A novel glycoside hydrolase 43-like enzyme from Clostridium boliviensis is an endo-xylanase and a candidate for xylooligosaccharide production from different xylan substrates.

An uncharacterized gene encoding a glycoside hydrolase family 43-like enzyme from Clostridium boliviensis strain E-1 was identified from genomic sequence data, and the encoded enzyme, CbE1Xyn43-l, was produced in Escherichia coli. CbE1Xyn43-l (52.9 kDa) is a two-domain endo-ß-xylanase consisting of a C-terminal CBM6 and a GH43-like catalytic domain. The positions of the catalytic dyad conserved in GH43, the catalytic base (Asp74), and proton donor (Glu240) were identified in alignments including GH43-enzymes of known 3D-structure from different subfamilies. CbE1Xyn43-l is active at pH 7.0-9.0, with optimum temperature at 65°C, and a more than 7 days' half-life in irreversible deactivation studies at this temperature. The enzyme hydrolyzed birchwood xylan, quinoa stalks glucuronoarabinoxylan, and wheat arabinoxylan with xylotriose and xylotetraose as major hydrolysis products. CbE1Xyn43-l also released xylobiose from pNPX2 with low turnover (kcat of 0.044 s-1) but was inactive on pNPX, showing that a degree of polymerization of three (DP3) was the smallest hydrolyzable substrate. Divalent ions affected the specific activity on xylan substrates, which dependent on the ion could be increased or decreased. In conclusion, CbE1Xyn43-l from C. boliviensis strain E-1 is the first characterized member of a large group of homologous hypothetical proteins annotated as GH43-like and is a thermostable endo-xylanase, producing xylooligosaccharides of high DP (xylotriose and xylotetraose) producer. IMPORTANCE: The genome of Clostridium boliviensis strain E-1 encodes a number of hypothetical enzymes, annotated as glycoside hydrolase-like but not classified in the Carbohydrate Active Enzyme Database (CAZy). A novel thermostable GH43-like enzyme is here characterized as an endo-ß-xylanase of interest in the production of prebiotic xylooligosaccharides (XOs) from different xylan sources. CbE1Xyn43-l is a two-domain enzyme composed of a catalytic GH43-l domain and a CBM6 domain, producing xylotriose as main XO product. The enzyme has homologs in many related Clostridium strains which may indicate a similar function and be a previously unknown type of endo-xylanase in this evolutionary lineage of microorganisms.

Novel thermostable GH5_34 arabinoxylanase with an atypical CBM6 displays activity on oat fiber xylan for prebiotic production.

Carbohydrate active enzymes are valuable tools in cereal processing to valorize underutilized side streams. By solubilizing hemicellulose and modifying the fiber structure, novel food products with increased nutritional value can be created. In this study, a novel GH5_34 subfamily arabinoxylanase from Herbinix hemicellulosilytica, HhXyn5A, was identified, produced and extensively characterized, for the intended exploitation in cereal processing to solubilize potential prebiotic fibers: arabinoxylo-oligosaccharides. The purified two-domain HhXyn5A (catalytic domain and CBM6) demonstrated high storage stability, showed a melting temperature Tm of 61°C and optimum reaction conditions were determined to 55°C and pH 6.5 on wheat arabinoxylan. HhXyn5A demonstrated activity on various commercial cereal arabinoxylans and produced prebiotic AXOS, whereas the sole catalytic domain of HhXyn5A did not demonstrate detectable activity. HhXyn5A demonstrated no side activity on oat ß-glucan. In contrast to the commercially available homolog CtXyn5A, HhXyn5A gave a more specific HPAEC-PAD oligosaccharide product profile when using wheat arabinoxylan and alkali extracted oat bran fibers as the substrate. Results from multiple sequence alignment of GH5_34 enzymes, homology modeling of HhXyn5A and docking simulations with ligands XXXA3, XXXA3XX and X5 concluded that the active site of HhXyl5A catalytic domain is highly conserved and can accommodate both shorter and longer ligands. However, significant structural dissimilarities between HhXyn5A and CtXyn5A in the binding cleft of CBM6, due to the lack of important ligand-interacting residues, is suggested to cause the observed differences in substrate specificity and product formation.

