Positional specifity of acetylxylan esterases on natural polysaccharide: An NMR study.
Uhliariková, I., Vršanská, M., McCleary, B. V. & Biely, P. (2013). Biochimica et Biophysica Acta (BBA), 1830(6), 3365–3372.
Background: Microbial degradation of acetylated plant hemicelluloses involves besides enzymes cleaving the glycosidic linkages also deacetylating enzymes. A detailed knowledge of the mode of action of these enzymes is important in view of the development of efficient bioconversion of plant materials that did not undergo alkaline pretreatment leading to hydrolysis of ester linkages. Methods: In this work deacetylation of hardwood acetylglucuronoxylan by acetylxylan esterases from Streptomyces lividans (carbohydrate esterase family 4) and Orpinomyces sp. (carbohydrate esterase family 6) was monitored by 1H-NMR spectroscopy. Results: The 1H-NMR resonances of all acetyl groups in the polysaccharide were fully assigned. The targets of both enzymes are 2- and 3-monoacetylated xylopyranosyl residues and, in the case of the Orpinomyces sp. enzyme, also the 2,3-di-O-acetylated xylopyranosyl residues. Both enzymes do not recognize as a substrate the 3-O-acetyl group on xylopyranosyl residues α-1,2-substituted with 4-O-methyl-D-glucuronic acid. Conclusions: The 1H-NMR spectroscopy approach to study positional and substrate specificity of AcXEs outlined in this work appears to be a simple way to characterize catalytic properties of enzymes belonging to various CE families.
CE16 on acetyl-4-O-methylglucuronoxylan and acetyl-galactoglucomannan.
Mai-Gisondi, G., Maaheimo, H., Chong, S. L., Hinz, S., Tenkanen, M. & Master, E. (2017). Biochimica et Biophysica Acta (BBA)-General Subjects, 1861(9), 2398-2405.
Background: The backbone structure of many hemicelluloses is acetylated, which presents a challenge when the objective is to convert corresponding polysaccharides to fermentable sugars or else recover hemicelluloses for biomaterial applications. Carbohydrate esterases (CE) can be harnessed to overcome these challenges. Methods: Enzymes from different CE families, MtAcE (CE1), OsAcXE (CE6), and MtAcE (CE16) were compared based on action and position preference towards acetyl-4-O-methylglucuronoxylan (MGX) and acetyl-galactoglucomannan (GGM). To determine corresponding positional preferences, the relative rate of acetyl group released by each enzyme was analyzed by real time 1H NMR. Results: AnAcXE (CE1) showed lowest specific activity towards MGX, where OsAcXE (CE6) and MtAcE were approximately four times more active than AnAcXE (CE1). MtAcE (CE16) was further distinguished by demonstrating 100 times higher activity on GGM compared to AnAcXE (CE1) and OsAcXE (CE6), and five times higher activity on GGM than MGX. Following 24 h incubation, all enzymes removed between 78 to 93% of total acetyl content from MGX and GGM, where MtAcE performed best on both substrates. Major Conclusions: Considering action on MGX, all esterases showed preference for doubly substituted xylopyranosyl residues (2,3-O-acetyl-Xylp). Considering action on GGM, OsAcXE (CE6) preferentially targeted 2-O-acetyl-mannopyranosyl residues (2-O-acetyl-Manp) whereas AnAcXE (CE1) demonstrated highest activity towards 3-O-acetyl-Manp positions; regiopreference of MtAcE (CE16) on GGM was less clear. General significance: The current comparative analysis identifies options to control the position of acetyl group release at initial stages of reaction, and enzyme combinations likely to accelerate deacetylation of major hemicellulose sources.
Enzymatic Systems for Cellulose Acetate Degradation.
Haske-Cornelius, O., Pellis, A., Tegl, G., Wurz, S., Saake, B., Ludwig, R., Sebastian, A., Gibson S. Nyanhongo, G. S. & Guebitz, G. M. (2017). Catalysts, 7(10), 287.
