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

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

It All Starts with a Sandwich: Identification of Sialidases with Trans-Glycosylation Activity

Sialidases (3.2.1.18) may exhibit trans-sialidase activity to catalyze sialylation of lactose if the active site topology is congruent with that of the Trypanosoma cruzi trans-sialidase (EC 2.4.1.-). The present work was undertaken to test the hypothesis that a particular aromatic sandwich structure of two amino acids proximal to the active site of the T. cruzi trans-sialidase infers trans-sialidase activity. On this basis, four enzymes with putative trans-sialidase activity were identified through an iterative alignment from 2909 native sialidases available in GenBank, which were cloned and expressed in Escherichia coli. Of these, one enzyme, SialH, derived from Haemophilus parasuis had an aromatic sandwich structure on the protein surface facing the end of the catalytic site (Phe168; Trp366), and was indeed found to exhibit trans-sialidase activity. SialH catalyzed production of the human milk oligosaccharide 3'-sialyllactose as well as the novel trans-sialylation product 3-sialyllactose using casein glycomacropeptide as sialyl donor and lactose as acceptor. The findings corroborated that Tyr119 and Trp312 in the T. cruzi trans-sialidase are part of an aromatic sandwich structure that confers trans-sialylation activity for lactose sialylation. The in silico identification of trans-glycosidase activity by rational active site topology alignment thus proved to be a quick tool for selecting putative trans-sialidases amongst a large group of glycosyl hydrolases. The approach moreover provided data that help understand structure-function relations of trans-sialidases

Characterization and immobilization of engineered sialidases from <em>Trypanosoma rangeli</em> for transsialylation

A sialidase (EC 3.2.1.18; GH 33) from non-pathogenic Trypanosoma rangeli has been engineered with the aim of improving its transsialylation activity. Recently, two engineered variants containing 15 and 16 amino acid substitutions, respectively, were found to exhibit significantly improved transsialylation activity: both had a 14 times higher ratio between transsialylation and hydrolysis products compared to the first reported mutant TrSA5mut. In the current work, these two variants, Tr15 and Tr16, were characterized in terms of pH optimum, thermal stability, effect of acceptor-to-donor ratio, and acceptor specificity for transsialylation using casein glycomacropeptide (CGMP) as sialyl donor and lactose or other human milk oligosaccharide core structures as acceptors. Both sialidase variants exhibited pH optima around pH 4.8. Thermal stability of each enzyme was comparable to that of previously developed T. rangeli sialidase variants and higher than that of the native transsialidase from T. cruzi (TcTS). As for other engineered T. rangeli sialidase variants and TcTS, the acceptor specificity was broad: lactose, galactooligosaccharides (GOS), xylooligosaccharides (XOS), and human milk oligosaccharide structures lacto-N-tetraose (LNT), lacto-N-fucopentaose (LNFP V), and lacto-N-neofucopentaose V (LNnFP V) were all sialylated by Tr15 and Tr16. An increase in acceptor-to-donor ratio from 2 to 10 had a positive effect on transsialylation. Both enzymes showed high preference for formation α(2,3)-linkages at the non-reducing end of lactose in the transsialylation. Tr15 was the most efficient enzyme in terms of transsialylation reaction rates and yield of 3’-sialyllactose. Finally, Tr15 was immobilized covalently on glyoxyl-functionalized silica, leading to a 1.5-fold increase in biocatalytic productivity (mg 3’-sialyllactose per mg enzyme) compared to free enzyme after 6 cycles of reuse. The use of glyoxyl-functionalized silica proved to be markedly better for immobilization than silica functionalized with (3-aminopropyl)triethoxysilane (APTES) and glutaraldehyde, which resulted in a biocatalytic productivity which was less than half of that obtained with free enzyme

Open Source Software Acquisition: Beyond the Business Case

Lately we have seen a growing interest from both public and private organisations to adopt Open Source Software (OSS), not only for a few, specific applications but also on a more general level throughout the organisation. As a consequence, the organisations’ decisions on adoption of OSS are becoming increasingly more important and complex. We present three perspectives organisations can employ in their decisions: seeing OSS acquisition as a business case, as COTS acquisition, and as architectural change within a governance framework. We present case studies of decisions on OSS adoption, and categorise the decision criteria we have found. Our results indicate that for large-scale adoption of OSS, focus will be on architectural considerations: enterprise-wide architectures will at first be a barrier, but in the long term OSS’s support of open standards can be a major enabler for OSS adoption. In contrast, in smaller organisations and in small-scale adoption of OSS, the cheap price of OSS is a major enabler, as it provides a good opportunity for experiments and short-term economic benefits. For small organisations these experiments can lead to development of a common IT-architecture, and in larger organisations OSS can be adopted in niche-areas, without significantly violating an existing IT-architecture

Enterprise Achitecture versus Total Cost of Ownership

Abstract: The paper presents an explorative study of Open Source Software (OSS) focusing on the managerial decisions for acquisition of OSS. Based on three case studies we argue that whereas small organizations often may chose adoption of OSS expecting significant cost savings, a major barrier for larger organizations’ adoption of OSS lies in the organizations’ consolidation of the enterprise architectures, in addition to that OSS will not be adopted before satisfactory delivery and procurement models for OSS are established. Keywords: Open Source Software, OSS, Enterprise Architecture, Total Cost of Ownership, Delivery and Procurement Model