Novel Function of CtXyn5A from Acetivibrio thermocellus: Dual Arabinoxylanase and Feruloyl Esterase Activity in the Same Active Site.

Enzymes' uncharacterised side activities can have significant effects on reaction products and yields. Hence, their identification and characterisation are crucial for the development of successful reaction systems. Here, we report the presence of feruloyl esterase activity in CtXyn5A from Acetivibrio thermocellus, besides its well-known arabinoxylanase activity, for the first time. Activity analysis of enzyme variants mutated in the catalytic nucleophile, Glu279, confirmed removal of all activity for E279A and E279L, and increased esterase activity while removing xylanase activity for E279S, thus allowing the proposal that both reaction types are catalysed in the same active site in two subsequential steps. The ferulic acid substituent is cleaved off first, followed by hydrolysis of the xylan backbone. The esterase activity on complex carbohydrates was found to be higher than that of a designated ferulic acid esterase (E-FAERU). Therefore, we conclude that the enzyme exhibits a dual function rather than an esterase side activity.

Novel xylan-degrading enzymes from polysaccharide utilizing loci of Prevotella copri DSM18205.

Prevotella copri is a bacterium that can be found in the human gastrointestinal tract (GIT). The role of P. copri in the GIT is unclear, and elevated numbers of the microbe have been reported both in dietary fiber-induced improvement in glucose metabolism but also in conjunction with certain inflammatory conditions. These findings raised our interest in investigating the possibility of P. copri to grow on xylan, and identify the enzyme systems playing a role in digestion of xylan-based dietary fibers. Two xylan degrading polysaccharide utilizing loci (PUL10 and 15) were found in the genome, with three and eight glycoside hydrolase (GH) -encoding genes, respectively. Three of them were successfully produced in Escherichia coli: One extracellular enzyme from GH43 (subfamily 12, in PUL10, 60 kDa) and two enzymes from PUL15, one extracellular GH10 (41 kDa), and one intracellular GH43 (subfamily 137 kDa). Based on our results, we propose that in PUL15, GH10 (1) is an extracellular endo-1,4-ß-xylanase, that hydrolazes mainly glucuronosylated xylan polymers to xylooligosaccharides (XOS); while, GH43_1 in the same PUL, is an intracellular ß-xylosidase, catalyzing complete hydrolysis of the XOS to xylose. In PUL10, the characterized GH43_12 is an arabinofuranosidase, with a role in degradation of arabinoxylan, catalyzing removal of arabinose-residues on xylan.

Engineering CGTase to improve synthesis of alkyl glycosides.

Alkyl glycoside surfactants with elongated carbohydrate chains are useful in different applications due to their improved biocompatibility. Cyclodextrin glucanotransferases can catalyze the elongation process through the coupling reaction. However, due to the presence of a hydrophobic tail, the interaction between an alkyl glycoside acceptor and the active site residues is weaker than the interaction with maltooligosaccharides at the corresponding site. Here we report the mutations of F197, G263 and E266 near the acceptor subsites in the CGTase CspCGT13 from Carboxydocella sp. The results showed that substitutions of both F197 and G263 were important for the binding of acceptor substrate dodecyl maltoside during coupling reaction. The double mutant F197Y/G263A showed enhanced coupling activity and displayed a 2-fold increase of the primary coupling product using γ-cyclodextrin as donor when compared to wildtype CspCGT13. Disproportionation activity was also reduced, which was also the case for another double mutant (F197Y/E266A) that however not showed the corresponding increase in coupling. A triple mutant F197Y/G263A/E266A maintained the increase in primary coupling product (1.8-fold increase) using dodecyl maltoside as acceptor, but disproportionation was approximately at the same level as in the double mutants. In addition, hydrolysis of starch was slightly increased by the F197Y and G263A substitutions, indicating that interactions at both positions influenced the selectivity between glycosyl and alkyl moieties.

