act on proteins and cellulose present in cell walls. Pectins were isolated from different plant byproducts, i.e., chicory roots, citrus peel, cauliflower florets and leaves, endive, and sugar beet pulps. Enzymatic extraction was performed at 50 degrees C for 4 h, in order to fulfill the conditions required for microbiological safety of extracted products. High methoxy (HM) pectins of high molar mass were extracted with three different enzyme mixtures. These pectins were subsequently demethylated with two pectin methyl esterases (PMEs), either the fungal PME from Aspergillus aculeatus or the orange PME. It was further demonstrated that high molar mass low methoxy (LM) pectins could also be extracted directly from cell walls by adding the fungal PME to the mixture of protease and cellulase. Moreover, health benefit pectic oligosaccharides, the so-called modified hairy regions, were obtained after enzymatic treatment of the residue recovered after pectin extraction. The enzymatic method demonstrates that it is possible to convert vegetable byproducts into high-added value compounds, such as pectins and pectic oligosaccharides, and thus considerably reduce the amount of these residues generated by food industries.

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.