Cellulose acetate (CA)-based materials, like cigarette filters, contribute to landscape pollution challenging municipal authorities and manufacturers. This study investigates the potential of enzymes to degrade CA and to be potentially incorporated into the respective materials, enhancing biodegradation. Deacetylation studies based on Liquid Chromatography-Mass Spectrometry-Time of Flight (LC-MS-TOF), High Performance Liquid Chromatography (HPLC), and spectrophotometric analysis showed that the tested esterases were able to deacetylate the plasticizer triacetin (glycerol triacetate) and glucose pentaacetate (cellulose acetate model compound). The most effective esterases for deacetylation belong to the enzyme family 2 (AXE55, AXE 53, GAE), they deacetylated CA with a degree of acetylation of up to 1.8. A combination of esterases and cellulases showed synergistic effects, the absolute glucose recovery for CA 1.8 was increased from 15% to 28% when an enzymatic deacetylation was performed. Lytic polysaccharide monooxygenase (LPMO), and cellobiohydrolase were able to cleave cellulose acetates with a degree of acetylation of up to 1.4, whereas chitinase showed no activity. In general, the degree of substitution, chain length, and acetyl group distribution were found to affect CA degradation. This study shows that, for a successful enzyme-based deacetylation system, a cocktail of enzymes, which will randomly cleave and generate shorter CA fragments, is the most suitable.
Action of different types of endoxylanases on eucalyptus xylan in situ.
Puchart, V., Fraňová, L., Krogh, K. B. M., Hoff, T. & Biely, P. (2018). Applied Microbiology and Biotechnology, 1-12.
Most studies of the mode of action of industrially important endoxylanases have been done on alkali extracted-plant xylan. In just few cases, the native form of the polysaccharide, acetylated xylan, was used as a substrate. In this work action of xylanases belonging to three glycoside hydrolase families, GH10, GH11, and GH30 was investigated on acetylglucuronoxylan directly in hardwood cell walls. Powdered eucalyptus wood was used as xylanase substrate. Enzyme-generated fragments were characterized by TLC, MALDI ToF MS, and NMR spectroscopy. All three xylanases generated from eucalyptus wood powder acetylated xylooligosaccharides. Those released by GH10 enzyme were the shortest, and those released by GH30 xylanase were of the largest diversity. For GH30 xylanase the 4-O-methyl-D-glucuronic acid (MeGlcA) side residues function as substrate specificity determinants regardless the acetylation of the neighboring hydroxyl group. Much simpler xylooligosaccharide patterns were observed when xylanases were applied in combination with carbohydrate esterase family 6 acetylxylan esterase. In the presence of the esterase, all aldouronic acids remained 3-O-acetylated on the xylopyranosyl (Xylp) residue substituted with MeGlcA. The 3-O-acetyl group, in contrast to the acetyl groups of otherwise unsubstituted Xylp residues, does not affect the mode of action of endoxylanases, but contributes to recalcitrance of the acidic xylan fragments. The results confirm importance of acetylxylan esterases in microbial degradation of acetylated hardwood glucuronoxylan. They also point to still unresolved question of efficient enzymatic removal of the 3-O-acetyl group on MeGlcA-substituted Xylp residues negatively affecting the saccharification yields.
Comprehensive evaluation of combining hydrothermal pretreatment (autohydrolysis) with enzymatic hydrolysis for efficient release of monosaccharides and ferulic acid from corn bran.
iang, K., Li, L., Long, L. & Ding, S. (2018). Industrial Crops & Products, 113, 348-357.
The combination of hydrothermal pretreatment (autohydrolysis) and enzymatic hydrolysis was comprehensively evaluated for the efficient release of monosaccharides and ferulic acid from corn bran. Arabinan was depolymerized and solubilized more easily during autohydrolysis compared to xylan, esterified ferulic acid, and the acetyl group. Also, the enzymatic xylose yield showed strong linear correlation with arabinan, ferulic acid, and acetic acid content in autohydrolysis residues while correlations between enzymatic glucose yield and hemicellulose contents were separated into two stages with different slopes. The addition of a few debranching enzymes to commercial cellulase and xylanase only slightly enhanced enzymatic hydrolysis of autohydrolysis residues, whereas an enzyme blend from Aspergillus oryzae and Eupenicillium parvum showed a significant synergistic effect. Desirable combined hydrolysis yields of glucose (72.26%), xylose (75.87%), arabinose (76.95%), and ferulic acid (74.13%) were obtained after autohydrolysis at 165°C for 40 min and subsequent hydrolysis by an equal mixture blend produced by A. oryzae and E. parvum at an enzyme loading dosage of 14.1 mg protein/g dry destarched corn bran.