Synthesis of novel oligomeric anionic alkyl glycosides using laccase/TEMPO oxidation and cyclodextrin glucanotransferase (CGTase)-catalyzed transglycosylation.

Modification of alkyl glycosides, to alter their properties and widen the scope of potential applications, is of considerable interest. Here, we report the synthesis of new anionic alkyl glycosides with long carbohydrate chains, using two different approaches: laccase/2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) oxidation of a long-carbohydrate-chain alkyl glycoside and cyclodextrin glucanotransferase (CGTase)-catalyzed elongation of anionic alkyl glycosides. The laccase/TEMPO oxidation of dodecyl ß- d-maltooctaoside proceeded efficiently with the formation of aldehyde and acid products. However, depolymerization occurred to a large extent, limiting the product yield and purity. On the other hand, CGTase-catalyzed coupling/disproportionation reactions with α-cyclodextrin and dodecyl ß- d-maltoside diuronic acid (DDM-2COOH) or octyl ß- d-glucuronic acid (OG-COOH) as substrates gave high conversions, especially when the CGTase Toruzyme was used. It was found that pH had a strong influence on both the enzyme activity and the acceptor specificity. With non-ionic substrates (dodecyl ß- d-maltoside and octyl ß- d-glucoside), Toruzyme exhibited high catalytic activity at pH 5-6, but for the acidic substrates (DDM-2COOH and OG-COOH) the activity was highest at pH 4. This is most likely due to the enzyme favoring the protonated forms of DDM-2COOH and OG-COOH, which exist at lower pH (pKa about 3).

Chemoenzymatic synthesis of the pH responsive surfactant octyl ß-D-glucopyranoside uronic acid.

Methodology was developed to expand the range of benign alkyl glycoside surfactants to include also anionic types. This was demonstrated possible through conversion of the glycoside to its carboxyl derivative. Specifically, octyl ß-D-glucopyranoside (OG) was oxidised to the corresponding uronic acid (octyl ß-D-glucopyranoside uronic acid, OG-COOH) using the catalyst system T. versicolor laccase/2,2,6,6-tetramethylpiperidinyloxy (TEMPO) and oxygen from air as oxidant. The effects of oxygen supply methodology, concentrations of laccase, TEMPO and OG as well as reaction temperature were evaluated. At 10 mM substrate concentration, the substrate was almost quantitatively converted into product, and even at a substrate concentration of 60 mM, 85% conversion was reached within 24 h. The surfactant properties of OG-COOH were markedly dependent on pH. Foaming was only observed at low pH, while no foam was formed at pH values above 5.0. Thus, OG-COOH can be an attractive low-foaming surfactant, for example for cleaning applications and emulsification, in a wide pH range (pH 1.5-10.0).

Xylo- and arabinoxylooligosaccharides from wheat bran by endoxylanases, utilisation by probiotic bacteria, and structural studies of the enzymes.

Xylooligosaccharides (XOS) and arabinoxylooligosaccharides (AXOS) were produced from the insoluble arabinoxylan fraction of pretreated wheat bran by endoxylanases. The glycoside hydrolase (GH) family 10 xylanases GsXyn10A from Geobacillus stearothermophilus and RmXyn10A-CM from Rhodothermus marinus produced the AXOS A3X, A2XX and A2 + 3XX in addition to XOS. RmXyn10A-CM also produced XA2 + 3XX due to its non-conserved aglycone region accommodating additional arabinose substitutions in subsite +2. The GH11 enzymes, Pentopan from Thermomyces lanuginosus and NpXyn11A from Neocallimastix patriciarum had minor structural differences affecting hydrogen bonds in subsites -3 and +3, with similar hydrolysis profiles producing XA3XX as major AXOS and minor amounts of XA2XX but different ratios of X3/X2. In vitro analysis of the prebiotic properties of (A)XOS produced by Pentopan revealed nearly complete uptake of X2 and X3 by the probiotic bacteria Lactobacillus brevis and Bifidobacterium adolescentis. In contrast to previous reports, the GH43 arabinofuranosidase BaAXHd-3 from B. adolescentis cleaved α-1,3-linked arabinose on some single substituted AXOS.

