Cutin depolymerization

Fig. 6. Proposed chemical structures of isolated soluble products of lime cutin depolymerization with TMSil (bottom) and pancreatic lipase (top)...

The polyester domains of suberized walls can also be depolymerized using chemical and/or enzymatic approaches similar to those used for cutin. The aromatic domains are far more difficult to depolymerize as C-C and C-O-C crosslinks are probably present in such domains. Therefore, more drastic degradation procedures such as nitrobenzene, CuO oxidation, or thioglycolic... 

Fig. 3. (Top left) Chemical methods used to depolymerize the polyesters. (Top right) Thin-layer and gas-liquid chromatograms (as trimethylsilyl derivatives) of the monomer mixture obtained from the cutin of peach fruits by LiAlD4 treatment. In the thin-layer chromatogram the five major spots are, from the bottom, C18 tetraol, C16 triol, and C18 triol (unresolved), diols, and primary alcohol. Nx = C16 alcohol N2= C18 alcohol Mj = C16 diol M2 = C18 diol D = C16 triol D2 and D3 = unsaturated and saturated C18 triol, respectively, T4 and T2, unsaturated and saturated C18 tetraol, respectively. (Bottom) Mass spectrum of component D3 in the gas chromatogram. BSA = bis-N,O-trimethylsilyl acetamide...

The biosynthetic origin of the depolymerization-resistant core of cutin (cutan) remains to be established. The early observation that linoleic acid and linolenic acid were preferentially incorporated into the non-depolymerizable core of cutin in apple skin slices suggested that the ether-linked or C-C-linked core might arise preferentially from the czs-l,4-pentadiene system [31]. The insoluble residue, that contained the label from the incorporated polyunsaturated C18 acids, released the label upon treatment with HI, supporting the notion that some of those aliphatic chains were held together by ether bonds. More recently,... 

High-resolution 13C NMR studies have been conducted on intact cuticles from limes, suberized cell walls from potatoes, and insoluble residues that remain after chemical depolymerization treatments of these materials. Identification and quantitation of the major functional moieties in cutin and suberin have been accomplished with cross-polarization magic-angle spinning as well as direct polarization methods. Evidence for polyester crosslinks and details of the interactions among polyester, wax, and cell-wall components have come from a variety of spin-relaxation measurements. Structural models for these protective plant biopolymers have been evaluated in light of the NMR results. 

Isolation of the Biopolyesters. Cutin was obtained from the skin of limes using published methods (8,9). The final solvent extractions were omitted in studies of cutin-wax interactions. Typically, 20 limes provided 800 mg of powdered polymer. Suberized cell walls were isolated from wound-healing potatoes after seven days of growth (10), with a yield of 4.5 g from 22 kg of potatoes. Chemical depolymerization of both polyesters was accomplished via transesterification with BF3/CH3OH (11). 

Figure 4. 31.94 MHz 13C NMR data for intact lime cutin (bottom) and the solid residue of a depolymerization treatment (top). Both spectra were obtained with a 1H-13C contact time of 1.0 ms, repetition rate of 1.0 s, spinning rate of 3.0 kHz, a H decoupling field of 60 kHz, and a line broadening of 20 Hz. (For the chosen contact time, peak intensities within each spectrum reflect the approximate numbers of each carbon type.) Only the intact cutin spectrum retained signal intensity near 30 ppm when decoupling was delayed before acquisition (13,14).

Suberized Cell Walls. An analogous set of CPMAS experiments is presented for suberin in Figure 6. Because this polymer is an integral part of the plant cell wall, the 13C NMR spectrum had contributions from both polysaccharide and polyester components. Chemical-shift assignments, summarized in Table IV, demonstrated the feasibility of identifying major polyester and sugar moieties despite serious spectral overlap. Semiquantitative estimates for the various carbon types indicated that, as compared with cutin, the suberin polyester had dramatically fewer aliphatic and more aromatic residues. A similar observation was made previously for the soluble depolymerization products of these plant polymers (1,8,11). 

Preliminary structural studies of cutin and suberin breakdown involved examination of 13C NMR spectra for insoluble residues that were resistant to chemical depolymerization. In cutin samples, flexible CH2 moieties in particular were removed by such treatments, but CHOCOR crosslinks and polysaccharide impurities were retained preferentially. A concomitant narrowing of NMR spectral lines suggested that the treatments produced more homogeneous polyester structures in both cases. Our current studies of cu-ticular breakdown also employ selective depolymerization strategies with appropriate enzymes (1,28). 

TLC is commonly used for the separation of different classes of wax components or for analysis of monomers from cutin and suberin depolymerization. A typical separation is shown in Fig. 6.12. By such methods, it is possible to separate hydrocarbons, wax esters, primary alcohols, secondary alcohols and /8-diketones from plant waxes (von Wettstein-Knowles, 1979). Products of hydrogenolysis from cutin can be separated by TLC into alkan-l-ols, alkane-a,ft>-diols, Cis triols, Ci6 triols and Cis tetrols (Kolattukudy, 1980). Unsaturated components can be resolved by argentation-TLC (Tulloch, 1976) and threo or erythro diastereoisomers separated by boric acid/silica gel TLC (Eglinton and Hunneman, 1968). Straight-chain compounds can be preferentially removed from branched compounds as their urea complexes (Kolattukudy, 1980). 

Hydroxy acids are major aliphatic components of cutin and suberin and these are readily identified by GLC-mass spectrometry. Major ions generated from the usual cutin components are listed in Kolattukudy (1977). The position of the hydroxyl group in the chain is easily seen because cleavage occurs on either side of the substituent (Fig. 6.13). The rather simple phenolic compounds yielded by reductive depolymerization of cutin and suberin are also very... 

Benitez et al. [82], recently reported the synthesis of a polyester resembling cutin, a natural polymer whose structure is close to that of aliphatic suberin [83], by a circular approach, which consisted in depolymerizing cutin through ester cleavage and then submitting the ensuing monomer mixture to a chemical polyesterification process. 

Purdy RE, Kolattukudy PE (1973) Depolymerization of a hydroxy fatty-acid biopolymer, cutin, by an extracellular enzyme fl om Fusarium solani pisi—isolation and some properties of the enzyme. Arch Biochem Biophys 159 61-69... 

Fig. 6.4.8. Cross-polarization/magic-angle spinning NMR spectra of cutin from the fruit peel of Malus pumila suberin from the bark of Pseudotsuga menziesiiy suberin from the periderm of the tuber of Solarium tuberosum and the residue left after L1A1H4 depolymerization of S. tuberosum suberin. (Spectra courtesy of Regional NMR Center, Fort Collins, Colorado.)...

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