Multiple Reaction Monitoring for quantitative laccase kinetics by LC-MS

AbstractLaccases (EC 1.10.3.2) are enzymes known for their ability to catalyse the oxidation of phenolic compounds using molecular oxygen as the final electron acceptor. Lignin is a natural phenylpropanoids biopolymer whose degradation in nature is thought to be aided by enzymatic oxidation by laccases. Laccase activity is often measured spectrophotometrically on compounds such as syringaldazine and ABTS which poorly relate to lignin. We employed natural phenolic hydroxycinnamates having different degree of methoxylations, p-coumaric, ferulic and sinapic acid, and a lignin model OH-dilignol compound as substrates to assess enzyme kinetics by HPLC-MS on two fungal laccases Trametes versicolor laccase, Tv and Ganoderma lucidum laccase, Gl. The method allowed accurate kinetic measurements and detailed insight into the product profiles of both laccases. Both Tv and Gl laccase are active on the hydroxycinnammates and show a preference for substrate with methoxylations. Product profiles were dominated by the presence of dimeric and trimeric species already after 10 minutes of reaction and similar profiles were obtained with the two laccases. This new HPLC-MS method is highly suitable and accurate as a new method for assaying laccase activity on genuine phenolic substrates, as well as a tool for examining laccase oxidation product profiles.</jats:p

The natural catalytic function of <i>CuG</i>E glucuronoyl esterase in hydrolysis of genuine lignin-carbohydrate complexes from birch

Abstract Background Glucuronoyl esterases belong to carbohydrate esterase family 15 and catalyze de-esterification. Their natural function is presumed to be cleavage of ester linkages in lignin–carbohydrate complexes particularly those linking lignin and glucuronoyl residues in xylans in hardwood. Results Here, we show for the first time a detailed product profile of aldouronic acids released from birchwood lignin by a glucuronoyl esterase from the white-rot fungus Cerrena unicolor (CuGE). CuGE releases substrate for GH10 endo-xylanase which results in significantly increased product release compared to the action of endo-xylanase alone. CuGE also releases neutral xylo-oligosaccharides that can be ascribed to the enzymes feruloyl esterase side activity as demonstrated by release of ferulic acid from insoluble wheat arabinoxylan. Conclusion The data verify the enzyme’s unique ability to catalyze removal of all glucuronoxylan associated with lignin and we propose that this is a direct result of enzymatic cleavage of the ester bonds connecting glucuronoxylan to lignin via 4-O-methyl glucuronoyl-ester linkages. This function appears important for the fungal organism’s ability to effectively utilize all available carbohydrates in lignocellulosic substrates. In bioprocess perspectives, this enzyme is a clear candidate for polishing lignin for residual carbohydrates to achieve pure, native lignin fractions after minimal pretreatment

Increased trans-glycosylation activity of hexosaminidases for synthesis of human milk oligosaccharides

It is well known that the composition of human breast milk differs significantly from the one of ordinary bovine milk. Especially the presence of sialylated and fucosylated oligosaccharides contributes to its health and development promoting features for newborn infants. [1] Nevertheless, not all newborns and especially premature infants sometimes cannot be breast fed for different reasons. For those children it is important that they receive a proper balanced formula product containing the above mentioned human milk oligosaccharides (HMOs). With respect to this we are developing new enzymatic routes for synthesis of sialylated and fucosylated oligosaccharides, which can be used as functional ingredient for infant formula. In a previous work two candidate hexoasaminidases (both belonging to the GH20 family) were identified from a metagenomic library, which were able to synthesize the basic HMO backbone structure, Lacto-N-triose II, from chitobiose and lactose by trans-glycosylation. [2] Since the yields using these enzymes were low (2% for hex1 and 8% for hex2 based on the donor substrate chitobiose) we wanted to increase their trans-glycosylation activity to increase their applicability for a feasible process. It was decided to follow a rational design approach first to keep the screening effort low. Therefore peptide pattern recognition (PPR) [3] analysis was performed on the whole GH20 CAZy family (approx. 3000 sequences) to identify other enzymes with potential trans-glycosylation activity based on relatedness. By phylogenetic analysis of the group containing the two known enzymes (approx. 1000 sequences) and subsequent alignment of the closely related sequences a loop insertion close to the active site was identified. Homology modelling revealed that introduction of this loop structure into hex1 and hex2 would lead to a significantly narrower active site and therefore contribute to exclusion of water from the active site, which is a well-known strategy to increase trans-glycosylation activity. The proposed loop mutants were then expressed, purified and characterized towards trans-glycosylation activity. For hex2 it turned out that none of the loop mutants showed an improved trans-glycosylation activity compared to the wild-type. But for hex1 three out of four showed an up to seven-fold improved trans-glycosylation activity compared to the wild-type, which refers even to a higher trans-glycosylation activity than previously observed for the hex2 wild-type. [4] In conclusion we succeeded in engineering an enzyme towards increased trans-glycosylation activity using a custom-made rational approach utilizing available sequence analysis methods. [1] L. Bode, Glycobiology 2012, 22, 1147–1162. [2] C. Nyffenegger, R. T. Nordvang, B. Zeuner, M. Łężyk, E. Difilippo, M. J. Logtenberg, H. A. Schols, A. S. Meyer, J. D. Mikkelsen, Appl. Microbiol. Biotechnol. 2015, 99, 7997–8009. [3] P. K. Busk, L. Lange, Appl. Environ. Microbiol. 2013, 79, 3380–3391. [4] S. B. Jamek, J. Muschiol, J. Holck, P. K. Busk, L. Lange, J. D. Mikkelsen, A. S. Meyer, 2017, manuscript submitted