Endo-xylanases as tools for production of substituted xylooligosaccharides with prebiotic properties.

Xylan has a main chain consisting of ß-1,4-linked xylose residues with diverse substituents. Endoxylanases cleave the xylan chain at cleavage sites determined by the substitution pattern and thus give different oligosaccharide product patterns. Most known endoxylanases belong to glycoside hydrolase (GH) families 10 and 11. These enzymes work well on unsubstituted xylan but accept substituents in certain subsites. The GH11 enzymes are more restricted by substituents, but on the other hand, they are normally more active than the GH10 enzymes on insoluble substrates, because of their smaller size. GH5 endoxylanases accept arabinose substituents in several subsites and require it in the - 1 subsite. This specificity makes the GH5 endoxylanases very useful for degradation of highly arabinose-substituted xylans and for the selective production of arabinoxylooligosaccharides, without formation of unsubstituted xylooligosaccharides. The GH30 endoxylanases have a related type of specificity in that they require a uronic acid substituent in the - 2 subsite, which makes them very useful for the production of uronic acid substituted oligosaccharides. The ability of dietary xylooligosaccharides to function as prebiotics in humans is governed by their substitution patterns. Endoxylanases are thus excellent tools to tailor prebiotic oligosaccharides to stimulate various types of intestinal bacteria and to cause fermentation in different parts of the gastrointestinal tract. Continuously increasing knowledge on the function of the gut microbiota and discoveries of novel endoxylanases increase the possibilities to achieve health-promoting effects.

ß-Mannanase-catalyzed synthesis of alkyl mannooligosides.

ß-Mannanases catalyze the conversion and modification of ß-mannans and may, in addition to hydrolysis, also be capable of transglycosylation which can result in enzymatic synthesis of novel glycoconjugates. Using alcohols as glycosyl acceptors (alcoholysis), ß-mannanases can potentially be used to synthesize alkyl glycosides, biodegradable surfactants, from renewable ß-mannans. In this paper, we investigate the synthesis of alkyl mannooligosides using glycoside hydrolase family 5 ß-mannanases from the fungi Trichoderma reesei (TrMan5A and TrMan5A-R171K) and Aspergillus nidulans (AnMan5C). To evaluate ß-mannanase alcoholysis capacity, a novel mass spectrometry-based method was developed that allows for relative comparison of the formation of alcoholysis products using different enzymes or reaction conditions. Differences in alcoholysis capacity and potential secondary hydrolysis of alkyl mannooligosides were observed when comparing alcoholysis catalyzed by the three ß-mannanases using methanol or 1-hexanol as acceptor. Among the three ß-mannanases studied, TrMan5A was the most efficient in producing hexyl mannooligosides with 1-hexanol as acceptor. Hexyl mannooligosides were synthesized using TrMan5A and purified using high-performance liquid chromatography. The data suggests a high selectivity of TrMan5A for 1-hexanol as acceptor over water. The synthesized hexyl mannooligosides were structurally characterized using nuclear magnetic resonance, with results in agreement with their predicted ß-conformation. The surfactant properties of the synthesized hexyl mannooligosides were evaluated using tensiometry, showing that they have similar micelle-forming properties as commercially available hexyl glucosides. The present paper demonstrates the possibility of using ß-mannanases for alkyl glycoside synthesis and increases the potential utilization of renewable ß-mannans.

