In a first step, deacetylation of glucose pentaacetate (GPA) and triacetin (TA) by different esterases was investigated. Hydrolysis of triacetin was studied since this compound is used as plasticizer in many CA materials, while hydrolysis may have an impact on biodegradation of CA [16
]. Although several enzymes were able to completely deacetylate both compounds, there were significant differences between the individual enzymes (Figure 2
). Major differences were seen for acetyl xylan esterases (AXE) 35 and AXE O), which deacetylated GPA to 80% and 90%, respectively, but had only minor effects on triacetin. Classified as AXE, these enzymes clearly prefer sugar-bound esters compared to aliphatic esters, like in triacetin. On the other hand, cutinase (CUT) showed similar activities on both substrates and was previously described to cleave ester bonds in hydrophobic aromatic and aliphatic polyesters [17
]. Moreover, different applications of cutinase are known, e.g., a wild-type cutinase was reported to esterify the hydroxyl groups of cellulose [18
]. This makes cutinase a promising enzyme in cellulose acetate treatment.
AXE 53 and glucomannan acetyl esterase (GAE) showed high capacities to deacetylate both substrates. Especially for GAE, 60% of triacetin were deacetylated in 2 min. For AXE’s deacetylation of different model compounds, representing acetylated polysaccharides and non-polysaccharide compounds, was reported before for chitosan, chitin [19
], and cephalosporin-C [20
On the other hand, AXE 34 only weakly deacetylated both substrates. Altaner et al. found evidence for regioselectivity of esterases derived from different families. They claim that enzymes of the carbohydrate esterase (CE) family 1 exclusively deacetylated CA in the C2- and C3-carbon positions, without cleaving C6 in the sugar. Furthermore, they postulate differences in cleavability for different substitution positions on the polymer chain [21
]. Complete deacetylation was observed for pectin acetyl esterase PAE, GAE, AXE O, and AXE 53. For some enzymes, e.g., AXE 34 only one acetyl group of GPA was attacked. For triacetin, the degrees of deacetylation in Figure 2
are arranged into three groups corresponding to the three acetyl groups of the molecule. This indicates cleavage of only one ester bond for AXE O, AXE 34, and AXE 35. Two bonds were cleaved for CE 265, CUT, and AXE 36. All other enzymes deacetylated triacetin completely. An influence of the enzyme family for this behavior is not visible. Apart from GPA, enzymatic hydrolysis of acetylated oligomers, namely cellobiose octaacetate and cellohexose eicosaacetate, was investigated. Within 68 h only GAE (45%) AXE 55 (14%), and CUT 1 (13.5%) showed significant deacetylation of cellohexaose eicosaacetate. All other tested enzymes reached deacetylation degrees lower than 4%. Additionally, for GPA, an influence of the enzyme family cannot be stated.
In nature, glucomannan acetyl esterase is part of the wood degradation process. O
-Acetylgalactoglucomannans (AcGGM) are the principal hemicellulose components in softwoods. They are mainly water insoluble, but their acetylation pattern influences their behavior in water [22
]. The structure of AcGGM is a linear backbone of (1→4)-linked β-d
-mannopyranosyl and (1→4)-linked β-d
-glucopyranosyl units, with (1→6)-linked α-d-galactopyranosyl units. Naturally, the mannose subunits can be acetylated at C-2 and C-3, however, via chemical acetylation galactose and glucose subunits were also acetylated [23
]. It was shown that linear oligosaccharides from AcGGM could be obtained when an acetyl mannan esterases and a α-galactosidases were used in combination [22
]. It was also suggested that galactomannan deacetylation is an inherent property of some AXEs [11
]. Due to catalytic similarities between acetyl xylan esterases and glucomannan esterases, the latter enzyme is of interest for degradation processes of acetylcellulose.