Three in One: Temperature, Solvent and Catalytic Stability by Engineering the Cofactor-Binding Element of Amine Transaminase.

Amine transaminase (ATA) catalyse enantioselectively the direct amination of ketones, but insufficient stability during catalysis limits their industrial applicability. Recently, we revealed that ATAs suffer from substrate-induced inactivation mechanism involving dissociation of the enzyme-cofactor intermediate. Here, we report on engineering the cofactor-ring-binding element, which also shapes the active-site entrance. Only two point mutations in this motif improved temperature and catalytic stability in both biphasic media and organic solvent. Thermodynamic analysis revealed a higher melting point for the enzyme-cofactor intermediate. The high cofactor affinity eliminates the need for pyridoxal 5'-phosphate supply, thus making large-scale reactions more cost effective. This is the first report on stabilising a tetrameric ATA by mutating a single structural element. As this structural "hotspot" is a common feature of other transaminases it could serve as a general engineering target.

Development of in situ product removal strategies in biocatalysis applying scaled-down unit operations.

An experimental platform based on scaled-down unit operations combined in a plug-and-play manner enables easy and highly flexible testing of advanced biocatalytic process options such as in situ product removal (ISPR) process strategies. In such a platform, it is possible to compartmentalize different process steps while operating it as a combined system, giving the possibility to test and characterize the performance of novel process concepts and biocatalysts with minimal influence of inhibitory products. Here the capabilities of performing process development by applying scaled-down unit operations are highlighted through a case study investigating the asymmetric synthesis of 1-methyl-3-phenylpropylamine (MPPA) using ω-transaminase, an enzyme in the sub-family of amino transferases (ATAs). An on-line HPLC system was applied to avoid manual sample handling and to semi-automatically characterize ω-transaminases in a scaled-down packed-bed reactor (PBR) module, showing MPPA as a strong inhibitor. To overcome the inhibition, a two-step liquid-liquid extraction (LLE) ISPR concept was tested using scaled-down unit operations combined in a plug-and-play manner. Through the tested ISPR concept, it was possible to continuously feed the main substrate benzylacetone (BA) and extract the main product MPPA throughout the reaction, thereby overcoming the challenges of low substrate solubility and product inhibition. The tested ISPR concept achieved a product concentration of 26.5 gMPPA · L-1 , a purity up to 70% gMPPA · gtot-1 and a recovery in the range of 80% mol · mol-1 of MPPA in 20 h, with the possibility to increase the concentration, purity, and recovery further. Biotechnol. Bioeng. 2017;114: 600-609. © 2016 Wiley Periodicals, Inc.

Comparison of lipases and glycoside hydrolases as catalysts in synthesis reactions.

Lipases and glycoside hydrolases have large similarities concerning reaction mechanisms. Acyl-enzyme intermediates are formed during lipase-catalyzed reactions and in an analogous way, retaining glycoside hydrolases form glycosyl-enzyme intermediates during catalysis. In both cases, the covalent enzyme intermediates can react with water or other nucleophiles containing hydroxyl groups. Simple alcohols are accepted as nucleophiles by both types of enzymes. Lipases are used very successfully in synthesis applications due to their efficiency in catalyzing reversed hydrolysis and transesterification reactions. On the other hand, synthesis applications of glycoside hydrolases are much less developed. Here, important similarities and differences between the enzyme groups are reviewed and approaches to reach high synthesis yields are discussed. Useful strategies include the use of low-water media, high nucleophile concentrations, as well as protein engineering to modify the selectivity of the enzymes. The transglycosylases, hydrolases which naturally catalyze mainly transfer reactions, are of special interest and might be useful guides for engineering of other hydrolases.

Eliminating hydrolytic activity without affecting the transglycosylation of a GH1 ß-glucosidase.

Unveiling the determinants for transferase and hydrolase activity in glycoside hydrolases would allow using their vast diversity for creating novel transglycosylases, thereby unlocking an extensive toolbox for carbohydrate chemists. Three different amino acid substitutions at position 220 of a GH1 ß-glucosidase from Thermotoga neapolitana caused an increase of the ratio of transglycosylation to hydrolysis (r s/r h) from 0.33 to 1.45-2.71. Further increase in r s/r h was achieved by modulation of pH of the reaction medium. The wild-type enzyme had a pH optimum for both hydrolysis and transglycosylation around 6 and reduced activity at higher pH. Interestingly, the mutants had constant transglycosylation activity over a broad pH range (5-10), while the hydrolytic activity was largely eliminated at pH 10. The results demonstrate that a combination of protein engineering and medium engineering can be used to eliminate the hydrolytic activity without affecting the transglycosylation activity of a glycoside hydrolase. The underlying factors for this success are pursued, and perturbations of the catalytic acid/base in combination with flexibility are shown to be important factors.

Characterization of a family 43 ß-xylosidase from the xylooligosaccharide utilizing putative probiotic Weissella sp. strain 92.

In this work, we present the first XOS degrading glycoside hydrolase from Weissella, WXyn43, a two-domain enzyme from GH43. The gene was amplified from genomic DNA of the XOS utilizing Weissella strain 92, classified under the species-pair Weissella cibaria/W.confusa, and expressed in Escherichia coli. The enzyme is lacking a putative signal peptide and is, from a homology model, shown to be composed of an N-terminal 5-fold ß-propeller catalytic domain and a C-terminal ß-sandwich domain of unknown function. WXyn43 hydrolyzed short (1-4)-ß-D-xylooligosaccharides, with similar kcat/KM for xylobiose (X2) and xylotriose (X3) and clearly lower efficiency in xylotetraose (X4) conversion. WXyn43 displays the highest reported kcat for conversion of X3 (900 s(-1) at 37 °C) and X4 (770 s(-1)), and kcat for hydrolysis of X2 (907 s(-1)) is comparable with or greater than the highest previously reported. The purified enzyme adopted a homotetrameric state in solution, while a truncated form with isolated N-terminal catalytic domain adopted a mixture of oligomeric states and lacked detectable activity. The homology model shows that residues from both domains are involved in monomer-monomer hydrogen bonds, while the bonds creating dimer-dimer interactions only involved residues from the N-terminal domain. Docking of X2 and X3 in the active site shows interactions corresponding to subsites -1 and +1, while presence of a third subsite is unclear, but interactions between a loop and the reducing-end xylose of X3 may be present.

A CGTase with high coupling activity using γ-cyclodextrin isolated from a novel strain clustering under the genus Carboxydocella.

Cyclodextrin glucanotransferases (CGTases; EC 2.4.1.19) have mainly been characterized for their ability to produce cyclodextrins (CDs) from starch in an intramolecular transglycosylation reaction (cyclization). However, this class of enzymes can also catalyze intermolecular transglycosylation via disproportionation or coupling reactions onto a wide array of acceptors and could therefore be valuable as a tool for glycosylation.In this paper, we report the gene isolation, via the CODEHOP strategy, expression and characterization of a novel CGTase (CspCGT13) from a Carboxydocella sp. This enzyme is the first glycoside hydrolase isolated from the genus, indicating starch degradation via cyclodextrin production in the Carboxydocella strain. The fundamental reactivities of this novel CGTase are characterized and compared with two commercial CGTases, assayed under identical condition, in order to facilitate interpretation of the results. The comparison showed that the enzyme, CspCGT13, displayed high coupling activity using γ-CD as donor, despite preferentially forming α- and ß-CD in the cyclization reaction using wheat starch as substrate. Comparison of subsite conservation within previously characterized CGTases showed significant sequence variation in subsites -3 and -7, which may be important for the coupling activity.

The addition of transglutaminase improves the physical quality of extruded fish feed.