In a next step, enzymatic hydrolysis of CA with different degrees of acetylation was investigated (Figure 3
). In general, the activity of the enzymes increased with decreasing degree of acetylation confirming the “protective” function of acetyl groups as reported before [11
]. Only for AXE 55, AXE 53, and GAE was deacetylation weaker for CA-DS 0.9 (degree of substitution) than for CA-DS 1.4, which belongs to the same family (Table 1
). Poutanen et al. reported the behavior of acetyl xylan esterases on their natural substrates. AXE prefers polymeric substrates, whereas acetyl esterase showed high affinity on acetyl xylobiose. Out of this, they claim a high deacetylation specificity, which depends on the specific position of the acetyl groups and not on the degree of polymerization. Using acetyl xylan esterase in combination with other enzymes they obtained a complete degradation of polymeric substrates to acetic acid and xylose using xylanase and β-xylosidase [24
]. Regioselectivity claimed for AXE’s natural model substrates explains the non-complete deacetylation of CA materials. Due to the higher number of acetyl groups per monomer, regioselectivity plays a more important role for CAs with a high degree of substitution. The probability to find a critical position blocked is, therefore, larger for highly-acetylated substrates.
Interestingly, most of the enzymes not belonging to family II showed almost slightly lower activity on CA-DS 1.4 compared to CA-DS 0.9, while the activity dramatically decreased for CA-DS 1.8. As mentioned before, family II enzymes showed regioselectivity for deacetylation. These three enzymes (AXE 55, AXE 53, GAE) had the highest deacetylation activity. A possible explanation might be given by the distribution of the acetyl groups across the polymer. Hence, for enzymes, cellulose acetates with different degrees of substitutions pose to be substrates with different properties. Based on the conserved motive, AXE 55, AXE 53, and GAE are representatives of the so-called GDSL-family or family II, and all esterases within lipolytic enzymes can be classified into the thirteen families [25
]. This family shares five highly-conserved homology blocks, which are important for their classification. GDSL hydrolases have a flexible active site and they can change conformation in the presence of different substrates [26
]. This flexibility might be an explanation for their good deacetylation efficiency over a broad range of substrates. Enzymes of this family were also reported to show broad regioselectivity, which probably makes them suitable for degradation of CAs with a heterogeneous acetylation pattern [27
Despite investigations for triacetin and glucose pentaacetate (Figure 2
), for PAE deacetylation was examined over time by using different cellulose acetate model compounds varying in degree of substitution (CA-DS 0.9, CA-DS 1.4, CA-DS 1.8) and real cigarette filter material (R. J. Reynolds Tobacco Company, Winston-Salem, NC, USA). Figure 4
compares deacetylation efficiencies for cellulose acetates over time. For the used model compounds, deacetylation decreased from 20% for low-acetylated substrate (CA 0.9) to 5% for highly-acetylated material (CA 1.8) after 165 h of incubation. Real filter material with a degree of substitution of 2.5 revealed a high resistance against enzymatic degradation with PAE, resulting in no detectable release of acetic acid. As shown in Figure 2
for glucose pentaacetate and triacetin, PAE exhibited high and medium ability to degrade small acetylated substrates, whereas larger substrates, like different cellulose acetates (Figure 4
), were less deacetylated even when the DS was lower. For the substrates reported in this study, the polymer size influenced the deacetylation efficiency of PAE to a great extent. The highest deacetylation efficiency was visible for CA-DS 0.9 within the first 2 h. This can be explained by auto-degradation of the polymer in combination with the enzyme action. For all other conditions, deacetylation was linear for the whole reaction time course.
The results of enzymatic deacetylation of a variety of substrates with different degrees of acetylation are summarized in Table 2
. None of the enzymes were able to deacetylate CA with a degree of substitution higher than 1.8. The most promising enzymes for the degradation of large and highly substituted polymers were of family II (AXE 55, AXE 53, GAE). Small- and highly-acetylated molecules, such as cellobiose octaacetate and cellohexose eicosaacetate, were less deacetylated than larger molecules with less acetyl groups per monomer. An increase in the number of glucose subunits from one (glucose pentaacetate) to six (cellohexaose eicosaacetate) strongly decreased deacetylation efficiency for all enzymes except for PAE and GAE (compare Figure 4
and Table 2
). For example, cellohexaose eicosaacetate was less deacetylated than glucose pentaacetate with a higher degree of substitution. Hence, it seems that for shorter molecules the degree of acetylation has a lower impact on deacetylation efficiency than the chain length.