OBJECTIVES: The possibility of improving the physical quality of extruded fish feed using transglutaminase (TGase) treatment at different stages of the production process was investigated. RESULTS: The addition of TGase to the raw material mix and processing under high and medium moisture conditions significantly increased (p > 0.05) the durability, hardness and elasticity of fish feed pellets. However, the water stability of feeds was only improved when the TGase was applied in the vacuum coater; when it was mixed with the dry raw materials, the water stability of the product decreased. By further optimization of the enzyme dose when added to the vacuum coater, an increase in pellet durability was observed at enzyme dosages between 2.5 and 5 g kg(-1). Application of TGase in the coating step allowed an 87.5 % decrease in the dose of enzyme in feed produced under high moisture conditions. CONCLUSIONS: TGase treatment improves the physical quality of extruded fish feed, and the importance of optimization of enzyme dosage and processing conditions was demonstrated.

Immobilisation and application of lipases in organic media.

Different methods of preparing lipases for use in organic media are critically reviewed. Solid lipase preparations can be made by typical immobilisation methods such as adsorption, entrapment, covalent coupling or cross-linking. Immobilisation is especially attractive for lipases because, in addition to the normal benefits of enzyme immobilisation, it can also lead to a considerable increase in catalytic activity, probably caused by conformational changes in the lipase molecules. Activation can be achieved, for example, using hydrophobic support materials or surfactants during the immobilisation procedure. Surfactants can also be used to solubilise lipases in organic media via the formation of hydrophobic ion pairs, surfactant-coated lipase or reversed micelles. Lipase preparation methods are discussed with regard to potential lipase inactivation and activation effects, mass transfer limitations, lipase stability and other features important for applications. The practical applications of lipases in organic media reviewed include ester synthesis, modification of triacylglycerols and phospholipids, fatty acid enrichment, enantiomer resolution, biodiesel production and acylation of carbohydrates and bioactive compounds.

Effects of Regioselectivity and Lipid Class Specificity of Lipases on Transesterification, Exemplified by Biodiesel Production.

Lipase-catalyzed ethanolysis of triolein was studied as a model for biodiesel production. Four lipases were immobilized on porous polypropylene, and ethanolysis reactions were carried out in methyl t-butyl ether. The reaction products were analyzed using gas chromatography. Three of the four lipases studied were efficient in the conversion of triolein to 2-monoolein, but slow in the final step of producing glycerol. However, Candida antarctica lipase B was slow in the conversion of triolein, but more efficient in the subsequent two steps than the other lipases. The 1,3-selectivity of the lipases was less pronounced for the monooleins than for triolein. Silica gel was investigated as a catalyst for acyl migration, showing an increase in biodiesel yield with three of the lipases, but a reduction in yield when C. antarctica lipase B was used. The highest biodiesel yield (96 %) was obtained with a combination of Rhizopus arrhizus lipase and C. antarctica lipase B.

Xylanases and high-degree wet milling improve soluble dietary fibre content in liquid oat base.

The growth of plant-based food and drink substitutes has led to increased interest in oat-based milk substitute as a dairy milk alternative. Conventional liquid oat base (LOB) production results in a fibre-rich insoluble by-product and loss of valuable macronutrients. This study investigates the use of xylanase enzymes to release insoluble arabinoxylan (AX) fibre and employs different degrees of milling in the LOB manufacturing process, with the aim to reduce insoluble waste and simultaneously increase soluble dietary fibre in oat-based milk substitutes. The combination of decreased mill gap space from 1 to 0.05 mm and addition of GH10 xylanase, resulted in a homogenous LOB product and solubilization of all available AX. Potential prebiotic arabinoxylooligosaccharides of DP3-7 from GH10 hydrolysis were identified using HPAEC-PAD and MS analysis. These findings demonstrate the value of utilizing xylanases and fine-milling in LOB manufacturing, offering a sustainable approach to maximize health benefits of oat-based beverages.