2.3. Lytic Polysaccharide Monooxygenase (LPMO) Hydrolysis of CA
Since the degree of enzymatic deacetylation decreased with increasing polymer chain length, it was speculated that cleavage of the polymer into smaller fragments prior to deacetylation increases CA degradation. Lytic polysaccharide monooxygenases (LPMOs) are known to attack cellulose by an oxidative mechanism, which cleaves the glycoside bond at either the C1 or the C4 [29
]. An interesting feature of LPMO is its flat substrate binding surface, which fits onto the cellulose surfaces and brings the active-site copper with an activated oxygen species into close contact with the species [30
]. This enzyme can theoretically bind at any position to cellulose to perform cleavage. Some LPMOs like LPMO-02916 (also known as LPMO 9C) from Neurospora crassa
can also act on the less-structured substrates, hemicellulose and cellooligosaccharides [31
], which suggests that LPMO-02916 might also be suitable to act on CA. In experiments where LPMO-02916 was incubated with a reductant and cellulose (PASC, Avicel, or steam-exploded spruce), fragments between two and five monomers were detected [31
]. CA with a DS of 0.9 and 1.4 was incubated separately with LPMO-02916 and cleavage fragments were analyzed. For CA with a DS of 0.9 after incubation with LPMO no fragments larger than five monomers and more than one acetyl group were detected. For CA 0.9, the number of different fragments was decreasing for increasing amount of acetyl groups. LPMO liberated fragments for both CA with DS 0.9 and 1.4 (Figure 6
), while no fragments were detected for CA with higher DS. Preferentially, fragments with a low DS were released. This indicates that acetyl groups interfere with LPMO’s substrate binding site or disturb the catalytic reaction. It also indicates that CA may not be uniformly acetylated, allowing the LPMO to cleave in those regions with a lower DS. In this context synergies of LPMOs in combination with other cellulose degrading enzymes were observed and are worth of further investigations [33
]. Cleavage of CA with DS 0.9 and 1.4 by LPMO was investigated by analyzing the released fragments (Figure 6
shows backbone cleavage by cellobiohydrolase I and chitinase after 145 h of reaction. Cleavage activity was monitored photometrically by detection of the reducing sugar ends using the dinitrosalicylic acid (DNS) method [32
]. For cellobiohydrolase I, the amount of reducing ends is increased by a factor of three for low-substituted material (CA-DS 0.9). Minor increases are visible for medium-substituted substrates (DS 1.4). No cleavage activity was measured for highly-substituted cellulose acetate (CA-DS 1.8). As visible for the deacetylation with esterases (Figure 3
), the decrease in activity, for highly-acetylated substrates, is also not linear for glycosidic-acting enzymes in backbone cleavage, with the substrates used here. Cellobiohydrolase I is an enzyme with broad product specificity, reported to bind only the hydrophobic parts of the cellulose crystal structure [34
]. Ike et al. reported chitinase activity for cellobiohydrolase I [35
] and Textor et al. mentioned the cleavage of carboxymethyl cellulose by the enzyme. This presence of a cellulose binding module (CBM) and low end-product inhibition promise applicability of cellobiohydrolase I in industrial degradation processes [36
]. The ability of cellobiohydrolases to decrease the degree of polymerization was reported before by Saake et al. for low- and medium-acetylated substrates, based on size exclusion chromatography of acetylated cellulose [37
Due to the chemical similarities of the polymers chitin and cellulose acetate, chitinase was tested on its ability to cleave glycosidic bonds in different cellulose acetates. Chitinase only showed small changes for CA-DS 0.9 and no shift in absorbance for other substrates. The N
-acetamide group seems to be essential for the ability of chitinase to detect glycosidic bonds. The binding ability for a chitinase on chitin and cellulose was determined to be equal for both polymers [38
]. The effects of reduced binding affinity to substrates with at least one acetyl group per monomer might be the reason for reduced backbone cleavage of cellobiohydrolase for CA 1.4 and CA 1.8.